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appreciate the time taken to perform your review(s) successfully. mahsa abassi kabir abdullahi teferi m. abera oyekola v. abiri olusola a. adejumo olukemi adekanmbi adeyemi adeyemo ezekiel rachel akinkunmi george alemnji r. joseph alexis william k. ampofo timothy amukele kingsley anukam mustafa arasa kevin arien george aryee rosemary a. audu george awungafac funmilola ayeni françois–xavier babin tony badrick fay betsou stephanie bispo debrah i. boeras andre st. aubyn bowers qi chen linda cherepow nelson chimbiya richard b. clark oksana debrah kassaye t. desta alpha a. diallo wayne dimech jienchi dorward robert eley mohammed a. elsheikh zahra eslamirad emmanuel fajardo samuel a. fayemiwo noah fongwen adeola fowotade tiago selbach garcia maria gazouli lisa gerwing-adima philip m. giffard mark a. giffen jr kjell grankvist alex greninger tomáš hanke lucia hans bernice harris william p. hausdorff marianne k. henderson pamela hepple susan horton ekua houphouet offiong f. ikpatt zeynep ozturk inal md. sadequl islam sam kariuki aderemi kehinde stephen b. kennedy luc kestens charles kiyaga jerzy klijanienko kekoura kourouma chika kusano george boateng kyei majda laboudi sylvia lacourse william lali kim lewis raphael lihana direk limmathurotsakul smijlika de lussigny megha maheshwari alexander martin-odoom talkmore maruta patrick mateta kamran mirza meade morgan anneta f. naidoo nicaise ndembi brooke nichols nicholas i. nii-trebi jean n. nikiema patrick njukeng michael a. noble jacinta nwogu iheanyi o. okonko ahmed olowo-okere sien ombelet pascale ondoa japheth opintan philip o. oshun collins o. otieno chin-yih ou pragyan s. panda ursula panzer ketan patel mario plebani nira pollock oluwafemi akinyele popoola philomena raftery michele ramsay sarah riley lucy j. robertson julia k. rohr debra de assis rosa ahmed saleh david jahbiuk sambian paul sandstrom christine e. schaner-tooley mary e. schmitz heidi schütt-gerowitt yan shen adebayo shittu cassandra soo derek r. stein timothy su anthony p. sunjaya catherine g. sutcliffe tomas szotkowski rosemary tambouret maria a. telles merih tesfazghi ajaykumar k. thirumala ralph timperi cristina touriño florette treurnicht raquel villegas lara vojnov hongping wei shannon whitmer peter r. williamson feng xu nicole young peter young anissa zaouak http://www.ajlmonline.org� https://ajlmonline.org� https://ajlmonline.org� https://ajlmonline.org� https://ajlmonline.org/index.php/ajlm/user https://ajlmonline.org/index.php/ajlm/user mailto:publishing@aosis.co.za ajlm 9(2)_2020_contents.indd http://www.ajlmonline.org open access table of contents i lessons from the field driving the usage of tuberculosis diagnostic data through capacity building in lowand middle-income countries natasha gous, alaine u. nyaruhirira, bradford cunningham, chris macek african journal of laboratory medicine | vol 9, no 2 | a1092 | 18 november 2020 review article pooled testing: a tool to increase efficiency of infant hiv diagnosis and virological monitoring wolfgang preiser, gert u. van zyl african journal of laboratory medicine | vol 9, no 2 | a1035 | 11 august 2020 opinion paper building diagnostic systems in sierra leone: the role of point-of-care devices in laboratory strengthening rashid ansumana, fatmata bah, kan biao, doris harding, mohamed b. jalloh, ann h. kelly, francess koker, zikan koroma, mambu momoh, mohamed h. rogers, james rogers, alice street, eva vernooij, isatta wurie african journal of laboratory medicine | vol 9, no 2 | a1029 | 01 april 2020 opinion paper nigeria’s efforts to strengthen laboratory diagnostics – why access to reliable and affordable diagnostics is key to building resilient laboratory systems dhamari naidoo, chikwe ihekweazu african journal of laboratory medicine | vol 9, no 2 | a1019 | 26 august 2020 opinion paper preparing national tiered laboratory systems and networks to advance diagnostics in africa and meet the continent’s health agenda: insights into priority areas for improvement pascale ondoa, nqobile ndlovu, mah-sere keita, marguerite massingaloembe, yenew kebede, collins odhiambo, teferi mekonen, aytenew ashenafi, amha kebede, john nkengasong african journal of laboratory medicine | vol 9, no 2 | a1103 | 21 september 2020 35 41 48 53 58 page i of i table of contents i editorial connected diagnostics systems: the future of disease control in africa noah fongwen, debi boeras, rosanna w. peeling, timothy amukele african journal of laboratory medicine | vol 9, no 2 | a1365 | 21 december 2020 original research field evaluation of a mobile biosafety laboratory in senegal to strengthen rapid disease outbreak response and monitoring cheikh fall, aurélie cappuyns, oumar faye, steven pauwels, gamou fall, ndongo dia, moussa m. diagne, cheikh t. diagne, makhtar niang, alassane mbengue, martin faye, idrissa dieng, babacar gningue, abdoulaye bousso, ousmane faye, rudi pauwels, amadou a. sall african journal of laboratory medicine | vol 9, no 2 | a1041 | 20 august 2020 lessons from the field timely delivery of laboratory efficiency information, part i: developing an interactive turnaround time dashboard at a high-volume laboratory naseem cassim, manfred e. tepper, lindi m. coetzee, deborah k. glencross african journal of laboratory medicine | vol 9, no 2 | a947 | 29 april 2020 lessons from the field timely delivery of laboratory efficiency information, part ii: assessing the impact of a turn-around time dashboard at a high-volume laboratory naseem cassim, lindi m. coetzee, manfred e.e. tepper, louella perelson, deborah k. glencross african journal of laboratory medicine | vol 9, no 2 | a948 | 29 april 2020 lessons from the field an open-source molecular diagnostic platform approach for outbreak and epidemic preparedness devy m. emperador, laura t. mazzola, cassandra kelly-cirino african journal of laboratory medicine | vol 9, no 2 | a1017 | 28 september 2020 1 3 12 21 29 vol 9, no 2 (2020) special collection: the future of diagnostics issn: 2225-2002 (print) | issn: 2225-2010 (online)african journal of laboratory medicine abstract introduction ethical considerations case presentation management and outcome discussion acknowledgements references about the author(s) barend mitton department of medical microbiology, university of pretoria, pretoria, south africa tshwane academic division, department of medical microbiology, national health laboratory service, pretoria, south africa roxanne rule department of medical microbiology, university of pretoria, pretoria, south africa tshwane academic division, department of medical microbiology, national health laboratory service, pretoria, south africa nontombi mbelle department of medical microbiology, university of pretoria, pretoria, south africa tshwane academic division, department of medical microbiology, national health laboratory service, pretoria, south africa wesley van hougenhouck-tulleken division of nephrology, department of internal medicine, university of pretoria, pretoria, south africa department of internal medicine, steve biko academic hospital, pretoria, south africa mohamed said department of medical microbiology, university of pretoria, pretoria, south africa tshwane academic division, department of medical microbiology, national health laboratory service, pretoria, south africa citation mitton b, rule r, mbelle n, van hougenhouck-tulleken w, said m. post-procedural bacillus cereus septic arthritis in a patient with systemic lupus erythematosus. afr j lab med. 2020;9(1), a1119 https://doi.org/10.4102/ajlm.v9i1.1119 case study post-procedural bacillus cereus septic arthritis in a patient with systemic lupus erythematosus barend mitton, roxanne rule, nontombi mbelle, wesley van hougenhouck-tulleken, mohamed said received: 06 nov. 2019; accepted: 27 may 2020; published: 20 aug. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract introduction: bacillus species are often considered as contaminants when cultured from clinical samples. bacillus cereus may be a pathogen in certain circumstances and is known to cause musculoskeletal infections. this report aims to educate clinicians and clinical microbiology laboratories on b. cereus musculoskeletal infections and to heighten awareness that bacillus species should not always be dismissed as contaminants. case presentation: we report the case of a patient who presented to a tertiary hospital in pretoria, south africa, in november 2018 with b. cereus septic arthritis and underlying systemic lupus erythematosus (sle). the isolate would otherwise have been dismissed as a contaminant had it not been for the crucial interaction between the laboratory and the treating clinicians. to our knowledge, this is the first case report of septic arthritis caused by b. cereus in an sle patient where the organism was cultured from the joint specimen. identification of the organism was performed using matrix-assisted laser desorption/ionisation mass spectrometry. management and outcome: definitive treatment was with intravenous vancomycin, continued for four weeks, in addition to arthroscopy and management of the underlying sle. the patient had a good clinical outcome and regained full mobility. conclusion: musculoskeletal infections, specifically septic arthritis caused by b. cereus, are exceedingly rare infections. immune suppression, trauma, prosthetic implants and invasive procedures are important risk factors for b. cereus musculoskeletal infections. close collaboration with a multi-disciplinary team approach will effect the best outcome for complicated patients with b. cereus infections. keywords: bacillus cereus; septic arthritis; systemic lupus erythematosus; matrix-assisted laser desorption/ionisation mass spectrometry; maldi-tof ms; musculoskeletal infection; arthroscopy. introduction as the vast majority of bacillus species are non-pathogenic and ubiquitous in the environment, many clinical microbiologists and clinicians dismiss bacillus species cultured from clinical specimens as contaminants. occasionally, this results in a missed diagnosis and inappropriate clinical decision-making. this case is important, because it illustrates the importance of communication between clinicians and the clinical laboratory staff in determining the significance of culture results. this report aims to educate healthcare workers on bacillus cereus joint infections and further endeavours to assist healthcare practitioners in distinguishing when this organism should be dismissed as a contaminant and when it should be considered as a pathogen. ethical considerations written, informed consent was obtained from the patient. this research was approved by the university of pretoria, faculty of health sciences, research ethics committee (ethics reference number 133/2019). case presentation in november 2018, a 32-year-old male was referred to a tertiary academic hospital in pretoria, south africa, with a non-resolving septic arthritis of his right knee. the patient presented to a secondary hospital 10 days prior with a tender, swollen right knee, with no history of trauma. he underwent an arthroscopy at that centre and received intravenous amoxicillin-clavulanic acid, with a suboptimal response. in addition, he developed symptoms suggestive of systemic lupus erythematosus (sle), which included polyarthritis, xerostomia, raynaud’s phenomenon, proteinuria and confusion. on examination he was haemodynamically stable with a pulse rate of 100 beats per minute, a blood pressure of 117/81 mmhg and a temperature of 36.5 °c. he had asymmetric polyarthritis, involving the right elbow, left wrist, right knee and both ankles. the right knee was the worst affected, with swelling, erythema and tenderness on examination. an emergency arthroscopy of the right knee was performed, revealing a purulent effusion. intravenous ceftriaxone (1 g twice daily) was started empirically. x-rays of all affected joints revealed no accompanying osteomyelitis. no other imaging of the joints was done. the diagnosis of sle was confirmed based on a systemic lupus international collaborating clinics score1 of five (anti-nuclear antibody positive, lupus nephritis class 3, arthritis, low c3 and neurologic sle). laboratory investigations admission blood test revealed a white cell count of 8.04 × 109 cells/l with neutrophilia (73%), a c-reactive protein of 107 mg/l, a positive anti-nuclear antibody (titre 160) and a low c3 (0.50 g/l). in addition, an epstein–barr virus viraemia of 530 copies/ml was found. admission blood cultures had no growth. a pus sample taken during arthroscopy showed numerous gram-positive bacilli on the direct gram stain. culture revealed large, flat, grey, beta-haemolytic colonies on 5% horse blood agar (figure 1), which also grew on chocolate agar and macconkey agar. this isolate was identified as bacillus species and reported as a possible contaminant. the patient had a poor clinical response to the empiric ceftriaxone after 6 days of treatment, and a request was made by the attending clinicians for antimicrobial susceptibility testing on the isolate. the isolate was therefore referred for further identification to species level using matrix-assisted laser desorption/ionisation mass spectrometry by means of vitek® ms (biomérieux, marcy l’etoile, france), instrument software version 1.5.0.4, myla® version 4.5.1 (biomérieux), knowledge base (database) version 3.2. the isolate was identified as bacillus cereus. antibiotic susceptibilities were performed by etest® (biomérieux, marcy l’etoile, france) and interpreted using the clinical & laboratory standards institute m45 (2015) breakpoints.2 the isolate was resistant to penicillin (minimum inhibitory concentration [mic] 32 µg/ml), and cefotaxime (mic 32 µg/ml), but susceptible to vancomycin (mic 4 µg/ml) and imipenem (mic 4 µg/ml). figure 1: typical morphology of bacillus cereus on 5% horse blood agar (pretoria, south africa, 19 march 2019). management and outcome based on the report, the patient was started on intravenous vancomycin 1 g twice daily. the dose was adjusted to achieve a target vancomycin blood trough level of 15 mg/ml – 20 mg/ml. trough levels were monitored roughly every 3 days over the treatment period, and the dosage adjusted accordingly. no vancomycin-related adverse events occurred during this time. vancomycin was continued for 4 weeks; over this period, the pain and swelling improved dramatically, inflammatory markers normalised and the patient regained mobility. management of the sle included prednisone, mycophenolate mofetil and chloroquine, with good response. discussion bacillus species are gram-positive, aerobic or facultative anaerobic sporulating bacilli, which are ubiquitous in the environment.3 over 100 species are known to belong to the genus.3 bacillus cereus is a common cause of food poisoning and occasionally causes opportunistic infections, usually in vulnerable hosts. these infections include ophthalmic infections, wound infections, septicaemia, endocarditis, meningitis, necrotising pneumonia and orthopaedic infections.3,4 important virulence factors of b. cereus include production of toxins and the formation of biofilms and spores.4 except for b. cereus and b. anthracis, the genus bacillus is rarely associated with disease.3,5,6,7 therefore, bacillus species are often reported as contaminants, even when cultured from sterile specimens. this may result in a delay in correct diagnosis and inappropriate treatment. bacillus cereus grows well on most routine culture media such as blood agar (figure 1), chocolate agar and macconkey agar. routine laboratory tests will reveal large boxcar-shaped, gram-positive bacilli (figure 2) that are catalase positive. however, identification to species level requires specialised techniques such as matrix-assisted laser desorption/ionisation mass spectrometry or molecular techniques.7 figure 2: microscopic image at 1000 × magnification showing boxcar-shaped, gram-positive bacilli, typical of bacillus cereus (pretoria, south africa, 19 march 2019). musculoskeletal infections caused by b. cereus are rare but have been previously reported in literature.4,5,6,7 åkesson, hedströum and ripa.4 reported 12 cases of b. cereus orthopaedic infections, all occurring in post-operative or post-traumatic wounds, whilst dubouix et al.5 reported 41 cases with b. cereus wound infections associated with open fractures. gallo et al.6 reported two cases of b. cereus prosthesis-related septic arthritis, which were culture negative but identified using pcr-mass-spectrometric-technology and fluorescence in situ hybridisation of tissue. ha et al.7 reported a single case of late prosthetic joint infection with b. cereus that occurred 13 years after total hip replacement surgery, confirmed by 16s ribosomal ribonucleic acid (rrna) sequencing. in total, we found 56 cases reported over 25 years, highlighting the rarity of b. cereus musculoskeletal infections and emphasising the difficulty in making a definitive diagnosis. the scarcity of these infections may actually be the result of under-reporting, as bacillus species are often reported as contaminants. bacillus cereus produces β-lactamases and is resistant to most β-lactam antibiotics, except carbapenems.3 classically, bacillus species are susceptible to vancomycin, aminoglycosides and fluoroquinolones, whilst resistance to erythromycin, tetracycline and even carbapenems have been reported.3 the empiric antibiotic of choice for invasive b. cereus infections is vancomycin.3,7 in addition to administering antibiotics, it is imperative to obtain source control at the infected site, as b. cereus is known to form biofilm. the removal of implanted medical devices may be necessary.7 the concomitant sle may be considered a notable risk factor for invasive b. cereus infection in this patient. it is uncertain if the epstein–barr virus viraemia played a significant role in predisposing the patient further to this infection. however, both sle and epstein–barr virus infection are known to be immune–modulatory, down-regulating the innate and humoral systems and placing the patient at increased risk of opportunistic infections.8,9 an additional risk factor was the preceding arthroscopy, which may have introduced spores into the joint space. to our knowledge, this is the first case of septic arthritis in an sle patient where the organism was cultured from the joint specimen. the communication between the clinical and microbiology teams ensured that the organism was identified to species level and that antibiotic susceptibility testing was performed, resulting in a favourable outcome for the patient. conclusion bacillus species are often regarded as contaminants and receive little attention from the medical community. in certain high-risk patient groups, however, b. cereus may be a formidable pathogen. clinical microbiology laboratorians and clinicians should have a high index of suspicion in these patients and identify bacillus species in cultures from sterile sites to species level as well as perform antibiotic susceptibility when b. cereus is identified. immune suppression, trauma, prosthetic implants and invasive procedures are important risk factors for b. cereus musculoskeletal infections. bacillus cereus is universally resistant to most β-lactam antibiotics, with the exception of carbapenems. treatment with vancomycin was successful in the case described. close collaboration with a multi-disciplinary team approach will effect the best outcome for complicated patients with b. cereus infections. acknowledgements competing interests the authors have declared that no competing interests exist. authors’ contributions b.m. designed the data collection tools, collected case data, analysed the data, and drafted and revised the paper. b.m. was also the guarantor. r.r. collected case data, and drafted and revised the paper. n.m. drafted and revised the paper. w.v.h-t. collected case data, analysed the data, and drafted and revised the paper. m.s. designed the data collection tools, analysed the data, and drafted and revised the paper. sources of support this research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references petri m, orbai a–m, alarcón gs, et al. derivation and validation of the systemic lupus international collaborating clinics classification criteria for systemic lupus erythematosus. arthritis rheum. 2012;64(8):2677–2686. https://doi.org/10.1002/art.34473 clinical and laboratory standards institute (clsi). methods for antimicrobial dilution and disk susceptibility testing of infrequently isolated or fastidious bacteria. 3rd ed. clsi guideline m45. wayne, pa: clinical and laboratory standards institute; 2015. bottone ej. bacillus cereus, a volatile human pathogen. clin mircobiol rev. 2010;23(2):382–398. https://doi.org/10.1128/cmr.00073-09 åkesson a, hedströum så, ripa t. bacillus cereus: a significant pathogen in postoperative and post-traumatic wounds on orthopaedic wards. scand j infect dis. 1991;23(1):71–77. https://doi.org/10.3109/00365549109023377 dubouix a, bonnet e, alvarez m, et al. bacillus cereus infections in traumatology – orthopaedics department: retrospective investigation and improvement of healthcare practices. j infect. 2005;50(1):22–30. https://doi.org/10.1016/j.jinf.2004.05.012 gallo ph, melton-kreft r, nistico l, et al. demonstration of bacillus cereus in orthopaedic-implant-related infection with use of a multi-primer polymerase chain reaction-mass spectrometric assay: report of two cases. j bone joint surg. 2011;93(15):e85. https://doi.org/10.2106/jbjs.j.01181 ha j, park yj, kim yj, oh hc. late prosthetic joint infection and bacteremia by bacillus cereus confirmed by 16s rrna sequencing and hip joint tissue pathology. ann clin microbiol. 2016;19(2):54–57. https://doi.org/10.5145/acm.2016.19.2.54 ning s. innate immune modulation in ebv infection. herpesviridae. 2011;2(1):1. https://doi.org/10.1186/2042-4280-2-1 tikly m, navarra sv. lupus in the developing world – is it any different? best pract res clin rheumatol. 2008;22(4):643–655. https://doi.org/10.1016/j.berh.2008.05.003 what is the problem? blood donation shortages what are the causes of reduced blood supply and donations? what is the impact of decreased blood donations? how do we address blood shortages? acknowledgements references about the author(s) kenneth b. david faculty of pharmaceutical sciences, kaduna state university, kaduna, nigeriahull york medical school, university of hull, hull, united kingdom knovicks simfukwe school of veterinary medicine, university of zambia, lusaka, zambia mohamed b. musa faculty of pharmacy, omdurman islamic university, khartoum, sudan steven munharo training and research unit of excellence, college of medicine, university of malawi, blantyre, malawi don e. lucero-prisno iii department of global health and development, london school of hygiene and tropical medicine, london, united kingdom citation david kb, simfukwe k, musa mb, munharo s, lucero-prisno iii de. impact of covid-19 on blood donation and supply in africa. afr j lab med. 2021;10(1), a1408 https://doi.org/10.4102/ajlm.v10i1.1408 opinion paper impact of covid-19 on blood donation and supply in africa kenneth b. david, knovicks simfukwe, mohamed b. musa, steven munharo, don e. lucero-prisno iii received: 27 sept. 2020; accepted: 23 june 2021; published: 25 oct. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. what is the problem? blood donation shortages as the coronavirus disease 2019 (covid-19) continues to spread in africa, unprecedented disruptions at all levels of human endeavours including healthcare delivery systems have been recorded.1 one of the major areas of healthcare systems affected is blood supply – a commodity needed for the survival of many patients. blood and blood products such as cryoprecipitate, plasma, immune globulins, platelets, etc., are required for the management of medical conditions including but not limited to trauma, renal impairment, cancer, sickle cell anaemia, haemorrhagic shock, and other medical conditions related to acute or chronic loss of blood. despite the covid-19 pandemic, health problems and accidents occur including maternal morbidities, malnutrition, blood-transmitted infectious diseases (hiv and aids, hepatitis c virus infection, hepatitis b virus infection, syphilis and anaemia-inducing infectious diseases such as malaria, which is particularly worse in rainy seasons in countries such as malawi and nigeria,2 kidney disease, liver disease, and cancer, etc.). hence, blood donation shortages caused by the covid-19 pandemic are wrecking africa’s already overwhelmed blood transfusion services and are a guaranteed threat to a positive patient outcome, particularly for children under the age of 5, who are the recipients of 54% of the 118.5 million blood collected in low-income countries.3 in countries that rely on voluntary blood donations, particularly from students, the trends of blood supply and donation are likely to decline in many other countries if the covid-19 pandemic continues. this will consequently and adversely affect beneficiaries in various hospitals, particularly in countries whose sole blood donors are volunteers and students.4 what are the causes of reduced blood supply and donations? blood transfusions save lives, hence the need to maintain an adequate supply of blood. blood donors have made it possible for blood to be available for transfusion to other patients. these donors can either be family members, voluntary non-renumerated donors, or paid donors. the world health organization preferentially recommends voluntary non-renumerated donors as the major source of blood for transfusion purposes.5,6 the uncertainty in the pattern of blood demand and supply and the inadequacy of human resources (which can be reduced due to ill health) are some of the most pressing challenges posed by the covid-19 pandemic.7 the major contributors to low donations in africa are restricted movements, lockdowns, and closure of blood donation institutions.8 others include fear of contracting covid-19 by both donors and healthcare workers due to limited knowledge on severe acute respiratory syndrome coronavirus 2 (sars-cov-2) transmission. in zambia, for instance, there was a sharp decline in the number of blood donations because donors were afraid that by donating their blood, they could contract the virus. the lockdown imposed in the country was also a precipitating factor.5 however, the challenge might be more severe in countries where blood donation is not a culture. for instance, in kenya, less than 1% of the 47 million kenyans donated blood in the 2018/2019 year.9 in south africa, there was a decreased blood supply and increased blood demand when schools and colleges were shut down because over 30% of the below 1% daily blood donors are younger than 25 years old.10 what is the impact of decreased blood donations? covid-19 has caused some blood transfusion centres to miss their blood collection targets. for instance, the zambia national blood transfusion services target for the first quarter of 2020 was 18 750 units, but the institution barely collected 6516 units, representing 34.7% of the initial target.5 the botswana blood transfusion service had enjoyed an annual collection of 25 000 units, but due to covid-19 preventive measures, only 11 000 units had been collected as of may 2020. this implied an impending decrease in the units of blood obtainable in a pandemic-affected year.8 the national blood transfusion services in botswana was able to collect only about 23 000 units of blood and blood products against the 45 000 units set as the target for the year 2020.11 in kenya, covid-19 has also reduced blood collection manpower (staff) due to ill health, social distancing measures placed in all institutions, and the lack of sufficient effective protective equipment to ensure staff and client safety.9 a study conducted at a teaching hospital in nigeria revealed that 71.4% of patients with various types of malignancies such as acute myeloid leukaemia, bladder cancer, extra-orbital malignancy, prostate cancer, and lung cancer died due to the lockdown imposed in the country which disrupted the availability of blood donors.12 to this end, transfusion institutions – both hospital and independent blood transfusion units – have a key role to play in monitoring the demand and supply of blood and blood products to ensure that these products are adequately available. how do we address blood shortages? when considering issues related to the availability of blood and blood products for clinical management, the principle of demand and supply should be evaluated.13 decrease demand proactive measures aimed at reducing demand and increasing the supply of blood and blood products must be implemented, particularly, at this time of the covid-19 pandemic. these include patient blood management and strengthened cross-matching protocols for allogenic blood transfusion to reduce repeat transfusion.10 increase supply to maintain supply, intense mobilisation campaigns and safer donation protocols are required. mobilisation campaigns should be aimed at creating awareness: educating the populace on the importance of blood donations, debunking covid-19-related blood donation myths, and sharing extra safety and screening measures instituted in the facilities.14 unlike the recorded decline in the number of donated blood and blood products in africa, india for instance devised ways to boost the confidence level of donors for outdoor blood donation drives during the lockdown period. this allowed the blood bank to receive adequate supplies of blood amid the pandemic.14 some of the measures taken to boost the confidence of donors during the pandemic included the education of both staff and blood donors on covid-19 preventive measures, the observation of safety protocols while conveying the donors to the venue of the blood donation drive, the provision of face masks and other personal protective equipment to all donors and staff, the compulsory use of hand sanitisers, and the use of infrared thermometers to check the body temperature of all the donors. also, the use of ‘namaste’ as the mode of greetings to avoid handshakes was reinforced.14 like other coronaviruses, sars-cov-2 infects the upper and lower respiratory tract and its rna is shed into the serum or plasma of infected persons. thus, some studies reported that sars-cov-2 can be detected in either blood plasma or serum.15,16 theoretically, this implies that blood recipients can be infected with sars-cov-2 if they receive infected blood or blood products. however, wang and his co-authors in 202015 disagreed with this theoretical claim by saying: ‘to date, there are no reported cases of sars-cov-2 transmission by any blood product but transfusion transmission cannot yet be completely excluded’. also, the world health organization has stated that the role of blood-borne transmission of sars-cov-2 is uncertain as the very low viral titres in plasma suggest a low risk of blood-borne transmission of the virus.17 fortunately, there has not been any reported case of sars-cov-2 transmission via transfusion in any country.15 consequently, it is not mandated for blood donors to get tested for sars-cov-2.17 however, because of the high number of asymptomatic covid-19 infections, the safety of blood donors and staff needs to be ensured. aside from the routine screening for infectious diseases, the screening of potential donors for covid-19 exposure and symptoms should be done before donation, that is, at the time of donor clearance. the world health organization recommends that donor restrictions be modified according to the covid-19 community transmission status so as not to worsen the unavailability of donated blood. some of the guidelines include sensitising donors about self-deferring upon the onset of symptoms, and instructing donors to inform the blood collection centre if they develop a respiratory illness. deferring lasts for 14 days once a positive test is confirmed and delaying lasts for another 14 days after recovering from covid-19 symptoms.18,19 improve management finally, blood inventory management should be implemented to prevent stockouts in emergency cases, collected blood and blood products should be harnessed judiciously.14 conclusion covid-19 is a threat to blood transfusion services in many countries, particularly lower-income countries. with a recorded decline in blood collection rates and missed targets, the impact casts a shadow on health and health outcomes amid the covid-19 pandemic. covid-19 offers african governments a chance to intensify efforts to effectively tackle all the uncertainties posed by the pandemic,20 implement responsive systems, and strategise effective ways to reach yearly targets even in pandemics.21 with steps taken to judiciously utilise available blood and broaden the sources of blood donation amid the pandemic, the impact of covid-19 will be mitigated. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions k.b.d. and d.e.l-p. iii conceived the idea. k.b.d., k.s., m.b.m. and s.m. wrote the draft of the manuscript. d.e.l-p. iii reviewed and assisted with data collection and the language edit. all of the authors have read and agreed to the final manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. data availability data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references david kb, thomas n, solomon jk. epidemiology of covid-19 in africa: daily cumulative index and mortality rate. int j infect control. 2020;16(2). https://doi.org/10.3396/ijic.v16i2.008.20 cho hj, koo jw, roh sk, et al. covid-19 transmission and blood transfusion: a case report. j infect public health. 2020;13(11):1678–1679. https://doi.org/10.1016/j.jiph.2020.05.001 who. blood safety and availability [homepage on the internet]. 2020 [cited 2020 may 3]. available from: https://www.who.int/news-room/fact-sheets/detail/blood-safety-and-availability wang y, han w, pan l, et al. impact of covid-19 on blood centres in zhejiang province china. vox sang. 2020;115(6):502–506. https://doi.org/10.1111/vox.12931 who. blood transfusion safety [homepage on the internet]. 2021 [cited 2021 feb 14]. available from: https://www.who.int/bloodsafety/voluntary_donation/en/ allain jp, sarkodie f, boateng p, asenso k, kyeremateng e, owusu-ofori s. a pool of repeat blood donors can be generated with little expense to the blood center in sub-saharan africa. transfusion (paris). 2008;48:735–741. https://doi.org/10.1111/j.1537-2995.2007.01599.x kasanga m, mudenda s, gondwe t, chileshe m, solochi b, wu jian. impact of covid-19 on blood donation and transfusion services at lusaka provincial blood transfusion centre, zambia. pan afr med j. 2020;35(2):74. https://doi.org/10.11604/pamj.supp.2020.35.2.23975 ngwako, t. botswana blood donation decline amid covid-19 [homepage on the internet]. allafrica. [cited 2021 apr 24]. available from: https://allafrica.com/stories/202006020164.html mwai p. why has kenya been facing serious shortages of human blood? [homepage on the internet]. 2020 [cited 2020 may 3]. available from: https://www.bbc.com/news/world-africa-51458114 kaserer a, rössler j, braun j, et al. impact of a patient blood management monitoring and feedback programme on allogeneic blood transfusions and related costs. anaesthesia. 2019;74:1534–1541. https://doi.org/10.1111/anae.14816 swanka c. botswana’s blood bank half empty [homepage on the internet]. [cited 2021 apr 24]. available from: https://www.google.com/amp/s/www.sundaystandard.info/botswanas-blood-bank-half-empty/%3famp/ ugwu ao, madu aj, efobi cc, ibegbulam og. pattern of blood donation and characteristics of blood donors in enugu, southeast nigeria. niger. j clin pract. 2018;21:1438–1443. himan y, patidar gk, arora s. covid-19 pandemicresponse to challenges by blood transfusion services in india: a review report. isbt science series. 2020;15(4):365–373. https://doi.org/10.1111/voxs.12563 gupta am, ojha s, poojary m, sumathi sh, nagaraju p, dhokle r. organization of the outdoor blood donation drives amid novel coronavirus pandemic and national lockdown: an experience from a tertiary care oncology institution in india. transfus apher sci. 2020;59(5):102878. https://doi.org/10.1016/j.transci.2020.102878 wang w, xu y, gao r, et al. detection of sars-cov-2 in different types of clinical specimens. jama. 2020;323(18):1843–1844. https://doi.org/10.1001/jama.2020.3786 chang l, zhao l, gong h, wang l, wang l. severe acute respiratory syndrome coronavirus 2 rna detected in blood donations. emerg infect dis. 2020;26:1631–1633. https://doi.org/10.3201/eid2607.200839 who. transmission of sars-cov-2: implications for infection prevention precautions [homepage on the internet]. scientific brief. 2020 [cited 2020 may 3]. available from: https://apps.who.int/iris/rest/bitstreams/1286634/retrieve adebisi ya, oke gi, ademola ps, chinemelum ig, ogunkola io, lucero-prisno iii de. sars-cov-2 diagnostic testing in africa: needs and challenges. pan afr med j. 2020;35(2):4. https://doi.org/10.11604/pamj.2020.35.4.22703 aneke jc, okocha ce. blood transfusion safety; current status and challenges in nigeria. asian j transfus sci. 2017;11(1):1–5. https://doi.org/10.4103/0973-6247.200781 stanweth sj, new hv, apelseth to, et al. effects of the covid-19 pandemic on supply and use of blood for transfusion. lancet haematol. 2020. https://doi.org/10.1016/s2352-3026(20)30186-1 lucero-prisno de 3rd, adebisi ya, lin x. current efforts and challenges facing responses to 2019-ncov in africa. glob health res policy. 2020;5:21. https://doi.org/10.1186/s41256-020-00148-1 abstract introduction ethical considerations case presentation management and outcomes discussion acknowledgements references about the author(s) katherine e. hodkinson department of molecular medicine and haematology, university of the witwatersrand, johannesburg, south africa national health laboratory services, johannesburg, south africa yvonne perner national health laboratory services, johannesburg, south africa department of anatomical pathology, university of the witwatersrand, johannesburg, south africa deborah k. glencross national health laboratory services, johannesburg, south africa department of haematology and molecular medicine, university of the witwatersrand, johannesburg, south africa tracey wiggill national health laboratory services, johannesburg, south africa department of haematology and molecular medicine, university of the witwatersrand, johannesburg, south africa adam botha national health laboratory services, johannesburg, south africa department of anatomical pathology, university of the witwatersrand, johannesburg, south africa janet poole department of paediatric oncology, university of the witwatersrand, johannesburg, south africa department of paediatric oncology, charlotte maxeke johannesburg academic hospital, johannesburg, south africa citation hodkinson ke, perner y, glencross dk, wiggill t, botha a, poole j. extranodal presentation of a lymphoma with precursor b-cell phenotype and translocation t(8;14) in south africa. afr j lab med. 2022;11(1), a1355. https://doi.org/10.4102/ajlm.v11i1.1355 case study extranodal presentation of a lymphoma with precursor b-cell phenotype and translocation t(8;14) in south africa katherine e. hodkinson, yvonne perner, deborah k. glencross, tracey wiggill, adam botha, janet poole received: 12 aug. 2020; accepted: 14 sept. 2021; published: 31 jan. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract introduction: a rare entity of a b-cell malignancy with precursor b-cell phenotype and concomitant translocation t(8;14) or variant myc translocation exists. these cases show clinical, pathological and molecular overlap between precursor b-lymphoblastic leukaemia or lymphoma and burkitt leukaemia or lymphoma (bll). case presentation: we report a case from february 2019 at the charlotte maxeke johannesburg academic hospital, south africa, of a 9-month-old infant with a predominantly extracranial soft tissue mass showing extradural extension. there was no involvement of the peripheral blood or bone marrow. fine needle aspiration and tru-cut biopsy of the soft tissue scalp mass showed the tumour to be of precursor b-cell phenotype. contrastingly, an immunophenotypic assessment revealed a high s-phase fraction and raised concern for bll. this prompted testing for the translocation t(8;14) by fluorescence in-situ hybridisation analysis, which confirmed this aberration. management and outcome: based on the published experience of other centres, the patient was initiated on a bll protocol. despite an excellent clinical response, the patient succumbed to neutropenic sepsis six months after diagnosis. conclusion: leukaemia or lymphoma with translocation t(8;14) or variant myc translocation and precursor b-cell phenotype is a rare entity with a varied clinical presentation. this poses a challenge for diagnosis and classification and a clinical dilemma for the choice of treatment. keywords: burkitt leukaemia/lymphoma; b-lymphoblastic leukaemia/lymphoma; translocation t(8;14); variant myc translocations; s-phase fraction; terminal deoxynucleotidyl transferase (tdt); extranodal. introduction the presence of the translocation t(8;14) or variant myc translocation in leukaemia with a precursor b-cell phenotype has been described in approximately 2% of paediatric cases.1,2 rarer still is a pure lymphomatous version (extranodal or nodal), which, to the best of our knowledge, has only been reported twice in the literature, one of which was in a paediatric patient.3,4 this entity has overlapping clinical and pathological features with burkitt leukaemia or lymphoma (bll) and precursor b-lymphoblastic leukaemia or lymphoma (b-all). it has been described across a spectrum of ages ranging from 32 months to 64 years, in both male and female patients. peripheral blood and bone marrow involvement have been reported in the majority of cases, with isolated extranodal disease reported in only a few.1,3,4,5 the immunophenotype is that of a precursor b-cell, with the expression of cd19, cd10 and terminal deoxynucleotidyl transferase (tdt), varied expression of cd34, and no expression of both surface light chain and cd20. molecular studies have confirmed the involvement of the myc gene in a translocation t(8;14) or variant translocation, which is the molecular hallmark of bll.2 this genetic aberration has largely directed the choice of therapy towards a mature b-cell or burkitt-like protocol in almost all reported cases. it is uncertain whether this represents the best therapeutic approach as there is very limited data on both the duration of remission in these patients and the exact underlying molecular behaviour of this tumour. this case study highlights this rare entity, as well as its associated diagnostic and therapeutic challenges. ethical considerations parental informed consent was provided for this case report. the ethical clearance was obtained from the university of the witwatersrand human research ethics committee (medical), johannesburg, south africa, under the approval number m190356. patient results were de-identified and stored in a secure database to ensure patient confidentiality. case presentation a 9-month-old male patient from angola presented to the charlotte maxeke johannesburg academic hospital, south africa, in february 2019, with a 3–4-week history of progressive bilateral proptosis and scalp masses. the computed tomography scan identified bilateral occipital and temporal scalp masses with extracranial extension into the orbital cavities and sinuses, and extradural extension into the anterior cranial fossa. there was no hepatosplenomegaly or lymphadenopathy. a fine needle aspiration sample of the soft tissue scalp mass was submitted for flow cytometry, and a tru-cut biopsy (manufacturer unknown) for histological assessment at the national health laboratory service. as part of the staging work up, bone marrow aspiration and a trephine biopsy were performed to exclude infiltration of the marrow. laboratory methods flow cytometry was performed on the fine needle aspiration sample from the scalp mass on a facs calibur instrument (bd biosciences, san jose, california, united states), using the paint-a-gate (becton dickinson, bd biosciences, san jose, california, united states) and modfit (verity software house, topsham, maine, united states) software. the following antibodies were used: cd19 fitc (becton dickinson, bd biosciences, san jose, california, united states), cd10 pe (beckman coulter inc., brea, california, united states), cd45 percp (becton dickinson, bd biosciences, san jose, california, united states), cd34 apc (becton dickinson, bd biosciences, san jose, california, united states), cd117 pe (beckman coulter inc., brea, california, united states), hla-dr fitc (becton dickinson, bd biosciences, san jose, california, united states), cd13 pe (beckman coulter inc., brea, california, united states), kappa fitc (dako, glostrup, denmark), lambda pe (dako, glostrup, denmark) and cytoplasmic tdt fitc (dako, glostrup, denmark). immunohistochemical work up was performed on the tru-cut biopsy from the scalp mass using the following stains: cd20 (dako, glostrup, denmark), pax5 (dako, glostrup, denmark), cd10 (leica biosystems, newcastle upon tyne, united kingdom), tdt (cell marque, rocklin, california, united states), cd34 (dako, glostrup, denmark), eber ish (roche, mannheim, germany), bcl2 (dako, glostrup, denmark), and ki67 (dako, glostrup, denmark). fluorescence in-situ hybridisation for the detection of translocation t(8;14) was performed using a vysis lsi myc/igh/cep8 tri-colour dual fusion probe (abbott, chicago, illinois, united states), with orange, green and aqua signals reflecting the myc, igh and centromere 8 regions. laboratory results histological examination of the tru-cut biopsy of the scalp mass revealed intermediate-sized tumour cells with irregular nuclear contours, a high nuclear-cytoplasmic ratio, dispersed nuclear chromatin, and one to four inconspicuous nucleoli present within a prominent background of tingible body macrophages (figure 1). the tumour immunophenotype based on flow cytometry and immunohistochemistry was that of a precursor b-cell, with the expression of tdt, absence of surface light chains and a high ki67 index (table 1, table 2 and figure 2). contrastingly, the high s-phase fraction detected was in a range that is typically seen in burkitt leukaemia.6 given the latter, fluorescence in-situ hybridisation analysis was requested and found to be positive for the translocation t(8;14)(q24;q32) (figure 3). figure 1: tru-cut biopsy of scalp mass of a 9-month-old male patient who presented to the charlotte maxeke johannesburg academic hospital in february 2019. image shows diffuse infiltration by intermediate-sized tumour cells with dispersed nuclear chromatin, irregular nuclear contours and one to four inconspicuous nucleoli. numerous tingible body macrophages impart a starry sky pattern. 7 mitotic figures per 40 hpf. haematoxylin & eosin (h&e) stain at ×40 magnification. figure 2: terminal deoxynucleotidyl transferase (tdt) immunohistochemical stain of tru-cut biopsy of scalp mass from a 9-month-old male patient who presented to the charlotte maxeke johannesburg academic hospital in february 2019. image shows ~40% positivity in the tumour. ×40 magnification. figure 3: fluorescence in-situ hybridisation analysis of fine needle aspirate from a 9-month-old male patient who presented to the charlotte maxeke johannesburg academic hospital in february 2019. image shows translocation t(8;14)(q24;q32) using the vysis lsi myc/igh/cep8 tri-colour dual fusion probe, as well as a 2f1o1g2a pattern: 2 fusion, 1 orange, 1 green, 2 aqua signals. the orange, green and aqua signals represent the myc, igh and centromere 8 regions. table 1: laboratory results at presentation of a 9-month-old male patient who presented to the charlotte maxeke johannesburg academic hospital in february 2019. table 2: laboratory results at presentation of a 9-month-old male patient who presented to the charlotte maxeke johannesburg academic hospital in february 2019. management and outcomes the patient was initiated on a bll regimen as per the fab lmb 95 protocol with excellent clinical response.7 there was complete resolution of the proptosis and the scalp masses were no longer clinically evident. sadly, the patient succumbed to neutropenic sepsis six months after diagnosis. discussion the co-existence of the translocation t(8;14) or variant myc translocation with a precursor b-cell phenotype in an extranodal malignancy is extremely uncommon and has been reported in only one paediatric and one adult patient in the literature.3,4 these individuals were both female and had no laboratory evidence of either peripheral blood or bone marrow involvement at presentation. the youngest, a 5-year-old, presented with abdominal, orbital and mandibular masses.4 the second, a 64-year-old, had multiple extranodal lesions and a chronic disease course.3 similarly, the clinical presentation of our patient was with isolated extranodal disease. this pattern of involvement is often seen in burkitt lymphoma and thus formed part of the differential diagnosis in our patient. laboratory work up demonstrated the co-expression of cd19 and cd10 by the tumour, with no expression of cd34 or light chains. because of this, the expression of tdt was assessed. terminal deoxynucleotidyl transferase is a dna polymerase that functions to provide junctional diversity in both b-cell and t-cell receptor genes at the precursor cell stage.8 it is a marker of immaturity and its expression in mature neoplasms such as bll would be aberrant. terminal deoxynucleotidyl transferase was found to be positive in ~30% – 40% of the tumour on both the fine needle aspiration and tru-cut biopsy specimens. while these results pointed towards a tumour at a precursor cell stage of development, the s-phase fraction analysis found the tumour proliferative activity to be very high. this measurement is performed by flow cytometry and uses dna content to determine the proportion of cells in each phase of the cell cycle. studies performed at our centre have shown the s-phase fraction of b-all to be characteristically around 10, whereas a fraction of more than 30, as seen in our patient, would be more typical of bll.6 comparisons could not be made with the other cases in the literature as s-phase fraction analysis was not reported. the ki67 index detects a protein associated with cellular proliferation, which is present in all active stages of the cell cycle. given that the ki67 index and s-phase analysis measure different aspects of proliferation, these parameters are not always comparable. notably, the ki67 index is unhelpful in distinguishing b-all and bll as values of over 95% can be anticipated in both.6 despite the conflicting laboratory findings in our patient, the high s-phase fraction in the context of extranodal disease prompted the testing for, and confirmation of, the translocation t(8;14). b-cell lymphoma 2 (bcl2) expression is typically seen in acute lymphoblastic leukaemia and the possibility of a double-hit lymphoma was excluded by the presence of tdt.2 s-phase fraction analysis may therefore serve as an early indicator of this disease entity, albeit further investigation of its clinical utility is required. in summary, a combination of diagnostic modalities with evaluation by a multidisciplinary team is required to confirm this rare entity. we propose a laboratory approach to the diagnosis of lymphomas with features of b-all and bll (figure 4). figure 4: laboratory approach to the diagnosis of lymphoma with features of precursor b-lymphoblastic leukaemia or lymphoma and burkitt leukaemia or lymphoma. the therapeutic management of bll differs significantly from b-all. the former requires high-intensity pulsed chemotherapy that is tailored to the high proliferative rate of the tumour. furthermore, the duration of therapy is shorter as compared to the extended maintenance used in b-all.7 our patient was instituted on a bll treatment protocol, in line with the therapeutic approach described in the literature.1,4,5 there are however limited cases and minimal long-term follow-up data from which meaningful conclusions can be drawn. on a molecular level, there is a lack of consensus as to whether this entity represents a precursor b-cell at an intermediate stage of maturation and with aberrant myc expression, or a mature b-cell with a less differentiated immunophenotype. furthermore, questions have been raised as to whether or not the presence of the translocation t(8;14) translates into an actual proliferation advantage. in an attempt to address these questions, one study analysed the molecular characteristics of neoplasms positive for ig-myc rearrangement and with precursor b-cell phenotype in 12 patients. aberrant variable diversity joining recombination was shown in five patients and activating nand k-ras mutations were detected in seven; all neoplasms had a dna methylation profile that clustered with that of precursor b-cells. in light of these findings, the authors of the study posed the question as to whether or not cases with myc rearrangement better fit a b-all profile.9 what is apparent from the literature is that further studies are required into the molecular behaviour of this entity to determine the prognostic implications and best therapy. conclusion leukaemia or lymphoma with translocation t(8;14) or variant myc translocation and precursor b-cell phenotype is a rare entity with varied clinical presentation. awareness of this entity and a high index of suspicion are required by both clinicians (haematologists, oncologists) and pathologists to prevent a misdiagnosis. further studies into the molecular and clinical behaviour of this tumour are required to optimise the therapeutic approach. acknowledgements thanking both ashleigh forsman and tshilidzi dzivhani from the somatic cell genetics unit (department of molecular medicine and haematology, national health laboratory service, johannesburg) for the analysis of the fluorescence in-situ hybridisation. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions k.e.h. drafted the manuscript. all authors were involved in the conceptualisation, structuring and editing of the final manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the author. references navid f, mosijczuk ad, head dr, et al. acute lymphoblastic leukemia with the (8;14)(q24;q32) translocation and fab l3 morphology associated with a b-precursor immunophenotype: the pediatric oncology group experience. leukemia. 1999;13(1):135–141. https://doi.org/10.1038/sj.leu.2401244 swerdlow sh, campo e, pileri sa, et al. the 2016 revision of the world health organization classification of lymphoid neoplasms. blood. 2016;127(20):2375–2390. https://doi.org/10.1182/blood-2016-01-643569 shiratori s, kondo t, fujisawa s, et al. c-myc rearrangement in b-cell lymphoblastic lymphoma with the involvement of multiple extranodal lesions. leuk lymphoma. 2011;52(4):716–718. https://doi.org/10.3109/10428194.2010.551158 meznarich j, miles r, paxton cn, afify z. pediatric b-cell lymphoma with lymphoblastic morphology, tdt expression, myc rearrangement, and features overlapping with burkitt lymphoma. pediatr blood cancer. 2016;63(5):938–940. https://doi.org/10.1002/pbc.25907 sakaguchi k, imamura t, ishimaru s, et al. nationwide study of pediatric b-cell precursor acute lymphoblastic leukemia with chromosome 8q24/myc rearrangement in japan. pediatric blood cancer. 2020;67(7):e28341. https://doi.org/10.1002/pbc.28341 glencross dk. flow cytometry in diagnostic haematopathology. johannesburg: university of the witwatersrand; 1992. patte c, auperin a, gerrard m, et al. results of the randomized international fab/lmb96 trial for intermediate risk b-cell non-hodgkin lymphoma in children and adolescents: it is possible to reduce treatment for the early responding patients. blood. 2007;109(7):2773–2780. https://doi.org/10.1182/blood-2006-07-036673 mccaffrey r, harrison ta, parkman r, baltimore d. terminal deoxynucleotidyl transferase activity in human leukemic cells and in normal human thymocytes. n engl j med. 1975;292(15):775–780. https://doi.org/10.1056/nejm197504102921504 wagener r, lópez c, kleinheinz k, et al. ig-myc (+) neoplasms with precursor b-cell phenotype are molecularly distinct from burkitt lymphomas. blood. 2018;132(21):2280–2285. https://doi.org/10.1182/blood-2018-03-842088 abstract introduction methods results discussion acknowledgements references about the author(s) howard newman national health laboratory service, virology, port elizabeth, south africa department of pathology, division of medical virology, stellenbosch university, cape town, south africa faculty of health sciences, nelson mandela university, port elizabeth, south africa donald tshabalala department of paediatrics, nelson mandela academic hospital, mthatha, south africa department of paediatrics, walter sisulu university, mthatha, south africa sikhumbuzo mabunda department of public health, walter sisulu university, mthatha, south africa mpumalanga department of health, nelspruit, south africa nokwazi nkosi department of pathology, division of medical virology, stellenbosch university, cape town, south africa national health laboratory service, tygerberg academic hospital, cape town, south africa candice carelson national health laboratory service, virology, port elizabeth, south africa citation newman h, tshabalala d, mabunda s, et al. rapid testing for respiratory syncytial virus in a resource-limited paediatric intensive care setting. afr j lab med. 2020;9(1), a1084. https://doi.org/10.4102/ajlm.v9i1.1084 brief report rapid testing for respiratory syncytial virus in a resource-limited paediatric intensive care setting howard newman, donald tshabalala, sikhumbuzo mabunda, nokwazi nkosi, candice carelson received: 27 aug. 2019; accepted: 12 aug. 2020; published: 08 dec. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract we analysed the performance characteristics of the respiratory syncytial virus lateral flow rapid antigen assay in use when compared to a multiplex polymerase chain reaction for detection of respiratory viruses. the study was conducted at a tertiary paediatric hospital in port elizabeth, south africa, from 01 january 2017 to 31 december 2018. we found the clinical sensitivity (36.8%) of the rapid test to be too low for routine diagnostic use. knowledge of assay performance characteristics of rapid tests are important for appropriate interpretation of rapid test results. keywords: respiratory syncytial virus; rapid antigen tests; respiratory viruses; respiratory multiplex polymerase chain reaction; assay performance characteristics. introduction respiratory syncytial virus (rsv) infection is a common cause of casualty visits and hospital admissions in infants.1,2,3,4 in 2005, a meta-analysis reported that between 66 000 and 160 000 children under age 5 years died of rsv infection worldwide.4 during the south african rsv season, 50% – 60% of all respiratory admissions in children are due to rsv.5 data from the 2016 pneumonia surveillance programme in south africa showed that 17% of enrolled patients tested positive for rsv, with a case fatality rate of less than 1%.6 rsv is highly contagious and numerous hospital outbreaks have been reported in multiple age groups,7 thus necessitating appropriate infection prevention and control measures. for timeous initiation of appropriate infection prevention and control measures, rapid laboratory confirmation of rsv is essential. options for laboratory testing include virus isolation, rapid antigen tests, direct fluorescent antibody tests and molecular methods such as polymerase chain reaction (pcr).8,9 rapid assays utilising antigen capture technology, that can be performed in less than 30 min, are available.10 numerous commercial assays exist, ranging in sensitivity from approximately 50% to 96% in children, while the majority of assays have specificity above 97%.1,11,12,13,14,15,16 the sensitivity of rapid antigen assays for rsv is affected by several factors; decreased sensitivity was noted in patients with a prolonged duration of symptoms at the time of testing, infection with subtype b, and older age.13,14,15,16 nucleic acid amplification tests have been shown to be superior to classical methods such as virus isolation and direct fluorescent antibody testing.17,18 not only is pcr more sensitive and specific than conventional methods, but it is also more amenable to ‘multiplexing’, thus allowing for the simultaneous detection of a panel of common respiratory viruses.19 one limitation of molecular testing is its potentially prohibitive cost. this is of particular concern in resource-limited settings. in addition, the laboratory turn-around time may not be rapid enough to aid clinical decision making, especially when samples have to be referred to distant laboratories.20 the commercial rapid antigen rsv assay used in the virology laboratory of the national health laboratory service in port elizabeth, south africa, is the rsv respi-strip by coris bioconcept (gembloux, belgium). it has a reported sensitivity of approximately 80%, with a specificity of greater than 95%.16 when compared to direct fluorescent antibody tests, the rsv respi-strip was found to be 92% sensitive and 98% specific.21 if the reported sensitivity of the assay of 80% is accurate, while not ideal, this may still be a clinically useful test in resource-limited settings, since positive cases would be detected 80% of the time, allowing for patient cohorting and isolation, and thereby limiting nosocomial transmission.7 for respiratory multiplex pcr testing, the virology laboratory in port elizabeth refers specimens to another national health laboratory service virology laboratory at tygerberg hospital in cape town, south africa. this laboratory makes use of the seeplex® rv16 assay by seegene (seoul, south korea). the sensitivity and specificity for the individual viruses varies. for rsv, the sensitivity and specificity is reported to be above 90% when compared with singleplex or duplex pcr.17 since patients admitted to our paediatric intensive care unit (icu) for suspected lower respiratory tract infection are all tested for rsv with a rapid antigen assay, in addition to testing for a panel of common respiratory viruses (including rsv subtypes a and b) by pcr, we compared the two assays to determine the sensitivity and specificity of the rsv respi-strip assay. we additionally sought to describe the common respiratory viruses detected in our icu setting. methods ethical considerations this study received ethical clearance from the human research committee of the faculty of health sciences, walter sisulu university (reference 043/2018). study design this was a descriptive, retrospective cross-sectional study analysing laboratory reports for rsv rapid antigen and multiplex pcr tests for respiratory viruses. results from the rsv rapid antigen assay were compared to results from the multiplex pcr assay, thus allowing for the calculation of performance characteristics of the rapid antigen assay. factors associated with false-negative rsv rapid antigen results were analysed. sample selection the study population comprised all paediatric patients admitted to icu at dora nginza hospital in port elizabeth, eastern cape province, south africa, who had respiratory samples taken for laboratory investigation of viral infections from 1 january 2017 to 31 december 2018. rapid testing for rsv is performed locally by the virology laboratory of the national health laboratory service in port elizabeth, south africa, with the nasopharyngeal aspirates being the specimen matrix. in addition, remnant specimen is referred to another virology laboratory (cape town, south africa) within the national health laboratory service, for multiplex pcr. this allowed for retrospective analysis of laboratory data. data analysis all variables were captured and coded in microsoft excel 2013 (microsoft corporation, seattle, washington, united states) and exported to stata 14.1 for analysis (stata corp lp, college station, texas, united states). the distribution of age in days (numerical variable) was explored using the shapiro wilk test, and age was further converted into a categorical variable in months. clinical sensitivity and specificity of the rsv respi-strip rapid antigen assay was calculated by comparison against the multiplex pcr (seeplex® rv16). categorical variables are presented using frequency tables, percentages and graphs. bivariate logistic regression models were used to determine associations of false rsv rapid antigen results with risk factors such as age, sex, hiv co-infection, rsv subtype, and co-infections with other viruses or bacteria. the odds ratio was the relative measure of association used. the 95% confidence interval was used to estimate the precision of estimates. the level of statistical significance was set at 5% (p-value ≤ 0.05). results test reports from a total of 152 participants were obtained and included in the study. eighty-one of the participants (53.3%) were female, and 121 were under the age of one year. table 1 shows all the demographic characteristics considered during this study. table 1: demographic characteristics of the study population from dora nginza hospital, port elizabeth, south africa, 2017–2018. results from the multiplex pcr assay show that rhinovirus was the most common virus detected (n = 55), followed by adenovirus (n = 30), rsv (n = 19) and enterovirus (n = 17), with the remainder of the viruses occurring less commonly (figure 1). respiratory syncytial virus subtype a was detected much more frequently than subtype b. figure 1: prevalence of the various respiratory viruses detected in paediatric patients from dora nginza hospital, port elizabeth, south africa, 2017–2018. when comparing the rsv rapid antigen assay to the multiplex pcr assay, the former was found to be 36.8% sensitive (table 2). table 2: retrospective analysis of laboratory data from doran nginza hospital, port elizabeth, south africa, 2017–2018. when analysing factors associated with false-negative rsv rapid antigen results, only viral–viral co-infection and infection with rsv subtype a were statistically significant associations (table 3). table 3: factors associated with false-negative respiratory syncytial virus rapid antigen results from paediatric patients at dora nginza hospital, port elizabeth, south africa, 2017–2018. discussion to the best of our knowledge, this is the first study comparing a rsv rapid antigen assay to a multiplex pcr assay that includes rsv as a target. in addition, it is also the first study to report on the prevalence of the common respiratory viruses found in our local paediatric icu. we found the clinical sensitivity of the rsv rapid antigen assay (rsv respi-strip by coris bioconcept) to be 36.84%; rhinovirus was the most commonly detected virus on the respiratory multiplex pcr assay. although the majority of respiratory admissions worldwide are due to rsv,5 the most common viruses detected in our setting were rhinovirus (n = 55) and adenovirus (n = 30), followed by rsv (n = 19) and enterovirus (n = 17). the main aim of this study was to determine the performance characteristics of the rsv rapid antigen assay in use (rsv respi-strip) by comparison to the respiratory multiplex pcr assay (seeplex® rv16 assay), which includes rsv as a target. the sensitivity of the rsv rapid antigen assay was found to be 36.84% (confidence interval: 16.29% – 61.64%). even at the upper end of the confidence interval, this sensitivity is too low for routine diagnostic use. while the specificity of above 90% may be sufficient for a rapid antigen assay, the low sensitivity would have resulted in many cases being undiagnosed, thus defeating the main purpose of using a rapid test, which is to allow for early institution of infection prevention and control measures to mitigate against the well-known nosocomial outbreak risk.7 potential reasons for the discrepancy in performance of this rapid test in our setting compared to the manufacturer’s claims are manifold. rapid antigen assays are generally less sensitive than pcr. this can be compounded by low viral loads in the clinical samples, as may be the case with upper respiratory tract infections only. it was beyond the scope of this study to differentiate patients based on severity of disease. the relatively small sample size may also account for the discrepancy in clinical sensitivity. the bivariate logistic regression analysis revealed a statistically significant association between false-negative rsv rapid antigen tests and viral co-infection, and infection with rsv subtype a. in contrast, a previous study showed that infection with rsv subtype b was associated with decreased sensitivity of rapid antigen assays.15 cytomegalovirus and hiv positivity were more likely to be associated with false rsv rapid antigen results, as well as more likely to be associated with false-negative results specifically. neither of these associations was statistically significant. limitations the main limitation of this study is the small sample size as a result of resource constraints, resulting in wider-than-ideal confidence intervals in the calculation of clinical sensitivity of the rsv rapid antigen test. however, even at the upper limit of the confidence intervals, the main conclusion that the sensitivity and positive predictive value are not acceptable for routine diagnostic use, still holds. conclusion this study highlights the importance of ongoing monitoring of newly introduced assays, as verification experiments may not always determine whether an assay will perform optimally in real-world conditions. based on the results of this study, the rsv rapid antigen assay in use (rsv respi-strip) was discontinued due to an unacceptably high rate of false results. in conclusion, we have demonstrated the common respiratory viruses found in our local paediatric icu setting. in addition, we report on the poor performance of the rsv rapid antigen assay in use and the potential factors associated with false results, with only viral co-infections and infection with rsv subtype a being statistically significant. clinicians should have an idea of the sensitivity and specificity of the rapid tests in use to allow for the appropriate interpretation of results. acknowledgements we would like to express our gratitude to all the staff at the virology laboratory of the national health laboratory service, port elizabeth, south africa, who assisted in collection of data. competing interests the authors have declared that no competing interests exist. authors’ contributions h.n. initiated the study. d.t., s.m., n.n. and c.c. assisted in producing the protocol. d.t. oversaw the ethics application. c.c. and h.n. handled the data collection. s.m. performed the data analysis. h.n., d.t., s.m., n.n. and c.c. wrote and revised the manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references chartrand c, tremblay n, renaud c, papenburg j. diagnostic accuracy of rapid antigen detection tests for respiratory syncytial virus infection: systematic review and meta-analysis. j clin microbiol. 2015;53(12):3738–3749. https://doi.org/10.1128/jcm.01816-15 green rj, zar hj, jeena pm, madhi sa, lewis h. south african guideline for the diagnosis, management and prevention of acute viral bronchiolitis in children. s afr med j. 2010;100(5):320–325. https://doi.org/10.7196/samj.4016 madhi sa, cutland cl, downs s, et al. burden of respiratory syncytial virus infection in south african human immunodeficiency virus (hiv)-infected and hiv-uninfected pregnant and postpartum women: a longitudinal cohort study. clin infect dis. 2018;66(11):1658–1665. https://doi.org/10.1093/cid/cix1088 nair h, nokes dj, gessner bd, et al. global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis. lancet. 2010;375(9725):1545–1555. https://doi.org/10.1016/s0140-6736(10)60206-1 moyes j, cohen c, pretorius m, et al. epidemiology of respiratory syncytial virus-associated acute lower respiratory tract infection hospitalizations among hiv-infected and hiv-uninfected south african children, 2010–2011. j infect dis. 2013;208(suppl. 3):s217–s226. https://doi.org/10.1093/infdis/jit479 walaza s, cohen c, treurnicht f, et al. epidemiology of respiratory pathogens from influenza-like illness and pneumonia surveillance programmes, south africa, 2016. commun dis surveill bull nicd. 2017;15(1):9–25. french ce, mckenzie bc, coope c, et al. risk of nosocomial respiratory syncytial virus infection and effectiveness of control measures to prevent transmission events: a systematic review. influ other respir viruses. 2016;10(4):268–290. https://doi.org/10.1111/irv.12379 ahluwalia g, embree j, mcnicol p, law b, hammond gw. comparison of nasopharyngeal aspirate and nasopharyngeal swab specimens for respiratory syncytial virus diagnosis by cell culture, indirect immunofluorescence assay, and enzyme-linked immunosorbent assay. j clin microbiol. 1987;25(5):763–767. https://doi.org/10.1128/jcm.25.5.763-767.1987 griffiths c, drews sj, marchant dj. respiratory syncytial virus: infection, detection, and new options for prevention and treatment. clin microbiol rev. 2017;30(1):277–319. https://doi.org/10.1128/cmr.00010-16 aslanzadeh j, zheng x, li h, et al. prospective evaluation of rapid antigen tests for diagnosis of respiratory syncytial virus and human metapneumovirus infections. j clin microbiol. 2008;46(5):1682–1685. https://doi.org/10.1128/jcm.00008-08 leonardi gp, wilson am, dauz m, zuretti ar. evaluation of respiratory syncytial virus (rsv) direct antigen detection assays for use in point-of-care testing. j virol methods. 2015;213:131–134. https://doi.org/10.1016/j.jviromet.2014.11.016 cruz at, cazacu ac, greer jm, demmler gj. performance of a rapid assay (binax now) for detection of respiratory syncytial virus at a children’s hospital over a 3-year period. j clin microbiol. 2007;45(6):1993–1995. https://doi.org/10.1128/jcm.00279-07 miernyk k, bulkow l, debyle c, et al. performance of a rapid antigen test (binax now® rsv) for diagnosis of respiratory syncytial virus compared with real-time polymerase chain reaction in a pediatric population. j clin virol. 2011;50(3):240–243. https://doi.org/10.1016/j.jcv.2010.11.011 schauer u, ihorst g, rohwedder a, et al. evaluation of respiratory syncytial virus detection by rapid antigen tests in childhood. klin padiatr. 2007;219(4):212–216. https://doi.org/10.1055/s-2006-933530 papenburg j, buckeridge dl, de serres g, boivin g. host and viral factors affecting clinical performance of a rapid diagnostic test for respiratory syncytial virus in hospitalized children. j pediatr. 2013;163(3):911–913. https://doi.org/10.1016/j.jpeds.2013.03.067 blyth cc, booy r, dwyer de. point of care testing: diagnosis outside the virology laboratory. in: stephenson jr, warnes a, editors. diagnostic virology protocols. methods in molecular biology, vol. 665. new jersey: humana press, 2011; pp. 415–433. https://doi.org/10.1007/978-1-60761-817-1 pillet s, lardeux m, dina j, et al. comparative evaluation of six commercialized multiplex pcr kits for the diagnosis of respiratory infections. plos one. 2013;8(8):e72174. https://doi.org/10.1371/journal.pone.0072174 gharabaghi f, hawan a, drews sj, richardson se. evaluation of multiple commercial molecular and conventional diagnostic assays for the detection of respiratory viruses in children. clin microbiol infect. 2011;17(12):1900–1906. https://doi.org/10.1111/j.1469-0691.2011.03529.x somerville lk, ratnamohan vm, dwyer de, kok j. molecular diagnosis of respiratory viruses. pathology. 2015;47(3):243–249. https://doi.org/10.1097/pat.0000000000000240 mills jm, harper j, broomfield d, templeton ke. rapid testing for respiratory syncytial virus in a paediatric emergency department: benefits for infection control and bed management. j hosp infect. 2011;77(3):248–251. https://doi.org/10.1016/j.jhin.2010.11.019 gregson d, lloyd t, buchan s, church d. comparison of the rsv respi-strip with direct fluorescent-antigen detection for diagnosis of respiratory syncytial virus infection in pediatric patients. j clin microbiol. 2005;43(11):5782–5783. https://doi.org/10.1128/jcm.43.11.5782-5783.2005 article information author: henry a. mbah1 affiliation: 1senior technical advisor lab, fhi-360, nigeria correspondence to: henry mbah postal address: fhi-360, plot 1073-a1, j.s. tarka street, godab plaza, area 3 garki-abuja, nigeria dates: received: 06 aug. 2013 accepted: 03 apr. 2014 published: 18 aug. 2014 how to cite this article: mbah ha. phlebotomy and quality in the african laboratory. afr j lab med. 2014;3(1), art. #132, 4 pages. http://dx.doi.org/10.4102/ ajlm.v3i1.132 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. phlebotomy and quality in the african laboratory in this opinion paper... open access • abstract • introduction    • phlebotomy and the error-prone pre-analytical stage    • phlebotomy has been undervalued worldwide    • current practice in africa       • staff       • space       • quality of specimen       • logistics       • identification       • safety       • documentation       • training    • existing complexity in the setup    • recommendations • conclusion • acknowledgements    • competing interest • references abstract top ↑ phlebotomy, the act of drawing blood through venepuncture, is one of the most common medical procedures in healthcare, as well as being a basis for diagnosis and treatment. a review of the available research has highlighted the dearth of information on the phlebotomy practice in africa. several studies elsewhere have shown that the pre-analytical phase (patient preparation, specimen collection and identification, transportation, preparation for analysis and storage) is the most error-prone process in laboratory medicine. the validity of any laboratory test result hinges on specimen quality; thus, as the push for laboratory quality improvement in africa gathers momentum, the practice of phlebotomy should be subjected to critical appraisal. this article offers several suggestions for the improvement of phlebotomy in africa. introduction top ↑ medical laboratory services, despite playing a pivotal role in africa’s healthcare system, have suffered gross neglect for decades.1,2 government investment in laboratories is often inadequate, resulting in substandard laboratory infrastructure; poorly-trained and unmotivated staff; and a limited scope of testing services. furthermore, it is widely understood that insufficient investment in the laboratory can have a negative effect on the quality of testing services, impacting the overall quality of healthcare. over the past five years, efforts have been made to improve laboratory services in africa. in january 2008, the consensus meeting on clinical laboratory testing harmonization and standardization convened governments, agencies and development partners in maputo, mozambique.3 the meeting established the maputo declaration on strengthening of laboratory systems, aimed at improving clinical laboratory services in developing countries.3 subsequent meetings held in 2008 on african laboratory medicine in lyon, france, yaoundé, cameroon and dakar, senegal formulated strategies for laboratory strengthening and arrived at several landmark achievements.3 in 2009, the world health organization, regional office for africa (who-afro) and partners launched the stepwise laboratory quality improvement process towards accreditation (slipta) to help laboratories in resource-limited settings strive toward international accreditation.3,4 the african society for laboratory medicine (aslm) was launched in 2011 and aslm’s first international conference for laboratory medicine was held in 2012. phlebotomy and the error-prone pre-analytical stage phlebotomy, also called venesection or venotomy, is the incision into a vein for the purpose of drawing blood5 that is used for laboratory analysis, diagnosis, transfusions and research. the person who performs phlebotomy is called a phlebotomist. phlebotomists in africa are also commonly responsible for collecting and properly packaging specimens (blood, sputum, urine, other body fluids, tissues, etc.), accepting incoming specimens and routing specimens to the proper section for analysis. the significant role phlebotomists play in the delivery of essential healthcare services has been described numerous times in existing literature.6,7,8,9phlebotomists are often the only laboratory professional a patient will meet during a hospital stay.accurate and precise laboratory test results depend on properly-performed phlebotomy in order to obtain high-quality specimens. the most well-trained testing staff, using the most sophisticated instruments, cannot produce accurate results from a poorly-collected specimen. traditionally, the laboratory testing process is divided into three phases: pre-analytical, analytical, and post-analytical. there have also been suggestions that the pre-analytical phase be divided into a ‘pre-pre-analytical phase’ and a ‘true pre-analytical phase’.10 phlebotomy falls within the realm of the pre-pre-analytical phase, which includes steps (test requesting, patient and sample identification, sample collection and sample transportation) that may neither be performed in the laboratory nor undertaken by laboratory personnel. the post/pre-analytical phase, which is carried out in the laboratory after specimen reception, involves the steps required to prepare samples for analysis (centrifugation, aliquoting and sorting). several studies suggest that in laboratory diagnosis, most errors occur within the pre-analytical phase (46% – 70%), followed by the post-analytical phases (18% – 47%), with the fewest errors occurring in the analytical phase (7% – 13%).11,12,13 the most frequent pre-analytical errors in laboratory medicine include: missing sample and/or test request; incorrect or missing identification; in vitro haemolysis; undue clotting; use of the wrong container; contamination from infusion route; insufficient sample; inappropriate blood-to-anticoagulant ratio; insufficient mixing of the sample; inappropriate transport; and incorrect storage conditions.12 phlebotomy has been undervalued worldwide historically, the critical role of phlebotomy has been overlooked,14 having been suggested as the most underestimated procedure in healthcare.6,14,15 for example, although most employers in the united states (us) require valid certification or a licence issued by an accredited phlebotomy training programme or a professional body such as the american society of phlebotomy technicians (aspt), only five us states mandate phlebotomy certification for practise.16 according to a recent survey on phlebotomy practice in 28 european countries, 21% of the countries do not require specific training for phlebotomy; national phlebotomy guidelines are available in only 25% of the countries; and only 36% have specific training available as a continuous educational resource.17 in many countries (and most countries in europe), there are no professional phlebotomists.13 phlebotomy is performed by doctors, nurses, laboratory staff and other healthcare professionals. current practice in africa anecdotal evidence (i.e., accounts from individual healthcare workers) from countries such as cameroon, chad, côte d’ivoire, kenya and nigeria indicates a high prevalence of suboptimal phlebotomy practices. unfortunately, because of the paucity of published information on phlebotomy practice in africa, this paper refers to information from europe and the us, where causes of phlebotomy service issues have been well researched and may be similar to those faced in resource-limited settings. staff in many facilities, laboratory phlebotomy staff draw blood from outpatients whereas doctors and nurses usually draw blood from inpatients. less oversight from the laboratory could contribute to service quality issues if medical staff have multiple tasks to perform simultaneously. use of trained phlebotomy-specific personnel may greatly reduce pre-analytical error rates.in many facilities across africa, resource constraints require that staff be cross-trained for multiple tasks, possibly eroding specific skills and resulting in excessive workloads. furthermore, the practice of rotating staff through different facilities likely decreases institutional expertise, especially if adequate planning and training are not performed well in advance. a study in europe found that the rates of pre-analytical errors are higher for inpatients than outpatients, for whom procedures are performed by personnel under direct laboratory control.18 a publication from the us concluded that phlebotomists are preferred over nurses and physicians for blood draws.19 increasingly, phlebotomy skills are being diluted in african healthcare settings through the implementation of task shifting and multi-skilled staffing strategies. thus, a decrease in phlebotomy expertise is exposing an increasing number of facilities to serious underlying safety problems and the likelihood of liabilities. space in most cases, dedicated space is not available for specimen collection; blood is drawn in patient waiting areas, laboratory result-collecting areas, in the heart of the laboratory, or in corridors and passageways, without demarcation. thus, confidentiality may be compromised. quality of specimen the correct order of blood draw is not well understood and recommended volumes are not always considered.20 blood may be drawn from intravenous infusion devices and needles may be withdrawn with the tourniquet still in place. there may also be inadequate quality checks by supervisors. logistics materials and supplies are often inadequate; blood is frequently drawn into an ordinary syringe and moved, exposed, from the wards to the laboratory. using ordinary syringes, the volume and proportion of mixture with anticoagulant and other additives relative to the intended test may be ignored. in the wards in particular, piercing the skin with ordinary needles and failure to put pressure on the puncture site, have most likely caused risky situations in which blood leaks down the patient’s arm onto the bed and clothing. identification poor labelling is a major source of concern.20 labelling is done locally, by matching the patient name with a number written with wax marker, coloured pencil, or marker onto the tubes and, in some cases, on the rubber tube caps only. this may lead to a mix-up of specimens during processing when, in some cases, the tops are removed before centrifugation. some technicians may claim they still know which tube belongs to which patient, which is highly unlikely. transfusion-related deaths21 and undue surgeries22 have been traced back to patientor specimen-identification errors. one laboratory even reported pregnancy in a male patient. safety some staff members may not wear or change gloves23 between patients, either because of limited supply or lack of training and supervision. in some cases, staff members have expressed concern that they would not be able to feel a vein with gloves on. touching the incision site to find the vein after alcohol swabbing is common23 and can expose the patient to risk of infection. sharps containers and colour-coded waste bins are often absent; it is not uncommon to find only a single trash bin without lining for general use. such practices have exposed healthcare workers to needle-stick injuries and sharps injuries.24,25 documentation standard operating procedures (sops) are not readily available. where they exist, sops are too often locked up and not easily accessible to all staff members who perform phlebotomy. in the wards, clinical sops are severely lacking. policies are rarely available and job aids are uncommon, or there is no appropriate place to affix them. training special training, continuous and refresher training and certification or competency assessment in phlebotomy are rarely mandatory. it is worth noting that in south africa, continuous phlebotomy training and certification is required. in donor-funded antiretroviral therapy (art) programmes, the drive to put as many persons as possible on art may overshadow plans for phlebotomy training. ironically, the quality of an art programme hinges on the entry point of sample collection and testing. existing complexity in the setup although the importance and vulnerability of the preand post-analytical phases have been acknowledged for many years, current quality management programmes still tend to focus on the performance and efficiency of analytical processes and activities within the direct control of the laboratory. there are concerns on the thoroughness of coverage of the pre-analytical phase in the major accreditation schemes. the current tiered quality improvement scheme, slipta, focuses on resource management covering nine of the 14 requirements considered by iso 15189/2007 on pre-examination procedures.26 despite the coverage by iso 15189/2007, pre-analytical errors remain, even in an accredited laboratory.27 to date, the pre-analytical variables that lie outside the direct control or supervision of laboratory personnel are difficult to monitor, as tools and policies are not fully standardised or harmonised worldwide. recommendations some suggestions are proposed to stimulate thoughts and actions that will help to improve phlebotomy practice and reduce pre-analytical errors in laboratory medicine. firstly, clear written procedures from existing guidelines should be developed.28,29 phlebotomy techniques should be standardised and sops, operative guidelines and preventive and reporting policies widely disseminated. secondly, a dedicated phlebotomist should be appointed, if possible, and/or specific healthcare professional training, continuous education and routine competency assessment should be enhanced. quality indicators focusing on the pre-analytical phase should be adapted, implemented and monitored in quality improvement projects.30 external quality assurance programmes should be modified to check the entire examination process, including pre-analytical and post-analytical procedures. phlebotomy services in health facilities should be centralised and communication amongst healthcare professionals and interdepartmental cooperation should be improved. finally, field studies should be undertaken in order to address the dearth of research in the area of phlebotomy practice and pre-analytical errors in africa. conclusion top ↑ in some african countries, the quality of phlebotomy, the entry point to laboratory testing, is inadequate. this important period in the struggle for laboratory quality improvement in africa provides a window of opportunity to enhance strategies to improve phlebotomy practices. acknowledgements top ↑ special thanks are extended to professor julius n. anyu of the department of public administration at the university of the district of columbia, usa and miss ashley mbah, a high school junior at st. thomas more academy, magnolia, delaware, usa, for reviewing this article. the views expressed herein are those of the author and do not necessarily reflect those of fhi-360. competing interest the author declares that he has no financial or personal relationship(s) which may have inappropriately influenced him in writing this article. references top ↑ 1.petti ca, polage cr, quinn tc, et al. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377–382. http://dx.doi.org/10.1086/4993632.nkengasong jn, nsubuga p, nwanyanwu o, et al. laboratory systems and services are critical in global health: time to end the neglect? am j clin pathol. 2010;134(3):368–373. http://dx.doi.org/10.1309/ajcpmpsinq9brmu6 3.gershy-damet g-m, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm 4.yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3):401–409. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj 5.stedman t. stedman’s medical dictionary for the health professions and nursing. 7th ed. philadelphia: lippincott williams & wilkins; 2012. 6.waheed u, ansari ma, zaheer ha. phlebotomy as the backbone of the laboratory. lab med. 2013;44(1):e69–e71. 7.sembiante t. phlebotomy : the vital link between the patient and the lab. med lab obs. 2004;36(2):46. 8.ogden-grable h. phlebotomists: laboratory ambassadors. lab med. 2007;38:571. 9.balance lo. lab management. phlebotomy tips: 10 sure-fire formulas for success. med lab obs [article on the internet]. c2011 [cited 2014 jul 07]. available from: http://www.mlo-online.com/articles/201105/phlebotomy-tips-rn10-sure-fire-formulas-for-success.php 10.plebani m. pre-analytical errors and patient safety. j med biochem. 2012;31(4):265–270. http://dx.doi.org/10.2478/v10011-012-0014-1 11.carraro p, plebani m. errors in a stat laboratory: types and frequencies 10 years later. clin chem. 2007;53(7):1338–1342. http://dx.doi.org/10.1373/clinchem.2007.088344 12.lippi g, chance jj, church s, et al. preanalytical quality improvement: from dream to reality. clin chem lab med. 2011;49(7):1113–1126. http://dx.doi.org/10.1515/cclm.2011.600 13.carraro p, zago t, plebani m. exploring the initial steps of the testing process: frequency and nature of pre-preanalytic errors. clin chem. 2012;58(3):638–642. http://dx.doi.org/10.1373/clinchem.2011.175711 14.southwick k. back to the drawing board: hospitals rethink their phlebotomy staffing practices. cap today. 2001;15(2):12–21. 15.edwards m. coalition strives for phlebotomy personnel standards. med lab obs. 2005;37(10):24–25. 16.phlebotek. states that require phlebotomy certification or license [page on the internet]. c2012 [cited 2014 feb 18]. available from: http://phlebotek.com/education/states-that-require-phlebotomy-certification-or-license/ 17.simundic am, cornes m, grankvist k, et al. survey of national guidelines, education and training on phlebotomy in 28 european countries: an original report by the european federation of clinical chemistry and laboratory medicine (eflm) working group for the preanalytical phase (wg-pa). clin chem lab med. 2013;51(8):1585–1593. 18.lippi g, blanckaert n, bonini p, et al. haemolysis: an overview of the leading cause of unsuitable specimens in clinical laboratories. clin chem lab med. 2008;46(6):764–772. http://dx.doi.org/10.1515/cclm.2008.170 19.caruana c. the laboratory test begins with phlebotomy. lab med. 2003;34(8): 566–576. http://dx.doi.org/10.1309/095mc7vc1hycl1cj 20.makubi an, meda c, magesa a, et al. audit of clinical-laboratory practices in haematology and blood transfusion at muhimbili national hospital in tanzania. tanzan j health res. 2012;14(4):1–8. 21.myhre ba, mcruer d. human error − a significant cause of transfusion mortality. transfusion. 2000;40(7):879–885. http://dx.doi.org/10.1046/j.1537-2995.2000.40070879.x 22.chassin mr, becher ec. the wrong patient. ann intern med. 2002; 136(11):826–833 [comment in rice f. the wrong patient. ann intern med. 2003;138(6):518, author reply 518–519]. http://dx.doi.org/10.7326/0003-4819-136-11-200206040-00012 23.wondimu h, addis z, moges f, et al. bacteriological safety of blood collected for transfusion at university of gondar hospital blood bank, northwest ethiopia. isrn hematol. 2013; 308204:7 pages. 24.nsubuga fm, jaakkola ms. needle stick injuries among nurses in sub-saharan africa. trop med int health. 2005;10(8):773–781. http://dx.doi.org/10.1111/j.1365-3156.2005.01453.x 25.kebede g, molla m, sharma hr. needle stick and sharps injuries among health care workers in gondar city, ethiopia. saf sci. 2012;50(4):1093–1097. http://dx.doi.org/10.1016/j.ssci.2011.11.017 26.datema ta, oskam l, van beers sm, et al. critical review of the stepwise laboratory improvement process towards accreditation (slipta): suggestions for harmonization, implementation and improvement. trop med int health. 2011;17(3):361–367. 27.simundic am, nikolac n, vukasovic i, et al. the prevalence of preanalytical errors in a croatian iso 15189 accredited laboratory. clin chem lab med. 2010;48(7): 1009–1014. http://dx.doi.org/10.1515/cclm.2010.221 28.world health organization. who guidelines on drawing blood : best practices in phlebotomy. geneva: world health organization; 2010. 29.ernst d. procedures for the collection of diagnostic blood specimens by venipuncture. wayne, pa: clinical and laboratory standards institute; 2007. 30.plebani m, chiozza ml, sciacovelli l. towards harmonization of quality indicators in laboratory medicine. clin chem lab med. 2013;51(1):187–195. http://dx.doi.org/10.1515/cclm-2012-0582 abstract introduction methods results discussion acknowledgements references about the author(s) mmachuene i. hlahla department of forensic pathology, faculty of health sciences, university of limpopo, polokwane, south africa moshibudi j. selatole department of forensic pathology, faculty of health sciences, university of limpopo, polokwane, south africa citation hlahla mi, selatole mj. could ante-mortem computed tomography be useful in forensic pathology of traumatic intracranial haemorrhage? afr j lab med. 2021;10(1), a1040. https://doi.org/10.4102/ajlm.v10i1.1040 original research could ante-mortem computed tomography be useful in forensic pathology of traumatic intracranial haemorrhage? mmachuene i. hlahla, moshibudi j. selatole received: 24 apr. 2019; accepted: 25 feb. 2021; published: 29 july 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: imaging techniques have proven valuable in forensic pathology practice, with computed tomography being preferred for forensic use. in the era of virtual autopsy and a lowto middle-income, resource-constrained country, a question arises as to whether ante-mortem computed tomography (act) could be cost-effective by reducing the number of invasive autopsies performed. objective: the objective of this study was to assess the usefulness of act in forensic pathology by examining discrepancy rates between act scans and autopsy findings in cases of deceased individuals with traumatic intracranial haemorrhages and assess factors associated with discrepancies. methods: eighty-five cases of act and autopsy reports from 01 january 2014 to 31 december 2016 from the polokwane forensic pathology laboratory, south africa, were analysed retrospectively. using cohen’s kappa statistics, measures of agreement and resultant discrepancy rates were determined. also, the discrepancy patterns for each identified factor was also analysed. results: the discrepancy rate between act and autopsy detection of haemorrhage was 24.71% while diagnostic categorisation of haemorrhage was 55.3%. classification discrepancy was most observed in subarachnoid haemorrhages and least observed in extradural haemorrhages. a markedly reduced level of consciousness, hospital stay beyond two weeks and three or fewer years of doctors’ experience contributed to classification discrepancies. conclusion: ante-mortem computed tomography should be used only as an adjunct to autopsy findings. however, the low discrepancy rate seen for extradural haemorrhages implies that act may be useful in the forensic diagnosis of extradural haemorrhages. keywords: forensic imaging; ante-mortem computed tomography; traumatic intracranial hemorrhage; forensic autopsy. introduction imaging techniques have in recent years been found to be greatly useful in forensic pathology.1,2 these modalities not only serve to augment but lower the invasive autopsies performed.3 the latter is convenient for various reasons, including religious and economic reasons. computed tomography (ct) has been used as the preferred imaging modality in forensic imaging.4 though in the era of virtual autopsies, the usefulness of ante-mortem computed tomography (act) compared to the inexpensive invasive autopsy in a middle-income country, such as south africa, must be justified. thus, it is necessary to determine whether act will be an augmentation or a cost-effective alternative to autopsy. head injuries are common, usually carry a high mortality rate, and are therefore important in forensic pathology practice. while post-mortem ct has been found useful in determining the cause of death in cases of traumatic intracranial haematomas,2,5 there is limited empirical evidence for the use of act scan findings to this effect. discrepancies between clinical or ante-mortem ct scan and autopsy findings exist and are common.6 according to bruno et al.,7 radiologic interpretations cannot be programmed because interpretation is subject to a variety of factors such as doctors’ (radiologists’ and pathologists’) expertise and complex psychophysiological and cognitive level of the patient (e.g. level of consciousness). thus, act interpretation errors are inevitable. this study assessed the rate and pattern of discrepancies between act scan and conventional autopsy findings of intracranial haemorrhages in cases of traumatic head injury submitted for autopsy at a facility in a rural south africa province. methods ethical considerations ethical clearance for the study was secured from turfloop research and ethics committee (trec/268/2017: pg). consent was not applicable as we were not working on human subjects but case reports, permission for which was obtained from the limpopo provincial department of health. the case files were coded with numbers and names of the deceased were not recorded. study design this quantitative descriptive study retrospectively analysed 85 cases of deceased individuals across three years from 01 january 2014 to 31 december 2016. cases sustained head injuries, underwent act imaging and had no surgical intervention after being referred to an academic hospital. deceased cases were subjected to autopsy procedure as per legal requirement at the attached forensic pathology facility in limpopo, a rural province of south africa. data collection data were obtained from post-mortem reports from the forensic pathology laboratory while ct scan reports, clinical data and human resource records were obtained from the pietersburg hospital records section and radiology department. autopsy findings served as a reference point because studies have demonstrated that it remains superior to act scan for the detection of brain injuries.1,8 the majority (n = 80) of the cases had only one act scan done and the rest a second; only the first scan reports were therefore used in the study. the following intracranial haemorrhages were assessed from these reports: epidural, subdural and subarachnoid haemorrhages. data analysis data were captured and analysed using microsoft excel (microsoft office professional plus 2013; microsoft corp., redmond, washington, united states) and international business machines corporation statistical package for the social sciences version 23 (armonk, new york, united states) running under microsoft windows (microsoft corp., redmond, washington, united states). cross-tabulations were used to establish the percentage agreement between act scan and autopsy findings of extradural haemorrhage (edh), subdural haemorrhage (sdh) and subarachnoid haemorrhage (sah) single or combination occurrence. if there is agreement between the act scan and autopsy findings the individual case scored 1, but if there is a disagreement between the act scan and autopsy findings, the case scored 0. levels of agreement and resultant discrepancy rates were determined using cohen’s kappa statistics. the pattern of discrepancies for identified factors such as the level of consciousness by glasgow coma scale, the length of hospital stay, the experience of the clinician (radiologist and pathologist) and the site of haemorrhage was also evaluated. results were considered statistically significant when p was less than 0.05. results agreement in the detection and diagnosis of haemorrhages in 75.29% (64/85) of cases, the act scan and autopsy agreed on the presence or absence of haemorrhage with a kappa coefficient of 0.3834 (table 1). the remaining 24.71% represents the overall discrepancy rate between the act scan and autopsy detection of haemorrhage. table 1: agreement between autopsy and ante-mortem computed tomography findings in the detection of intracranial haemorrhages, south africa, 2014–2016. the highest agreement between the act scan and autopsy finding was recorded in the diagnosis of edh (agreement 90.59%, kappa coefficient 0.5823 and discrepancy rate 9.41%) (table 1). with regard to sdh, the agreement was 74.12% with a kappa coefficient of 0.4857 and a discrepancy rate of 25.88% (table 1). the lowest agreement of 65.88% with a kappa coefficient of 0.3219 and the highest discrepancy rate of 34.12% was found for sah (table 1). in 38 of the 85 cases (44.7%; 25 with and 13 without haemorrhage), both methods agreed in the diagnostic intracranial haemorrhage category (i.e. single, binary or ternary combinations of haemorrhages), denoting a diagnostic category discrepancy of 55.3% (table 2 and table 3). act agreed in 7 of the 11 cases categorised as singular sah by autopsy (bolded) but reclassified three cases as having binary haemorrhage (sah and sdh) and one case as absent (no haemorrhage). for 25 cases classified as ‘absent’ (having no haemorrhage) by autopsy, act agreed in 13 (bolded) cases but categorised the remaining 12 as having different haemorrhages. table 2: summary of diagnostic category for intracranial haemorrhage, south africa, 2014–2016. table 3: summary of misclassification of detection category agreement and discrepancies observed for haemorrhages, south africa, 2014–2016. pattern of discrepancies with regard to identified factors the analysis revealed that most discrepant intracranial haemorrhage diagnoses (5 out of 8 cases for edh; 15 out of 22 cases for sdh; 19 out of 29 cases for sah) were seen in patients with markedly low levels of consciousness, denoting severe traumatic head injury (figure 1). figure 1: discrepancies and level of consciousness, south africa, 2014–2016. 1 = mild (gcs 15–13), 2 = moderate (gcs 12–9), 3 = severe (gcs ≤ 8), 0 = unspecified. half (4/8) of the discrepant cases for edh were found to have been admitted on average for a day or less (figure 2). on the other hand, most discrepancies for sah were seen in cases that were admitted for two weeks or less (9/29) and four weeks or more (10/29) on average, while for sdh the majority (9/22) stayed in the hospital for more than a month. figure 2: discrepancies and length of admission, south africa, 2014–2016. in addition, more discrepant case diagnoses were seen with a radiologist or pathologist who had less than three years of working experience (figure 3). figure 3: discrepancies and level of experience (in years), south africa, 2014–2016. discussion the current study found a significant difference in haemorrhage detection between act and autopsy. individual intracranial haemorrhage detection discrepancies ranged from 9.41% to 34.12%, with sah carrying the highest rate. this is consistent with what has been reported by a number of previous studies that collectively reveal discrepancy rates ranging from 9% to 39%.5,9 there are varying findings concerning discrepancy rates for sah with the majority of previous studies showing high discrepancy10,11,12 in keeping with the current study. the study also found that in general act had a diagnostic accuracy of 44.7%. as such, the high level of discrepancy for sah was attributable to misclassification, which may mean misdiagnosis, and to a combination of haemorrhages that could have masked the sah. also, a high sah discrepancy rate (79% of sah cases) was noted with prolonged length of hospital stay, probably due to haemorrhage resorption with time. a progressive clearance of red blood cells in the cerebrospinal fluid results in approximately 50% of sahs not being visualised after one week of occurrence,13,14 and this process can be shorter or longer.15 this implies that the majority of the sah previously diagnosed under an act scan may after approximately three weeks not be seen in an autopsy if the patient demises. this is dependent on the amount of sah in the leptomeninges. this was evident from the current study where the act scan superseded autopsy in the diagnosis of sah, which was consistent with previous studies.11,14 the high level of agreement of agreement (90.59%) observed for edh diagnosis (with high true negative rate) was corroborated in a similar study comparing act and autopsy findings11 and when comparing post-mortem ct diagnoses to autopsy results.9 we opine that the high level of discrepancy in cases admitted for a day or less may be because it takes more than a day for the typical shape of the haemorrhage to appear. more discrepancies were noted in cases with markedly low levels of consciousness, denoting severe injuries. this may be because critically ill patients may not assume specific positions required to visualise the haemorrhage, particularly when the haemorrhage is small in size, a notion also suggested by liisanantti and ala-kokko.5 practitioners (radiologists and pathologists) with a low level of experience made more discrepant diagnoses. although most studies support that experience appears to decrease discrepancies,16,17 they also agree that there are multifactorial and complex factors that can result in those discrepancies. some stipulate that the main reason for the discrepancies in lower postgraduate trainees has been identified as a lack of knowledge.18,19 doctors with low levels of experience are typically registrars and junior medical officers. lee et al.20 report that discrepancies tend to be higher among registrars, owing to ‘physical discomfort, eye strain and lack of motivation’, which intensify by the end of the workday. moreover, registrars and medical officers work overtime, during which they have to work overnight. this may result in focusing difficulty and hence reduced diagnostic or detection accuracy.21,22 length of admission has also been shown to affect agreement for sdh. the study showed that the greater part of the discrepant sdh cases were seen in patients who were admitted for more than a month on average. a possible reason could be the effects of the healing processes, which can happen in thinner blood collection cases such that after a month the haemorrhage may be enclosed or completely absorbed.23,24 brain slicing intervals during autopsies are usually not the same as the intervals used during act imaging and this may also account for the general discrepancy rate. delayed intracranial haemorrhage could occur after the act scan is obtained and could also account for the discrepancies as most of the cases did not undergo a subsequent act scan. a useful scenario to study this would have been to have the act and autopsy on the same day. limitations the major limitation of this study is the small sample size. moreover, the study addressed only three types of brain haemorrhages (edh, sdh and sah); therefore, the findings cannot be generalised to forensic pathology practice, or any other type of intracranial haemorrhage. further research with a larger sample size and broader scope is recommended. conclusion the overall detection discrepancy rate of 24.74% and ct diagnostic accuracy of 44.7% implies that act scans may not be used as an altervative to reduce the number of autopsies performed at the mentioned facility but can only be used as an adjunct to autopsy. however, the low discrepancy rate in edh, especially after a day of admission, implies that act may be useful for the diagnosis of this haemorrhage in the forensic setting. a markedly reduced level of consciousness, length of hospital stay depending on the type of haemorrhage and three or fewer years of doctors’ experience all contributed to discrepancies observed between act and autopsy findings. the study employed a limited sample and thus calls for more similar studies in both high and lowand middle-income countries. acknowledgements we would like to thank mr p. mphekgwana (university of limpopo) and mr a. poopedi (pietersburg provincial hospital) for statistical analyses. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions m.i.h. conducted the research and produced a mini-dissertation. m.j.s. supervised m.i.h. and prepared the publication manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references jalalzadeh h, giannakopoulos gf, berger fh, fronczek j, van de goot frw, reijnders uj. post-mortem imaging compared with autopsy in trauma victims – a systematic review. forensic sci int. 2015;257(december):29–48. https://doi.org/10.1016/j.forsciint.2015.07.026 kasahara s, makino y, hayakawa m, yajima d, ito h, iwase h. diagnosable and non-diagnosable causes of death by postmortem computedtomography: a review of 339 forensic cases. leg med. 2012;14(5):239–245. https://doi.org/10.1016/j.legalmed.2012.03.007 yaniv g, guranda l, or j, zaitsev k, konen e, hiss j. correlation between radiological and pathological findings for a sudden death incident in the emergency department. isr med assoc j. 2011;13(11):707–708. baglivo m, winklhofer s, hatchd gm, ampanozi g, thali mj, ruder td. the rise of forensic and post-mortem radiology – analysis of the literature between the year 2000 and 2011. j forensic radiol imaging. 2013;1(1):3–9. https://doi.org/10.1016/j.jofri.2012.10.003 liisanantti jh, ala-kokko ti. the impact of antemortem computed tomographic scanning on postmortem examination rate and frequency of missed diagnosis – a retrospective analysis of postmortem examination data. j crit care. 2015;30(6):1420.e1–1420.e4. https://doi.org/10.1016/j.jcrc.2015.08.024 brady ap. error and discrepancy in radiology: inevitable or avoidable? insights imaging. 2017;8(1):171–182. https://doi.org/10.1007/s13244-016-0534-1 bruno ma, walker ea, abujudeh hh. understanding and confronting our mistakes: the epidemiology of error in radiology and strategies for error reduction. radiographics. 2015;35(6):1668–1676. https://doi.org/10.1148/rg.2015150023 panda a, kumar a, gamanagatti s, mishra b. virtopsy computed tomography in trauma: normal postmortem changes and pathologic spectrum of findings. curr probl diagn radiol. 2015;44(5):391–406. https://doi.org/10.1067/j.cpradiol.2015.03.005 leth pm, struckmann h, lauritsen j. interobserver agreement of the injury diagnoses obtained by postmortem computed tomography of traffic fatality victims and a comparison with autopsy results. forensic sci int. 2013;225(1–3):15–19. https://doi.org/10.1016/j.forsciint.2012.03.028 graziani g, tal s, adelman a, kugel c, bdolah-abram t, krispin a. usefulness of unenhanced post mortem computed tomography – findings in postmortem non-contrast computed tomography of the head, neck and spine compared to traditional medicolegal autopsy. j forensic leg med. 2018;55(april):105–111. https://doi.org/10.1016/j.jflm.2018.02.022 panzer s, covaliov l, augat p, peschel o. traumatic brain injury: comparison between autopsy and ante-mortem ct. j forensic leg med. 2017;52(november):62–69. https://doi.org/10.1016/j.jflm.2017.08.007 sharma r, murari a. a comparative evaluation of ct scan findings and post mortem examination findings in head injuries. indian j forensic med toxicol. 2006;4(2):2–4. jones hr jr, srinivasan j, allam g, baker r. netter’s neurology [homepage on the internet]. 2nd ed. philadelphia, pa: elsevier saunders; 2012 [cited 2018 mar 12]. available from: https://www.elsevier.com/books/the-netter-collection-of-medical-illustrations-nervous-system-volume-7-part-1-brain/jones-jr/978-1-4160-6387-2 kidwell cs, wintermark m. imaging of intracranial hemorrhage. lancet neurol. 2008;7(3):256–267. https://doi.org/10.1016/s1474-4422(08)70041-3 daroff rb, jakovic j, mazzotta tc, pomeroy sc. bradley’s neurology in clinical practice. 7th ed. philadelphia, pa: elsevier; 2016. bruni sg, bartlett e, yu e. factors involved in discrepant preliminary radiology resident interpretations of neuroradiological imaging studies: a retrospective analysis. ajr am j roentgenol. 2012;198(6):1367–1374. https://doi.org/10.2214/ajr.11.7525 mellnick v, raptis c, mcwilliams s, picus d, wahl r. on-call radiology resident discrepancies: categorization by patient location and severity. j am coll radiol. 2016;13(10):1233–1238. https://doi.org/10.1016/j.jacr.2016.04.020 brady ap, ó’laoide r, mccarthy p, mcdermott r. discrepancy and error in radiology: concepts, causes and consequences. ulster med j. 2012;81(1):3–9. filograna l, tartaglione t, filograna e, cittadini f, oliva a, pascali vl. computed tomography (ct) virtual autopsy and classical autopsy discrepancies: radiologist’s error or a demonstration of post-mortem multi-detector computed tomography (mdct) limitation? forensic sci int. 2010;195(1–3):13–17. https://doi.org/10.1016/j.forsciint.2009.11.001 lee cs, nagy pg, weaver js, newman-toker de. cognitive and system factors contributing to diagnostic errors in radiology. ajr am j roentgenol. 2013;201(3):611–617. https://doi.org/10.2214/ajr.12.10375 krupinski ea, berbaum ks, caldwell rt, schartz km, kim j. long radiology workdays reduce detection and accommodation accuracy. j am coll radiol. 2010;7(9):698–704. https://doi.org/10.1016/j.jacr.2010.03.004 waite s, scott j, gale b, fuchs t, kolla s, reede d. interpretive error in radiology. ajr am j roentgenol. 2017;208(4):739–749. https://doi.org/10.2214/ajr.16.16963 itabashi hh, andrews jm, tomiyasu u, erlich ss, sathyavagiswaran l. forensic neuropathology – a practical review of the fundamentals. burlington, vt: academic press; 2007. saukko p, knight b. knight’s forensic pathology. 4th ed. new york, ny: crc press; 2016. abstract background description of the intervention lessons learnt recommendations acknowledgements references about the author(s) patrick a. njukeng global health systems solutions, douala, cameroon charles njumkeng global health systems solutions, douala, cameroon callistus ntongowa global health systems solutions, douala, cameroon mohammed abdulaziz africa centres for disease control and prevention, addis ababa, ethiopia citation njukeng pa, njumkeng c, ntongowa c, abdulaziz m. strengthening laboratory networks in the central africa region: a milestone for epidemic preparedness and response. afr j lab med. 2022;11(1), a1492. https://doi.org/10.4102/ajlm.v11i1.1492 lessons from the field strengthening laboratory networks in the central africa region: a milestone for epidemic preparedness and response patrick a. njukeng, charles njumkeng, callistus ntongowa, mohammed abdulaziz received: 08 dec. 2020; accepted: 11 feb. 2022; published: 19 may 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: health systems in the central africa region are among the weakest and least funded in the world. the lack of laboratory networks and adequately trained personnel with clearly defined responsibilities has hampered the implementation of laboratory quality improvement programmes. global health systems solutions (ghss) obtained a grant from the africa centres for disease control and prevention to develop laboratory networks for disease surveillance and strengthen the quality of laboratory testing in the central africa region. intervention: one year after the grant was awarded on 01 october 2018, ghss has launched a regional integrated surveillance and laboratory network (rislnet) for central africa and developed national laboratory strategic plans and policies for member states, eight frameworks and guideline documents, as well as a website for rislnet central africa. ghss has also launched an extension for community health outcomes platform to supervise laboratories enrolled for accreditation, installed a basic laboratory information system (blis) in four laboratories in four member states, and trained 247 laboratory personnel and laboratory experts on blis, quality assurance, external quality assurance, strengthening laboratory management towards accreditation (slmta), quality management systems, and equipment maintenance and calibration. lessons learnt: participating laboratories now serve as reference laboratories for covid-19 testing in various countries. point-of-care testing, using the genexpert platform, has been the central strategy for the scale-up of covid-19 testing in the central africa region. recommendations: expanding slmta to other laboratories within central africa will significantly improve the quality management of laboratories for a better healthcare system. keywords: covid 19; laboratory; health system; strengthening; network; quality; management; response. background clinical laboratories form an essential component of the health system. in addition to providing test results for disease diagnosis and guiding treatment by detecting drug resistance, they form the backbone of disease surveillance and public health response to outbreaks and epidemics.1,2,3 efforts to strengthen health systems should therefore focus on laboratory services and systems as they provide primary information that informs decision making for best healthcare outcomes.4 the role of the laboratory in responding to epidemics is undoubtedly a vital one judging from the lessons learned in recent years. the 2014 ebola outbreak in west africa revealed the need to organise and maintain laboratory systems or networks that can rapidly and effectively adjust to carry out new diagnostic testing or laboratory services in response to large-scale epidemics.5 another lesson learned from the ebola outbreak was the need to set up a platform that enables planning and preparedness activities to be rapidly adapted to emerging pathogens or outbreaks.6 the current coronavirus disease 2019 (covid-19) pandemic has further emphasised the overarching role of quality laboratory systems in disease outbreak preparedness and response in africa and, specifically, in the central africa region. health systems in the central africa region are among the weakest in the world and have received the least funding support when compared to other regions.7,8 however, funding sources and advanced testing technologies are increasingly being made available to laboratories in the region from bodies such as the african union/africa centres for disease control and prevention (cdc), african society for laboratory medicine, the world health organization’s regional office for africa and the united states cdc, among others. nonetheless, laboratory quality improvement is still hindered by the lack of adequately trained personnel with clearly defined responsibilities, as well as the lack of well-established organisations and laboratory networks. to promote the strengthening of laboratory systems, the network of national public health institutes needs to be strengthened through continuous advocacy and staff capacity development. global health systems solutions (ghss), an international non-governmental organisation based in cameroon, obtained a grant from africa cdc to establish and strengthen public health laboratory systems and networks in the central africa region. ghss worked in close collaboration with africa cdc and the regional collaboration center to achieve this strategic vision of the africa cdc. the principal goal of the african union/africa cdc grant was to develop laboratory networks for disease surveillance and strengthen the quality of laboratory testing across nine countries in the central africa region, namely gabon, cameroon, central african republic, chad, congo brazzaville, equatorial guinea, burundi, democratic republic of congo, and sao tome and principe. this goal was predicated on three key objectives. the first objective was to develop a framework and statute for the central africa regional integrated surveillance and laboratory network (rislnet) that defines its function and operations, permitting the establishment of a website to share framework documents and experiences between the different countries. the second objective was to implement a quality management system (qms) towards the accreditation of laboratories in the region with emphasis on the quality of point-of-care (poc) testing, biosafety and biosecurity, equipment maintenance, sample referral systems and antimicrobial resistance surveillance. the final objective was to support selected member states to develop the laboratory components of regional proposals to address antimicrobial resistance. this article discusses the activities in the first year of the project implementation and the role of the implementation in the fight against covid-19, which is currently the greatest global challenge. description of the intervention ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. development of a framework and statute for the rislnet two workshops were organised in july 2018 and march 2019 to accomplish this task. the first workshop brought countries together within the circumscription of the central africa regional collaboration centre of africa cdc, and this was a key step towards setting up a network of health institutes and laboratories within the region. in this forum, experts from the human, animal and environmental health sectors of the nine countries came together in a workshop in brazzaville, gabon, and began to share contacts, create linkages and share information and experiences within the region. the workshop, which ended with the launching of rislnet for the central africa region under the patronage of the minister of health of the republic of congo, also saw the inauguration of bureau members for the central africa rislnet. a second workshop was organised in malabo, equatorial guinea, to guide the development of the national laboratory strategic plan and policies for member states. to foster the development of a framework and statute for the central africa rislnet, frameworks and guidelines were developed that included: a framework and statute for rislnet, a sample transport system framework for the central africa rislnet, a quality manual template for laboratory testing, and guidelines for laboratory biosafety and biosecurity. a website was also developed for the central africa rislnet to enhance the sharing of public health information, laboratory quality best practices and laboratory quality documents, as well as to encourage networking among public health laboratories in the central africa region. mapping and linkage of centres of excellence and laboratories in the region using the extension for community health outcomes platform the extension for community health outcomes (echo) was launched at ghss’ head office in douala, cameroon, to support the mentorship and supervision of seven laboratories enrolled for accreditation. these laboratories were linked and coordinated together with centres of excellence and public health laboratories for event-based and laboratory-based disease surveillance. they include laboratoire l’hôpital general de reference national, n’djamena, chad; laboratoire de l’hôpital prince regent charles, bujumbura, burundi; laboratoire national de sante publique, brazzaville, congo brazzaville; laboratorio national de referencia para tuberculose sao tome, sao tome and principe; international centre of medical research of franceville, franceville, gabon; laboratorio castroverde, malabo, equatorial guinea; and laboratoire national de biologie clinique et de sante publique, bangui, central african republic. the sample transport and information sharing systems between these laboratories and countries’ centres of excellence and national reference laboratories have been linked to echo. implementation of qms and biosafety in the region a training workshop was held in n’djamena, chad, from 22 july 2019 to 26 july 2019. biologists, laboratory managers, and laboratory technicians were trained on laboratory qms and the implementation of immediate improvement projects to speed up the world health organization’s regional office for africa accreditation process. the training served as a springboard for international organization for standardization-15189 accreditation, as 21 participants were trained on productivity management, quality assurance, documents and records management, and the use of the strengthening laboratory management towards accreditation (slmta) toolbox. development of a framework for implementing qms for poc testing this activity was designed to increase the accuracy and reliability of poc testing, which is useful for the rapid detection of endemic and outbreak diseases. a 5-day training of trainers was conducted from 28 october 2019 to 01 november 2019 in libreville, gabon, to build the capacity of 26 healthcare personnel to support the institutionalisation and sustainability of poc testing. this activity aimed to ensure that quality assurance officers are well equipped with the skills, knowledge and abilities necessary to perform assigned tasks. the world health organization/cdc-approved poc comprehensive quality assurance training package was used, with emphasis on the stepwise process for improving the quality of hiv-related point-of-care testing checklist.9 implementation of the computing for good (c4g) blis eight hospital laboratories, one public health laboratory and one reference laboratory each in burundi, congo, chad, and sao tome and principe, benefited from the computing for good (c4g) basic laboratory information systems (blis) network. after a baseline survey, gaps identified in each of the laboratories were addressed to allow the implementation of the blis network, and the ghss team embarked on the implementation phase to install and implement a c4g blis network in these laboratories to improve turn-around times and to support clinical decision making. the ghss team also purchased and installed information technology materials and network connectivity to support the management of c4g blis in the laboratories. the laboratory staff were then trained on the use of blis for the proper management of laboratory data and the day-to-day activities of the laboratory. lessons learnt the covid-19 disease is entirely new, and the characteristics of its causative virus, the severe acute respiratory syndrome coronavirus-2, are not fully understood.10 drug development typically takes years of scientific studies, and unfortunately, the new coronavirus has not given the world that length of time. at the start of the pandemic, there was no defined guideline available for the management of the disease. the echo platform provides practitioners with the opportunity to come together weekly to share experiences from the treatment centres in different countries. this will enhance patient management as expertise on better patient management strategies will easily be shared among different countries. this platform will also permit various treatment centres to discuss treatment challenges and receive feedback and contributions from colleagues. there is a great need to boost fragile health systems like those in the central africa region for better disease surveillance and outbreak response. a body such as ghss with vast experience in supporting health systems in central africa can utilise the echo platform established in the region to support the respective countries as they respond to this pandemic. it can guide infection prevention and control and virtual training on biosafety practices and sample collection, packaging and transportation for healthcare workers via the echo platform, all of which are crucial in the fight against covid-19. this platform is now used regularly by ghss to strengthen the quality of testing in cameroon and the democratic republic of congo. the rislnet initiative, after one year of its implementation, has been very helpful in the fight against the covid-19 outbreak. at a moment when health systems across the globe are facing significant challenges, it is the product of the rislnet initiative (the elected centres of excellence, referral network, trained personnel, and developed guidance documents) that has formed the basis of the sub-region’s strategy in the fight against covid-19. as covid-19 testing is not entirely decentralised within member states, the sample transport system framework for rislnet central africa can easily be adapted to the covid-19 sample transport network within member states. furthermore, the laboratory testing quality manual template put in place will guide good laboratory practice for covid-19 testing and ensure timely, accurate and reliable results. the guidelines for laboratory biosafety and biosecurity should provide an appropriate foundation for the implementation of safety practices for the protection of frontline healthcare workers who are most at risk. most importantly, the rislnet website11 has been essential in the sharing of information of public health interest, laboratory quality best practices, and laboratory quality documents. the website contains the emergency numbers of member states, provides updates on the covid-19 situation and enhances the sharing of covid-19 strategic documents, recommendations, guidelines, feedback from meetings, etc. the slmta programme was established because the united states cdc, world health organization’s regional office for africa and stakeholders recognised the poor state of medical laboratory systems in africa and prioritised the need to strengthen them to fight multiple diseases.4 few laboratories in central africa recognise the importance of developing qmss, laboratory strategic plans, and protocols that facilitate continuous laboratory quality improvement for patient care. the implementation of slmta in africa has improved laboratory quality, biosafety, standardisation, maintenance, record keeping, and reporting in africa, all of which are crucial to the optimal function of laboratories.12,13 the laboratories that participated in the rislnet initiative were mentored for accreditation using the slmta toolkit. it is therefore not surprising that these laboratories now serve as reference laboratories for covid-19 testing in their respective countries. the countries would have found it challenging to quickly respond to the covid-19 outbreak if the laboratories had not received training on qms, quality assurance, biosafety and biosecurity, and equipment maintenance and calibration. also, poc testing has been at the centre of the strategy to scale-up covid-19 testing in central africa. notably, genexpert, which is currently being used in scaling up covid-19 testing in central africa, was the focus of this implementation. the participants trained in the implementation of qms for poc testing are now adequately equipped to lead their countries in the scale-up of covid-19 testing. participants were also trained on capacity development for healthcare personnel to support the institutionalisation and sustainability of poc testing. thus, they can easily initiate, implement, evaluate and provide corrective action to the various poc sites carrying out covid-19 diagnoses. the use of a c4g blis network in these laboratories will improve turn-around times for covid-19 testing, which is very critical for clinical decision making, surveillance efforts, and the enforcement of preventive measures. the provision of information technology materials and network connectivity to support the management of c4g blis will facilitate data sharing and communication between the testing laboratories and collection sites. laboratory staff trained on the proper use of blis can properly manage testing data and rapidly generate and report testing statistics to health authorities for quick decision making. the impact of these project implementation activities on the regional response to covid-19 cannot be understated but may be difficult to quantify. nonetheless, lessons learned in the past have shown that strengthening laboratory systems is the best strategy for outbreak preparedness and response. during the ebola epidemic in west africa between 2013 and 2016, laboratory systems were overwhelmed, and the medical world saw the need to rethink the future of global laboratory medicine. in west africa, the laboratory system could not diagnose haemorrhagic fevers and other diseases and, as a result, the public health experts failed to notice the outbreak in time.5 resultant from weak laboratory systems, public health authorities in the west africa region continues to miss ebola cases, leading to ebola illnesses and deaths erroneously attributed to other infections.14 similarly, the world health organization was only notified of the ebola outbreak characterised by fever, severe diarrhoea, vomiting, and a high fatality rate in guinea in march 2014; however, epidemiological investigations linked laboratory-confirmed cases with a presumed first fatal case of the outbreak in december 2013.15,16 thus, weak laboratory systems frustrate surveillance systems and decrease the quality of patient care, which are integral parts of the preventive and therapeutic interventions needed in disease control. recommendations expanding slmta to other laboratories within member states in the central africa region will significantly improve the quality management in these laboratories and result in a better healthcare system. the laboratories enrolled in this programme can be further supported through supportive site visits or coaching through the echo platform; this will improve their effectiveness in responding to covid-19 testing challenges. outlining a clear plan to support these sites will help sustain efforts towards achieving accreditation and boost their current role in the fight against covid-19. more funding is required for these efforts and the improvement of health systems in central africa. acknowledgements the authors express sincere gratitude to the funding bodies (africa cdc and african union) and all the authorities of central africa. we acknowledge the various laboratories involved and the personnel who led the implementation at different levels. we equally applaud the diverse team of experts at ghss for their relentless efforts and commitment. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions p.a.n. conceived, designed and supervised the project implementation. c. njumkeng participated in the field implementation of the project and drafted the paper. c. ntongowa coordinated the project implementation. m.a. directed the project implementation and reviewed and corrected the final manuscript. all authors read and approved the final manuscript. sources of support this project was funded by the african union commission and the africa centres for disease control and prevention. however, the results and conclusions made in this publication are made by the authors and do not represent the official position of the african union commission and africa centres for disease control and prevention. data availability data sharing is not applicable to this article. disclaimer all authors were involved in year one activity implementation of laboratory networks for disease surveillance and strengthening of the quality of laboratory testing in central africa. references panteghini m. the future of laboratory medicine: understanding the new pressures. clin biochem rev. 2004;25(4):207–215. wians fh. clinical laboratory tests: which, why, and what do the results mean? lab med. 2009;40:105–113. https://doi.org/10.1309/lm4o4l0hhutwwudd gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. https://doi.org/10.1309/ajcptuuc2v1wjqbm nkengasong jn. a shifting paradigm in strengthening laboratory health systems for global health: acting now, acting collectively, but acting differently. am j clin pathol. 2010;134(3):359–360. https://doi.org/10.1309/ajcpy5asuejyq5rk sealy tk, erickson br, taboy ch, et al. laboratory response to ebola – west africa and united states. mmwr suppl. 2016;65(suppl-3):44–49. https://doi.org/10.15585/mmwr.su6503a7 oleribe oo, salako bl, ka mm, et al. ebola virus disease epidemic in west africa: lessons learned and issues arising from west african countries. clin med (london, england). 2015;15(1):54–57. https://doi.org/10.7861/clinmedicine.15-1-54 world health organization. world health statistics 2020: monitoring health for the sdgs, sustainable development goals. geneva: world health organization; 2020. whitworth ja, kokwaro g, kinyanjui s, et al. strengthening capacity for health research in africa. lancet (london, england). 2008;372(9649):1590–1593. https://doi.org/10.1016/s0140-6736(08)61660-8 stepwise process for improving the quality of hiv related point-of-care testing (spi-poct). checklist spi-poct checklist (instrument based) version 2.0 9/16/2014. geneva: world health organization. sharma a, tiwari s, deb mk, marty jl. severe acute respiratory syndrome coronavirus-2 (sars-cov-2): a global pandemic and treatment strategies. int j antimicrob agents. 2020;56(2):106054. https://doi.org/10.1016/j.ijantimicag.2020.106054 regional integrated surveillance and laboratory network (rislnet) [homepage on the internet]. [cited 2020 sept 7]. available from: https://africacdc.org/rislnet/ nkengasong jn, nsubuga p, nwanyanwu o, et al. laboratory systems and services are critical in global health: time to end the neglect? am j clin pathol. 2010;134(3):368–373. https://doi.org/10.1309/ajcpmpsinq9brmu6 carter jy. external quality assessment in resource–limited countries. biochem med (zagreb). 2017;27(1):97–109. https://doi.org/10.11613/bm.2017.013 juan do, greenberg la, ishaan kd, et al. building laboratory capacity to strengthen health systems the partners in health experience. clin lab med. 2017;38(1):101–117. https://doi.org/10.1016/j.cll.2017.10.008 baize s, pannetier d, oestereich l, et al. emergence of zaire ebola virus disease in guinea. n engl j med. 2014;371(15):1418–1425. https://doi.org/10.1056/nejmoa1404505 sack k, fink s, belluck p, et al. how ebola roared back [homepage on the internet]. new york times, 2014 [cited 2020 sept 7]. available from: https://www.nytimes.com/2014/12/30/health/how-ebola-roared-back.html introduction challenges in expanding and enhancing yellow fever laboratory diagnostics building the lassa fever laboratory network during an epidemic access to diagnostics and laboratory systems integration acknowledgements references about the author(s) dhamari naidoo who health emergency program, infectious hazard management, world health organisation, abuja, nigeria chikwe ihekweazu nigeria centre for disease control, abuja, nigeria citation naidoo d, ihekweazu c. nigeria’s efforts to strengthen laboratory diagnostics – why access to reliable and affordable diagnostics is key to building resilient laboratory systems. afr j lab med. 2020;9(2), a1019 https://doi.org/10.4102/ajlm.v9i2.1019 opinion paper nigeria’s efforts to strengthen laboratory diagnostics – why access to reliable and affordable diagnostics is key to building resilient laboratory systems dhamari naidoo, chikwe ihekweazu received: 30 mar. 2019; accepted: 27 may 2020; published: 26 aug. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction since 2016, the nigeria centre for disease control (ncdc) has been strengthening its technical capacities as the country’s national public health institute.1 one key area receiving considerable attention has been the strengthening of laboratory diagnostic and networking capabilities for diseases of public health importance. previous investments in strengthening and financing laboratory capacities have focused almost entirely on single disease programmes. in just over two years, the ncdc has made progress in changing this situation with the operationalisation of a new national reference laboratory. the national reference laboratory has the diagnostic capacity for national priority diseases, including yellow fever (yf), lassa fever, monkeypox, cerebrospinal meningitis, cholera, influenza and other enteric pathogens. in addition, a specimen referral system has been established to cover the large geographic expanse of the country and a national laboratory network supporting 41 facilities. the national laboratory network integrates vertical disease networks for the standardisation of testing algorithms and improvement of coordination and functionality.2 in 2017 alone, the ncdc responded to widespread outbreaks of cerebrospinal meningitis, monkeypox, yf, lassa fever and cholera.1 with several public health challenges, including increasing disease outbreaks and emerging antimicrobial resistance,3 efficient coordination and integration of surveillance and laboratory systems is of critical importance. improving access to diagnostics at the country level is a fundamental component of building laboratory systems critical for disease surveillance and timely outbreak detection and containment, as well as improved patient management.4 advocacy on the importance of diagnostic development for outbreak preparedness is occurring through global strategies such as the world health organization’s (who) research and development blueprint and eliminating yellow fever epidemics strategy.5,6 however, challenges still remain in accessing diagnostic tests that are reliable, available through local production or procurement, and easily implementable and scalable during emergencies.7 this is becoming more apparent as countries struggle to leverage laboratory capacity from established vertical disease programmes, such as polio, hiv and tuberculosis, for emerging public health threats. the limited global investments to increase access to diagnostics across a wider range of diseases of public health concern, rather than individual diseases, will continue to limit the capacity of african countries to develop resilient and disease-wide public health systems.4 this report summarises the challenges and early successes of laboratory integration across two disease networks in nigeria. challenges in expanding and enhancing yellow fever laboratory diagnostics since 2016, the re-emergence of yf has caused outbreaks in angola, brazil, chad, the democratic republic of congo, ghana, guinea and uganda.8 this prompted the who to launch the eliminating yellow fever epidemics strategy to reduce the risk of yf in high-risk countries through strengthening outbreak detection, response and prevention.9 in 2017, yf re-emerged in nigeria, 17 years since the last reported case.10 localised outbreaks continue to be reported and, as of march 2019, nigeria had seen approximately 4100 suspect cases, of which 139 were confirmed in 17 states throughout the country11 (figure 1). figure 1: weekly trends of yellow fever outbreak in nigeria from 2017 to 2019. the nigerian yf laboratory network, established in 2006, consists of four subnational laboratories located in lagos, kaduna, abuja and gombe (figure 2), with capacity to implement imunoglobulin m (igm) detection, the standardised african yf laboratory diagnostic algorithm. the national laboratories receive reagents for laboratory-developed, enzyme-linked immunosorbent assays through two sources: the institut pasteur of dakar and the united states centres for disease control and prevention.12 due to operational differences between the laboratory-developed and commercial elisa protocols, implementation is challenging and requires continuous training for the national staff. following recommended guidelines, presumptive positive samples from any of the four national laboratories have to be shipped to the who’s regional reference laboratory at the institut pasteur of dakar in senegal, where additional testing is conducted. this is done to exclude vaccine-induced imunoglobulin m antibody positivity and cross-reactivity because of infections with other flaviviruses – a limitation of serological testing.8 figure 2: geographic location and specimen referral catchment areas for the lassa fever and yellow fever laboratories. (a) lassa fever laboratories, (b) yellow fever laboratories. the response to the ongoing outbreak has been challenged by limited access to reagents. between 2016 and august 2017, no yellow fever testing occurred in nigeria because of a lack of reagents.7 when the outbreak was first detected in kwara state, nigeria, in august 2017, the same month that reagents became available, it took one month to confirm the laboratory result at the regional reference laboratory. the index case reported the onset of symptoms on 16 august 2017 and the final laboratory confirmation was only received on 12 september 2017.9 delayed outbreak detection and reporting of cases has an impact on the approval of vaccine requests from the international coordination group on vaccine provision13 and planning for reactive vaccination campaigns for outbreak control. the importance of rapid confirmation was recently highlighted during the widespread yf outbreak in edo state, nigeria, in 2018. clusters of cases were reported from four local government areas on 14 november 2018. on 21 november 2018, the ncdc was informed of nine patient samples that had tested positive for yf by quantitative reverse transcription polymerase chain reaction (qrt-pcr) at the african center of excellence for genomics of infectious disease, a non-yf-network laboratory. these yf cases were found in patients initially referred to the irrua specialist teaching hospital with suspected lassa fever. after lassa fever tests were negative, the patients were tested for yf following continued clinical presentation with typical viral haemorrhagic signs and symptoms.14 immediately after receiving the report of positive results, the samples were sent to the institut pasteur of dakar on 29 november 2018 for re-confirmation. the first set of results yf confirming both imunoglobulin m and qrt-pcr results were received from the institut pasteur of dakar on 7 december 2018. the plaque reduction neutralisation test results were shared on 31 december 2018. however, the outbreak was declared on 24 november 2018, based on the qrt-pcr results. declaring the outbreak using the molecular results allowed for the immediate deployment of rapid response teams and the initiation of a request for vaccines from the international coordination group on vaccine provision. however, the challenge faced by national authorities was two fold – accepting test results carried out by non-who-recognised yf-network laboratory; and by qrt-pcr, a method not routinely used for confirmation at the national level. molecular testing has not been effectively operationalised in the african region.4,15 in response, the national yf testing algorithm was updated to include molecular testing, and the national lassa fever laboratories were registered in a yf qrt-pcr quality-assurance programme, a critical step required to demonstrate technical capacity to detect yf virus infection. overall, two critical bottlenecks hamper the strengthening of the national yf laboratory network. these are limited access to clinically validated or regulatory approved molecular and serologic tests either through the who network or through commercial manufacturers; and the limited capacity to perform in-country confirmatory diagnosis by plaque reduction neutralisation test and differential diagnosis for flaviviruses.8 the eliminating yellow fever epidemics strategy is currently addressing the shortfalls in diagnostics through updating diagnostic algorithms, working with industries and partners to fast-track diagnostic development and evaluations, and creating an international procurement and supply chain. to operationalise these changes at the country level, increased support and funding is required for training, procurement of diagnostic tests for yf and other flaviviruses, and access to quality control materials for the development of national external quality assessment activities. building the lassa fever laboratory network during an epidemic in 2018, nigeria recorded an unusually active epidemic season of lassa fever. a total of 3498 suspect cases were reported, of which 633 were laboratory confirmed. several factors, such as increased disease awareness, improved surveillance and laboratory capacity, were thought to have contributed to the increased number of confirmed cases.3 before establishing national diagnostic capacity, testing was performed outside nigeria, in kenema, sierra leone. between 2005 and 2012, diagnostic capacity was established at the lagos university teaching hospital and the irrua specialist teaching hospital. the irrua specialist teaching hospital accounted for over 90% of the testing workload, as it provided diagnostic support for an endemic region in nigeria.16 the 2018 epidemic season marked the beginning of a collaboration with the international community under the framework of the who’s research and development blueprint to improve lassa fever detection, treatment and prevention.17 one of the biggest gains made in 2018 was the rapid expansion of national diagnostic capacity, with the establishment of two additional testing facilities at the ncdc national reference laboratory in abuja and the virology laboratory at the federal teaching hospital in abakaliki, ebonyi state (figure 2). the expansion was attributable to the availability of funding during the outbreak from donors and the who through the federal government to the ncdc. this enabled the procurement of equipment and reagents, the building of infrastructure and increased political commitment from the federal and state governments of nigeria. a key factor in the early success of the network was the adoption of a standardised testing algorithm based on a 2-gene target testing strategy using qrt-pcr, with a combination of both commercial(realstar® lassa fever rt pcr kit version 1.0, altona diagnostics, hamburg, germany) and laboratory-developed tests being used to detect all known lineages of lassa fever virus.16 published diagnostic literatures showed limited availability of commercial assays for lassa fever diagnosis; none was who approved.17 however, through the expedited who research and development pathway, a new kit-based format of the laboratory-developed test was evaluated in nigeria and submitted for review under the who’s expert review panel for diagnostics at the end of 2018. because of comparable analytical data between the laboratory-developed and commercial kits, improved usability and an easier procurement process, the new kit format (realstar® lassa fever rt pcr kit version 2.0, altona diagnostics, hamburg, germany) was adopted within the laboratory network despite the need to perform the assay as two singleplex tests. despite the remaining challenges, such as difficulties in importing large quantities of diagnostic kits considered as dual-use by german and european union legislation, and sustainable funding to maintain national testing capacity at the current levels, the collaborations with technical partners such as the bernhard nocht institute of tropical medicine, the foundation for innovative new diagnostics and the who under the who research and development blueprint initiative, proved to be instrumental in capacity building and development of national priorities for operational research. this multi-partner collaboration has delivered tangible benefits to the country by improving diagnostic preparedness for lassa fever and demonstrating that investments made during outbreaks improve health systems. access to diagnostics and laboratory systems integration as the ncdc continues its journey to deliver on its mandate, stronger integration of surveillance and laboratory systems becomes critical for outbreak detection and response. laboratory network integration will facilitate integrated electronic reporting systems, improve the use of diagnostic testing to guide patient care across all levels of the health system, and allow for better financial planning to allocate resources required to maintain operational activities in the face of dwindling external funding. to improve nigeria’s yf and lassa fever outbreak detection and response, there is the need to integrate both systems networks into an efficient and effective laboratory surveillance system. to achieve this, yf would need to be included as part of a panel of recommended tests for a differential diagnosis on patients who test negative for lassa fever. to operationalise this differential testing, access to commercial yf molecular assays with clinical validation data is required.8 further product development is needed for safe and reliable lassa fever point-of-care testing to improve access to diagnostic testing for patients in all hospital facilities across nigeria. the experience of nigeria has shown that restrictive access to diagnostic tests as a result of unequal global investment in diagnostic development hinders the building of resilient and integrated laboratory systems. stronger leadership is required from the who to address these challenges. greater progress will be made in building laboratory systems at the country level when the framework is built on (1) multi-partner engagement with joint leadership and collaboration with the ministries of health and national public health institutes of affected countries, (2) international and domestic funding that ensures the sustainability of diagnostic testing to promote continuous product development and (3) a revised african regional laboratory strategy that promotes diagnostic stewardship and the uptake of new diagnostics for multi-pathogen testing to reduce vertical disease structures. this is not only important for nigeria but for the entire continent. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions d.n. and c.i. contributed equally to the writing of this manuscript. ethical considerations ethical clearance is not required for this opinion article. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references njidda am, oyebanji o, obasanya j, et al. the nigeria centre for disease control. bmj glob health. 2018;3(2):e000712. https://doi.org/10.1136/bmjgh-2018-000712 nigeria centre for disease control [homepage on the internet]. c2018 [cited 2020 jan 19]. available from: https://ncdc.gov.ng/reports/annualreports ilori ea, frank c, dan-nwafor cc, et al. increase in lassa fever cases in nigeria, january–march 2018. emerg infect dis. 2019;24(5):1026–1027. https://doi.org/10.3201/eid2505.181247 onyebujoh pc, thirumala ak, ndihokubwayo j-b. integrating laboratory networks, surveillance systems and public health institutes in africa. afr j lab med. 2016;5(3):a431. https://doi.org/10.4102/ajlm.v5i3.431 kieny mp, salama p. who r&d blueprint: a global coordination mechanism for r&d preparedness. lancet. 2017;389(10088):2469–2470. https://doi.org/10.1016/s0140-6736(17)31635-5 world health organization (who). the eye strategy addresses three important global health agendas [homepage on the internet]. c2017 [cited 2020 jan 19]. available from: https://www.who.int/csr/disease/yellowfev/eye-strategy-and-global-agendas/en/ kelly-cirino cd, nkengasong j, kettler h, et al. importance of diagnostics in epidemic and pandemic preparedness. bmj glob heal. 2019;4(suppl 2):e001179. https://doi.org/10.1136/bmjgh-2018-001179 domingo c, charrel rn, schmidt-chanasit j, zeller h, reusken c. yellow fever in the diagnostics laboratory. emerg microbes infect. 2018;7(1):1–15. https://doi.org/10.1038/s41426-018-0128-8 world health organization (who). weekly epidemiological record, 10 august 2018, vol. 93, 32 (pp. 409–416) [homepage on the internet]. c2018 [cited 2019 mar 23]. available from: https://www.who.int/wer/2018/wer9332/en/ world health organization (who). 2000 – yellow fever in nigeria [homepage on the internet]. c2015 [cited 2019 mar 25]. available from: https://www.who.int/csr/don/2000_05_19/en/ nigeria centre for disease control. an update on yellow fever outbreak in nigeria [homepage on the internet]. c2018 [cited 2019 mar 23]. available from: https://ncdc.gov.ng/diseases/sitreps/?cat=10&name=anupdateofyellowfeveroutbreakinnigeria martin da, muth da, brown t, johnson aj, karabatsos n, roehrig jt. standardization of immunoglobulin m capture enzyme-linked immunosorbent assays for routine diagnosis of arboviral infections. j clin microbiol. 2000;38(5):1823–1826. https://doi.org/10.1128/jcm.38.5.1823-1826.2000 world health organization (who). international coordinating group (icg) on vaccine provision [homepage on the internet]. c2016 [cited 2020 jan 19]. available from: https://www.who.int/csr/disease/icg/qa/en/ nigeria centre for disease control. response to yellow fever cases in edo state [homepage on the internet]. c2018 [cited 2019 mar 17]. available from: https://ncdc.gov.ng/news/157/response-to-yellow-fever-cases-in-edo-state best m, sakande j. practical recommendations for strengthening national and regional laboratory networks in africa in the global health security era. afr j lab med. 2016;5(3):a471. https://doi.org/10.4102/ajlm.v5i3.471 asogun da, adomeh di, ehimuan j, et al. molecular diagnostics for lassa fever at irrua specialist teaching hospital, nigeria: lessons learnt from two years of laboratory operation. plos negl trop dis. 2012;6(9):e1839. https://doi.org/10.1371/journal.pntd.0001839 mazzola lt, kelly-cirino c. diagnostics for lassa fever virus: a genetically diverse pathogen found in low-resource settings. bmj glob health. 2019;4(suppl 2):e001116. http://doi.org/10.1136/bmjgh-2018-001116 abstract introduction methods results discussion acknowledgements references about the author(s) mostafa vaghari-tabari department of clinical biochemistry and laboratory medicine, tabriz university of medical sciences, tabriz, iran liver and gastrointestinal diseases research center, tabriz university of medical sciences, tabriz, iran soheila moein molecular medicine research center, hormozgan university of medical sciences, bandar abbas, iran department of biochemistry, faculty of medicine, hormozgan university of medical sciences, bandar abbas, iran durdi qujeq cellular and molecular biology research center (cmbrc), health research insititute, babol university of medical sciences, babol, iran department of clinical biochemistry, babol university of medical sciences, babol, iran mehrdad kashifard department of internal medicine, gastroenterology division, ayatollah rouhani hospital, babol university of medical sciences, babol, iran haydeh alaoddolehei department of hematology and medical laboratory sciences, para medical faculty, babol university of medical sciences, babol, iran karimollah hajian-tilaki department of biostatistics and epidemiology, babol university of medical sciences, babol, iran citation vaghari-tabari m, moein s, qujeq d, kashifard m, alaoddolehei h, hajian-tilaki k. sensitivity and specificity of mean platelet volume as a laboratory marker for irritable bowel syndrome: can it be added to rome criteria? afr j lab med. 2020;9(1), a1001. https://doi.org/10.4102/ajlm.v9i1.1001 original research sensitivity and specificity of mean platelet volume as a laboratory marker for irritable bowel syndrome: can it be added to rome criteria? mostafa vaghari-tabari, soheila moein, durdi qujeq, mehrdad kashifard, haydeh alaoddolehei, karimollah hajian-tilaki received: 24 feb. 2019; accepted: 14 sept. 2020; published: 21 dec. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: irritable bowel syndrome (ibs) is a functional gastrointestinal disorder. objective: this study aimed to evaluate red blood cell distribution width (rdw) and mean platelet volume (mpv) as laboratory markers to discriminate ibs patients from both healthy controls and patients with inflammatory bowel disease (ibd). methods: this case-control study enrolled patients referred to ayatollah rouhani hospital, endoscopy department, babol, iran, for colonoscopy examination from 2015 to 2017. fifty ibs patients were selected from among patients who had undergone a normal colonoscopy and showed symptoms matching the rome iii criteria. fifty healthy participants and 50 ibd patients, matched for sex and age, were also enrolled in this study. both rdw and mpv were measured and analysed by independent sample t-test and receiver operating characteristic curve analysis. a p-value of less than 0.05 was considered statistically significant. results: while rdw was higher and mpv was lower among ibs patients compared to healthy controls (p = 0.047 and p = 0.001), there were no significant differences in rdw or mpv levels between ibs and ibd patients. the area under the curve of rdw in the discrimination between ibs and ibd was 0.620 (p = 0.039), and the area under the curve of mpv in the discrimination between healthy controls and ibs patients was 0.801 (p = 0.001). conclusion: mean platelet volume is potentially a useful laboratory marker for distinguishing between ibs patients and healthy individuals. red blood cell distribution width should be considered as a potential marker to distinguish among ibs and ibd patients. keywords: ibs; irritable bowel syndrome; inflammatory bowel disease; red blood cell distribution width; mean platelet volume. introduction bowel disorders have been categorised into organic and functional diseases. organic diseases have observable and measurable disease processes. for example, these diseases may be associated with tissue damage. inflammatory bowel disease (ibd) and colorectal cancer are examples of important organic intestinal diseases. there are no organic pathologies such as masses and ulcers in intestinal functional disorders.1 irritable bowel syndrome (ibs) is one of the most common functional gastrointestinal disorders associated with abdominal pain and a range of other symptoms like stomach cramps, bloating, diarrhoea, constipation, or alternate periods of diarrhoea and constipation.2 the exact pathogenesis of ibs is not clear but it is believed that impairment in the brain-gut axis causes ibs. it seems that multiple factors, including environmental factors such as diet, stress, and intrinsic factors, such as epigenetics and genetics, are involved in ibs pathogenesis and can affect the brain-gut axis.2 irritable bowel syndrome symptoms and severity vary from one individual to another. irritable bowel syndrome is commonly diagnosed by clinical signs and symptoms according to the rome iii criteria.3 although colonoscopy is not required in the diagnosis of ibs, a colonoscopy can be requested to rule out organic diseases such as ibd in patients with rectal bleeding. based on the rome iii criteria proposed in 2006, an ibs patient is one who has had recurrent discomfort or abdominal pain 3 days per month in the last 3 months and who has met two or more of the following criteria: decrease in pain or discomfort after defecation, change in stool frequency or change in stool form.4 according to signs and symptoms, ibs is divided into four groups, namely: ibs-d—diarrhoea is the predominant symptom; ibs-c—constipation is the predominant symptom; ibs-m—alternating periods of diarrhoea and constipation is a predominant symptom; ibs-u—no predominant symptom is experinced.5,6 in addition to the rome iii criteria, a simple laboratory test with acceptable sensitivity and specificity will aid the diagnosis of ibs. the effectiveness of laboratory tests for ibs diagnosis is poorly investigated. however, some studies have shown that red blood cell distribution width (rdw) and mean platelet volume (mpv) may be useful for ibs diagnosis.7,8 both rdw and mpv are hematologic markers with clinical significance and are routinely included in the complete blood count test. not only is rdw an indicator of erythrocyte size variation and conventionally used for categorisation of anaemia,9 it is also high in some clinical conditions such as autoimmune disease, liver disorder and sickle cell disease.9,10 some studies have demonstrated high rdw in ibd.11,12 meanwhile, mpv has been traditionally used for examination of platelet production status in the bone marrow and has clinical importance in some circumstances13; mpv levels may also be altered in hypertension, diabetes and ibd.14 irritable bowel syndrome is an organic intestinal disease with two major subtypes: ulcerative colitis and crohn’s disease.15 its clinical signs and symptoms have been shown to have a significant overlap with ibs.5 some studies of ibd have demonstrated that mpv can be used for assessment of disease activity.14,16,17 although ibs is conventionally diagnosed based on clinical symptoms, a further laboratory test can be done to increase the validity of the diagnosis. additionally, patients may need a colonoscopy, for example colonoscopy can be requested for patients who have a family history of ibd. however, this could be avoided if an available laboratory test that accurately discriminates ibs from an organic disorder like ibd. this would also reduce the incidences of colonoscopy. thus this study evaluated the utility of rdw and mpv as potential laboratory biomarkers to discriminate between ibs and healthy controls as well as for distinguishing among ibs and ibd patients. methods ethical considerations this retrospective study is a part of master of science (msc) thesis project (no. 6793) and has conformed to the standards of the world medical association, as embodied in the declaration of helsinki and the protocol was approved by the hormozgan university of medical sciences ethical committee (ir.hums.rec.94.182). all patients signed the written informed consent forms provided by hormozgan university of medical sciences and agreed that their medical information could be used in this study. these forms contained information about the project and how patient information was used. to protect patients’ personal information, a special code was assigned to each patient’s information. study design and sample selection all participants were over 18 years of age and were referred to a gastroenterologist at the ayatollah rouhani hospital, babol in northern iran. the consultation period was from september 2015 to january 2017. irritable bowel syndrome case group these were patients whose clinical symptoms matched the rome iii criteria, who had normal c-reactive protein (crp) and erythrocyte sedimentation rate without organic disorders, confirmed by colonoscopy examination. patients’ symptoms included abdominal pain, diarrhoea, constipation and other ibs symptoms. primary reasons for colonoscopy included rectal bleeding, family history of colorectal malignancy and positive stool occult blood test. colonoscopy examination was done by expert gastroenterologists using an olympus colonoscopy instrument (olympus, tokyo, japan). irritable bowel syndrome patients with any of the following criteria were excluded: abnormal haemoglobin level, iron deficiency, abnormal blood smear microscopic analysis, any type of cancer, diverticular disease, history of colorectal surgery, any type of blood disease, diabetes, cardiovascular dysfunction, liver and kidney disease, any type of infectious disease, any type of congenital disease or use of non-steroidal anti-inflammatory drugs (e.g. using aspirin before blood sampling and colonoscopy examination). a case group of 50 patients, 23 women and 27 men, met the inclusion criteria and were enrolled as ibs patients. among these ibs patients, 18 patients had ibs-d, 17 patients had ibs-c and 15 patients had ibs-m. inflammatory bowel disease case group these were patients diagnosed with ibd by colonoscopy and approved by histopathologic methods. inflammatory bowel disease patients who met the following criteria were excluded: previous colorectal surgery, diverticular disease, cardiovascular disease, cancers, infectious disease, liver diseases, kidney diseases, congenital blood disorders, diabetes and taking non-steroidal anti-inflammatory drugs before blood sampling. fifty ibd patients (14 of them had crohn’s disease and others had ulcerative colitis) were matched for sex and age. among these ibd patients, 25 patients had active disease and 25 patients were in clinical remission. control group the control group consisted of 50 healthy volunteers, matched for sex and age, who were referred to the laboratory of ayatollah rouhani hospital, in babol in northern iran. the healthy participants were selected after consultation with a gastroenterologist. all did not have a colonoscopy examination, clinical signs and symptoms of ibs or a history of ibd. healthy individuals were excluded if they had: any systemic condition, any type of anaemia or blood disorder, abnormal haemoglobin, abnormal crp or erythrocyte sedimentation rate levels, abnormal blood smear, abnormal iron levels or ibs sign or symptoms. laboratory measurements a venous blood sample was taken from each participant’s arm into ethylenediaminetetraacetic acid-tubes for complete blood count testing. for a precise selection of patients and controls routine laboratory analysis including complete blood count, erythrocyte sedimentation rate, crp, iron profile and blood smear microscopic analysis was performed for all participants. red blood cell distribution width and mpv were determined simultaneously with complete blood count in a sysmex cell counter instrument (sysmex, kobe, japan). the crp, serum iron and total iron-binding (tibc) levels were quantitatively measured in an auto-analyser instrument (hitachi, tokyo, japan). the following assay kits were used: bionic crp kit (bionic, tehran, iran), serum iron kit (darman faraz kave, tehran, iran) and tibc kit (darman faraz kave, tehran, iran). serum ferritin level was measured by the enzyme-linked immunosorbent assay method according to the ferritin enzyme-linked immunosorbent assay kit instructions (pishtaz teb, tehran, iran) and using an rt2100c enzyme-linked immunosorbent assay reader instrument (raytolife, hamburg, germany). erythrocyte sedimentation rate was measured by the conventional westergren method. in this method, 2 ml of blood sample collected in a tube containing 0.4 ml of sodium citrate was transferred to a standard westergren-katz tube. the tube was set to stand for 1 h, for erythrocytes to settle. the column of separated plasma was measured, along with the rate of settling in millimetres per hour. statistical analysis the data obtained were analysed using an ibm statistical package for the social sciences (spss software version 17, armonk, new york, united states). the independent sample t-test was used for comparing variable means between groups. the receiver operating characteristic analysis was also used for comparing the utility of mpv and rdw for discrimination between ibs patients and healthy controls as well as for discrimination between ibs and ibd patients. p-values less than 0.05 were considered as statistically significant. results our data demonstrated that the mpv level was significantly reduced while the rdw level was significantly elevated in ibs patients when compared with those of healthy subjects, but the levels of the same parameters were not significantly different between ibs and ibd patients (table 1). table 1: demographic and laboratory characteristics of patient and control groups, babol, iran, 2015–2017. receiver operating characteristic analysis showed that mpv has a larger area under the curve (0. 801, p = 0.001) than rdw (figure 1, figure 2, table 2) in differentiating between the healthy control and ibs patients. mean platelet volume had a 68% sensitivity and 88% specificity at 9.55 femtoliter. figure 1: receiver operating characteristic curve analysis to evaluate mean platelet volume utility in discriminating between healthy controls and irritable bowel syndrome patients, babol, iran, 2015–2017. figure 2: receiver operating characteristic curve analysis to evaluate red blood cell distribution width utility in discriminating between irritable bowel syndrome patients and healthy controls, babol, iran, 2015–2017. table 2: receiver operating characteristic curves for comparison of mean platelet volume and red blood cell distribution width utility in discriminating between healthy controls and ibs patients, babol, iran, 2015–2017. receiver operating characteristic analysis showed that rdw compared to mpv had a larger but not significant area under the curve of 0.620 (p = 0.039) for differentiating between ibs and ibd patients (figure 3, figure 4, table 3). the best cut-off point for rdw was a value of 13.05%, with 72% sensitivity and 56% specificity, to help discriminate ibd patients from ibs patients. figure 3: receiver operating characteristic curve analysis to evaluate mean platelet volume utility in discriminating between irritable bowel syndrome and inflammatory bowel disease patients, babol, iran, 2015–2017. figure 4: receiver operating characteristic curve analysis to evaluate red blood cell distribution width utility in discriminating between inflammatory bowel disease and irritable bowel syndrome patients, babol, iran 2015–2017. table 3: receiver operating characteristic curves for comparison of mean platelet volume and red blood cell distribution width utility in discriminating between irritable bowel syndrome and inflammatory bowel disease patients, babol, iran, 2015–2017. discussion our results showed that there was not a significant difference in rdw and mpv between ibs and ibd patients. although average rdw among ibs patients was lower compared with rdw among ibd patients, the difference was not statistically significant (p = 0.068). however, this difference could become meaningful if the sample size were increased. our data also showed that, in comparison with healthy controls, the mean mpv was lower while rdw was higher among ibs patients, which could be due to subclinical inflammation in ibs patients. subclinical inflammation in ibs has been previously reported in some studies,18,19 and it is well known that mpv and rdw can be altered in inflammatory conditions.11,12,14 our findings augment the hypothesis that subclinical inflammation has a role to play in ibs. however, further studies in this regard are needed. our findings, in contrast to a study conducted in turkey, showed that the mpv levels were higher among ibs patients than among healthy controls.7 this inconsistency may be due to the difference in study populations or inclusion criteria. in our study, the results of receiver operating characteristic curve analysis showed that the area under the curve, sensitivity and specificity of mpv for ibs diagnosis are acceptable. the use of receiver operating characteristic curve analysis to evaluate the utility of mpv in ibs diagnosis has not been previously presented by other studies; thus, a direct comparison of our results with other studies, results was not possible. inflammatory bowel disease is a complex gastrointestinal disease and multiple factors are involved in its pathogenesis.20,21,22 according to our results, rdw has a relatively low specificity for differentiating between ibd and ibs patients. our results are similar to a study conducted in hungary that reported 92% specificity and 78% sensitivity for rdw (cut-off: 13.4%) in active crohn’s disease diagnosis. these researchers also reported that in the majority of ibs patients rdw was normal. they however did not evaluate the specificity and sensitivity of rdw in discriminating between ibd and ibs.23 further studies are needed to evaluate the utility of rdw as a diagnostic marker. there are very few studies investigating laboratory diagnosis of ibs and it is proposed that the mpv may be used as an ibs biomarker. a mean platelet volume test in addition to the clinical parameters of the rome criteria can improve the diagnosis of ibs. limitations our study has some limitations. firstly, a major study limitation is that our sample size was relatively low because the aim of the study was primarily to evaluate the utility of rdw and mpv for laboratory diagnosis of ibs. budget and time limitations further limited the possible sample size. the second limitation of our study was that we could not assess the other laboratory factors to discriminate between healthy controls and ibs patients. however, we used rigorous inclusion and exclusion criteria for patient selection in an attempt to mitigate the influence of confounding conditions. conclusion red blood cell distribution width was higher while the mpv level was lower among ibs patients compared to healthy controls, although the same parameters did not differ significantly when compared with ibd patients. mean platelet volume is a potential marker with adequate sensitivity and specificity to discriminate healthy controls from ibs patients. however, it is not useful for discriminating between ibd and ibs patients. red blood cell distribution width could be a potential marker for differentiation between ibd and ibs. further studies with a sufficiently large sample size are needed for a comprehensive evaluation of the utility of these potential biomarkers to discriminate between healthy controls, ibs patients and ibd patients. acknowledgements the authors express their thanks to hormozgan university of medical sciences. the authors also thank the personnel of the endoscopy department of ayatollah rouhani hospital and the laboratory personnel. moreover, the authors wish to express their gratitude to dr javad shokri-shirvani, dr seyed saeid mohammadi, tooba yousefi, koroush rasoulpour, hamed ghasem tabar and maryam ghasemnejad for their helpful and professional assistance. competing interests the authors have declared that no competing interests exist. authors’ contributions d.q. designed the experiments. m.v.-t. performed the experiments. k.h.-t. analysed the data and s.m. contributed to the writing and revising of the manuscript. m.k. selected the patients based on colonoscopy examination and h.a. helped in the performance of haematology tests and analysed these tests. sources of support this project was funded by prof. soheila moein. also, this investigation was partly supported by the grant no. 9437 from the research council of hormozgan university of medical sciences. data availability statement our data are available from the corresponding author upon reasonable request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references drossman da. functional gi disorders: what’s in a name? gastroenterology. 2005;128(7):1771–1772. https://doi.org/10.1053/j.gastro.2005.04.020 gazouli m, wouters mm, kapur-pojskic l, et al. lessons learned-resolving the enigma of genetic factors in ibs. nat rev gastroenterol hepatol. 2016;13(2):77–87. https://doi.org/10.1038/nrgastro.2015.206 saha l. irritable bowel syndrome: pathogenesis, diagnosis, treatment, and evidence-based medicine. world j gastroenterol. 2014;20(22):6759–6773. https://doi.org/10.3748/wjg.v20.i22.6759 shih dq, kwan ly. all roads lead to rome: update on rome iii criteria and new treatment options. gastroenterol rep. 2007;1(2):56–65. lacy be, mearin f, chang l, et al. bowel disorders. gastroenterology. 2016;150(6):1393–1407. https://doi.org/10.1053/j.gastro.2016.02.031 enck p, aziz q, barbara g, et al. irritable bowel syndrome. nat rev dis prim. 2016;2:16014. https://doi.org/10.1038/nrdp.2016.14 aktas g, alcelik a, tekce bk, tekelioglu v, sit m, savli h. red cell distribution width and mean platelet volume in patients with irritable bowel syndrome. prz gastroenterol. 2014;9(3):160–163. https://doi.org/10.5114/pg.2014.43578 coskun a, yavasoglu i, sargin g, et al. the role of mean platelet volume in patients with non-specific abdominal pain in an emergency department. prz gastroenterol. 2015;10(3):156–159. https://doi.org/10.5114/pg.2015.49042 lippi g, plebani m. red blood cell distribution width (rdw) and human pathology. one size fits all. clin chem lab med. 2014;52(9):1247–1249. https://doi.org/10.1515/cclm-2014-0585 sahli ca, bibi a, ouali f, et al. red cell indices: differentiation between β-thalassemia trait and iron deficiency anemia and application to sickle-cell disease and sickle-cell thalassemia. clin chem lab med. 2013;51(11):2115–2124. https://doi.org/10.1515/cclm-2013-0354 arhan m, önal i̇k, taş a, et al. the role of red cell distribution width as a marker in inflammatory bowel disease. turk j med sci. 2011;41(2):227–234. yeşil a, senateş e, bayoğlu i, erdem ed, demirtunç r, kurdaş övünç a. red cell distribution width: a novel marker of activity in inflammatory bowel disease. gut liver. 2011;5(4):460–467. https://doi.org/10.5009/gnl.2011.5.4.460 bancroft aj, abel ew, mclaren m, belch jj. mean platelet volume is a useful parameter: a reproducible routine method using a modified coulter thrombocytometer. platelets. 2000;11(7):379–387. https://doi.org/10.1080/09537100020008311 gasparyan ay, ayvazyan l, mikhailidis dp, kitas gd. mean platelet volume: a link between thrombosis and inflammation? curr pharm des. 2011;17(1):47–58. https://doi.org/10.2174/138161211795049804 moein s, vaghari-tabari m, qujeq d, majidinia m, nabavi sm, yousefi b. mirnas and inflammatory bowel disease: an interesting new story. j cell physiol. 2019;234(4):3277–3293. https://doi.org/10.1002/jcp.27173 kapsoritakis an, koukourakis mi, sfiridaki a, et al. mean platelet volume: a useful marker of inflammatory bowel disease activity. am j gastroenterol. 2001;96(3):776–781. https://doi.org/10.1111/j.1572-0241.2001.03621.x yüksel o, helvacı k, başar ö, et al. an overlooked indicator of disease activity in ulcerative colitis: mean platelet volume. platelets. 2009;20(4):277–281. https://doi.org/10.1080/09537100902856781 de silva ap, nandasiri sd, hewavisenthi j, et al. subclinical mucosal inflammation in diarrhea-predominant irritable bowel syndrome (ibs) in a tropical setting. scand j gastroenterol. 2012;47(6):619–624. https://doi.org/10.3109/00365521.2012.666672 tornblom h, lindberg g, nyberg b, veress b. full-thickness biopsy of the jejunum reveals inflammation and enteric neuropathy in irritable bowel syndrome. gastroenterology. 2002;123(6):1972–1979. https://doi.org/10.1053/gast.2002.37059 vaghari-tabari m, moein s, qujeq d, kashifard m, hajian-tilaki k. positive correlation of fecal calprotectin with serum antioxidant enzymes in patients with inflammatory bowel disease: accidental numerical correlation or a new finding? am j med sci. 2018;355(5):449–455. https://doi.org/10.1016/j.amjms.2017.12.009 mohammadi e, qujeq d, taheri h, hajian-tilaki k. evaluation of serum trace element levels and superoxide dismutase activity in patients with inflammatory bowel disease: translating basic research into clinical application. biol trace elem res. 2017;177(2):235–240. https://doi.org/10.1007/s12011-016-0891-0 de souza hs, fiocchi c. immunopathogenesis of ibd: current state of the art. nat rev gastroenterol hepatol. 2016;13(1):13–27. https://doi.org/10.1038/nrgastro.2015.186 farkas k, papp m, nyári t, et al. red blood cell distribution width in combination with serological markers can help in the differentiation between crohn’s disease and ulcerative colitis. open gastroenterol j. 2010;4:1–4. https://doi.org/10.2174/1874259901004010001 article information authors: ameh james1,2 kingsley ochei1 nnamdi emenyonu3 lovett lawson3 affiliations: 1family health international 360, abuja, nigeria 2keystone laboratories international, diagnostic unit, abuja, nigeria 3zankli medical centre, plot 1021, b5, shehu yaradua way, abuja, nigeria correspondence to: ameh james email: ameh.james@research.usc.edu.au postal address: molecular engineering research laboratory, university of the sunshine coast, maroochydore dc, qld 4558, australia dates: received: 22 may 2013 accepted: 27 aug. 2015 published: 18 nov. 2015 how to cite this article: james a, ochei k, emenyonu n, lawson l. improved sensitivity, safety and laboratory turnaround time in the diagnosis of pulmonary tuberculosis by use of bleach sedimentation. afr j lab med. 2015;4(1), art. #117, 5 pages. http://dx.doi.org/10.4102/ajlm.v4i1.117 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. improved sensitivity, safety and laboratory turnaround time in the diagnosis of pulmonary tuberculosis by use of bleach sedimentation in this original research... open access • abstract • introduction • methods    • settings and patient recruitment    • sample collection    • direct microscopy    • bleach sedimentation    • microscopic examination and interpretation    • sputum decontamination (modified petroff method), culture and isolation of m. tuberculosis    • statistical analyses    • ethical considerations • results • discussion    • limitations    • recommendations    • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: inadequate diagnostic processes and human resources in laboratories contribute to a high burden of tuberculosis (tb) in lowand middle-income countries. direct smear microscopy is relied on for tb diagnosis; however, sensitivity rates vary. to improve sensitivity of direct microscopy, the researchers employed several approaches, including sputum digestion and concentration of acid-fast bacilli (afb), a technique which uses commercial bleach. objectives: this study compared methods used to diagnose active mycobacterium tuberculosis infections. methods: three sputum specimens were collected from each of 340 participants in abuja, nigeria, over two consecutive days. direct microscopy was performed on all specimens; following microscopy, one specimen from each patient was selected randomly for bleach sedimentation and one for lowenstein-jensen culture. results: direct microscopy produced 28.8% afb-positive results, whilst bleach sedimentation resulted in 30.3%. when compared with the cultures, 26.5% were afb true positive using direct microscopy and 27.1% using bleach sedimentation. whilst the specificity rate between these two methods was not statistically significant (p = 0.548), the sensitivity rate was significant (p = 0.004). conclusion: based on these results, bleach increases the sensitivity of microscopy compared with direct smear and has similar specificity. when diagnosing new cases of pulmonary tb, one bleach-digested smear is as sensitive as three direct smears, reducing waiting times for patients and ensuring the safety of laboratory technicians. introduction top ↑ pulmonary tuberculosis (tb) has a large impact on developing country populations, especially in sub-saharan africa where its burden has been increased by the rapid spread of human immunodeficiency virus (hiv). hiv infection impairs cell-mediated immunity, which provides an opportunity for the reactivation of tb, making individuals living with hiv more susceptible to this pathogen. furthermore, hiv can reduce the sputum positivity rate, leading to false sputum-smear-negative tb.1,2,3 tb can be caused by any member of the mycobacterium tuberculosis complex (m. tuberculosis, m. bovis, m. africanum, m. caprae, m. microti, m. cannettii and m. pinnipedi). the factors responsible for the high burden of tb in lowand middle-income countries (lmic) include poor diagnostic processes and inadequate human resources in laboratories. the most widely-available diagnostic test in these settings is direct smear microscopy (hereafter called direct microscopy), which is used for tb diagnosis. this test is cheap, simple and highly specific for mycobacterium tuberculosis.4 in 1995, through the directly observed therapy short (dots) course strategy, the world health organization (who) set a global target for 2005 to detect 70% of new smear-positive cases.5 unfortunately, this target has not been met, in part due to inaccurate diagnoses.6 several investigators reported varying sensitivity rates of direct microscopy, ranging from 20% – 60% in some settings to 80% in another setting.7,8,9,10,11,12,13, 14,15 this finding has led to the search for alternative techniques to improve the sensitivity rate of direct microscopy, resulting in the development of several methods to optimise the procedure. amongst these approaches is the sputum digestion and concentration of acid-fast bacilli (afb) using commercial bleach (sodium hypochlorite), a widely-available household commodity, instead of the standard sodium hydroxide (naoh) concentration method. the technique has been shown to improve the clarity of the smears and increase the yield of bacilli for easy detection.16 to achieve these benefits, investigators used centrifugation and sedimentation concentration methods. bleach centrifugation and sedimentation studies have been widely reported and reviewed by different authors to determine the suitability of the method for tb diagnosis in lmic.6,17,18 only a few reports that used lowenstein-jensen (lj) culture are available and none of the studies were conducted in nigeria.19,20 the only published study in nigeria utilised the bactec mgit 960 (beckton dickinson, franklin lakes, new jersey, united states), but because of a lack of required technology the researchers could not differentiate between the species of m. tuberculosis complex.21 furthermore, most published studies compared either bleach centrifugation or sedimentation with direct microscopy rather than with the gold standard of mycobacterial culture.13,22,23,24 additionally, in a recent review by cattamanchi et al., lack of validation of these studies has been challenged.18 as a result, this study was conducted to compare the method of bleach sedimentation for less than one hour with direct microscopy and lj culture. this study reports the sensitivity, specificity and positive and negative predictive values of bleach sedimentation and direct microscopy as compared to lj culture, the reference standard. methods top ↑ settings and patient recruitment the dots clinics in six different government-owned and managed health facilities across the federal capital territory (fct) in abuja, nigeria, referred 340 patients to the zankli medical centre. the referring health facilities included maitama district hospital, asokoro district hospital, wuse general hospital, gwagwalada specialist hospital, kubwa general hospital and gwarimpa general hospital. the participants, 192 men and 148 women aged between 10 and 64 years, were prospectively enrolled in the study between november 2004 and july 2005. the participants referred from the six sites were assessed for suspected pulmonary tb. participants who did not submit three specimens over a two-day period and participants receiving anti-tb treatment were excluded from the study. sample collection each of the 340 participants submitted three sputum specimens over two consecutive days. in total, 1020 specimens were collected. the first specimen was collected during the patients’ first visit to zankli medical centre, whilst the second was collected by the patients at their homes. patients were given instructions on how to collect an appropriate specimen for diagnosis of pulmonary tb; this process included taking the sample early in the morning before brushing the teeth. the third specimen was taken at the zankli medical centre when patients delivered their second specimen. the two specimens taken at the zankli medical centre were produced by patients in an open and well-ventilated area of the facility. laboratory technicians performed direct microscopy on all specimens collected and randomly selected one specimen for bleach sedimentation and one specimen for lj culturing. all diagnostic tests gave conclusive results for the 340 participants. direct microscopy smears (1 cm × 2 cm) were made from the purulent sputum, air-dried and heat-fixed on a hot plate at 85 °c for two to three minutes, then stained by the ziehl-neelsen (zn) method (1% filtered carbol-fuchsin and 0.1% malachite green or methylene blue). bleach sedimentation an equal volume of undiluted commercial bleach (5% sodium hypochlorite) was added to the remaining part of the specimen. the specimen cup was tightly closed and the contents were vigorously shaken by hand for about 20 seconds; the cup was then placed at an angle of 45 ° and remained, undisturbed, at room temperature (18 °c – 30 °c) for 30 minutes. the sediment was gently withdrawn using a disposable pasteur pipette and a drop of the deposit was transferred to a slide. this was used to make a smear of approximately 1 cm × 2 cm. the smear was air-dried, heat-fixed and stained using the zn method. microscopic examination and interpretation both the direct and bleach smears were read using the oil immersion lens (×100) of an ordinary light microscope by experienced microscopists who were blinded to the results. slides were read again in the case of discordant results. for both direct and bleach slides, positive and negative smears were defined according to the national tuberculosis and leprosy control program's afb grading system (table 1).7 a patient was reported smear-positive for tb if at least one to nine afb were seen in 100 high-power fields. hence, the study reported on the number of patients diagnosed with active m. tuberculosis infections. table 1: guide to acid-fast bacilli (afb) microscopy interpretation according to the national tuberculosis and leprosy control program's afb grading system.7 sputum decontamination (modified petroff method), culture and isolation of m. tuberculosis sputum for the lj culture technique was selected randomly from the participants’ three specimens. an equal volume of 4% sodium hydroxide was added to 5 ml of sputum in a 30 ml screw-cap tube. this tube was capped tightly and shaken to digest the sputum; thereafter, the tube stood at room temperature for 15 minutes with occasional shaking. the mixture was centrifuged at 3000 revolutions per minute for 15 minutes. the supernatant was carefully decanted, after which the deposit was resuspended with 15 ml of sterile normal saline and re-centrifuged at the same rate. the supernatant was removed and the tube sediment of the second centrifugation was inoculated on an lj agar slope and incubated at 37 °c ± 2 °c. for the first three days, the specimen was observed daily for signs of possible contamination. at weekly intervals over the following six to 10 weeks, the culture was examined regularly for the isolation of m. tuberculosis. positive and negative growth controls were always included, using wild strains of m. tuberculosis complex and sterile distilled water, respectively. the isolates were identified as mycobacterium species by the nitrate reduction test, catalase heat-labile test and zn smear method. statistical analyses the proportions (sensitivity, specificity and negative and positive predictive values) were calculated using standard definitions.25 the estimated proportions were then compared using the two-sample test of proportions for large samples (using the normal approximation to the binomial distribution) and estimating the confidence intervals in the process. in particular, an immediate form of the two-sample test of proportions was applied using the prtesti command in stata software version 11 (stata corp lp, college station, texas, united states). a p-value of < 0.05 was considered statistically significant. ethical considerations verbal informed consent was obtained from the participants and ethical approval granted by the ethical committee of the fct hospital management board and the zankli medical centre. results top ↑ of the 340 participants evaluated for tb, 28.8% were afb-positive using direct smear and 30.3% using bleach sedimentation (table 2). when compared with mycobacterial culture (figure 1), the gold standard, 26.5% of samples were true positive for afb using direct smear and 27.1% using bleach sedimentation (table 3). this comparison determined the sensitivity and specificity rates of the two methods (table 4). whilst the difference in the specificity rate between the two evaluated methods was not significantly significant (p = 0.548), the difference between sensitivity rates was significant (p = 0.004), indicating that bleach sedimentation is more sensitive. furthermore, unlike the difference between positive predictive values (p = 0.542), the difference in the negative predictive values was statistically significant (p = 0.003), demonstrating that the bleach sedimentation method more accurately identified the afb-negative participants who were not infected with m. tuberculosis, in contrast to those with false-negative results. figure 1: positive (a) and negative (b) lowenstein-jensen cultures for m. tuberculosis. table 2: results of ziehl-neelsen microscopy for acid-fast bacilli using both direct and bleach sedimentation-treated sputa, abuja, nigeria, november 2004 to july 2005. table 3: comparison of ziehl-neelsen microscopy for acid-fast bacilli using direct and bleach sedimentation-treated sputa with culture on lowenstein-jensen media following sodium hydroxide decontamination, abuja, nigeria, november 2004 to july 2005. table 4: diagnostic accuracy of direct microscopy and bleach sedimentation-treated sputa compared with lowenstein-jensen culture following sodium hydroxide decontamination, abuja, nigeria, november 2004 to july 2005. discussion top ↑ in this study, researchers found that bleach increased the sensitivity of microscopy compared with the direct smear and had similar specificity. this finding supports the outcomes reported in other studies that have evaluated bleach sedimentation whilst using culture as a reference standard.10,19,20 despite its drawbacks, direct microscopy remains the cornerstone of tb diagnosis in developing countries, particularly in sub-saharan africa. direct microscopy has low sensitivity, as described in a review of 14 studies.6 however, in this study, direct microscopy showed a relative increase in sensitivity. this finding cannot be extrapolated; however, as it was likely the result of the setting in which this study was conducted: a research laboratory with greater time resources than routine diagnostic laboratories, particularly government-owned or public health facilities. though not a problem in this study, an additional drawback of direct microscopy without bleach sedimentation is that it requires the submission of three specimens, which can result in increased dropout rates. many patients are unable to afford the cost of travelling to a health centre to submit one or multiple samples.26 therefore, the failure to attain the who's global target of 70% case detection is not surprising, given that the burden of tb is in developing countries where direct microscopy is still routinely used for diagnosis. the use of bleach sedimentation, requiring only one specimen, has several benefits, such as greatly reducing the workload of overextended laboratories in resource-poor settings and reducing the long turnaround time associated with both direct microscopy and the overnight sedimentation method employed in other studies.10,19 additional advantages associated with bleach sedimentation are safety, ease of manipulation and cost-effectiveness.21 significantly, bleach is readily available even in remote parts of the developing world, whereas sodium hydroxide, traditionally used in laboratories, may be difficult or even impossible to acquire. one study showed that the use of 3% bleach for 20 minutes completely sterilises sputum containing m. tuberculosis.27 however, a previous study found total sterilisation in only 93.6% of afb-positive sputa after treatment with 5% bleach for between 15 minutes and three hours.28 regardless, the use of household-strength bleach in processing sputa for afb microscopy has significant laboratory safety advantages, as it sterilises the majority of processed sputa, reducing technicians’ risk of exposure to afb. limitations this study was conducted in a controlled laboratory, unlike a typical public health laboratory where laboratorians are expected to meet a particular turnaround time. thus, this study was carefully carried out without any ‘time pressure’. it is suggested that a similar study should be performed in a typical routine diagnostic laboratory. recommendations this study recommends the use of bleach for sputum microscopy, as it helps to increase the sensitivity of the test and reduces the work load on the laboratory. it also offers protection for the laboratory personnel's against the bacteria. conclusion in conclusion, in settings with a high tb burden, bleach sedimentation may improve sensitivity and laboratory safety in the diagnosis of m. tuberculosis and reduce waiting periods for test results. evidence shows that one bleach-digested sputum smear may be more sensitive than three direct smears in the detection of new cases of pulmonary tb. this diagnostic test could increase the detection rate of new cases of tb in rural areas in developing countries. acknowledgements top ↑ we wish to thank the patients recruited for this study and james jafali (medical research council, the gambia) for providing statistical support without financial compensation. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions a.j. (family health international 360 and keystone laboratories international, diagnostic unit) performed the literature search, contributed to data analysis and interpretation and drafted the manuscript. k.o. (family health international 360) conceived and designed the study, acquired the samples and data and revised the manuscript. n.e. (zankli medical centre) contributed to the study design, acquired the samples and data and revised the manuscript. l.l. (zankli medical centre) contributed to the study design and revised the manuscript. all authors gave final approval of the version to be published and agree to be accountable for the accuracy and integrity of the work. references top ↑ world health organization. global tuberculosis control: surveillance, planning, financing. who report 2005 [homepage on the internet]. c2014 [updated 2014; cited 2014 jul 9]. available from: http://www.who.int/tb/publications/global_report/2005/en/;now available from: http://www.hst.org.za/sites/default/files/tb2005who.pdf. harries ad, maher d, nunn p. an approach to the problems of diagnosing and treating adult smear-negative pulmonary tuberculosis in high-hiv-prevalence settings in sub-saharan africa. bull world health organ. 1998;76(6):651–662. pmid: 10191561. cantwell mf, binkin nj. impact of hiv on tuberculosis in sub-saharan africa: a regional perspective. int j tuberc lung dis. 1997;1(3):205–214. pmid: 9432365. perkins md. new diagnostic tools for tuberculosis. int j tuberc lung dis. 2000;4(12 suppl 2):s182–s188. pmid: 11144551. dye c, hosseini m, watt c. did we reach the 2005 targets for tuberculosis control? bull world health organ. 2007;85(5):325–420. pmid: 17639221. steingart kr, ng v, henry m, et al. sputum processing methods to improve the sensitivity of smear microscopy for tuberculosis: a systematic review. lancet infect dis. 2006;6(10):664–674. pmid: 17008175, http://dx.doi.org/10.1016/s1473-3099(06)70602-8 james a, abba su, ibrahim a, et al. improving the case detection of pulmonary tuberculosis by bleach microscopy method in the north west of nigeria. jmld. 2013;4(3):34–37. angeby ka, alvarado-gálvez c, pineda-garcía l, et al. improved sputum microscopy for a more sensitive diagnosis of pulmonary tuberculosis. int j tuberc lung dis. 2000;4(7):684–687. pmid: 10907772. bruchfeld j, aderaye g, palme ib, et al. sputum concentration improves diagnosis of tuberculosis in a setting with a high prevalence of hiv. trans r soc trop med hyg. 2000;94(6):677–680. pmid: 11198655, http://dx.doi.org/10.1016/s0035-9203(00)90230-x farnia p, mohammadi f, zarifi z, et al. improving sensitivity of direct microscopy for detection of acid-fast bacilli in sputum: use of chitin in mucus digestion. j clin microbiol. 2002;40(2):508–511. pmid: 11825964, http://dx.doi.org/10.1128/jcm.40.2.508-511.2002 gebre n, karlsson u, jönsson g, et al. improved microscopical diagnosis of pulmonary tuberculosis in developing countries. trans r soc trop med hyg. 1995;89(2):191–193. pmid: 7539954, http://dx.doi.org/10.1016/0035-9203(95)90491-3 vasanthakumari r. concentrated sputum smear microscopy: a simple approach to better case detection in pulmonary tuberculosis. indian j tuberc. 1988;35:80–82. wilkinson d, sturm aw. diagnosing tuberculosis in a resource-poor setting: the value of sputum concentration. trans r soc trop med hyg. 1997;91(4):420–421. pmid: 9373638, http://dx.doi.org/10.1016/s0035-9203(97)90263-7 allwood m, lee y, salaniponi f, et al. case finding with a single sputum sample and household bleach. int j tuberc lung dis. 1997;1(suppl 1):s144. naganathan n, ganapathy kt, rajalakshmi r. evaluation of sputum smears prepared by different methods. indian j med res. 1979;69:893–900. ji. lawson l, yassin ma, ramsay a, et al. short-term bleach digestion of sputum in the diagnosis of pulmonary tuberculosis in patients co-infected with hiv. tuberculosis. 2007;87(4):368–372. pmid: 17392025. angeby ka, hoffner se, diwan vk. should the ‘bleach microscopy method’ be recommended for improved case detection of tuberculosis? literature review and key person analysis. int j tuberc lung dis. 2004;8(7):806–815. pmid: 15260270. cattamanchi a, davis jl, pai m, et al. does bleach processing increase the accuracy of sputum smear microscopy for diagnosing pulmonary tuberculosis? j clin microbiol. 2010;48(7):2433–2439. pmid: 20421442, http://dx.doi.org/10.1128/jcm.00208-10 frimpong eh, adukpo r, owusu-darko k. evaluation of two novel ziehl-neelsen methods for tuberculosis diagnosis. west afr j med. 2005;24(4):316–320. pmid: 16483048. merid y, yassin ma, yamuah l, et al. validation of bleach-treated smears for the diagnosis of pulmonary tuberculosis. int j tuber lung dis. 2009;13(1):136–141. pmid: 19105892. lawson l, yassin ma, ramsay a, et al. microbiological validation of smear microscopy after sputum digestion with bleach; a step closer to a one-stop diagnosis of pulmonary tuberculosis. tuberculosis. 2006;86(1):34–40. pmid: 16263328, http://dx.doi.org/10.1016/j.tube.2005.06.003 gebre-selassie s. evaluation of the concentration sputum smear technique for the laboratory diagnosis of pulmonary tuberculosis. trop. doct. 2003;33(3):160–162. pmid: 12870603, http://dx.doi.org/10.1177/004947550303300313 miörner h, ganlöv g, yohannes z, et al. improved sensitivity of direct microscopy for acid-fast bacilli: sedimentation as an alternative to centrifugation for concentration of tubercle bacilli. j clin microbiol. 1996;34(12):3206–3207. pmid: 8940473. bonnet m, ramsay a, githui w, et al. bleach sedimentation: an opportunity to optimize smear microscopy for tuberculosis diagnosis in settings of high prevalence of hiv. clin infect dis. 2008;46(11):1710–1716. pmid: 18444789, http://dx.doi.org/10.1086/587891 drewe ja, tomlinson aj, walker nj, et al. diagnostic accuracy and optimal use of three tests for tuberculosis in live badgers. plos one. 2010;5(6):e11196. pmid: 20585404, http://dx.doi.org/10.1371/journal.pone.0011196 squire sb, belaye ak, kashoti a, et al. ‘lost’ smear-positive pulmonary tuberculosis cases: where are they and why did we lose them? int j tuberc lung dis. 2005;9(1):25–31. pmid: 15675546. chew r, calderón c, schumacher s, et al. evaluation of bleach-sedimentation for sterilising and concentrating mycobacterium tuberculosis in sputum specimens. bmc infect dis. 2011;11:269. pmid: 21985457, http://dx.doi.org/10.1186/1471-2334-11-269 githui wa, matu sw, tunge n, et al. biocidal effect of bleach on mycobacterium tuberculosis: a safety measure. int. j tuberc lung dis. 2007;11(7):798–802. pmid: 17609057. background complexities of naat selection challenges of robust and rapid naat evaluations conclusion acknowledgements references about the author(s) lesley e. scott department of molecular medicine and haematology, school of pathology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa lara d. noble department of molecular medicine and haematology, school of pathology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa ashika singh-moodley department of molecular medicine and haematology, school of pathology, faculty of health sciences, university of the witwatersrand, johannesburg, south africanational priority programme, national laboratory services, johannesburg, south africa trish kahamba department of molecular medicine and haematology, school of pathology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa diana r. hardie division of medical virology, faculty of health sciences, university of cape town, cape town, south africa wolfgang preiser division of medical virology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa national health laboratory service, cape town, south africa wendy s. stevens department of molecular medicine and haematology, school of pathology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa national priority programme, national laboratory services, johannesburg, south africa citation scott le, noble ld, singh-moodley a, et al. challenges and complexities in evaluating severe acute respiratory syndrome coronavirus 2 molecular diagnostics during the covid-19 pandemic. afr j lab med. 2022;11(1), a1429. https://doi.org/10.4102/ajlm.v11i1.1429 opinion paper challenges and complexities in evaluating severe acute respiratory syndrome coronavirus 2 molecular diagnostics during the covid-19 pandemic lesley e. scott, lara d. noble, ashika singh-moodley, trish kahamba, diana r. hardie, wolfgang preiser, wendy s. stevens received: 15 oct. 2021; accepted: 07 feb. 2022; published: 26 apr. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. background severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the causative agent of coronavirus disease 2019 (covid-19), was first identified in wuhan, china, in december 2019.1 since the world health organization (who) declared covid-19 a global pandemic on 11 march 2020,2 more than 280 million cases of infection and over 5 million deaths had been reported globally by 31 december 2021.3 the primary method of diagnosing infection with sars-cov-2 is by nucleic acid amplification technology (naat). diagnostic assays have become available at an impressive rate, with a total of 514 molecular assays listed by december 2021.4 regulatory bodies in australia, brazil, canada, europe, japan and the united states, and organisations such as the who, the united states food and drug administration, the foundation for innovative new diagnostics (find), and the united states centers for disease control and prevention (cdc) provide ongoing updates on assay performance. many assays, however, remain unavailable in certain geographic regions; in-house assays are in use that are not accessible to these evaluation bodies, and the ongoing emergency use need has meant that national programmes have had to perform their own evaluations. furthermore, in mid-2020, limited guidance was available for performing such evaluations and equally limited guidance existed on acceptance criteria or patient use cases.5 the find had verified 21 naat assay manufacturer claims6 (< 8% of available assays by july 2020), and the who target product profile (tpp) version 0.1 was only released in august 2020.7 through emergency validations in south africa, numerous complexities of molecular testing were quickly realised. many of these align with those described elsewhere,8,9 but additional difficulties arose due to drastic local lockdown measures, and the experience of using assays during lockdown yielded several relevant insights. complexities of naat selection the entire pathology value chain needs to be considered when selecting suitable naats. each step contributes to meaningful and valid test results: patient sampling, specimen type, specimen collection and handling (stability), viral rna purification, amplification and detection, and result interpretation (figure 1). specimen collection and handling challenges include swab type, collection site, collection and pre-treatment methods, and transport. swabs are manufactured from different materials (cotton, nylon, polyester, rayon and foam) and may be spun or flocked.10,11 thus, swabs differ in absorption properties, viral capture and release. swab shafts may be made from wood, plastic or aluminium, and some of these materials are incompatible with certain collection and laboratory techniques (e.g. wooden shafts are unsuitable for paediatric sampling and may inhibit naat).10 commonly used swab collection sites are the nasopharynx, mid-turbinate region, nasal cavity or oropharynx, but the method used for a particular specimen is often not documented or made known to a testing laboratory. alternative specimen types include saliva, gargle, sputum and faecal material. furthermore, multiple swabs may be combined in a single testing vial. after collection, the swabs may be transported dry or in plain saline, phosphate-buffered saline, or various preservation media (e.g. viral transport media). the medium may vary in volume (1 ml – 3 ml), impacting viral concentration. specimens are transported at 2 °c – 8°c or at ambient temperature, which, in some settings, means high humidity and temperature. certain testing laboratories insist on inactivation of viral swabs for biosafety, which could include chemical or heat pre-treatment. figure 1: considerations for severe acute respiratory syndrome coronavirus 2 molecular assays across the pathology value chain. viral rna extraction (or purification) is prone to variability. the majority of rna purification methods rely on automated magnetic bead extraction performed on automated platforms (e.g. nuclisens® easymag® [biomérieux, johannesburg, south africa]). these differ in processing capacity, input volume, purified rna elution yield and turnaround time (tat). manual methods range from spin columns (e.g. rneasy mini spin [qiagen, hilden, germany]), which rely on centrifugation, to crude approaches that are used where no extraction instrumentation is available. the latter include direct lysis-to-polymerase chain reaction (pcr) kits (e.g. lyra® direct sars-cov-2 assay [quidel®, san diego, california, united states]), proprietary lysis buffers (e.g. bosphore ex-tract dry swab rna [anatolia geneworks, istanbul, turkey]) or simply heat (where rapid sample preparation is needed).12 these procedures aim to disrupt viral particles to release rna and inactivate inhibitory enzymes. added variables with the lysis approach could be a specimen’s exposure time to the buffer and buffer compatibility with pcr. the primary specimen input testing volume also differs between technologies: 300 µl added directly to an xpert® xpress® sars-cov-2 cartridge (cepheid, sunnyvale, california, united states), 400 µl to the cobas® sars-cov-2 assay (roche, basel, switzer;and) and 500 µl to the realtime sars-cov-2 assay (abbott, chicago, illinois, united states). similarly, most rna extraction platforms require input of anywhere between 100 µl and 300 µl of raw specimen. furthermore, for a direct lysis-to-pcr assay, input volume can range from 10 µl preservation buffer (nagene [diagnóstica longwood, zaragoza, spain]) to 30 µl raw specimen (smartchek®), and ideally the patient swab should be added directly to the proprietary medium. the naats used are also variable. they are performed on a variety of platforms that may be closed (dedicated) (e.g. m2000® [abbott, chicago, illinois, united states]) or open (‘plug-and-play’ approach subsequent to extraction using different systems and instruments). the latter requires compatibility with real-time thermocycler instruments (e.g. cfx96 touch™ [biorad, hercules, california, united states], quantstudio [thermo fisher scientific, waltham, massachusetts, united states]). throughput ranges from a single test at a time (e.g. genexpert® [cepheid, sunnyvale, california, united states]) to 1000–1500/day (e.g. cobas® [roche, basel, switzerland]). many of the open-platform thermocyclers use 96-well plates and may require specimen batching. as with specimen input volume, the rna input volume also varies between assays (e.g. 8 µl for the allplex sars-cov-2 assay [seegene, seoul, south korea], 10 µl for the taqpath™ sars-cov-2 assay [thermo fisher scientific, waltham, massachusetts, united states], and 14 µl for the perkinelmer™ sars-cov-2 rt-qpcr reagent kit ce-ivd [perkinelmer™, waltham, massachusetts, united states]. a number of novel assays have rna input volumes as low as 5 µl. the differences in volume for extraction or pcr may influence theoretical limits of detection. in addition to these complexities, the sars-cov-2 genes (envelope [e], nucleocapsid [n], spike [s], membrane [m], open reading frames 1a and 1b [orf1ab], rnase-dependent rna polymerase [rdrp]) or combination of genes targeted by naat are inconsistent. these have been reported to affect overall assay sensitivity and specificity,13,14 and may also be impacted by viral genetic evolution that can affect gene targets.15 furthermore, there is the complex layer of different diagnostic algorithms implemented within countries that often change over time. for example, some settings report specimen results based on a single gene target, while others require two or more gene targets or result confirmation by a second naat method using a different gene target. automated software algorithms are not always available for all pcr platforms, and result analysis may require skilled user input (e.g. visual interpretation of amplification curves or adjustment of threshold settings and thus cycle thresholds [ct]). this adds another, potentially subjective, layer of variability. challenges of robust and rapid naat evaluations the complex variables described above come to the fore when clear, comparative standardised evaluations are conducted. such evaluations generally had to be performed in parallel with managing covid-19 testing emergencies. in our case, the most challenging issue experienced early in the pandemic was access to sufficient numbers of relevant clinical specimens available for inclusion in evaluation challenge panels. the volume of extracted rna per specimen was often only sufficient to test a limited number of naats. thus, constant panel ‘manufacture’ was required, with the potential to introduce variability (e.g. different specimens) across evaluations. the sample size, the numbers of positive and negative panel specimens, and the range in viral concentration (high, medium or low) of challenge specimens may influence sensitivity scores. the last addresses the need for assays to correctly identify differences in patient risk stratification: while there does not appear to be a difference in median ct between symptomatic and asymptomatic patients,16 a lower ct (higher viral load [vl]) in real-time pcr assays may indicate increased virus transmissibility16,17 and may impact patient outcomes.17,18 the impact of sars-cov-2 variants on technologies15 should also be considered, as specimens selected from waves of infection driven by different variants could affect the technology (e.g. s gene target failure of the taqpath sars-cov-2 assay [thermo fisher scientific, waltham, massachusetts, united states] with the alpha19 and omicron20 variants). these challenges contribute to generating variability in the assay performance outcome and may make the difference between accepting different assays and criteria. false negative results can arise from low levels of virus due to patient or sample characteristics (i.e. either biologically or artefactually), degraded viral rna, viral genetic variation or presence of inhibitors. false positive results can arise from inaccurate ct threshold settings, and contamination during processing. the correct placement of naat technologies should also be considered. a less sensitive assay may be valuable in settings that receive specimens from patients with high vl concentrations (low ct values), that is, emergency settings or referral centres. in contrast, community or mobile screening and testing sites will require assays with greater sensitivity. in addition to clinical specimens, several types of reference materials (plasmids, biomimetic standards, or viable, inactivated or lysed virus) have become commercially available (e.g. accuplex™ sars-cov-2 reference material kit [seracare, milford, massachusetts, united states]), although these may not always be compatible with naat primer or probe sequences. access to locally manufactured sars-cov-2 viral culture supernatant is an asset to expanding an evaluation panel, independent of naat target genes. culture dilutions may be used to measure assay precision, linearity and, potentially, the limit of detection. another limitation is often kit size, where only a limited number of specimens can be tested per kit supplied or where single-plex pcr assays limit the number of specimens in a test run (e.g. single-plex with 3 gene targets can only assess 32 specimens per 96-well rna extraction plate including controls). other considerations determined during performance evaluation are assay ease of use, time to reportable result and throughput. general considerations, often listed in a tpp are: testing footprint, number of testing steps, operator skills required to perform the test, minimum pipetting volume, biosafety requirements, internal controls, positive and negative batch controls, reagent storage stability, reagent reconstitution required, training needs, maintenance, additional consumables and connectivity options, to name a few. moreover, it is important to select suitable comparator technology, a limitation also described by axell-house et al.21 this may be based on who recommendations, or, under emergency use, current in-county methodology already approved for standard of care (soc). in our case, at least seven soc technologies are in use to report patient results across south africa, and residual specimens from these assays were available for challenge panels. the goal of performance evaluation is to determine whether a new method or technology is as good as soc and use thereof will not alter patient care. however, this does pose a challenge if soc specimens are in a different format or rna degrades during specimen storage. our group obtained institutional review board approval to use residual patient specimens for evaluations with an informed consent waiver. where fresh or paired specimens were needed, a full clinical trial was required with informed consent, which increased the time needed and cost of evaluations. once an evaluation is completed, method comparison involves determining accuracy (sensitivity and specificity) or agreement, precision (reproducibility), linearity and limit of detection. recommendations are made based on analytical performance, as well as ease of use. in the absence of acceptance criteria guidelines, we investigated these parameters for an initial 24 assays using a standardised evaluation protocol approach as described in table 1. over and above the already mentioned complexities and challenges, it became apparent that single method comparison acceptance criteria might not be applicable to all assays. we identified four types or categories of assays submitted for evaluation: (1) closed systems (incorporating rna extraction and naat), (2) standalone pcr kits to be performed off already purified rna, (3) direct lyse-to-pcr, where no front-end rna purification or extraction is available or where there is a need for rapid testing, and (4) standalone rna extraction or purification kits. the 24 assays were among groups 1 (n = 9), 2 (n = 12) and 3 (n = 3), with the predominant viral gene targets being n (35.0%), followed by orf1ab (25.0%), e (20%), s (10.0%) and rdrp (10.0%). a sensitivity score of > 90% was achieved by 62.5% (15/24) of assays and > 95% sensitivity achieved by 37.5% (9/24) compared to soc. those that employ direct lyse-to-pcr (group 3) were below the acceptance criteria by at least a 20% drop in median sensitivity (72.0%) compared to the median sensitivity in group 1 (95%) and group 2 (92.0%). the clinical specimens used in these assays’ evaluation panels covered a range in sars-cov-2 vl, with a median target ct = 27. group 3 performed well on specimens with high vl (ct < 30), and hence another challenge is not only in the choice of range in clinical specimens being evaluated, but potentially the need to apply different acceptance criteria, bearing in mind that group 3 assays are designed for use where no front-end rna extraction (or rapid) system is available. the reduced sensitivity needs to be weighed against the rapid availability of a result which would likely identify patients who are newly symptomatic and at the height of being infectious. therefore, the diagnostic dilemma facing regulators and end users could be ‘no available test’ or ‘specimens routed a further distance (with ensuing longer tat) to a testing laboratory with access to front-end rna extraction technology’, or a ‘less sensitive test with potentially shorter tat and greater clinical relevance’. table 1: key evaluation features of a protocol applied under emergency use and acceptance criteria. conclusion selecting clinically relevant, laboratory-compatible and well-performing sars-cov-2 naat assays or systems for patient care is complex and subject to several challenges, as also highlighted elsewhere.9,22 current evaluation protocols are not robustly performed under emergency circumstances,21 and performance acceptance criteria and technology placement may require flexibility.23 assays themselves have also improved with time. added layers of complexity that we experienced were donated tests that did not always follow required regulatory pathways prior to implementation, and suppliers requesting evaluation from multiple laboratories in the hope of improving their performance score. however, having a new test ready for use is only half the battle, and still requires rapid implementation and scale-up, with further factors requiring investigation such as cost, supply chain management, training, quality assessment, interfacing to existing laboratory information systems, compatibility to existing testing landscapes, continuous quality monitoring and post-market surveillance, to name a few. acknowledgements network of evaluating laboratories: national health laboratory service (lucia hans, kim steegen, irene ketseoglou, puleng marokane, pedro da silva, somayya sarang, koleka mlisana), national institute of communicable diseases (olga perovic, mignon du plessis), clinical laboratory service (blessing kadira, lyndel singh, timothy moshema). developers of viral culture stock: universities of stellenbosch and the western cape (tasnim suliman), national health laboratory service (bavesh kana, bhavna gordhan) and harsha desai and mohapi mokone (programme managers engaging with suppliers and regulators). competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions l.e.s., l.d.n., a.s.-m., t.k., d.r.h., w.p. and w.s.s. contributed equally to the design and implementation of the research, to the analysis of the results and to the writing of the article. ethical considerations the use of residual blood, swab and universal transport medium specimens for research and development of molecular and serology assays for sars-cov-2 was approved by the university of the witwatersrand human research ethics committee (medical): m1911201. sources of support w.s.s., l.e.s., l.d.n. and t.k. are supported by funding received from the bill and melinda gates foundation through the innovation in laboratory engineered accelerated diagnostics investment (grant number opp1171455). data availability data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references zhu n, zhang d, wang w, et al. a novel coronavirus from patients with pneumonia in china, 2019. new engl j med. 2020;382(8):727–733. https://doi.org/10.1056/nejmoa2001017 world health organization. who director-general’s opening remarks at the media briefing on covid-19 – 11 march 2020 [homepage on the internet]. 2020 [cited 2020 apr 2]. available from: https://www.who.int/director-general/speeches/detail/who-director-general-s-opening-remarks-at-the-media-briefing-on-covid-19---11-march-2020 center for systems science and engineering (csse) at johns hopkins university (jhu). covid-19 dashboard [homepage on the internet]. © 2022 [cited 2021 dec 31]. available from: https://coronavirus.jhu.edu/map.html foundation for innovative diagnostics (find). covid-19 test directory [homepage on the internet]. © 2022 [cited 2021 dec 28]. available from: https://www.finddx.org/test-directory/ shuren j, stenzel t. covid-19 molecular diagnostic testing – lessons learned. n engl j med. 2020;383:e97. https://doi.org/10.1056/nejmp2023830 find dx. sars-cov-2 molecular assay evaluation results (3 july 2020) [homepage on the internet]. 2020 [cited 2020 jul 27]. available from: https://www.finddx.org/covid-19/sarscov2-eval-molecular/molecular-eval-results/ world health organization. covid-19 target product profiles for priority diagnostics to support response to the covid-19 pandemic v.0.1 [homepage on the internet]. 2020 [cited 2020 aug 08]. available from: https://www.who.int/publications/m/item/covid-19-target-product-profiles-for-priority-diagnostics-to-support-response-to-the-covid-19-pandemic-v.0.1 feng w, newbigging am, le c, et al. molecular diagnosis of covid-19: challenges and research needs. anal chem. 2020;92(15):10196–10209. https://doi.org/10.1021/acs.analchem.0c02060 vandenberg o, martiny d, rochas o, van belkum a, kozlakidis z. considerations for diagnostic covid-19 tests. nat rev microbiol. 2021;19(3):171–183. https://doi.org/10.1038/s41579-020-00461-z public health england. guidance covid-19: guidance on alternative swab types and transport media [homepage on the internet]. [cited 2021 dec 22]. available from: https://www.gov.uk/government/publications/wuhan-novel-coronavirus-guidance-for-clinical-diagnostic-laboratories/covid-19-guidance-for-alternative-swab-types-and-transport-media kahamba tr, noble l, stevens w, scott l. comparison of three nasopharyngeal swab types and the impact of physiochemical properties for optimal sars-cov-2 detection. j vaccines vaccin. 2020;s6:005. https://doi.org/10.1101/2020.10.21.20206078 marais g, naidoo m, hsiao n-y, valley-omar z, smuts h, hardie d. the implementation of a rapid sample preparation method for the detection of sars-cov-2 in a diagnostic laboratory in south africa 2020. plos one. 2020 oct 20;15(10):e0241029. https://doi.org/10.1371/journal.pone.0241029. ecollection 2020 li d, zhang j, li j. primer design for quantitative real-time pcr for the emerging coronavirus sars-cov-2. theranostics. 2020;10(16):7150–7162. https://doi.org/10.7150/thno.47649 gand m, vanneste k, thomas i, et al. use of whole genome sequencing data for a first in silico specificity evaluation of the rt-qpcr assays used for sars-cov-2 detection. int j mol sci. 2020;21(15):5585. https://doi.org/10.3390/ijms21155585 world health organization. tracking sars-cov-2 variants [homepage on the internet]. 2020 [cited 2021 dec 17]. available from: https://www.who.int/en/activities/tracking-sars-cov-2-variants/ singanayagam a, patel m, charlett a, et al. duration of infectiousness and correlation with rt-pcr cycle threshold values in cases of covid-19, england, january to may 2020. euro surveill. 2020;25(32):pii=2001483. https://doi.org/10.2807/1560-7917.es.2020.25.32.2001483 rao sn, manissero d, steele vr, pareja j. a systematic review of the clinical utility of cycle threshold values in the context of covid-19. infect dis ther. 2020;9(3):573–586. https://doi.org/10.1007/s40121-020-00324-3 prebensen c, hre plm, jonassen c, et al. sars-cov-2 rna in plasma is associated with icu admission and mortality in patients hospitalized with covid-19. clin infect dis. 2020;73(3):e799–e802. https://doi.org/10.1093/cid/ciaa1338 european centres for disease prevention and control. rapid increase of a sars-cov-2 variant with multiple spike protein mutations observed in the united kingdom (20 december 2020) [homepage on the internet]. 2020 [cited 2021 jan 12]. available from: https://www.ecdc.europa.eu/sites/default/files/documents/sars-cov-2-variant-multiple-spike-protein-mutations-united-kingdom.pdf national institute for communicable diseases. sars-cov-2 sequencing update 26 november 2021. prepared by the national institute for communicable diseases (nicd) of the national health laboratory (nhls) on behalf of the network for genomics surveillance in south africa (ngs-sa) [homepage on the internet]. 2021 [cited 2021 nov 27]. availalbe from: https://www.nicd.ac.za/wp-content/uploads/2021/11/update-of-sa-sequencing-data-from-gisaid-26-nov_final.pdf axell-house db, lavingia r, rafferty m, clark e, amirian es, chiao ey. the estimation of diagnostic accuracy of tests for covid-19: a scoping review. j infect. 2020;81(5):681–697. https://doi.org/10.1016/j.jinf.2020.08.043 yuzhong xu mc, xinchun chen, jialou zhu. current approach in laboratory testing for sars-cov-2. int j infect dis. 2020;100:7–9. https://doi.org/10.1016/j.ijid.2020.08.041 mina mj, parker r, larremore db. rethinking covid-19 test sensitivity – a strategy for containment. n engl j med. 2020;383:e120. https://doi.org/10.1056/nejmp2025631 south african health products regulatory authority. md018: specification criteria for covid-19 molecular test kits [homepage on the internet]. [cited 2020 sep 14]. available from: http://www.sahpra.org.za/wp-content/uploads/2020/07/md018-specifications-molecular-test-kits-v1-22072020.pdf article information authors: ankie van den broek1 coosje j. tuijn2 lisette van ’t klooster2 elizabeth msoka3 marion sumari-de boer3 jaffu chilongola4 linda oskam2 affiliations: 1royal tropical institute (kit) health, the netherlands2royal tropical institute (kit), biomedical research, the netherlands 3kilimanjaro clinical research institute (kcri), tanzania 4kilimanjaro christian medical university college, tumaini university makumira correspondence to: ankie van den broek postal address: royal tropical institute, kit health, po 95001, 1090 ha, amsterdam, the netherlands dates: received: 10 july 2013 accepted: 25 feb. 2014 published: 24 july 2014 how to cite this article: van den broek a, tuijn cj, van ’t klooster l, msoka e, sumari-de boer m, chilongola j, oskam l. understanding the interface between clinical and laboratory staff. afr j lab med. 2014;3(1), art. #127, 6 pages. http://dx.doi.org/10.4102/ ajlm.v3i1.127 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. understanding the interface between clinical and laboratory staff in this original research... open access • abstract • introduction • research method and design    • literature search    • analysis of guidelines    • testing • results    • the conceptual model       • inner circle: three phases where communication takes place       • outer circle: factors influencing relationships and communication       • organisational factors       • personal factors       • relationship between organisational factors and personal factors       • context       • analytical framework • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: the interface between clinicians and laboratory staff is where the two meet and work together to provide quality care to their clients (patients). effectiveness of the interface depends on the way the two groups of professionals relate to and communicate with each other. the number and type of tests requested and the use of the test results for clinical decision making can be influenced by the interface between clinicians and laboratory staff. a model to understand the factors and dynamics around the interface is lacking.objectives: to propose a new conceptual model to gain insight and analyse factors that influence the laboratory–clinical staff interface. methods: to develop the conceptual model, a literature study was performed, regulatory guidelines and standards for laboratories were analysed and discussions were held with experts on the topic. result: a conceptual model and analytical framework provided good guidance in understanding and assessing the organisational and personal factors shaping the interface. the model was based on three elements: (1) the three phases of communication (pre-analytical, analytical and post-analytical); (2) the organisational and personal factors of interaction; and (3) the socio-political, economic and cultural context in which clinicians and laboratory staff operate. conclusion: assessment of the interface between clinicians and laboratory workers can be performed in a systematic way. applying this model will provide information to managers of health institutions and heads of laboratories and clinical departments about what happens when clinicians and laboratory staff interact, thus aiding them in designing strategies to improve this interface. introduction top ↑ diagnostic tests requested by clinicians are performed by laboratory staff and provide clinicians and patients with the test results that are required for clinical decision making. the contribution of laboratory services to clinical decision making not only depends on the performance of the laboratory itself, but also on the behaviour of clinicians with regard to requesting tests and using the results.1 request behaviour is influenced by the interactions between these two health cadres.2 a study by bridges et al.3 on interprofessional collaboration highlighted factors that shape the interface, including responsibility, accountability, coordination, communication, cooperation, assertiveness, autonomy, mutual trust and respect. in low-income countries, little research has been performed to understand elements that shape the quality of the interface between clinicians and laboratory workers and their influence on the quality of care. carter et al.4 mention the lack of communication between clinicians and laboratory services, giving various reasons for this from the perspective of the clinicians and the laboratory staff. according to this article, clinicians are not accustomed to teamwork and the laboratory staff may not recognise the clinical importance of their findings, either for clinical decision making or for patient management.4assessment of the interface between clinicians and laboratory workers will help health workers and their managers understand how factors related to the organisational culture and the personalities of the staff members have an impact on the interface and, therefore, the effectiveness and quality of service delivery. research method and design top ↑ the conceptual model depicted in figure 1 was developed after completion of a literature search and an analysis of regulatory guidelines and standards for laboratories, followed by a thorough discussion between clinical and laboratory experts within the team. following this discussion, the study team returned to the literature to search for missing information. figure 1: conceptual model of the various factors that shape the interface between clinical and laboratory staff. literature search a literature search was performed using a non-systematic approach, as it was known beforehand that minimal peer-reviewed literature would be available and important data could be found in grey literature. the first literature search (scopus) yielded 59 relevant peer-reviewed articles, from which 14 were selected as appropriate. articles were searched for that could provide information about the interactions and interface between laboratory and clinical services by using different search terms referring to the services or to the staff working at these services. the search strategy included the following terms: ‘laboratory services’ and ‘health systems’; ‘laboratory services’ and ‘human resources’ or ‘laboratory personnel’ or ‘laboratory staff’ or ‘laboratory workforce’; ‘laboratory services’ and (‘role’ or ‘impact’) and ‘health care’ or ‘health services’; ‘laboratory services’ and (‘physicians’ or ‘clinician’ or ‘nurses’ or ‘health manpower’ or ‘health personnel’ or ‘medical staff’ or ‘nursing staff’ or ‘patients’) and (‘consumer satisfaction’ or ‘satisfaction’ or ‘dissatisfaction’ or ‘interaction’ or ‘opinion’ or ‘attitude of health personnel’); ‘laboratory services’ and (‘essential’ or ‘rational’) and ‘health care’ or ‘hospital’ or ‘hospitals’ or ‘clinic’ or ‘clinics’ or ‘medical centre’ or ‘medical centres’. the second search performed focused on peer-reviewed articles and grey literature from 1995 onwards using google scholar, isi web of knowledge, pubmed, the royal tropical institute website, science direct, scopus and the world health organization website. the following search terms were used: ‘attitude of health personnel’; ‘laboratories/utilization’; ‘physicians/psychology’; ‘trust’ and ‘clinician’ or ‘health workers’; ‘laboratory quality’; ‘national laboratory guideline’; ‘laboratory strengthening declaration’. the term ‘interface’ was not used in the search strategy as it was not expected to increase the number of articles related to the interface between laboratory workforce and clinicians; when both types of health workers are mentioned, all articles concerning this interface appear. adding the term ‘interface’ would yield the retrieval of articles that discuss the interface between the functioning of the laboratory and computerised systems used in the laboratory. this search was followed by the snowball literature search method. analysis of guidelines the analysis of regulatory guidelines and standards for laboratories (international organization for standardization [iso] 15189,5 iso 228696 and iso 9001;7 clinical and laboratory standards institute [clsi] gp 26;8 joint commission international [jci]9) provided information on the proposed daily practices for human resource management and relationship building between the laboratory and clinical departments. the interface between the clinicians and laboratory staff is addressed in these documents. an analysis showed that maintaining the relations with customers and monitoring of the customer satisfaction (clinicians are considered to be customers of the laboratory) are both included in several of these regulatory guidelines (table 1). table 1: issues in iso 15189, iso 22869, iso 9001, clsi gp 26 and jci guidelines that refer to the interface between the laboratory and clinical departments. the importance of including the interface in regulatory guidelines and standards for laboratories is also recognised by yao et al.,10 who mention the consultation of the client and client satisfaction surveys as being key areas on the road to laboratory accreditation. based on the findings from the literature search, intensive discussions were held amongst the clinical and laboratory experts in the study team. it was determined that the quality of the interface is related to the moment when interaction happens, the organisational culture of the health facility and the personalities of the clinicians and the laboratory staff. testing the conceptual model was tested during a mixed-method field study in four health facilities in moshi district of kilimanjaro region, in tanzania. this field study is described in ‘the interface between clinicians and laboratory staff: a field study in northern tanzania’ by tuijn et al.11 results top ↑ the conceptual model the conceptual model is based on three elements: (1) the phases during which communication takes place; (2) the organisational and personal factors that influence the interface; and (3) the social, political, cultural and economic context in which the health facility operates. inner circle: three phases where communication takes place plebani12 argues that clinicians and laboratory staff interact during the preand post-analytical phases; this concept was taken as being the starting point of the framework. a third phase was added to plebani’s concept, namely, the analytical phase. application of plebani’s concept to the analytical phase refers to communication that takes place during the performance of a test or a range of tests; for example, a laboratory worker may ask for clarification on the sample type, sample volume, or information regarding the patient’s situation in order to understand the test results or to suggest additional tests. incidentally, the clinician can be present when a test is performed and the outcome can be discussed based on observations by both health cadres (although this does not happen often). in this way, the phases are linked to the time frame in which the clinician (on behalf of the patient) asks for the diagnostic test, waits for the test results and develops a plan after receiving the results. in all three phases, information sharing between clinicians and laboratory staff can take place. during these phases, a number of activities and interactions take place, as are listed in table 2. this list of activities is based on the knowledge and experience of clinicians and laboratory scientists in the research team. table 2: activities and interactions taking place between clinicians and laboratory staff during the pre-analytical, analytical and the post-analytical phases. outer circle: factors influencing relationships and communication communication between clinicians and laboratory staff is influenced by: (1) organisational factors, such as the management (rules, guidelines, meetings) and the identity of the organisation; and (2) personal factors (knowledge, attitude, competencies) visualised in the outer circle of the conceptual model. in some cases, no literature could be found to support the opinion that certain factors influence the dynamics in the interface. as explained in the methodology, the factors adopted in the model were based on the experiences of the study coordinators. organisational factors identity of the organisation: identity can influence many aspects in an organisation such as conditions for thinking and learning; possibilities for open communication between management and staff; and communication between health cadres working in various departments of the health facility. for this article, the identity of a health organisation in a low-income country was defined based on its position in the health system (primary, secondary or tertiary level); the ownership of the organisation (public, private – faith-based or secular); and the organisation’s profit status (not-for-profit or corporate). it was assumed that the identity of the organisation has a bearing on the level of knowledge of clinicians and laboratory staff. for example, the level of education and opportunities for continuous professional development are different for staff working in referral hospitals compared with staff working in primary healthcare facilities. monitoring and supervision of health staff can be organised differently in public and private health facilities.the identity of an organisation can also relate to the availability of resources, such as equipment, consumables and human resources, as well as to leadership and management styles, thus influencing the motivation of the health staff. it can also influence the personalities and the relationships between people working in an organisation: in faith-based organisations, it is likely that staff have activities in common outside working hours (e.g. church activities) that can influence the communication channels in the health facility. although the literature study did not provide information related to the identity of the organisation, this factor was added to the framework as it was assumed that this is an important variable for the interface between different departments in a health institution. style of management: the management can influence the interface between the clinical and laboratory departments in a health institution in three ways: 1. provision of rules and guidelines for the requesting of diagnostic tests (selecting the correct test, following correct test requisition methods, being aware of the availability of tests) and reporting of test results. here. the role of an ‘intermediate health worker’, often a nurse, is important to take into consideration, but was not noted in the literature search. however from the field study it was learned that a nurse is often responsible for taking samples to the laboratory and collecting the test results, as well as transferring them to the clinician. 2. facilitation of mandatory and voluntary meetings in which clinical and laboratory staff participate and interact. 3. the style of management that influences the performance and motivation of health workers by mechanisms such as availability of job descriptions, supportive supervision of staff, implementation of staff appraisals, provision of opportunities for continuous education and career development and demonstration of appreciation for the staff’s work. these mechanisms also influence the interaction between different cadres of health workers that need each other’s competencies in order to perform their work. personal factors the personal factors that influence the interface are the individual competencies of the health workers, including knowledge, attitude and skills, as well as issues relating to the professionalism and professional education of the clinical and laboratory staff. in the literature, several examples are provided regarding personal factors that influence the laboratory–clinician interface. table 3 provides an overview of the evidence found in the literature regarding these personal factors. table 3: evidence found in peer-reviewed and grey literature on personal factors that have an influence on the interface between clinicians and laboratory staff. phases. the individual competencies of clinical and laboratory staff can be insufficient because of lack of updates on national policies and guidelines, lack of supervision and coaching and lack of motivation to understand the impact of inaccuracy on the quality of care. in the field study described by tuijn et al.,11evidence was found suggesting that lack of competencies can also be a result of a low level of education, especially amongst laboratory workers. many tasks in the laboratory are performed by laboratory attendants without formal training. during the field study, it was determined that the needs and wishes of patients could influence clinicians’ test-requesting behaviour or their use of test results. when waiting times at the laboratory were long, clinicians did not ask for a repeat test, as it could be inconvenient for the patient. when a clinician was influenced in his request behaviour, it was classified as a personal factor. however, management can have an impact on personal factors when, through guidelines and supervision, these issues are discussed. issues related to professional education and professionalism are: (1) the different professional viewpoints of clinicians and laboratory workers: the laboratory worker is focused on the outcome of the test as the gold standard for treatment whilst the clinician values it as additional information to confirm or complete the clinical diagnosis; (2) the institutionalised professional positions of both cadres in the health system and health facility in which clinicians occupy a higher position in the hierarchy; and (3) trust in the quality of the work of the other health professional: for the clinician to make decisions regarding diagnosis and treatment and for the laboratory worker to make decisions regarding the test procedures and interpretation of results, each needs to rely on the competence of the other. relationship between organisational factors and personal factors organisational factors and personal factors are partly interdependent. management of the health facility can influence the individual factors and professionalism through attrition and deployment policies, supervisory mechanisms and opportunities for continuous professional education. the management style can also impact staff motivation and the acceptance and ability of the various health professionals to communicate with persons in different positions and different fields of expertise. the identity of the health facility can also have an influence on the type of job applicants and on personal contacts between staff members (e.g. workers at faith-based hospitals often meet during church activities). context in the outer square of the conceptual model is the context in which the health institution operates. social, cultural, political and economic factors have an impact on the hospital and its health workers. many low-income countries experience a severe shortage of human resources for health, leading to understaffing or employment of insufficiently-qualified staff in several departments of the hospital.13,14,15,16,17,18,19,20 the field study confirmed the shortage of qualified laboratory staff, leading to a situation where staff with less education take on responsibilities that include communicating with highly-educated clinicians. analytical framework we developed an analytical framework that serves as a guide when assessing the interface between laboratory and clinical staff. in this framework, all the factors and activities that influence the dynamics around the interface in the three phases are brought together. this analytical framework was used and is explained in more detail in the field study, ‘the interface between clinicians and laboratory staff: a field study in northern tanzania’, by tuijn et al.11 discussion top ↑ this model is new and was developed for health services in low-income countries, which face challenges with regard to service delivery in resource-constrained settings. the conceptual model and the framework provide an overview of factors that determine the interface between clinicians and laboratory workers. all of the factors that provided the basis for this conceptual model informed the study team about the complexity of this interface, but also showed that a well-functioning interface can contribute to quality of care. in low-income countries, little attention is given to this interface, even though the high workload for many of the health workers requires the efficient use of all services and thus efficient cooperation between services to provide quality care. by creating awareness in both groups and improving the interface between the clinicians and laboratory staff, use of laboratory services can be optimised, enabling clinicians to make better diagnoses and treatment plans for their patients. in developing this model, it was determined that the identified peer-reviewed articles do not provide information regarding all factors that influence an effective interface; for example, the interdepartmental management was hardly discussed. however, the importance of interdepartmental management is partly addressed in the reviewed regulatory standards for laboratories, which mention development and/or monitoring of relationships with clients, including physicians, as well as policies regarding interdepartmental meetings. several factors which were included in the model were not mentioned in either peer-reviewed articles or regulatory standards, for example, general personal factors such as age and gender, and cultural factors such as hierarchy in relationships or family ties, but it was assumed that these factors influence staff attitudes and they were thus included in the framework. the pilot field study provided indications that these factors influence the communication between clinicians and laboratory staff, but the study was too small to make firm conclusions about this issue. the complexity of the interface, which is influenced by organisational and personal factors as well as the health facility’s context, calls for a holistic analysis involving all stakeholders (clinicians, laboratory staff, intermediate health workers such as nurses, the management and patients or clients). this first field study has demonstrated the robustness of this conceptual model. it enables analysis of the factors that shape an effective interface. the plan is to perform an assessment of the interface with a sample of 20 health facilities to increase the evidence on these factors. outcomes of such a study may motivate managers of health institutions and heads of laboratories and clinical departments to invest in analysis of interdepartmental interaction so that, based on their findings, they can design strategies to improve the interface in their settings. conclusion top ↑ a new conceptual model has been developed to assess the interface between laboratory and clinical staff in low income countries. acknowledgements top ↑ we kindly acknowledge marjolein dieleman, royal tropical institute (kit) health, development policy and practice, for her valuable input and critical assessment of this article. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions a.vdb. (royal tropical institute [kit] health), c.j.t. (royal tropical institute [kit], biomedical research), l.vtk. (royal tropical institute [kit], biomedical research) and l.o. (royal tropical institute [kit], biomedical research) developed the conceptual model. a.vdb., c.j.t, e.m. (kilimanjaro clinical research institute), m.s-db (kilimanjaro clinical research institute) and j.c. (kilimanjaro christian medical university college) participated in the field study to test the model. a.vdb. drafted the article. all authors contributed to and approved the final manuscript. references top ↑ 1.cohen gm. access to diagnostics in support of hiv/aids and tuberculosis treatment in developing countries. aids. 2007;21(suppl 4):s81–s87. http://dx.doi.org/10.1097/01.aids.0000279710.47298.5c 2.polage cr, bedu-addo g, owusu-ofori a, et al. laboratory use in ghana: physician perception and practice. am j trop med hyg. 2006;75(3):526–531. 3.bridges dr, davidson ra, odegard ps, et al. interprofessional collaboration: three best practice models of interprofessional education. med educ online. 2011;16:10. http://dx.doi.org/10.3402/meo.v16i0.6035 4.carter j, müller-stöver i, östensen h, et al. good clinical diagnostic practice: a guide for clinicians in developing countries to the clinical diagnosis of disease and to making proper use of clinical diagnostic services. world health organization regional office for the eastern mediterranean: cairo; 2005. isbn: 978-92-9021-393-2. available from: http://www.scribd.com/doc/160701374/dsa236-goof-clinical-diagnostic-practice-who-pdf 5.international organization for standardization. iso15189:2007. medical laboratories – particular requirements for quality and competence [page on the internet]. 2007 [cited 2014 mar 30]. available from: http://www.iso.org/iso/catalogue_detail?csnumber=42641 6.international organization for standardization. iso/tr 22869:2005. medical laboratories – guidance on laboratory implementation of iso 15189:2003 [page on the internet]. 2005 [cited 2014 mar 30]. available from: http://www.iso.org/iso/home/store/catalogue_tc/catalogue_detail.htm?csnumber=35172 7.international organization for standardization. iso 9001:2008. quality management systems – requirements [page on internet]. 2008 [cited 2014 mar 30]. available from: http://www.iso.org/iso/catalogue_detail?csnumber=4648 8.linical laboratory standards institute. gp26-a3. application of a quality management system model for laboratory services, approved guideline – third edition. nccls: wayne, pa; 2004. available from: http://isoforlab.com/phocadownload/csli/gp26-a3.pdf 9.joint commission international. accreditation standards for clinical laboratories. 2nd ed. joint commission international: oakbrook terrace, il; 2010. 10.yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3):401–409. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj 11.tuijn cj, msoka e, mushi dl, et al. the interface between clinicians and laboratory staff: a field study in northern tanzania. afr j lab med. 2014;3(1), art. in press. 12.plebani m. exploring the iceberg of errors in laboratory medicine. clin chim acta. 2009;404(1):16–23. http://dx.doi.org/10.1016/j.cca.2009.03.022 13.nuwaha f. the challenge of chloroquine-resistant malaria in sub-saharan africa. health policy plan. 2001:16(1):1–12. http://dx.doi.org/10.1093/heapol/16.1.1 14.mepham so, squire sb, chisuwo l, et al. utilisation of laboratory services by health workers in a district hospital in malawi. j clin pathol. 2009;62(10):935–938. doi: 10.1136/jcp.2009.069062938 15.world health organization. strategic approach for the strengthening of laboratory services for tuberculosis control, 2006–2009 [document on the internet]. 2006 [cited 2014 mar 30]. available from: http://apps.who.int/iris/bitstream/10665/69303/1/who_htm_tb_2006.364_eng.pdf?ua=1 http://dx.doi.org/10.1093/heapol/czm046 17.butao d, chafulumira f, felling b, et al. malawi: laboratory services and supply chain assessment. usaid/deliver project: arlington, va; 2009. 18.barat l, chipipa j, kolczak m, et al. does the availability of blood slide microscopy for malaria at health centers improve the management of persons with fever in zambia? am j trop med hyg. 1999;60(6):1024–1030. 19.may ta, clancy m, critchfield j, et al. reducing unnecessary inpatient laboratory testing in a teaching hospital. am j clin pathol. 2006;126(2):200–206. http://dx.doi.org/10.1309/wp59ym73l6cegx2f 20.world health organization. the world health report 2006 – working together for health. who: geneva; 2006. available from: http://www.who.int/whr/2006/en/ abstract introduction methods results discussion acknowledgements references about the author(s) adedayo o. faneye department of virology, faculty of basic medical sciences, university of ibadan, ibadan, nigeria oyeteju s. babalola department of virology, faculty of basic medical sciences, university of ibadan, ibadan, nigeria georgina n. odaibo department of virology, faculty of basic medical sciences, university of ibadan, ibadan, nigeria juwon arotiba department of oral and maxilofacial surgery, faculty of dentistry, university of ibadan, ibadan, nigeria olufemi d. olaleye department of virology, faculty of basic medical sciences, university of ibadan, ibadan, nigeria citation faneye ao, babalola os, odaibo gn, arotiba j, olaleye od. oral human papilloma virus infection among dental clinic attendees in ibadan, nigeria. afr j lab med. 2022;11(1), a1555. https://doi.org/10.4102/ajlm.v11i1.1555 original research oral human papilloma virus infection among dental clinic attendees in ibadan, nigeria adedayo o. faneye, oyeteju s. babalola, georgina n. odaibo, juwon arotiba, olufemi d. olaleye received: 17 feb. 2021; accepted: 26 may 2022; published: 25 nov. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: human papilloma virus (hpv) is associated with a subset of oropharyngeal squamous cell carcinoma and mouth or throat warts. however, there is currently limited information about oral hpv infections in nigeria. objective: this study aimed to provide information on the occurrence and circulating genotypes of hpv among patients attending three (one government and two private) dental clinics in ibadan, nigeria. methods: an oral swab was collected from 231 dental clinic attendees in ibadan between january 2016 and march 2017 and tested for hpv dna by polymerase chain reaction targeting the e6/7 genes of the virus. results: twenty-three of the 231 swab samples were hpv dna positive comprising 16 mono-infections and seven co-infections in 13 males and ten females. genotype 16 was present in ten patients, genotype 6/11 in five, genotype 18 and genotype 33 in four each, genotype 31 in three and genotype 39 in one. twenty-one cases were high-risk hpv genotypes, while two were low-risk. samples had co-infection and five had low risk type 6/11 either as single or as co-infection. persons who had engaged in oral sex as well as those aged 21-30 years has significantly higher prevalence. conclusion: this study showed that although hpv genotype 16 is the most common type among dental clinic attendees in ibadan, other genotypes are also circulating and that oral sex is a risk factor for the infection. therefore, introducing a multivalent hpv vaccine will reduce the risk of hpv-associated oropharyngeal carcinoma and other cancers in nigeria. keywords: oral hpv infection; dental clinic attendees; molecular detection; hpv vaccine; nigeria. introduction human papilloma viruses (hpv) are associated with anogenital cancer, including vulval, penile, vaginal, anal, and cervical cancer. these viruses have also been associated with a subset of head and neck cancers causing oropharyngeal squamous cell carcinoma.1 apart from hpv genotypes associated with oropharyngeal carcinoma, infections with a few other genotypes of hpv can cause warts in the mouth or throat.2 oral hpv infection is a common infection worldwide. a 4.9% oral hpv infection prevalence among healthy individuals worldwide has been reported. southern europe has the highest oral hpv prevalence (9.5%), while central america has 6.6% and east asia 0.6%. from the few studies in africa, the prevalence of oral hpv ranges from 2.3% to 6.5%.3 the situation of hiv infection has caused an increase in the prevalence of oral hpv infection, especially among hiv-infected individuals in developed countries.4 however, information on the effect of hiv on the prevalence of oral hpv in africa, and nigeria in particular, is limited. human papilloma viruses are small, non-enveloped, double-stranded dna viruses belonging to the papovaviridae family. the family has five major genera: alpha, beta, gamma, mu, and nu, with those in the alpha genus having the most medical importance. human papilloma viruses are further categorised into high risk or low risk based on their epidemiological associated malignancies.5,6,7,8 there are 14 hpv genotypes classified as high risk and they include hpv types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68, while genotypes 6, 11, 42, 43 and 44 are classified as low-risk types. human papilloma virus infection of the mouth and the oropharynx, like hpv infection of the uterine cervix, is associated with high-risk sexual behaviours (orogenital sex). high-risk hpv genotypes, especially hpv-16, are present in many oral and oropharyngeal squamous cell carcinoma where, in some cases, they play an essential aetiological role.7 presently, the prevalence of anogenital hpv infection in africa ranges from 9.8% in central africa to 19.5% in western africa9; in nigeria, it ranges from 3.5% to 28.8%. the common anogenital hpv genotypes circulating in nigeria are hpv types 16, 18, 31, 35, 33, 39, 45, 51, 52, 56, 59, 70 and 82,10 while those associated with cancer in nigeria include genotypes 16, 18, 33, 35, 45, 52 and 56.10 although various hpv strains are circulating in nigeria, the easily available vaccine is only against genotypes 16 and 18. yet, the available hpv vaccine is not included in the national vaccination programme, and only a few informed individuals make private arrangements for the vaccination. the population of people making this arrangement is also low due to cost and awareness11,12; thus, the hpv vaccine coverage in nigeria is meagre. there is limited information on the prevalence and circulating genotypes of oral hpv in africa and especially nigeria. this information is essential to know if available vaccines will be effective in preventing oral hpv infection and associated oral cancers in nigeria. this study was carried out to provide information on oral hpv and the circulating genotypes among patients presenting in dental clinics in ibadan, nigeria. methods ethical considerations this study was approved by the ethics review committee of the university of ibadan and university college hospital ibadan with approval number 160057, and participants provided written informed consent. confidentiality of the study participants was maintained; participants’ identities were codified, and only the authorised party could link the identity of the participants to the given code when the result is given to the dentist to notify the participants of their result. study design this study was a descriptive cross-sectional study carried out among 231 patients attending three dental clinics comprising one government (university college hospital ibadan dental clinic) and two private dental clinics in ibadan, oyo state, nigeria, from august 2016 to february 2017. government dental clinics in ibadan provide dental services for the population of ibadan and its environment. the minimum sample size calculated was 196 but a total of 231 individuals was recruited for the study. the sample size was determined using the formula: a prevalence of 15%13 at 95% confidence interval and 0.02 level of variability of the target population (n = the sample size, a = 1.96, p = prevalence, q = 1 – p, and d = 0.05). study population a total of 231 individuals who consented and were aged 18 years or older were recruited for the study. the participants were first-time attendees of dental clinics (one public and two private) in ibadan, oyo state, nigeria, presenting with various dental complaints. a convenient sampling method was used.14 individuals were excluded if they were unable or unwilling to provide consent or were in severe pain or with symptoms of oropharyngeal cancer. sample collection and processing socio-demographic and risk behaviour information (presence of oral warts, smoking, number of sexual partners, previous oral infection, and engaging in oral sex) was collected from each participant using a semi-structured questionnaire. a dental technician trained in collecting oral swabs for hpv testing collected oral swab samples from all participants. briefly, the tip of the swab stick was rubbed several times in the mouth and base of the tongue. the swab sticks were then placed in vials containing 500 µl of transport medium containing minimum essential medium and 2% bovine serum albumin with antibiotics (gentamicin) in a secured capped vial. samples were stored and transported at 4 °c to the laboratory. initial processing in the laboratory was done within 24 h of sample collection, and included vortexing each vial containing transport medium, removing the swab; the medium was aliquoted and stored at –80 °c until analysed for hpv dna by polymerase chain reaction (pcr). laboratory analysis total dna was extracted from each sample using a commercially available dna purification kit (jena bioscience, jena, germany) per the manufacturer’s instruction. the extracted dna was tested for the presence of the e6/e7 hpv viral gene by pcr using previously described primers (gp e6-3f, gp-e6-5b and gp-e6-6b) and protocol.15 amplification was achieved using a abi 9700 geneamp thermal cycler (applied biosystems®, waltham, massachusetts, united states) using the following cycling condition: 5 min at 95 °c for dna denaturation followed by 65 cycles of 30 s of denaturation at 95 °c, 30 s of annealing at 45 °c, 30 s of elongation at 68 °c and a final elongation of 5 min at 72 °c. the amplified products (602 base pairs – 666 base pairs) were detected using agarose gel electrophoresis. human papilloma viruses isolates were typed using genotype specific primers targeting the e6/e7 hpv virus gene as previously described15 (table 1). the primers used could not differentiate between genotypes 6 and 11, thus, it is referred to as genotype 6/11. table 1: list of primres used and the amplicon size. data analysis the data anlalysis was done using statistical package for social sciences (spss) version 18.2 (ibm corp. armonk, new york, united states). all the data generated were analysed using descriptive statistics such as mean and standard deviation and results were analysed using chi-square at α = 0.05. results characteristics of the study population a total of 231 study participants were recruited for this study, out of which 129 (55.9%) were female. their mean age was 42.59 (range: 18–62 years) (table 2). all the participants reported having sexual experience, while 46 (20.0%) had engaged in oral sex. thirty-six (15.6%) of the participants had warts, either oral or genital, while 24 (10.4%) were smokers. fifty-four (23.4%) of the participants had more than one sexual partner, and none had received any hpv vaccination. table 2: age distribution of dental clinic attendees in ibadan, nigeria, january 2016 to march 2017. human papilloma virus dna prevalence and genotypes of the 231 oral swabs tested, 23 (9.9%) had hpv dna, of which 21 were high-risk and two were low-risk hpv. of the 23 hpv dna-positive participants, 13 (56.5%) were male and 10 (43.5%) were female. also, 18 (78.3%) of the 23 participants with hpv had previously engaged in oral sex. participants in the age group 21–40 years had the highest rate of positivity (14.5%), whereas those aged 60 years or older had the lowest rate (2.1%) (p = 0.241) (table 3). table 3: distribution of human papilloma virus infection among dental clinic attendees in ibadan, nigeria, january 2016 to march 2017. of the 23 participants with hpv infection, 16 had mono infections while seven had co-infections with either other high-risk or low-risk genotypes. the most common genotypes detected in this study were genotypes 16 and 18. hpv genotype 16 was detected in ten participants, eight of which were single infection and one co-infection with genotype 33, the other one were the low risk group (6/11). genotype 18 was also detected in four participants, of which three were single infection and one co-infection with genotype 31. genotypes 31 and 33 were each detected in three participants, two as single infection and one each as co-infection with other genotype. genotype 39 was the least prevalent genotype detected in only one participants. a total of 21 participants had high-risk hpv dna. discussion the prevalence of oral hpv infection and the circulating genotypes among dental clinic attendees in ibadan, nigeria, was determined in this study. an oral hpv prevalence of 9.9% was observed. previous reports on the rates of oral hpv infection among asymptomatic individuals varied between different geographic areas. it ranged from 12.0% in south africa to 5.0% in the united states. the prevalence obtained among dental clinic attendees in this study is lower than the 12% reported from south africa among individuals attending hiv testing centres13 and higher than the 7.3% and 6.9% reported from the general population in the united states.16,17 the differences obtained could be due to the population tested. the higher prevalence in the south african population could be due to the underlying hiv infection. it was also noted that the prevalence of oral hpv among hiv-positive individuals in the united states is also high. the primers used for the detection of hpv dna in this study target the e6/7 region of the virus. this region is part of the viral oncogenes and is usually integrated into the host genome. thus, primers targeting this region are more sensitive than the l1 region most commonly used. other studies have shown that pcr protocols targeting the l1 region of the hpv genome are likely to miss some infections if the viral dna has been integrated, as the l1 region of the viral genome is usually deleted during viral integration.18,19 in addition, the detection of the e6/7 genes of the hpv can suggest a persistent hpv infection, which could be used to predict people at risk of oropharyngeal carcinoma. in this study, people aged 60 years and older had the lowest prevalence (2.1%) of hpv infection, while those in the age group of 21–40 years had the highest rate (14.5%) (table 3). the 21–40 age group is the most sexually active age group; thus, the pattern may result from sexual activities and engagement in oral sex among the persons within this age range. studies have shown that this is the age group most likely to engage in oral sex. in addition, oral sex was not as common in the past as it is now. this may be why the prevalence of oral hpv infection is higher among younger participants. the age distribution found in this study is similar to what was reported by antonsson et al.20 in australia but different from the pattern reported in the united states by sanders et al.,16 where the highest prevalence (11.3%) was observed among the age group 55–64 years. gender differences have been observed in the distribution of oral hpv.8,9 in this study, there are no significant differences in the gender distribution of hpv dna.21,20 furthermore, findings from this study showed that participants with more than one sexual partner had a higher prevalence of oral hpv infection. positive oral hpv testing is significantly positively correlated with an increase in vaginal sexual partners.21 there was also no significant difference in prevalence of hpv dna as per marital status of the participants in this study, although kero et al.22 reported that stable marital relationships protect men from oral and genital hpv infection. this study also noted that participants who engaged in oral sex had a significantly higher prevalence of oral hpv infection. oral sex is a major route for hpv transmission.21,23,24 although oral sex and having multiple sexual partners are shown to be significantly associated with oral hpv positivity, other behaviours such as kissing can transmit hpv.25 this study detected low risk hpv genotype 6/11 and high-risk genotypes 16, 18, 31, 33 and 39 among dental clinic attendees in oyo state, nigeria. among the previously reported circulating genital hpv types among women in ibadan, nigeria, according to nejo et al. and thomas et al.26,27 the most common hpv genotype (type 16) in this study is similar to the findings of thomas et al. but different from the report of nejo et al., which reported hpv genotype 31 as the most prevalent. human papilloma viruses genotype 16 has also been reported from other parts of the world as the most common oral hpv type. finally, the inclusion of the hpv vaccine into the national vaccination programme to cover both boys and girls in nigeria will reduce the prevalence of oral hpv infection and, ultimately, hpv-associated oropharyngeal cancer in nigeria. presently, the most common vaccine in nigeria (cervarix marketed by glaxosmithkline) targets hpv 16 and 18 and gardasil (marketed by merk & co.) targets only hpv 16, 18, 6 and 11 whereas this study and previous nigerian studies have identified other genotypes. thus, introducing hpv vaccine that will cover other circulating strains in addition to the ones they cover presently will make the vaccination programme more effective. the study by herrero et al.28 has demonstrated the efficacy of hpv vaccination in reducing prevalence of oral hpv after four years of vaccinating women in costa rica. limitations patients with symptoms of oropharyngeal cancer were not included in this study to identify the virus genotypes associated with oropharyngeal cancer in this region. information about the number of kissing partners of the participants was also not accessed in the study. the size of the study population is also a limitation of the study. conclusion this study describes a high prevalence of oral hpv infection among dental clinic attendees in ibadan, oyo state, nigeria, by detecting hpv dna from the oral swabs of the study participants using pcr. a prevalence of 9.9% of hpv infection was identified and also showed that hpv type 16 and 18 are the most common types detected among the study participants. oral sex was also significantly associated with hpv infection. the hpv vaccine for use in nigeria should cover at least the commonest circulating genotypes to reduce the risk of hpv-associated oropharyngeal and other cancers in nigeria. acknowledgements we are grateful to the members of staff of oyo state dental clinic, ibadan, for their assistance in collecting samples analysed, as well as the participants in this study. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them, as well as the participants in writing this article. authors’ contributions a.o.f. was responsible for conceptualisation of the idea, sample collection, sample analysis, writing of the draft manuscript and manuscript review. o.s.b. contributed to conceptualisation of the idea, sample collection, sample analysis and manuscript review. g.n.o. and o.d.o. were involved in conceptualisation of the idea, sample analysis and manuscript review, while j.a. contributed to conceptualisation of the idea, sample collection and manuscript review. sources of support this study was partly funded by the college of medicine seed award 2014. the manuscript writing of the project described was supported by the medical education partnership initiative in nigeria project funded by fogarty international, the office of aids research, and the national human genome research institute of the united states national institutes of health, the health resources and services administration and the office of the united states global aids coordinator under award number r24tw008878. data availability the authors confirm that the data supporting the findings of this study are available within the article. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. the content is solely the responsibility of the authors and does not necessarily represent the official views of the funding organisations. references gillison ml, koch wm, capone rb, et al. evidence for a causal association between human papillomavirus and a subset of head and neck cancers. j natl cancer inst. 2000;92(9):709–720. https://doi.org/10.1093/jnci/92.9.709 greenspan d, de villiers em, greenspan js, de souza yg, zur hausen h. unusual hpv types in oral warts in association with hiv infection. j oral pathol. 1988;17(9–10):482–488. https://doi.org/10.1111/j.1600-0714.1988.tb01321.x wood nh, makua ks, lebelo rl, et al. human papillomavirus prevalence in oral and oropharyngeal rinse and gargle specimens of dental patients and of an hiv-positive cohort from pretoria, south africa. adv virol. 2020;2020:2395219. https://doi.org/10.1155/2020/2395219 beachler dc, weber km, margolick jb, et al. risk factors for oral hpv infection among a high prevalence population of hiv-positive and at-risk hiv-negative adults. cancer epidemiol biomarkers prev. 2012;21(1):122–133. https://doi.org/10.1158/1055-9965.epi-11-0734 cogliano v, baan r, straif k, grosse y, secretan b, el ghissassi f. carcinogenicity of human papillomaviruses. lancet oncol. 2005;6(4):204. https://doi.org/10.1016/s1470-2045(05)70086-3 kurose k, terai m, soedarsono n, et al. low prevalence of hpv infection and its natural history in normal oral mucosa among volunteers on miyako island, japan. oral surg oral med oral pathol oral radiol endod. 2004;98(1):91–96. https://doi.org/10.1016/j.tripleo.2003.12.029 d’souza g, kreimer ar, viscidi r, et al. case-control study of human papillomavirus and oropharyngeal cancer. n engl j med. 2007;356(19):1944–1956. https://doi.org/10.1056/nejmoa065497 de villiers e-m, gunst k, stein h, scherübl h. esophageal squamous cell cancer in patients with head and neck cancer: prevalence of human papillomavirus dna sequences. int j cancer. 2004;109(2):253–258. https://doi.org/10.1002/ijc.11685 bruni l, albero g, serrano b, et al. human papillomavirus and related diseases in africa. summary report [homepage on the internet]. 2021 [cited 2017 mar 29]. available from: http://www.hpvcentre.net/statistics/reports/xfx.pdf bruni l, barrionuevo-rosas l, albero g, et al. executive summary [document on the internet]. ico/iarc information centre hpv cancer, hpv information centre; 2017 [cited 2018 july 15]. available from: http://www.hpvcentre.net/statistics/reports/nga.pdf world health organization. human papillomavirus (hpv) and cervical cancer: fact sheets [homepage on the internet]. 2019 [cited 2019 sep 11]. available from: https://www.who.int/news-room/fact-sheets/detail/human-papillomavirus-(hpv)-and-cervical-cancer world health organization regional office for africa. nigeria’s call to action – time to eliminate cervical cancer in nigeria [homepage on the internet]. 2019 [cited 2021 june 8]. available from: https://www.afro.who.int/news/nigerias-call-action-time-eliminate-cervical-cancer-nigeria vogt sl, gravitt pe, martinson na, hoffmann j, d’souza g. concordant oral-genital hpv infection in south africa couples: evidence for transmission. front oncol. 2013;3:303. https://doi.org/10.3389/fonc.2013.00303 taherdoost h. sampling methods in research methodology: how to choose a sampling tech-nique for research [homepage on the internet]. 2016 [cited 2021 may 20]. available from: https://hal.archives-ouvertes.fr/hal-02546796 sotlar k, stubner a, diemer d, et al. detection of high-risk human papillomavirus e6 and e7 oncogene transcripts in cervica scrapes by nested rt-polymerase chain reaction. j med virol. 2004;74(1):107–116. https://doi.org/10.1002/jmv.20153 sanders ae, slade gd, patton ll. national prevalence of oral hpv infection and related risk factors in the u.s. adult population. oral dis. 2012;18(5):430–441. https://doi.org/10.1111/j.1601-0825.2011.01892.x gillison ml, broutian t, pickard rkl, et al. prevalence of oral hpv infection in the united states, 2009–2010. jama. 2012;307(7):693–703. https://doi.org/10.1001/jama.2012.101 schneider-maunoury s, croissant o, orth g. integration of human papillomavirus type 16 dna sequences: a possible early event in the progression of genital tumors. j virol. 1987;61(10):3295–3298. https://doi.org/10.1128/jvi.61.10.3295-3298.1987 de andrea m, kiyono t, pal a, kundu r. human papillomavirus e6 and e7: the cervical cancer hallmarks and targets for therapy. front microbiol. 2020;10:3116. https://doi.org/10.3389/fmicb.2019.03116 antonsson a, cornford m, perry s, davis m, dunne mp, whiteman dc. prevalence and risk factors for oral hpv infection in young australians. plos one. 2014;9(3):e91761. https://doi.org/10.1371/journal.pone.0091761 dalla torre d, burtscher d, sölder e, widschwendter a, rasse m, puelacher w. the impact of sexual behavior on oral hpv infections in young unvaccinated adults. clin oral investig. 2016;20:1551–1557. https://doi.org/10.1007/s00784-015-1633-y kero km, rautava j, syrjänen k, kortekangas-savolainen o, grenman s, syrjänen s. stable marital relationship protects men from oral and genital hpv infections. eur j clin microbiol infect dis. 2014;33(7):1211–1221. https://doi.org/10.1007/s10096-014-2061-7 chung ch, bagheri a, d’souza g. epidemiology of oral human papillomavirus infection. oral oncol. 2014;50(5):364–369. https://doi.org/10.1016/j.oraloncology.2013.09.003 d’souza g, cullen k, bowie j, thorpe r, fakhry c. differences in oral sexual behaviors by gender, age, and race explain observed differences in prevalence of oral human papillomavirus infection. plos one. 2014;9(1):e86023. https://doi.org/10.1371/journal.pone.0086023 touyz lzg. kissing and hpv: honest popular visions, the human papilloma virus, and cancers. curr oncol. 2014;21(3):e515. https://doi.org/10.3747/co.21.1970 nejo yt, olaleye do, odaibo gn. molecular characterisation of genital human papillomavirus among women in southwestern, nigeria. plos one. 2019;14(11). https://doi.org/10.1371/journal.pone.0224748 thomas jo, herrero r, omigbodun aa, et al. prevalence of papillomavirus infection in women in ibadan, nigeria : a population-based study. 2004:638–645. https://doi.org/10.1038/sj.bjc.6601515 herrero r, quint w, hildesheim a, et al. reduced prevalence of oral human papillomavirus (hpv) 4 years after bivalent hpv vaccination in a randomized clinical trial in costa rica. plos one. 2013;8(7). https://doi.org/10.1371/journal.pone.0068329 abstract introduction methods results discussion acknowledgements references about the author(s) seth a. attoh j.m. wadhwani department of anatomical pathology, 37 military hospital, accra, ghana frederick hobenu j.m. wadhwani department of anatomical pathology, 37 military hospital, accra, ghana lawrence edusei department of pathology, korle-bu teaching hospital, accra, ghana kwasi agyeman-bediako j.m. wadhwani department of anatomical pathology, 37 military hospital, accra, ghana clement t. laryea department of medicine, 37 military hospital, accra, ghana edward o. nyarko public health division, 37 military hospital, accra, ghana michael k. amedi department of radiology, 37 military hospital, accra, ghana richard h. asmah department of molecular biology, university of health and allied sciences, ho, ghana edward asumanu department of surgery, 37 military hospital, accra, ghana mary mcaddy j.m. wadhwani department of anatomical pathology, 37 military hospital, accra, ghana anthony maison j.m. wadhwani department of anatomical pathology, 37 military hospital, accra, ghana godwin nyarko j.m. wadhwani department of anatomical pathology, 37 military hospital, accra, ghana raymond d. fatchu department of pathology, 37 military hospital, accra, ghana kafui akakpo department of pathology, university of cape coast, cape coast, ghana citation attoh sa, hobenu f, edusei l, et al. postmortem diagnosis of covid-19: antemortem challenges of three cases at the 37 military hospital, accra, ghana. afr j lab med. 2020;9(1), a1290. https://doi.org/10.4102/ajlm.v9i1.1290 original research postmortem diagnosis of covid-19: antemortem challenges of three cases at the 37 military hospital, accra, ghana seth a. attoh, frederick hobenu, lawrence edusei, kwasi agyeman-bediako, clement t. laryea, edward o. nyarko, michael k. amedi, richard h. asmah, edward asumanu, mary mcaddy, anthony maison, godwin nyarko, raymond d. fatchu, kafui akakpo received: 03 june 2020; accepted: 21 aug. 2020; published: 03 nov. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: consistency among clinical symptoms, laboratory results and autopsy findings can be a quality measure in the diagnosis of coronavirus disease 2019 (covid-19). there have been classic clinical cases that have met the case definition of covid-19 but real-time reverse-transcription polymerase chain reaction (rrt-pcr) tests of nasopharyngeal swabs were negative. objectives: this study aimed to share pathological observations of autopsies performed at the 37 military hospital’s department of anatomical pathology on three presumed covid-19 cases in accra, ghana. method: complete autopsies with detailed gross and histopathological analysis were conducted between april 2020 and may 2020 on three suspected covid-19 cases, of which two had initial negative (rrt-pcr) nasopharyngeal tests. postmortem bronchopulmonary samples of two cases were collected and tested by rrt-pcr for severe acute respiratory syndrome coronavirus 2 (sars-cov-2). results: the two postmortem bronchopulmonary samples tested for sars-cov-2 by rrt-pcr were positive. though no postmortem bronchopulmonary sample was taken from the third case, a close contact tested positive for sars-cov-2 in later contact tracing. for all three cases, lung histopathological findings were consistent with acute respiratory distress syndrome. conclusion: the outcome of covid-19 testing is dependent on the sample type and accuracy of sampling amongst other factors. histopathological findings vary and may be dependent on a patient’s modifying factors, as well as the duration of infection. more autopsies are required to fully understand the pathogenesis of this disease in ghanaians. keywords: covid-19; autopsy; postmortem diagnosis; false-negative; ghana. introduction the global estimate of confirmed cases of coronavirus disease 2019 (covid-19) as of 27 may 2020 stood at over 5.5 million in approximately 213 countries and territories with over 349 190 deaths, giving a mortality rate of 15.7%.1 in ghana, the first confirmed covid-19 case was reported on 12 march 2020. as at the end of may 2020, over 7303 cases, 34 deaths and 2412 recoveries had been recorded.2 covid-19 emerged from wuhan, hubei province, china in december 2019, and is clinically associated with viral pneumonia.3,4 clinical, laboratory and radiological features for covid-19 are non-specific; features are similar to other respiratory tract infections.5 thus, mild symptoms of covid-19 such as fever, cough, dyspnoea, myalgia and fatigue were initially treated and managed as pneumonia symptoms by healthcare workers. the world health orginization (who) situation report 94 defines a suspected covid-19 case as: [a] person presenting with acute respiratory illness (fever and at least one sign/symptom of respiratory disease, e.g. cough, shortness of breath) and no aetiology that fully explains the clinical presentation; or a patient with an acute respiratory illness and has been in contact with a confirmed or probable case of covid-19 in the last 14 days before the onset of symptoms; or a patient with severe acute respiratory illness (fever and at least one sign/symptom of respiratory disease, e.g. cough shortness of breath; and requiring hospitalization) and in the absence of an alternative diagnosis that fully explains the clinical presentation. (pp. 11, 12) the clinical presentation of covid-19 infection varies from mild to moderate to severe. the severe presentation is reportedly characterized by acute respiratory distress syndrome. in line with this, diffuse alveolar damage (dad) is a reported postmortem pathological feature of covid-19; dad is the histological correlate of acute respiratory distress syndrome. in the acute stage, dad is characterized by hyaline membrane formation in the alveoli and the organizing stage by interstitial widening due to oedema and fibroblast proliferation.7 however, there is an instance where dad was not reported.8 other pathological features that have been reported in the lung include: oedema, fibrinous exudates, reactive hyperplasia limited to some type ii pneumocytes and patchy inflammatory changes with scattered multinucleated giant cells.7 others report pathological similarities between these coronavirus infections: covid-19, severe acute respiratory syndrome (sars) and middle eastern respiratory syndrome.3 though other pathological features have been reported in other organs, including the liver and heart, the changes in these organs have been less significant than those reported in the lung. the prominent role of endotheliitis in covid-19 infection was highlighted in one such publication that reported the presence of viral inclusion in endothelial cells.9 the authors suggested that infection with sars coronavirus 2 (sars-cov-2) results in endotheliitis in several organs from the involvement of the virus and as part of the host inflammatory response. the authors hypothesized that a strategy that targets endotheliitis in susceptible patients (people with diabetes, hypertension, obesity) is likely to improve survival in such patients.9 among nucleic acid tests, the polymerase chain reaction (pcr) method is considered the ‘gold standard’ for the detection of covid-19, and it is characterized by rapid detection, high sensitivity and high specificity.10 the specificity of most real-time reverse-transcription (rrt) pcr tests is estimated at 100%, because the design of the primers is specific for the genome sequence of sars-cov-2. however, false-negative reports may occur and have been associated with intended and non-intended activities during case detection, patient preparation, sample collection, packaging, storage, transport and reporting.11 the nasopharyngeal swabs or other upper respiratory tract specimens, including throat swabs or, more recently, saliva are the commonly used specimens for rrt-pcr diagnosis of covid-19.11 howbeit, the positivity rates of these samples vary; bronchoalveolar lavage has the highest positivity rate. in one study of 205 confirmed covid-19 infections, the rrt-pcr positivity rate of bronchoalveolar lavage, sputum, nasal and oropharyngeal specimens were 93%, 72%, 63% and 32%, respectively.3 although confirmed deaths due to covid-19 are being recorded, autopsy findings of covid-19 reported deaths in africa, including ghana, are largely unavailable. this report shares pathological observations of autopsies performed at the 37 military hospital’s j.m. wadhwani department of anatomical pathology on three presumed covid-19 cases. it also highlights the challenges associated with managing presumed cases. methods ethical considerations due to the emergency nature of the pandemic, ethical review and approval was not conducted. however, all identifiers were removed from the report and verbal consent was also sought from the relations of the deceased, where required. study site the 37 military hospital, a 600-bed tertiary facility, is one of the largest hospitals in ghana. annually, the j.m. wadhwani department of anatomical pathology of the hospital conducts approximately 1500 hospital, medico-legal or coroner’s autopsies in its standard morgue facility. the notices of death were received by the department of anatomical pathology for two presumed covid-19 cases who were being managed within the 37 military hospital. also received at the department was a case referred from a peripheral hospital for a medico-legal or coroner’s autopsy to determine the cause of death. all cases were received between april 2020 and may 2020 and met the clinical case definition of covid-19; antemortem sars-cov-2 tests of the two tested cases were negative. all cases were transported to the morgue under covid-19 recommended protocols as published by the who.12 a complete autopsy was conducted on all cases with detailed gross and histopathological analysis. examinations were performed in the department’s state-of-the-art morgue following guidelines for performing autopsies on presumed covid-19 cases. except in the coroner’s autopsy case, postmortem bronchopulmonary samples were collected, immediately placed in viral transport media and sent to the noguchi memorial institute for medical research where rrt-pcr was performed. selected organs (lungs, heart, brain, kidneys, liver, spleen) were sampled and fixed in 10% buffered formalin for histopathological studies. organ slides were made, stained with haematoxylin and eosin and examined by certified histopathologists. tissue sections were retained in formalin and blocks appropriately stored. findings from the autopsy were corroborated by both clinical presentations and laboratory outcomes. results case 1 the first case was a man with hypertension and diabetes who was in his late thirties. he had a non-productive cough, fever (39.5 °c) and had experienced breathlessness for six days before hospital admission. he alleged that he had no contact with a confirmed or probable covid-19 case. a provisional diagnosis of covid-19 was made, the patient was isolated, and a nasopharyngeal swab was taken for sars-cov-2 testing. the sars-cov-2 test result came in after his demise and was negative. therefore, a coroner’s autopsy was requested and was performed to find out the cause of death (table 1). no postmortem bronchopulmonary specimen was taken. however, following contact tracing, it was discovered that he had prior contact with a confirmed covid-19 patient and that one of his caregivers tested positive for sars-cov-2. table 1: autopsy findings of three presumed covid-19 cases at the 37 military hospital, accra, ghana, april 2020 to may 2020. microscopic sections of the lungs show autolytic changes with bacterial colonization of the bronchioles (figure 1). the prominent pathological findings in fairly preserved areas of the lungs were severe oedema and dad with prominent hyaline membrane formation, infiltration of the interstitium by macrophages and scattered multinucleated giant cells. also, there was evidence of pneumonic changes in the lungs: moderate dense inflammatory cell exudate. in the final autopsy report, the cause of death was listed as bronchopneumonia most likely due to or as a consequence of covid-19; diabetes and hypertension were contributory causes. figure 1: haematoxylin and eosin staining of lung tissue samples from a 38 year old male patient presumed to have covid-19 (37 military hospital, accra, ghana, april 2020). case 1: (a) autolytic changes with bacterial colonization in the smaller airways (blue arrows) ×100. diffuse alveolar damage is noted with hyaline membrane formation (green arrows) ×100. (b) higher magnification showing hyaline membrane (black arrows) and alveolar macrophages (blue arrow) ×400. (c) diffuse alveolar damage with prominent hyaline membrane formation (black arrow) ×100. (d) hyaline membrane formation (black arrows) ×400. (e) pneumonic changes with mixed inflammatory cells exudate (blue arrow). hyaline membrane (black arrow) ×100. (f) area of microthrombi in smaller pulmonary capillaries ×400. case 2 the second case was a 60-year-old woman with hypertension who was morbidly obese and had in the past week been diagnosed with diabetes mellitus. she had a three-week history of worsening breathing difficulty, one day of unproductive cough and no history of asthma. she allegedly had no contact with a confirmed or probable case of covid-19. she was being managed for bronchitis or asthma with no improvement. an initial diagnosis of pulmonary thromboembolism was made. differential diagnoses were congestive cardiac failure due to hypertension or covid-19 infection. she was therefore transferred from the emergency centre to the isolation unit. she was managed with intravenous fluids, anticoagulants, antibiotics, insulin and anti-hypertensive medications. a nasopharyngeal swab was taken for a covid-19 test three days after admission. she experienced palpitation and dyspnoea but no orthopnoea, paroxysmal nocturnal dyspnoea, pedal oedema, calf tenderness or fever. she had an oxygen saturation of 85% at room air and 95% at non-rebreather oxygen, a normal pulse and a blood pressure of less than or equal to 140/90 mmhg throughout admission. she had a marginally increased neutrophil-white blood cell count and a high d-dimer of 1.26 ug/ml (fibrinogen equivalent unit; normal limit 0.0–0.5). her liver function test showed deranged liver enzymes. however, renal function tests, c-reactive protein (3.6) and troponin were normal. a computerised tomogram scan was done, the pulmonary vessels were reported as normal (figure 2). figure 2: high resolution computerised tomography scan of a 60 year old female patient with covid-19 (37 military hospital, accra, ghana, april 2020). case 2: axial image showing traction bronchiectasis in an area of ground-glass opacities (red arrows) and bilateral ‘crazy paving’ opacities (blue arrows). the pcr results for covid-19 were returned as negative three days after sample collection. she was therefore transferred from the isolation unit to a general medical ward. she later suddenly developed severe respiratory distress, started desaturating and later died. an academic autopsy was ordered by the attending clinical team who queried the pcr covid-19 result (table 1). a postmortem bilateral lung parenchymal swab was taken for sars-cov-2 testing. microscopic examination of the lungs showed severe congestion with foci of haemorrhage. there was a proliferation of fibroblasts and infiltration of macrophages within the interstitium and in the alveolar space; dad with characteristic hyaline membranes in the alveoli; and interstitial fibrosis and oedema. fibrin thrombi, mostly located in the subpleural region were noted. the liver and spleen were poorly preserved. microthrombi were also noted in some of the glomeruli (figure 3). in the final autopsy report, the cause of death was listed as acute pulmonary embolism due to or as a consequence of covid-19 pneumonia; diabetes mellitus and hypertensive heart disease were contributing causes. figure 3: haematoxylin and eosin staining of lung tissue samples from a 60 year old female patient with covid-19 (37 military hospital, accra, ghana, april 2020). case 2: (a) alveolar sacs filled with alveolar macrophages (green arrow) and multinucleated giant cells (blue arrow) ×400. (b) fibrous tissue proliferation in alveolar sacs (black arrow). also noted are alveolar macrophages (blue arrow) ×400. (b) fibroblast proliferation (green arrow) ×400. (d) diffuse alveolar damage with prominent hyaline membrane formation (black arrow) x400. (e) thrombus at the glomerulus (blue arrow) ×400. (f) thrombus in a small pulmonary artery (blue arrow) ×100. case 3 the third case was a 55 year old man with hypertension and diabetes who had a seven-day history of non-productive cough and dyspnoea. he had a fever, general weakness and headache but no chest pains, sore throat, leg swelling, paroxysmal nocturnal dyspnoea or orthopnoea. he had a recent travel history that suggested a covid-19 exposure. the man was in respiratory distress; oxygen saturation56% on room air, 82% on non-rebreather oxygen and his temperature was 37.6 °c. his blood pressure was 175/94 mmhg, and his pulse rate was 112 beats per minute regular. he had no pallor of mucous membranes, not jaundiced and hydration was fair. examinations of the chest revealed reduced air entry in lung bases with crepitations. there were bronchial breath sounds over lower zones. heart sounds i and ii were present, normal and no murmurs were heard. no bi-pedal oedema was also noticed. the abdomen was soft, non-tender and there was no organomegaly. a diagnosis of bilateral pneumonia to rule out covid-19 in a patient with diabetes and hypertension was made. pulmonary embolism was a differential diagnosis. he was managed on intravenous fluids, anticoagulants, antibiotics, insulin and anti-hypertensive medications. he had a low haemoglobin (11.5g/dl) and a high white blood cell count (15.58 × 1010/l) with platelets at 185 × 1010/l. liver and renal function tests were normal. a plain chest x-ray and a computerised tomography scan were requested (figure 4). later that day, his breathing became laboured and uneven. his temperature was 36.6 °c, pulse rate 100 beats per minute; respiratory rate was 29 per minute; oxygen saturation was 96% on intranasal oxygen. his blood pressure shot up to 220/140 mmhg but was controlled by intravenous labetalol. figure 4: radiological images of a 55 year old male patient with covid-19 (37 military hospital, accra, ghana, may 2020). case 3: (a) postero-anterior chest x-ray showing bilateral ground-glass opacification and right upper and middle zone peripheral patchy opacities. (b) high resolution computerised tomography scan, coronal view, showing bilateral, ground-glass opacities with thickened interlobular and intralobular lines with ‘crazy paving’ appearance. bilateral peripheral and subpleural opacities (red arrows). a nasopharyngeal swab for sars-cov-2 testing was to be collected the following day but the patient passed before the sample could be taken. an academic autopsy was therefore ordered by the attending clinical team (table 1). postmortem bilateral lung parenchymal swabs were positive for sars-cov-2. microscopic examination of the lungs showed severe congestion with haemorrhages. there were severe pulmonary oedema and moderately dense macrophage exudate. diffuse alveolar damage with hyaline membrane formation was striking, and large pneumocytes showing enlarged nuclei and granular amphophilic cytoplasm were present. microthrombi were also noted in some smaller pulmonary capillaries (figure 5). in the final autopsy report, the cause of death was listed as covid-19 pneumonia with diabetes mellitus and hypertension as contributory. figure 5: haematoxylin and eosin staining of lung tissue samples from a 55 year old male patient with covid-19 (37 military hospital, accra, ghana, may 2020). case 3: (a) severe pulmonary oedema, diffuse alveolar damage with hyaline membrane formation (blue arrow) ×100. (b) severe pulmonary oedema, diffuse alveolar damage with hyaline membrane formation (blue arrow) ×100. (c) large pneumocytes (blue arrow) ×400. (d) microthrombi in small pulmonary arteries (black arrows) ×100. (e) higher magnification (x400) of image (d) showing microthrombi (blue arrow) ×100. (f) interstitial widening, pulmonary oedema and prominent hyaline membranes (blue arrow). discussion though thousands of covid-19 cases have been reported in ghana with more than 30 deaths, no autopsies performed on such cases have been published in the medical literature. postmortem samples were taken for covid–19 testing in two of the cases that were autopsied. both lung parenchymal samples were positive for sars-cov-2. in two cases, an earlier test using a nasopharyngeal swab sample was negative. this suggests that there is the possibility of false-negative results and that there is a proportion of covid-19 patients who, because of their false-negative test results, will be managed in health facilities without protective protocols. similarly, for those that die without being tested or have false-negative test results, their remains will be handled without the necessary safety precautions. to ensure the safety of workers and prevent unconscious exposure, health facilities and funeral homes should ensure that adequate safety measures are put in place during this pandemic. in one case, the antemortem nasopharyngeal sample was negative for sars-cov-2, whereas the postmortem bronchopulmonary sample was positive for sars-cov-2. such false-negative errors for covid-19 may be the result of pre-analytical activities, such as a poorly collected sample, wrong sample labelling, mislabeling, interference from medications and improper storage or transport.11,13 it is therefore essential that great emphasis is made in ensuring adherence to proper sample collection and processing protocols across the entire cascade of activities in the diagnosis and reporting of covid-19. considering the inevitable occurrence of deaths within the global covid-19 pandemic, it is crucial for anatomical pathologists, while following due safety protocols, to look out for evidence of covid-19 in all autopsy cases. the most significant pathological findings in our patients were in the lungs. this finding is similar to previous findings in other covid-19 autopsies. in our patients, significant pathological findings that were present in all three patients included severe oedema, congestion, haemorrhages, the proliferation of pneumocytes, scattered multinucleated giant cells and dad with hyaline membrane formation.3,8,14 other pathological features including the proliferation of fibroblasts with interstitial fibrosis and microthrombi in the kidneys were seen in one patient. again, in a previous report, one patient had patchy inflammatory infiltrates that suggested a pneumonic process.4 though all our patients had diabetes, we did not find the reported endotheliitis that has been reported to be present in some diabetic individuals. although molecular testing for case 1 was not done, dad was observed. this combined with the anecdotal evidence – contact with a covid-19 patient and a later positive covid-19 test of his caretaker – is suggestive of covid-19. the death was thus ascribed to covid-19 infection, in line with who6 situation report 94 for certifying deaths due to covid-19 which states that: [w]here a definite diagnosis of covid-19 cannot be made but it is suspected or likely (e.g. the circumstances are compelling within a reasonable degree of certainty), it is acceptable to report covid-19 on a death certificate as ‘probable’ or ‘presumed’. (p. 12) the pathological presentation observed in the lungs were consistent with similar cases reported in beijing and oklahoma by barton et al.,7 and xu et al.14 limitations bronchopulmonary sampling was not done for sars-cov-2 testing for case 1 as a result of the psychological unpreparedness of the autopsy team for covid-19 autopsies. additionally, no immunohistochemical staining was done for histopathological samples collected to show the presence and distribution of immune cells in the lungs. also, the quality of photomicrographs could have been better if the bodies were better preserved after death. conclusion findings from this study indicate that autopsies are capable of reporting evidence of covid-19 and provides insights for proper sample management for purposes of diagnosis. more so, the diagnosis of covid-19 at autopsy is relevant in situations where molecular testing such as rrt-pcr is not available to inform relevant public health action. bronchopulmonary samples for sars-cov-2 testing during postmortem should be taken for presumed covid-19 cases. more autopsies are required to fully understand the pathogenesis of this disease in ghanaians. = acknowledgements competing interests the authors have declared that no competing interest exist. authors’ contributions s.a.a. conceived the idea and performed autopsies; k.a. and r.d.f. developed the initial manuscript; f.h., l.e. and k.a.-b. reviewed slides; k.a.-b. assisted in autopsies; c.t.l. managed clinical cases; m.k.a. reported x-rays and computerised tomography scans; a.m. and g.n. processed tissues and prepared slides. e.o.n., r.h.a., e.a. and m.m. did the literature review and discussed the findings of the study. all authors reviewed the manuscript and provided feedback. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references who. who covid-19 dashboard who covid-19 dashboard [homepage on the internet]. who; 2020 [updated 27 may 2020; cited 2020 may 27]. available from: https://covid19.who.int/?gclid=cj0kcqjwn7j2brdrarisahjkxmychap3bruknnxltl7keoklhmv1v91uttjfrby-hruu5_zpj4fopcuaaidaealw_wcb ghs. ghana covid-19 updates [homepage on the internet]. ghana health service (ghs); 2020 [updated 26 may 2020; cited 2020 may 27]. available from: https://www.ghanahealthservice.org/covid19/latest.php huang c, wang y, li xl, ren l, zhao j, hu y. clinical features of patients infected with 2019 coronavirus in wuhan, china. lancet. 2020;395(10223):497–506. https://doi.org/10.1016/s0140-6736(20)30183-5 singhal t. a review of coronavirus disease-2019 (covid-19). indian j pediatr. 2020;87(4):286. https://doi.org/10.1007/s12098-020-03263-6 samsami m, bagherpour z, nematihonar b, tahmasbi h. covid-19 pneumonia in asymptomatic trauma patients; report of 8 cases. arch acad emerg med. 2020;8(1):e46. who coronavirus disease. (covid-19) situation report – 94. data as received by who from national authorities by 10:00 cest. 23 april 2020 [document on the internet]. 2019. [cited 2020 sept 27] available from: https://reliefweb.int/sites/reliefweb.int/files/resources/20200423-sitrep-94-covid-19. barton lm, duval ej, stroberg e, ghosh s, mukhopadhyay s. covid-19 autopsies, oklahoma, usa. am j clin pathol. 2020;153(6):725–733. https://doi.org/10.1093/ajcp/aqaa062 ng w-f, to k-f, lam wwl, ng t-k, lee kc. the comparative pathology of severe acute respiratory syndrome and avian influenza a subtype h5n1 – a review. hum pathol. 2006;37(4):390. https://doi.org/10.1016/j.humpath.2006.01.015 varga z, flammer a, steiger p, haberecker m, andermatt r, zinkernagel as. endothelial cell infection and endotheliitis in covid-19. lancet; 2020;395(10234):1417–1418. https://doi.org/10.1016/s0140-6736(20)30937-5 tahamtan a, ardebili a. real-time rt-pcr in covid-19 detection: issues affecting the results. expert rev mol diagn. 2020;20(5):453–454. https://doi.org/10.1080/14737159.2020.1757437 sethuraman n, jeremiah ss, ryo a. interpreting diagnostic tests for sars-cov-2. jama. 2020;323(22):2049–2051. https://doi.org/10.1001/jama.2020.8259 who. infection prevention and control for the safe management of a dead body in the context of covid-19: interim guidance. who; march 24, 2020 p. 6. https://www.who.int/publications/i/item/infection-prevention-and-control-for-the-safe-management-of-a-dead-body-in-the-context-of-covid-19-interim-guidance lippi g, simundic am, plebani m. potential preanalytical and analytical vulnerabilities in the laboratory diagnosis of coronavirus disease 2019 (covid-19). clin chem lab med. 2020;58(7):1070–1076. https://doi.org/10.1515/cclm-2020-0285 xu z, shi l, wang y, et al. pathological findings of covid-19 associated with acute respiratory distress syndrome. lancet respir med. 2020;8(4):422. https://doi.org/10.1016/s2213-2600(20)30076-x abstract introduction methods results discussion acknowledgements references about the author(s) james h. kimotho innovation technology transfer division, kenya medical research institute, nairobi, kenya abdiaziz a. gosar innovation technology transfer division, kenya medical research institute, nairobi, kenya ronald inyangala pharmacy and poisons board of kenya, nairobi, kenya paulyne wairimu pharmacy and poisons board of kenya, nairobi, kenya fred siyoi pharmacy and poisons board of kenya, nairobi, kenya damaris matoke-muhia centre of biotechnology research development, kenya medical research institute, nairobi, kenya cecilia wanjala innovation technology transfer division, kenya medical research institute, nairobi, kenya jeremiah zablon kenyatta national hospital, nairobi, kenya moses orina innovation technology transfer division, kenya medical research institute, nairobi, kenya lucy muita innovation technology transfer division, kenya medical research institute, nairobi, kenya jacqueline thiga innovation technology transfer division, kenya medical research institute, nairobi, kenya lameck nyabuti innovation technology transfer division, kenya medical research institute, nairobi, kenya eunice wainaina innovation technology transfer division, kenya medical research institute, nairobi, kenya joseph mwangi centre for virus research, kenya medical research institute, nairobi, kenya alice mumbi innovation technology transfer division, kenya medical research institute, nairobi, kenya samuel omari innovation technology transfer division, kenya medical research institute, nairobi, kenya ann wanjiru innovation technology transfer division, kenya medical research institute, nairobi, kenya samson m. nzou innovation technology transfer division, kenya medical research institute, nairobi, kenya missiani ochwoto innovation technology transfer division, kenya medical research institute, nairobi, kenya citation kimotho jh, gosar aa, inyangala r, et al. pre-evaluation assessment of serological-based covid-19 point-of-care lateral flow assays in kenya. afr j lab med. 2021;10(1), a1317. https://doi.org/10.4102/ajlm.v10i1.1317 original research pre-evaluation assessment of serological-based covid-19 point-of-care lateral flow assays in kenya james h. kimotho, abdiaziz a. gosar, ronald inyangala, paulyne wairimu, fred siyoi, damaris matoke-muhia, cecilia wanjala, jeremiah zablon, moses orina, lucy muita, jacqueline thiga, lameck nyabuti, eunice wainaina, joseph mwangi, alice mumbi, samuel omari, ann wanjiru, samson m. nzou, missiani ochwoto received: 25 june 2020; accepted: 23 june 2021; published: 17 sept. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: timely testing is a key determinant of management outcomes of coronavirus disease 2019 (covid-19). real-time reverse transcription polymerase chain reaction tests are currently the mainstay for covid-19 testing. however, serological point-of-care tests (pocts) can be useful in identifying asymptomatic and recovered cases, as well as herd immunity. objective: the aim of this study was to assess covid-19 pocts in kenya to support the emergency use authorisation of these tests. methods: between march 2020 and may 2020, 18 firms, of which 13 were from china, submitted their pocts to the national regulatory authority, the pharmacy and poison board, who in turn forwarded them to the kenya medical research institute for pre-evaluation assessment. the tests were run with real-time reverse transcription polymerase chain reaction covid-19-positive samples. pre-covid-19 plasma samples that were collected in june 2019 were used as negative samples. the shelf lives of the pocts ranged from 6 to 24 months. results: only nine (50%) tests had sensitivities ≥ 40% (range: 40% – 60%) and the ability of these tests to detect igm ranged from 0% to 50%. many (7/18; 38.9%) of the kits had very weak igm and igg band intensities (range: 2–3). conclusion: serological-based pocts available in kenya can only detect covid-19 in up to 60% of the infected population. keywords: covid-19; point-of-care; igm; igg; sensitivity; specificity; sars-cov-2. introduction the world is grappling with one of the worst disease pandemics ever experienced in over 100 years, the coronavirus disease 2019 (covid-19). according to a world health organization situation report, as of 07 june 2020, the number of covid-19 cases had reached 6 931 000 people globally, with more than 400 121 deaths. in kenya, the number of reported cases as of 06 june 2020 was 2600 with 83 deaths.1 real-time reverse transcription polymerase chain reaction (rrt-pcr) is currently the gold standard for severe acute respiratory syndrome coronavirus 2 (sars-cov-2) diagnosis as it is highly sensitive and relatively easy to develop.2 however, rrt-pcr test protocols are complex, expensive, mainly suited to advanced laboratories, and typically take 4–6 h to complete. moreover, these tests are manufactured by predominantly european and american companies that have adopted a ‘me first’ policy due to the high numbers of covid-19-related deaths in their own countries, thereby successfully eliminating the chances of these test kits flowing to africa and kenya in quantities sufficient for use.3,4 on the other hand, serological-based point-of-care tests (pocts) that take 5–15 min to complete can pick up asymptomatic or recovered cases of covid-19, making them suitable to support disease surveillance and the determination of herd immunity.5 however, these pocts have low overall sensitivities (34% – 80%) and specificities (70% – 100%) compared to covid-19 rrt-pcr as the gold standard.6 serological-based pocts detect plasma levels of immunoglobin g (igg), immunoglobin m (igm), and, sometimes, iga against sars-cov-2. the levels of igm are elevated during the first week after sars-cov-2 infection, peak at 2 weeks, and then reduce to baseline levels in most patients. on the other hand, igg is detectable after 1 week and is sustained at a high level for a long period.7,8,9 currently, the world health organization does not recommend the use of covid-19 serological tests in the clinical setting. however, the potential of the use of antigen-based pocts at triage to rapidly detect cases is acknowledged.10 to evaluate in vitro serological-based pocts, the world health organization recommends the use of a minimum of 200 prospective covid-19 specimens (100 confirmed rrt-pcr positive samples and 100 prospective specimens from patients with signs and symptoms suggestive of covid-19). at least 30 specimens should be rrt-pcr positive at the time of specimen collection and within a week of symptom onset. the remaining data may be supplemented during the review process. for diagnostic specificity, 200 individual specimens from symptomatic patients that tested negative to covid-19 by rrt-pcr and at least 1000 specimens from the general population collected before november 2019 are used. at least 50% of data are requested for submission.11 manufacturers of commercial covid-19 pocts submit their pocts to the pharmacy and poisons board (ppb), nairobi, kenya, for evaluation to facilitate and enhance the issuance of emergency use authorisation (eua). upon receiving the eua, the supplier of a poct may opt to submit it for the full evaluation. in the face of the pandemic, the regulatory pathway of eua bypasses the often longer, data scrutinising pre-evaluation process conducted by the regulator (the ppb) before allowing market authorisation to a manufacturer. owing to the novelty of the coronavirus and the devastating risk of death of infected persons, the need to avail the pocts and rrt-pcr to the general population was paramount. the need for laboratory performance pre-evaluation was identified as a key component required in ascertaining the quality of the pocts, thus helping to ensure that in the interim period of use of the pocts, performance, efficacy and safety standards are met. this study aimed to conduct a preliminary evaluation of commercial pocts that had been presented to the kenya medical research institute (kemri), nairobi, kenya, by the ppb. methods ethical considerations this study was initially approved by the scientific ethical review unit at kemri under protocol kemri/seru/cbrd/209/4008. it was also reviewed and approved by the kenyatta national hospital ethical review committee under protocol number p274/05/2020. covid-19 patients or subjects had to provide written informed consent before blood sample collection. all samples were anonymised. sample collection a total of 50 anonymised blood samples were collected through purposive sampling from patients (male and female) of all ages with active infection as determined by covid-19 rrt-pcr. the study subjects were from the isolation and quarantine centre at the kenyatta national hospital infectious diseases unit. after consenting, 5 ml blood samples were drawn from the subjects and transported to the kemri innovation and technology division. the serum was separated and stored at –20 °c until the day of the pre-evaluation assessment. eighteen covid-19 pocts were received from the ppb, the kenyan drug regulatory authority, who had received the tests from various clients for pre-evaluation assessment and subsequent registration for eua. the kits included in the study were rapid-format antibody-based pocts that were meant to detect igm or igm/igg antibodies to sars-cov-2 nucleocapsid or spike 1 or 2 proteins. pre-covid human de-identified archived blood samples were collected from national blood transfusion centres and were randomly selected for the pre-evaluation of kits. they were stored at –80 °c at the kemri innovation and technology division to serve as covid-negative samples. test evaluation the tests were run according to the manufacturers’ instructions under a biosafety cabinet. briefly, for lateral flow assays, the procedure was as follows: blood samples were spun in a centrifuge and the serum and plasma were separated. the test cassette was removed from foil and allowed to equilibrate to room temperature. one drop (about 10 μl) of plasma sample was loaded using a micropipette into the sample well in the cassette. thereafter, about 60 μl (2–3 drops) of the assay solution (chase buffer) was pipetted into the sample well in the device. after about 10 min, the results were interpreted. only tests in which the colour of the control line changed were considered valid, and if a coloured line was observed for igm or igg, the test was considered positive. the intensity of the colour was compared with that of the colour reference card and semi-quantified. the immunofluorescent protocol was as follows: 150 μl of detector diluent was transferred into a vial containing detector crystal. 10 μl of plasma sample was then added, mixed, and, using a micropipette, 75 μl of the mixture was pipetted into the sample well in the cartridge. after 10 min, the cartridge was placed into the immunofluorescent reader and the results were read and interpreted. determination of sensitivity and specificity of the assays data generated were recorded and analysed using microsoft excel 2010 (microsoft corporation, redmond, washington, united states) and stata version 14.1 (statacorp llc, brownsville, texas, unites states). the specificity of the assays was determined using a panel of 50 pre-covid-19 pandemic serum samples from june 2019. sensitivity was determined for each assay using 50 rrt-pcr sars-cov-2-positive samples. the rna for confirmatory tests were extracted using a qiaamp viral rna mini kit (qiagen, germantown, maryland, united states). the pcr was done on an applied biosystems quantstudio™ 5 (thermofisher scientific, waltham, massachusetts, united states) pcr machine using a sacace biotechnologies sars-cov-2 real-tm detection kit (scalabrini, como, italy) with their recommended cycling conditions set at 35 °c for 20 min for reverse transcription, initial pcr activation at 94 °c for 10 s, and five cycles at 64 °c for 25 s. the cycling step was done at 94 °c for 10 s followed by 64 °c for 25 s for 45 cycles. analysis of the results was done using quantstudio design and analysis software (thermofisher scientific, waltham, massachusetts, united states) and interpreted using the sacace sars-cov-2 real-tm result analysis guide. the antibody tests were considered positive when the control band and igm, igg or both bands were visible. diagnostic sensitivity (%) was calculated as a/(a + c) × 100 while diagnostic specificity (%) was calculated as d/(b + d) × 100, where a represents true positive results, c represents false negatives, b represents false positives and d represents true negatives. the pocts were considered to have a high sensitivity when able to correctly identify all positive samples in a panel. tests that only detected 40% or lower of the positive samples in the panel were deemed to have lower sensitivity as they would miss positives and give higher false negative rates. results the study involved a total of 18 pocts from different manufacturers. the number of kits submitted by each manufacturer differed as the national policy on the number of kits required for eua pre-evaluation assessment had not been established at the time. the shelf lives allocated to the kits were highly varied: 6 months (2/18; 11.1%), 12 months (8/18; 44.4%), 18 months (1/18; 5.6%), and 24 months (7/18; 38.9%) (table 1). many (7; 38.9%) of the kits were manufactured in march 2020 (and the study commenced in may 2020). thirteen (72.2%) of the kits analysed were manufactured in china, 2 (11.1%) in korea, 1 (5.6%) in canada, 1 (5.6%) in the united states, and 1 (5.6%) in malaysia. table 1: characteristics of covid-19 point-of-care test kits submitted to the kenya medical research institute by the pharmacy and poisons board, nairobi, kenya, for pre-evaluation assessment, march–may 2020. most of the tests (15/18; 83%) were based on covid-19 igg or igm detection on separate bands; one was based on covid-19 igg or igm detection on a combined band, while two could only detect igm. the signal from 17 tests could be detected by the naked eye, while the signal of one kit (id 11) could be detected only using a fluorometric machine which is a semi-automated in vitro diagnostic device that detects analytes through fluorescent scanning. diagnostic sensitivity of the point-of-care test kits kit 2 (igm) and kit 14 showed low sensitivities of 26.7% and 11.1%; kit 5 showed no activity; kit 11, which was the only kit with igg and igm combined in a single band test, showed high sensitivity (figure 1). of the total 18 kits, 6 (33.3%) kits had sensitivities of at least 50%: kits 7, 10, 12, 13, 16, and 18. five kits had diagnostic sensitivities between 40% and 50% (kits 3, 4, 6, 8 and 17), while kits 1, 9 and 15 had sensitivities between 30% and 40%. only kits manufactured in china and the united states managed to score more than 40% diagnostic sensitivity. figure 1: diagnostic sensitivity of the pre-assessed covid-19 point-of-care tests submitted to the kenya medical research institute by the pharmacy and poisons board, nairobi, kenya, for pre-evaluation assessment, march–may 2020. combined igg and igm sensitivity was assessed using rrt-pcr as standard. diagnostic sensitivity of the igg and igm separate bands the diagnostic sensitivity of the igg and igm bands were considered separately for all the pocts (figure 2). the igm band of kit 1 did not demonstrate any capacity to detect igm. kit 2, an igm-only kit, had a low sensitivity of 19% and a specificity of 95%. the sensitivity of kit 13 was 55% for igg and 40% for igm. kit 12 showed higher diagnostic sensitivity for igg (40%) than for igm (5%). kit 3 was the only kit that showed higher diagnostic sensitivity for igm (43%) than igg (19%). interestingly, five kits (kits 4, 6, 8, 10, and 18) showed equal diagnostic sensitivity for igg and igm. there was a weak positive correlation between the igm and igg diagnostic sensitivities of the kits (pearson’s correlation coefficient [r] = 0.2527; r2 = 0.0639; p = 0.45). figure 2: diagnostic sensitivity of the pre-assessed covid-19 point-of-care tests submitted to the kenya medical research institute by the pharmacy and poisons board, nairobi, kenya, for pre-evaluation assessment, march 2020 – may 2020. separate igg or igm sensitivities were assessed using rrt-pcr as standard. diagnostic specificity of the point-of-care test kits eight kits (kits 1, 3, 8, 9, 12, 14, 16, and 18) had a diagnostic specificity of 100%, while five kits had low diagnostic specificities of 81% (kit 7), 92% (kit 17), 94% (kit 13), and 95% (kits 2 and 15). the remaining five kits were not tested for diagnostic specificity as they had run out of stock. igm and igg band signal intensities of the point-of-care test kits the intensities of the igm and igg bands of the pocts were analysed with the assistance of a colour reference chart (figure 3). the average intensities of the observed bands of many (7/18; 38.8%) of the kits were at score 3 on the scale (figure 3). the intensities of the igg bands were generally stronger than those of igm. kits 3, 6, 8, 10, 12, 13, 16, 17, and 18 displayed the strongest intensities, which corresponded with their higher diagnostic sensitivities compared to the remaining kits. figure 3: colour intensity of the positive control signal for the igm and igg bands of point-of-care tests assessed at kenya medical research institute between march 2020 and may 2020. discussion this study carried out a pre-evaluation assessment of poct kits submitted by manufacturers to the ppb for pre-evaluation. out of the kits evaluated, 50% had sensitivities of ≥ 40%, and most of the kits had low band intensities. the kits with sensitivities of ≥ 40% were given emergency approval. currently, rrt-pcr tests are the mainstay of sars-cov-2 diagnosis; however, they have complex protocols, are expensive, and require advanced laboratory equipment.2 the main goal of introducing a poct is to avail quick test results to the healthcare workers or the patient to support fast clinical management decisions and, ultimately, improve patient outcomes and overall public health. it is hoped that pocts will be useful in identifying asymptomatic and recovered cases of covid-19 as well as herd immunity.12 the pre-evaluation assessment of pocts is crucial to mitigating the risks associated with the introduction of kits that can cause public anxiety and false alarm to the market. the regulatory pathway for the evaluation and assessment of pocts per the stipulated turnaround time for in vitro diagnostics takes 1–3 years; however, the covid-19 pandemic presented increased pressure and the need to grant marketing authorisation to manufacturers, necessitating the development of a pre-evaluation assessment policy. according to unpublished reports from the ppb, and as a pre-requisite measure for issuance of the eua, those kits without satisfactory sensitivity and specificity results must be subjected to further evaluation. the shelf lives of the pocts in this study ranged from 6 to 24 months. the manufacturers did not provide reports of their stability data or their plans for generating such data.13 the study showed that 50% of the pocts that were submitted for pre-evaluation assessment did not meet the criteria to proceed to full evaluation as they were unsatisfactory. of the 18 pocts, one had no activity at all, two igm-detecting tests had both low sensitivity and low specificity, and three tests displayed high false-positive values. only nine (50%) tests had sensitivities of ≥ 40% (range: 40% – 60%). the capacity of these nine tests to detect igm ranged from 0% to 50%. the majority (57.1%) of the tests displayed very weak visual igm band intensities (scale scores of 2–3). the sensitivities observed in this evaluation were lower than those reported elsewhere, and the ability of the tests to detect igm varied, with one test failing to capture any covid-19 igm. there was also a weak positive correlation between igm and igg sensitivity for the kits. the abbott id now covid-19 test (abbott laboratories, abbott park, illinois, united states) is said to be the most sensitive and specific poct on the global market, with a sensitivity of 80.4% and specificity of 95.9% with rrt-pcr as the standard.14,15 a study conducted to assess the quality of a poct based solely on pcr-positivity among anonymous blood donors in uppsala university, uppsala, sweden, reported a high performance, with sensitivities of 69% for igm and 93.1% for igg, and specificities of 100% for igm and 99.2% for igg.16 another study conducted in china among covid-19-positive patients established the sensitivity and specificity of the test they assessed to be 88.66% and 90.63%.17 a study done at guangzhou eighth people’s hospital, china, among patients with confirmed sars-cov-2 infection found that the combined detection of nucleocapsid-specific and spike-specific igm and igg could identify up to 75% of sars-cov-2-positive cases in the first week using an enzyme-linked immunosorbent assay test kit.18 in consonance with the findings of this study, the organization for economic co-operation and development has emphasised the need to recognise that pocts have not been fully developed for sars‑cov‑2 and their true clinical performance is mostly unknown.19 a satisfactory quality assessment of covid-19 diagnostics is very critical. recently, quality failure-related issues have been reported globally. these include instances such as india stopping the use of 500 000 pocts from two chinese firms for questionable efficacy,20 a report of initial pcr test kits that were contaminated with coronavirus synthetic materials in the united states,21 britain buying millions of pocts that did not work,22 first samples of pocts manufactured in india failing the quality tests,23 and spain withdrawing chinese-manufactured kits with a sensitivity of 30%.24 it is noteworthy that kit 11, which failed in this study, is from the same company that made the kits that failed in india. all kits need to be re-evaluated using standard covid-19 igg and igm to establish a gold standard poct. the kits assessed in this pre-evaluation should be limited to use in research and sero-surveillance only. furthermore, there is a need for continued evaluation of these kits using whole blood samples with larger sample sets of at least 400 covid-19-positive and negative samples. this would be a full evaluation of the kits and would increase the power of the study. there is also a need to formulate national and international policies on the determination of shelf lives of products that are developed during medical emergencies such as the covid-19 pandemic. limitations this study only used plasma matrix. there is a need to assess the performance of these poct kits using whole blood samples, especially whole blood from finger pricks and serum as per the manufacturers’ instructions. the sample size used in this study was less than the 400 samples recommended by the world health organization. the suppliers of the pocts could not provide the adequate number of kits required. conclusion there was very poor igm detection by most of the poct kits assessed and this could affect timely detection of covid-19 early infection and spread as compared to rrt-pcr. similarly, the serological-based test kits available in the country can only detect up to 60% of the infected population. acknowledgements this work was supported by prof. yeri kombe, director general, kenya medical research institute, to whom we are very grateful. we are also grateful to dr fred siyoi, chief executive officer, pharmacy and poisons board, kenya, for his unreserved support for this work. the authors would like to confirm that the people mentioned in this section have granted permission as required. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions j.h.k., a.a.g., r.i., p.w., f.s., d.m-m., c.w., j.z., m. orina, l.m., j.t., l.n., e.w., j.m., a.m., s.o., a.w., s.m.n. and m. ochwoto contributed in different capacities to the design and implementation of the research, to the analysis of the results and to the writing of the manuscript. sources of support this work was supported by the kenya medical research institute under the grant award kemri/cov/innov/001. data availability repository: figure share https://doi.org/10.608/m9figshare12727037. this project contains the following underlying data: data file 1 (data for true negatives). data file 2 (data for true positives). data file 3 (pre-evaluation raw data). data are available under the terms of creative commons by 4.0 and can be requested from the corresponding author. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency or the authors. references john hopkins university. epidemic map updated in real-time. baltimore, md: center for systems science and engineering; 2020. jeong sae-im. korea approves 2 more covid-19 detection kits for urgent use. korea biomed rev. 2020. liang m, du l, liu j, et al. sars patients-derived human recombinant antibodies to s and m proteins efficiently neutralize sars-coronavirus infectivity. biomed environ sci. 2005;186(6):363. sheridan c.coronavirus and the race to distribute reliable diagnostics. nat biotechnol. 2020;38:382–384. https://doi.org/10.1038/d41587-020-00002-2 hopkins j. serology-based tests for covid-19. baltimore, md: johns hopkins; 2020. zainol r, othman s, abdul s, et al. diagnostic performance of covid-19 serology assays. malaysian j pathol. 2020;42(1):13–21. vabret n, graham j, conor g, et al. immunology of covid-19 current state of the science. immunity. 2020;42(2):910–941. srikrishna d, dhillon rs, beier d. we need a cheap way to diagnose coronavirus. harv bus rev. 2020. hou h, wang t, zhang b, et al. detection of igm and igg antibodies in patients with coronavirus disease 2019. clin translational immunol. 2020;9(5):e1136. https://doi.org/10.1002/cti2.1136 world health organization. advice on the use of point-of-care immunodiagnostic tests for covid-19: scientific brief. geneva: world health organization; 2020. world health organization. instructions for submission requirements: in vitro diagnostics (ivds) detecting antibodies to sars-cov-2 virus. geneva: world health organization; 2020. drain p, hyle e, noubary f, et al. diagnostic point-of-care tests in resource-limited settings. lancet infect dis. 2014;14(3):239–249. https://doi.org/10.1016/s1473-3099(13)70250-0 world health organization (b). establishing stability of in vitro diagnostic medical devices. (no. who/bs/2017.2304). geneva: who; 2019. i. n. covid-19. abbott [homepage on the internet]. [cited 2020 june 14]. available from: https://www.alere.com/en/home/ hogan c, sahoo m, huang c, et al. five-minute point-of-care testing for sars-cov-2 not there yet. j clin virol. 2020;128:104410. https://doi.org/10.1016/j.jcv.2020.104410 hoffman t, nissen k, krambrich j, et al. evaluation of a covid-19 igm and igg rapid test; an efficient tool for assessment of past exposure to sars-cov-2. infect ecol epidemiol. 2020;10(1):1754538. https://doi.org/10.1080/20008686.2020.1754538 li z, yi y, luo x, et al. development and clinical application of a rapid igm-igg combined antibody test for sars-cov-2 infection diagnosis. j med virol. 2020;92(9):1518–1524. https://doi.org/10.1002/jmv.25727 sun b, feng y, zheng p, et al. kinetics of sars-cov-2 specific igm and igg responses in covid-19 patients. emerg microb infect. 2020;9(1):940–948. https://doi.org/10.1080/22221751.2020.1762515 oecd. home testing for covid-19: a way to lift confinement restrictions. oecd. paris; 2020. hindustan times new delhi china. concerned as india decides to stop use of chines covid-19 test kits. new delhi; 2020. new york times. cdc labs were contaminated delaying corona virus testing official say. new york; 2020. the sunday times. britain has millions of coronavirus antibody tests, but they don’t work. london; 2020. nidheesh la. first samples of hll’s india-made rapid testing fail quality test in kerala. new delhi: livemint; 2020. the guardian. coronavirus test kits withdrawn in spain over poor accuracy rate. madrid; 2020. abstract background description of the intervention lessons learnt recommendations acknowledgements references about the author(s) alban zohoun department of hematology, faculty of health sciences, national university hospital center hubert koutoukou maga, cotonou, benin army teaching hospital, university hospital center, cotonou, benin tatiana b. agbodandé department of hematology, faculty of health sciences, national university hospital center hubert koutoukou maga, cotonou, benin angélique kpadé army teaching hospital, university hospital center, cotonou, benin raliatou o. goga army teaching hospital, university hospital center, cotonou, benin rené gainsi army teaching hospital, university hospital center, cotonou, benin paul balè army teaching hospital, university hospital center, cotonou, benin bibata m. sambo army teaching hospital, university hospital center, cotonou, benin remi charlebois global scientific solutions for health (gsshealth), baltimore, maryland, united states rachel crane global scientific solutions for health (gsshealth), baltimore, maryland, united states michele merkel global scientific solutions for health (gsshealth), baltimore, maryland, united states ludovic anani department of hematology, faculty of health sciences, national university hospital center hubert koutoukou maga, cotonou, benin ekaterina milgotina global scientific solutions for health (gsshealth), baltimore, maryland, united states citation zohoun a, agbodandé tb, kpadé a, et al. from benchmarking to best practices: lessons from the laboratory quality improvement programme at the military teaching hospital in cotonou, benin. afr j lab med. 2021;10(1), a1057. https://doi.org/10.4102/ajlm.v10i1.1057 lessons from the field from benchmarking to best practices: lessons from the laboratory quality improvement programme at the military teaching hospital in cotonou, benin alban zohoun, tatiana b. agbodandé, angélique kpadé, raliatou o. goga, rené gainsi, paul balè, bibata m. sambo, remi charlebois, rachel crane, michele merkel, ludovic anani, ekaterina milgotina received: 06 june 2019; accepted: 14 sept. 2020; published: 11 feb. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: in 2015, the army teaching hospital–university teaching hospital (hia-chu [hôpital d’instruction des armées de cotonou centre hospitalier et universitaire]) laboratory in benin launched a quality improvement programme in alignment with the world health organization regional office for africa’s stepwise laboratory improvement process towards accreditation (slipta). among the sub-saharan african laboratories that have used slipta, few have been francophone countries, and fewer have belonged to a military health system. the purpose of this article was to outline the strategy, implementation, outcomes and military-specific challenges of the hia-chu laboratory quality improvement programme from 2015 to 2018. intervention: the strategy for the quality improvement programme included: external baseline slipta evaluation, creation of work plan based on slipta results, execution of improvement projects guided by work plan, assurance of accountability via regular meetings, training of personnel to improve personnel competencies, development of external stakeholder relationships for sustainability and external follow-up post-slipta evaluation. lessons learnt: over a period of 3 years, the hia-chu laboratory improved its slipta score by 29% through a quality improvement process guided by work plan implementation, quality management system documentation, introduction of new proficiency testing and internal quality control programmes, and enhancement of personnel competencies in technical and quality management through training. recommendations: the programme has yielded achievements, but consistent improvement efforts are necessary to address programme challenges and ensure continual increases in slipta scores. despite successes, military-specific challenges such as the high mobility of personnel have hindered programme progress. the authors recommend that further implementation research data be shared from programmes using slipta in under-represented settings such as military health systems. keywords: laboratory medicine; stepwise laboratory improvement process towards accreditation; slipta; quality improvement; laboratory quality improvement; military laboratory; quality assurance; slmta; strengthening laboratory management toward accreditation. background the benin army health service provides healthcare to army personnel, their families and civilians across benin. the benin army health service is involved in disaster response and supports the beninese public health system, with approximately 75% – 80% of its services provided to the civilian population. the army teaching hospital–university teaching hospital (hia-chu [hôpital d’instruction des armées de cotonou centre hospitalier et universitaire]) of the benin army health service in cotonou is a reference health centre known for the quality of its services and training. the hia-chu laboratory plays a leading role in the public healthcare continuum by providing diagnosis, screening, and initial and follow-up treatment as well as preventative care.1 therefore, the hia-chu laboratory prioritises quality, in that it promotes the accuracy, reliability and timeliness of results. in recent years, there has been an emphasis on improving medical laboratory services globally. health priorities have been set forth by public declarations, such as the international health regulations and the maputo declaration (2008), in which signatories pledged to address and strengthen laboratory and health services.2 the international standards organization (iso) 15198:2012 standard, ‘medical laboratories–particular requirements for quality and competence’, serves as a standard for the laboratory quality management system (qms) – a formalised system that outlines required structures and functions for medical laboratories. progress made on the iso 15189 standard can help a laboratory prepare for accreditation through a recognised agency; this allows the laboratory to demonstrate to clients, partners and staff that it has attained a high level of technical competence, thus instilling confidence in stakeholders on the accuracy and reliability of its results. in sub-saharan africa, the clinical laboratory remains a weak link in the healthcare chain.2,3 though progress has been made, most clinical laboratories in sub-saharan africa remain under-equipped, under-funded and far from attaining international norms and standards. few laboratories are accredited to international quality standards, and most of these internationally accredited laboratories are based in south africa.4 quality assurance, which comprises qms including the existence of a quality manual, use of internal quality controls (iqc) and participation in external quality assessment (eqa), is poorly implemented and often unavailable in many laboratories in sub-saharan africa.5,6,7 in the absence of quality assurance, there is the risk of laboratory errors, which could adversely impact patient care. to alleviate these challenges and support awareness of the importance of achieving quality standards in medical laboratories in sub-saharan africa, the world health organization regional office for africa developed in 2009 a phased laboratory quality management evaluation system called the stepwise laboratory quality improvement process toward accreditation (slipta).8 the african society of laboratory medicine serves as the secretariat of the world health organization regional office for africa slipta programme. the slipta framework guides improvement of performance, measures and evaluates the progress of laboratories towards iso 15189 international accreditation and awards a certificate of recognition from zero to five-star ratings.8,9 the hia-chu in cotonou, benin, has a staff consisting of military and civilian personnel and includes a clinical pathologist, 12 medical laboratory scientists and support staff. the services provided include medical fitness evaluation, disease prevention, treatment and care, and teaching and research in medical, biological, pharmaceutical, paramedical, odontological and veterinary specialities. laboratory services at hia-chu are versatile and the laboratory manages, on average, 20 000 patient files and 50 000 tests per year. in january 2015, the hia-chu laboratory service initiated, for the first time, a quality improvement (qi) programme in alignment with the slipta framework. the qi programme goal was to improve laboratory services related to disease diagnosis and monitoring via the implementation of qms using slipta framework. the qi programme aimed to provide training for laboratory personnel on quality management concepts and practices, monitor laboratory quality and adherence to quality systems, conduct laboratory test method validation and provide proficiency testing panels for hiv, tuberculosis and other critical tests – successful implementation of the qi programme ensures that laboratory stakeholders and end-users have confidence in laboratory data which informs clinical decisions and optimises patient care. the hia-chu laboratory qi programme was launched through a collaboration with the us department of defense hiv/aids prevention program (dhapp). as of 2015, the benin army health service already had a long-standing collaboration with dhapp, which had been funding laboratory equipment and reagents, specifically to support hiv diagnosis and treatment monitoring. over the years, both parties recognised that laboratory tests and equipment alone are not sufficient to demonstrate test result quality; tests and equipment must be accompanied by laboratory quality practices implemented by properly trained and motivated personnel. to address this recognised need, dhapp began supporting the benin army health service in the execution of the hia-chu laboratory qi programme in 2015, with implementation assistance provided by the united states-based global health company global scientific solutions for health (gsshealth). the purpose of this article is to report on the implementation of the qi programme at the hia-chu laboratory from 2015 to 2018. description of the intervention baseline slipta evaluation and work plan creation the laboratory qi programme began in january 2015 with initial programme planning among stakeholders (hia-chu leadership, dhapp, gsshealth) and the facilitation of a comprehensive baseline evaluation of the hia-chu laboratory by gsshealth using the world health organization regional office for africa slipta checklist, version 2:2015. the baseline slipta evaluation allowed the assessment of the 12 quality management sections: organisation and personnel, management reviews, process control and internal quality assessment and eqa, information management, corrective action, equipment, purchasing and inventory, documents and records, occurrence management and process improvement, internal audit, client management and customer service, and facilities and safety. following the evaluation, leaders from hia-chu, dhapp and gsshealth convened for a collaborative session to review findings, identify priority qi areas and develop a tailored qi approach for each priority area. a tailored qi approach was developed to strengthen hiv testing processes – the focus of the funding agency dhapp; it covered the identification of key quality indicators to track and improve upon over time and the development and execution of the qi work plan. the key indicators identified to measure laboratory improvement based on ease of collection and relevance to the work plan included: slipta: percentage improvement in total and by section. eqa: percentage score in pt programme. iqc: accuracy of quality control results. personnel training: percentage change in theoretical test scores for training workshops. laboratory documentation: number of documents created and adequately implemented to standardise laboratory practices and formalise the commitment to the qms. the laboratory-set targets were: continual improvement in the slipta score through the establishment and implementation of improvement projects tracked by work plans, participation in eqa and pt and improvement of performance where the score is less than 100%, participation in iqc activities and improvement of processes in case of discrepancies between expected and obtained results, strengthening of laboratory quality management processes guided by well defined documentation, and the advancement of laboratory personnel competencies through training and mentorship workshops. the hia-chu laboratory supervisor facilitated work plan development in collaboration with partners (dhapp and gsshealth) and laboratory staff. the work plan included high-level qi goals, specific objectives for each goal and details of specific, measurable, achievable, realistic, and timely, or ‘smart’, tasks and deliverables to meet set targets within defined timeframes (see figure 1). figure 1: example of a quality improvement work plan (first page) developed by the army teaching hospital–university teaching hospital laboratory, benin, 2016–2017. the work plan was progressively implemented and updated through improvement project execution, progress monitoring, ongoing data collection and review, and continual alignment with the funder’s priorities. staff were engaged in the work plan implementation through their appointments to specific qi projects with oversight by the laboratory supervisor, and through continuous engagement in recurring collaborative work plan update meetings to promote accountability. quality improvement activities conducted in the context of the work plan included: nomination of new qms personnel roles (quality manager, biosafety manager), targeted laboratory personnel training and mentorship, implementation of eqa and iqc, definition of essential qms processes and development and use of associated documentation, and coordination of a follow-up slipta evaluation to measure progress. lessons learnt from the baseline slipta evaluation, the identified priority issues, which correspond to low-scoring slipta sections, include the following: the lack of a quality manual or safety manual, the lack of standard operating procedures for all processes and technical procedures, the lack of pt and quality control for tests, the lack of equipment maintenance and repair logs, the lack of temperature monitoring processes, and the lack of mistakes or error logs. the initiation and implementation of a qi programme at the hia-chu laboratory yielded numerous positive changes as perceived by hia-chu hospital leadership, laboratory management and staff, and partners (dhapp and gsshealth). positive changes included the establishment of a laboratory quality management team, achievement of designated qi projects resulting in slipta score improvements, the improvement of iqc and eqa test result accuracy, the development and implementation of over 50 standard operating procedures, and the strengthening of personnel competencies through targeted qms training and mentorship. throughout the years of the qi programme, support from funding partner dhapp and implementing partner gsshealth ensured continuous evaluation of processes via the internal and external evaluations and the update of work plans and associated qi projects. staff training and work plan updating the engagement of laboratory staff was key to the establishment and implementation of the laboratory qi work plan at hia-chu. to encourage staff commitment to the qi approach and ensure staff in-depth orientation to quality management concepts and testing processes, the laboratory qi approach prioritised staff training and mentorship. over 50 technicians participated in training workshops on qms co-organised and co-facilitated by the benin military health system and gsshealth. all training sessions consisted of didactic and interactive sessions and tests were administered preand post-training to evaluate knowledge change among the trainees. the median change in test score from pre-training to post-training for all the trainees increased from 15% to 40% (figure 2). improvements in participants’ theory test results demonstrated improved understanding of qms and technical concepts and facilitated personnel participation in qi projects. figure 2: preand post-training workshop theoretical test scores of military laboratory staff from quality management (left panel) and biosafety (right panel) workshops, benin, 2015–2018. box plots illustrate the distribution of scores among trainees (red diamonds for pre-training and yellow diamonds for post-training) and grey horizontal lines with data labels indicate median score across all the trainees. the interquartile range shows how the data are dispersed by dividing the data into quartiles (depicted by the dark and grey horizontal lines). (a) qms training: test scores, (b) bs&s training: test scores. in the first year of the programme, two quality workshops were held for laboratory staff with gsshealth facilitators. the objectives of the workshops were to improve the competency of laboratory staff in the areas of quality assurance, qms and hiv and tuberculosis testing. these topic areas were priorities for the laboratory director and dhapp – the funder, whose focus is the prevention and control of hiv and hiv comorbidities. the first workshop took place in april 2015 with a focus on quality management concepts for equipment management, document writing and eqa. staff received guidance and mentorship on hiv rapid testing. a second workshop took place in september 2015, with a focus to increase staff competency on laboratory supply chain and stock management. at the end of the first year, a follow-up slipta evaluation was conducted. in 2016, the qi programme continued with an updated work plan, and laboratory staff participated in a national biosafety and biosecurity workshop hosted by the ministry of defense and the ministry of health. the second year concluded with a poster presentation by the hia-chu laboratory supervisor on the quality programme at the african society of laboratory medicine international conference in december 2016. in 2017, the laboratory quality team updated their quality work plan, worked with laboratory personnel on qi projects, implemented the use of quality controls for cd4 testing, and organised an internal slipta evaluation. in april 2017, one laboratory staff member from hia-chu was sponsored to attend a multi-country, 5-day training workshop in senegal. the workshop covered essential aspects of laboratory quality management, including document management and standard operating procedure writing; equipment management and maintenance; error occurrence management, prevention and corrective action, and quality indicators. finally, a follow-up external slipta evaluation was conducted in october 2017 to re-evaluate the laboratory system, measure progress since the previous external slipta evaluation, and update the qi programme. as the qi programme moved into its third year in 2018, the laboratory again updated its work plan, based on the previous evaluation, and revised and updated key documentation including the laboratory quality manual and biosafety manual. in april 2018, a joint ministry of defense and ministry of health laboratory biosafety and quality management workshop was organised, to increase staff competency on principles and iso standards for biosafety and biosecurity. through its ongoing commitment to training personnel in both technical and quality management topics, the laboratory has observed significant improvements in staff competencies and performance. for example, the slipta sections in which the laboratory achieved the most significant quality improvements correspond to the quality management topics covered during training workshops. data from the qi programme showed that post-training, personnel knowledge was improved and retained (figure 2). additionally, in 2015, staff undertook training on hiv rapid testing processes; thereafter, their hiv pt scores reached 100%. document creation and implementation the laboratory document management system has been developed gradually over time and has included the creation and implementation of a quality manual, a biosafety manual, a sample collection manual and more than 50 standard operating procedures, technical instructions, forms and logs (e.g. temperature monitoring logs, corrective and preventive action forms). the laboratory supervisor and designated quality manager took the lead in developing documents, executing a document management process, and introducing new documents into circulation in the laboratory. laboratory personnel were oriented to new documents and procedures during regular laboratory meetings, facilitating the understanding and proper use of new documents. the availability of procedures and other documents helped laboratory management ensure that personnel observed standardised processes across the pre-analytical, analytical and post-analytical phases, and simplified the training of new personnel. furthermore, to ensure that only correct and updated documents were available in the laboratory, processes to control document revision, approval and version release were instituted by the laboratory supervisor and quality team. external quality assessment and internal quality control results in september 2015, the hia-chu laboratory enrolled and participated in an annual pt programme for hiv serology with commercial eqa provider thistle quality assurance, and later with the benin ministry of health, to promote sustainability. during the four eqa events for hiv serology, nine technicians participated, and pt scores of 100% were obtained. no corrective action was needed to improve proficiency; laboratory personnel were nonetheless oriented on root-cause analysis, and corrective and preventive action in case of future instances of lower eqa scores. in 2018, the hia-chu laboratory participated in two commercial haematology eqa programmes with human quality assessment services. the laboratory results for the first haematology eqa test were in the acceptable range for all analytes except for monocytes or mid cells, due to a reporting error. as a corrective action, the laboratory established a system for the supervised recording of results, in which the laboratory section supervisor verifies the accuracy of test results entered manually by technicians, after which results are entered into a computer by a secretary and printed for final validation. this process has helped reduce transcription errors. the hia-chu also began an iqc programme in biochemistry for the first time, in which the laboratory used high, normal and low commercial controls on equipment daily. when control values are outside the acceptable range, the laboratory staff will conduct a root-cause investigation and analysis. elements to be investigated include reagents (conservation, expiration, etc.), room temperature, quality of distilled water, quality of the control product, etc. if the issue persists after the above steps, the equipment is then recalibrated. all corrective actions and measures taken are documented and recorded. improvement of slipta score overall improvement of the laboratory’s quality system was demonstrated by a 29% increase in slipta score, from 16% at baseline evaluation in 2015 to 45% at the follow-up evaluation in 2017 (see figure 3). figure 3: performance of the army teaching hospital–university teaching hospital laboratory in each section of the stepwise laboratory improvement process towards accreditation checklist, benin, 2016-2018. baseline evaluation, september 2015; follow-up evaluation 1, january 2016; follow-up evaluation 2, october 2017. since 2018, reliance on the ministry of health national hiv reference laboratory for the administration of an hiv serology pt programme has allowed the hia-chu laboratory and other military laboratories to ensure a more sustainable and affordable pt option. several important factors can explain the successes achieved during the qi programme. the laboratory leadership and staff benefited from the strong support of the hospital management, thus underpinning laboratory staff motivation to sustain quality improvements. also, the implementing partner provided support in the execution of work plans, drafting of procedures, funding of training visits and organisation of regular teleconferences to track progress. the implementing partner provided sound advice for the appropriation of the quality approach and the implementation of improvement steps. recommendations although slipta is an adaptable structure and was used to guide the hia-chu laboratory’s quality successes, its formal implementation in francophone african countries and military contexts has been limited.8 military health systems face unique challenges that impact their laboratory qi efforts; the challenges and associated responses of hia-chu laboratory may be relevant to other military laboratory programmes or to non-military laboratories that have limited funds to invest in qi efforts. regardless of improvements made, qi is a never-ending process and the sustainability of gains is not guaranteed.10 despite the quality advancements made over the past several years, challenges remain, requiring corrective action to ensure the efficacy of the ongoing programme.11,12,13 the issue of the high mobility of military personnel has made it difficult to fully and consistently integrate staff into quality laboratory operations and maintain the laboratory qms. as a result of high staff turnover – a key facet of the military system – all members of the starting laboratory quality team are no longer in service at the hia-chu laboratory in cotonou. continuous staff turnover in part explains the slowdown in progress observed between the 2016 and 2017 follow-up evaluations. staff mobility has also negatively impacted the timelines for the execution of planned activities such as management reviews, internal evaluations and inventory processes, for all of which personnel must be properly trained and oriented to ensure their execution per slipta and iso 15189 requirements. lesson learned the implementation of a qi approach supported by improved documentation and record-keeping has increased staff and end-user confidence in the reliability and reproducibility of laboratory test results. support from the hospital hierarchy is essential to ensure the necessary time and resources are allocated to quality improvement processes. for a qi initiative to be effective and sustainable, all laboratory personnel should be engaged in the process, with quality management roles well defined. staff mobility in military settings renders the ongoing implementation of a qi programme more challenging; military sites should take into account the need to engage all laboratory personnel in a qi mindset and should plan backup qi roles in case of personnel deployment. although the laboratory qi programme has incorporated training workshops, the programme would benefit from the establishment of a structured framework for continous personnel training allowing the laboratory to maintain a pool of trained and dedicated personnel. going forward, the ambition of hia-chu laboratory is to continue advancing in laboratory quality using the slipta guideline, to ultimately achieve a five-star slipta rating and become internationally accredited to the iso 15189 standard. the hia-chu laboratory also has the support of the benin military health system authorities to expand the qi programme to multiple laboratory sites, prioritise the accreditation of clinical laboratories, and to integrate accreditation programmes into health sector policy and development programmes. to date, the hia-chu laboratory has expanded the qi programme to military clinical laboratories in ouidah, porto-novo and parakou. military laboratory leaders envision the further expansion of the qi programme to additional military sites using a network approach. acknowledgements we hereby thank the united states department of defense hiv/aids prevention program for sponsoring the quality programme of the hia-chu laboratory. competing interests the authors have declared that no competing interest exists. authors’ contributions a.z. is the initiator of the study and wrote the manuscript. t.b.a., a.k., r.o.g., r.g., p.b., b.m.s., l.a., r. charlebois, r. crane, m.m. and e.m. reviewed, commented on and contributed to its improvement. ethical considerations the advice of an ethics committee was not required. the article is an account of an ongoing process and does not involve an investigation or technical manipulation. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references saliou p. le laboratoire en zone tropicale. bull soc pathol exot. 2006;99(5):409–417. https://doi.org/10.3185/pathexo2822 revitalised laboratory declaration to forge a path for action [homepage on the internet]. aslm; 2015 [cited 2019 dec 26]. available from: https://aslm.org/news-article/revitalised-laboratory-declaration-to-forge-a-path-for-action/ petti ca, polage cr, quinn tc. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377–382. https://doi.org/10.1086/499363 beyanga m, gerwing-adima l, jackson k, et al. implementation of the laboratory quality management system (iso 15189): experience from bugando medical centre clinical laboratory – mwanza, tanzania. afr j lab med. 2018;7(1):a657. https://doi.org/10.4102/ajlm.v7i1.657 mboup s, gershy-damet gm, touré kane c, bélec l. le défi de la formation dans le domaine du laboratoire de biologie clinique en afrique. med sante trop. 2014;24:237–240. https://doi.org/10.1684/mst.2014.0349 deom a. qualité et biologie dans quelque pays européens et américains. spec biol. 1996;15(78):17–23. gershy-damet gm, rotz p, cross d. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin patho. 2010;134(3):393–400. https://doi.org/10.1309/ajcptuuc2v1wjqbm ndihokubwayo jb, maruta t, ndlovu n, et al. implementation of the world health organization regional office for africa stepwise laboratory quality improvement process towards accreditation. afr j lab med. 2016;5(1):a280. https://doi.org/10.4102/ajlm.v5i1.280 mothabeng d, maruta t, lebina m, lewis k, wanyoike j, mengstu y. strengthening laboratory management towards accreditation: the lesotho experience. afr j lab med. 2012;1(1):a9. https://doi.org/10.4102/ajlm.v1i1.9 andiric lr, massambu cg. one laboratory’s progress toward accreditation in tanzania. afr j lab med. 2014;3(2):a202. https://doi.org/10.4102/ajlm.v3i2.202 yao k, luman et, slmta collaborating authors. evidence from 617 laboratories in 47 countries for slmta-driven improvement in quality management systems. afr j lab med. 2014;3(3):a262. https://doi.org/10.4102/ajlm.v3i2.262 bouchet b. iso 15189: 2012: quels changements pour les laboratoires africains? afr j lab med. 2015;4(1):a181. https://doi.org/10.4102/ajlm.v4i1.181 guevara g, gordon f, irving y, et al. the impact of slmta in improving laboratory quality systems in the caribbean region. afr j lab med. 2014;3(2):a199. https://doi.org/10.4102/ajlm.v3i2.199 abstract introduction methods results discussion acknowledgements references about the author(s) talkmore maruta laboratory department, african centres for disease control and prevention, lusaka, zambia sikhulile moyo laboratory department, botswana harvard aids institute partnership, gaborone, botswana citation maruta t, moyo s. impact of pre-covid-19 epidemic preparedness on the trajectory of the pandemic in african countries. afr j lab med. 2022;11(1), a1571. https://doi.org/10.4102/ajlm.v11i1.1571 original research impact of pre-covid-19 epidemic preparedness on the trajectory of the pandemic in african countries talkmore maruta, sikhulile moyo received: 28 feb. 2021; accepted: 02 feb. 2022; published: 31 mar. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the novel coronavirus disease 2019 (covid-19), declared a pandemic by the world health organization (who) in march 2020, has taught us about the importance of epidemic preparedness. objective: we analysed the pre-covid-19 preparedness of sub-saharan african countries and how this may have influenced the trajectory of covid-19 cases. methods: the who joint external evaluation (jee) tool and the global health security (ghs) index were used to determine the epidemic preparedness of countries in the who african region. the relationship between pre-covid-19 preparedness and the reported number of cases per million people was evaluated over the first 120 days of the first reported case in each country, between february 2020 and september 2020. results: the overall performance of the 42 countries was 40% in the 19 jee core capacities and 32% in the six ghs index indicators. at day 1, the mean number of cases per million population was significantly higher among countries rated as ‘prepared’ in the jee legislation, policy and finance (p = 0.03), ports of entry (p = 0.001), and international health regulation coordination, communication and advocacy (p = 0.03) categories. at day 90, countries rated as ‘prepared’ in the national laboratory systems (p = 0.05) and real-time surveillance (p = 0.04) jee categories had statistically significantly fewer cases per million population. conclusion: this analysis highlights the importance of building capacity for pandemic preparedness in africa. the who african region was not adequately prepared for the covid-19 pandemic as measured by the who jee tool and the ghs index. keywords: covid-19; preparedness; response; world health organization joint external evaluation; global health security index. introduction the rapid spread of the novel coronavirus disease 2019 (covid-19) prompted the world health organization (who) to declare it as a public health emergency of international concern in january 2020 and a global pandemic on 11 march 2020.1,2 the first covid-19 case in africa was reported by egypt on 14 february, and by 14 may, all african countries had reported at least one covid-19 case.3 by june 2020, 152 442 covid-19 cases and 4334 deaths (case fatality rate: 3%) had been reported by the 54 african countries.4 african countries instituted various public health and social measures to curb the transmission and allow them to prepare for the pandemic.5 although the coronavirus was novel, the emergency of its spread exposed the status of countries’ preparedness for threats posed by epidemics, disasters, and other events of public health concern. countries are expected to invest resources to limit the impact of disease outbreaks and natural disasters.6 epidemic preparedness is a measure of the capacity of countries to detect, report, and respond to outbreaks. to effectively respond to outbreaks, countries need to set up robust systems for surveillance and outbreak investigation with capabilities to rapidly identify, characterise, and track emerging infectious diseases.6 for a coordinated response, these systems need to function within strong national public health systems linked to an effective international system.6 the 2014 ebola outbreak in west africa infected 28 000 and killed 10 000 people in the three affected countries of guinea, liberia, and sierra leone, re-emphasising that epidemics can emerge unexpectedly and unprepared countries are a threat to all.7 the estimated combined loss was $2.8 billion in gross domestic product.7 this outbreak prompted globally coordinated efforts to ensure that countries strengthen their capacities as a way of ensuring that threats are limited and contained within borders when they occur. the international health regulations of 2005 (ihr 2005), among other innovations, was a means to ensure that countries are obliged to develop minimum capacities, defined as core public health capacities.8 due to the limited implementation of ihr 2005, especially in africa, the who developed the integrated disease surveillance and response (idsr) to improve surveillance by linking community, health facility, district, and national levels in a streamlined manner using a one health approach.9 however, by the 2012 deadline, only 58% of the signatories had developed national plans to meet ihr 2005 core capacity requirements, with as few as 10% indicating full implementation of the requirements. further to that, the global health security agenda was launched in 2014 to address the limited implementation of global commitments to building capacities for preparedness for epidemics and other events of public health concern using a multisectoral approach that includes the human, agriculture, animal, security, finance, border control, education, and research sectors.10 the impact of epidemics is well understood by governments.11 in addition to loss of lives, the cost to the broader economic and social sectors is a notable impact of epidemics, with epidemic response costlier than investing in preparedness. despite all this, the argument to invest in preparedness has not been won by many governments. ten years after the abuja declaration, where leaders of african union countries pledged to allocate at least 15% of their annual budget to the health sector, only 26 countries had increased the proportion of total government expenditures for health, with only one, tanzania, having achieved the 15% target.11 within that health budget, pandemic preparedness is often overlooked in favour of more immediate and visible curative demands.12 the covid-19 pandemic once again tested the epidemic preparedness of countries and the global community. in this report, we review the pre-covid-19 epidemic preparedness of countries in the who african region and its impact on how the virus spread, as well as the strength and effectiveness of the initial responses by countries and the global community. lessons learnt can be useful for the inevitable future pandemics. methods ethical considerations the study did not involve human or animal subjects and therefore no ethical approval or clearance was required. study population data from the who joint external evaluations (jee) conducted between 2016 and 2019 and the global health security (ghs) index conducted in 2019 were used to determine the levels of preparedness of countries in the who african region. forty-two countries from the who african region with complete scores for both the who jee and ghs index were included in the analysis. data on the reported number of cases per million people from day 1 (date of the first reported case; range: 28 february 2020 – 15 may 2020) to day 120 (120 days from the date of the first reported case; range: 26 june 2020 – 11 september 2020) in each of the 42 countries was also obtained and analysed. preparedness data sources world health organization jee the who jee is a process established by the who to assess countries’ capacities to prevent, detect, and rapidly respond to public health risks.13 the jee has two levels of assessment: an initial self-evaluation by the host country using local experts from all relevant sectors and an in-country evaluation conducted by an external team made up of multisectorial subject matter experts and peer countries. the process measures country-specific preparedness for events of public health concern across 19 technical areas, as well as progress made towards achieving ihr 2005 targets.13 the scored jee tool contains a set of questions to collect data on the status of implementation of the 19 technical areas, which are divided into 4 areas, namely prevent, detect, respond, and other ihr-related hazards (table 1).14 each question is scored from 1, indicating no evidence of implementation, to 5, indicating full implementation. table 1: sections and technical areas of the who jee tool. global health security index the ghs index is a project of the nuclear threat initiative and the johns hopkins center for health security that provides a recurring measure of the international capability for preventing, detecting, and rapidly responding to epidemic and pandemic threats.15 the ghs index tracks health security and related capabilities of the 195 countries that are signatories to the ihr 2005 guidelines and uses a framework with 140 questions organised into six categories, 34 indicators, and 85 sub-indicators (table 2). the ghs index was considered as an additional measure of countries’ preparedness as it also includes an assessment of the robustness of the broader healthcare system, national political and socio-economic risks, and adherence to international norms. table 2: categories, indicators, and sub-indicators of the ghs index. each country is assigned an overall score between 0% and 100% as a weighted sum of the six categories. to allow for comparisons, each category is normalised based on the sums of its indicators and sub-indicators. the ‘our world in data’ programme the ‘our world in data’ is a collaborative programme between the university of oxford and the global change data laboratory that, among others, has been collecting data on several areas related to the progress of the covid-19 pandemic across the globe.16 data on the number of daily confirmed covid-19 cases per million people from day 1 (date of the first reported case in each country) to day 120 (120 days from the date of the first reported case in each country) of the epidemic was collected from the our world in data database. countries’ responses during the first 120 days of the pandemic were taken as a reflection of their existing pre-covid-19 capacities and preparedness. the stringency index, which is tracked by ‘our world in data’, is also reported. the stringency index is a composite measure calculated from nine response indicators, including school closures, workplace closures, cancellation of public events, restrictions on public gatherings, closures of public transport, stay-at-home requirements, public information campaigns, restrictions on internal movements, and international travel controls and travel bans. strictness is measured on a scale of 0–100, with 100 being the strictest. determination of preparedness the determination of pre-covid-19 preparedness was based on the who jee and ghs indices. the who jee evaluations were conducted between 2016 and 2019. the total scores from each of the 19 technical areas assessed were converted to percentage scores. being prepared was defined as obtaining a 50% or greater score within the technical areas and overall. using data on related capabilities from the ghs index conducted in 2019, the weighted scores for each of the six categories were used to measure performance in each of the categories. the overall score was used as a measure of each country’s preparedness, where overall score = ∑ category scores, and category score = ∑ weighted indicator scores. the ghs index scoring system was adopted to rate the countries as having low scores (0.0% – 33.3%), moderate scores (33.4% – 66.6%) or high scores (66.7% – 100.0%). a concordance analysis, using cohen’s kappa, was used to determine the agreement between the who jee and the ghs index, with an agreement of 0.61 ≤ κ ≤ 0.80 considered as ‘substantial’.17 kendall’s coefficient of concordance was also calculated for concordance between the ratings using the jee and ghs index measurement scales. a kendall’s coefficient of concordance > 0.5 with an associated p < 0.05 was considered a strong agreement. stata® version 16 (statacorp llc, college station, texas, united states) was used for statistical analysis. p < 0.05 was considered statistically significant. student t-tests were used to compare the mean number of cases per million population by performance in the jee categories or ghs indices rating. results forty-two (89%) of the 47 member states of the who african region (who/afro) were included; the other five (11%) had no jee and ghs index data. of the 19 jee core capacities, only four (21%) categories, including immunisation, laboratory systems, real-time surveillance, and workforce development, had a mean score of at least 50%. the overall performance of the 42 countries in the 19 core capacities was below 50% (mean: 40%; standard deviation [s.d.]: 9) (table 3). table 3: performance of the 42 who african region member states in the 19 core capacities of the jees conducted between 2016 and 2019. in the ghs index evaluation, the overall performance of the 42 countries was below 50% in all the six categories of prevention, detection and reporting, rapid response, status of health systems, compliance with international norms and standards for biosafety and biosecurity, and risk and vulnerability of the country system (table 4). the overall performance was also low (mean: 32; s.d.: 7.1). table 4: performance of the 42 who african region member states in the ghs index evaluation conducted in 2019. using the jee-based ratings (not prepared: 0.0% – 49.0%, prepared: 50.0% – 100.0%), overall, the 42 countries were rated as ‘prepared’ in only five of the 19 technical areas, including immunisation (95.0%), real-time surveillance (81.0%), laboratory systems (62.0%), workforce development (60.0%), and reporting (55.0%). using the ghs index scoring system (low score: 0.0% – 33.3%; moderate score: 33.4% – 66.6%; high score: 66.7% – 100.0%), ≥ 50.0% of the countries had medium scores in three categories, including detection and reporting (n = 21; 50%), risk environment (n = 29; 70.0%), and compliance with international norms (n = 38; 90.0%). few member states had high scores, and these were in the risk environment (n = 1; 3.0%), compliance to international norms (n = 2; 5.0%), and detection and reporting (n = 2; 5.0%) categories. notably, no country had a low score for compliance with international norms; all had either high (n = 2) or moderate (n = 40) scores. there was a strong overall agreement between the jee and ghs index percentages (kendall’s coefficient of concordance = 0.7; p = 0.003). the rate of increase in the number of covid-19 cases from day 1 to day 90 was 14.3 cases per million people per week (n = 41, s.d.: 21.1, range: 0.6–114.0) and from day 90 to day 120 was 49.8 cases per million population per week (n = 42, s.d.: 106.8, range: 98.6–577.1). the stringency index, calculated as the mean score of the response indicators of school closures, workplace closures, public events, use of public transport, lockdowns, and internal and international travel, was evaluated. each indicator, taking a value between 0 and 100 (100 = strictest), increased from an average of 29.3% at day 1 to 64.1% by day 90, and slightly reduced to 60.1% by day 120 (figure 1). figure 1: average stringency index of 42 who african region countries at day 1 (28 february 2020 – 15 may 2020); and day 120 (120 days from the date of the first reported case; range: 26 june 2020 – 11 september 2020). there were no significant differences in reported cases per million people on day 1, day 90, and day 120 by performance rating in all the ghs index categories (table 5). at day 1, the mean number of covid-19 cases per million population was significantly higher among countries rated as ‘prepared’ based on overall jee scores compared to countries rated as ‘not prepared’ (p = 0.01) (table 6). the mean number of covid-19 cases per million population at day 1 was also significantly higher in the countries rated as ‘prepared’ in the following jee core capacities: legislation, policy and finance (3.0 vs 0.2; p = 0.03), international health regulation coordination, communication and advocacy (2.9 vs 0.3; p = 0.03), food safety (3.4 vs 0.2; p = 0.01), and ports of entry (4.8 vs 0.2; p = 0.001). conversely, at day 90, the mean number of covid-19 cases per million population was significantly higher in the countries rated as ‘not prepared’ in the core competencies of national laboratory systems (298.3 vs 121.5; p = 0.05) and availability of real-time surveillance (376.8 vs 145.3; p = 0.04). however, the mean number of covid-19 cases per million population was still significantly higher in the countries rated as ‘prepared’ in the jee immunisation category compared to the countries rated as ‘not prepared’ (1589.8 vs 579.4; p = 0.01). at day 120, the mean number of covid-19 cases per million population was significantly higher in the ‘not prepared’ countries in the jee immunisation category (1370.2 vs 370.3; p = 0.04) but higher in the ‘prepared’ countries in the jee preparedness (1167.0 vs 359.7; p = 0.04) and ports of entry (1046.1 vs 330.9; p = 0.02) categories. table 5: mean number of cases per million population and the performance of the 42 who african region countries in the ghs index evaluations conducted in 2019. table 6: mean number of cases per million population and performance of the 42 who african region countries in the jee conducted between 2016 and 2019. discussion at the onset of the pandemic in who/afro member countries on 14 february 2020, 44 countries had conducted and published their jee reports as an indicator of preparedness of their systems for any event of public health concern. although the 42 countries considered in this review scored below 50% on average, indicating a lack of preparedness, there were notable areas where systems were in place. these included structures for immunisation, laboratory systems, surveillance, and workforce development. over the years, many african countries have conducted and built systems for immunisation of children under five years, leading to successes like the elimination of the wild poliovirus.18 in addition, numerous outbreaks of cholera, ebola, influenza, rift valley fever and other endemic diseases like malaria may have contributed to the observed existence of surveillance systems. since 2009, who/afro, in collaboration with the africa society for laboratory medicine and other partners, has developed and implemented the strengthening laboratory management towards accreditation training programme and the strengthening laboratory quality improvement process towards accreditation programme across africa.19,20 this may have contributed to the preparedness of the laboratory systems as reported by the jee. nevertheless, who/afro countries were generally unprepared for a global pandemic as determined by both the who jee and ghs index assessments. there was a lack of preparedness in key core capacities such as legislation, coordination, preparedness, emergency response, medical countermeasures, risk communication, and ports of entry. in the early phases of the epidemic, all reported cases were imported through the various ports of entry. most countries did not have legislation to empower governments to institute some of the public health measures needed to control the epidemic, resulting in delays and court challenges in some instances.21 it took time to mobilise all the coordination mechanisms required, especially the activation of the emergency operation centres and the establishment of covid-19 task teams, resulting in disaggregated responses in the early days. although the concept of a rapid response team was known and, in some cases, documented, these had not been tested at the scale needed and the anxiety associated with the disease delayed the full activation and deployment of the rapid response teams. there are two additional areas measured by the ghs index that provide additional measures of preparedness. these include the country’s vulnerability to biological threats and the overall risk environment, as well as the sufficiency and robustness of health systems to treat the sick and protect health workers. the ghs index suggests that health systems in who/afro countries are underdeveloped, with the 42 countries having their lowest scores in the health systems category among the six indicators of preparedness. deficiencies identified in the health systems by the ghs index include poor infrastructure, lack of dedicated finance from fiscus, and lack of documented commitment to prioritising healthcare services for healthcare workers who participate in a public health response, among others.15 the ghs index also showed that who/afro did not have a conducive environment in terms of political systems and government effectiveness in dealing with epidemics, as indicated by the low score in the risk environment category.15 a sizeable number of countries in the region have political and security risks, including civil wars, political and economic instabilities, and other cross-boundary disputes that could undermine national capabilities to counter threats. the ebola epidemic in west africa was an example where accessibility to affected areas was hampered by civil strife.22 did the level of readiness of who/afro member countries as depicted by the who jee and ghs index have a bearing on how the epidemic spread to and within africa? the authors examined the onset of the epidemic and its progression from day 1, the date of the first reported case in each country, to day 120. this was done because, after 120 days, the status or progression of the epidemic may have been affected by the rapid changes adopted by the countries following the realisation of its possible social and economic impacts. preparedness, as measured by the ghs index indicators, was found to have no significant impact on the mean number of cases per million population from the onset of the epidemic until day 120. however, at the onset of the pandemic (day 1), the mean number of cases was statistically significantly higher among countries rated as ‘prepared’ compared to countries rated as ‘not prepared’ based on overall jee scores and scores in the food safety, ports of entry, legislation, policy and finance, and international health regulation jee core capacities. with an average overall jee score of 40%, the poor status of overall preparedness of countries in the region seems to have had an impact on the onset and spread of the disease in the region. coordination, legislation, and advocacy are key to mounting a response in the event of a threat like the covid-19 pandemic. all initial cases in the region were imported through the various ports of entry, which, besides the airports, are largely very porous. the designated ports of entry were not adequately prepared, with no designated screening and isolation infrastructure or adequate and appropriately trained personnel resources to deal with covid-19.23 this calls for the strengthening of early warning systems similar to the who global influenza surveillance and response system.24 as the epidemic progressed, countries began to mobilise resources and set coordination structures, including the activation of emergency operation centres and the establishment of covid-19 coordination mechanisms like task force teams that were reporting to the highest offices in the country. implementation of public health measures, including lockdowns, was the most immediate response for most countries. this is reflected in the reported increase in stringency index from 29.3% at the onset to 64.1% by day 90. early in the pandemic, the who established the identification of cases through laboratory testing as central to the response.25 consequently, most countries prioritised localising diagnostic capacity and, with support from the who and the africa centres for disease control and prevention, the number of countries able to confirm covid-19 increased from 2, when the first case was reported in africa on 14 february 2020, to 24 countries able to confirm severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection by 20 february 2020.26 this supports the observed higher rating of the laboratory system capacity in most countries 120 days into the pandemic. limitations the findings of the study may be limited by inconsistent reporting in the early days of the pandemic as countries were establishing various capacities. through external funding, many countries made rapid changes within the first few months of detecting the first cases. this may have altered the relationship between baseline preparedness rating and the mean number of cases at day 90 and day 120 after the first case. we did not have enough data to adjust for these rapid interventions. despite the weaknesses of the data collection tools, the data generated using the jee and ghs index was critical in drawing international attention to the critical areas for building the capacity of nations to prevent, detect, and respond to epidemic threats. conclusion this analysis highlights the importance of building capacity for pandemic preparedness and response at multiple levels. at the onset of the covid-19 pandemic in february 2020, who/afro member countries were not adequately prepared as measured by the who jee and the ghs index. countries’ ratings in the legislation, policy and finance, and ihr coordination, communication and advocacy who jee categories, as well as in the health systems ghs index category, were not optimal. given all the lessons learnt during the covid-19 pandemic, including the rapid global spread and emergence of variants of concern, critical areas that predict the successful handling of epidemics need to be assessed. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions t.m. initiated the study and prepared the first draft of the manuscript. t.m. and s.m. analysed the data and reviewed and revised the manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the data that support the findings of this study are available on request from the corresponding author, t.m. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the affiliated agency of the authors. references word health organization emergency committee. statement on the second meeting of the international health regulations (2005) emergency committee regarding the outbreak of novel coronavirus (covid-19) [homepage on the internet]. geneva: who; 2020 [cited 2020 feb 1]. available from: https://www.who.int/news-room/detail/30-01-2020-statement-on-the-second-meeting-of-the-international-health-regulations-(2005)-emergency-committee-regarding-the-outbreak-of-novel-coronavirus-(covid-19) world health organization. director general’s opening remarks at the media briefing on covid-19 [homepage on the internet]. [cited 2020 mar 11]. available from: https://www.who.int/dg/speeches/detail/who-director-general-s-opening-remarks-at-the-media-briefing-on-covid-19---11-march-2020 marius g, giulia p, francesco p, et al. preparedness and vulnerability of african countries against importations of covid-19: a modelling study. lancet. 2020;395(10227):871–877. https://doi.org/10.1016/s0140-6736(20)30411-6 africa centres for disease control and prevention. coronavirus disease 2019 (covid-19): latest updates on the covid-19 crisis, 2020 [homepage on the internet]. [cited 2020 may 15]. available from: https://africacdc.org/covid-19/ talisuna ao, okiro ea, yahaya aa, et al. spatial and temporal distribution of infectious disease epidemics, disasters and other potential public health emergencies in the world health organisation africa region, 2016–2018. global health. 2020;16:9. https://doi.org/10.1186/s12992-019-0540-4 world health organization. a safer future: global public health security in the 21st century, 2007 [homepage on the internet]. [cited 2020 may 22]. available from: https://www.who.int/whr/2007/whr07_en.pdf centres for disease control and prevention, ‘2014–2016 ebola outbreak in west africa’ [homepage on the internet]. 2016 [cited 2021 jan 07]. available from: www.cdc.gov/vhf/ebola/history/2014-2016-outbreak/index.html world health organization. international health regulations [homepage on the internet]. 3rd ed. 2005 [cited 2021 jan 07]. available from: https://apps.who.int/iris/bitstream/handle/10665/246107/9789241580496-eng.pdf?sequence=1 world health organization. technical guidelines for integrated disease surveillance and response in the african region [homepage on the internet]. 2nd ed. 2017 [cited 2021 jan 07]. available from: https://www.afro.who.int/sites/default/files/2017-06/idsr-technical-guidelines_final_2010_0.pdf global health security agenda. a partnership against global health threats [homepage on the internet]. 2020 [cited 2021 jan 07]. available from: https://ghsagenda.org/ world health organization. the abuja declaration: ten years on [homepage on the internet]. 2011 [cited 2021 jan 07]. available from: https://www.who.int/healthsystems/publications/abuja_report_aug_2011.pdf?ua=1 world bank. from panic and neglect to investing in health security: financing pandemic preparedness at a national level [homepage on the internet]. 2018 [cited 2021 jan 07]. available from: http://documents.worldbank.org/curated/en/979591495652724770/pdf/115271-revised-final-iwg-report-3-5-18.pdf world health organization. strengthening health security by implementing the international health regulations (2005). joint external evaluation (jee) mission reports [homepage on the internet]. [cited 2020 jul 2]. available from: https://www.who.int/ihr/procedures/mission-reports/en/ world health organization. joint external evaluation tool and process overview [homepage on the internet]. [cited 2020 jul 2]. available from: https://apps.who.int/iris/bitstream/handle/10665/252755/who-hse-gcr-2016.18-eng.pdf?sequence=1 global health security index. 2019. building collective action and accountability [homepage on the internet]. [cited 2020 jul 2]. available from: https://www.ghsindex.org/ our world data. total confirmed covid-19 cases, permission people [homepage on the internet]. [cited 2020 jun 28]. available from: https://ourworldindata.org/grapher/total-confirmed-cases-of-covid-19-per-million-people?region=africa watson pf, petrie a. method agreement analysis: a review of correct methodology. sciencedirect. 2020;73(9):1167–1170. https://doi.org/10.1016/j.theriogenology.2010.01.003 world health organization. africa eradicates wild poliovirus [homepage on the internet]. [cited 2020 jan 12]. available from: https://www.afro.who.int/news/africa-eradicates-wild-poliovirus yao k, maruta t, luman et, nkengasong jn. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(2):194. https://doi.org/10.4102/ajlm.v3i2.194 ndihokubwayo j, maruta t, ndlovu n, et al. implementation of the world health organization regional office for africa stepwise laboratory quality improvement process towards accreditation. afr j lab med. 2016;5(1):a280. https://doi.org/10.4102/ajlm.v5i1.280 annayath m, noor zk. analyzing barriers for implementation of public health and social measures to prevent the transmission of covid-19 disease using dematel method. diabetes metab syndr. 2020;14(5):887–892. https://doi.org/10.1016/j.dsx.2020.06.024 rohan h, mckay g. the ebola outbreak in the democratic republic of the congo: why there is no ‘silver bullet’. nat immunol. 2020;21:591–594. https://doi.org/10.1038/s41590-020-0675-8 maxmen a. has covid taught us anything about pandemic preparedness? nature. 2021;596:332–335. https://doi.org/10.1038/d41586-021-02217-y world health organization. laboratory testing strategy recommendations for covid-19 [homepage on the internet]. interim guidance. [cited 2020 apr 01]. available from: https://apps.who.int/iris/bitstream/handle/10665/331509/who-covid-19-lab_testing-2020.1-eng.pdf?sequence=1&isallowed=y reliefweb. news and press release – 20 february 2020 [homepage on the internet]. [cited 2020 apr 08]. available from: https://reliefweb.int/report/world/more-20-african-countries-can-now-test-coronavirus-disease maruta t, kebede y. the evolution of sars-cov-2 testing in africa: observations from the first 1 million cases. south afr j pub health. 2020;4(4):106–110. diagnostic systems not diagnostics the hidden burdens of technology what is diagnosis for? acknowledgements references about the author(s) rashid ansumana school of community health sciences, njala university, bo, sierra leone fatmata bah kings sierra leone partnership, king’s centre for global health and health partnerships, freetown, sierra leone kan biao sierra leone-china friendship biological safety laboratory, chinese center for disease control and prevention, beijing, china national institute for communicable disease control and prevention, beijing, china doris harding public health laboratories, sierra leone ministry of health and sanitation, freetown, sierra leone mohamed b. jalloh department of community health, faculty of clinical sciences, college of medicine and allied health sciences, university of sierra leone, freetown, sierra leone ann h. kelly global health and social medicine, kings college london, london, united kingdom francess koker kings sierra leone partnership, king’s centre for global health and health partnerships, freetown, sierra leone zikan koroma sierra leone-china friendship biological safety laboratory, chinese center for disease control and prevention, beijing, china clinical laboratories and national coordinator biobanking and biosecurity, sierra leone ministry of health and sanitation, freetown, sierra leone mambu momoh kenema government hospital, viral hemorrhagic fever consortium, kenema, sierra leone school of nursing and medical laboratory sciences, eastern polytechnic, kenema, sierra leone mohamed h. rogers college of medicine and allied health sciences, university of sierra leone, freetown, sierra leone james rogers laboratory technical working group, sierra leone ministry of health and sanitation, freetown, sierra leone alice street school of social and political sciences, university of edinburgh, edinburgh, united kingdom eva vernooij school of social and political sciences, university of edinburgh, edinburgh, united kingdom isatta wurie college of medicine and allied health sciences, university of sierra leone, freetown, sierra leone citation ansumana r, bah, f, biao k, et al. building diagnostic systems in sierra leone: the role of point-of-care devices in laboratory strengthening. afr j lab med. 2020;9(2), a1029. https://doi.org/10.4102/ajlm.v9i2.1029 opinion paper building diagnostic systems in sierra leone: the role of point-of-care devices in laboratory strengthening rashid ansumana, fatmata bah, kan biao, doris harding, mohamed b. jalloh, ann h. kelly, francess koker, zikan koroma, mambu momoh, mohamed h. rogers, james rogers, alice street, eva vernooij, isatta wurie received: 12 apr. 2019; accepted: 14 jan. 2020; published: 01 apr. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the 2014–2016 ebola virus disease (evd) outbreak highlighted the vital importance of investing in west africa’s laboratory infrastructure and systems. in the absence of facilities capable of handling highly infectious pathogens, the national response across the region was hamstrung by costly delays in case identification and blind spots in epidemiological surveillance. the rapid development of evd diagnostic tools that could speed up testing and be used at or near the point of care became a public health priority. to expedite their deployment, a series of cross-sectional research and development initiatives were launched including innovative financing mechanisms, data-sharing platforms, public-private partnerships and accelerated regulatory pathways.1 a number of novel evd diagnostics were trialled in sierra leone.2,3,4,5,6,7 guided by a national testing algorithm, rapid diagnostic tests (rdts) such as reebov (corgenix, broomfield, colorado, united states) and the oraquick (orasure technologies, bethlehem, pennsylvania, united states), intended for use at the primary care level, and automated real-time reverse transcription polymerase chain reaction (rt-pcr), primarily run on the benchtop, underwent rapid validation and performance evaluation through the world health organization’s emergency use and assessment listing mechanism.8,9,10,11 the reebov test was temporarily deployed in a field setting in sierra leone and another rapid test, the dstl evd rapid diagnostic antigen test, developed by the defence science technology laboratory in the united kingdom, was validated in hospital and primary care settings.6 however, biosafety concerns, related to challenges in procuring, distributing and providing training for the use of personal protective equipment to safely draw, handle and dispose of blood samples suspected of being infected with ebola virus, in addition to imperfect sensitivity and specificity, meant rdts were rarely used outside the laboratory. instead, they were deployed at laboratories in key areas of the country alongside automated rt-pcr machines, such as genexpert (cepheid, ab, sunnyvale, california, united states) and biofire film array (biofire diagnostics, salt lake city, utah, united states), that could quickly confirm their results. in addition to commercially available platforms, international public health agencies and non-governmental organisations brought their own in-house assays, some of which came to provide a point of reference for field evaluations of novel tests.12 collectively, these advances in rapid and proximate point-of-care diagnostic capacity helped bring the outbreak to an end and became the cornerstone of sierra leone’s enhanced surveillance plan, intended to sustain a ‘resilient zero’ number of cases.13 in the aftermath of ebola, some of these devices and machines were left in clinical and surveillance laboratories as part of the post-ebola effort to improve disease detection and response capabilities. an rt-pcr machine in the biosafety level 3 laboratory supported by the china center for disease control and prevention (china cdc) currently serves the freetown area, while a genexpert machine for point-of-care testing supports upcountry evd surveillance in the bo and makeni government laboratories. while important for future outbreak response, the availability of evd diagnostics is clearly only a minor part of public health emergency resilience.14 the handover of epidemic preparedness from international partners to national institutions offers a unique opportunity to think through the broader, system-level needs for the accurate and rapid diagnosis of infectious diseases. critically, more attention is needed to grasp how national strategic plans for strengthening laboratory infrastructure can be best adapted to and advanced by the 2024 global health security agenda.15 global health and policy debates, largely dominated by institutions in europe and the united states, have focused on the development of new diagnostic technologies appropriate for resource-poor settings, which generally means that tests should be affordable, easy to use, rapid, and available at the point of care.16,17,18 less prominent in these discussions are the voices of experts in laboratory medicine from the countries for which the rapid tests are designed. the experience of sierra leonean laboratory medicine experts, both during and after the outbreak, offers a valuable, under-recognised and much-needed perspective on the role of point-of-care tests and diagnostic innovation in global health. on 04 march 2019, a multidisciplinary group of policymakers, biomedical and social scientists and local experts in the field of laboratory medicine convened to discuss the role of point-of-care diagnostic devices in outbreaks and their integration with healthcare infrastructure in sierra leone. the meeting was organised by the diadev research project, funded by the european research council under the horizon 2020 framework, which investigates the development of point-of-care diagnostic devices in global health and their role in transforming health systems in resource-constrained settings (www.diadev.eu). the meeting featured short presentations as well as a panel discussion with sierra leonean laboratory scientists and health workers, who shared their first-hand experience using rapid diagnostic tests during the ebola outbreak. three key insights from the day are highlighted here as disruptive and constructive contributions to debates about diagnostic futures in africa. diagnostic systems not diagnostics point-of-care devices are often championed as solutions for places with weak or no laboratory infrastructure. for evd alone, there are up to 27 assays at different stages of development or use, including 9 antigen-based rapid tests and 18 rt-pcr assays for evd.19 but the lower sensitivity of point-of-care devices means they are often integrated into complex algorithms that require confirmatory testing prior to treatment, which necessitates a wider public health laboratory network. moreover, even in the case of tests approved for stand-alone use, routine quality assurance systems require proficiency training and regular cross-checking of samples by a reference laboratory. lessons from sierra leone show that effective use of point-of-care devices also depends on the existence of regulatory capacity to safeguard the quality of point-of-care devices. many rdts on the sierra leonean market are of questionable quality and have not passed through the pharmacy board, the agency that regulates medicines and diagnostics. representatives of the ministry of health and sanitation underscored that, while the central public health reference laboratory is mandated with overseeing quality assurance and post-market validation of diagnostic tests for specific diseases such as hiv, there is a need to expand regulatory and quality assurance systems for all diagnostics, especially those for the national priority special pathogens. one current priority for the central public health reference laboratory is to improve the post-market regulatory control by re-introducing lot verification testing of diagnostic devices before and after they are distributed to clinical settings. enhancing these systems will curtail the supply of low-standard tests and also empower national clinical and public health systems to demand more rigorously tested and, arguably, superior products from international vendors. moreover, further investigation is needed into how these devices are being used in clinical practice and their capacity to improve patient care. for example, in the country’s main referral hospital, rapid tests for hepatitis b, helicobacter pylori and urine analysis, are used inside the clinical laboratory, but routine reporting systems in the laboratory mean results are only given back to clinicians the next day, even when the test result is ready earlier, impacting the ability of point-of-care tests to reduce result turn-around times. the migration of rapid diagnostic technologies into clinical laboratories also poses the risk that technologies designed to provide preliminary clinical diagnosis in places without laboratories actually replace and diminish existing laboratory capacity. point-of-care tests do not present an alternative to investment in central laboratories, which remain essential for quality assurance, confirmatory testing and research.20 particularly among patient populations likely to be afflicted by more than one disease, the availability of a comprehensive suite of basic laboratory tests located at a primary care level, as outlined by the world health organization’s recently published essential diagnostics list,21 is critical to the establishment of person-centred healthcare. rather than asking whether we should invest in laboratory strengthening or point-of-care tests, we need to look at how we can best build diagnostic systems and what role new diagnostic technologies might play in strengthening those systems. the world health organization framework for health systems strengthening has drawn critical attention to the six technical ‘building blocks’ of health systems: service delivery, health workforce, information, medical products, financing, and leadership and governance.22 all these areas are fundamental to the operation of a diagnostic system, whether this means training the health workforce in the use of new diagnostic devices, ensuring health information systems are aligned with the data from new diagnostic devices, securing supply systems to ensure reagents and other equipment are at hand, or building quality assurance systems that connect peripheral health facilities to central laboratories. but while the world health organization framework focuses on technical elements, our on-the-ground experience of laboratory medicine in sierra leone emphasises the relationships between people, technologies and infrastructure that enable diagnostic systems to work. these relationships are social and political, as well as technical, and their understanding requires attention to the interaction between local and global normative frameworks and value systems in addition to formal management structures.23 building on the work of social scientists working in this area, we propose that systems thinking entails a shift away from a focus on diagnostic technologies to understanding the way relationships between people, technologies and infrastructure unfold within everyday diagnostic processes and practices.24 the hidden burdens of technology recent global health policy discussions about diagnosis in lowand middle-income countries have focused on the need to incentivise markets for new diagnostic technologies. point-of-care tests that can be sold as affordable products are often championed as simple and easy-to-use solutions for places with limited infrastructure. but the sierra leonean experience suggests that new technologies also place a significant un-costed burden on the health system and the people who work in it, reinforcing the existing findings from social science research that point-of-care tests in africa have unexpected, intensive infrastructure requirements.25,26 contributors to the workshop noted that the burden is most sharply felt in supply chains, especially around the need for a constant and reliable supply of reagents, and transportation costs for confirmatory testing as part of quality assurance. in a recent measles outbreak in sierra leone in 2018, limited resources for specimen transportation to the reference laboratory from districts, provided through the national surveillance programme, and the lack of a full complement of reagents for analysis, hampered timely diagnosis. in this instance, preliminary diagnosis with a point-of-care test may have provided a stopgap for a quicker response. however, the 2012–2013 cholera outbreak in sierra leone showed that point-of-care tests often lack the required sensitivity, with only 10% of cases confirmed positive by conventional culture in the bacteriology laboratory, resulting from weak international and national regulatory frameworks safeguarding the quality of cholera rdts.27 point-of-care tests with very high sensitivity and specificity need to be coupled with confirmatory testing infrastructure and proper coordination between government institutions with oversight roles to ensure that the right types of kits are used for routine diagnosis. beyond the compromises in accuracy that point-of-care test designs often entail, every new diagnostic device and each iteration of platforms already in use requires a retraining of health workers and laboratory staff, creating heavy logistical, administrative and financial costs on public health institutions. these expenditures are compounded by those associated with the routine operations of the laboratory, including the purchasing of equipment, reagents, storage of large volumes of tests that require refrigeration, everyday maintenance and, critically, management of non-biodegradable waste, generated by point-of-care tests such as the cartridges used in genexpert machines or the test cassettes holding reagent strips for viral hepatitis tests. in the past, these institutional overhead costs have been mainly supported by donors as part of short-term research budgets. to support efforts to improve quality-assured diagnostic operations, a clearer understanding is needed of the hidden costs of diagnostics and where they fit in the broader financial landscape of national laboratory infrastructure and systems. what is diagnosis for? diagnostic testing is not a medical intervention, but a means of generating information for evidence-based medical practice. the question we must ask is: what can be done with this information in this place, with these resources, by these people? the point of diagnosis is not just to know what disease someone has, but to be able to act on that knowledge – whether in terms of public health measures or therapeutic intervention. as the panellists in a round table discussion dedicated to reflections on the use of point-of-care tests during the evd outbreak made clear, detecting the presence of a pathogen is just one component in a larger set of testing needs. diagnosing evd is key to public health measures, such as the isolation of patients, safe burial of bodies and contact tracing, but for clinicians it is only the starting point for therapeutic intervention. when it comes to care, other tests, such as liver function or electrolytes are arguably more important and depend on a generalised laboratory capacity. for example, point-of-care bedside analysers, such as the i-stat (abbott, lake forest, illinois, united states), which can help monitor patients in the red zone of the treatment unit, were crucial to saving lives during the outbreak but received far less attention than ebola diagnostics. intermittent power outages can affect the validity of standard laboratory tests, such as blood culture, with dangerous implications for clinical outcomes during routine care practices but also poses a significant challenge for clinicians working during the outbreak. finally, as we look ahead towards building a sustainable healthcare laboratory system in sierra leone, we need to link the question of diagnostic use to that of diagnostic value, or ‘what worth does a specific diagnostic test have for the particular health system?’ at a cost-effectiveness level, answering this question might mean calculating in the hidden burdens that new technologies generate, including workload burdens involved in using tests and reporting results, burdens on patients to travel for testing, and burdens on the health system to provide training, quality assurance, regulation, procurement and supply, and waste disposal systems. some point-of-care tests may reduce the cost of care in wealthy countries but are a burden in resource-constrained countries such as sierra leone.28 for example, point-of-care pcr tests, such as the biofire film array system and genexpert machines, require the use of expensive cartridges that deter their routine use for testing; the cost of a biofire gastro-intestinal panel ($155.00) is higher than the minimum monthly wage in sierra leone.28 calculations of value would caution against the hasty introduction of new, more advanced testing devices for particular diseases, when local capacity for testing already exists. for instance, investing in simple and affordable technology such as ammonia solution for assessment of haemoglobin using a colorimeter may offer better value for money than handheld point-of-care haemoglobin meters with costly cuvettes (e.g. hemocue, ängelholm, sweden), which also require the training of laboratory staff and placement in coordinated systems. as another example, while biosequencing may be a compelling orientation for research on emerging infections, for clinical use in a resource-poor context, its running costs are plainly prohibitive. the value of a diagnostic test cannot be determined merely by the accuracy of the test or the global health priority of the pathogen but on the basis of local needs and consideration of the test’s clinical and operational benefits. the work that the viral hemorrhagic fever consortium did in partnering with a local institution in the design and development of an rdt for lassa fever virus is an example of how local priorities can be built into innovation processes from the outset and such local institutional partnerships are to be encouraged in the development of future diagnostic systems in african countries.29 a novel diagnostic test’s value, moreover, is not the same as its value for money. beyond a bottom-line economic analysis, the adjudication of diagnostic value requires attention to the everyday lives and work of patients, clinicians, nurses, laboratory technicians, surveillance officers and public health officials. conclusion point-of-care tests are never introduced in a vacuum. ebola brought visibility to the need for improved diagnostic systems in lowand middle-income countries, but even countries severely lacking in laboratory infrastructure have pre-existing and highly specific diagnostic needs and capacities. sierra leone had a national medical laboratory policy and five-year strategic plan in place when the ebola outbreak occurred. while the renewed focus on global health security strengthening and, by extension, laboratory system improvement, is welcome, it is critical that the national tools and plans put in place are aligned to any new diagnostic devices or laboratory strengthening initiatives and allow for national priorities to be addressed. point-of-care diagnostic devices are often championed for their ability to work anywhere, but technologies are never autonomous from the systems in which they are used. diagnostic innovation needs to start from existing diagnostic systems and national policies and plans. this requires the involvement of social science research in understanding the local context into which point-of-care testing devices are introduced and in identifying local priorities for strengthening diagnostic systems. global health research and development should be directed at making diagnostic systems become workable in their own right, rather than finding ways that an ever-increasing range of individual technologies can be best accommodated. post-ebola, sierra leone is focused on improving the resilience of healthcare delivery.30 this goal will require building a diagnostic system that can prepare for and respond to ‘health shocks’, such as outbreaks and natural disasters, and also withstand the chronic stresses that accompany long-term resource constraints.31 a systems approach that encompasses both emergency and long-term timeframes needs to be present at the outset of the diagnostic development process, not only at the point at which new technologies are deployed. most importantly, diagnostic futures need to be designed with the input of the people who work in and use them and they need to incorporate the insight that local experts have gained on the front line of global health innovation. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.s. was the project leader, co-wrote the manuscript and led responses to reviewers and the redrafting for publication. e.v. and a.h.k. organised the workshop on which this opinion piece is based, gave workshop presentations and co-wrote the manuscript. i.w., r.a. and m.b.j. provided conceptual contributions, gave workshop presentations on which the manuscript is based, and made significant critical revisions to the manuscript. f.b., k.b., d.h., f.k., z.k., m.m., m.h.r. and j.r. provided conceptual contributions and gave workshop presentations on which the manuscript is based. ethical considerations ethical approval for research on which this manuscript is based was granted by the research and research ethics integrity committee, school of social and political science, university of edinburgh on 14 september 2016 and by the office of the sierra leone ethics and scientific review committee on 27 september 2018. sources of support this project has received funding from the european research council under the european union’s horizon 2020 research and innovation programme under grant agreement no. 715450. the kings sierra leone partnership provided support in hosting and organising the workshop on which this opinion piece is based. ann h. kelly is funded by the national institute of health research (nihr) global health research unit on health system strengthening in sub-saharan africa, king’s college london (ghru 16/136/54) using aid from the uk government. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views expressed in this publication are those of the authors and do not directly represent the views of any funding or support agencies. references world health organization. emergency use and assessment listing procedure (eual) for candidate in vitro diagnostics (ivds) [homepage on the internet]. 2014; p. 1–9. available from: https://www.who.int/diagnostics_laboratory/eual/emergency/en/%0a biava m, colavita f, marzorati a, et al. evaluation of a rapid and sensitive rt-qpcr assay for the detection of ebola virus. j virol methods. 2018;252(november 2017):70–74. https://doi.org/10.1016/j.jviromet.2017.11.009 broadhurst mj, kelly jd, miller a, et al. reebov antigen rapid test kit for point-of-care and laboratory-based testing for ebola virus disease: a field validation study. lancet. 2015;386(9996):867–874. https://doi.org/10.1016/s0140-6736(15)61042-x leski ta, ansumana r, taitt cr, et al. use of the filmarray system for detection of zaire ebola virus in a small hospital in bo, sierra leone. j clin microbiol. 2015;53(7):2368–2370. https://doi.org/10.1128/jcm.00527-15 semper ae, broadhurst mj, richards j, et al. performance of the genexpert ebola assay for diagnosis of ebola virus disease in sierra leone: a field evaluation study. plos med. 2016;13(3):1–15. https://doi.org/10.1371/journal.pmed.1001980 walker nf, brown cs, youkee d, et al. evaluation of a point-of-care blood test for identification of ebola virus disease at ebola holding units, western area, sierra leone, january to february 2015. euro surveill. 2015;20(12):1–6. https://doi.org/10.2807/1560-7917.es2015.20.12.21073 weller sa, bailey d, matthews s, et al. evaluation of the biofire filmarray biothreat-e test (v2.5) for rapid identification of ebola virus disease in heat-treated blood samples obtained in sierra leone and the united kingdom. j clin microbiol. 2016;54(1):114–119. https://doi.org/10.1128/jcm.02287-15 world health organization. who emergency use assessment and listing for ebola virus disease ivds [homepage on the internet]. public report. oraquick® ebola rapid antigen test kit (cadaveric oral fluid and whole blood). 2016 [cited 2018 sep 30]. available from: http://www.who.int/diagnostics_laboratory/141002_revised_invitation-to_mx_of_ebola_virus_diagnostics_rc world health organization. who emergency use assessment and listing for evd ivds [homepage on the internet]. public report. product: filmarraytm biothreat-e. 2015 [cited 2018 sep 30]. available from: https://www.who.int/diagnostics_laboratory/procurement/150819_final_public_report_pqdx_0010-010-00.pdf?ua=1 world health organization. who emergency use assessment and listing for ebola virus disease ivds [homepage on the internet]. public report. reebov tm antigen rapid test kit. who; 2014 [cited 2018 sep 30]. available from: http://www.who.int/diagnostics_laboratory/141002_revised_invitation-to_mx_of_ebola_virus_diagnostics_rc world health organization. who emergency use assessment and listing for evd ivds [homepage on the internet]. public report. product: xpert® ebola assay. vol. 3. 2016. available from: http://www.who.int/medicines/news/public_consult_med_prods/en/ pollock nr, wonderly b. evaluating novel diagnostics in an outbreak setting: lessons learned from ebola. j clin microbiol. 2017;55(5):1255–1261. https://doi.org/10.1128/jcm.00053-17 world health organization. ebola outbreak to recovery sierra leone progress report [homepage on the internet]. 2015. available from: https://www.afro.who.int/publications/ebola-outbreak-recovery-sierra-leone-progress-report-2015 ravi sj, snyder mr, rivers c. review of international efforts to strengthen the global outbreak response system since the 2014–16 west africa ebola epidemic. health policy plan. 2018;34(1):1–8. https://doi.org/10.1093/heapol/czy102 global health security agenda. global health security agenda (ghsa) 2024 framework overview [homepage on the internet]. 2018. available from: https://www.ghsagenda.org/ghsa2024 chua ac, cunningham j, moussy f, perkins md, formenty p. the case for improved diagnostic tools to control ebola virus disease in west africa and how to get there. plos negl trop dis. 2015;9(6):4–9. https://doi.org/10.1371/journal.pntd.0003734 wu g, zaman mh. low-cost tools for diagnosing and monitoring hiv infection in low-resource settings. bull world health organ. 2015;90(12):914–920. https://doi.org/10.2471/blt.12.102780 oldach l, sall a, lehe j, fernandes p. pioneering new diagnostics: addressing challenges and implications for point-of-care testing in african settings [homepage on the internet]. republished 4 jan 2018 from aslm lab culture, february 2015, issue 13, pp. 11–21. available from: https://aslm.org/resource/pioneering-new-diagnostics-addressing-challenges-and-implications-for-point-of-care-testing-in-african-settings/ tembo j, simulundu e, changula k, et al. recent advances in the development and evaluation of molecular diagnostics for ebola virus disease. expert rev mol diagn [serial online]. 2019;19(4):325–340. available from: https://www.tandfonline.com/doi/full/10.1080/14737159.2019.1595592 jephcott fl, wood jln, cunningham aa. facility-based surveillance for emerging infectious diseases; diagnostic practices in rural west african hospital settings: observations from ghana. philos trans r soc b biol sci. 2017;372(1725). https://doi.org/10.1098/rstb.2016.0544 world health organization. who model list of essential in vitro diagnostics [homepage on the internet]. 2018 [cited 2019 jun 18]. available from: https://www.who.int/medical_devices/diagnostics/selection_in-vitro/edl-model-lists/en/ world health organization. everybody’s business: strengthening health systems to improve health outcomes: who’s framework for action. [homepage on the internet]. 2007. available from: http://www.who.int/healthsystems/strategy/everybodys_business.pdf gore r, parker r. analysing power and politics in health policies and systems. glob public health. 2019;14(4):481–488. https://doi.org/10.1080/17441692.2019.1575446 pai np, vadnais c, denkinger c, engel n, pai m. point-of-care testing for infectious diseases: diversity, complexity, and barriers in lowand middle-income countries. plos med [serial online]. 2012 [cited 2014 oct 17];9(9):e1001306. available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3433407&tool=pmcentrez&rendertype=abstract chandler cir, whitty cjm, ansah ek. how can malaria rapid diagnostic tests achieve their potential? a qualitative study of a trial at health facilities in ghana. malar j [serial online]. 2010;9:95. available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2859355&tool=pmcentrez&rendertype=abstract engel n, pant pai n. qualitative research on point-of-care testing strategies and programs for hiv. expert rev mol diagn [serial online]. 2014[cited 2014 nov 24];30:1–5. available from: http://www.ncbi.nlm.nih.gov/pubmed/25267607 ramamurthy t, balakrish niar g, quilici m-l. cholera surveillance, rapid diagnostics and laboratory networks. wkly epidemiol rec [serial online]. 2015;40(2). available from: https://apps.who.int/iris/handle/10665/242433 beal sg, tremblay ee, toffel s, velez l, rand h. a gastrointestinal pcr panel improves clinical management and lowers health care costs. j clin microbiol. 2018;56(1):1–9. https://doi.org/10.1128/jcm.01457-17 boisen ml, hartnett jn, shaffer jg, et al. field validation of recombinant antigen immunoassays for diagnosis of lassa fever. sci rep. 2018;8(1):1–14. https://doi.org/10.1038/s41598-018-24246-w wurie i. sierra leone laboratory systems – now and future. afr j lab med. 2016;5(3):a549. https://doi.org/10.4102/ajlm.v5i3.549 kruk me, ling ej, bitton a, et al. building resilient health systems: a proposal for a resilience index. bmj [serial online]. 2017;357(may):1–8. https://doi.org/10.1136/bmj.j2323 abstract background description of the intervention lessons learnt recommendations acknowledgements references about the author(s) seth attoh jm wadhwani department of anatomical pathology, pathology division laboratory, 37 military hospital, accra, ghana francis k.m. tetteh department of microbiology, pathology division laboratory, 37 military hospital, accra, ghana mary mcaddy jm wadhwani department of anatomical pathology, pathology division laboratory, 37 military hospital, accra, ghana kingsley ackah department of microbiology, pathology division laboratory, 37 military hospital, accra, ghana richmond kyei jm wadhwani department of anatomical pathology, pathology division laboratory, 37 military hospital, accra, ghana marcus moroti department of microbiology, pathology division laboratory, 37 military hospital, accra, ghana cynthia boateng department of chemical pathology, pathology division laboratory, 37 military hospital, accra, ghana laurinda adusu-donkor department of chemical pathology, pathology division laboratory, 37 military hospital, accra, ghana joseph boafo department of haematology, pathology division laboratory, 37 military hospital, accra, ghana alhassan yakubu department of quality assurance, pathology division laboratory, 37 military hospital, accra, ghana sarah kwao department of quality assurance, pathology division laboratory, 37 military hospital, accra, ghana emmanuel sarkodie department of haematology, pathology division laboratory, 37 military hospital, accra, ghana nana-banyin koranteng department of chemical pathology, pathology division laboratory, 37 military hospital, accra, ghana monica a. addo department of chemical pathology, pathology division laboratory, 37 military hospital, accra, ghana frederick hobenu jm wadhwani department of anatomical pathology, pathology division laboratory, 37 military hospital, accra, ghana kwasi agyeman-bediako jm wadhwani department of anatomical pathology, pathology division laboratory, 37 military hospital, accra, ghana raymond d. fatchu department of quality assurance, pathology division laboratory, 37 military hospital, accra, ghana citation attoh s, tetteh fkm, mcaddy m, et al. challenges with the pursuit of iso 15189 accreditation in a public health laboratory in ghana. afr j lab med. 2022;11(1), a1448. https://doi.org/10.4102/ajlm.v11i1.1448 lessons from the field challenges with the pursuit of iso 15189 accreditation in a public health laboratory in ghana seth attoh, francis k.m. tetteh, mary mcaddy, kingsley ackah, richmond kyei, marcus moroti, cynthia boateng, laurinda adusu-donkor, joseph boafo, alhassan yakubu, sarah kwao, emmanuel sarkodie, nana-banyin koranteng, monica a. addo, frederick hobenu, kwasi agyeman-bediako, raymond d. fatchu received: 10 nov. 2020; accepted: 04 apr. 2022; published: 19 july 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: accreditation is important for all medical laboratories, particularly public health laboratories in developing countries. several laboratories in ghana implemented the requirements of the international organization for standardization (iso) 15189 but were unable to proceed to accreditation. this article describes the challenges faced by the pathology division laboratory of the 37 military hospital, accra, ghana, during the acquisition of iso 15189 accreditation and suggests solutions for a better approach. intervention: following iso 15189 accreditation in 2017, an online survey was conducted between 01 and 30 march 2020 among the laboratory staff. respondents were required to grade, on a scale of 0 (least) to 5 (most), the extent to which 16 key challenges influenced the process of obtaining accreditation. key informant interviews were also held with laboratory personnel who were directly involved in the establishment of the quality management system in the laboratory and the accreditation acquisition process. lessons learnt: documentation, laboratory safety measures, laboratory management support, and reagent unavailability were estimated as the challenges that most affected the acquisition of laboratory accreditation. challenges such as poor communication, staff apathy and workload had the least effect on the accreditation process. there was no difference in challenges identified between persons who worked in the laboratory before or after accreditation (p = 0.11). recommendations: to surmount the anticipated challenges, there is the need for national strategic direction for laboratory accreditation, hospital and laboratory management support for the accreditation acquisition and maintenance processes, and sufficient technical assistance in the form of training and mentorship. keywords: iso 15189:2012; accreditation; strengthening laboratory management towards accreditation; laboratory; challenges. background the quest to meet international standards, gain international recognition and acceptability, win the trust and confidence of clinicians and patients, improve quality of care, and improve the capacity to measure performance and streamline operations is driving medical facilities to pursue international accreditation.1 accreditation and the pursuance of quality health systems in lowand middle-income countries have become critical in achieving universal health coverage.2 a well-structured and well-implemented healthcare system can result in better health outcomes and reduce health inequalities, hospitalisation rates, and hospitalisation costs.3,4 according to the world health organization, despite the huge investments made in the health sector globally, including the medical laboratory, health systems and laboratory practices still face significant challenges.5 in sub-saharan africa, several initiatives focused on medical laboratory improvement have resulted in few practical and sustainable outcomes.6,7 key factors that negatively influence the accreditation process include the lack of prioritisation of accreditation, inadequate allocation of resources for attaining and maintaining accreditation, poor understanding of the importance of accreditation by both laboratory personnel and health authorities, and the high cost of the process.8 between the years 2010 and 2013, ghana enrolled 15 public sector medical laboratories in the strengthening laboratory management towards accreditation programme to develop laboratory systems towards accreditation readiness and subsequent accreditation.9 at the close of the programme, three laboratories were awarded a four-star rating according to the strengthening laboratory management towards accreditation grading system.9 however, despite the quality improvement within the respective laboratories, none has proceeded to accreditation due to major gaps threatening the acquisition of accreditation. even for laboratories that have attained accreditation in other countries, weak hospital management support, inadequate documentation, inefficient equipment, irregular supply of reagents, little mentorship, and high staff turnover have been identified as bottlenecks to attaining and maintaining accreditation.10,11 as several african countries within the sub-region, including ghana, pursue healthcare reforms to achieve universal health coverage, the need for national accreditation policies and systems has become a priority for several ministries of health.12 the pathology division laboratory of the 37 military hospital became the first public sector laboratory to be accredited for methods in chemical pathology and haematology in accra, ghana, in 2017.8 learning from its experiences and challenges, the laboratory has maintained its accreditation status over the years while gradually expanding its scope to include other departments in the division. the microbiology department also subsequently obtained international organization for standardization (iso) 15189:2012 accreditation in 2019 from the southern african development community accreditation service. this article describes the challenges faced by the pathology division laboratory of the 37 military hospital in accra, ghana, in the pursuit of iso 15189:2012 accreditation and suggests solutions to the identified challenges. description of the intervention ethical considerations ethical clearance, with reference number 37mh-irb/ds/ipn/417/2020, was received from the institutional review board of the 37 military hospital, accra, ghana. participants were required to respond to a consent section before voluntarily completing the online questionnaire. to protect the privacy of participants, no personal information was collected. data collection the study was conducted in the pathology division laboratory of the 37 military hospital in accra, the capital of ghana, west africa, which received iso 15189 accreditation for haematology, chemical pathology, and microbiology test methods between 2017 and 2019. the laboratory also operates other departments, including histopathology, serology, and a blood transfusion service. key informant (ki) interviews were conducted with personnel who were directly involved in the establishment of the quality management system (qms) of the laboratory and the accreditation acquisition process. these kis included the officer-in-charge of the division, laboratory manager, quality manager, and heads of department for the haematology, chemical pathology, and microbiology departments. the kis were coded (ki-1, ki-2, etc.) to maintain confidentiality. interviews were digitally voice recorded after securing interviewee consent. interviews were conducted in english, transcribed verbatim immediately thereafter, and then reviewed by members of the research team to ensure their validity. the main themes explored covered personnel, documentation, and management. an electronic questionnaire was sent to all laboratory staff between 01 and 30 march 2020 to obtain staff opinions on the challenges with the laboratory’s accreditation acquisition process. the key challenges were selected based on other studies10 and the laboratory’s observed experiences during the iso 15189 implementation phase. these covered challenges related to documentation, laboratory management support, laboratory safety, reagent availability, equipment availability, mentorship, support from doctors, hospital management support, staff attrition, staff qualification, staff strength, laboratory information management system, support from nurses, workload, staff apathy, and communication. participation in the study was voluntary. participants were required to grade, on a scale of 0 (least challenging) to 5 (most challenging), the extent to which 16 key challenges impacted the accreditation acquisition process of the pathology division laboratory. the three most challenging and three least challenging aspects of the accreditation process were identified. the maximum score attributable to each challenge is 520 (total number of respondents [n = 104] multiplied by the maximum score applicable [n = 5]). data analysis data collected were processed and analysed in microsoft excel 2016 (microsoft corporation, redmond, washington, united states) and stata ic/16 (statacorp, college station, texas, united states). summary descriptive statistics were used to describe the characteristics of the data set obtained from survey respondents. the number of responses on each challenge to accreditation was also represented as percentages for each score category. a multivariate regression model was used to compare the scores of key challenges between personnel who joined the pathology division laboratory before accreditation versus after accreditation, adjusting for employee type, education, gender, and awareness of the accreditation status of the pathology division. p-values less than 0.0025 were considered statistically significant. lessons learnt a total of 106 of the 110 personnel working in the laboratory completed the structured questionnaire, representing a 96.4% response rate. there were two non-responses on time of joining the laboratory (before or after accreditation) and five non-responses on job title. more respondents (67/104, 64.4%) were present throughout the laboratory’s accreditation acquisition process compared to respondents (37/104, 35.6%) who joined the laboratory after the accreditation in 2017 (table 1). table 1: characteristics of study respondents (n = 106) at the pathology division laboratory, 37 military hospital, accra, ghana, november 2020. the pathway to iso 15189 accreditation is marked with several challenges, especially in resource-limited environments. this, however, does not make it an improbable cause. notable among the challenges to the accreditation process were laboratory personnel attrition, personnel attitude to change, service interruptions and logistic constraints, documentation, continuous external assessment outcomes, and size and complexity of the laboratory (figure 1). figure 1: scoring of key challenges to accreditation by laboratory staff at the pathology division laboratory, 37 military hospital, accra, ghana, november 2020. documentation, availability of reagents, workload, and staff strength were among the highest-rated challenges. these challenges are consistent across different laboratories.10 challenges such as nurses’ support, doctors’ support and staff qualification were rated among the least challenging. the role of laboratory management in ensuring that the requirements of the international standards are understood by laboratory personnel is important to ensure collective buy-in and conformance. where such inadequacies exist, mentorship would be needed to complement management roles in the application of the relevant standards.8 when controlled for employee type, education, gender, and awareness of the accreditation status of the pathology division, there was no statistically significant difference in responses between respondents who joined the laboratory before or after accreditation (table 2, figure 2). this is a clear indication that best practices implemented in the build-up to any accreditation process do not significantly change the laboratory’s processes, especially in laboratories where qms may be seen as a burden. this ease of transition may be associated with the conscious effort to involve personnel in the accreditation process, thus increasing their level of satisfaction and making them more committed to the process.13 when staff are committed and involved in the accreditation process from the beginning, good commitment is more likely to persist throughout the life cycle of the laboratory. figure 2: comparison of the scores of key challenges to accreditation between personnel who joined the pathology division laboratory, 37 military hospital, accra, ghana, before versus after accreditation, november 2020. table 2: multivariate regression model comparing the scores of key challenges between personnel who joined the pathology division laboratory, 37 military hospital, accra, ghana, before versus after accreditation, november 2020. key informant interviews staff attrition critical among the challenges to the accreditation process was laboratory personnel attrition. ki-1 acknowledged: ‘as a military facility, military personnel are moved every now and then on military and allied duties resulting in significant reduction in staff numbers and consequent increase in workload on the remaining personnel.’ (k1-1, male, laboratory scientist) as a result, persons trained and deemed competent in the quality system were lost and new persons introduced. this can be described as a ‘brain drain’ of laboratory personnel. according to ki-2 (female, laboratory manager), ‘between 2014 and 2017, five out of eight members of the laboratory quality steering committee members had been changed’. this certainly increased the amount of work the few remaining personnel had to do to support the quality system. to address this situation, the laboratory developed and deeply integrated a staff orientation and training programme to consistently update old and new staff with the status quo and requirements of the laboratory qmss. additionally, for every key appointment (e.g. quality manager, safety officer) there were two assigned deputies. although these deputies had technical roles, they also understudied the key officer in preparation for taking over when one was moved. the attrition of personnel may constitute a significant form of brain drain in a laboratory that has built the capacity of its personnel to implement the requirements of the qms. the effect of attrition is felt when the quality system is established around personalities and not as a functional system. establishing a functional system is to ensure that developed policies and procedures are followed as required. however, there is the need to maintain a good level of stability of key personnel such as the quality manager and the quality team players to maximise the human resource capacity. personnel attitude to changing work routines the attitude of personnel to changing work routines was another challenge that was apparent during the application process. according to ki-4: ‘laboratory personnel considered the accreditation process and its requirement as additional work burden which added very little immediate financial benefits to one’s pocket hence the lack of enthusiasm towards the process.’ (k1-4, female, laboratory scientist supervisor) we opine that the general perception among personnel is that the implementation of a quality system towards accreditation is entirely different from the ‘regular’ laboratory work. hence, staff are more likely to go about their routine duties and only implement quality system requirements if they remember to do so. both ki-3 and ki-4 emphasised: ‘the same personnel who are deployed on technical bench duties and processing of samples were the very staff involved in the numerous administrative and documentation tasks. they are simply overwhelmed with tasks and had the right to complain.’ (k1-3 & k1-4, laboratory scientist supervisors) the routine duties of laboratory staff and the quality requirements associated with iso accreditation are more interconnected than mutually exclusive as perceived. the perception of the two being separate stems from the situation where too much emphasis is placed on getting documentation done to the detriment of patient care or vice versa. additionally, lack of motivation coupled with the extra duties increases that perception gap. personnel are originally employed by their managers to run patient samples and churn out results. therefore, any additional duty is expected to come with some extra, usually financial, remuneration. where this fails to occur, as is the case in most public sector laboratories because they do not manage their own budget, enforcing such a change is met with firm resistance or indifference. this situation is however contrary to a study done in lebanon where accreditation was found to be an impetus for better performance.2 it took several one-on-one mentorship and coaching sessions and in-house continuous education on iso 15189 accreditation, the improvement process, the requirements of the stepwise laboratory improvement process towards accreditation checklist, good clinical laboratory practices, and laboratory qms to get a lot more personnel on board with the process. according to ki-2: ‘the concerns of laboratory personnel regardless of how often or how much they complain is good feedback. though we knew that indeed they were overwhelmed, everyone was overwhelmed. however, we initiated a staff of the month award scheme to motivate personnel.’ (ki-2, female, laboratory manager) this ‘staff of the month’ award included a lunch package for the winner and a crate of drinks for the entire department, as well as the display of the winner’s photograph at the front desk. service interruptions and logistic constraints interruptions in the continuity of laboratory service was considered one of the challenges to the accreditation process. ki-1 explained: ‘service interruptions usually occurred as a result of delays by suppliers to deliver reagents and other consumables, as well as bureaucratic procurement processes. these mostly resulted in reagent stock-outs. most of the reagents and consumables required for testing are imported and are very capital intensive. due to the amount involved and the foreign exchange transaction policies of the government of ghana, it became even more difficult to carry out such procurement.’ (k1-1, male, laboratory scientist) ki-1 acknowledged as a solution: ‘to ensure a more regular and continuous supply of reagents and consumables, some level of financial autonomy was given to the laboratory. an accountable imprest was also set aside for the laboratory. this brought to the fore the level of support offered by the hospital management.’ (k1-1, male, laboratory scientist) the level of support and trust provided sufficient drive for the staff and management of the laboratory to improve their interest in the whole process and secure the coveted accreditation. all kis agreed that the absence of a dedicated budget for procurement of laboratory logistics was the main reason for the service interruptions occurring in the laboratory. continuous external assessment outcomes in preparation for accreditation, self-checks and external assessments are needed to ascertain the readiness of the laboratory for the final accreditation assessment. according to ki-1: ‘we were subjected to several external assessments; however, it was as if the outcomes did us ‘more harm than good’. despite the efforts put into preparing for external assessments, the scores were low and staff were demoralised. different assessors almost reported different findings for the same requirement.’ (k1-1, male, laboratory scientist) all kis agreed that assessments (internal and external) are critical as required by the standard but that it is important to focus on managing the quality system rather than on the numbers or scores of an assessment. documentation constant documentation of activities is one of the key requirements of accreditations in general. the iso 15189 standard requires the generation of evidence of the implementation of laboratory policies, processes, and procedures. ki-3 said: ‘it is like you have to document everything you do or do not do, this is hard, this is not something we are or were used to, hence the massive resistance.’ (k1-3, laboratory scientist supervisor) in a time of evidence-based laboratory medicine, it is only imperative to document all activities as proof that they were carried out as required. the key role of conformance to documentation in any quality system cannot be overemphasised. ki-1 commented: ‘it is not possible for any laboratory to implement a quality system when the personnel who are the direct implementers of the system are not committed to the process. no matter how good the policies and processes are, if the people do not conform to it, it is useless.’ (k1-1, male, laboratory scientist) this represented one of the biggest challenges to manage in an environment where documentation and conformance are not part of the culture. group training and consistent one-on-one coaching with staff on the importance and benefits of documentation were conducted. most importantly, results of documentation (e.g. occurrence, quality indicators, temperature monitoring, etc.) were periodically reviewed and communicated to staff to help them appreciate the need for and benefits of keeping quality records. size and complexity of the laboratory ki-5 (laboratory scientist) stated that ‘the medical laboratory, to the ordinary person, may be perceived as a small space within which simple tests are performed’, and further opined that the converse rather is true, stating that ‘the laboratory is a large complex network of several units and departments involved in an interconnected series of test activities’. this was a major challenge. the laboratory has five testing departments, collectively performing over 100 test panels and generating results for over 300 analytes. ‘at a point we were confused, not knowing exactly what was going on because they were just too many’, ki-2 (laboratory manager) said. the initial attempt to get all methods accredited at once was not working. the burden of work was overwhelming due to the size and complexity of the laboratory. therefore, the laboratory adopted a stepwise approach to attaining its much-desired accreditation. this phased approach implied prioritising the methods to be accredited and working at accreditation in smaller chunks. chemical pathology and haematology were therefore chosen for the initial accreditation process, with the subsequent addition of microbiology a year later. no historical precedence all kis acknowledged that the accreditation of a public sector laboratory in ghana was considered highly improbable. this made it even more difficult for laboratory staff to be convinced that it was possible to be accredited as a public sector laboratory. as explained by ki-2: ‘staff always made reference to other bigger hospitals in ghana and in the west african sub-region that if those facilities were not accredited though better placed to be, how was it going to be possible for the military hospital which is resource-stricken be accredited.’ (k1-2, female, laboratory manager) however, it was clear that although there was no historical precedence, with the needed guidance and mentorship, it was possible to meet the requirements for accreditation. according to ki-1 and ki-5: ‘effective mentorship is a pivotal element for any public sector laboratory seeking accreditation. in the absence of a historical precedence, our mentorship program guided the development of policies and process and further assisted in the implementation, conformance and the review process.’ (k1-1 & k1-5, laboratory scientists) in a country where the motivation of the government towards laboratory accreditation is weak, securing accreditation for any public sector laboratory is usually a huge challenge.14 the inadequacies in the national support for accreditation result in the inadequate dedication of resources (human, capital and infrastructural) towards attaining and maintaining accreditation. while a national focus is required, hospital management support, including support from other healthcare workers such as doctors and nurses, cannot be overemphasised.8 management support plays a key role in the accreditation process in resource-limited settings, and the engagement of hospital management with laboratory management accelerates the accreditation acquisition process. perspectives about the challenges with securing accreditation vary between laboratory personnel and management staff. while laboratory personnel identified excessive documentation, weak laboratory management support, and inadequate safety as the main challenges to accreditation, management personnel identified staff attrition, personnel attitudes, and the size and complexity of the laboratory facility as the main challenges. however, both groups of personnel identified excessive documentation and service interruption as key challenges. there is a need for constant feedback across levels of hierarchy to get everyone on the same page in the quest for accreditation. that way, the resources, although limited, can be better apportioned to address common challenges. limitations the study was limited to staff of the pathology division laboratory so the perspectives of hospital management, other hospital staff and clients were not solicited. the ki interviews did not include personnel whose testing activities were not within the scope of accreditation. some bias may therefore be inherent in the responses. recommendations to make laboratory accreditation more relevant and, consequently, surmount the anticipated challenges, there is the need for national strategic direction for laboratory accreditation, hospital and laboratory management support for the accreditation acquisition and maintenance processes, and sufficient technical assistance in the form of training and mentorship. acknowledgements we are grateful to the management and staff of the entire pathology division of the 37 military hospital for their contribution and support in the conduct of this study. special thanks go to dr edward asumanu of the postgraduate college of the 37 military hospital for providing professional guidance. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions s.a. conceived the idea, l.a.-d. started the initial draft and the manuscript and was completed by r.d.f., f.k.m.t., k.a., r.k., m. mcaddy, m. moroti, c.b., j.b., a.y., s.k., e.s., n.-b.k., m.a.a., f.h., and k.a.-b. all authors reviewed the manuscript and provided feedback. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability data sharing is not applicable to this article. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references alemnji ga, zeh c, yao k, fonjungo pn, regional c, office k. strengthening national health laboratories in sub-saharan africa: a decade of remarkable progress. trop med int health. 2014;19(4):450–458. https://doi.org/10.1111/tmi.12269 el-jardali f, hemadeh r, jaafar m, et al. the impact of accreditation of primary healthcare centers: successes, challenges and policy implications as perceived by healthcare providers and directors in lebanon. bmc health serv res. 2014;14:86. https://doi.org/10.1186/1472-6963-14-86 schoen c, osborn r, doty mm, squires d, peugh j, applebaum s. a survey of primary care physicians in eleven countries, 2009: perspectives on care, costs, and experiences. health aff. 2009;28(6):w1171–w1183. https://doi.org/10.1377/hlthaff.28.6.w1171 department of health and ageing. primary health care reform in australia: report to support australia’s first national primary health care strategy. 2009; canberra: department of health and ageing. who. the world health report 2008 – primary healthcare: how wide is the gap between its agenda and implementation in 12 high-income health systems? health policy. 2012;7(3):38–58. https://doi.org/10.12927/hcpol.2013.22778 masanza mm, nqobile n, mukanga d, gitta sn. laboratory capacity building for the international health regulations (ihr[2005]) in resource-poor countries: the experience of the african field epidemiology network (afenet). bmc public health. 2010;10 suppl 1(suppl 1):1–7. https://doi.org/10.1186/1471-2458-10-s1-s8 gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. https://doi.org/10.1309/ajcptuuc2v1wjqbm attoh s, kodjoe it, boateng c, et al. lessons learnt of the first public sector iso 15189 accredited laboratory in ghana. postgrad med j ghana. 2020;9(1):1–5. nkrumah b, van der puije b, bekoe v, et al. building local human resources to implement slmta with limited donor funding: the ghana experience. afr j lab med. 2014;3(2):1–7. https://doi.org/10.4102/ajlm.v3i2.214 girma m, desale a, hassen f, sisay a, tsegaye a. survey-defined and interview-elicited challenges that faced ethiopian government hospital laboratories as they applied iso 15189 accreditation standards in resource-constrained settings in 2017. am j clin pathol. 2018;150(4):303–309. world health organization. regional office for the eastern mediterranean. (2004). quality improvement in primary health care: a practical guide. who regional publications, eastern mediterranean series (26). https://apps.who.int/iris/handle/10665/119694 mate ks, rooney al, supachutikul a, gyani g. accreditation as a path to achieving universal quality health coverage. global health. 2014;10(1):1–8. https://doi.org/10.1186/s12992-014-0068-6 paccioni a, sicotte c, champagne f. accreditation: a cultural control strategy. int j health care qual assur. 2008;21(2):146–158. https://doi.org/10.1108/09526860810859012 grochau ih, ten caten cs, de camargo forte mm. motivations, benefits and challenges on iso/iec 17025 accreditation of higher education institution laboratories. accredit qual assur. 2018;23(3):183–188. https://doi.org/10.1007/s00769-018-1317-9 abstract introduction methods results discussion acknowledgements references about the author(s) koumpingnin nebie national blood center of ouagadougou, ouagadougou, burkina faso laboratory of haematology, department of fundamental sciences, health sciences research and training unit, university joseph ki-zerbo, ouagadougou, burkina faso salam sawadogo national blood center of ouagadougou, ouagadougou, burkina faso laboratory of haematology, department of fundamental sciences, health sciences research and training unit, university joseph ki-zerbo, ouagadougou, burkina faso salifo sawadogo national institute for medical sciences, university nazi boni, bobo-dioulasso, burkina faso souro sanou teaching hospital, bobo-dioulasso, burkina faso jérôme koulidiati laboratory of haematology, department of fundamental sciences, health sciences research and training unit, university joseph ki-zerbo, ouagadougou, burkina faso yalgado ouedraogo teaching hospital, ouagadougou, burkina faso habi y.a. lengani yalgado ouedraogo teaching hospital, ouagadougou, burkina faso abdoul g. sawadogo national blood center of ouagadougou, ouagadougou, burkina faso jérôme babinet centre national de référence pour les groupes sanguins (cnrgs), national institute for blood transfusion, paris, france mohammed khalloufi french establishment of blood, bobigny, france saliou diop department of haematology, university cheikh anta diop, dakar, senegal eléonore kafando laboratory of haematology, department of fundamental sciences, health sciences research and training unit, university joseph ki-zerbo, ouagadougou, burkina faso laboratory of haematology, paediatric teaching hospital charles de gaulle, ouagadougou, burkina faso citation nebie k, sawadogo s, sawadogo s, et al. red blood cell alloimmunisation in multi-transfused patients from an haemodialysis service in burkina faso. afr j lab med. 2022;11(1), a1625. https://doi.org/10.4102/ajlm.v11i1.1625 original research red blood cell alloimmunisation in multi-transfused patients from an haemodialysis service in burkina faso koumpingnin nebie, salam sawadogo, salifo sawadogo, jérôme koulidiati, habi y.a. lengani, abdoul g. sawadogo, jérôme babinet, mohammed khalloufi, saliou diop, eléonore kafando received: 12 may 2021; accepted: 26 may 2022; published: 26 sept. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: in burkina faso, red blood cell (rbc) transfusion remains the crucial anaemia treatment following chronic renal failure (crf) as erythropoietin and its analogues are unavailable. however, blood group matching beyond the abo and rhesus is not common in burkina faso. thus, alloimmunisation is a potential issue for transfused patients. objective: our study aimed to identify anti-erythrocyte antibodies in multi-transfused crf patients at the yalgado ouedraogo teaching hospital, ouagadougou, burkina faso. methods: this cross-sectional study, conducted from october 2018 to november 2019, included crf patients who had received at least two rbc units. we screened patients for the presence of rbc antibodies using three commercial cells panels and identified antibody specificities for positive screenings using 11 cells panels for an indirect antiglobulin test (iat) in a low ionic strength microcolumn gel-card system. results: two hundred and thirty-five patients (45.1% female; average age: 41.5 years) were included. the median number of blood units received per patient was 10 (interquartile range: 5–20). the overall alloimmunisation rate was 5.9% (14/235). antibodies identified included: anti-d (1 case), anti-c (1 case), anti-d+c (4 cases), anti-cw (1 case), anti-e (1 case), anti-s (1 case) and anti-lea (1 case). in four positive patients, the specificity of the antibodies was indeterminate. no risk factors were associated with alloimmunisation. conclusion: in burkina faso, screening for rbc alloantibodies should be mandated for patients at risk. the high rate of indeterminate antibodies suggests the need to develop a local rbc antibody panel adapted to the local population. keywords: blood transfusion; alloimmunisation; rbc antibody; crf; burkina faso. introduction blood transfusion, specifically the transfusion of red blood cells (rbc), significantly contributes to the modern healthcare system. every day, blood transfusion saves lives in developing countries where acute anaemia caused by malaria, sickle cell disease (scd), pregnancy-related events and other trauma remains high or is on the rise. for example, in burkina faso, around 103 731 rbc units were used in 2017.1 however, this did not meet the transfusion needs of the country. moreover, this number is far lower than the theoretical needs of around 196 000–580 000 per the world health organization estimation method iii (i.e. 1% – 3% of the 19.5 million inhabitants).2 besides the chronic blood shortage, developing countries also face poor quality of blood products and their unsafe use.3 indeed, residual risks of transfusion-transmitted infections remain high4,5 due to inadequacies in blood donor selection and retention and laboratory screening of blood donations. furthermore, blood transfusion adverse events are underestimated due to the weakness or nonexistence of haemovigilance and quality management systems.6,7 finally, although transfusion-transmitted infections and major blood groups matching errors are worrying, blood transfusion safety issues, alloimmunisation and the occurrence of alloantibody are also pressing issues, especially in multi-transfused patients such as those undergoing haemodialysis for chronic renal failure (crf).4,8,9,10 anaemia is highly prevalent in end-stage renal disease patients, often non-regenerative normochromic normocytic anaemia caused by inadequate renal erythropoietin production. erythropoietin infusions or other erythropoietin-stimulating agents manage anaemia in end-stage renal disease patients. the united states food and drug administration recommends an erythropoietin haemoglobin target range of 100 g/l – 120 g/l11 and expressly states that erythropoietin-stimulating agents should be used to increase haemoglobin only to the level necessary to avoid transfusion.11,12,13 in 2016, an expert committee advocated for including erythropoietin-stimulating agents in the world health organization model list of essential medicines to reduce the need for transfusions in patients with end-stage chronic kidney disease. erythropoietin-stimulating agents prevent transfusion-related risks, facility requirements, and risk management costs in the event of possible harm (infections, haemosiderosis).14 however, erythropoietin-stimulating agents treatments are out of reach for most patients in our context. therefore, rbc transfusion is used to manage crf-related anaemia and scd patients. meanwhile, our country faces poor pre-transfusion compatibility practices; abo and rhd matching is the only mandatory screening for rbc transfusions. no other blood group is considered, and no alloantibody screening or compatibility test is performed.15 given this context, high rbc alloantibodies frequency is expected among transfused patients; however, there is a paucity of data on this. thus, this study determined the frequency of anti-erythrocyte alloimmunisation and identified alloantibody specificities among crf multi-transfused patients in yalgado ouedraogo teaching hospital, ouagadougou, burkina faso. methods ethical considerations both the yalgado ouedraogo teaching hospital direction and the internal ethical committee of the national blood transfusion centre (authorisation no. 015/cnts/dg/cirs, 03/23/2018) approved the study. the nurses and the medical doctor in charge of the interview and other data collection obtained verbal informed consent. also, the data were password protected and accessible only by the first author. results were shared with staff and patients and used to influence patients’ future transfusions. study setting this study was conducted in the nephrology and haemodialysis unit of the teaching hospital yalgado ouedraogo of ouagadougou, burkina faso, where about 400 patients with chronic kidney failure undergo haemodialysis yearly. we conducted this cross-sectional study from january 2018 to december 2019 and included haemodialysis end-stage chronic kidney failure patients who had ever received rbc transfusions at least twice. socio-demographic information, clinical data, and medical history of each included patient were recorded on a standardised survey form during an in-person interview with the medical doctor responsible for the study or trained nurses. data collected include gender, age, date of the first transfusion, date of the last transfusion received, number of transfusions, the total number of blood units received since crf started, and number and type of adverse reactions related to transfusions reported. additionally, the number of pregnancies, live and still births, abortions, and anti-d injection use were also reported for female patients. five mililitres of blood was drawn from each patient into ethylenediaminetetraacetic acid tubes. the sample was centrifuged, and the obtained plasma was used for alloantibodies screening and identification. testing methods we used the indirect antiglobulin test method with the gel column agglutination card technique (invitrogel ahg, mtc invitro diagnostics ag, bensheim, germany). in this technique, the gel column contains an anti-human antibody that traps irregular antibodies present in a patient’s plasma and fixed on rbc. agglutinated rbcs are trapped in the gel column, making the agglutination easy to read.16,17 in the first step, a panel of three rbc reagents (invitrocell screen i-ii-ii, mtc invitro diagnostics ag, bensheim, germany), that targets antigens d, c, c, e, e, v, cw, k, k, kpa, kpb, jsa, jsb, fya, fyb, jka, jkb, lea, leb, p1, m, n, s, s, lua, lub and xg*a antibodies, were used for screening. samples positive for any antibody were further tested to identify antibody specificity using an 11 rbc panel (invitrocell ident 11, mtc invitro diagnostics ag, bensheim, germany) that targets the same antigens in the screening stage. an enzyme-treated rbc panel was not used. statistical analyses we used epi-infotm software (version 7.2.2.2, centers for disease control and prevention, atlanta, georgia, united states) for data analysis. the frequencies and percentages are given with a 95% confidence interval. we used the chi-square test to compare proportions, and differences were considered significant for p < 0.05. results baseline characteristics during the study period, 235 patients with crf were included, comprising 45.1% (106/235) female patients. the mean age was 41.9 (standard deviation 14.5 years; median 41 years; range 15–86 years). the mean number of received rbc units was 18 units ranging from two to 160 rbc, while the median number of received blood units per patient was 10 (interquartile range: 5–20). about 55.2% (128/232) had received more than 10 rbc units (table 1). table 1: social and demographic characteristics of patients with chronic renal failure, yalgado ouedraogo teaching hospital of ouagadougou, burkina faso, 2018. red blood cell alloimmunisation prevalence of the 235 patients included, 14 had alloantibodies, representing an overall positivity rate of 5.9%. four of the 14 patients (28.6%) had indeterminate antibody specificity. in 10 patients, 14 antibodies were identified: 5 anti-d, 5 anti-c, 1 anti-e, 1 anti-cw, 1 anti s, 1 anti-lea; four patients were positive for both anti-d and anti-c (table 2). table 2: characteristics of patients with red blood cell alloimmunisation, yalgado ouedraogo teaching hospital of ouagadougou, burkina faso, 2018.† most antibodies (12 of 14; 85.7%) were of the anti-rh blood group antigens, with anti-d and anti-c being the most prevalent, each accounting for 35.7% (table 3). table 3: specificity and frequency of alloantibodies found among multi-transfused patients of the yalgado ouedraogo teaching hospital of ouagadougou, burkina faso, 2018.† red blood cell alloimmunisation risk factors there were no differences in the mean age (43.3 vs 41.8 years, p = 0.21) and the mean number of blood units received (13.0 vs 15.7 rbc units, p = 0.36) between immunised and non-immunised patients. the alloimmunisation rate was higher in patients who had received more than 10 rbc units (7.4% vs 2.3%, p = 0.12), but this difference was statistically insignificant. there were no other factors associated with alloantibodies (table 4). table 4: factors associated with alloimmunisation in multi-transfused patients with chronic renal failure, yalgado ouedraogo teaching hospital, ouagadougou, burkina faso, 2018. discussion our study aimed at determining the frequency and the specificity of alloantibodies among the multi-transfused haemodialysis crf patients at the teaching hospital yalgado ouedraogo of ouagadougou (burkina faso). we found an alloimmunisation rate of 5.9% with antibodies mainly of the anti-rh blood group antigens specificity. this study overviews of rbc immunological risks among patients with chronic diseases who are lifelong blood transfusion patients. although there have been recent changes in the blood transfusion system in burkina faso, including the replacement of multiple hospital-based blood banks with a centralised system, standardisation and harmonisation of practices,6,18,19,20 improved blood collection and infectious disease screening, some improvements towards managing blood recipients are necessary. for example, compactibility screening is still limited to the abo and rhd antigens, contrary to obtainable standards in high-income countries, where rare groups, at least rh-kell major antigens, are screened for before transfusion. moreover, in burkina faso, alloantibody screening tests and laboratory compatibility tests using at least an indirect antibody test as recommended is not implemented: the patient’s plasma and a sample of the rbc units are tested for agglutination on a glass surface. this study was the first in the country to use the gel column card method, one of the current best methods for alloantibody screening. nevertheless, the study presents some limitations as complementary antibody identification techniques, such as enzyme-treated red cells reagents panels (papain, bromelain or other) or wide-range panels, were not used. the lack of complementary identification can explain the high rate (4 of 14; 28.7%) of alloantibody undetermined specificity (inconclusive antibody identification).21 the overall alloimmunisation rate of 5.9% among crf patients undergoing haemodialysis on our study is consistent with the findings of two systematic reviews and meta-analysis studies conducted by ngoma et al. in 2015 and boateng et al. in 2019. these studies reported an overall alloimmunisation rate of 6.95% and 7.5% in sub-saharan africa.22,23 our results are similar to those of kafando et al., who found an alloimmunisation rate of 4.2% among children transfused with rh-kell unmatched blood units.24 alloimmunisation rates in the same range were reported in uganda (6.1%), rwanda (6.4%), sudan (4.0%) and tanzania (4.1%). however, some reported higher rates: uganda in 2010 (10.2%), mali in 2013 (10.3%) and nigeria in 2015 (9.3%).22,25,26,27,28 these results reflect the poor immunological safety of blood transfusions in sub-saharan africa, where blood transfusion is performed based only on the blood donor and recipient abo and rhd antigens matching. alloimmunisation rates observed in our study and other studies from sub-saharan africa are lower than those observed in europe and north america when they only screened for abo and rhd. prevalences ranged from 18% to 76% in the united kingdom and united states.29,30,31,32,33,34 in france, the rate was about 30%.35,36 despite the mandatory donor and recipient rh-kell antigens matching before transfusion in these developed countries, alloimmunisation rates in those settings are higher than ours.34,37,38 this serves as a reminder that the risk of alloimmunisation is multiparametric, depending on the population’s subgrouping or prevalent diseases.30,36 also, high rates of alloimmunisation could be due to antigen discrepancies between transfused rbc concentrates collected from donors with european ancestry and scd recipients who are often of sub-saharan african descent.36,37 a similar hypothesis was assumed in some other countries with multi-ethnic groups, such as iran.39 in burkina faso, blood group antigen distribution is established for abo and rhd within blood donors and patients.40,41 there is no data about rh subgroups or other important rbc antigens. it is known that significant differences in the distribution of blood group antigens within the country’s natural ethnic groups could exist. sawadogo et al. found that the phenotype o was more frequent in the central-west, central and east regions corresponding to ‘mossi’, ‘gourounsi’, and ‘gourmantché’ areas, whereas the phenotype a and ab were more prevalent in ‘boucle du mouhoun’ and ‘hauts-bassins’ regions and the ‘bwaba’ and ‘bobo’ areas. the phenotype o negative was infrequent in ‘bwaba’.40 these studies suggest that in burkina faso, with more than 50 ethnic groups, dominant blood groups vary between or are specific to particular ethnic groups. thus, new studies should be conducted to establish blood subgroup frequencies and rbc matching strategies in the country. in our study, the antibody specificity of four participants of 14 (28.6%) was indeterminate. this impairment could be due to the discrepancy between the european-sourced red cell reagent panel and our population. this situation highlights the need to implement local panels for rbc alloantibodies testing as with some other lowand middle-income countries.42,43,44 furthermore, boateng et al. claim that creating and maintaining a database of phenotyped blood donors will facilitate the selection of matched blood components for emergency transfusions as seen in sub-saharan africa and help locally manufacture rbc reagents. thus, rbc alloantibodies screening may become more economical and sustainable for multi-transfused patients, particularly patients with scd in this zone.22 the majority (85.7%) of the alloantibodies found in this study were anti-rh group antigens. anti-d and anti-c antibodies accounted for 35.7%, followed by anti-e and anti-cw. in a previous study of children who received transfusions in burkina faso, anti-c and anti-e were the most frequent.24 in our study, the two mainly represented antibodies were co-associated (anti-d+c) in 4 of 14 patients. the predominance of rh group antibodies was also reported in some other west african countries, as well as in côte d’ivoire,45 mali,26 senegal46 and nigeria,27,47 but in these studies, anti-e was the most often encountered when compared to anti-d and anti-c. surprisingly, we found rh anti-d antibodies, which could be due to errors occurring during patients’ blood typing. our hospital has reported as many as 46 blood typing errors yearly (unpublished data). another hypothesis is that partial rhd antigen carriage is frequent in individuals with african ancestry. in this case, an rhd-positive patient can develop alloantibodies after receiving rhd-positive rbc, as reported by chou et al.30,48 the same hypothesis applies to partial c carriers.49 this study tried to identify the risk factors associated with alloimmunisation. neither gender, age, nor the number of blood units received was associated with alloimmunisation in our study. however, ifeoma et al.47 in nigeria, senghor et al.46 in senegal and natukunda et al.50 in uganda have associated these factors with alloimmunisation; the small size of our sample might have prevented the observation of such associations. limitations one limitation of this study was that we could not screen for antibodies within a reasonable time after each transfusion. for many patients, screening occurred months or years after the last transfusion event. this delay may have impacted the alloimmunisation rate. conclusion this study showed that rbc alloimmunisation is a reality in multi-transfused patients in burkina faso. therefore, exhaustive donor-patient blood matching beyond abo and rhd is necessary for lifelong transfused patients, such as crf and scd patients. further investigations are needed to efficiently establish the distribution of rbc antigens and phenotypes among blood donors and patients in the country, which may facilitate rbc reagent manufacturing. acknowledgements salfo kellé for patients interview, data and sample collection. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions k.n. designed the study, collected data, participated in samples testing, contributed to data analysis and drafted the manuscript. salam sawadogo, salifo sawadogo, j.k., j.b., h.y.a.l. and a.g.s. contributed to designing the study, data analysis and interpretation. m.k., s.d. and e.k. critically reviewed and revised the manuscript. all of the authors approved the final version of the manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability data are available from the corresponding author, k.n., upon request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references centre national de transfusion sanguine (cnts). annuaire statistiques de la transfusion sanguine 2017. ouagadougou: cnts/ministère de la santé, 2016; p. 128. report no.: n001. institut national de la statistique et de la démographie (insd). projections démographiques de 2007 à 2020, par région et province [homepage on the internet]. ouagadougou: institut national de la statistique et de la démographie, 2009 [cited 2019 july 23]; p. 69. available from: http://www.insd.bf/n/contenu/autres_publications/projections_demographiques_sous_nationales_2007-2020.pdf barro l, drew vj, poda gg, et al. blood transfusion in sub-saharan africa: understanding the missing gap and responding to present and future challenges. vox sang. 2018;113(8):726–736. https://doi.org/10.1111/vox.12705 yooda ap, sawadogo s, soubeiga st, et al. residual risk of hiv, hcv, and hbv transmission by blood transfusion between 2015 and 2017 at the regional blood transfusion center of ouagadougou, burkina faso. j blood med. 2019;10:53–58. https://doi.org/10.2147/jbm.s189079 yooda ap, soubeiga st, nebie ky, et al. impact of multiplex pcr in reducing the risk of residual transfusion-transmitted human immunodeficiency and hepatitis b and c viruses in burkina faso. mediterr j hematol infect dis. 2018;10(1):e2018041. https://doi.org/10.4084/mjhid.2018.041 nébié k, ouattara s, sanou m, et al. poor procedures and quality control among nonaffiliated blood centers in burkina faso: an argument for expanding the reach of the national blood transfusion center. transfusion. 2011;51(7 pt 2):1613–1618. https://doi.org/10.1111/j.1537-2995.2011.03222.x sawadogo s, nebie k, millogo t, et al. traceability of blood transfusions and reporting of adverse reactions in developing countries: a six-year postpilot phase experience in burkina faso. adv hematol. 2018;2018:1–9. https://doi.org/10.1155/2018/7938130 shander a. emerging risks and outcomes of blood transfusion in surgery. semin hematology. 2004;41:117–124. https://doi.org/10.1053/j.seminhematol.2003.11.023 yooda ap, nebie k, tranchot-diallo j, et al. evaluation of two serological screening kits for hepatitis c virus infection at the regional blood transfusion center of ouagadougou, burkina faso. adv infect dis. 2020;10(05):216–227. https://doi.org/10.4236/aid.2020.105019 atterbury c, wilkinson j. blood transfusion. nursing standard. 2000;14(34):47–52. https://doi.org/10.7748/ns2000.05.14.34.47.c2837 fishbane s, nissenson ar. the new fda label for erythropoietin treatment: how does it affect hemoglobin target? kidney int. 2007;72(7):806–813. https://doi.org/10.1038/sj.ki.5002401 shah hh, fishbane s. biosimilar erythropoiesis-stimulating agents in chronic kidney disease. adv chronic kidney dis. 2019;26(4):267–271. https://doi.org/10.1053/j.ackd.2019.04.007 nguyen tv, goldfarb ds. implications of a reduction in the hemoglobin target in erythropoiesis-stimulating agent-treated hemodialysis patients. nephron extra. 2011;1(1):212–216. https://doi.org/10.1159/000334228 banzi r, gerardi c. 2016–2017 application for erythropoietin-stimulating agents. geneva: world health organization; 2016. minstère de la santé. arrêté portant directives nationales de bonnes pratiques transfusionnelles. n2014-589/ms juin 4. ouagadougou: minstère de la santé; 2014; p. 89. eggington ja, bromilow im, duguid jkm. the use of pooled red cells and column techniques for routine red cell antibody detection. transfus med. 1996;6(4):345–349. https://doi.org/10.1111/j.1365-3148.1996.tb00094.x blomme s, de maertelaere e, verhoye e. a comparison of three column agglutination tests for red blood cell alloantibody identification. bmc res notes. 2020;13(1):129. https://doi.org/10.1186/s13104-020-04974-x dahourou h, tapko j-b, kienou k, nebie k, sanou m. recruitment of blood donors in burkina faso: how to avoid donations from family members? biologicals. 2010;38(1):39–42. https://doi.org/10.1016/j.biologicals.2009.10.017 dahourou h, tapko j-b, nebie y, et al. mise en place de l’hémovigilance en afrique subsaharienne. transfusion clinique et biologique. 2012;19(1):39–45. https://doi.org/10.1016/j.tracli.2011.11.001 world health organization. twenty-eigth world health assembly, geneva, 13–30 may 1975 wha28.72 utilisation and supply of human blood and blood products [homepage on the internet]. [cited 2021 apr 14]. available from: https://www.who.int/bloodsafety/en/wha28.72.pdf?ua=1 hill bc, hanna ca, adamski j, pham hp, marques mb, williams la. ficin-treated red cells help identify clinically significant alloantibodies masked as reactions of undetermined specificity in gel microtubes. lab med. 2017;48(1):24–28. https://doi.org/10.1093/labmed/lmw062 boateng la, ngoma am, bates i, schonewille h. red blood cell alloimmunization in transfused patients with sickle cell disease in sub-saharan africa: a systematic review and meta-analysis. transfus med rev. 2019;33(3):162–169. https://doi.org/10.1016/j.tmrv.2019.06.003 ngoma am, mutombo pb, ikeda k, nollet ke, natukunda b, ohto h. red blood cell alloimmunisation in transfused patients in sub-saharan africa: a systematic review and meta-analysis. transfus apher sci. 2016;54(2):296–302. https://doi.org/10.1016/j.transci.2015.10.017 kafando e, wandji nana lr, domo y, nebie y, obiri-yeboah d, simporé j. incompatible blood transfusion in children in burkina faso. open j hematol. 2017;6. https://doi.org/10.13055/ojhmt_8_1_1.170123 natukunda b, schonewille h, ndugwa c, brand a. red blood cell alloimmunisation in sickle cell disease patients in uganda. transfusion. 2010;50(1):20–25. https://doi.org/10.1111/j.1537-2995.2009.02435.x baby m, fongoro s, cissé m, et al. fréquence de l’allo-immunisation érythrocytaire chez les malades polytransfusés au centre hospitalo-universitaire du point g, bamako, mali. transfusion clinique et biologique. 2010;17(4):218–222. https://doi.org/10.1016/j.tracli.2010.06.026 ugwu n, awodu o, bazuaye g, okoye a. red cell alloimmunisation in multi-transfused patients with sickle cell anemia in benin city, nigeria. niger j clin pract. 2015;18(4):522–526. https://doi.org/10.4103/1119-3077.154204 ndahimana e, gothot a, gerard c, et al. risk of red blood cell alloimmunisation in rwanda: assessment of pretransfusion cross-match techniques used in district hospitals. east afr med j. 2013;90(4):124–129. chou st, liem ri, thompson aa. challenges of alloimmunisation in patients with haemoglobinopathies. br j haematol. 2012;159(4):394–404. https://doi.org/10.1111/bjh.12061 chou st, jackson t, vege s, smith-whitley k, friedman df, westhoff cm. high prevalence of red blood cell alloimmunisation in sickle cell disease despite transfusion from rh-matched minority donors. blood. 2013;122(6):1062–1071. https://doi.org/10.1182/blood-2013-03-490623 aygun b, padmanabhan s, paley c, chandrasekaran v. clinical significance of rbc alloantibodies and autoantibodies in sickle cell patients who received transfusions. transfusion. 2002;42(1):37–43. https://doi.org/10.1046/j.1537-2995.2002.00007.x vichinsky ep, earles a, johnson ra, hoag ms, williams a, lubin b. alloimmunization in sickle cell anemia and transfusion of racially unmatched blood. n engl j med. 1990;322(23):1617–1621. https://doi.org/10.1056/nejm199006073222301 badjie ksw, tauscher cd, van buskirk cm, et al. red blood cell phenotype matching for various ethnic groups. immunohematology. 2011;27(1):12–19. https://doi.org/10.21307/immunohematology-2019-169 zheng y, maitta rw. alloimmunisation rates of sickle cell disease patients in the united states differ from those in other geographical regions. transfus med. 2016;26(3):225–230. https://doi.org/10.1111/tme.12314 norol f, nadjahi j, bachir d, et al. transfusion et alloimmunisation chez les patients drépanocytaires. transfusion clinique et biologique. 1994;1(1):27–34. https://doi.org/10.1016/s1246-7820(05)80054-0 noizat-pirenne f. relevance of blood groups in transfusion of sickle cell disease patients. comptes rendus – biologies. 2013;336(3):152–158. https://doi.org/10.1016/j.crvi.2012.09.011 meunier n, rodet m, bonin p, et al. étude d’une cohorte de 206 patients drépanocytaires adultes transfusés: immunisation, risque transfusionnel et ressources en concentrés globulaires. transfusion clinique et biologique. 2008;15(6):377–382. https://doi.org/10.1016/j.tracli.2008.10.002 allali s, peyrard t, amiranoff d, et al. prevalence and risk factors for red blood cell alloimmunisation in 175 children with sickle cell disease in a french university hospital reference centre. br j haematol. 2017;177(4):641–647. https://doi.org/10.1111/bjh.14609 sarihi r, amirizadeh n, oodi a, azarkeivan a. distribution of red blood cell alloantibodies among transfusion-dependent β-thalassemia patients in different population of iran: effect of ethnicity. hemoglobin. 2020;44(1):31–36. https://doi.org/10.1080/03630269.2019.1709205 sawadogo s, nebie k, millogo t, et al. distribution of abo and rhd blood group antigens in blood donors in burkina faso. int j immunogenet. 2019;46(1):1–6. https://doi.org/10.1111/iji.12408 kouloudiati j, miningou m, sawadogo s, et al. prévalence des groupes sanguins érythrocytaires des systèmes abo et rhésus d au laboratoire du centre médical du camp général aboubacar sangoulé lamizana de ouagadougou (burkina faso). médecine d’afrique noire. 2020;67(4):175–182. salamat n, bhatti fa, yaqub m, hafeez m, hussain a, ziaullah null. indigenous development of antibody screening cell panels at armed forces institute of transfusion (afit). j pak med assoc. 2005;55(10):439–443. sawierucha j, posset m, hähnel v, johnson cl, hutchinson ja, ahrens n. comparison of two column agglutination tests for red blood cell antibody testing. plos one. 2018;13(12):e0210099. https://doi.org/10.1371/journal.pone.0210099 yu y, ma c, sun x, et al. frequencies of red blood cell major blood group antigens and phenotypes in the chinese han population from mainland china. int j immunogenet. 2016;43(4):226–235. https://doi.org/10.1111/iji.12277 sekongo ym, kouacou ap, kouamenan s, et al. allo-immunisation anti-érythrocytaire chez les drépanocytaires suivis dans l’unité de thérapeutique transfusionnelle du centre national de transfusion sanguine de côte d’ivoire. transfusion clinique et biologique. 2015;22(4):244–245. https://doi.org/10.1016/j.tracli.2015.06.098 senghor ab, seck m, faye bf, et al. séroprévalence virale et allo-immunisation post-transfusionnelle chez les patients suivis pour syndrome drépanocytaire majeur. transfusion clinique et biologique. 2017;24(3):355. https://doi.org/10.1016/j.tracli.2017.06.234 obi ei, pughikumo co, oko-jaja ri. red blood cell alloimmunisation in multi-transfused patients with chronic kidney disease in port harcourt, south-south nigeria. afr health sci. 2018;18(4):979. https://doi.org/10.4314/ahs.v18i4.18 ipe ts, wilkes jj, hartung hd, westhoff cm, chou st, friedman df. severe hemolytic transfusion reaction due to anti-d in a d+ patient with sickle cell disease. j pediatr hematol oncol. 2015;37(2):e135–e137. https://doi.org/10.1097/mph.0000000000000241 tournamille c, meunier-costes n, costes b, et al. partial c antigen in sickle cell disease patients: clinical relevance and prevention of alloimmunisation. transfusion. 2010;50(1):13–19. https://doi.org/10.1111/j.1537-2995.2009.02382.x natukunda b, mugyenyi g, brand a, schonewille h. maternal red blood cell alloimmunisation in south western uganda. transfus med. 2011;21(4):262–266. https://doi.org/10.1111/j.1365-3148.2011.01073.x abstract introduction methods results discussion acknowledgements references about the author(s) naseem cassim department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa national priority programme, national health laboratory service, johannesburg, south africa lindi m. coetzee department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa national priority programme, national health laboratory service, johannesburg, south africa abel l. makuraj national priority programme, national health laboratory service, johannesburg, south africa wendy s. stevens department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa national priority programme, national health laboratory service, johannesburg, south africa deborah k. glencross department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa national priority programme, national health laboratory service, johannesburg, south africa citation cassim n, coetzee lm, makuraj al, stevens ws, glencross dk. establishing the cost of xpert mtb/rif mobile testing in high-burden peri-mining communities in south africa. afr j lab med. 2021;10(1), a1229 https://doi.org/10.4102/ajlm.v10i1.1229 original research establishing the cost of xpert mtb/rif mobile testing in high-burden peri-mining communities in south africa naseem cassim, lindi m. coetzee, abel l. makuraj, wendy s. stevens, deborah k. glencross received: 16 sept. 2020; accepted: 16 july 2021; published: 30 nov. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: globally, tuberculosis remains a major cause of mortality, with an estimated 1.3 million deaths per annum. the xpert mtb/rif assay is used as the initial diagnostic test in the tuberculosis diagnostic algorithm. to extend the national tuberculosis testing programme in south africa, mobile units fitted with the genexpert equipment were introduced to high-burden peri-mining communities. objective: this study sought to assess the cost of mobile testing compared to traditional laboratory-based testing in a peri-mining community setting. methods: actual cost data for mobile and laboratory-based xpert mtb/rif testing from 2018 were analysed using a bottom-up ingredients-based approach to establish the annual equivalent cost and the cost per result. historical cost data were obtained from supplier quotations and the local enterprise resource planning system. costs were obtained in rand and reported in united states dollars (usd). results: the mobile units performed 4866 tests with an overall cost per result of $49.16. staffing accounted for 30.7% of this cost, while reagents and laboratory equipment accounted for 20.7% and 20.8%. the cost per result of traditional laboratory-based testing was $15.44 us dollars (usd). the cost for identifying a tuberculosis-positive result using mobile testing was $439.58 usd per case, compared to $164.95 usd with laboratory-based testing. conclusion: mobile testing is substantially more expensive than traditional laboratory services but offers benefits for rapid tuberculosis case detection and same-day antiretroviral therapy initiation. mobile tuberculosis testing should however be reserved for high-burden communities with limited access to laboratory testing where immediate intervention can benefit patient outcomes. keywords: genexpert test, tuberculosis screening, mobile testing, costing. introduction globally, tuberculosis is one of the top 10 causes of mortality.1 in 2017, tuberculosis infected about 10 million individuals and accounted for an estimated 1.3 million deaths among hiv-negative people, with an additional 300 000 deaths among people living with hiv.1 the epidemiology of tuberculosis varies widely between countries. in 2017, the tuberculosis incidence in most high-income countries was under 10 tuberculosis cases per 100 000 population compared to between 150 and 400 tuberculosis cases per 100 000 population in most of the top 30 high-burden countries.1,2 countries such as south africa (567), mozambique (551) and the philippines (554) reported over 500 cases per 100 000 population.1 as reported by the world health organization, there were 227 224 new cases of tuberculosis in south africa in 2017. although 322 000 cases of active tuberculosis were diagnosed in 2017 in south africa, only 65% of the cases were bacteriologically confirmed, with a treatment coverage of 68% (95% confidence interval [ci]: 51–96).3 clinically, a patient is suspected of having tuberculosis based on the following symptoms: persistent cough of 2 weeks or more, persistent cough of any duration for hiv-positive individuals, fever for over 2 weeks, night sweats, and unexplained weight loss (≥ 1.5 kg within 1 month).4 tuberculosis can present with different symptoms and atypical radiologic findings, and the pathological diagnosis has historically been based on acid-fast bacilli smear microscopy and bacteriological culture.5 the latter has a higher sensitivity for diagnosing and confirming active tuberculosis than acid-fast bacilli smear microscopy.5 the development of polymerase chain reaction tuberculosis assays has improved tuberculosis diagnosis and facilitates early treatment initiation by significantly reducing the time to result to 2 h, compared to 6 months for bacteriological culture.6 in south africa, the xpert mtb/rif polymerase chain reaction assay (cepheid, california, united states) is used routinely for tuberculosis diagnosis using patient sputum. test results, which determine the therapeutic intervention and management in line with the diagnostic algorithm, are returned within two days.7 tuberculosis incidence rates globally are especially high in the mining sector. in gold mines around the world, an estimated 3000 per 100 000 population are infected.8 in south africa, the mining sector accounted for 7.5% of the national gross domestic product, employing 495 592 workers in 2014.9 mining activities and environments are associated with a high risk of hiv and tuberculosis transmission and the migration of miners to their place of work is known to disrupt tuberculosis detection and care.10,11 given the higher rates of tuberculosis transmission in mines, it is anticipated that the communities where miners live, the so-called peri-mining communities, would also have higher tuberculosis incidence rates. due to the higher burden of disease among miners, a framework to address tuberculosis in the mining sector was developed for the southern african development community in 2014.11 in march 2015, a comprehensive tuberculosis campaign targeted at inmates in correctional services prisons, mine workers and peri-mining communities was launched in south africa under the banner ‘ending sa [south africa] tuberculosis epidemic: accelerating the response in key populations’.12 in response to this call and through the support of the global fund, the national health laboratory service and its clinical partner, the aurum institute, introduced a funded mobile genexpert testing facility to improve tuberculosis diagnosis in peri-mining communities.13 this initiative aimed to increase resources to deal with three of the world’s most devastating diseases (hiv and aids, tuberculosis and malaria) by focusing on the areas of greatest need.13 mobile testing was targeted at communities with a high burden of disease (high tuberculosis or hiv prevalence) and little or no access to laboratory testing facilities. these included remote areas of the north west and limpopo provinces in south africa between 2016 and 2019.13 the step-by-step approach to introducing mobile testing included identification of testing needs, execution of a feasibility study, procurement of funding, conducting of the necessary steps and processes to prepare for testing (setup of vehicles and equipment), assay verification, training, competency assessment, identification of measurable outcomes for monitoring, and commencement of testing. various studies have demonstrated that mobile testing is feasible, improves access to diagnostics, and may improve linkage to care and decrease time to treatment.14,15,16,17 a local study has reported that linkage to tuberculosis treatment was not associated with either sex or service type (mobile versus stand-alone), but older patients were less likely to be linked to tuberculosis treatment.15 mobile testing for hiv, tuberculosis and, more recently, severe acute respiratory syndrome coronavirus 2 can bring diagnostics to where it is needed in high-burden or outbreak communities.18 as previously reported in a local study to evaluate mobile versus traditional laboratory cd4 testing, mobile diagnostics could be substantially more expensive.19 mobile testing is not widely used in south africa, with its use limited to pilot projects or funded studies. however, it should be possible to integrate mobile testing as part of a national tiered laboratory network to extend services20 and absorb the higher cost of mobile testing into the national laboratory expenditure allocations. there is limited local data on the cost to provide mobile xpert mtb/rif testing in high-burden communities. only one local study reported that the cost to detect one tuberculosis case was $1117.00 united states dollars (usd)based on 1385 patients enrolled.16 the paucity of local data for mobile tuberculosis testing highlights the need for a comprehensive costing study, which could inform the modalities of providing these services and identify scenarios that are best suited for on-site testing. the objective of this study was to determine the cost per result and cost per positive result of mobile xpert mtb/rif testing and to compare it to the cost of traditional laboratory-based testing. methods ethical considerations ethics clearance was obtained from the university of the witwatersrand (reference number: m160978). our study did not contain any patient identifiers. no patient consent was required. context the national health laboratory service implemented mobile testing in three high-tuberculosis-burden districts in south africa (kenneth kaunda, north west, waterberg, limpopo, and sekhukhune, limpopo). traditional laboratory-based testing was conducted at the potchefstroom laboratory, a clinical pathology district laboratory offering a basic repertoire of testing, including tuberculosis testing, in the kenneth kaunda district. costing methodology the costing analysis was undertaken using microsoft excel (redmond, washington, united states).21 a bottom-up costing approach was used to determine the cost per result from a provider perspective; all costs are reported for the national health laboratory service as the provider of mobile tuberculosis testing. all costs (excluding value-added tax) were obtained in south african rand and reported in united states dollars, with an exchange rate of r14.4838 south african rand (zar) to the dollar.22 the main outcome of interest was the cost per result. the ingredients-based costing approach established annual equivalent costs (aec) for the following categories of mobile testing: staff (medical technologist and driver), reagents, external quality assurance, vehicle purchase, vehicle operations, laboratory equipment, and coordinator costs to manage testing. for the costing of the traditional laboratory-based xpert mtb/rif testing, we reported the following cost categories: staff (medical technologist), reagents, external quality assurance, laboratory equipment, courier logistics, and coordinator costs to manage testing. all laboratory equipment was purchased outright. for traditional laboratory testing, a placement agreement includes the costs for regular maintenance and servicing of the analyser. all data are reported for the 2018 calendar year. the consolidated health economic evaluation reporting standards checklist was used in the preparation of the manuscript.23 for laboratory equipment costing, useful life, which refers to the projected lifespan of depreciable equipment, was set at seven years, with a discount rate of 4%. for the calculation of staff costs, we determined the full-time equivalent hours (the number of hours worked by an employee divided by the number of hours worked by a full-time employee) based on the amount of time employees were assigned to mobile testing and multiplied this by the annual cost to company salary scales to determine the aec. reagent and test consumable costs were obtained from quotations received from the oracle enterprise resource planning system used by the national health laboratory service, and the aec was determined using annual test volumes.24 for external quality assurance, the frequency of panel testing and the number of samples prepared were used to calculate the aec per site, that is, panels were sent out quarterly, with three samples per instrument. the aecs for vehicle purchase, vehicle operations, laboratory equipment and the coordinator costs were also determined and are described in more detail below. start-up costs were defined as all aecs associated with the purchase of the mobile vehicle and laboratory equipment. the total cost per result minus the contribution of start-up costs was also determined. we reported the cost per positive result (the cost to find one tuberculosis-positive case) for both mobile and laboratory tuberculosis testing. this was calculated as the aec divided by the number of tuberculosis-positive results. for mobile testing, it was also possible to use the clinical outcomes data to estimate the diagnostic cost per tuberculosis-positive patient, as well as the cost per patient initiated on treatment (calculated as aec divided by the number of people that received treatment). mobile xpert mtb/rif costing the costs for the initial start-up of the mobile service were determined and included the costs for the purchase of the vehicles, modifications made to the mobile units (benches, air conditioning), and purchase and placement of equipment on the mobile units. the mobile units were equipped with genexpert platform instruments (cepheid, sunnyvale, california, united states). this is an automated real-time polymerase chain reaction test for the simultaneous detection of tuberculosis and rifampicin resistance.25 four genexpert instruments, as well as one computer per analyser, were placed in each mobile unit for a combined daily testing capacity of 64 samples. operational vehicle costs were included in the cost per result and comprised maintenance, fuel, repairs, and annual licensing costs. additional operational costs included costs for procurement of reagents, consumables, specimen collection and quality control materials (internal and external schemes), as well as other miscellaneous costs such as for printing of results. each mobile testing unit required a driver and a medical technologist. the percentage of time spent offering mobile testing was used for full-time equivalent calculations, ranging from 40% to 80%. the cost to company salary for a coordinator was calculated using historical expenditure data. the aecs for travel, office setup, miscellaneous costs and coordinator costs were also determined (total aec divided by the number of mobile testing sites). the test volumes and number of positive results for each mobile unit were reported using bar charts, with the total cost per result presented as a line chart on the secondary y-axis. the cost per result without start-up costs and the cost per kilometre were also reported. the number of site visits and kilometres travelled were indicated as text on the charts. for the three mobile units, we reported the correlation between the cost per result and distance travelled. laboratory-based xpert mtb/rif comparative costing as a comparator, the cost per result was determined for traditional laboratory-based xpert mtb/rif testing. initial laboratory setup included the installation of the four genexpert systems (cepheid, sunnyvale, california, united states) (capacity of 64 samples per day), an air conditioner, a level two biosafety hood and a vortex mixer. operational costs included costs to procure reagents, consumables, specimen collection materials, quality control materials (internal and external), printer cartridges and paper. the assumptions for these operational costs were similar to those for mobile testing. the staff complement required to perform mobile testing included a medical technologist and a laboratory manager, who provided minimal supervision. the technologists performed other testing in addition to xpert mtb/rif. the costs of the business management unit (coordinator costs) in the north west province were determined and included the following personnel: business manager, secretary, quality assurance coordinator, human resources officers, training staff, and other support staff. to determine the coordinator costs per result, the aec was divided by the annual test volume for the province. for the courier costs, the annual expenditure for the laboratory was used. results the three mobile units performed 4866 tuberculosis tests, of which the majority were performed by mobile unit 1 (68.7%). the mobile units covered a total distance of 64 605 km, with mobile units 3 and 1 contributing 73.7% of all travel. a total of 258 healthcare facilities were visited, evenly distributed between the three units. there were 544 tuberculosis-positive samples reported, with an overall tuberculosis positivity of 11.2%. the tuberculosis positivity was 9.6% for mobile unit 1, 16.6% for mobile unit 2, and 10.7% for mobile unit 3. for the period reported, 11 603 tests were done at the potchefstroom laboratory, of which 1086 were positive (9.4%). mobile testing costs the overall cost per result for mobile testing was $49.16 usd with an aec of $239 130.00 usd (table 1). without the start-up costs, the overall cost per result decreased to $31.11 usd. a breakdown of cost contributors showed that staff accounted for 30.7%, primarily due to the cost per result ($11.69 usd; 23.8%) of the medical technologist performing the test. reagents accounted for 20.7% ($10.16 usd), while vehicle operation costs made up 3.6% ($1.76 usd) of the overall cost per result. specimen collection and external quality control only contributed 0.5% ($0.27 usd) to the final cost per result. the aec for reagents, staffing and laboratory equipment made up 72.2% of the total cost. the start-up costs, which comprised the costs to purchase the mobile vehicle and laboratory equipment, accounted for 36.7% ($87 804.00 usd) of the total cost of mobile testing. these initial costs need to be considered when mobile units are rolled out without links to an established laboratory network or testing programme. the cost per result for the three mobile units ranged from $30.22 usd to $154.31 usd. without the start-up costs, the cost per result ranged from $21.47 usd to $95.06 usd (figure 1). figure 1: number of tuberculosis tests performed (dark blue bars) by mobile xpert mtb/rif testing units in high-burden peri-mining communities in south africa, 2018. positive results (red bars) are reported on the primary y-axis. on the secondary y-axis, the green line indicates the total cost per result in usd, the purple line indicates the total cost per result less start-up costs, and the orange line indicates the cost per kilometre travelled. the number of site visits for testing and the total distance travelled for those visits are indicated as text for each mobile unit. table 1: comparison of cost per result between mobile xpert mtb/rif testing in high-burden peri-mining communities and traditional laboratory-based xpert mtb/rif testing offered at a laboratory in the kenneth kaunda district in south africa, 2018. effect of distance travelled on the cost per result the three mobile units covered distances of 21 766 km, 16 985 km and 25 854 km. the estimated overall cost per kilometre was $2.34 usd, with mobile unit 2 accounting for the highest cost per kilometre ($8.91 usd). the number of health clinics visited by the mobile units ranged from 79 to 90 clinics. the correlation between the cost per result and distance travelled was not statistically significant (p = 0.053), with a perfect negative correlation reported (−1.0000). cost per positive tuberculosis result the aec for offering mobile testing was $239 130.78 usd to produce 4866 results. there were 544 positive results (11.2%), with 300 patients documented as having received tuberculosis treatment (55.1%). the cost to find one positive tuberculosis case using mobile testing was $439.58 usd and the cost of initiating a positive patient on treatment was $797.10 usd (table 1). comparative costing analysis the overall cost per result for laboratory-based xpert mtb/rif testing was $15.44 usd (table 1). equipment for laboratory testing is procured through a national tender process, that is, there are no costs for installation and maintenance of adequate testing platforms. reagent costs were similar to that of mobile testing and accounted for 65.8% of the total cost per result. staff costs contributed $1.62 usd (10.5%) to the cost per result. for specimen collection materials, the cost per result was $0.34 usd (2.2%); for test consumables, the cost was $1.61 usd (10.4%); for external quality assurance, the cost was $0.02 usd (0.1%); for laboratory equipment, the cost was $1.37 usd (8.9%); for the coordinator, the cost was $0.06 usd (0.4%). the courier costs contributed $0.26 usd (1.7%) to the total cost per result. the aec for laboratory-based testing was $179 132.08 usd to produce 11 603 results. the cost to find one positive tuberculosis case was $164.95 usd. unfortunately, the number of patients with a laboratory test result who received tuberculosis treatment was not available. discussion mobile diagnostics for high burden diseases such as tuberculosis can provide significant public health and epidemiological value in regions where individuals do not have easy access to laboratory facilities. overall, the average cost per result for all three mobile units was $49.16 usd. however, the cost per result ranged from $30.22 usd to $154.31 usd, highlighting differences in how and where mobile testing was offered. the biggest contributors to cost differences were test volumes and distance travelled. for example, mobile unit 1 performed the most testing with short travel distances and reported the lowest cost per result. in contrast, mobile unit 3 served a very remote area with longer travel times and had the highest cost per result. staff, reagents, laboratory equipment and vehicle purchase contributed 88.1% of the total cost per result. this indicates that the majority of costs associated with mobile testing are not flexible, and suggests that the cost of mobile testing could only be reduced by increasing test volumes, reducing input costs or widening the test repertoire. test volumes could be increased by identifying clinical settings with higher test volumes that would maximise the use of mobile testing. test volumes are however limited by the daily throughput of the testing platform and space on the mobile units for multiple units of the test platforms. negotiations with suppliers could result in lower reagent and consumable pricing. in addition, by adding mobile testing to the existing traditional laboratory national tenders, the placement agreement for reagents and analysers could be extended to mobile testing. the higher test volumes would lower the unit costs of the traditional laboratory supply chain management agreements and, by extension, benefit mobile testing. various point-of-care platforms with a very small footprint could be used to offer additional routine haematology and chemical pathology mobile testing.26 these could be used to facilitate the fast-tracking of antiretroviral therapy for patients with tuberculosis and hiv.27 a wide range of tuberculosis positivity rates were reported for the three mobile units in this study. this highlights the importance of identifying high-burden settings with high tuberculosis prevalence for effective deployment of mobile testing. the reported cost to find a single tuberculosis-positive case would vary substantially based on the setting where testing is offered. offering mobile testing in high-burden areas with a large population would substantially reduce the overall diagnostic cost and simultaneously offer immediate access to treatment. the higher cost of mobile testing should be weighed against the impact of earlier diagnosis, improved coverage, same-day treatment and care, as well as reduced loss to follow-up.17,28,29,30,31 mobile testing as an extension of laboratory testing could also see its higher costs offset by high volume laboratory testing, as bulk testing is still reserved for the laboratory service. the findings of this study confirmed that mobile testing is 3.2 times more expensive than conventional laboratory testing on the same genexpert testing platform. some of the reasons for the higher cost per result for mobile testing include lower test volumes, lost time due to travel to the health facility, and the impact of the clinical workflow on sample collection. an earlier study conducted to determine the cost of providing mobile cd4 testing in pixley ka seme in the northern cape of south africa also reported a substantially higher cost for mobile testing versus laboratory testing.19 in such remote areas, the cost of mobile testing should be weighed against improving sample collection and distribution routes to the nearest testing laboratory. for mobile tuberculosis testing, scenarios should be identified that match the increased costs of mobile testing with improved patient outcomes such as rapid tuberculosis case identification and same-day antiretroviral therapy initiation. a clinical outcome study should be embedded within any future mobile testing to assess the impact on patient outcomes. similarly, detailed cost-effectiveness studies are needed to provide evidence of how mobile tuberculosis testing can save lives and fully realise the potential of targeting high-risk groups. limitations this study used actual costs from the 2018 calendar year that would be more accurate than a desktop exercise. however, some staffing estimates are based on the typical number of days of mobile testing and this could have underestimated the costs. more so, the costs reported are based on the clinical referral of patients for testing. in a different clinical scenario with higher patient volumes, the costs could be very different. there are several assumptions made for this costing analysis that could have affected the reported cost per result. the number of xpert platforms in each mobile unit, the level and type of staff employed (technologist versus technician), full-time equivalent assumptions, and the exclusion of some costs, such as overheads, would affect the reported cost per result. conclusion this study reported that mobile tuberculosis testing is more expensive than traditional laboratory testing. however, mobile testing holds the potential to offer rapid tuberculosis case detection and improve coverage and diagnostics in communities with a high burden of disease. furthermore, mobiles could be dovetailed to be used to deliver same-day antiretroviral therapy initiation. further cost-effectiveness studies are needed using the patient outcome data reported. acknowledgements the authors thank the staff that operated the mobile units. we also wish to thank the global fund for making this project possible and the aurum institute (clinical partner). competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions n.c. and l.m.c. designed the study, developed the methodology and conducted the research. n.c. conducted the costing analysis. a.l.m. provided the data required for the costing analysis. d.k.g. and w.s.s. provided editorial comments and technical input. d.k.g. supervised the study by providing leadership and oversight as the project leader. all authors reviewed the results and contributed to the manuscript development. sources of support no funding was obtained for this study. the global fund to fight aids, tuberculosis and malaria covered the cost of mobile testing in the peri-mining communities (zaf-c-ndoh [national department of health]). data availability the authors do not have permission to share the data used for this study. disclaimer the authors declare that the views expressed in the submitted article are our own and not the official position of any institution or funder. references world health organization (who). global tuberculosis report [homepage on the internet]. geneva: world health organization; 2018 [cited 2019 jul 29]. available from: https://www.who.int/tb/publications/global_report/en/ world health organization (who). use of high burden country lists for tb by who in the post-2015 era: summary [homepage on the internet]. geneva: world health organization; 2015 [cited 2019 jul 29]. available from: https://www.who.int/tb/publications/global_report/high_tb_burdencountrylists2016-2020.pdf world health organization (who). south africa: tuberculosis profile [homepage on the internet]. geneva: world health organization; 2017 [cited 2019 jul 29]. available from: https://extranet.who.int/sree/reports?op=replet&name=/who_hq_reports/g2/prod/ext/tbcountryprofile&iso2=za&outtype=pdf national department of health (ndoh). national tuberculosis management guidelines [homepage on the internet]. pretoria: national department of health; 2014 [cited 2019 aug 01]. available from: http://www.tbonline.info/media/uploads/documents/ntcp_adult_tb-guidelines-27.5.2014.pdf ryu yj. diagnosis of pulmonary tuberculosis: recent advances and diagnostic algorithms. tuberc respir dis (seoul). 2015;78(2):64–71. https://doi.org.10.4046/trd.2015.78.2.64 singer-leshinsky s. pulmonary tuberculosis: improving diagnosis and management. jaapa. 2016;29(2):20–25. https://doi.org.10.1097/01.jaa.0000476207.96819.a7 national department of health (ndoh). national consolidated guidelines for the prevention of mother-to-child transmission of hiv (pmtct) and the management of hiv in children, adolescents and adults [homepage on the internet]. pretoria, 2015 [cited 2019 jun 10]; p. 128. available from: http://www.health.gov.za/index.php/2014-03-17-09-09-38/policies-and-guidelines/category/230-2015p?download=937:national-art-guidelines-2015final chang st, chihota vn, fielding kl, et al. small contribution of gold mines to the ongoing tuberculosis epidemic in south africa: a modeling-based study. bmc med. 2018;16(1):52. https://doi.org.10.1186/s12916-018-1037-3 u.s. department of the interior, u.s. geological survey. 2014 minerals yearbook [homepage on the internet]. u.s. department of the interior u.s. geological survey; 2017 [cited 2019 aug 01]. available from: https://s3-us-west-2.amazonaws.com/prd-wret/assets/palladium/production/mineral-pubs/country/2014/myb3-2014-sf.pdf stuckler d, basu s, mckee m, lurie m. mining and risk of tuberculosis in sub-saharan africa. am j public health. 2011;101(3):524–530. https://doi.org.10.2105/ajph.2009.175646 churchyard gj, mametja ld, mvusi l, et al. tuberculosis control in south africa: successes, challenges and recommendations. s afr med j. 2014;104(3):244–248. https://doi.org.10.7196/samj.7689 south african government. ending south africa’s tb epidemic: accelerating our response in key populations [homepage on the internet]. pretoria: south african government; 2015 [cited 2019 aug 01]. available from: https://www.gov.za/world-tb-day-2015 national department of health (ndoh). global fund grants operations manual for national department of health [homepage on the internet]. pretoria: national department of health; 2016 [cited 2019 aug 01]. available from: https://www.theglobalfund.org/media/6586/oig_gf-oig-17-014_report_en.pdf sloot r, glenshaw mt, van niekerk m, meehan s-a. rapid point-of-care cd4 testing at mobile units and linkage to hiv care: an evaluation of community-based mobile hiv testing services in south africa. bmc public health. 2020;20(1):528. https://doi.org.10.1186/s12889-020-08643-3 meehan s-a, sloot r, draper hr, naidoo p, burger r, beyers n. factors associated with linkage to hiv care and tb treatment at community-based hiv testing services in cape town, south africa. plos one. 2018;13(4):e0195208. https://doi.org.10.1371/journal.pone.0195208 kranzer k, lawn sd, meyer-rath g, et al. feasibility, yield, and cost of active tuberculosis case finding linked to a mobile hiv service in cape town, south africa: a cross-sectional study. plos med. 2012;9(8):e1001281. https://doi.org.10.1371/journal.pmed.1001281 bassett iv, forman ls, govere s, et al. test and treat tb: a pilot trial of genexpert mtb/rif screening on a mobile hiv testing unit in south africa. bmc infect dis. 2019;19(1):110. https://doi.org.10.1186/s12879-019-3738-4 abdool karim ss. the south african response to the pandemic. n engl j med. 2020;382(24):e95. https://doi.org.10.1056/nejmc2014960 coetzee lm, cassim n, glencross dk. a cost analyses of mobile laboratory cd4 testing in a national health insurance (nhi) pilot site [homepage on the internet]. cape town: african society for laboratory medicine; 2012 [cited 2020 dec 12]. available from: https://www.researchgate.net/publication/267651724_a_cost_analyses_of_mobile_laboratory_cd4_testing_in_a_national_health_insurance_nhi_pilot_site glencross dk, coetzee lm, cassim n. an integrated tiered service delivery model (itsdm) based on local cd4 testing demands can improve turn-around times and save costs whilst ensuring accessible and scalable cd4 services across a national programme. plos one. 2014;9(12):e114727. https://doi.org.10.1371/journal.pone.0114727 microsoft corporation. office 365. redmond, wa: microsoft corporation, 2019. standard bank of south africa (sbsa). forex closing indication rates for 24 april 2019 as at 16:08 [homepage on the internet]. johannesburg: standard bank of south africa; 2019 [cited 2019 apr 24]. available from: https://ws15.standardbank.co.za/finsnapshot/getforexservlet husereau d, drummond m, petrou s, et al. consolidated health economic evaluation reporting standards (cheers) statement. int j technol assess health care. 2013;29(2):117–122. https://doi.org.10.1017/s0266462313000160 national health laboratory service (nhls). annual report 2017/18 [homepage on the internet]. johannesburg: national health laboratory service; 2018 [cited 2019 oct 08]. available from: https://nationalgovernment.co.za/entity_annual/1714/2018-national-health-laboratory-service-(nhls)-annual-report.pdf schnippel k, meyer-rath g, long l, et al. scaling up xpert mtb/rif technology: the costs of laboratoryvs. clinic-based roll-out in south africa. trop med int health. 2012;17(9):1142–1151. https://doi.org.10.1111/j.1365-3156.2012.03028.x martin cl. i-stat – combining chemistry and haematology in poct. clin biochem rev. 2010;31(3):81–84. cassim n, coetzee lm, stevens ws, glencross dk. addressing antiretroviral therapy-related diagnostic coverage gaps across south africa using a programmatic approach. afr j lab med. 2018;7(1):681. https://doi.org.10.4102/ajlm.v7i1.681 govindasamy d, van schaik n, kranzer k, wood r, mathews c, bekker lg. linkage to hiv care from a mobile testing unit in south africa by different cd4 count strata. j acquir immune defic syndr. 2011;58(3):344–352. https://doi.org.10.1097/qai.0b013e31822e0c4c grolla a, jones s, kobinger g, et al. flexibility of mobile laboratory unit in support of patient management during the 2007 ebola-zaire outbreak in the democratic republic of congo. zoonoses public health. 2012;59 suppl 2:151–157. https://doi.org.10.1111/j.1863-2378.2012.01477.x larson ba, schnippel k, ndibongo b, et al. rapid point-of-care cd4 testing at mobile hiv testing sites to increase linkage to care: an evaluation of a pilot program in south africa. j acquir immune defic syndr. 2012;61(2):e13–e17. https://doi.org.10.1097/qai.0b013e31825eec60 van schaik n, kranzer k, wood r, bekker lg. earlier hiv diagnosis – are mobile services the answer? s afr med j. 2010;100(10):671–674. https://doi.org.10.7196/samj.4162 abstract introduction methods results discussion acknowledgements references about the author(s) arielle g. hernandez division of hematology, cincinnati children’s hospital medical center, cincinnati, ohio, united states global health center, cincinnati children’s hospital medical center, cincinnati, ohio, united states department of epidemiology, human genetics and environmental sciences, school of public health, university of texas health science center, houston, texas, united states charles kiyaga central public health laboratories, ministry of health, kampala, uganda thad a. howard division of hematology, cincinnati children’s hospital medical center, cincinnati, ohio, united states global health center, cincinnati children’s hospital medical center, cincinnati, ohio, united states isaac ssewanyana central public health laboratories, ministry of health, kampala, uganda grace ndeezi department of paediatrics and child health, makerere university college of health sciences, kampala, uganda jane r. aceng ministry of health, kampala, uganda russell e. ware division of hematology, cincinnati children’s hospital medical center, cincinnati, ohio, united states global health center, cincinnati children’s hospital medical center, cincinnati, ohio, united states department of pediatrics, university of cincinnati college of medicine, cincinnati, ohio, united states citation hernandez ag, kiyaga c, howard ta, et al. operational analysis of the national sickle cell screening programme in the republic of uganda. afr j lab med. 2021;10(1), a1303. https://doi.org/10.4102/ajlm.v10i1.1303 original research operational analysis of the national sickle cell screening programme in the republic of uganda arielle g. hernandez, charles kiyaga, thad a. howard, isaac ssewanyana, grace ndeezi, jane r. aceng, russell e. ware received: 16 june 2020; accepted: 13 apr. 2021; published: 12 aug. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: sickle cell anaemia is a common global life-threatening haematological disorder. most affected births occur in sub-saharan africa where children usually go undiagnosed and die early in life. uganda’s national sickle cell screening programme was developed in response to a 2014 sickle cell surveillance study that documented a high disease prevalence. objective: this study describes the temporal and financial aspects of uganda’s 2014–2019 sickle cell screening programme. methods: national sickle cell screening data from uganda’s central public health laboratories were used to calculate turn-around times (tats) from sample collection to delivery, testing, and result reporting for blood samples collected from february 2014 to march 2019. the parameters affecting specific tats were assessed. the exact programme expenditures were analysed to determine cost per test and per positive sickle cell disease case detected. results: a total of 278 651 samples were analysed. the median tat from sample collection to laboratory receipt was 8 days (interquartile range [iqr]: 6–12), receipt to testing was 3 days (iqr: 1–7), and testing to result reporting was 6 days (iqr: 3–12). altogether, the sample continuum averaged 16 days (iqr: 11–24). lower level healthcare facilities were associated with longer sample delivery tats. calendar months (january and december) and larger sample volumes impacted testing and result reporting tats. the cost per test was $4.46 (united states dollars [usd]) and $483.74 usd per positive case detected. conclusion: uganda’s sickle cell screening programme is efficient and cost-effective. universal newborn screening is the best strategy for detecting sickle cell anaemia in uganda. keywords: uganda; sickle cell disease; newborn screening; turn-around time; cost-effectiveness. introduction sickle cell anaemia is a monogenic haematological disorder that manifests as a devastating systemic disease with high morbidity and early mortality. the pathophysiology begins in infancy with acute and life-threatening complications, which presents as increased susceptibility to infections, chronic haemolytic anaemia, and painful vaso-occlusive events.1,2 sickle cell anaemia is the most prevalent haemoglobinopathy that impacts more than 20 million people worldwide, with an estimated 312 302 babies born with the disease each year.3 over 75% of the world’s annual sickle cell anaemia births occur in sub-saharan africa where resources for providing early detection and care are most constrained,3 leading to the deaths of more than 500 affected children per day.4 the world health organization and other international groups have begun to recognise sickle cell anaemia as a major public health concern around the globe, but particularly in sub-saharan africa where there is a lack of government programmes to address this overwhelming and growing burden of disease.5,6,7,8 these organisations have identified the need to implement affordable evidence-based strategies that can be sustainably woven into existing healthcare systems, highlighting newborn screening as a priority intervention.6 evidence from both high-resource and limited-resource countries have shown that newborn screening for sickle cell anaemia can significantly decrease morbidity and mortality by enabling early initiation of penicillin prophylaxis and pneumococcal immunisations as primary prevention.9,10,11,12 however, the accurate diagnosis of sickle cell anaemia in limited-resource settings still faces major challenges related to high equipment and reagent costs, education and training of healthcare providers, and appropriate medical interventions. newborn sickle cell screening requires efficient laboratory methodologies and infrastructure for an early and accurate diagnosis to reduce preventable deaths among children born with sickle cell disease and to support the ultimate goal of universal screening of all babies across sub-saharan africa. the uganda sickle surveillance study (us3) commenced in 2014 using a simple but innovative approach to screen children for the sickle cell trait and disease on a national level.13 residual dried blood spot cards collected across the country as part of the early infant hiv detection programme were tested. isoelectric focusing (ief) was conducted on about 100 000 samples over a cross-section of one year and found an overall high prevalence of sickle cell trait at 13.1% and disease at 0.7%, but with a disproportionate distribution across the country. broader sickle cell screening has been initiated since 2015, strategically focused on the highest burden districts. to expedite early detection and facilitate linkage to care for affected infants, it is crucial to identify where delays in diagnosis occur. here, we describe a detailed operational analysis of the temporal and financial aspects of sickle cell screening in uganda. we documented the turn-around times (tats) for sickle cell screening, starting from sample collection to arrival, testing, and result reporting at the national centralised laboratory. to examine the cost implications of integrated sickle cell screening, we calculated the cost per test, based on exact expenditures from the us3, and made estimates of the cost-effectiveness of sickle cell screening in uganda. methods ethical considerations the initial us3 research proposal was approved by the school of medicine research ethics committee at makerere university (no. 2012-138) in kampala, uganda, on 14 august 2012, with continuous annual renewal through august 2021. approval from the uganda national council of science and technology was also completed. the proposal included an objective to conduct a cost-effectiveness analysis of the sample collection and testing process. as described in the us3 manuscript,13 the uganda ministry of health waived informed consent for all samples tested as part of us3. data integrity and patient privacy were assured through the use of de-identified specimen numbers maintained in a secured database. integrated screening us3 was conducted from february 2014 to march 2015 to determine the prevalence of sickle cell trait and disease in uganda. a foundational goal of the study was building local sickle cell laboratory capacity within the ministry of health’s existing centralised laboratory infrastructure and determining the feasibility of the scale-up of newborn screening to the national level. at the central public health laboratories (cphl) in kampala, uganda, a partnership with cincinnati children’s hospital medical center led to the construction of a new sickle cell laboratory that was outfitted with ief equipment, and local personnel were recruited and trained on a standardised haemoglobin electrophoresis protocol. as previously described for the early infant hiv detection programme, blood was collected from hiv-exposed infants and young children across the country using standard dried blood spot cards.14 each health facility populated a dispatch form with demographic information and testing requests. samples were transferred to the cphl via the national sample transport system.14 in the us3, all samples requesting hiv testing were also queued for sickle cell testing; results were entered into the centralised database and disseminated back to health facilities and onward to caretakers. following the us3, the dispatch form included an option for sickle cell testing only. turn-around time analysis the cphl database collects laboratory data on all samples received and processed; data for this analysis were abstracted for children aged 0–24 months who underwent routine sickle cell screening over five years, from 01 february 2014 to 31 march 2019. inbound tat was defined as the total number of days between sample collection at health facilities and the result dispatch from the cphl back to healthcare providers. three phases were defined within inbound tat: (1) sample delivery phase tat is the time between sample collection and receipt at the cphl, (2) sample testing phase tat is the time between receipt at the cphl and date of testing, and (3) sample result reporting phase tat is the time between testing and result dispatch from the cphl. outbound tat, defined as the number of days between result dispatch and result receipt or subsequent action by the healthcare providers, was not assessed in this analysis because those data points are not currently collected by the cphl. median tats were calculated for inbound tat and its three phases: sample delivery, testing, and result reporting using stata statistical software (statacorp llc. 2019. release 16. college station, texas, united states). the tat calculation included weekends and holidays because the cphl operates 24 hours a day, 7 days a week, 365 days a year. summary statistics included medians, 25% and 75% interquartile ranges (iqr), and frequencies. programme year 1 was the period of us3, followed by years 2 to 5 for every 12 months through march 2019. there were two distinct cohorts for sickle cell screening, based on how the sickle cell testing service was requested. the sickle/hiv co-testing cohort included all samples in the database that had both a sickle cell result and an hiv result. the sickle specific testing cohort included all samples that had a sickle cell result only.15 phase-specific parameters were determined for sample delivery, testing, and result reporting tats to assess different parts of the inbound sample continuum. for the sample delivery phase tat, parameters included geographical region, health facility level, programme year, collection month, and testing cohort. for the sample testing phase tat, parameters were programme year, testing month, and testing cohort. for the sample result reporting phase tat, parameters were programme year, dispatch month, and testing cohort. analysis of collection month was conducted using only year 5 data (01 april 2018 to 31 march 2019) to better understand recent temporal trends in tat. although there is no numerical value for laboratory efficiency or success, tat is considered an indicator of laboratory performance, where the shorter the tat the more efficient a laboratory is in producing results promptly. cost analysis the cost analysis of sickle cell screening was conducted by using microsoft excel (microsoft, corp., redmond, washington, united states) included the cost per test by ief and cost per positive case detected. costs were assessed by detailed analysis of all us3 expenditures over 13 months (01 february 2014 to 31 march 2015), using actual procurement documents and vendor price sheets for ief equipment, reagents, and consumables, plus cphl labour costs, including salary and other employment costs. costs that were shared with other cphl programmes and the health facilities were not included, such as the dried blood spot kits, sample transportation, local healthcare provider salaries, and screening-related training. expenses incurred by cincinnati children’s hospital medical center for travel and initial training of cphl sickle cell laboratory personnel were also excluded. cost for equipment, reagents, and consumables was reported in united states dollars (usd) according to actual costs using out-of-country and in-country vendors. for labour costs, annual salaries were expressed in uganda shillings and converted to usd using the purchasing power parity exchange rate.16 the national sickle cell disease prevalence data for 2014–2019 and the calculated cost per test were used to generate the cost per sickle cell disease case detected by region within uganda. results sickle cell testing turn-around time between 01 february 2014, and 31 march 2019, a total of 324 356 samples were collected and tested at the cphl. samples were excluded from analysis if they were outside the study date range, beyond 24 months of age, or collected and tested outside of routine sickle cell screening. the number of samples included in the final analysis was 278 651. in the subsequent analysis, only samples with present and plausible dates for collection, receipt, testing, or result dispatch were included depending on the tat being calculated. accordingly, there were 265 766 samples included in the inbound tat calculation, 276 521 samples for sample delivery tat, 263 417 samples for sample testing tat, and 267 325 samples for sample result reporting tat. the overall median inbound tat, from sample collection to result dispatch, was 16 days (iqr: 11–24) (table 1). most of the samples (n = 228 684, ~86%) did not exceed an inbound tat of one month. programme year 1 had the largest sample volume (n = 90 872), followed by almost 70 000 samples in year 4. the south western region had the shortest median inbound tat of 13 days (iqr: 9–20), while the mid-western region had the longest tat of 18 days (iqr: 12–26). the sickle cell/hiv co-testing cohort had a slightly shorter inbound tat (15 days) than the sickle specific cohort (17 days). table 1: characteristics of sickle cell samples tested in uganda from 01 february 2014 to 31 march 2019. for the sample delivery phase, the median tat was 8 days (iqr: 6–12). parameters affecting sample delivery tat included the region of the requesting hospital and the level of healthcare provided (table 2). the kampala region, where the cphl is located, had the shortest median sample delivery time of 6 days (iqr: 4–9), while six regions had a median sample delivery time of 9 days (table 2). regional referral hospitals had a shorter median sample delivery tat of 7 days (iqr: 5–9) compared to the lower level health centres ii, iii, and iv. the collection month of december 2018 had the longest median tat of 10 days (iqr: 5–18) compared to other collection months. table 2: sample delivery phase turn-around time for sickle cell samples collected in uganda, by parameter, february 2014 to march 2019. once the sample arrived at the cphl, the median sample testing tat was 3 days (iqr: 1–7) and for sample result reporting tat, it was 6 days (iqr: 3–12). for sample testing phase tat, programme year 4 had the longest median tat of 7 days (iqr: 4–14) (table 3). median sample testing and sample result reporting tats were highest in the months of december 2018 and january 2019 (table 3 and table 4). the two testing cohorts had similar median sample testing and sample result reporting tats. table 3: sample testing phase turn-around time for sickle cell samples collected in uganda, by parameter, february 2014 to march 2019. table 4: sample result reporting phase turn-around time for sickle cell samples collected in uganda, by parameter, february 2014 to march 2019. sickle cell screening costs inputs included in the cost analyses were exact direct expenditures for equipment, reagents, consumables, and labour for 99 243 us3 samples tested by ief from a dried blood spot at the cphl over 13 months (table 5). the total equipment costs were annualised and calculated at $0.94 usd per test. reagent costs and consumables were calculated at $1.04 usd and $0.15 usd per test. the annual salary for laboratory personnel included gross pay, national social security fund 10% monthly tax, customary ‘13th month’ end-of-year compensation, and medical insurance per standard ministry of health employee contracts. labour costs were $2.33 usd per test, for an overall cost per test of $4.46 usd. personnel costs made up more than half of the cost per test total at 52%, followed by reagents (23%), equipment (21%), and consumables (3%). table 5: costs for sickle cell screening and testing for 99 243 children in uganda from the us3 project over 13 months, february 2014 to march 2015. in uganda, the highest burden of sickle cell disease is observed in the east central (1.6%) and mid-northern (1.5%) regions (table 6). using the calculated total cost per test and national prevalence data, the cost per sickle cell disease case detected was $483.74 usd (table 6). when stratified by region, however, the cost per positive case detected ranged widely from $278.07 usd in the east central region to $2607.19 usd in the south western region. table 6: cost per positive sickle cell disease case in uganda, by region from february 2014 to march 2019. discussion for any newborn screening programme, reduction of sample tat is an important intervention to ensure that poor health outcomes or preventable deaths are not the results of laboratory or operational delays. identification of the specific barriers related to longer tat and implementation of measures to mitigate those causes are essential to strengthen screening programmes. in this first detailed operational analysis of tat for sickle cell screening in uganda, the total duration between sample collection at the health facility to the dispatch of results from the cphl averaged 16 days (figure 1). multiple parameters affected this time interval, including the health facility level, programme year, collection month, and testing cohort. however, the vast majority (~86%) had results sent by cphl back to the local healthcare facilities within one month of receipt (table 1), which we propose as the maximal tat threshold before putting sickle cell disease patients at risk by delaying diagnosis and treatment. penicillin prophylaxis and pneumococcal immunisations are expected to be administered by three months of age, and these tat results are sufficient to meet those goals.9,10,11,12 figure 1: uganda central public health laboratories sample continuum and turn-around time phases, with results shown as the median in days (25% – 75% iqr) from february 2014 to march 2019. for the uganda sickle cell screening programme, sample delivery accounted for most of the inbound tat (figure 1). lower health facilities likely have greater barriers to timely delivery, such as delayed collection of their samples for transport (table 2). within each district, hub motorbike riders perform a weekly pickup from the more rural locations of health centres ii, iii, and iv in comparison to regional referral hospitals or district hospitals that experience daily pickup. it is also possible that samples are not readily available for transport at the time the hub motorbike riders come for collection, or some facilities are missed due to inclement weather or mechanical problems. to address these issues, a twice-weekly sample pickup with validation measures, such as timestamps, should be put into place. this should also be supplemented with ongoing training regarding sample preparation and the importance of timely sample transport for all personnel involved in the process. after sample receipt at the cphl, sample testing was relatively rapid with an average of only 3 days (figure 1). this phase involves the routing of samples for sickle cell testing to the laboratory, punching and eluting of dried blood spots, testing by ief by highly trained personnel, and entering of results into database. based on the short tat, this internal process can be deemed efficient. however, when looking at the parameter of the programme year, it does appear that longer tat in this phase is associated with periods of increased sample volumes (table 3). this may be the cause of reagent stock out or point to the need for more cross-training of cphl personnel on the ief procedure to support the sickle cell laboratory. as the programme expands toward the eventual goal of universal newborn screening, more human resources will be necessary to handle the increased number of samples. following sample testing, result reporting took an average of 6 days (figure 1). altogether, the time that samples spent at the cphl was prolonged. delays with the dispatch of results could be sample retesting or stock out of printing supplies. it was also observed that the testing and dispatch months of december 2018 and january 2019 had distinctly longer tats (table 4). this may be linked to personnel leave due to the holidays, thus increasing or staggering support in the sickle cell laboratory during this time could help avoid lengthy delays. in a previous study by kiyaga et al. at the uganda cphl regarding tat of early infant hiv detection screening, the average sample delivery tat was 12 days, sample processing was 2 days, and the overall average tat from sample collection to reporting to the patient was 26 days.14 as part of this study, short message service printers were piloted to allow test results to be transmitted immediately to healthcare facilities after testing, which further reduced the overall tat to 14 days.14 in the united states, the national newborn screening and global resource center reported newborn bloodspot screening tat to be within 10 to 14 days from sample collection, with considerable variation by state.17 to overcome these differences in state programmes, the united states department of health and human services advisory committee on heritable disorders in newborns and children recently set out updated newborn screening timeliness goals specifying that tests should be completed within 7 days of birth.18 in our study, we found that tat for sickle cell screening was shorter in all inbound phases compared to what was previously reported for uganda’s early infant hiv detection programme, and exhibited overall improved time efficiency for the entire cphl sample continuum (median 16 days vs 26 days). the current programme is approaching a comparable timeframe to that of the long-standing united states newborn sickle cell screening programme and will likely continue to evolve and improve over time. information on outbound tat, which includes time to family notification and matriculation to care, could not be examined in this study, because those data are currently not collected by the cphl. however, with greater visibility and a push toward newborn screening in sub-saharan african countries, steered by the new american society of hematology african screening consortium,19 outbound tat and individual patient follow-up data will be coveted information. we predict that tat will greatly impact healthcare provider and patient utilisation of sickle cell screening, as well as overall satisfaction with the programme in uganda. most importantly, prolonged tat very likely postpones life-saving treatments for patients with the disease, if the diagnosis is not established and communicated promptly. also, delayed result reporting can lead to affected infants being lost to follow-up and may require a replacement test. improving tat helps ensure that caretakers collect their results on time and minimises the need for inefficient and expensive repeat testing. ultimately, making improvements in all tat phases for sickle cell screening is essential to optimise infant health outcomes, cost-effectiveness, and screening satisfaction for healthcare providers and patients in uganda. a major strength of this analysis was the complete national representative long-term data from the cphls centralised database, which allowed us to investigate previously undocumented parameters that affect sickle cell testing tat in uganda. limitations include the access to only inbound tat data, which limits our ability to connect our findings to patient indicators such as notification and receipt of sickle cell results, followed by care and management of affected patients. another limitation is the lack of recommendations for sickle cell testing tat by the uganda ministry of health, other regional programmes, or by international bodies such as the world health organization for a comparison of efficiency. however, our data provided the first detailed time analysis of sickle cell screening in sub-saharan africa, to enable monitoring and evaluation of future interventions and other programmes. analysis of the direct costs of a sickle cell screening programme in uganda showed that the cost per test by ief using dried blood spots was $4.46 usd. in angola, the cost per infant screened in a newborn screening programme that used ief was $15.36 usd, or $7.42 usd when considering just the inputs of laboratory personnel, reagents, consumables, and equipment as in our study.20 the cost of the screening test for sickle cell disease performed by ief in the united states was $2.29 usd and approximately $4.50 usd (reported as £3.51) in the united kingdom.21,22 the united states and united kingdom have addressed the cost‐effectiveness of newborn screening for sickle cell disease for universal and targeted programmes. both nations have justified the greater cost incurred by screening every newborn for sickle cell disease, because it identifies all infants with the disease, prevents more deaths, and has demonstrated better outcomes for patients.21,22,23 in africa, modelling simulations have found universal newborn sickle cell screening to be extremely cost-effective, especially in countries with a high disease prevalence of 0.2% – 0.5%.24,25 yet, no federal newborn sickle cell screening currently exists in any country in sub-saharan africa, despite the well-evidenced economic and humanitarian basis for such programmes. in uganda, the estimated costs per sickle cell disease case detected among the 10 regions varied from $278.07 usd in the east central to nine times that in the south western region ($2607.19 usd) (table 6). these results show that newborn screening in regions with low sickle cell disease prevalence would result in a higher cost per positive case detected compared with screening focused in high-burden areas. however, because uganda is a country with a high overall disease prevalence of 0.9%,26 universal newborn screening would still be the most economical and impactful public health strategy for sickle cell disease across the entire country. limitations this analysis only considered costs and numbers of cases detected for partial cost-effectiveness analysis. more extensive studies with patient follow-up data will be vital to provide evidence that screening is effective in reducing sickle cell morbidity and mortality, the benefits and harms of screening, and the long-term cost-effectiveness of a newborn sickle cell screening programme in uganda. conclusion this study provides a contemporary and detailed description of the time and costs of sickle cell screening in uganda. analysis of the different phases of tat highlights areas for improvement to reduce the number of samples with excessive delays, and to strengthen the overall integrated cphl sample continuum. the lack of sickle cell testing guidelines limits our ability to compare these results to screening and care standards; however, this study documents that the cphl centralised database can be used to accurately monitor and manage tat for sickle cell screening and can identify factors affecting tat at different phases to prompt targeted improvements. this study also shows that sickle cell testing by ief is provided at under $5.00 usd, and the strategy of universal newborn screening is cost-effective to save and improve the lives of thousands of individuals with sickle cell disease in uganda. acknowledgements we are very thankful to the uganda ministry of health leadership and the central public health laboratories personnel for their contribution and dedication to the sickle cell screening programme. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.g.h., c.k., t.a.h., i.s., g.n., j.r.a. and r.e.w. substantially contributed to the design and implementation of the research, to the analysis of the results, and to the writing of the manuscript. sources of support this work was financially supported by the cincinnati children’s research foundation. data availability these data are not publicly available. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references ware re, de montalembert m, tshilolo l, abboud mr. sickle cell disease. lancet. 2017;390(10091):311–323. https://doi.org/10.1016/s0140-6736(17)30193-9 steinberg mh, rodgers gp. pathophysiology of sickle cell disease: role of cellular and genetic modifiers. semin hematol. 2001;38(4):299–306. https://doi.org/10.1053/shem.2001.27570 piel fb, patil ap, howes re, et al. global epidemiology of sickle haemoglobin in neonates: a contemporary geostatistical model-based map and population estimates. lancet. 2013;381(9861):142–151. https://doi.org/10.1016/s0140-6736(12)61229-x mcgann pt. time to invest in sickle cell anemia as a global health priority. pediatrics. 2016;137(6):e20160348. https://doi.org/10.1542/peds.2016-0348 united nations. general assembly resolution on recognition of sickle cell anaemia as a public health problem. a/res/63/237. geneva: united nations; 2008. world health organization. sickle-cell disease: a strategy for the who african region. report afr/rc60/8. geneva: who; 2010. world health organization. sickle-cell anaemia. report a59/9. geneva: who; 2006. african union. assembly decision on the proposal on sickle cell anaemia. assembly/au/dec.81 (v). addis ababa: african union. gaston mh, verter ji, woods g, et al. prophylaxis with oral penicillin in children with sickle cell anemia. a randomized trial. n engl j med. 1986;314(25):1593–1599. https://doi.org/10.1056/nejm198606193142501 quinn ct, rogers zr, mccavit tl, buchanan gr. improved survival of children and adolescents with sickle cell disease. blood. 2010;115(17):3447–3452. https://doi.org/10.1182/blood-2009-07-233700 ware re. is sickle cell anemia a neglected tropical disease? plos negl trop dis. 2013;7(5):e2120. https://doi.org/10.1371/journal.pntd.0002120 lee a, thomas p, cupidore l, serjeant b, serjeant g. improved survival in homozygous sickle cell disease: lessons from a cohort study. bmj. 1995;311(7020):1600–1602. https://doi.org/10.1136/bmj.311.7020.1600 ndeezi g, kiyaga c, hernandez ag, et al. burden of sickle cell trait and disease in the uganda sickle surveillance study (us3): a cross-sectional study. lancet glob health. 2016;4(3):e195–e200. https://doi.org/10.1016/s2214-109x(15)00288-0 kiyaga c, sendagire h, joseph e, et al. uganda’s new national laboratory sample transport system: a successful model for improving access to diagnostic services for early infant hiv diagnosis and other programs. plos one. 2013;8:e78609. https://doi.org/10.1371/journal.pone.0078609 hernandez ag, kiyaga c, howard ta, ssewanyana i, ndeezi g, aceng jr, ware re. trends in sickle cell trait and disease screening in the republic of uganda, 2014-2019. trop med int health. 2021 jan;26(1):23–32. https://doi.org/10.1111/tmi.13506. epub 2020 dec 14. pmid: 33151598. world bank official exchange rate [homepage on the internet]. [cited 2019 nov 01]. available from: http://data.worldbank.org/indicator/pa.nus.fcrf united states national newborn screening & global resource center [homepage on the internet]. [cited 2019 nov 01]. available from: https://genes-r-us.uthscsa.edu/parentfaq.htm united states health resources & services administration newborn screening timeliness goals [homepage on the internet]. [cited 2019 nov 01]. available from: https://www.hrsa.gov/advisory-committees/heritable-disorders/newborn-screening-timeliness.html american society of hematology sickle cell disease initiative [homepage on the internet]. [cited 2019 nov 01]. available from: https://www.hematology.org/advocacy/sickle-cell/ mcgann pt, grosse sd, santos b, et al. a cost-effectiveness analysis of a pilot neonatal screening program for sickle cell anemia in the republic of angola. j pediatr. 2015;167(6):1314–1319. https://doi.org/10.1016/j.jpeds.2015.08.068 panepinto ja, magid d, rewers mj, lane pa. universal versus targeted screening of infants for sickle cell disease: a cost-effectiveness analysis. j pediatr. 2000;136(2):201–208. cronin ek, normand c, henthorn js, hickman m, davies sc. costing model for neonatal screening and diagnosis of haemoglobinopathies. arch dis child fetal neonatal ed. 1998;79(3):f161–f167. grosse sd, olney rs, baily ma. the cost effectiveness of universal versus selective newborn screening for sickle cell disease in the us and the uk: a critique. appl health econ health policy. 2005;4(4):239–247. kuznik a, habib ag, munube d, lamorde m. newborn screening and prophylactic interventions for sickle cell disease in 47 countries in sub-saharan africa: a cost-effectiveness analysis. bmc health serv res. 2016;16:304. https://doi.org/10.1186/s12913-016-1572-6 davies sc, cronin e, gill m, greengross p, hickman m, normand c. screening for sickle cell disease and thalassaemia: a systematic review with supplementary research. health technol assess. 2000;4(3):i–v, 1–99. review. https://doi.org/10.3310/hta4030 kiyaga c, hernandez ag, ssewanyana i, et al. sickle cell screening in uganda: high burden, human immunodeficiency virus comorbidity, and genetic modifiers. pediatr blood cancer. 2019;66(8):e27807. https://doi.org/10.1002/pbc.27807 ajlm 8(1)_2019_contents.indd http://www.ajlmonline.org open access table of contents i original research morphological patterns of anaemia among pregnant women from sudan abuobieda b. abusharib african journal of laboratory medicine | vol 8, no 1 | a743 | 14 october 2019 original research haematological values in a healthy adult population in yaoundé, cameroon martin e. oloume, abas mouliom, bernard f. melingui, suzanne belinga, julie s. nana, mathurin tejiokem, francoise n. sack, jeanne manga, annie r. epote african journal of laboratory medicine | vol 8, no 1 | a852 | 31 october 2019 original research onsite healthcare worker acceptability and performance of the point-of-care pima cd4 assay in dar es salaam, tanzania mary e. schmitz, karen chang, nichole arnett, luciana kohatsu, ruth lemwayi, michael mwasekaga, john nkengasong, omotayo bolu, fausta mosha, larry westerman african journal of laboratory medicine | vol 8, no 1 | a740 | 21 november 2019 original research assessment of the aquios flow cytometer – an automated sample preparation system for cd4 lymphocyte panleucogating enumeration daniel rhodes, guislaine carcelain, mike keeney, christophe parizot, dominika benjamins, laurine genesta, jin zhang, justin rohrbach, denise lawrie, deborah k. glencross african journal of laboratory medicine | vol 8, no 1 | a804 | 05 december 2019 original research resazurin microtitre plate assay and sensititre® mycotb for detection of mycobacterium tuberculosis resistance in a high tuberculosis resistance setting prenika jaglal, melendhran pillay, koleka mlisana african journal of laboratory medicine | vol 8, no 1 | a840 | 13 december 2019 lessons from the field development and validation of a high performance liquid chromatography method to determine nevirapine in plasma in a resource-limited setting faithful makita-chingombe, andrew j. ocque, robin difrancesco, charles maponga, farai muzambi, tsitsi g. monera-penduka, tinashe mudzviti, takudzwa j. mtisi, gene d. morse african journal of laboratory medicine | vol 8, no 1 | a880 | 16 may 2019 lessons from the field strengthening laboratory capacity for detection of respiratory viral pathogens through the global health security agenda (ghsa) framework brett whitaker, karen a. alroy, erica guthrie, sarah schildecker, susan hiers, jill woodard, s. arunmozhi balajee african journal of laboratory medicine | vol 8, no 1 | a861 | 18 july 2019 48 55 61 69 79 88 95 page i of ii table of contents i editorial extending the breadth of african laboratory medicine iruka n. okeke african journal of laboratory medicine | vol 8, no 1 | a1128 | 05 december 2019 review article review of molecular subtyping methodologies used to investigate outbreaks due to multidrug-resistant enteric bacterial pathogens in sub-saharan africa anthony m. smith african journal of laboratory medicine | vol 8, no 1 | a760 | 14 march 2019 original research implementation and evaluation of the presto combined qualitative real-time assay for chlamydia trachomatis and neisseria gonorrhoeae in rwanda vicky cuylaerts, irith de baetselier, claude m. muvunyi, lambert mwambarange, hilde smet, john rusine, viateur musengamana, janneke van de wijgert, tania crucitti african journal of laboratory medicine | vol 8, no 1 | a739 | 18 april 2019 original research molecular detection of enterovirus d68 among children with acute respiratory tract infection in ghana joyce a. kubi, mohamed mutocheluh, joseph h.k. bonney, william k. ampofo, john k. odoom african journal of laboratory medicine | vol 8, no 1 | a732 | 26 june 2019 original research molecular confirmation and phylogeny of lassa fever virus in benin republic 2014–2016 olumuyiwa b. salu, ayorinde b. james, honoré s. bankolé, jijoho m. agbla, magloire da silva, fernand gbaguidi, christian f. loko, sunday a. omilabu african journal of laboratory medicine | vol 8, no 1 | a803 | 22 august 2019 original research organisational management of hospital blood transfusion services in nairobi county, kenya: evidence of implementation anne n. mbuthia, eunice m. mwangi, musa o. ong’ombe african journal of laboratory medicine | vol 8, no 1 | a676 | 26 august 2019 original research the use of real-time polymerase chain reaction and an adenosine deaminase assay for diagnosing pleural tuberculosis mulalo molaudzi, julitha molepo african journal of laboratory medicine | vol 8, no 1 | a731 | 28 august 2019 original research xpert® mtb/rif assay on formalin-fixed paraffin-embedded tissues in the diagnosis of extrapulmonary tuberculosis allan n. njau, samuel m. gakinya, shahin sayed, zahir moloo african journal of laboratory medicine | vol 8, no 1 | a748 | 18 september 2019 1 3 13 19 24 30 38 43 vol 8, no 1 (2019) issn: 2225-2002 (print) | issn: 2225-2010 (online)african journal of laboratory medicine http://www.ajlmonline.org open access table of contents ii lessons from the field blueprint for building a biorepository in a resource-limited setting that follows international best practices alash’le g. abimiku, talishea croxton, petronilla j. ozumba, ndidi agala, olasinbo balogun, emmanuel jonathan, enzenwa onyemata, kachimi ndifon, sunji nadoma, thankgod anazodo, sam peters, christine m. beiswanger african journal of laboratory medicine | vol 8, no 1 | a722 | 28 august 2019 lessons from the field medical laboratory accreditation in a resource-limited district health centre laboratory, addis ababa, ethiopia daniel m. desalegn, boja d. taddese, nebiyou yemanebrhane, mulye s. getahun, kumera t. kitila, tariku t. dinku, kassahun d. asferie, elizabeth a. wolde, gemechis b. tura, tilahun b. mersha, alemayhu w. rorissa, daniel d. wondimagegnehu, tinsae k. hailu, abrham t. bika african journal of laboratory medicine | vol 8, no 1 | a793 | 19 september 2019 lessons from the field tropical laboratory initiative: an innovative model for laboratory medicine in rural areas zelda r. moran, atta b. frimpong, pablo castañeda-casado, francis k. frimpong, manuela b. de lorenzo, yanis ben amor african journal of laboratory medicine | vol 8, no 1 | a922 | 26 september 2019 lessons from the field a comprehensive district-level laboratory intervention after the ebola epidemic in sierra leone annelies w. mesman, musa bangura, sahr m. kanawa, joseph s. gassimu, kerry l. dierberg, mohamed m. sheku, j. daniel orozco, regan h. marsh african journal of laboratory medicine | vol 8, no 1 | a885 | 22 october 2019 lessons from the field design and implementation of a clinical laboratory information system in a low-resource setting timothy m. mtonga, faheema e. choonara, jeremy u. espino, chimwemwe kachaje, kenneth kapundi, takondwa e. mengezi, soyapi l. mumba, gerald p. douglas african journal of laboratory medicine | vol 8, no 1 | a841 | 28 october 2019 case study coexistence of kaposi sarcoma and molluscum contagiosum on the same site in a hiv-aids patient: a very rare occurrence kabir abdullahi, yahaya mohammed, saddiku a. sahabi, mahmood m. dalhat african journal of laboratory medicine | vol 8, no 1 | a747 | 29 april 2019 case study late diagnosis of multidrug-resistant tuberculosis in a child at dr george mukhari academic hospital, ga-rankuwa, south africa: a case report bakani a. siwele, ndivhuho a. makhado, matodzi t. mariba african journal of laboratory medicine | vol 8, no 1 | a783 | 29 july 2019 99 111 116 123 130 137 140 case study atypical presentation of herpes simplex virus type 1 infection in paediatric burns patients in a large tertiary hospital, south africa mpho l. sikhosana, asma salloo, monica birkhead, kerrigan mccarthy african journal of laboratory medicine | vol 8, no 1 | a916 | 23 october 2019 brief report whole genome sequencing for drug resistance determination in mycobacterium tuberculosis shaheed v. omar, lavania joseph, halima m. said, farzana ismail, nabila ismail, thabisile l. gwala, nazir a. ismail african journal of laboratory medicine | vol 8, no 1 | a801 | 21 february 2019 brief report utilisation of fine needle aspiration cytology and biopsy in sokoto, nigeria: a five-year review kabir abdullahi, mohammed umar, saddiku m. sahabi african journal of laboratory medicine | vol 8, no 1 | a809 | 30 may 2019 brief report seroprevalence of hepatitis b virus co-infection among hiv-1-positive patients in north-central nigeria: the urgent need for surveillance terver m. akindigh, abba o. joseph, chrisitiana o. robert, ocheme j. okojokwu, juliet n. okechalu, aje j. anejo-okopi african journal of laboratory medicine | vol 8, no 1 | a622 | 27 june 2019 brief report potential role of lu/bcam in hiv-related atherosclerosis modisa s. motswaledi, ishmael kasvosve, oluwafemi o. oguntibeju african journal of laboratory medicine | vol 8, no 1 | a792 | 30 september 2019 brief report hyperuricaemia is associated with dyslipidemia but not hba1c among type 2 diabetes mellitus patients in botswana ellen gobusamang, naledi g. nyepetsi, modisa s. motswaledi, ishmael kasvosve african journal of laboratory medicine | vol 8, no 1 | a786 | 07 november 2019 correction corrigendum: antimicrobial resistance surveillance in ethiopia: implementation experiences and lessons learned rajiha a. ibrahim, amete m. teshale, surafel f. dinku, negga a. abera, abebe a. negeri, feven g. desta, eyasu t. seyum, adugna w. gemeda, wubshet m. keficho african journal of laboratory medicine | vol 8, no 1 | a1109 | 06 december 2019 reviewer acknowledgement african journal of laboratory medicine | vol 8, no 1 | a1136 | 12 december 2019 145 149 154 157 161 165 169 170 page ii of ii article information authors: christopher kariuki1 john m. kagira2 victor mwadime1 maina ngotho1 affiliations: 1institute of primate research, kenya 2jomo kenyatta university of agriculture and technology, kenya correspondence to: christopher kariuki email: chriskinya@gmail.com postal address: po box 24481, karen 00502, nairobi, kenya dates: received: 27 aug. 2013 accepted: 18 aug. 2015 published: 05 oct. 2015 how to cite this article: kariuki c, kagira jm, mwadime v, ngotho m. virulence and pathogenicity of three trypanosoma brucei rhodesiense stabilates in a swiss white mouse model. afr j lab med. 2015;4(1), art. #137, 8 pages. http://dx.doi.org/10.4102/ajlm.v4i1.137 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. virulence and pathogenicity of three trypanosoma brucei rhodesiense stabilates in a swiss white mouse model in this original research... open access • abstract • introduction • methods    • animals    • trypanosomes    • experimental design    • statistical analyses    • ethical considerations • results    • parasitaemia and gross clinical observations    • parasite virulence and mouse survival    • parasite morphology    • histopathological findings • discussion    • limitations of the study    • recommendations • conclusion • trustworthiness    • reliability and validity of the research • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: a key objective in basic research on human african trypanosomiasis (hat) is developing a cheap and reliable experimental model of the disease for use in pathogenesis and drug studies. objective: with a view to improving current models, a study was undertaken to characterise the virulence and pathogenicity of three trypanosoma brucei rhodesiense stabilates, labelled as international livestock research institute (ilri)-2918, ilri-3953, and institute of primate research (ipr)-001, infected into swiss white mice. methods: swiss white mice were infected intraperitoneally with trypanosomes and observed for parasitaemia using wet blood smears obtained by tail snipping. induction of late-stage disease was undertaken using diminazene aceturate (40 mg/kg, berenil) with curative treatment done using melarsoprol (3.6 mg/kg, arsobal). results: the prepatent period for the stabilates ranged from three to four days with mean peak parasitaemia ranging from log10 6.40 to 8.36. first peak parasitaemia for all stabilates varied between six and seven days post infection (dpi) followed by secondary latency in ilri-2918 (15–17 dpi) and ipr-001 (17–19 dpi). survival times ranged from six dpi (ilri-3953) to 86 dpi (ipr-001). hindleg paresis was observed in both ilri-3953 (at peak parasitaemia) and ilri-2918 (after relapse parasitaemia). mice infected with ipr-001 survived until 54 dpi when curative treatment was undertaken. conclusions: this study demonstrated that the stabilates ilri-2918 and ilri-3953 were unsuitable for modelling late-stage hat in mice. the stabilate ipr-001 demonstrated the potential to induce chronic trypanosomiasis in swiss white mice for use in development of a late-stage model of hat. introduction top ↑ human african trypanosomiasis (hat), or sleeping sickness, is a parasitic infectious disease caused by infection with the haemoprotozoans trypanosoma brucei gambiense (chronic form/gambian hat) or t. b. rhodesiense (acute form/rhodesian hat), two morphologically-identical but epidemiologically-distinct subspecies of t. brucei. both parasites are transmitted cyclically by haematophagous tsetse flies of the genus glossina and are restricted to discrete foci within sub-saharan africa.1,2,3,4,5,6 hat is the archetype of a neglected disease, affecting the poorest people in africa.7 the disease runs an intricate course which can result in death if not promptly managed.8 the disease pathology consists of two distinct stages. during the early (haemolymphatic) stage, trypanosomes are present in the lymphatic fluid and blood, as well as the extravascular spaces of most organs. in the late (meningoencephalitic) stage, parasites are detected within the central nervous system (cns), where they cannot be treated by drugs that are incapable of crossing the blood-brain barrier.3 one of the major objectives in current basic research on hat is the development of a cheap and reliable experimental model of the disease that can be utilised to both increase our understanding of the disease and allow for the development of drug regimens for the management of late-stage hat.9 however, the development of such a model has proven to be difficult as most t. brucei subspecies tend to cause an acute rather than chronic infection in experimental animals.10,11 presently, drug efficacy trials against late-stage hat are performed in both murine models (mus musculus) and in non-human primates such as vervet monkeys (chlorocebus aethiops).9,12,13,14,15,16,17 whilst the vervet monkey model of rhodesian hat has been widely used in a number of drug evaluation and pathogenesis studies,9 it possesses unique disadvantages for the purposes of basic research. primates represent an expensive and ethically-challenging model, whose use is only justifiable under the auspices of avoiding unnecessary human involvement in most studies. on the other hand, most mouse models of rhodesian hat have been extensively developed using the non-human infective t. b. brucei, which may possess dissimilar pathogenicity and drug susceptibility profiles when compared to the human-infective t. b. rhodesiense or t. b. gambiens e.11 in particular, these mouse models, being non-human infective and parasite-based, may not depict accurately the cns involvement by human-infective parasites.10,11,12,15 thus, mouse models for rhodesian hat, particularly those based on t. b. rhodesiense, are essential in order to provide a more correct depiction of the disease's pathogenicity and drug susceptibility profiles.11 as different parasite strains from a particular subspecies may have dissimilar disease progression, it is of interest to perform preliminary studies that compare a number of strains in order to have a proper baseline for future studies involving the selected parasite strains. this study aimed to characterise the virulence and pathology of three t. b. rhodesiense stabilates, international livestock research institute (ilri)-2918, ilri-3953, and institute of primate research (ipr)-001, the target parasites for establishment of the rodent and primate models of hat, in outbred swiss white mice. to the best of the authors’ knowledge, these parasites have not yet been evaluated comparatively. methods top ↑ animals as the pathogenicity and virulence of two of the trypanosome subspecies used were known (ilri-2918 and ilri-3953), available data from previous studies were used to estimate the sample size per group.11,18,19,20 swiss white mice of either sex (n = 108), weighing 25–30 grams, were obtained from the rodent facility at the ipr and provided with mouse feed (mice cubes®, unga feeds, nairobi, kenya) and water ad libitum. the mice were kept in 14 cm x 30 cm x 15 cm macrolone cages. they were maintained at an ambient temperature of between 20 and 25°c and wood shavings (various timber mills, embulbul, kajiado, kenya) were used as bedding. trypanosomes stabilate ilri-2918 was clonally derived from kenya trypanosomiasis research institute (ketri)-2772, which was isolated from a natural infection in a human in alupe, busia, kenya and designated 2772.18 stabilate ilri-3953 was originally referred to as uganda trypanosomiasis organisation (utro)-310185, which was isolated from a naturally infected human in bugiri, uganda in 1982. the parasite has been passaged in mice and designated ilri-3953.19,20 stabilate ipr-001 was obtained from a cerebrospinal fluid sample from an infected human in bugiri, uganda. after three passages in outbred swiss white mice at the ipr, the stabilate was designated ipr-001. for the current study, all stabilates were obtained from storage in liquid nitrogen (–196 °c), thawed and diluted with phosphate saline glucose (psg) buffer (ph 8.0), then injected intraperitoneally (0.2 ml each) into three swiss white female mice that were immunosuppressed via irradiation with caesium chloride at 600 rads for five minutes. upon the onset and rise of parasitaemia, the imunosuppressed mice were euthanised and their blood collected via cardiac puncture. the collected blood was then diluted to a parasitaemia of approximately 104 trypanosomes/0.2 ml using psg, after which the diluted blood was injected intraperitoneally (ip; 0.2 ml each) into the 108 mice included in the study, as described below. six mice were used as positive controls and injected with psg buffer only (0.2 ml ip). experimental design the experiment was designed and executed as indicated in table 1. after infection, experimental mice were observed daily for parasitaemia using wet blood smears obtained by tail snipping. at the same time, thin blood smears were also prepared and giemsa stained for observation of pleomorphism via microscopy, as described by kagira et al.11 the parasitaemia was then estimated using the method described by herbert and lumsden.21 table 1: experimental design. for both ilri-2918 and ipr-001-infected mice, all surviving mice (both experimentally infected and controls) were treated with diminazene aceturate (40 mg/kg ip, berenil®, intervet, south africa) at 21 days post infection (dpi) as described previously by kagira et al.11 at this stage, the parasites were assumed to have invaded the cns. the diminazene aceturate therefore only clears the haemolymphatic trypanosome complement, because it cannot cross the blood-brain barrier in sufficient quantities to clear the cns. mice were then observed daily for relapse parasitaemia using wet smears obtained via tail snipping. relapse detection sensitivity was enhanced by examining the collected blood using the woo method.22 upon relapse of parasitaemia, all surviving mice (both experimentally infected and controls) were treated with melarsoprol (3.6 mg/kg ip, arsobal®, sanofi-aventis, paris, france) for four days consecutively. six mice per stabilate were euthanised at set time points (dpi 7, 14 and 21). all their major organs (heart, liver, spleen, brain, lungs and kidneys) were harvested and perfused with citrate saline buffer via the left ventricle through the hepatic portal vein until all the blood was completely drained. they were then preserved in 10% neutral buffered formalin for histopathology. statistical analyses data were managed and analysed using ms excel (microsoft excel® 2007, microsoft corporation, redmond, washington, united states). the means for parasitaemia and parasite morphology were calculated using the average function in ms excel and variability was calculated using the standard error of the mean function in microsoft excel, ‘= (stdev(a1:a2))/(sqrt(count(a1:a2))’, where a1 represents the first value in the series and a2 represents the last value in the series. ethical considerations all experiments were carried out at the ipr in karen, nairobi, kenya. the experiments were conducted in accordance with protocols approved and authorised by the institutional review committee of the institute. results top ↑ parasitaemia and gross clinical observations in ilri-2918-infected mice, patency was demonstrated at five dpi (figure 1). this was estimated at log10 3.07. all of the patent mice were active and had smooth coats at the onset of patency. first peak parasitaemia was observed at seven dpi (figure 1) and the mean peak parasitaemia was estimated at log10 6.4. secondary latency was observed at 17 dpi (figure 1), with mean peak parasitaemia estimated at log10 4.87. following treatment at 21 dpi with diminazene aceturate, parasitaemia dropped below microscopically-detectable levels between 22 and 28 dpi (figure 1). relapse parasitaemia was observed on 50 dpi. the parasitaemia at the onset of relapse was log10 0.6. figure 1: mean parasitaemia pattern for t. b. rhodesiense ilri-2918 infection in swiss white mice. the first peak occurred at seven dpi; the second peak occurred at 17 dpi. mean parasitaemia at the first peak was estimated at log10 6.4. mean parasitaemia at the second peak was estimated at log10 4.87. following treatment at 21 dpi with diminazene aceturate (40 mg/kg ip, berenil®, intervet, south africa), parasitaemia dropped below microscopically-detectable levels between 22 and 28 dpi. relapse parasitaemia was observed on 50 dpi. the parasitaemia at the onset of relapse was log10 0.6. from 10 dpi, the mice began to develop ruffled coats, lower appetite and lower activity levels. these symptoms occurred at varying levels of parasitaemia, with some groups experiencing the symptoms during the rising phase of parasitaemia and others during the dropping phase of the first parasitaemia peak. peri-orbital oedema also occurred during the rising and dropping phases of parasitaemia, with three mice having one or both eyes shut at all times. these symptoms persisted until the second rising phase of parasitaemia, at which time they disappeared and the mice appeared active and once again had smooth coats. at 26 and 27 dpi, hindleg paresis was observed in two mice. these mice were euthanised and their major organs collected for histopathology. at 27 dpi, after treatment with diminazene aceturate at 21 dpi, the surviving mice again had lowered activity and ruffled coats. the prognosis of these mice, however, improved steadily and the clinical symptoms slowly dissipated, disappearing by 31 dpi. at 50 dpi, all surviving mice relapsed into parasitaemia at log10 0.6 and at 51 dpi, the mice succumbed to hindleg paresis and were subsequently euthanised. in ilri-3953-infected mice, patency was demonstrated from three dpi, with peak parasitaemia observed at seven dpi (figure 2). this was estimated at log10 8.36. all of the patent mice were active and had smooth coats at the onset of patency. mice began to develop ruffled coats, lower activity level and paresis from seven dpi. one mouse also exhibited peri-orbital oedema at peak parasitaemia (log10 8.4; seven dpi). as the parasitaemia rose, mice developed hindleg paresis and were found dead in their cages, with some deaths occurring at six dpi. by 10 dpi, all experimental mice had been euthanised or were found dead in their cages, necessitating a termination of the experiment on this isolate. figure 2: mean parasitaemia pattern for t. b. rhodesiense ilri-3953 infection in swiss white mice. the first peak occurred at seven dpi. mean parasitaemia at the first peak was estimated at log10 8.36. all mice were found dead in their cages or were euthanised by 10 dpi. in ipr-001-infected mice, patency was demonstrated from three to five dpi (figure 3). all of the patent mice were active at the onset of patency. the first peak of parasitaemia was observed at six dpi (log10 8.36) and the second peak parasitaemia occurred at 18 dpi (log10 8.81). ruffled coats, lower appetite and lower activity level were observed from as early as the first parasitaemia peak. after treatment with diminazene aceturate, parasitaemia dropped below microscopically-detectable levels and clinical symptoms abated. relapse parasitaemia occurred at 39 dpi (log10 1.62) and peaked at 54 dpi (log10 6.8). following treatment with melarsoprol, parasitaemia was again observed to drop below microscopically-detectable levels until the termination of the experiment at 86 dpi. figure 3: mean parasitaemia pattern for t. b. rhodesiense ipr-001 infection in swiss white mice. the first peak occurred at six dpi; the second peak occurred at 18 dpi. mean parasitaemia at the first peak was estimated at log10 8.36. mean parasitaemia at the second peak was estimated at log10 8.81. following treatment at 21 dpi with diminazene aceturate (40 mg/kg i.p., berenil®, intervet, south africa), parasitaemia dropped below microscopically-detectable levels between 22 and 25 dpi. relapse parasitaemia was observed to occur at 39 dpi (log10 1.62) and peaked at 54 dpi (log10 6.8). following treatment with melarsoprol (3.6 mg/kg ip, arsobal®, sanofi-aventis, paris, france), parasitaemia was again observed to drop below microscopically-detectable levels until the termination of the experiment at 86 dpi. parasite virulence and mouse survival in ilri-2918-infected mice, survival was constant (100%) up to and after treatment with diminazene aceturate at 21 dpi (figure 4). upon treatment, mice developed hindleg paresis (50% of the experimental population at the time), at which time they were euthanised. upon relapse of parasitaemia, the remaining mice succumbed to the parasite, thereby causing termination of the experiment. figure 4: mean survival for ilri-2918, ilri-3953 and ipr-001 infected mice. in ilri-2918 infected mice, survival was 100% until after treatment with diminazene aceturate at 21 dpi, whereupon 50% developed hindleg paresis and were euthanised with the rest of the population succumbing to the parasite after parasitaemia relapse. in ilri-3953 infected mice, mortality was 100% by 10 dpi. in ipr-001 infected mice, survival was 97% before treatment with diminazene aceturate at 21 dpi, dropping and stabilising at 38% (24–31 dpi), followed by 35% (32–44 dpi), then 31% (45–56 dpi) and finally stabilising after treatment with melarsoprol to 27% (57–79 dpi). all mice surviving after the treatment were euthanised. in ilri-3953-infected mice, mortality increased with the rise in parasitaemia, with mice rendered moribund as the experiment progressed. by 10 dpi, all mice were dead in their cages or had been euthanised after hindleg paresis was observed. in ipr-001-infected mice, mortality occurred even before treatment with diminazene aceturate at 21 dpi. approximately 3% of the experimental population succumbed to the stabilate's parasitaemia, with 97% surviving before treatment with diminazene aceturate at 21 dpi. the survival dropped further through 38% (24–31 dpi), to 35% (32–44 dpi), then 31% (45–56 dpi), before finally stabilising after treatment with melarsoprol to 27% (57–79 dpi), whereupon it remained stable until the end of the experiment. at the end of the experiment, the surviving mice were euthanised and their organs harvested for histopathology. parasite morphology examination of the giemsa-stained thin blood smears indicated that pleomorphism occurred in two of the parasite strains used, namely, ilri-2918 and ipr-001. in these stabilates, two trypomastigote forms – long slender and short stumpy – were observed during the course of infection. from five dpi in ilri-2918 and three dpi in ipr-001, there was a predominance of long slender trypomastigotes (97.4% in ilri-2918 and 83.7% in ipr-001), with few short stumpy trypomastigotes observed (2.6% in ilri-2918 and 16.3% in ipr-001). for both strains, observation of the different morphological forms ceased at 23 dpi after treatment with diminazene aceturate at 21 dpi. in ilri-2918-infected mice, there was an increase in short stumpy forms (18.6%) with a decline in long slender forms (83.7%) (figure 5) during the first parasitaemia peak at seven dpi, compared to the populations at the beginning of the observation period. at eight dpi, there was a great increase in the number of short stumpy trypomastigotes (64.6%), with a decline in the long slender forms (35.4%). at the second peak (at 17–19 dpi), there was a predominance of short stumpy forms (up to 92.4%) and a decline in long slender forms (7.6%). figure 5: parasite morphology pattern for ilri-2918 infection in swiss white mice. during the first parasitaemia peak at seven dpi, there was an increase in short stumpy forms (18.6%) with a decline in long slender forms (83.7%). at the second peak at 17–19 dpi, there was a predominance of short stumpy forms (up to 92.4%) and a decline of long slender forms (7.6%). in ipr-001-infected mice, during the first peak at six dpi, there was a notable increase in the number of short stumpy trypomastigotes (33.6%) and a decline in the long slender forms (66.4%) compared to the populations at the beginning of the observation period (figure 6). in this isolate as well, at seven dpi, there was a great increase in the number of short stumpy trypomastigotes (54.4%) and a decline in the long slender forms (45.6%). at the second peak, at 18 dpi, there was a predominance of short stumpy forms (74.9%) and a decline of long slender forms (25.1%). however, the greatest increase in short stumpy forms during the ipr-001 infection occurred between 20 and 23 dpi (from 94.6% – 95.2%). figure 6: parasite morphology pattern for ipr-001 infection in swiss white mice. during the first peak at six dpi, there was a notable increase in the number of short stumpy trypomastigotes (33.6%) and a decline in the long slender forms (66.4%) compared to the populations at the beginning of the observation period. at the second peak at 18 dpi, there was a predominance of short stumpy forms (74.9%) and a decline of long slender forms (25.1%). histopathological findings gross anatomy observation revealed enlargement of the spleen and hyperaemia in the major organs. histopathological observations of the brain included discrete inflammation, with the foci of meningitis characterised by progressive mononuclear cell infiltration, perivascular cuffing (figure 7) and hyperaemia of meningeal blood vessels. these observations were seen to progress over time in all observed samples from experimental mice. infiltration of the brain parenchyma with mononuclear cells was rare and only began to be prominent in the late stages of infection. the choroid plexus was prominent in infected mice and contained inflammatory exudates, mainly of mononuclear cells. figure 7: perivascular cuffing (indicated by the shaded arrow) in a parasite-relapse mouse brain sample. in all groups of experimental mice, most organs were characterised by interstitial mononuclear cell infiltration into the parenchyma and perivascular cuffing. the most affected organs were the liver, spleen, heart and lungs. there were no marked differences between ilri-2918 and ipr-001-infected mice. the germinal centres of the spleen were active and contained many immature lymphocytes and plasma cells, especially in samples obtained at 14 and 21 dpi, although these cells were reduced in number by 28 dpi. the lung alveoli, as well as the interseptal spaces, were characterised by infiltration with inflammatory exudates and cells, including lymphocytes, macrophages and a few neutrophils. this was more prominent in samples from mice euthanised after 21 dpi. the liver indicated extensive lymphocytic infiltration coupled with necrosis of hepatocytes, especially in mice euthanised after 28 dpi. discussion top ↑ of the three stabilates investigated, we found that two possessed the necessary parasitaemic characteristics of a typical hat infection. both ilri-2918 and ipr-001 had a mean pre-patent period and parasitaemia pattern similar to that reported by other researchers.11 the survival times for these two stabilates and the pathological changes they induced in experimental animals demonstrated that these parasite strains are capable of causing a chronic infection. this is a feature particularly useful in models for drug efficacy testing. some of the symptoms observed in mice infected with ilri-2918 and ipr-001, particularly peri-orbital oedema, have also been described in higher primate models of hat.13,23 the deaths occurring at 31 dpi for ilri-2918 and at 21 dpi for ipr-001 could have resulted from an immunological crisis arising after massive trypanolysis occasioned by treatment with diminazene aceturate. the mortality and/or morbidity observed in trypanosome-infected mice could be attributed primarily to self-inflicted damage by a disproportionate immune and/or innate response.24 mortality and morbidity could also be caused by the immuno-depressant capabilities of a trypanosome infection on a mouse strain. this may have led to opportunistic infections, leading to the death of mice infected with a trypanosome strain.25 in this study, the parasitaemia pattern showed a predominance of two different morphological types of trypomastigotes, namely, short stumpy and long slender forms. in both ilri-2918 and ipr-001 infections, there was a predominance of short stumpy forms during or shortly after peak parasitaemia. this phenomenon was observed at both parasitaemia peaks and appeared to be alleviated by treatment with diminazene aceturate treatment. however, for both strains, the situation was reversed during the rising phase of the parasitaemia. in this case, the short stumpy form declined and the long slender form predominated. this observation is in line with what has been reported by others.11 pleomorphism is considered a pre-adaptation to transmission to the tsetse fly, with long slender, multiplicative forms being transformed into short stumpy, non-multiplicative forms capable of survival in the tsetse fly midgut.26 this process, which is a host antibody-independent response, is probably mediated by a density-sensing mechanism.27 the pathological changes described in most organs appeared to be characteristic of t. brucei infections. unlike most other trypanozoon genus members, t. brucei is particularly tissue invasive, causing cellular infiltration into the parenchyma of most organs.11,13 the cellular reactions we observed in most organs showed that a lymphoid immune reaction is critical in the pathogenesis of t. b. rhodesiense infections, as described previously by others.11,13,28 in the brain, it has been shown that the inflammatory reaction begins as meningitis and progresses into the virchow-robin spaces before spreading to the parenchyma.11 whilst the successful induction of the meningoencaphalitic stage was assumed here (because of the relapsing parasitaemia after treatment with diminazene aceturate, which clears only the haemolymphatic component of the parasite), further proof in terms of demonstration of trypanosomes within the cns, coupled with appearance of anti-trypanosomal antibodies as described by previous studies,29,30 would greatly improve the value of the reported model. in addition, the role played by the particular mouse strain can influence the disease progression and pathology encountered. this has been shown by others, with certain strains such as balb/c mice showing the greatest susceptibility, whereas c57bl/6 mice show the greatest tolerance.25,31 given that the mice used in this study were classified as outbred swiss white, there could be a significant deviation, particularly in the disease pathology, when the model is established with inbred strains. most murine models of trypanosomiasis for drug efficacy trials rely on the assumption that trypanosomes have invaded the brain parenchyma by 21 dpi.10,21 in this study, the same assumption was made with subcurative treatment with diminazene aceturate. relapses occurred in both ilri-2918and ipr-001-infected mice, coupled with severe meningoencephalitis as has been reported previously.10,11,32 limitations of the study as described in the preceding section, there were a number of limitations observed which can be summarised as follows. firstly, the strain of mouse greatly influences the disease progression and pathology encountered. there could thus be deviations if the model is established using a different mouse strain, particularly an inbred one. secondly, the study assumed the occurrence of cns involvement by relapse of parasitaemia after haemolymphatic clearance with diminazene aceturate, as has been done in previous studies. however, assessment of plasma concentrations of acute-phase protein, c-reactive protein and haptoglobin could also be used as a marker for experimental infections.16,33 thirdly, it is difficult to interpret brain pathology in terms of relapse following treatment with trypanocidals, as they, too, have been shown to induce brain pathology. another point of contention when using trypanocidals to stage the disease is the danger that subcurative treatment of a parasitic infection may lead to emergence of drug resistance, a position which would complicate the interpretation of efficacy data if the drug under investigation has a similar mode of action.11 finally, the parasites used in this study have not yet been characterised genetically. recommendations in order to circumvent the preceding limitations, an initial baseline study is required for any novel mouse model utilising the described parasite isolates, incorporating more extensive measures of parasitaemia as well as parasite staging. full genetic characterisation of the parasites used here is needed in order to ascertain the identity of the isolates during future experiments and in order to account for variations observed in subsequent studies. because of the heterogenous nature of outbred swiss mice, there may be significant deviations in results from mouse studies using the same isolates in the future. for this reason, studies using the more homogenous inbred mouse strains, which may not be susceptible to the isolates used here, are recommended in order to establish the late-stage hat model. conclusion top ↑ in conclusion, this study investigated infection with three different t. b. rhodesiense strains, ilri-2918, ilri-3953 and ipr-001, which led to the occurrence of acute, hyper-acute and chronic infections, respectively. ilri-2918 showed indications of causing a chronic infection in mice; however, its relapse parasitaemia induced hindleg paresis and death. this strain of the parasite was therefore classified as causing an acute infection in mice and was regarded by the investigators as being unsuitable for the development of a late-stage model of hat. ilri-3953 was observed to be highly virulent, with all the experimental mice found dead in their cages or humanely euthanised by 10 dpi. upon further passaging in immunocompetent swiss white mice (unpublished data), the parasite showed no indications of reducing its virulence. therefore, the parasite was classified as hyper-acute and unsuitable for the development of a late-stage model of hat. ipr-001, a field-isolated parasite, showed indications of causing a chronic infection in mice. the first peak of parasitaemia rose to log10 8.36 at six dpi. after subcurative treatment, mice relapsed and survived in good physical condition/health until curative treatment. this parasite was therefore classified as causing chronic infection and is proposed as the parasite of choice for further work in developing a late-stage model of hat. trustworthiness top ↑ in as far as the authors are concerned, and in view of the outcome of the t. b. rhodesiense infections in the mouse studies, the results obtained in this study appear to compare well with those arising from t. b. brucei and other related studies. reliability and validity of the research the authors consider the experimental design of this study to be reliable and valid for the purpose of establishing a mouse model in outbred swiss white mice. the procedures utilised in this study have been tested and validated in other studies as cited in this article. acknowledgements top ↑ this project was funded by the director of the ipr. the authors are grateful to the technical assistance provided by alex gaithuma, thomas adino and claire n. kimani of the department of animal sciences, ipr. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions m.n. (ipr) was the project leader and, along with j.m.k. (jomo kenyatta university of agriculture and technology), was in charge of animal welfare. m.n., c.k. (ipr) and j.m.k. were responsible for the experimental and project design. c.k., v.m. (ipr) and j.m.k. performed the experiments, whilst c.k. and j.m.k. performed the data analysis. all authors participated in the writing of the manuscript. references top ↑ kennedy pge. human african trypanosomiasis of the cns: current issues and challenges. j clin invest. 2004;113(4):496–504. http://dx.doi.org/10.1172/jci200421052 kennedy pge. diagnostic and neuropathogenesis issues in human african trypanosomiasis. int j parasitol. 2006;36(5):505–512. http://dx.doi.org/10.1016/j.ijpara.2006.01.012 kennedy pge. the continuing problem of human african trypanosomiasis (sleeping sickness). ann neurol. 2008;64(2):116–126. http://dx.doi.org/10.1002/ana.21429 fèvre em, odiit m, coleman pg, et al. estimating the burden of rhodesiense sleeping sickness during an outbreak in serere, eastern uganda. bmc public health. 2008:8:96. http://dx.doi.org/10.1186/1471-2458-8-96 simarro pp, jannin j, cattand p. eliminating human african trypanosomiasis: where do we stand and what comes next? plos med. 2008;5(2):e55. http://dx.doi.org/10.1371/journal.pmed.0050055 brun r, blum j, chappuis f, et al. human african trypanosomiasis. lancet. 2010;375(9709):148–159. http://dx.doi.org/10.1016/s0140-6736(09)60829-1 fèvre em, wissmann bv, welburn sc, et al. the burden of human african trypanosomiasis. plos negl trop dis. 2008;2(12):e333. http://dx.doi.org/10.1371/journal.pntd.0000333 bouteille b, oukem o, bisser s, et al. treatment perspectives for human african trypanosomiasis. fundam clin pharmacol. 2003;17(2):171–181. http://dx.doi.org/10.1046/j.1472-8206.2003.00167.x farah io, ngotho m, kariuki t, et al. animal models of tropical parasitic diseases. in: hau j & van hoosier gl, editors. handbook of laboratory animal science. florida: crc press, 2004; 3, p. 169–223. http://dx.doi.org/10.1201/9781420039627.ch9 jennings fw, whitelaw dd, urquhart gm. the relationship between duration of infection with trypanosoma brucei in mice and the efficacy of chemotherapy. parasitology. 1977;75(2):143–153. http://dx.doi.org/10.1017/s0031182000062284 kagira, j. m., ngotho, m. & thuita, j. development of a rodent model for late stage rhodesian sleeping sickness. j protozool res. 2007;17:48–56. jennings fw, hunter ca, kennedy pge, et al. chemotherapy of trypanosoma brucei infection of the central nervous system: the use of a rapid chemotherapeutic regimen and the development of post-treatment encephalopathies. trans r soc trop med hyg. 1993;87(2):224–226. http://dx.doi.org/10.1016/0035-9203(93)90502-h maina n, ngotho jm, were t, et al. proinflammatory cytokine expression in the early phase of trypanosoma brucei rhodesiense infection in vervet monkeys (cercopithecus aethiops). infect immun. 2004;72(5):3063–3065. http://dx.doi.org/10.1128/iai.72.5.3063-3065.2004 kagira jm, ngotho m, thuita jk, et al. hematological changes in vervet monkeys (chlorocebus aethiops) during eight months’ adaptation to captivity. am j primatol. 2007;69(9):1053–1063. http://dx.doi.org/10.1002/ajp.20422 kennedy pge. animal models of human african trypanosomosis – very useful or too far removed ? trans r soc trop med hyg. 2007;101(11):1061–1062. http://dx.doi.org/10.1016/j.trstmh.2007.05.001 ngure rm, ndungu jm, ngotho jm, et al. biochemical changes in the plasma of vervet monkeys (chlorocebus aethiops) experimentally infected with trypanosoma brucei rhodesiense. j cell anim biol. 2008;2(7):150–157. ngotho m, kagira jm, kariuki c, et al. influence of trypanocidal therapy on the haematology of vervet monkeys experimentally infected with trypanosoma brucei rhodesiense. acta trop. 2011;119(1):14–18. http://dx.doi.org/10.1016/j.actatropica.2011.02.013 clarkson ab. development of a new chemotherapy for human african trypanosomias using an animal model: suramin with dl-alpha-difluoromethylornithine (dfmo) [research proposal on the internet]. c1988 [cited 2012 august 31]. available from: http://www.dtic.mil/dtic/tr/fulltext/u2/a229593.pdf morty re, pellé r, vadász i, et al. oligopeptidase b from trypanosoma evansi. a parasite peptidase that inactivates a trial natriuretic factor in the bloodstream of infected hosts. j biol chem. 2005;280(12):10925–10937. http://dx.doi.org/10.1074/jbc.m410066200 morty re, vadász i, bulau p, et al. tropolysin, a new oligopeptidase from african trypanosomes. biochemistry. 2005;44(44):14658–14669. http://dx.doi.org/10.1021/bi051035k herbert wj, lumsden wh. trypanosoma brucei: a rapid ‘matching’ method for estimating the host's parasitemia. exp parasitol. 1976;40(3):427–431. http://dx.doi.org/10.1016/0014-4894(76)90110-7 woo pt. the haematocrit centrifuge technique for the diagnosis of african trypanosomiasis. acta trop. 1970;27(4):384–386. burudi em, karanja sm, njue ai, et al. establishment of a partly dfmo-sensitive primate model of trypanosoma rhodesiense sleeping sickness. acta trop. 1995;59(1):71–73. http://dx.doi.org/10.1016/0001-706x(94)00081-b naessens j. bovine trypanotolerance: a natural ability to prevent severe anaemia and haemophagocytic syndrome? int j parasitol. 2006;36(5):521–528. http://dx.doi.org/10.1016/j.ijpara.2006.02.012 antoine-moussiaux n, magez s, desmecht d. contributions of experimental mouse models to the understanding of african trypanosomiasis. trends parasitol. 2008;24(9):411–418. http://dx.doi.org/10.1016/j.pt.2008.05.010 matthews kr, gull k. evidence for an interplay between cell cycle progression and the initiation of differentiation between life cycle forms of african trypanosomes. j cell biol. 1994;125(5):1147–1156. http://dx.doi.org/10.1083/jcb.125.5.1147 reuner b, vassella e, yutzy b, et al. cell density triggers slender to stumpy differentiation of trypanosoma brucei bloodstream forms in culture. mol biochem parasitol. 1997;90(1):269–280. http://dx.doi.org/10.1016/s0166-6851(97)00160-6 thuita jk, kagira jm, mwangangi d, et al. trypanosoma brucei rhodesiense transmitted by a single tsetse fly bite in vervet monkeys as a model of human african trypanosomiasis. plos negl trop dis. 2008;2(5):e238. http://dx.doi.org/10.1371/journal.pntd.0000238 poltera aa, hochmann a, rudin w, et al. trypanosoma brucei brucei: a model for cerebral trypanosomiasis in mice – an immunological, histological and electronmicroscopic study. clin exp immunol. 1980;40(3):496–507. stoppini l, buchs pa, brun r, et al. infection of organotypic slice cultures from rat central nervous tissue with trypanosoma brucei brucei. int j med microbiol. 2000;290(1):105–113. http://dx.doi.org/10.1016/s1438-4221(00)80113-7 maina n, kagira jm, mäser p, et al. genotypic and phenotypic characterization of trypanosoma brucei gambiense isolates from ibba, south sudan, an area of high melarsoprol treatment failure rate. acta trop. 2007;104(2–3):84–90. http://dx.doi.org/10.1016/j.actatropica.2007.07.007 jennings fw, gray gd. relapsed parasitaemia following chemotherapy of chronic t. brucei infections in mice and its relation to cerebral trypanosomes. contrib microbiol immunol. 1983;7:147–154. ndung’u jm, eckersall pd, jennings fw. elevation of the concentration of acute phase proteins in dogs infected with trypanosoma brucei. acta trop. 1991;49(2);77–86. abstract introduction methods results discussion acknowledgements references about the author(s) foluke a. fasola department of haematology, college of medicine, university of ibadan, ibadan, nigeria department of haematology, university college hospital, ibadan, nigeria adeola a. fowotade department of medical microbiology & parasitology, college of medicine, university of ibadan, ibadan, nigeria adedayo o. faneye department of virology, college of medicine, university of ibadan, ibadan, nigeria adeyeni adeleke department of haematology, college of medicine, university of ibadan, ibadan, nigeria citation fasola fa, fowotade aa, faneye ao, adeleke a. prevalence of hepatitis b virus core antibodies among blood donors in nigeria: implications for blood safety. afr j lab med. 2022;11(1), a1434. https://doi.org/10.4102/ajlm.v11i1.1434 original research prevalence of hepatitis b virus core antibodies among blood donors in nigeria: implications for blood safety foluke a. fasola, adeola a. fowotade, adedayo o. faneye, adeyeni adeleke received: 23 oct. 2020; accepted: 20 apr. 2022; published: 26 july 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: anti-hepatitis b core antibody (anti-hbc) testing improves transfusion safety by detecting past and current hepatitis b virus (hbv) infection while detecting hepatitis b surface antigen (hbsag) in serology-negative hbv infection. however, occult hbv infection (obi) (serum or liver hbv dna-positive but hbsag-negative) remains unaddressed among replacement blood donors – family members or friends who donate to replace blood transfused to a relative. objective: this study assessed risk factors for a positive anti-hbc test among donors with obi and determined the anti-hbc-positive status of replacement donors. methods: the study was conducted at the university college hospital blood bank, ibadan, nigeria, using blood samples collected from blood donors between april 2019 and may 2019. donors were screened for hbsag by rapid diagnostic test (rdt) and enzyme-linked immunosorbent assay (elisa) and anti-hbc by elisa, while hbv dna was detected using a semi-nested polymerase chain reaction. results: of the 274 participants, 15 (5.5%) were hbsag-positive by rdt and 36 (13.1%) by elisa, while 133 (48.5%) were anti-hbc positive. out of 232 hbsag-negative donors, 107 (46.1%) were anti-hbc positive. of the 107 hbsag-negative but anti-hbc-positive samples, only one (0.93%) was hbv dna-positive. the hbv dna-positive donor was hbsag-negative by both rdt and elisa tests. conclusion: this study establishes a potential risk for hbv transmission from isolated anti-hbc-positive donors to blood recipients. hbc immunoglobulin (antibody) m testing to identify blood units requiring further screening with polymerase chain reaction to detect obi can prevent hbv transmission through blood transfusion. keywords: anti-hbc antibodies; donors; blood safety; hbv dna; occult hbv. introduction blood transfusion is an indispensable life-saving therapy for patient management; however, one of its adverse effects is the potential for transmitting infections. hepatitis b virus (hbv) is the most frequent transfusion-transmitted viral infection.1 in sub-saharan africa, the median risk of hbv infection following blood transfusion is 4.3/1000 units2 and the risk from blood screened by enzyme immunoassay for recipients younger than ten years old is 1:326.3 due to its public health burden, tackling hbv is a global health strategy for achieving the 2030 agenda for sustainable development.4 when an individual has an acute-phase infection, the first viral antigen to appear in the blood is hepatitis b surface antigen (hbsag), which persists even in the chronic phase but is undetectable when the virus is cleared from the blood. thus, hbsag is the hbv infection detection marker. the hbv genome is enclosed within a ‘core particle’ made of hepatitis b core protein or antigen (hbc). while the host is clearing the hbsag by developing hbsag antibodies (anti-hbs), the host also produces hbc immunoglobulin (antibody) m. during this period, known as the ‘window period’, the host is infectious, and the serological marker for infection is anti-hbcigm,5 as the period is associated with undetectable levels of both hbsag and anti-hbsag. blood donors at this infection stage (tail end carriers) can transmit hbv.6 progression of hbv infection to the chronic state is associated with the presence of both immunoglobin g (igg) and immunoglobin m (igm) anti-hbc against hbc. the natural course of infection in persons with chronic hbv infection could terminate as ‘inactive carriers’ with occult hbv infection (obi), defined as the existence of low-level hbv dna in the serum (< 200 iu/ml), cells of the lymphatic (immune) system, or hepatic tissue of patients with serological markers of the previous infection (anti-hbc or anti-hbs positive) and the absence of serum hbsag.7,8 hence, positive anti-hbc is considered a key obi marker.9 most blood bank screening panels consist of hbsag and anti-hbc total (igm + igg).10 the anti-hbc total (igm + igg) is used to detect both previous and current hbv infection. the hbsag, anti-hbc, and hbv nucleic acid tests (nat) are often used in blood banks in high-income countries to detect infection, during the window period, obi, and genetic and antigenic viral variants, thus ensuring optimal blood safety levels. a substantial percentage of blood recipients are likely to be the immunosuppressed, women, and children.11 transfusion of blood from obi donors to pregnant women can increase vertical transmission of viral hepatitis. vertically infected babies are prone to long-term complications of hbv infection, such as liver disease. sadly, the university college hospital (uch) blood bank in ibadan, nigeria, cannot detect hbv infection in blood donors with undetectable hbsag. in the uch blood bank, as in many developing countries, most blood units are from replacement donors, against the recommended voluntary blood donors for safe blood supply. replacement blood donors are family members or friends who donate blood to replace blood transfused relatives, in our setting pregnant relatives. thus, the inability to detect hbv infection in blood donors with undetectable hbsag implies a possible risk of post-transfusion hepatitis from anti-hbc-positive individuals. therefore, it is imperative to determine the anti-hbc positivity status of replacement donors who constitute the most significant proportion of our blood donors5,10 as with nigerian hospital-based blood banks.12 anti-hepatitis b core antibody tests and nat are excluded in our blood donor algorithm; therefore, this study aimed to assess the risk factors for positive anti-hbc in donors and the anti-hbc status of replacement donors to improve blood safety. methods ethical considerations the university of ibadan/university college hospital (ui/uch) ethics review team approved this research (19/0204). all recruited participants gave written informed consent. the confidentiality of participants was maintained by coding the samples, and only authorised personnel had access to participants’ identities. study location and participants the research was conducted at the blood bank of uch in ibadan, nigeria, an 850-bed hospital with 163 examination couches, excluding private suites. this blood bank collects, stores, and processes blood. the target population were replacement blood donors whom the patient or patient’s relatives recruited to replace blood used. only donors who presented blood donation forms indicating the patient on whose behalf blood was being donated were eligible. all donors were recruited from april 2019 to may 2019. in our blood bank, donors were allowed to donate blood if: donors were between 18 and 65 years, weighed 50 kg and above, passed the copper sulphate test and the verbal questioning stage, and were negative for transfusion-transmitted infections, including syphilis, hiv, hbv and hepatitis c virus. donors were excluded from this study if they: voluntarily came to donate blood, had a tattoo (exclusive of tribal marks obtained from infancy), failed the copper sulphate screening test or failed routine eligibility questioning by blood bank staff (which includes questions about previous blood donations, breastfeeding and menstruation status for female donors, presence of any health condition such as hypertension or diabetes, and hiv and hepatitis c virus status). in this study, a questionnaire was administered to include consecutive potential replacement blood donors who had passed the copper sulphate test and blood bank staff eligibility oral questioning stage. the questionnaire was translated into the native language (yoruba) for non-english-speaking participants and collected the patient’s demographic characteristics and knowledge of and risk factors of hbv. four questions tested hbv knowledge with ‘yes’ or ‘no’ questions. each ‘yes’ response scored 1, while each ‘no’ response scored 0. knowledge was graded from 0 to 4 based on increasing correct responses, that is, 1 = only one yes, 2 = two yeses, 3 = three yeses and 4 = four yeses. sample collection six millilitres of venous blood were collected from each donor into sterile plain bottles labelled with the donor’s identity number. the blood was allowed to stand for 45 min, and then the serum was separated by centrifugation daily. the serum was then stored in two aliquots at –80 °c. the samples were thawed to room temperature for each laboratory analysis. the serological tests were performed a week after collecting samples, and the polymerase chain reaction (pcr) test was carried out in july 2019, two months after the collection of samples. serology the blood bank’s screening algorithm identifies a blood donor as hbv-positive using hbsag rapid diagnostic test (rdt) kit and a conventional semi-automated enzyme-linked immunosorbent assay (elisa). the bio-check rapid kit test (bio-check, san francisco, california, united states) employs lateral chromatographic flow, while the monolisa™ hbs ag ultra elisa kit (bio-rad, marnes-la-coquette, france) employs a qualitative one-step solid-phase enzyme-linked immunoassay technique of the ‘sandwich’ type. each test kit came with positive and negative controls used in each assay. first, the rapid test was administered to all donors pre-donation. donors who were hbsag-negative by the rapid test were further tested with the semi-automated elisa test. then, using one of the two aliquots of sera stored, all samples were tested further for anti-hbc using the hbcab elisa kit (melsin medical co., ltd, changchun, china). the anti-hbc assay measures the total anti-hbc, including the igg and igm hbc antibodies. hepatitis b virus dna detection sera from blood donors that were anti-hbc positive were tested for hbv dna with primers targeting the hbv pre-s gene in a semi-nested pcr protocol as described below. briefly, the total hbv dna was extracted from the samples using a dna extraction method previously described by wang et al.12 the hbv pre-s gene was amplified in a semi-nested pcr using three primers. the 979 (5ʹcaaaagacccacaattctttgacatactttccaa3ʹ) and sf (5ʹgtgtcttggccaaaattcgcagt3ʹ) primers were used in the first pcr run, while the primers 979 and mc-f (5ʹtcggatccggtatgttgcccgtttgtc3ʹ) were used in the second pcr round. the cycling conditions used are as follows: 95 °c for 5 min; 40 cycles of 95 °c for 30 s, 60 °c for 45 s, and 72 °c for 45 s; and a final extension of 72 °c for 7 min. the amplicon of ~550 base pairs was analysed by gel electrophoresis using 2% (weight/volume) agarose gel and stained with sybr stain (jena biosciences, jena, germany). amplicons were visualised in an ultraviolet illuminator. the negative control was a previously known hbv-negative sample, while the positive control was a previously confirmed hbv dna-positive sample. occult hbv infection was defined as hbv dna-positive, and serology hbsag-negative and anti-hbc positive. data analysis statistical package for social sciences version 20 (ibm corp., chicago, illinois, united states) was used. frequencies and percentages in tables or charts were used to present the demographic characteristics, risk factors for hbv infection among donors, and hepatitis b screening results for participants. means and standard deviations were used to summarise continuous variables such as the age of participants. odds ratios (ors) and 95% confidence intervals (cis) were calculated to test for the association between risk factors and anti-hbc positivity. the chi-square was also used to test the association between anti-hbc, socio-demographic features and risk factors of the participants. the statistical significance was set at p < 0.05. results socio-demographic and risk factors for acquisition of hepatitis b virus infection from blood donors the 274 participants included in the study ranged from 18 to 62 years (mean age of 32.0 ± 8.86 years). approximately half of the participants, 157 (57.3%), were married, and 25 (9.1%) had multiple sexual partners, while 14 (5.1%) had been previously treated for a sexually transmitted disease (table 1). twenty-eight (10.2%) participants had scarification or tribal marks, 87 (31.8%) shared sharp objects (pedicure, manicure and use of razor blade), and 9 (3.3%) had previously used sex performance enhancement recreational drugs. furthermore, 18 (6.6%) participants had been previously transfused with blood, 7 (2.2%) had jaundice in the last year, and 57 (20.8%) had been asked to do a test for hepatitis b before coming to donate blood. participants’ hbv knowledge scores were: zero, 120 (46.9%); one, 29 (11.3%); two, 21 (8.1%); three, 57 (22.3%); and four, 29 (11.3%). table 1: demographic characteristics of 274 blood donors in ibadan, nigeria, between april 2019 and may 2019. risk factors for hepatitis b virus and anti-hepatitis b core protein or antigen positivity a total of 133 blood donors were anti-hbc positive. the proportion of participants aged 18–35 years with a positive anti-hbc (42.2%) was lower than the proportion of participants above 36 years with a positive anti-hbc (61.8%). donors showed a declining prevalence of anti-hbc with an increasing hbv knowledge score (p = 0.046). the odds of being hbcab positive were 0.35 times less likely among blood donors who had a knowledge score of 4 than blood donors who had a knowledge score of 0 (95% ci: 0.14; 0.84). all 9 participants who used recreational drugs had a positive anti-hbc compared to 117/252 (46.8%) of participants who did not use (p = 0.001). in contrast, there was a lower proportion of participants who shared sharp objects with positive anti-hbc, 34 (39.1%), compared to 96 (52.9%) for those who did not share (table 2). the sharp objects shared included blades and sharps used for a pedicure, manicure, beauty treatment, and shaving hair. the age, gender, educational level, number of sexual partners, history of sexually transmitted disease transfusion and jaundice did not show any significant statistical relationship to anti-hbc positivity. thirteen donors had prior hbv vaccination. five (33.3%) of the hbv vaccinated donors were positive for anti-hbc, while 10 (66.7%) were negative. however, the difference was not statistically significant. the odds of being hbcab positive were 2.22 times more likely among blood donors over 35 years old than those aged 18–35 years (95% ci: 1.32; 3.72). the odds of being hbcab positive were 0.35 times less likely among blood donors who had a knowledge score of 4 than blood donors who had a knowledge score of 0 (95% ci: 0.14; 0.84). the relative risk (rr) of blood donors being hbcab positive was 2.14 times higher among blood donors who had ever used recreational drugs before or during sex than in blood donors who never used recreational drugs before or during sex (95% ci: 1.88; 2.43). there is a 2.23 times higher likelihood of anti-hbc positivity for blood donors who had ever been transfused with blood. blood donors who shared sharp objects were 0.57 times less likely to be hbcab positive compared to donors who do not share sharp objects (95% ci of or: 0.34; 0.96). the comparison of the different risk factors with hbcab is shown in figure 1. figure 1: risk factors for hepatitis b virus infection among 133 donors with positive anti-hepatitis b core protein or antigen in ibadan, nigeria, between april 2019 and may 2019. table 2: comparison of hepatitis b virus risk factors among anti-hepatitis b core-positive and -negative blood donors in ibadan, nigeria, between april and may 2019. occult hepatitis b virus infection of the 274 blood donors, 42 (15.3%) cases were hbsag-positive by both or either rdt or elisa: 9 (3.28%) by both rdt and elisa, 6 (2.19%) by rdt only, and 27 (9.85%) by elisa only; 232 (84.7%) were hbsag-negative (rdt and elisa negative). almost half (133/274; 48.5%) of the donors were anti-hbc positive, while 26/274 (9.5%) donors were hbsag-positive and anti-hbc positive. among the 259 participants who tested negative for hepatitis b by rdt, 121 (46.7%) had a positive anti-hbc result, while 110 (46.2%) of the 238 participants who tested negative for hbsag by elisa test had a positive anti-hbc (table 3). table 3: patterns of anti-hepatitis b core results for rapid and enzyme-linked immunosorbent assay tests for hepatitis b surface antigen and relationship of the two hepatitis b surface antigen detection assays in ibadan, nigeria, between april 2019 and may 2019. of the 232 blood donors that were hbsag-negative by any method, 107 (46.1%) were positive for anti-hbc. however, only one case was anti-hbc positive, hbsag-negative and hbv dna-positive (0.93%). discussion this study on hbc antibodies among replacement blood donors, who form a significant proportion of blood donors in nigeria, showed that 48.8% were positive for total anti-hbc (igg and igm), and over 60.0% of the donors had a tertiary education level, with 47.0% of the donors having an hbv infection knowledge score of 0. in addition, the risk factors for hbv acquisition among anti-hbc-positive donors were being older than 35 years, having poor knowledge of the hbv transmission route, and the use of sex enhancement recreational drugs. an inconsistent positivity rate was observed when rdt and elisa were used to screen for hbsag among blood donors. this incongruity in the results suggests that it is better to use both methods in screening blood for transfusion to reduce the escape of hbv-positive blood into the transfusion pool, especially when hbv dna screening is not in use. testing for anti-hbc has been used in blood transfusion in low hbv-endemic regions to minimise the incidence of post-transfusion hepatitis following transfusion of hbsag-negative blood. the assay identifies chronically infected low-level hbv donors.13 the prevalence of anti-hbc (35.7%) among our hbsag-negative blood donors is high. other studies have reported lower anti-hbc prevalence in low and intermediate hbv-endemic regions and in some high hbv-endemic regions like india14 and north africa.15 the higher prevalence of anti-hbc (35.7%) among the blood donors may be attributed to a higher number of blood donors who had current or past exposure to hbv since anti-hbc persists for life even though the hbv infection is resolved. the possibility of obi should be considered in hbsag-negative but anti-hbc-positive blood donors. occult hbv is more frequently diagnosed in anti-hbc-positive individuals than in anti-hbc-negative individuals.16,17 occult hbv has been reported to range from 3.0% to 17.0% in nigeria.18,19 the prevalence of anti-hbc antibody positivity among blood donors varies between and within countries, depending on the assay method. the anti-hbc test lacks specificity as test reagents reactivity varies between manufacturers, which may affect between-study comparisons.20 depending on the assay type and screening algorithms, false-reactivity rates ranged between 16.0% and 75.0%.21 the confirmatory algorithm is complex. however, a positive anti-hbc in a high hbv-endemic region establishes a history of hbv infection and portends a high risk of obi with the possibility of deferring the blood donor. japhet et al. from nigeria reported a lower anti-hbc prevalence of 13.0% and a higher prevalence of 19.6% hbsag.22 the lower anti-hbc prevalence was probably caused by the igm anti-hbc test used in that study, which detects acute infections but misses chronic hbv carriers, whereas the current study detected total anti-hbc (igm and igg). the shortcoming of the total anti-hbc study is that it includes all blood donors who had been exposed to hbv including those with resolved infection. this shortcoming may exaggerate the prevalence of hbv infection in high-prevalence regions, thereby shrinking the size of the eligible blood donor pool. dhawan et al. observed a significantly higher anti-hbc (8.4% vs 6.9%) among replacement donors than among voluntary donors in india. however, a wide range of prevalence of anti-hbc positivity has been reported among indian donors, and this has been attributed to the use of assays with varying sensitivities in different studies for screening the donors and the nature of the study population.23 this study suggests that donors who had ever used recreational drugs are likely to be positive for anti-hbc. therefore, stringent blood donor screening criteria using a standard questionnaire that provides confidentiality for donors in addition to educational materials to eliminate donors who had ever used recreational drugs may significantly reduce hbv-positive donors irrespective of whether they are voluntary or replacement blood donors. comparing this study with other studies that investigated total anti-hbc in nigeria, the prevalence of 48.5% is higher than 32.5% by ogunfemi et al. in ilorin,24 but lower than 60.7% and 90.0% from the studies by akinbami et al. in lagos18 and ojo et al. in ife.25 the 90.0% prevalence was reported pre-hbv vaccination. levels of anti-hbc have been shown to decrease in the donor population; this could be attributed to acquired immunity from vaccination and increased awareness.26 similarly, in germany, the trend for anti-hbc-positive status was 1.17% in 2007 but decreased to 0.72% in 2015.27 summarily, the variations in prevalence have been associated with the different assays used, study design and hbv endemicity. some studies investigated anti-hbc in hbsag-negative donors, while some studies investigated in both hbsag-negative and hbsag-positive donors.23,25,26,27 total anti-hbc+/hbsagdonors of 107 (46.1%) in our study is higher than igm anti-hbc+/hbsagdonors of 49 (18.1%) and 20 (4.4%) for maiduguri28 and ile-ife,29 nigeria. the higher anti-hbc+/hbsagdonors in our study could suggest a higher potential for hbv blood transmission from donors. however, this may not be the case because high anti-hbc prevalence does not always imply high obi frequency.27 although other studies reported a lower prevalence of anti-hbc in some nigerian blood centres, the anti-hbc prevalence in those blood centres is still significant to cause an increase in donor deferral rate and reduce blood supply if anti-hbc testing is added to the blood donor screening algorithm. the anti-hbc test included in screening blood donors in hbv non-endemic countries is not routinely used in hbv-endemic countries because many blood products would be discarded due to positive result even though most of the blood would be safe for transfusion.20 total anti-hbc-positivity was significantly higher among donors older than 35 years compared to younger donors, which is contrary to the statistically non-significant difference observed in ilorin, nigeria.23,24 studies from bangladesh, italy, and korea also reported high anti-hbc-positive status in older donors, which was attributed to the acquisition of hbv infections in earlier years when hbv was highly endemic.30,31,32 although the prevalence of hbv infection is still high in nigeria, declining rates have been reported.33 this study suggests that donors younger than 35 years may be less likely to have hbv infection or be an occult hbv carrier. occupation and educational level did not significantly affect the anti-hbc status of the donors. however, a good knowledge of the virus transmission was associated with a lower anti-hbc-positive rate, which suggests that ignorance about the virus increases vulnerability to infection. therefore, health education to increase awareness of hbv transmission and prevention could reduce infection among the future donor population. the finding of lower anti-hbc antibodies among those who share sharp objects in salons may not have any obvious explanation, but olayinka et al. reported in a study in nigeria that public barbing salon clipper cuts, manicure and pedicure cuts, and scarification were not significantly associated with the presence of hbsag.34 all blood donors who used recreational drugs were anti-hbc positive. blood donors who use recreational drugs and are positive should be identified and permanently deferred from donating blood. surprisingly, five blood donors who had hbv vaccination were anti-hbc positive. vaccine recipients who develop anti-hbc might not have responded to the vaccine. it may also be indicative of hbv infection with escape mutants.20,33 the anti-hbsag (anti-hbs) titre of the vaccinated donors was not determined, so we could not confirm if the vaccination provoked an immune response in them. sharing of sharp objects, being sexually active, the number of sex partners and unprotected sex evidenced by those with an sexually transmitted disease are risk factors for hbv infection35,36 but were not evident in our study. this might be due to the targeted study population and the study’s small sample size. the finding in this study does not rule out the danger associated with collecting blood from donors with risk factors. recruiting donors with risk factors is strongly discouraged, especially where exhaustive screening tests are not available. the screening of the donors with anti-hbc-positive and hbsag-negative sera for hbv dna showed that occult infection was present in one of the 107 donors (0.93%). the presence of hbv dna in 0.93% of our serologically screened donors suggests that there is a potential for transmission of hepatitis b to blood recipients. this is higher than 0.56% of blood donors in cameroon35 and 0.5% in ghana,36,37 but less than 11.54% – 14.5% reported in blood donors in egypt.37,38 this study establishes that hbv infection may occur among recipients of blood from donors that are isolated anti-hbc positive. it might be difficult to sustain the blood supply if all the 48.5% of the blood donors who have anti-hbc in their blood are deferred or rejected. implementing anti-hbc testing could make blood safer. since exclusion of all anti-hbc-positive donors may reduce the availability of safe blood in the blood banks of countries with high hbv prevalence, and screening all blood donors with nat to determine the hbv dna status may be too expensive in resource-limited regions, an algorithm that attempts to detect occult hbv is being suggested. the complementary use of both rdt and elisa for hbsag screening of blood donors should continue for maximum identification of hbsag-positive donors. the recommended algorithm includes testing all hbsag-negative blood (by both rdt and elisa) for antibodies to hbc and subjecting anti-hbc-positive blood to nat to identify potential infectious blood units. this will improve the safety of the blood supply and reduce costs while capturing samples that are likely to have undetected threats. limitations the confirmatory algorithms for true positivity of anti-hbc include secondary testing with an alternative elisa, testing for anti-hbs, anti-hbe or hbeag. the most frequently used is anti-hbs. however, this study did not investigate the anti-hbs titre of the participants. this might affect the significance of the observed anti-hbc and hbv dna positivity in the donor population. conclusion almost half of the donors in this study were anti-hbc positive, suggesting that a large proportion of the donors had been exposed to hbv. moreover, donors who were anti-hbc positive and hbsag-negative could have hbv dna. we propose a testing algorithm that can be utilised in hbv-endemic, resource-limited settings. all donors should be tested for hbsag (by both elisa and rdt); those who are hbsag-negative should be further tested for anti-hbc, while only those who are both hbsag-negative and anti-hbc positive should undergo mini-pool nat. this algorithm optimises blood safety and prevents hbv transmission from infected blood units. acknowledgements we acknowledge the cooperation and support of the blood bank donor section staff in carrying out this study. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions f.a.f. was involved in the conception and design of the study, analysis and interpretation of data, drafting of the article, and final manuscript review. f.a.f., a.a.f., a.o.f. and a.a. were involved in laboratory analysis. a.a.f. was involved in the design of the study. a.o.f. was involved in the analysis of data and review of the manuscript. a.a. was involved in the administration of questionnaires and sample collection. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability data that support the findings of this study are available from the corresponding author, f.f., upon reasonable request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references calderon gm, gonzález-velázquez f, gonzález-bonilla cr, et al. prevalence and risk factors of hepatitis c virus, hepatitis b virus, and human immunodeficiency virus in multiply transfused recipients in mexico. transfusion. 2009;49(10):2200–2207. https://doi.org/10.1111/j.1537-2995.2009.02248.x jayaraman s, chalabi z, perel p, guerriero c, roberts i. the risk of transfusion-transmitted infections in sub-saharan africa. transfusion. 2010;50(2):433–442. https://doi.org/10.1111/j.1537-2995.2009.002402.x allain j-p, cox l. challenges in hepatitis b detection among blood donors. curr opin hematol. 2011;18(6):461–466. https://doi.org/10.1097/moh.0b013e32834bac10 world health organization. global health sector strategy on viral hepatitis 2016 – 2021 towards ending viral hepatitis. geneva: world health organization; 2016. krajden m, mcnabb g, art b, petric m. the laboratory diagnosis of hepatitis b virus diagnostic l’hépatite b en laboratoire. can j infect dis med microbiol. 2005;16(2):450574. https://doi.org/10.1155/2005/450574 hoofnagle jh. posttransfusion hepatitis b. transfusion. 1990;30(5):384–386. zobeiri m. occult hepatitis b: clinical viewpoint and management. hepat res treat. 2013;2013:259148. https://doi.org/10.1155/2013/259148 mak ly, wong dkh, pollicino t, raimondo g, hollinger fb, yuen mf. occult hepatitis b infection and hepatocellular carcinoma: epidemiology, virology, hepatocarcinogenesis and clinical significance. j hepatol. 2020;73(4):952–964. https://doi.org/10.1016/j.jhep.2020.05.042 esposito a, sabia c, iannone c, nicoletti gf, sommese l, napoli c. occult hepatitis infection in transfusion medicine: screening policy and assessment of current use of anti-hbc testing. transfus med hemother. 2017;44(4):263–272. https://doi.org/10.1159/000460301 kupek e. residual risk of hepatitis-b-infected blood donations: estimation methods and perspectives. int sch res notices. 2013;2013:839896. https://doi.org/10.5402/2013/839896 barro l, drew vj, poda gg, et al. blood transfusion in sub-saharan africa: understanding the missing gap and responding to present and future challenges. vox sang. 2018;113(8):726–736. https://doi.org/10.1111/vox.12705 wang t, wang l, zhang j, dong w. a simplified universal genomic dna extraction protocol suitable for pcr. genet mol res. 2011;10(1):519–525. https://doi.org/10.4238/vol10-1gmr1055 fiedler sa, oberle d, churdy m, et al. effectiveness of blood donor screening by hiv, hcv, hbv-nat assays, as well as hbsag and anti-hbc immunoassays in germany (2008–2015). vox sang. 2019;114(5):443–445. https://doi.org/10.1111/vox.12770 maheswari ks, arun r, arunugam p. the prevalence of the hepatitis b core antibody and the occult hepatitis b infection among voluntary blood donors in chennai, india. j. clin. diagnostic res. 2012 dec;6(10):1710–1712. https://doi.org/10.7860/jcdr/2012/4826.2636 mohamed s, ezzadin f, faisal i, nagi g, kamel a, fatma a. anti-hbc and hbv-dna among blood donors in north africa; western libya. international blood research & reviews. 2015;3:152–159. https://doi.org/10.9734/ibrr/2015/18364 arora s, doda v, kirtania t. sensitivity of individual donor nucleic acid testing (nat) for the detection of hepatitis b infection by studying diluted nat yield samples. blood transfus. 2015;13(2):227–232. de la fuente ra, gutiérrez ml, garcia-samaniego j, fernández-rodriguez c, lledó jl, castellano g. pathogenesis of occult chronic hepatitis b virus infection. world j gastroenterol. 2011;17(12):1543–1548. https://doi.org/10.3748/wjg.v17.i12.1543 akinbami aa, oshinaike oo, dosunmu oa, et al. seroprevalence of hepatitis b e antigen (hbe antigen) and b core antibodies (igg anti-hbcore and igm antihbcore) among hepatitis b surface antigen positive blood donors at a tertiary centre in nigeria. bmc res notes. 2012;5(1):167. https://doi.org/10.1186/1756-0500-5-167 oluyinka oo, tong hv, bui tien s, et al. occult hepatitis b virus infection in nigerian blood donors and hepatitis b virus transmission risks. plos one. 2015;10(7):e0131912. https://doi.org/10.1371/journal.pone.0131912 seo dh, whang dh, song ey, han ks. occult hepatitis b virus infection and blood transfusion. world j hepatol. 2015;7(3):600–606. https://doi.org/10.4254/wjh.v7.i3.600 candotti d, laperche s. hepatitis b virus blood screening: need for reappraisal of blood safety measures? front med (lausanne). 2018;5:29. https://doi.org/10.3389/fmed.2018.00029 japhet mo, adesina oa, donbraye e, adewumi mo. hepatitis b core igm antibody (anti-hbcigm) among hepatitis b surface antigen (hbsag) negative blood donors in nigeria. virol j. 2011;8(1):513. https://doi.org/10.1186/1743-422x-8-513 dhawan h-k, marwaha n, sharma r-r, et al. anti-hbc screening in indian blood donors: still an unresolved issue. world j gastroenterol. 2008;14(34):5327–5330. https://doi.org/10.3748/wjg.14.5327 ogunfemi mk, olawumi ho, olokoba ab, et al. prevalence of antibody to hepatitis b core antigen among hepatitis b surface antigen-negative blood donors in ilorin, nigeria: a cross-sectional study. malawi med j. 2017;29(1):7. https://doi.org/10.4314/mmj.v29i1.7 ojo os, thursz m, thomas hc, et al. hepatitis b virus markers, hepatitis d virus antigen and hepatitis c virus antibodies in nigerian patients with chronic liver disease. east afr med j. 1995;72(11):719–721. niederhauser c. reducing the risk of hepatitis b virus transfusion-transmitted infection. j blood med. 2011;2:91–102. https://doi.org/10.2147/jbm.s12899 houareau c, offergeld r. anti-hbc screening is it worth the effort? results of a 10-year surveillance programme covering more than 30 million donations in germany. vox sang. 2019;114(5):459–466. https://doi.org/10.1111/vox.12781 jeremiah za, idris h, ajayi bb, ezimah acu, malah mb, baba mm. isolated antihbc-igm antibody among blood donors in the semi-arid region of nigeria. hum antibodies. 2011;20(3–4):77–82. https://doi.org/10.3233/hab-2011-0242 salawu l, adegoke ao, aboderin ao, huraina ha. hepatitis b viral markers in surface antigen negative blood donors: the need to look beyond antibody negativity. west afr j med. 2011;30(4):292–295. shil n, biswas j, khatun a, et al. incidence of anti-hbc antibody (igg and igm) among hbsag negative apparently healthy blood donors. bangabandhu sheikh mujib med univ j. 2016;9(4):201–204. https://doi.org/10.3329/bsmmuj.v9i4.30241 romanò l, velati c, cambiè g, fomiatti l, galli c, zanetti ar. hepatitis b virus infection among first-time blood donors in italy: prevalence and correlates between serological patterns and occult infection. blood transfus. 2013;11(2): 281–288. lim ya, yoon s. an experience of use of anti-hbc and anti hbs for blood donor screening tests at a tertiary hospital blood center in korea. korean j lab med. 2009;(1):59–65. https://doi.org/10.3343/kjlm.2009.29.1.59 olayinka at, oyemakinde a, balogun ms, et al. seroprevalence of hepatitis b infection in nigeria: a national survey. am j trop med hyg. 2016;95(4):902–907. https://doi.org/10.4269/ajtmh.15-0874 musa bm, bussell s, borodo mm, samaila aa, femi ol. prevalence of hepatitis b virus infection in nigeria, 2000–2013: a systematic review and meta-analysis. niger j clin pr. 2015;18(2):163–172. https://doi.org/10.4103/1119-3077.151035 fopa d, candotti d, tagny ct, et al. occult hepatitis b infection among blood donors from yaoundé, cameroon. blood transfus. 2019;17(6):403–408. allain j-p, candotti d, soldan k, et al. the risk of hepatitis b virus infection by transfusion in kumasi, ghana. blood. 2003;101(6):2419–2425. https://doi.org/10.1182/blood-2002-04-1084 el-zayadi ar, ibrahim eh, badran hm, et al. anti-hbc screening in egyptian blood donors reduces the risk of hepatitis b virus transmission. transfus med. 2008;18(1):55–61. https://doi.org/10.1111/j.1365-3148.2007.00806.x mahmoud ai, elsherbiny nm, afifi na, ahmed bm, yasin as. occult hepatitis b infection among blood donors in al azhar university hospital, upper egypt: the current status after 25 years of vaccine introduction. egypt j immunol. 2018;25(1):45–56. abstract introduction methods results discussion acknowledgements references about the author(s) andré trollip foundation for innovative new diagnostics (find) south africa, cape town, south africa renuka gadde becton, dickinson & company, franklin lakes, new jersey, united states tjeerd datema datos, leiden, the netherlands kamau gatwechi becton, dickinson & company, nairobi, kenya linda oskam datos, leiden, the netherlands zachary katz foundation for innovative new diagnostics (find), geneva, switzerland andrew whitelaw department of pathology, faculty of medicine and health sciences, stellenbosch university, south africa national health laboratory service, tygerberg hospital, cape town, south africa peter kinyanjui national public health laboratory, kenyatta national hospital, nairobi, kenya patrick njukeng global health systems solutions, isokolo, cameroon dawit a. wendifraw national clinical bacteriology and mycology reference laboratory, ethiopian public health institute, addis ababa, ethiopia ibrahimm mugerwa ministry of health, national health laboratories and diagnostic services-amr-national coordination centre, kampala, uganda grace najjuka national health laboratories and diagnostic services, kampala, uganda nicholas dayie department of medical microbiology, university of ghana medical school, accra, ghana japheth a. opintan department of medical microbiology, university of ghana medical school, accra, ghana heidi albert foundation for innovative new diagnostics (find) south africa, cape town, south africa citation trollip a, gadde r, datema t, et al. implementation of a customised antimicrobial resistance laboratory scorecard in cameroon, ethiopia and kenya. afr j lab med. 2022;11(1), a1476. https://doi.org/10.4102/ajlm.v11i1.1476 original research implementation of a customised antimicrobial resistance laboratory scorecard in cameroon, ethiopia and kenya andré trollip, renuka gadde, tjeerd datema, kamau gatwechi, linda oskam, zachary katz, andrew whitelaw, peter kinyanjui, patrick njukeng, dawit a. wendifraw, ibrahimm mugerwa, grace najjuka, nicholas dayie, japheth a. opintan, heidi albert received: 30 nov. 2020; accepted: 11 mar. 2022; published: 20 june 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: in low-resource settings, antimicrobial resistance (amr) is detected by traditional culture-based methods and ensuring the quality of such services is a challenge. the amr scorecard provides laboratories with a technical assessment tool for strengthening the quality of bacterial culture, identification, and antimicrobial testing procedures. objective: to evaluate the performance of the amr scorecard in 11 pilot laboratory evaluations in three countries also assessed with the stepwise laboratory quality improvement process towards accreditation (slipta) checklist. methods: pilot laboratory evaluations were conducted in cameroon, ethiopia and kenya between february 2019 and march 2019. assessors with previous slipta and microbiology experience were trained. assessors performed the laboratory assessments using the slipta and amr scorecard tools. results: weaknesses in technical procedures and the quality management systems were identified in all areas and all laboratories. safety had the highest mean performance score (slipta: 68%; amr scorecard: 73%) while management review had the lowest (slipta: 32%; amr scorecard: 8%) across all laboratories. the amr scorecard scores were generally consistent with slipta scores. the amr scorecard identified technical weaknesses in amr testing, and slipta identified weaknesses in the quality management systems in the laboratories. conclusion: since the amr scorecard identified important gaps in amr testing not detected by slipta, it is recommended that microbiology laboratories use slipta and the amr scorecard in parallel when preparing for accreditation. expanding the use of the amr scorecard is a priority to address the need for quality clinical microbiology laboratory services in support of optimal patient care and amr surveillance. keywords: antimicrobial resistance; laboratory; clinical; blood; urine; faeces. introduction antimicrobial resistance (amr) is a global problem, with resistant infections currently claiming at least 50 000 lives each year across europe and the united states alone and hundreds of thousands more in other areas of the world.1 knowledge of amr patterns is essential for optimal individual patient care, antimicrobial stewardship and amr surveillance.2 reviews of available data from africa have found a high level of resistance to commonly used antibiotics in the region.3,4 despite nine new african countries joining the world health organization’s global antimicrobial resistance surveillance system in 2020/2021, amr data are generally lacking in many lowand middle-income countries (lmics).5 in addition to this limited availability, there are also concerns over the quality of existing amr data.6 during the current coronavirus disease 2019 pandemic, antimicrobial stewardship activities have been impacted globally, requiring coordinated strategies to inform actions to reduce the potential longer-term impact on amr.7 while recent advances in molecular methods to detect amr are being increasingly implemented in high-income settings,8,9 these are not available in many lmics, and traditional culture-based diagnostic methods, performed by clinical microbiology services, remain the gold standard. ensuring the quality of such services is a challenge because, in addition to the pre-analytical, analytical and post-analytical phases that take place within the laboratory, there are numerous other key drivers of overall diagnostic quality,10 including clinical question formulation and test selection, test ordering, sample collection and transportation to the laboratory, testing and results reporting, test results interpretation, and patient follow-up for clinical management or referral for further testing. laboratories with weak systems have higher levels of errors, which can affect patient care and undermine the confidence that clinicians have in laboratory services.11,12 to address the coronavirus disease 2019 pandemic, laboratories are receiving new molecular and point-of-care technologies, thus increasing the number of samples processed and reinforcing the need for high-performing and high-quality laboratories and systems, particularly in lmics. significant advances have been made towards improving laboratory capacity and quality in disease areas such as hiv and tuberculosis. one example is the stepwise laboratory quality improvement process towards accreditation (slipta) initiative developed by the united states centers for disease control and prevention in collaboration with the american society for clinical pathology, the clinton health access initiative, and the world health organization regional office for africa to promote the uptake of quality improvement initiatives in lmic laboratories.13,14,15 however, only a few clinical microbiology laboratories in lmics have achieved any form of accreditation, that is, a formal recognition that their quality management system (qms) complies with international standards.16,17,18 although the slipta initiative has, to some extent, facilitated laboratory improvement in africa, it may not specifically address the quality of processes in amr laboratories, including sample culture, species identification, and susceptibility testing. while implementing qms elements is critical, improving compliance with a technical standard of testing is equally important. in recognition of the potential gap in the quality of amr-related testing, we developed the amr laboratory scorecard (amr scorecard),19 which aims to improve the appropriate use of diagnostics to identify pathogens and guide patient treatment and management, and to optimise the surveillance and early detection of amr. the amr scorecard focuses on priority specimen types such as blood, urine, and faecal samples, and includes the culture, pathogen detection, species identification, and antimicrobial susceptibility testing (ast) processes. the amr scorecard is designed to assess these technical processes for the priority pathogens reported to the global antimicrobial resistance surveillance system. this includes pathogens associated with hospital and community-acquired infections in which amr is reportedly increasing, threatening the use of key drugs.5 in this article, we describe the performance of the developed amr scorecard during pilot laboratory evaluations in three countries. methods ethical considerations this article describes the performance of the amr scorecard during pilot evaluations and does not require ethical clearance. this article followed all ethical standards for research without direct contact with human or animal subjects. study design pilot evaluations of the amr scorecard were conducted in cameroon, ethiopia, and kenya between february and march 2019. the countries were selected based on their enrolment in the global antimicrobial resistance surveillance system and engagement with becton dickinson or foundation for innovative new diagnostics (find), the global alliance for diagnostics, in ongoing laboratory strengthening activities. the laboratories in these five countries are representative of microbiology services across the diagnostic network from central level laboratories to district-level laboratories. assessors were selected and trained on the use of the amr scorecard before performing the pilot laboratory assessments. antimicrobial resistance scorecard the amr scorecard was developed based on the latest guidance and requirements for amr testing obtained from a review of existing tools, checklists, and guidelines, including those of the healthcare-associated infection surveillance india20 and the united states centers for disease control and prevention.21 the amr scorecard is based on the world health organization regional office for africa slipta checklist version 2:2015 and incorporates clinical microbiology laboratory-specific requirements linked to sub-clauses in the slipta checklist. it consists of three scorecard modules that are used to assess the technical procedures for processing blood, urine, and faecal samples. assessment of technical procedures includes questions to determine if isolation procedures (e.g. ‘are media used for primary culture of faeces incubated at 35 °c – 37 °c for at least 18 hours?’), identification procedures (e.g. ‘are gram stains performed for all blood cultures showing any sign of positive growth [e.g. turbidity, haemolysis, or gas production]?’) and ast procedures (e.g. ‘does the laboratory use combination disk test or another equivalent method for carbapenemase screening?’) are being performed according to microbiology best practices. the amr scorecard is designed to be used as a stand-alone internal assessment tool or as part of a comprehensive slipta assessment to ensure the application of slipta requirements to these test methods. the amr scorecard uses the same scoring convention and the same 12-section structure as the slipta checklist. individual amr testing modules are scored according to the percentage of requirements met in each modular checklist. however, unlike slipta, whose laboratory assessment is based on the international standards organization (iso) 15 18922 standard, the amr scorecard assessment compares technical laboratory practices against best practices for microbiology and amr. an amr scorecard etool was developed to supplement the hard-copy technical modules and slipta checklist and to allow automated analyses and reporting of the laboratory assessments.22 the etool consists of a general amr testing spreadsheet for recording responses to questions common to all the technical modules, spreadsheets for information specific to faeces, urine and blood, a spreadsheet for previous audit information, and a summary spreadsheet with automated analysis and visualisation of the technical assessment scores. a spreadsheet corresponding to the 12 sections of slipta is also provided to allow simultaneous slipta evaluations. the summary spreadsheet with automated analysis provides a detailed overview and visualisation of the slipta assessment scores. assessor training trainee assessors who had microbiology experience and had participated in slipta assessments in their laboratories (but were not necessarily african society for laboratory medicine slipta certified) were chosen to attend the training workshops. training on the amr scorecard was conducted in ethiopia from 11 to 13 february 2019, in kenya from 25 to 26 february 2019, in uganda on 18 february 2020, and in ghana on 26 february 2020. facilitators trained two assessors from cameroon, eight assessors from ethiopia, and six assessors from kenya on the interpretation of amr scorecard questions, the use of the etool, and the procedures for conducting the assessments. as all the trainee assessors were already familiar with the use of the slipta checklist, training on slipta was not provided. theoretical training was supplemented by practical assessments of three facilities (two in ethiopia and one in kenya). the national or reference laboratories were chosen for the practical assessment to provide trainee assessors exposure to all the procedures evaluated using the scorecard (table 1). all three laboratories performed basic urine and faeces culture. automated blood cultures were performed by two laboratories, one in ethiopia and one in kenya. the reference laboratories in ethiopia and kenya are iso 15189:2012 certified and perform automated identification and ast. the practical assessments were overseen by the facilitators. due to time constraints, slipta assessments were not performed during the training assessments. table 1: antimicrobial resistance laboratory scorecard assessment activities in 14 laboratories in cameroon, ethiopia, and kenya between february 2019 and march 2019. pilot laboratory assessments before commencing the pilot assessments at the respective laboratories, assessors introduced the amr scorecard to the laboratory head and quality officer. all the assessments were conducted using the slipta checklist and the amr scorecard and transferred to the etool for further analysis. assessors evaluated the laboratory operations based on the slipta checklist and amr scorecard items, recording scores for each item and documenting findings in detail. during the assessment, assessors reviewed laboratory documentation to verify that policies, manuals, and standard operating procedures were complete, current, and accurate. they also reviewed records and observed laboratory procedures to verify that amr policies were being followed and that laboratory procedures used were appropriate for the testing performed. in addition, the assessors determined the availability of functional and well-maintained equipment, reviewed data on the number of processed samples, isolates, contaminated cultures, and negative cultures for each sample type, and reviewed the internal quality control and external quality assessment results. these data provided assessors with an overview of the laboratories’ operations and allowed the identification of systemic technical issues not easily determined using slipta alone. following the assessments, the assessors provided feedback to the laboratory head, quality officer, and laboratory technologists. non-conformities with the iso 15189:2012 standard identified by slipta23 and non-conformities with microbiology best practices identified by the amr scorecard were tabulated and presented to the laboratory along with copies of the completed checklists. data management the results of the pilot laboratory assessments were transferred to the etool, which then automatically calculated the scores and totals for each section and generated a bar graph of laboratory performance by section. the amr scorecard results for blood, faeces, and urine were analysed with the slipta scores. results antimicrobial resistance laboratory scorecard pilot assessments in the pilot assessments conducted in this study, weaknesses in technical procedures and the qms were identified in all areas and all laboratories (figure 1). the mean amr scorecard assessment scores ranged between 8% (section 2: management reviews) and 73% (section 12: facilities and safety), and the mean slipta scores ranged between 32% (section 2: management reviews) and 68% (section 12: facilities and safety). figure 1: mean performance scores of 11 microbiology laboratories assessed with the antimicrobial resistance laboratory scorecard and slipta in cameroon, ethiopia, and kenya between february 2019 and march 2019. based on the amr scorecard assessments, all 11 laboratories performed best with all sample types in section 12: facilities and safety (range: 25% – 100%; mean: 73%). the weakest performance for all sample types in all laboratories was in section 2: management reviews (range: 0% – 13%; mean: 8%), followed by section 11: occurrence management (range: 0% – 71%; mean: 12%), and section 6: evaluations and audits (range: 0% – 100%; mean: 22%). technical issues with the processing of all sample types (isolation, identification, and ast) were identified in section 8: process control and internal and external quality assessment of the amr scorecard (range: 3% – 72%; mean: 46%). in 10 of 11 laboratories, data on the number of isolated pathogens and cumulative ast patterns were not collected and reported to the relevant oversight committees such as the antimicrobial stewardship committee, or hospital surveillance or outbreak team. technical issues with the processing of urine samples included failure to perform cell counts or wet preparations (6 of 11 laboratories), lack of rejection criteria (8 of 11 laboratories), lack of quality controls for media (4 of 11 laboratories), lack of antibiotic discs for ast (9 of 11 laboratories), and failure to use purity plates or standardised inocula for ast (7 of 11 laboratories). technical issues with the processing of faeces samples included failure to perform wet preparations for parasites (5 of 11 laboratories), shortage of selenite f broth and lack of sub-culture testing (8 of 11 laboratories), and failure to perform serological identification of either salmonella or shigella species (5 of 11 laboratories). technical issues with the processing of blood samples included failure to perform extended-spectrum beta-lactamase and carbapenemase detection tests (11 of 11 laboratories). as with the results of the amr scorecard assessments, the slipta assessment also identified section 12: facilities and safety to be the strongest area (range: 40% – 95%; mean: 68%) in all the laboratories, followed by section 3: organization and personnel (range: 9% – 91%; mean: 57%) and section 7: purchasing and inventory (range: 0% – 88%; mean: 56%). the weakest area was section 2: management reviews (range: 0% – 43%; mean: 28%). some reasons for low slipta scores included failure to conduct regular management reviews or audits (6 of 11 laboratories), and failure to collect and analyse quality indicators (6 of 11 laboratories). weaknesses specific to the qms were identified using slipta as these are not assessed by the amr scorecard. for example, the content of the quality manual (documents and records) was identified as a weakness in 7 of 11 laboratories. comparisons between the amr scorecard and slipta scores the assessment scores obtained using the amr scorecard and slipta were disaggregated by laboratory area and laboratory level (tables 2–4). two laboratories were designated as central, five as regional and four as district laboratories. the central level laboratories had a mean amr scorecard assessment score of 37% and a mean slipta score of 45%. the regional-level laboratories had a mean amr scorecard assessment score of 40% and a mean slipta score of 55%. the district-level laboratories had a mean amr scorecard assessment score of 29% and a mean slipta score of 39%. table 2: stepwise laboratory quality improvement process towards accreditation and amr laboratory scorecard mean assessment scores in two central microbiology laboratories (a and b) in cameroon and ethiopia between february 2019 and march 2019. table 3: stepwise laboratory quality improvement process towards accreditation and amr laboratory scorecard mean assessment scores in five regional microbiology laboratories (a–e) in cameroon, ethiopia, and kenya between february 2019 and march 2019. table 4: stepwise laboratory quality improvement process towards accreditation and amr laboratory scorecard mean assessment scores in four district microbiology laboratories (a–d) in cameroon, ethiopia, and kenya between february 2019 and march 2019. at least one laboratory performed poorly at each level. at the central level, laboratory a had the lowest performance, with a mean amr scorecard assessment score of 30% and a mean slipta score of 35%. at the regional-level, laboratory a had a mean amr scorecard assessment score of 6% and a mean slipta score of 8%. at the district-level, laboratory a had a mean amr scorecard assessment score of 15% and a mean slipta score of 17%. as these results suggest, the overall amr scorecard scores were similar to the overall slipta scores, irrespective of the laboratory performance. however, differences between the slipta and amr scorecard assessment scores in the different areas of the laboratory were noted at all laboratory levels. at the central laboratories, the largest differences between the amr scorecard and slipta scores occurred in section 2: management reviews (0% vs 25%), section 9: information management (42% vs 63%) and section 10: corrective action (10% vs 39%). at the regional laboratories, the largest differences between the amr scorecard and slipta scores occurred in section 1: documents and records (33% vs 59%), section 2: management reviews (10% vs 36%), section 9: information management (22% vs 60%), and section 11: occurrence management (7% vs 50%). finally, at the district laboratories, the largest differences between the amr scorecard and slipta scores occurred in section 1: documents and records (26% vs 47%), section 4: client management (17% vs 40%), and section 7: purchasing and inventory (18% vs 48%). where there were large differences (> 20%) in the scores, the slipta score was always higher. discussion quality clinical microbiology services are an essential element of the amr response that enable the appropriate use of antibiotics, improve amr surveillance, and reduce the development of resistance.24 while implementing qms elements in the microbiology laboratory is critical, improving compliance to a technical standard of testing is equally important. the amr scorecard provides laboratories with a specific technical assessment tool for strengthening the quality of culture, identification, and ast laboratory procedures. in the pilot assessments conducted in this study, the amr scorecard scores generally correlated well with the slipta scores. safety in the laboratory has been identified as an increasingly important subject because of the emergence of highly infectious diseases, including coronavirus disease 2019. although safety has traditionally been regarded as a low-priority issue in developing countries,25 in both the amr scorecard and slipta assessments, section 12: facilities and safety was found to be the strongest area in the laboratories. this focus on safety, even in laboratories with weak systems (e.g. regional laboratory a), is encouraging. the slipta assessments identified weaknesses in the qms of the microbiology laboratories assessed. in 6 of the 11 laboratories, weaknesses identified included failure to conduct regular management reviews or audits. these findings are consistent with previous reports that some of the weakest areas in the laboratory are internal auditing and the collection of quality indicator data.11,15,16,26 overall, six laboratories (55%) received zero stars (< 55%) using the official slipta system, with only two laboratories (18%) scoring two stars (between 65% and 74%; regional laboratory b) and three stars (between 75% and 84%; regional laboratory d). the slipta scores in this study are consistent with the slipta scores from 47 countries worldwide, including 23 countries in africa, assessed using the strengthening laboratory management toward accreditation methodology prior to the initiation of laboratory strengthening activities (i.e. at baseline). yao et al.16 found that the mean score in these laboratories at baseline was 39% (median 37%), with 84% of the laboratories scoring zero stars (i.e. score < 55%). it has been suggested that microbiology laboratories are trailing other clinical laboratories in achieving accreditation.27 the overall poor performance recorded at each laboratory level suggests that providing quality microbiology services is a challenge across the tiered network. the exclusion of national or reference microbiology laboratories in the assessments likely resulted in the lower overall scores observed in the pilot. while national or reference microbiology laboratories were assessed using the amr scorecard during the training, slipta assessments were not conducted due to time constraints and thus the amr scorecard results were not included in this report. in settings with limited resources, strengthening technical testing and qms (including accreditation) is often initiated at the national level, suggesting that overall scores may have been higher had they been included. by providing a specific technical focus, the amr scorecard identified important gaps in amr technical testing not detected by slipta alone. the amr scorecard assesses the step-by-step procedures for sample processing, bacterial isolation and identification, and ast. approximately 44% of the amr scorecard focuses on these procedures in contrast to slipta which has a limited focus on technical procedures. topics covered by slipta are also covered by the amr scorecard but the latter focuses on the specific details related to amr. for example, slipta assesses whether standard operating procedures for laboratory functions and technical and managerial procedures are available, while the amr scorecard assesses whether the laboratory has, for example, documented procedures for microscopic examination and urine cell count. as the amr scorecard identified important gaps in amr testing not detected by slipta alone, and slipta identified specific weaknesses in the qms that were beyond the scope of the amr scorecard, it is recommended that microbiology laboratories that require a comprehensive assessment or are developing their qms through continuous improvement toward accreditation be assessed using both the slipta and the amr scorecard in parallel. thus, the amr scorecard can be used as an entry point into the qms journey, with laboratories choosing to apply for slipta certification (or iso certification) after reaching a satisfactory level (equivalent to 3–4 stars on slipta). it should be noted that the official star recognition system provided by african society for laboratory medicine can only be obtained through the slipta certification provided by the african society for laboratory medicine secretariat.28 the importance of data collection and analysis is also highlighted in several questions in the amr scorecard (e.g. ‘are the following performance indicators collected – number and percentage of urine cultures with cell counts > 105 cells/ml?’) and assessors are encouraged to assist laboratories to collect and analyse their data. the clinical laboratory is a major source of healthcare data that can be used to inform health system-wide actions meant to improve diagnostic test utilisation, service efficiency, and patient outcomes.29 in these evaluations, cumulative quality indicator data on isolated pathogens and ast were not collected by 10 of the 11 laboratories assessed. this was due to the lack of automated instruments or an electronic laboratory information system, meaning that staff were required to manually record and calculate isolation rates and ast patterns. in one laboratory, the compilation of data by the assessors exposed a very low isolation rate of enteric pathogens that required further investigation. quality indicator data, if available, could have been used to identify the cause of the low isolation rate, thereby improving the quality of enteric bacterial culture. in addition to improving the quality of laboratory testing, laboratory data can also be used to influence clinical decisions. for example, cumulative data on pathogens and ast results can be used to inform treatment guidelines. however, for laboratory data to impact health systems in such a way, the laboratory needs to carefully consider how the data are collated, communicated, and disseminated. data from the assessments in this pilot revealed that 10 of the 11 laboratories failed to collect data on cumulative ast patterns and report these data to oversight committees, thereby missing the opportunity to inform antimicrobial stewardship decisions with laboratory data. in the pilot assessments, where there were differences between the amr scorecard assessment and slipta scores, the slipta scores were consistently higher. in addition to the differences in the content of the two assessment tools, other factors may contribute to this finding. first, the laboratories included in the pilot were not part of any active and ongoing programmes to improve laboratory quality such as the strengthening laboratory management toward accreditation programme.15 only 2 of the 11 laboratories reported a previous slipta assessment. it is expected that laboratories participating in quality improvement programmes are more likely to score higher on both assessment tools. second, the amr scorecard is based on microbiology best practices and not on an iso standard such as slipta. without a standard to guide preparations, it may not be clear to laboratories what requirements need to be in place to assure quality amr testing. in this respect, the amr scorecard has a role to play in educating laboratories regarding the technical requirements for amr testing. there were several challenges to implementing the amr scorecard in the initial cohort of laboratories, including procurement of funding support for quality improvement and provision of cover for the trainers and mentors during programme-related absences. mentoring of laboratories has been reported to be an important component of successful and sustainable quality improvement initiatives;30 thus, it is important to ensure that enough resources in terms of funding and personnel are put in place to allow mentoring to take place. ideally, laboratories should be mentored by reference laboratories within the amr surveillance network. based on feedback from facilitators and assessors, the amr scorecard needed to be revised to strengthen identified weaknesses. suggested changes included revising the language of some questions and adding ‘not applicable’ options to others. two additional questions to determine whether laboratories were performing extended-spectrum beta-lactamase and carbapenemase screening on faecal samples were added to the faeces module. this increased the score of the faeces module by four points. the total score of the blood and urine modules remained unaltered in the revised amr scorecard. in 2020, the structure of the amr scorecard was changed, and additional scorecards were added to allow for assessments of other sample types including pulmonary, genital, and wound samples. evaluations of the revised amr scorecard were performed in three laboratories in ghana and uganda. limitations the microbiology laboratories selected for the pilot of the amr scorecard do not represent the scope of microbiology technical abilities across africa. while care was taken during the selection of laboratories for the study, the amr scorecard may be less useful in identifying and addressing gaps that impact the quality of testing in certain settings. in addition, all assessments, except those performed in cameroon, included find and becton dickinson facilitators. as these facilitators were involved in the development of the amr scorecard, they may have influenced the outcomes of the assessments in favour of the amr scorecard. conclusion this study showed that a customised scorecard to guide the establishment and strengthening of amr testing quality in resource-limited settings can assist in identifying and addressing quality gaps. the amr scorecard, used in conjunction with slipta, found important gaps in the procedures for identification and ast of priority pathogens that were not identified by slipta alone. expanding the use of this scorecard will help address the need for quality clinical microbiology laboratory services to support optimal patient care and amr surveillance. acknowledgements the authors are grateful to the ethiopia public health institute, kenya national public health laboratory service, and the ministry of health, cameroon. we wish to thank john nkengasong from the africa centres for disease control and prevention (africa) for his contribution to the conceptualisation of the amr scorecard and his guidance and support during its development. we are also grateful to cecilia ferreyra and cassandra kelly-cirino from find, and courtney maus, namita singh, kartik sharma, christine claire cruz, nermin hamurcu, and nuphar rozen-adler from becton dickinson for their support on this project. we wish to acknowledge the participating laboratories and facilities, the fleming fund, and uk united kingdom aid direct for support of the uganda and ghana sites, as well as the assessment teams in cameroon, ethiopia, and kenya. editorial assistance for later drafts was provided by rachel wright, phd, funded by find. competing interests the authors have the following competing interests: becton dickinson and find provided support for this study in the form of salaries for employees and financial support to the partner organisations and assessors. foundation for innovative new diagnostics and becton dickinson jointly developed the amr scorecard. foundation for innovative new diagnostics has partnership agreements with various diagnostic manufacturers for the development and implementation of amr diagnostic tools. foundation for innovative new diagnostics receives funding from multiple public and private donors for tuberculosis projects in technology development and clinical research. limited funding is occasionally provided by industry partners; acceptance of these funds is subject to review by an independent scientific advisory committee or another independent review body. becton dickinson is a global medical technology company that manufactures and supplies products and solutions pertaining to medical discovery, diagnostics, and the delivery of care. becton dickinson global health has longstanding collaborations with health agencies and is currently implementing projects in africa to strengthen laboratory quality including the use of the amr scorecard and improve laboratory capacity, including installation of becton dickinson equipment and providing broader systems. authors’ contributions a.t., t.d., l.o., a.w., k.g., z.k., r.g., and h.a. contributed to the development of the amr scorecard. a.t., d.a.w., g.n., j.a.o., k.g., i.m., p.k., p.n., and n.d. were responsible for the assessments. a.t., h.a. k.g., and r.g. were responsible for data analysis and preparation of the manuscript. all authors reviewed the manuscript and agreed with its content. sources of support this study was funded by becton dickinson and find through a grant from the department for international development, united kingdom. data availability the recorded assessment results and data sets generated or analysed during the current study are available from the corresponding author, a.t., on request. disclaimer the findings and conclusions in this publication are those of the authors and do not necessarily represent the official position of becton dickinson or find. references o’neill j. review on antimicrobial resistance: tackling a crisis for the health and wealth of nations [homepage on the internet]. 2014 [cited 2020 nov]. available from: https://amr-review.org/sites/default/files/amrreviewpaper-tacklingacrisisforthehealthandwealthofnations_1.pdf tangcharoensathien v, chanvatika s, sommanustweechaia a. complex determinants of inappropriate use of antibiotics. bull world health organ. 2018;96:141–144. https://doi.org/10.2471/blt.17.199687 ampaire l, muhindo a, orikiriza p, mwanga-amumpaire j, bebell l, boum y. a review of antimicrobial resistance in east africa. afr j lab med. 2016;5(1):432. https://doi.org/10.4102/ajlm.v5i1.432 leopold sj, van leth f, tarekegn h, schultsz c. antimicrobial resistance among clinically relevant bacterial isolates in sub-saharan africa: a systematic review. j antimicrob chemother. 2014;69(9):2337–2353. https://doi.org/10.1093/jac/dku176 who. global antimicrobial resistance and use surveillance system (glass) report 2021. geneva: world health organization; 2021. tadesse b, ashley e, ongarello s, et al. antimicrobial resistance in africa: a systematic review. bmc infect dis. 2017;17(1):616. https://doi.org/10.1186/s12879-017-2713-1 rawson tm, ming d, ahmad r, et al. antimicrobial use, drug-resistant infections and covid-19. nat rev microbiol. 2020;18:409–410. https://doi.org/10.1038/s41579-020-0395-y anjum m, zankari e, hasman h. molecular methods for detection of antimicrobial resistance. microbiol spectr. 2017;5(6):1–17. https://doi.org/10.1128/microbiolspec.arba-0011-2017 boolchandani m, d’souza a, dantas g. sequencing-based methods and resources to study antimicrobial resistance. nat rev genet. 2018;20(6):356–370. https://doi.org/10.1038/s41576-019-0108-4 mesfin e, taye b, belay g, ashenafi a, girma v. factors affecting quality of laboratory services in public and private health facilities in addis ababa, ethiopia. ejifcc. 2017;28(3):205–223. albert h, trollip a, erni d, et al. developing a customised approach for strengthening tuberculosis laboratory quality management systems toward accreditation. afr j lab med. 2017;6(2):576. https://doi.org/10.4102/ajlm.v6i2.576 peter tf, rotz pd, blair dh, khine a-a, freeman rr, murtagh mm. impact of laboratory accreditation on patient care and the health system. am j clin pathol. 2010; 134(4):550–555. https://doi.org/10.1309/ajcph1skq1hnwghf alemnji ga, zeh c, yao k, fonjungo pn. strengthening national health laboratories in sub-saharan africa: a decade of remarkable progress. trop med int health. 2014;19(4):450–458. https://doi.org/10.1111/tmi.12269 gershy-damet gm, rotz p, cross d et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. https://doi.org/10.1309/ajcptuuc2v1wjqbm yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3):401–409. https://doi.org/10.1309/ajcpnbbl53fwuiqj yao k, luman et, slmta collaborating authors. evidence from 617 laboratories in 47 countries for slmta-driven improvement in quality management systems. afr j lab med. 2014;3(2):262. https://doi.org/10.4102/ajlm.v3i2.262 kibet e, moloo z, ojwang p, sayed s, mbuthia a, adam r. measurement of improvement achieved by participation in international laboratory accreditation in sub-saharan africa: the aga khan university hospital nairobi experience. am j clin pathol. 2014;141(2):188–195. https://doi.org/10.1309/ajcpv8a9mrwhgxef petti ca, polage cr, quinn tc, et al. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377–382. https://doi.org/10.1086/499363 find. amr laboratory scorecard package [homepage on the internet]. [cited 2020 nov]. available from: https://www.finddx.org/amr-lab-scorecard-package/ healthcare associated infection surveillance india. all india institute of medical sciences (aiims), centers for disease control and prevention (cdc), indian council of medical research (icmr). laboratory antibacterial resistance surveillance readiness tool [homepage on the internet]. 2017 [cited 2020 nov]. available from: https://www.haisindia.com/upload/fileuploads/1527836102_lab.%20assessment.pdf centers for disease control and prevention. lab assessment of antibiotic resistance testing capacity (laarc) [homepage on the internet]. 2021 [cited 2020 nov]. available from: https://www.cdc.gov/drugresistance/intl-activities/laarc.html iso 15189. medical laboratories – requirements for quality and competence. geneva: international organization for standardization; 2012. african society for laboratory medicine. who afro slipta checklist [homepage on the internet]. 2007 [cited 2020 nov]. available from: http://www.afro.who.int/en/clusters-a-programmes/hss/blood-safety-laboratories-a-health-technology/blt-highlights/3859-who-guide-for-the-stepwise-laboratory-improvement-process-towards-accreditation-in-the-african-region-with-checklist.html wilson ml. assuring the quality of clinical microbiology test results. clin infect dis. 2008;47(8):1077–1082. https://doi.org/10.1086/592071 ejilemele aa, ojule ac. health and safety in clinical laboratories in developing countries: safety considerations. niger j med. 2004;13(2):182–188. maina rn, mengo dm, mohamud ad, et al. progressing beyond slmta: are internal audits and corrective action the key drivers of quality improvement? afr j lab med. 2014;3(2):222. https://doi.org/10.4102/ajlm.v3i2.222 jacobs j, hardy l, semret m, et al. diagnostic bacteriology in district hospitals in sub-saharan africa: at the forefront of the containment of antimicrobial resistance. front med (lausanne). 2019;6:205. https://doi.org/10.1086/592071 african society for laboratory medicine. slipta [homepage on the internet]. 2021. available from: https://aslm.org/what-we-do/#slipta shirts b, jackson b, baird g, et al. clinical laboratory analytics: challenges and promise for an emerging discipline. j pathol inform. 2015;6:9. https://doi.org/10.4103/2153-3539.151919 maruta t, motebang d, mathabo l, rotz pj, wanyoike j, peter t. impact of mentorship on who-afro strengthening laboratory quality improvement process towards accreditation (slipta). afr j lab med. 2012;1(1):6. https://doi.org/10.4102/ajlm.v1i1.6 abstract introduction methods results discussion acknowledgements references about the author(s) dawood da costa division of medical microbiology and immunology, department of pathology, faculty of medical and health sciences, stellenbosch university, cape town, south africa national health laboratory service, tygerberg hospital, cape town, south africa pieter nel division of medical microbiology and immunology, department of pathology, faculty of medical and health sciences, stellenbosch university, cape town, south africa national health laboratory service, tygerberg hospital, cape town, south africa citation da costa d, nel p. re-decontamination of liquid mycobacterial cultures: additional mycobacterium tuberculosis yield in the era of xpert mtb/rif ultra in cape town, south africa. afr j lab med. 2021;10(1), a1529 https://doi.org/10.4102/ajlm.v10i1.1529 brief report re-decontamination of liquid mycobacterial cultures: additional mycobacterium tuberculosis yield in the era of xpert mtb/rif ultra in cape town, south africa dawood da costa, pieter nel received: 15 jan. 2021; accepted: 26 july 2021; published: 10 dec. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract a retrospective review of liquid mycobacterial cultures was performed at a laboratory in south africa from 01 january 2018 to 31 december 2018 to assess the increased yield in detecting mycobacterium tuberculosis complex following sample re-decontamination. only 9 of 99 (9%) re-decontaminated samples were culture positive for m. tuberculosis complex. xpert mtb/rif ultra, concurrently performed on 7 of the 9 samples, detected m. tuberculosis complex in all but 1 sample. re-decontamination of non-sterile samples did not increase the m. tuberculosis complex yield enough to offset the financial costs and additional labour in a laboratory that utilises the xpert mtb/rif ultra system as a first-line diagnostic modality. keywords: tuberculosis culture, liquid mycobacterial growth indicator tube culture, decontamination, re-decontamination, xpert mtb/rif ultra. introduction tuberculosis is caused by mycobacterium tuberculosis, which belongs to a group of closely related, slow-growing mycobacteria collectively referred to as the m. tuberculosis complex. in 2018, there were an estimated 10 million cases of tuberculosis globally, with seven million cases notified and 1.45 million tuberculosis-related deaths1. south africa, in the same year, had an estimated 301 000 new tuberculosis cases1. detection of m. tuberculosis complex by culture method remains the gold standard for confirming tuberculosis disease.2 it can detect as few as 10 m. tuberculosis complex colony-forming units (cfu) per ml in clinical specimens.3 liquid culture increases the number of bacteriologically confirmed cases of tuberculosis by 20% – 30%, even when rapid, sensitive nucleic acid amplification tests such as the xpert ultra (cepheid, sunnyvale, california, united states)3,4 are used. culture is advantageous as it allows for phenotypic drug susceptibility testing of the isolates. processing of specimens for culture is, however, costly, labour intensive, expertise requiring, and time-consuming (requiring up to six weeks to process to completion) owing to the slow proliferation time of m. tuberculosis complex.5,6,7 the xpert ultra assay is a closed, cartridge-based, nucleic acid real-time polymerase chain reaction system that allows for the simultaneous detection of m. tuberculosis complex and rifampicin susceptibility in under 2 h. it can be performed directly on clinical samples and has a 5 cfu/ml – 25 cfu/ml limit of detection; but, it has a lower sensitivity than tuberculosis culture in samples from people living with hiv and on samples where no acid-fast bacilli (afb) are visualised on microscopy (smear negative).3,4 clinical specimens from non-sterile sites that are submitted to the mycobacteriology laboratory may be contaminated by other more rapid-growing bacteria.5,8 these specimens undergo a digestion-decontamination procedure as recommended by the world health organization. the n-acetyl-l-cysteine sodium hydroxide digestion-decontamination allows m. tuberculosis to be cultured in a liquid culture medium despite reducing the organism viability by between 20% and 30%.3,6,8,9 a proportion of cultures will however remain contaminated despite standard decontamination procedures. an acceptable rate for contaminated cultures in liquid media is 5% – 8%.5,6,7,8,10 global recommendations for re-decontamination exist,7 but no published data on an acceptable compliance level for re-decontamination could be found. the world health organization recommends that re-decontamination of contaminated liquid culture be performed when the first of two submitted cultures is ‘positive for m. tuberculosis and contaminated’ and the specimen requires drug susceptibility testing, while the second culture is negative for m. tuberculosis, and if the mycobacterial protein antigen 64 result is indeterminate, owing to the presence of contaminants. the aims and objectives of the study were to determine the contamination rate at the tygerberg hospital (tbh) mycobacteriology laboratory; assess the increase in m. tuberculosis complex yield following re-decontamination in samples that had undergone xpert ultra testing; to assess laboratory non-compliance rates with regard to the recommended sample re-decontamination protocol; and to perform a cost analysis of the re-decontamination of specimens. methods ethical considerations ethical approval for this laboratory-based study was obtained from the health research ethics committee, stellenbosch university, south africa, (project identification: 14939, hrec x20/04/016). informed consent was waived by the stellenbosch university ethics committee for this laboratory-based study. data remained confidential throughout the study. study setting this study was conducted at south africa’s national health laboratory service (nhls) division of medical microbiology and immunology mycobacteriology laboratory located in tbh. annually, the tbh mycobacteriology laboratory processes approximately 10 000 specimens for m. tuberculosis complex culture from tbh. all samples from non-sterile sites for m. tuberculosis complex culture undergo decontamination with 1.25% n-acetyl-l-cysteine sodium hydroxide and the decontaminated samples are processed according to the becton-dickinson mycobacterial growth indicator tube (mgit) testing protocol.5 specimen re-decontamination is performed on specimens from anatomical sites that are not easily obtainable or contaminated specimens that are microscopy positive for afb. easily obtainable anatomical specimens from non-sterile sites, such as urine and sputum specimens, are not re-decontaminated.11,12 study design and data analysis a retrospective study was conducted to determine the number of m. tuberculosis complex cultures performed from 01 january 2018 to 31 december 2018. data were electronically extracted from the nhls central data warehouse into a microsoft excel (microsoft office 2016, microsoft corporation, redmond, washington, united states) datasheet. results were anonymised and stratified according to culture status: negative, positive or contaminated. contaminated samples underwent further analysis to determine eligibility for re-decontamination; compliance rates were calculated, and results were verified on the nhls database, trakcare webview (trakcare lab version l6.10, 2012, intersystems corporation, cambridge, massachusetts, united states). results a total of 9585 m. tuberculosis complex cultures were performed; 8049 (82.1%) were culture-negative, 912 (9.3%) were positive for m. tuberculosis complex, 31 (0.3%) were positive for non-tuberculous mycobacteria, and 593 (6.0%) were contaminated. a total of 139 samples were assessed for re-decontamination, of which 99 (71%) were appropriately re-decontaminated, 37 (29%) were appropriately denied re-decontamination (due to having multiple samples incubating), and 3 (2.2%) were inappropriately denied re-decontamination. three samples were eligible for re-decontamination but did not undergo re-decontamination: two were sputum samples, in which afb was observed on the contaminated culture and had xpert ultra testing which detected rifampicin-susceptible m. tuberculosis complex, and one was a bronchoalveolar lavage sample in which a second sample was contaminated. non-compliance to re-decontamination was low, with 97.8% of samples correctly assessed for re-decontamination. of the 99 re-decontaminated samples, 75 (76%) were culture-negative, 5 (5%) contaminated, 10 (10%) positive for non-tuberculous mycobacteria and nine (9%) were positive for m. tuberculosis complex. of 99 re-decontaminated samples, 89 samples were from anatomical sites not easily obtainable and 10 were samples that were microscopy positive for afb. of the 10 contaminated samples that were microscopy positive for afb and that underwent re-decontamination, all were sputum samples; non-tuberculosis mycobacteria were isolated from three samples and m. tuberculosis was isolated from seven. only six of the seven samples that had m. tuberculosis complex isolated on culture underwent xpert ultra testing and were all positive for m. tuberculosis complex. in summary, only 9 of 99 (9%) re-decontaminated samples were culture positive for m. tuberculosis complex. on xpert ultra testing, 6 of the 9 (67%) tested positive for m. tuberculosis complex, 1 (11%) tested negative for m. tuberculosis complex, and 2 (22%) did not undergo xpert ultra testing (but were smear positive for afb on an auramine stain). discussion we found that 6% of all specimens undergoing mycobacterial culture at the tbh mycobacteriology laboratory were contaminated, which is in keeping with internationally accepted contamination standards of 5% – 8% in liquid media.5,6,7,8,10 non-compliance to the recommended re-decontamination standard operating procedure was low at 2.2%. these three samples were not re-decontaminated as the xpert ultra had detected m. tuberculosis complex on two samples, and the third sample had additional specimens still incubating. we found that of the seven re-decontaminated samples that were microscopy positive for afb and culture positive for m. tuberculosis complex, six (86%) were also xpert ultra positive for m. tuberculosis complex. a diagnostic accuracy study of the xpert ultra by dorman et al., with study participants from south africa, found the sensitivity for smear-positive m. tuberculosis complex to be 99%4 suggesting that xpert ultra would likely have detected m. tuberculosis complex in the sample that was not tested. in our setting, re-decontamination of samples that have undergone xpert ultra testing only yielded one additional m. tuberculosis complex isolate and although the sample size to identify the additional positive m. tuberculosis complex yield is small, to our knowledge this is the first study assessing the additional m. tuberculosis complex yield in re-decontaminated samples. the correlation between xpert ultra and m. tuberculosis complex positivity in re-decontaminated samples also reflects the excellent utility of xpert ultra testing as the initial diagnostic test in the south african national tuberculosis-testing algorithm. this finding is likely due to the xpert ultra’s lower m. tuberculosis complex detection limit of 5 cfu/ml – 25 cfu/ml compared to its predecessor xpert mtb/rif.3 currently, the cost of re-decontamination and additional liquid mgit culture at tbh nhls amounts to r79.22 (south african rand; approximately, $5.00 united states dollars) per sample. when considering the low additional m. tuberculosis complex yield, added labour, and long turnaround time to final culture result, re-decontamination is not a cost-effective option in the setting where xpert ultra is used as the initial diagnostic test. limitations this was a laboratory-based study using routinely available data. the treatment status of patients who submitted samples could not be obtained, which may have impacted on m. tuberculosis complex yield following re-decontamination. owing to the small sample size eligible for re-decontamination, and varying laboratory decontamination protocols, the findings in this study may not allow generalisation of our findings to other centres. conclusion the poor increase in yield of m. tuberculosis complex after re-decontamination of samples reflects the efficiency of the south africa tuberculosis-testing algorithm, which employs xpert ultra testing, that has low limit of detection. in our high-burden tuberculosis setting, routine re-decontamination is not cost-effective and not recommended in specimens that have undergone xpert ultra testing. acknowledgements we thank the nhls for access to their electronic central data warehouse and mr j. goodway for electronic data extraction. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions d.d.c. was responsible for the concept and study design, analysis and interpretation of data, and writing of the manuscript. d.d.c. and p.n. were involved in the critical revision and approval of the final manuscript. sources of support the authors received no financial support for the research, authorship or publication of this article. data availability the data that support the findings of this study are available from the corresponding author, d.d.c., upon reasonable request. disclaimer the views expressed in the submitted article are the authors’ own and not an official position of the institution. references world health organization. global tuberculosis report. geneva, switzerland: who; 2019. lewinsohn dm, leonard mk, lobue pa, et al. official american thoracic society/infectious diseases society of america/centers for disease control and prevention clinical practice guidelines: diagnosis of tuberculosis in adults and children. clin infect dis. 2017;64(2):e1–e33. https://doi/org/10.1093/cid/ciw694 chakravorty s, simmons am, rowneki m, et al. the new xpert mtb/rif ultra: improving detection of mycobacterium tuberculosis and resistance to rifampin in an assay suitable for point-of-care testing. mbio. 2017;8(4):1–12. https://doi/org/10.1128/mbio.00812-17 dorman se, schumacher sg, alland d, et al. xpert mtb/rif ultra for detection of mycobacterium tuberculosis and rifampicin resistance: a prospective multicentre diagnostic accuracy study. lancet infect dis. 2018;18(1):76–84. https://doi/org/10.1016/s1473-3099(17)30691-6 siddiqi sh, rüsch-gerdes s. mgit procedure manual for bactec mgit960 tb system. geneva, switzerland: foundation for innovative new diagnostics; 2006. kent pt, kubica gp. public health mycobacteriology: a guide for the level iii laboratory. atlanta, georgia: centre for disease control; 1985. fitz-gerald m, lumb r, who global tb programme. laboratory safety – the handbook global edition [homepage on the internet]. geneva: gli working group secretariat; 2019. [cited 2020 oct 23]. available from: http://www.stoptb.org/wg/gli garcia ls. clinical microbiology procedures handbook. third edit. (garcia ls, ed.). santa monica, california: asm press; 2007. mtafya b, sabiiti w, sabi i, et al. molecular bacterial load assays concurs with culture on naoh-induced loss of mycobacterium tuberculosis viability. j clin microbiol. 57(7). https://doi/org/10.1093/jac/dku415 stop tb partnership (world health organization). childhood tb subgroup. global laboratory initiative advancing tb diagnosis. mycobacteriology laboratory manual. first edit. geneva, switzerland: global laboratory initiative; 2014. goodway j, rautenbach c. processing of positive mgit vials: national health laboratory service tygerberg laboratory standard operating procedure mic1223. cape town, south africa: nhls; 2019. rautenbach c, goodway j. tb cultures: national health laboratory service tygerberg laboratory standard operating procedure mic1222. cape town, south africa: nhls; 2019. abstract introduction methods results discussion acknowledgements references about the author(s) felicity gopolang department of global health, schools of public health and medicine, international training and education center for health (i-tech), university of washington, seattle, washington, united states fales zulu-mwamba laboratory services unit, directorate of clinical care and diagnostic services, ministry of health zambia, lusaka, zambia davy nsama laboratory services unit, directorate of clinical care and diagnostic services, ministry of health zambia, lusaka, zambia annika kruuner zambart, lusaka, zambia dailes nsofwa laboratory quality management systems, centers for disease control and prevention (cdc) zambia, lusaka, zambia ishmael kasvosve faculty of health sciences, university of botswana, gaborone, botswana royce gomo immunogene labs, ruwa, zimbabwe tiny motlhabane medical laboratory technology department, institute of health sciences, gaborone, botswana bhavna chohan department of global health, schools of public health and medicine, international training and education center for health (i-tech), university of washington, seattle, washington, united states olusegun soge department of global health, schools of public health and medicine, international training and education center for health (i-tech), university of washington, seattle, washington, united states daniel osterhage department of global health, schools of public health and medicine, international training and education center for health (i-tech), university of washington, seattle, washington, united states nancy campbell department of global health, schools of public health and medicine, international training and education center for health (i-tech), university of washington, seattle, washington, united states michael noble department of pathology and laboratory medicine, university of british columbia, vancouver, british columbia, canada ann downer department of global health, schools of public health and medicine, international training and education center for health (i-tech), university of washington, seattle, washington, united states jean-frederic flandin department of global health, schools of public health and medicine, international training and education center for health (i-tech), university of washington, seattle, washington, united states anya nartker department of global health, schools of public health and medicine, international training and education center for health (i-tech), university of washington, seattle, washington, united states catherine koehn department of global health, schools of public health and medicine, international training and education center for health (i-tech), university of washington, seattle, washington, united states linda k. nonde hiv and aids twinning center program, american international health alliance (aiha), lusaka, zambia aaron shibemba laboratory services unit, directorate of clinical care and diagnostic services, ministry of health zambia, lusaka, zambia clement b. ndongmo center for disease control and prevention (cdc) zambia, lusaka, zambia martin steinau center for disease control and prevention (cdc) zambia, lusaka, zambia lucy a. perrone department of global health, schools of public health and medicine, international training and education center for health (i-tech), university of washington, seattle, washington, united states citation gopolang f, zulu-mwamba f, nsama d, et al. improving laboratory quality and capacity through leadership and management training: lessons from zambia 2016–2018. afr j lab med. 2021;10(1), a1225. https://doi.org/10.4102/ajlm.v10i1.1225 original research improving laboratory quality and capacity through leadership and management training: lessons from zambia 2016–2018 felicity gopolang, fales zulu-mwamba, davy nsama, annika kruuner, dailes nsofwa, ishmael kasvosve, royce gomo, tiny motlhabane, bhavna chohan, olusegun soge, daniel osterhage, nancy campbell, michael noble, ann downer, jean-frederic flandin, anya nartker, catherine koehn, linda k. nonde, aaron shibemba, clement b. ndongmo, martin steinau, lucy a. perrone received: 17 mar. 2020; accepted: 22 feb. 2021; published: 30 apr. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: competent leadership and management are imperative for delivering quality laboratory services; however, few laboratory managers receive job-specific training in organisational management and leadership. objective: to develop and evaluate participants’ competencies in organisational leadership and management as measured through learner and laboratory quality improvement assessments. methods: this professional development programme employed a mentored, blended learning approach, utilising in-person didactic and online training, with the practical application of a capstone project in the laboratories. programme impact was evaluated through a series of preand post-laboartory assessments using the stepwise laboratory improvement process towards accreditation checklist, as well as learner-competency assessments through online quizzes and discussions. results: from 2016 to 2018, 31 managers and quality officers from 16 individual laboratories graduated from the programme having completed capstone projects addressing areas in the entire laboratory testing process. laboratories increased their compliance with the international organization for standardization 15189 standard and all but two laboratories significantly increased their accreditation scores. two laboratories gained three stars, two laboratories gained two stars, and five laboratories gained one star. five laboratories subsequently achieved international organization for standardization 15189 accreditation in 2019. conclusion: this programme taught leadership theory to laboratory managers and allowed them to implement leadership and management practices in the laboratory setting. programmes such as this complement existing laboratory quality management training programmes such as strengthening laboratory management toward accreditation. keywords: leadership; quality management; workforce development. introduction medical laboratories are a critical component of healthcare because they provide essential data for effective patient care, pathogen detection, disease surveillance and response. enabling access to quality laboratory services is a challenge in low-resource settings1 and many laboratories in resource-constrained countries provide poor quality diagnostic testing with incorrect, unreliable, or significantly delayed test results. competent laboratory management and leadership are vital for delivering quality laboratory services and laboratories need leaders who can utilise their resources effectively in a variable healthcare environment.2 these leadership skills are required to effect beneficial change in complex healthcare settings3 and work effectively across disciplines; however, they are not skills that laboratory managers (lms) commonly cultivate during conventional academic programmes.4 few laboratory supervisors ever receive formal laboratory management and leadership training for their roles.5,6,7 effective laboratory quality management requires that laboratory supervisors have not only the technical knowledge of quality management systems (qms) and national and international standards for medical laboratory quality such as the international organization for standardization (iso)15189 but also the strong leadership and managerial skills to lead their staff and drive accreditation efforts.8,9,10,11,12 the strengthening laboratory management toward accreditation (slmta) programme was launched in 2009,13,14 and provides iterative quality management training to hundreds of laboratory personnel. slmta programme addresses common workforce knowledge gaps in resource-constrained settings via a multi-workshop implementation model. the guide for the stepwise laboratory improvement process towards accreditation15 checklist was endorsed by the world health organization regional office for africa in 2011. it serves as a benchmarking tool to monitor laboratory conformity to the iso15189 quality standard and the slmta programme. as of 2019, slmta has been implemented in 1368 laboratories globally and of these 191 (7.16%)16 have been iso15189 accredited. while significant progress has been made in laboratory quality through qms training programmes such as slmta in the last decade,17 strengthening the impact of these programmes across the continent and increasing representation of the laboratory sector in the upper levels of healthcare governance requires further investments in leadership and management training for lms and directors.18 public health leadership training is evidently beneficial to clinical practitioners and policymakers19,20 and there is a need for wider access to similar programmes for laboratory professionals.21 however, there are limited formal leadership programmes available.22,23,24,25,26,27 to address this gap, the certificate program in laboratory leadership and management28 was developed in 2013 at the university of washington in consultation with global laboratory practice experts. the goal of the certificate program is to build a scalable professional development programme aimed at building the leadership and management skills of laboratory staff in supervisory positions. the participant criteria ultimately ensured that participants were in the leadership position to make substantive and impactful improvements in their laboratory’s testing quality and operations. the programme was implemented in zambia for two years starting in 2016 to strengthen leadership and management competencies of lms and quality assurance officers from key tertiary public and military hospital laboratories. also, this programme aimed to improve the laboratories’ quality of diagnostic services and their compliance with the stepwise laboratory improvement process towards accreditation (slipta) checklist towards achieving iso15189 accreditation. we aim to describe the effectiveness of this laboratory leadership programme in two zambian cohorts, using the laboratories’ compliance with the slipta checklist as the main outcome measure. methods ethical considerations approval to conduct the study was received from the human research ethics committee, university of new england (approval number he13-240). program design and implementation the certificate program was implemented in two cohorts from 2016–2018; each programme cohort completed course and project works in 9 months. this culturally appropriate and effective25 programme employed a mentored, competency-based,29 blended learning approach. it was designed for adult learners and courses were delivered in-person and online. participants delivered a capstone project, which is an individualised, practical application of a quality improvement (qi) project (figure 1). in each cohort, two in-person sessions bookended the online coursework. the in-person sessions served as the programme orientation and finale sessions. figure 1: structural overview for the laboratory leadership and management programme in zambia, 2016–2018. two cohorts of participants from 16 laboratories across zambia participated from 2016–2018, each cohort taking 9 months to complete the programme work. both programme years utilised a similar approach to adult experiential learning, utilising a blended solution of online and face-to-face instruction, a robust online discussion board as well as close faculty and mentorship support for individual capstone projects conducted at participant’s home laboratories. orientation and finale sessions were conducted in lusaka. seventeen laboratory managers from 16 laboratories completed the 2016–2017 programme and completed 16 unique capstone projects. for the second cohort, 16 laboratory managers and 15 quality managers completed the programme and conducted 15 unique capstone projects. the orientation session introduced participants to the programme structure, content, learning goals and expectations, mentor-participant guidelines, the online learning management system (lms; canvas™ learning management system, london),30 and the laboratory assessment and audit tools to be used (e.g. slipta).15 following the orientation, participants returned to their worksites where they discussed the programme with their staff and chief medical superintendent before starting the baseline audit process and online coursework. the results of the baseline audits identified the cp focus area and provided a guideline for the development of cp work plans. the curriculum for the 2016 cohort included five courses from the university of washington delivered sequentially, the first on laboratory quality and systems (delivered in-person), followed by laboratory leadership, laboratory management, communicating laboratory information, and implementing diagnostic technology. the latter topics were delivered online via the canvas lms. each online course was four weeks long and included 20 h – 25 h (~5–6 h/week) of mixed media instruction and a weekly discussion. each course was followed by a 2–3-week instruction intermission during which participants submitted their cp-related assignments. the cp was a customisable qi project designed and implemented by participants at their laboratories with close support from mentors and faculty. the cp process began after the orientation session, with a baseline laboratory audit conducted over a period of 1 week using the slipta checklist.15 through their cps, participants were to exemplify team leadership and improve teamwork through delegation and a system of accountability. the curriculum for the second cohort (2017–2018) included a university of british columbia quality assurance online curriculum for quality assurance officers. this online course was delivered in seven online modules via the blackboard lms system (blackboard inc., washington, district of columbia, united states) and conveyed traditional qms principles of shewhart,31,32 deming,33,34,35,36 crosby,37 and juran38,39 with additional perspectives by faculty. curriculum courses delivered instructions necessary for compliance with the iso quality and competence (iso15189) requirements and expectations for medical laboratories. the lms in the second cohort undertook advanced training based on kouzes and posner’s textbook and workbook ‘the leadership challenge’.40 the lms participated in the online coursework concurrently with the quality assurance officers and conducted joint cps in their home laboratories. both programme cohorts ended with an in-person meeting where participants presented their cps to their peers, mentors and faculty and received a programme completion certificate. participant and mentor selection the programme was specially designed for quality assurance officers and lms who are currently working in a managerial role in a health laboratory; participants and mentors were selected by the programme’s selection committee following specific eligibility criteria. mentors had an average of 20 years’ experience in the clinical laboratory field and were paired with up to seven participants. mentors provided on-site and remote coaching using various communication channels, including the lms discussion board, email, skype (microsoft corp., redmond, washington, united states), and whatsapp™ (facebook inc., menlo park, california, united states) calls. mentors provided step-by-step support and motivated participants to apply knowledge gained from the global classroom to address management challenges such as staff resistance to change, particularly from some long-serving staff members. mentors also reinforced messages of individual leadership and accountability by encouraging laboratory staff at all service levels to implement smaller qi projects. the staff were to identify gaps related to the lm’s cp and led efforts to find solutions. mentors also coached participants to organise and conduct meetings with the laboratory staff, the quality team, and the senior hospital administrators. these proposed meetings were aimed at engaging all laboratory staff and the hospital administration with the implementation and review of the laboratory improvement program. learner and programme evaluation the programme was evaluated based on both learner and facility impact. learner outcome metrics included self-rated competency and graded assessments including graded participation in the weekly online discussion board accompanying each course, course exams and cp-related assignments (analysis of laboratory audit result, cp project proposal, work plan development, implementation update, final report and project presentation).41 course surveys, exit interviews, and facility pre-programme and post-programme slipta checklist audits conducted by the ministry of health were also used to evaluate the programme. at the end of each programme year, via an online programme survey, qualitative programme feedbacks were received from the participants on various aspects of the programme. participants identified the most valued aspect of the programme and evaluated the curriculum quality, the cp process, and the mentor’s support. also, a post-programme evaluation survey was conducted by an independent organisation in 2018.42 the survey utilised a likert scale rating system to collect anonymised data from both cohorts on how participants felt their abilities had changed since they graduated from the programme. all quantitative and qualitative evaluation data were collected from survey responders and analysed using excel software (microsoft corp., redmond, washington, united states). results demographics and graduation rate participants of both programmes were selected using established eligibility criteria from key laboratory facilities as indicated by the zambia ministry of health (table 1). overall, 31 individuals completed the programme with 16/17 (94%) graduating in 2016 and 26/31 (84%) in 2018. these graduates (25 men and 6 women) conducted their programme work at 16 individual hospital laboratories from all nine provinces in zambia (table 2). nine lms completed both cohorts. eight mentors from zambia, botswana, and zimbabwe (three men, five women) supported each paired programme participant for an average of 3 h per week. table 1: participant and mentor selection criteria, zambia, 2016–2018. table 2: programme demographics, zambia, 2016–2018. capstone project scope and success thirty-one cps were completed by graduates in these two years and the cp topics addressed a range of issues on the total laboratory testing process (table 3). in addition to these formal projects, supplemental qi projects were undertaken by other staff in the laboratory adjacent to the cp’s topical area. these supplementary projects which were undertaken by the general laboratory staff also contributed to the improved laboratory performance and addressed issues such as updating standard operating procedures to minimise specimen cross-contamination, implementing new duty rosters for daily equipment maintenance activities during public holidays and weekends and phlebotomy service task-shifting. the smaller qi projects strengthened both the internal and external laboratory communication channels and improved laboratory safety via the introduction of hand-washing facilities, controlled laboratory access, and routine class ii biological safety cabinet smoke tests. table 3: capstone project topic areas, zambia, 2016–2018.† quality improvement progress the ministry of health conducted baseline (the beginning of each programme year) and exit (the end of the 9-month programme) slipta audits. both audits were utilised as benchmarking tools to measure the impact of the cp. after the programme period in 2018, the slipta checklist audit scores of 14 out of the 16 participating laboratories (87.5%) increased (figure 2), with nine laboratories also improving their slipta star rating. two laboratories gained three slipta stars, another two gained two slipta stars, and five gained one slipta star. of the seven other laboratories, six maintained their star rating while one laboratory lost a star rating. three laboratories achieved five slipta stars by the end of the programme and five of these participating laboratories have achieved iso15189 accreditation16 at the time of this writing. figure 2: changes in laboratory audit scores before (2016) and after (2018) the laboratory leadership and management programme in zambia, 2016-2018. participants and representatives from the ministry of health conducted baseline stepwise laboratory improvement program towards accreditation audits of each laboratory at the beginning (2016) and end of the programme (2018). audit scores are shown as whole numbers with a maximum score of 275 points. learner satisfaction participants self-reported significant improvements of key competencies as a result of the programme as indicated by internal (table 4) and external surveys (figure 3). all participants reported improvement in their leadership and management knowledge and skills as well as laboratory practice compliance. more than 95% reported improved competencies in various other laboratory abilities such as critical analyses and interpretation of laboratory data, communication, collaboration with clinicians on result utilisation, improvement of laboratory practice compliance and accountability in line with national and international standards, implementation of essential quality assurance practices (timeliness, reliability and accuracy of testing), and application of leadership and management skills. participants also involved other laboratory staff in learning by downloading recorded lectures for others to watch offline as a team and discussed weekly topics as a group. the programme was also highly rated by mentors as indicated from both internally and externally conducted surveys. figure 3: participants’ self-perceived changes in key abilities after laboratory leadership and management programme in zambia, 2016–2018. a likert scale-based survey conducted of all programme graduates was conducted in 2018 by an external organisation. graduates of the programme self-reported key changes in abilities as a result of the programme (n = 24 respondants) and percentage of each response were calculated and shown here. table 4: qualitative feedback from participants about the programme, zambia, 2016–2018. discussion this programme set out to improve the leadership and quality management skills of a cohort of laboratory supervisors in zambia including improving their competencies in management, communication, policy development, laboratory data analysis, and international quality management principles to improve the laboratories’ ability to deliver quality clinical and public health services. the blended learning programme was successful in achieving a > 80% graduation target rate for both cohorts. participants indicated in surveys that the programme improved their leadership and management skills and subsequently their laboratory’s performance. all respondents reported that they thought the programme applied to their work and that they would recommend the programme to their peers. the continuous support and motivation from faculty and mentors43,44,45 ensured participants were supported during the entire programme period. also, the employment of an effective and reliable online lms to deliver high-quality asynchronous online courses and support a robust real-time discussion board to foster the cultivation of a strong community of practice among each cohort contributed to the high retention rate observed. the online discussion board was utilised daily for communication and enabled participants to share best laboratory leadership, management and advocacy practices with their peers and receive valuable feedback. importantly, the programme was valued by participants because it delivered both theoretical and practical applications of effective laboratory leadership and management. the cp was a unique component of the programme, unifying the entire laboratory around a common goal and fostering a strong working relationship between the management team and the technical staff. this process resonated the importance of strong teams in furthering an organisation’s mission. this blended learning programme is therefore unique in that the modular online curriculum is adaptable to any environment, allowing for customisation with location-specific needs and inclusion of a global audience and experts, regardless of the time zone. the potential for local ownership and expansion of this programme is immense as evidenced by the breadth of project topics participants undertook as well as the adjacent qi projects. the projects improved participant’s leadership and management skills as well as their laboratory’s qms in line with iso15189 (as measured by the preand post-programme slipta audits). the projects also addressed internal indicators of laboratory quality such as specimen rejection rate, turnaround time and client satisfaction. all participating laboratories demonstrated qi; however, not all these improvements are captured by the slipta audits. notably, the structured programme content and sustained faculty and mentor engagement are implicated in these observed laboratory qms improvements and contributed to the international recognition of three participating laboratories. as of the time of this writing, five of these participating laboratories have now achieved iso15189 accreditation. some challenges were encountered in the two-and-a-half-year programme. specifically, management staff changes in some facilities within the two programme periods challenged the continuum of qi. particularly, staff turnover in 2017 correlated with lower qi in many of the participating laboratories. also, other implementing partners at times were simultaneously present on-site with programme mentors which reduced available contact time with mentees. mentors expressed challenges such as mentees not responding to communications and availing themselves during distant mentorship. personal time management was the only participant self-reported challenge; concurrently meeting programme and work responsibilities was demanding for participants. as such, future efforts will be made by the programme developers to condense the programme length based on feedback, offer all of the coursework online to minimise on-site time, and include content pertaining to personnel time management and motivation particularly when there are competing interests. distance learning programmes that include significant components of field or work-based training are proving to be highly effective in fostering practical competency development and behaviour change in learners46 and the results of this programme for lms is no exception. importantly, the modular online curriculum and blended format of the programme is permissive of adoption and adaptation by local institutions such as universities and professional associations. adoption and implementation by local organisations will have added benefit to laboratory professionals either by contributing to university degrees such as diplomas or continuing education credits as part of an annual licensure programme or career advancement points for leadership positions. cost elements of the programme include faculty time, mentor honorarium, data plans for participants, lms maintenance and logistical costs for on-site coaching and in-person meetings. programme implementation costs could likely be reduced should the programme be converted to a completely online programme including mentorship. however, the impact and quality of an entirely online programme are yet to be evaluated. although massive open online courses offer exciting opportunities to distribute knowledge on a massive and global scale, a full understanding of their effectiveness to deliver competency-based training to healthcare professionals remains limited and further research is warranted.47 the value of laboratory leadership programmes such as the one we describe here are starting to receive greater attention from the public health practice community48 and should be supported alongside other efforts to strengthen national laboratory systems.49 limitations this programme was limited to a selected group of participants from zambia who were selected based on their position in their organisation or their occupation. as such, success in this programme was dependent on staff continuity in the programme and vulnerable to disruptions caused by staff reassignment. should the programme become more financially sustainable through user fees, the global audience could be expanded and no longer tied to priority facilities as determined by external donors. recommendations leadership and management training, such as this training programme, is highly recommended as it can lead to measurable impacts in the laboratory. leadership and management training programmes such as this programme are highly recommended to complement existing qms training programmes such as slmta. professional development programmes for healthcare practitioners delivered through online learning platforms should also include an applied project where learned theory from the global classroom can be applied to the job. conclusion this programme affirms the impact of formal leadership and management training on laboratory capacity and builds on previous investments to improve quality, system operability, and preparedness. the programme emphasised the functional practices of organisational leadership and effectively supplemented quality management training programmes; it can be implemented alongside other efforts to strengthen national laboratory systems. acknowledgements we express our appreciation to the hospitals, laboratory leadership, and staff at the 16 laboratories that participated in this programme over the two years. we also thank carl henn, audra stark, dickson kaoma, naomi mwanza, justina mthoniswa, and esther pandawe at aiha for assistance with local logistics support for course delivery and mentoring visits. we are grateful to alec mcgee, debbie confer, elizabeth scott, laura livingston, tom furtwangler, caitlyn bradburn, chris joss, lindsay mumm, leah klug, candice moss, solmaz shotorbani, jennifer hubber, jessica mcpherson, jennifer antilla, ali mokdad, olivier defawe, ellen wilcox, carlyn collins, patricia sadate-ngatchou, and robert martin for their contributions in programme development. we thank cardea services for the programme impact evaluation. we also thank subject matter experts from the university of washington, united states centers for disease control and prevention, sandia national laboratories, association of public health laboratories, washington state department of health, world health organization, university of british columbia, george washington university, and path. we thank anne fox for assistance with graphic design. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions f.g. developed the manuscript draft as the lead author, as well as reviewing and editing towards finalisation of the article. t.m. contributed to the review and editing of the manuscript draft. b.c. was a regional mentor and capstone project. d.o. was responsible for the background literature review and initial identification of target journals. j.-f.f. contributed to editing the manuscript. r.g. contributed (technical review and editing) to the final version of the manuscript. f.z.-m. participated as a mentor and provided input on the manuscript. d. nsama assisted to obtain access to resources and monitored the progress. c.k. performed programmatic, fiscal and operational management, and provided review of the final manuscript. n.c. was lead teacher in the leadership course, coached managers in developing a vision, mission and values for their teams as well as understanding how to motivate team members to engage in the changes needed to a more an effective and efficient laboratory. m.s. coordinated and oversaw the programme and edited the manuscript. a.n. reviewed the manuscript. o.s. contributed to the development and review of the manuscript. c.b.n. contributed to the manuscript writing, reading and approval to the final version. a.d. reviewed and approved the final version. i.k. reviewed and edited the manuscript draft. a.k. reviewed the manuscript draft. l.k.n. reviewed the manuscript draft. d. nsofwa reviewed the manuscript draft. m.n. was the course developer and lead faculty for the university of british columbia quality management course and mentored all quality managers in the programme in 2017. a.s. wrote the manuscript together with f.g. with input from co-authors. sources of support funding for this work was provided by the president’s emergency plan for aids relief through the united states department of health and human services, health resources and services administration under the terms of a cooperative agreement (u97ha04128) awarded to aiha. data availability data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the findings and conclusions of this report are those of the authors and do not necessarily represent the official position of the funding agencies. references martin r, barnhart s. global laboratory systems development: needs and approaches. infect dis clin north am. 2011;25(3):677–691. https://doi.org/10.1016/j.idc.2011.05.001 olmsted ss, moore m, meili rc, et al. strengthening laboratory systems in resource-limited settings. am j clin pathol. 2010 sep;134(3):374–380. https://doi.org/10.1309/ajcpdqosb7qr5glr smith t, stankunas m, czabanowska k, de jong n, o’connor s, fowler davis s. principles of all-inclusive public health: developing a public health leadership curriculum. public health. 2015 feb;129(2):182–184. https://doi.org/10.1016/j.puhe.2014.12.001 renner as, bennett s. developing the next generation of laboratory leaders [homepage on the internet]. clinical laboratory news 2015 nov 01 [cited 2019 jul 23]. available from: https://www.aacc.org/publications/cln/articles/2015/november/developing-the-next-generation-of-laboratory-leaders deboy jm, beck aj, boulton ml, kim dh, wichman md, luedtke pf. core courses in public health laboratory science and practice: findings from 2006 and 2011 surveys. public health rep. 2013;128 suppl 2(suppl 2):105–114. https://doi.org/10.1177/00333549131280s215 kasvosve i, ledikwe jh, phumaphi o, et al. continuing professional development training needs of medical laboratory personnel in botswana. hum resour health. 2014 aug;12:46. https://doi.org/10.1186/1478-4491-12-46 report of the who laboratory leadership and management training programme meeting, lyon, france, 12–13 may 2011. lyon: world health organization; 2011. international organization for standardization (iso). iso 15189: 2012 medical laboratories – requirements for quality and competence. geneva: international organization for standardization; 2012. albert h, de dieu iragena j, kao k, erni d, mekonen t, onyebujoh pc. implementation of quality management systems and progress towards accreditation of national tuberculosis reference laboratories in africa. afr j lab med. 2017;6(2):a490. https://doi.org/10.4102/ajlm.v6i2.490 andric lr, massambu cg. one laboratory’s progress towards accreditation in tanzania. afr j med. 2014;3(2), art. #202, 4p. https://doi.org/10.4102/ajlm.v3i2.202 mothabeng d, maruta t, lebina m, lewis k, wanyoike j, mengstu y. strengthening laboratory management towards accreditation: the lesotho experience. afr j lab med. 2012;1(1), art. #9, 7p. https://doi.org/10.4102/ajlm.v1i1.9 viegas so, azam k, madeira c, et al. mozambique’s journey towards accreditation of the national tuberculosis reference laboratory. afr j lab med. 2017;6(2):a491. https://doi.org/10.4102/ajlm.v6i2.491 yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3):401–409. https://doi.org/10.1309/ajcpnbbl53fwuiqj world health organization regional office for africa. strengthening laboratory management towards accreditation [homepage on the internet]. [cited 2019 jul 23]. available from: https://slmta.org/ world health organization regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation (slipta) in the african region [homepage on the internet]. version 2:2015. [cited 2019 jul 23]. available from: https://www.afro.who.int/sites/default/files/2017-06/slipta-checkist0711.pdf slmta.org. laboratories that have achieved accreditation [homepage on the internet]. [cited 2020 jun 25]. available from: https://slmta.org/accredited-labs/ nkengasong jn, mbopi-keou fx, peeling rw, yao k, zeh ce, schneidman m. laboratory medicine in africa since 2008: then, now, and the future. lancet infect dis. 2018 nov;18(11):e362–e367. https://doi.org/10.1016/s1473-3099(18)30120-8 alemnji ga, zeh c, yao k, fonjungo pn. strengthening national health laboratories in sub-saharan africa: a decade of remarkable progress. trop med int health. 2014 apr;19(4):450–458. https://doi.org/10.1111/tmi.12269 doherty j, gilson l, shung-king m. achievements and challenges in developing health leadership in south africa: the experience of the oliver tambo fellowship programme 2008–2014. health policy plan. 2018;33(suppl_2):ii50–ii64. https://doi.org/10.1093/heapol/czx155 nakanjako d, namagala e, semeere a, et al. global health leadership training in resource-limited settings: a collaborative approach by academic institutions and local health care programs in uganda. hum resour health. 2015;13:87. https://doi.org/10.1186/s12960-015-0087-2 best m, sakande j. practical recommendations for strengthening national and regional laboratory networks in africa in the global health security era. afr j lab med. 2016;5(3), a471. https://doi.org/10.4102/ajlm.v5i3.471 laboratory leadership curriculum [homepage on the internet]. chicago, il: clinical laboratory management association; 2015 [cited 2019 jul 23]. available from: http://www.clma.org/p/cm/ld/fid=18 lab management university [homepage on the internet]. chicago, il: american society for clinical pathology; 2015 [cited 2019 jul 23]. available from: http://www.ascp.org/lmu laboratory leadership service (lls) [homepage on the internet]. atlanta, ga: centers for disease control and prevention; 2015 [cited 2019 jul 23]. available from: https://www.cdc.gov/lls/index.html perrone la, confer d, scott e, et al. implementation of a mentored professional development programme in laboratory leadership and management in the middle east and north africa. east mediterr health j. 2016;22(11):832–839. https://doi.org/10.26719/2016.22.11.832 clinical laboratory leadership and management certificate program [homepage on the internet]. washington, dc: american association of clinical chemistry. [cited 2019 jul 23]. available from: https://www.aacc.org/education-and-career/online-certificate-programs/certificate-programs/clinical-laboratory-leadership-and-management-certificate-program emerging leader program [homepage on the internet]. washington, dc: association of public health laboratories. [cited 2019 jul 23]. available from: https://www.aphl.org/professional_development/pages/elp.aspx certificate program in laboratory leadership and management [homepage on the internet]. university of washington, i-tech. [cited 2019 jul 23]. available from: https://edgh.washington.edu/project/lab-certificate ned-sykes r, johnson c, ridderhof jc, perlman e, pollock a, deboy jm. competency guidelines for public health laboratory professionals: cdc and the association of public health laboratories. morb mortal wkly rep. 2015;64(1):1–81. instructure. canvas learning management system. salt lake city, ut: instructure corporation; 2016. [cited 2019 jul 23]. available from: http://www.canvaslms.com shewhart wa. economic control of quality of manufactured product/50th anniversary commemorative issue. milwaukee, wi: american society for quality; 1980. shewhart wa. statistical method from the viewpoint of quality control. washington. the graduate school, the department of agriculture.; 1939. deming we. the essential deming: leadership principles from the father of quality. new york, ny: mcgraw-hill; 2012 dec 11. deming.org. the fourteen points for management [homepage on the internet]. [cited 2020 jun 20]. available from: https://www.deming.org/theman/theories/fourteenpoints deming we. out of the crisis. cambridge, ma: the massachusetts institute of technology (mit) press; 2018. deming we. quality, productivity and competitive position. cambridge, ma: massachusetts institute of technology; 1982. crosby pb. quality is free. the art of making quality certain. new york, ny: mcgraw-hill; 1979. juran j, godfrey a. juran’s quality handbook. 5th ed. new york, ny: mcgraw-hill; 2000. juran jm. managerial breakthrough: a new concept of the manager’s job. new york, ny: mcgraw-hill companies; 1964. kouzes jm, posher bz. the leadership challenge: how to make extraordinary things happen in organizations. 6th ed. somerset: john wiley& sons inc.; 2017. kirkpatrick dl, kirkpatrick jd. evaluating training programs: the four levels. 3rd ed. san francisco, ca: berrett-koehler; 2006. cardia services. [cited 2019 jul 23]. available from: http://www.cardeaservices.org/ kapanka ar. journey to the millennium: mentoring in the clinical laboratory. mlo med lab obs. 1998 may;30(5):44–46. beck sj, laudicina rj. passing the torch: mentoring the next generation of laboratory professionals. clin lab sci. 2001;14(1):33–36. laudicina rj. mentoring for retention and advancement in the multigenerational clinical laboratory. clin lab sci. 2001;14(1):48–52. joynes, c. distance learning for health: what works a global review of accredited post-qualification training programmes for health workers in low and middle-income countries [homepage on the internet]. [cited 2020 jun 20]. london international development centre (ldic); 2011. available from: https://lidc.ac.uk/_assets/dl4h%20main%20report.pdf rowe m, osadnik cr, pritchard s, maloney s. these may not be the courses you are seeking: a systematic review of open online courses in health professions education. bmc med educ. 2019 sep 14;19(1):356. https://doi.org/10.1186/s12909-019-1774-9 albetkova a, isadore j, ridderhof j, et al. critical gaps in laboratory leadership to meet global health security goals. bull world health organ. 2017;95(8):547–547a. https://doi.org/10.2471/blt.17.195883 opio a, wafula w, amone j, kajumbula h, nkengasong jn. country leadership and policy are critical factors for implementing laboratory accreditation in developing countries: a study on uganda. am j clin pathol. 2010;134(3):381–387. https://doi.org/10.1309/ajcp6kmotclisgj3 acknowledgements references about the author(s) noah fongwen international diagnostics centre, london school of hygiene and tropical medicine, london, united kingdom debi boeras global health impact group, atlanta, georgia, united states rosanna w. peeling international diagnostics centre, london school of hygiene and tropical medicine, london, united kingdom timothy amukele department of pathology, johns hopkins school of medicine, johns hopkins university, baltimore, maryland, united states citation fongwen n, boeras d, peeling rw, amukele t. connected diagnostics systems: the future of disease control in africa. afr j lab med. 2020;9(2), a1365. https://doi.org/10.4102/ajlm.v9i2.1365 editorial connected diagnostics systems: the future of disease control in africa noah fongwen, debi boeras, rosanna w. peeling, timothy amukele copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the coronavirus disease 2019 (covid-19) pandemic has ushered us into a new era of global public health urgency, with diagnostics and laboratory medicine at its centre.1 to take advantage of this focus on diagnostics to leapfrog some of the barriers in lowand middle-income countries, we must first understand what is feasible and effective in our setting. this african journal of laboratory medicine’s special issue on the ‘future of diagnostics’ offers us the opportunity to examine innovations in diagnostics that are already afoot in africa. these innovations cover all three phases of testing and include a discussion of open (rather than closed) reagent systems for molecular testing, a tool for assessing the diagnostic capacity of a country, nigeria’s experience establishing laboratory networks, tuberculosis data analytics, and mobile testing, reporting and surveillance – among others. the global competition for covid-19 reagents and molecular tests has taught us that african communities must strive for self-reliance; this self-reliance starts long before testing in the laboratory begins.2 three articles in this issue address the need for changes outside the laboratory to support a better future for diagnostics. the article by emperador et al. encourages the design and deployment of open reagent systems for molecular testing rather than the closed ones we have now.3 they also discuss some ways to address the changes in reimbursement and ownership that will be needed to support such a system.3 the article by ondoa et al. presents the initial africa-wide roll-out of a powerful new tool, the joint external evaluation tool, which allows a country-wide comprehensive evaluation of diagnostic laboratory gaps and capabilities.4 finally, the article by naidoo and ihekweazu describes how nigeria built a national referral laboratory and established a country-wide specimen referral network to support national priority diseases including yellow fever, lassa fever, monkeypox, cerebrospinal meningitis, cholera, influenza and other enteric pathogens.5 networks of expert diagnostic laboratory sites for diseases of epidemic potential need to be established in a pan-african manner, so that specimens can be tested efficiently and reliably bio-banked to support local development and evaluation of new tests.6 the ability to develop and manufacture diagnostics in africa is the future of disease control in africa.7 the rapid spread of covid-19 means that countries need real-time smart data systems for evidence-based decisions on public health measures and travel restrictions – as illustrated in three articles in this issue. first, cassim et al. report on the use of an interactive dashboard at a high-volume laboratory that, when coupled with good management, dramatically improved the percentage of turn-around times within the expected range from 10% to 90%.8,9 gous and macek describe a new in-person and remote training programme, the tb data fellowship, which builds a tableau-based tuberculosis analytics capacity in lowand middle-income countries.10 rapid advances in data connectivity and digital computing, coupled with increased access to internet coverage and portable communications such as mobile phones in africa, provide us with a window of opportunity to adapt these technologies to strengthen diagnostic systems for the surveillance of diseases of epidemic potential, as well as for hiv, tuberculosis and neglected tropical diseases. a well-connected diagnostic system will cause a seismic shift in the way disease control is tackled in africa. mobile phones and other mobile surveillance systems present some examples of changes in the status quo. in senegal, epidemiological and diagnostic data on influenza-like illnesses from sentinel sites were collected and reported by phone through encrypted short message service to the ministry of health.11 peaks in the incidence of febrile syndromes were detected and the specific cause identified, leading to early notification of public health officials at the ministry of health.11 fall et al. report in this issue on a successful pilot evaluation of the use of a mobile van equipped with a biosafety laboratory (complete with internal negative pressure chambers) that was deployed for outbreak investigations.12 in tanzania, the control of neglected tropical diseases has taken a new leap forward by using mobile phones in early reporting of potential rabies cases, thus increasing compliance with post-exposure prophylaxis.13 in south africa, researchers in the africa health research institute have developed a user-friendly mobile phone diagnostic test for hiv that uses a camera and an online app that allows for self-testing and interpretation of results.14 whether this will translate to an increase in linkage to care is still being studied,15 but such an application can provide useful state-of-the art data on the transmission dynamics of hiv within communities, and has the potential to accelerate elimination efforts for hiv and other infectious diseases like syphilis and hepatitis. in malawi, the ministry of health has partnered with village reach to establish a nation-wide health information hotline called health centre by phone.16 such innovation has been critical in disseminating useful health information and advice to the lowest level of the health system and dispelling myths and rumours during the covid-19 pandemic. this special issue has highlighted that africa is a continent of qualified laboratory professionals and innovators. as new diagnostics are being developed on the continent, test developers should consider data connectivity as an essential component of any final product. connectivity of new and existing point-of-care diagnostics should be fully functional and integrated with laboratory services to form connected diagnostic systems that will support disease surveillance and outbreak responses. mobile phones are ubiquitous. we use them to access massive amounts of information and trust them with our mobile banking. they are powerful pocket computers and we should consider how we can use them to take healthcare to the last mile. the time to consider how we should harness this wealth of technology for the future of disease control in africa is now. acknowledgements competing interests the authors have declared that no competing interests exist. authors’ contributions all authors contributed equally to this work. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references lippi g, plebani m. the critical role of laboratory medicine during coronavirus disease 2019 (covid-19) and other viral outbreaks. clin chem lab med. 2020;58(7):1063–1069. https://doi.org/10.1515/cclm-2020-0240 nkengasong j. let africa into the market for covid-19 diagnostics. nature. 2020;580:565. https://doi.org/10.1038/d41586-020-01265-0 emperador dm, mazzola lt, kelly-cirino cd. an open source molecular diagnostic platform approach for outbreak and epidemic preparedness [lessons from the field]. afr j lab med. 2020;9(2):a1017. https://doi.org/10.4102/ajlm.v9i2.1017 ondoa p, ndlovu n, keita m-s, et al. preparing national tiered laboratory systems and networks to advance diagnostics in africa to meet the continent’s health agenda: insights into priority areas for improvement. afr j lab med. 2020;9(2):a1103. https://doi.org/10.4102/ajlm.v9i2.1103 naidoo d, ihekweazu c. nigeria’s efforts to strengthen laboratory diagnostics – why access to reliable and affordable diagnostics is key to building resilient laboratory systems. afr j lab med. 2020;9(2):1019. https://doi.org/10.4102/ajlm.v9i2.1019 peeling rw, boeras d, wilder-smith a, sall a, nkengasong j. need for sustainable biobanking networks for covid-19 and other diseases of epidemic potential. lancet infect dis. 2020;3099(20):1–6. https://doi.org/10.1016/s1473-3099(20)30461-8 organization for economic cooperation and development. africa’s response to covid-19: what roles for trade manufacturing and intellectual-property [homepage on the internet] [cited 2020 jun 23]. available from: http://www.oecd.org/coronavirus/policy-responses/africa-s-response-to-covid-19-what-roles-for-trade-manufacturing-and-intellectual-property-73d0dfaf/ cassim n, tepper me, coetzee lm, glencross dk. timely delivery of laboratory efficiency information, part i: developing an interactive turnaround time dashboard at a high-volume laboratory. afr j lab med. 2020;9(2):a947. https://doi.org/10.4102/ajlm.v9i2.947 cassim n, coetzee lm, tepper me, perelson l, glencross dk. timely delivery of laboratory efficiency information, part ii: assessing the impact of a turn-around time dashboard at a high-volume laboratory. afr j lab med. 2020;9(2):a948. https://doi.org/10.4102/ajlm.v9i2.948 gous n, nyaruhirira au, cunningham b, macek c. driving the usage of tb diagnostic data through capacity building in lowand middle-income countries [lessons from the field]. afr j lab med. 2020;9(2):a1092. https://doi.org/10.4102/ajlm.v9i2.1092 dia n, sarr fd, thiam d, et al. influenza-like illnesses in senegal: not only focus on influenza viruses. plos one. 2014;9(3):e93227. https://doi.org/10.1371/journal.pone.0093227 fall c, cappuyns a, faye o, et al. field evaluation of a mobile biosafety laboratory in senegal to strengthen rapid disease outbreak response and monitoring. afr j lab med. 2020;9(2):a1041. https://doi.org/10.4102/ajlm.v9i2.1041 mtema z, changalucha j, cleaveland s, et al. mobile phones as surveillance tools: implementing and evaluating a large-scale intersectoral surveillance system for rabies in tanzania. plos med. 2016;13(4):1–12. https://doi.org/10.1371/journal.pmed.1002002 i-sense united kingdom. using mobile technology to test and treat those hardest hit by hiv [homepage on the internet] [cited 2016 jun]. available from: https://www.i-sense.org.uk/news/using-mobile-technologies-test-and-treat-those-hardest-hit-hiv venter w, coleman j, chan vl, et al. improving linkage to hiv care through mobile phone apps: randomized controlled trial. jmir mhealth uhealth. 2018;6(7):e155. https://doi.org/10.2196/mhealth.8376 blauvelt c, west m, maxim l, et al. scaling up a health and nutrition hotline in malawi: the benefits of multisectoral collaboration. bmj. 2018;363:k4590. https://doi.org/10.1136/bmj.k4590 abstract introduction methods results discussion acknowledgements references about the author(s) barbara tornimbene amr division, surveillance, prevention and control department, world health organization, geneva, switzerland sergey eremin amr division, surveillance, prevention and control department, world health organization, geneva, switzerland reuben abednego national health laboratory quality assurance and training centre (nhlqatc), tanzania, dar es salaam, united republic of tanzania elamin o. abualas national public health laboratory, federal ministry of health, khartoum, sudan ilhem boutiba faculty of medicine, university of tunis el manar, tunis, tunisia abiodun egwuenu nigeria center for disease control, abuja, nigeria walter fuller antimicrobial resistance (amr) world health organization, regional office for africa, brazzaville, congo laetitia gahimbare antimicrobial resistance (amr) world health organization, regional office for africa, brazzaville, congo susan githii national microbiology reference lab, national public health laboratories, nairobi, kenya watipaso kasambara ministry of health, lilongwe, malawi chileshe lukwesa-musyani lusaka district laboratory, university of zambia, lusaka, zambia fidy a. miamina department of health watch, epidemiological surveillance and response (dvsser), antananarivo, madagascar sekesai mtapuri-zinyowera national microbiology reference laboratory, zimbabwe, harare, zimbabwe grace najjuka department of microbiology, joint clinical research centre (jcrc), kampala, uganda olga perovic centre for healthcare-associated infections, antimicrobial resistance and mycoses (charm), johannesburg, south africa bassem zayed world health organization, regional office for east mediterranean, cairo, egypt yahaya a. ahmed world health organization, regional office for africa, brazzaville, congo maha t. ismail world health organization, regional office for east mediterranean, cairo, egypt carmem l. pessoa da silva amr division, surveillance, prevention and control department, world health organization, geneva, switzerland citation tornimbene b, eremin s, abednego r, et al. global antimicrobial resistance and use surveillance system on the african continent: early implementation 2017–2019. afr j lab med. 2022;11(1), a1594. https://doi.org/10.4102/ajlm.v11i1.1594 original research global antimicrobial resistance and use surveillance system on the african continent: early implementation 2017–2019 barbara tornimbene, sergey eremin, reuben abednego, elamin o. abualas, ilhem boutiba, abiodun egwuenu, walter fuller, laetitia gahimbare, susan githii, watipaso kasambara, chileshe lukwesa-musyani, fidy a. miamina, sekesai mtapuri-zinyowera, grace najjuka, olga perovic, bassem zayed, yahaya a. ahmed, maha t. ismail, carmem l. pessoa da silva received: 28 mar. 2021; accepted: 20 apr. 2022; published: 31 aug. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: antimicrobial resistance (amr) is becoming a critical public health issue globally. the world health organization launched the global antimicrobial resistance and use surveillance system (glass) to support the strengthening of the amr evidence base. objective: the article describes the evolution of national amr surveillance systems and amr data reporting of countries in the african continent between 2017 and 2019, and the constraints, perceived impact and value of the participation in glass. methods: data on implementation of national surveillance systems and amr rates were submitted to glass between 2017 and 2019 and summarised though descriptive statistics. the information on constraints and perceived impact and value in glass participation was collected though a set of questionnaires. results: between 2017 and 2019, egypt, ethiopia, madagascar, malawi, mali, mozambique, nigeria, south africa, sudan, tunisia, uganda and zambia submitted data to glass. the main constraints listed are linked to scarce laboratory capacity and capability, limited staffing, budget issues, and data management. moreover, while the data are not yet nationally representative, high resistance rates were reported to commonly-used antibiotics, as the emerging resistance to last treatment options. conclusion: despite the limitations, more and more countries in the african continent are working towards reaching a status that will enable them to report amr data in a complete and systematic manner. future improvements involve the expansion of routine surveillance capacity for several countries and the implementation of surveys that allow to effectively define the magnitude of amr in the continent. keywords: amr; surveillance; africa; implementation; who. introduction antimicrobial resistance (amr) is defined as ‘the presence of resistance to antimicrobial medicines in infectious agents such as bacteria, viruses, fungi and parasites’1. this resistance can be inherent or acquired by the inappropriate use of medicines. recent studies position amr as a leading cause of death around the world, with the highest burdens in low-resource settings.2 africa remains the continent most afflicted by infectious diseases and amr can dramatically hamper treatment effectiveness and greatly amplify disease burden and its complications.3 a 2017 systematic review from tadesse et al.,4 documenting the status of amr in africa, found that ‘recent amr data was not available for more than 40% of countries’. in countries for which data were available, ‘the level of resistance to commonly prescribed antibiotics was significant’, and that the ‘quality of microbiological data is of serious concern’.3 a second systematic review, also published in 2017 and targeting west africa, found amr to be common in this subregion. it particularly occurred in hospitalised patients with bloodstream infections (bsi), and both outpatient and hospitalised patients with urinary tract infection (uti).5 two reviews of amr in sub-saharan africa and one review in east africa also revealed ‘a high prevalence of amr to commonly-used antibiotics in clinical bacterial isolates’.6,7 the studies also flagged the flaws in available data and the challenges faced in lowand lower middle-income countries when implementing amr surveillance. in 2015, the world health organization (who) launched the global antimicrobial resistance and use surveillance system (glass) to support strengthening of the amr evidence base. as stated in the glass report 2020, glass encourages countries to move to surveillance approaches based on systems that includes epidemiological, clinical, and population-level data, rather than only on laboratory data. in addition to data collection, glass promotes strengthening of national amr surveillance systems to ensure that reliable and representative information is produced. it is supported by who collaborating centres network, involving strong commitment from participating countries and close collaborations with who headquarter, regional and country offices.8 during the early implementation phase (2015–2019), glass focused on collecting information on the status of existing or newly developed national amr surveillance systems and to provide a standardised approach to the collection of amr rates for selected bacteria causing generic infections in humans. the first data call was opened in may 2017, and it recurs every year in the same period, between 1 may and 31 august. this article aims to describe the evolution of national amr surveillance systems and amr data reporting capacity of african countries participating in glass for the first three annual glass data calls (2017–2019), together with a summary of reported amr rates for selected indicators. the article also describes, for a subset of african countries, the constraints, perceived impact, and value linked to reporting data to glass. methods ethical considerations ethics approval was not required for this study. each country has its own ethical approval for amr surveillance and in many cases routine surveillance does not require ethical clearance. data sources the glass database is the source of information of countries participation and reporting, implementation of the national surveillance system, and amr rates. the data were submitted by counties during three data calls, between 2017 and 2019. the information on constraints and perceived impact and value in glass participation was collected though a set of questions sent via email to countries’ amr national focal points (nfps) and who regional offices’ staff in charge of amr activities. information gathered thorough global antimicrobial resistance and use surveillance system data calls the glass amr data call is open yearly between may and august, and countries submit the information on the implementation of the national amr surveillance systems for the data call year, and amr rates for the previous year. information on the implementation of the national surveillance system in this article, key indicators on the implementation of the national surveillance system are summarised and presented to reflect changes during three data calls (2017–2019). as stated in the glass report 2020,7 glass collects information on the implementation of national amr surveillance systems through a standardised questionnaire filled in every year by the nfp. a set of indicators is then used to measure the development and strengthening of national amr surveillance:7 the establishment of a national coordinating centre and the national reference laboratory. these two bodies are in charge of data management and capacity building, and are key to the coordination of the national systems. number of surveillance sites and local laboratories performing antimicrobial susceptibility testing (ast) that report amr data to glass. this information allows a better understanding of the structure and capacity of the national surveillance system structure. surveillance sites can be hospitals, clinics, or in-and outpatient community healthcare facilities with access to relevant epidemiological and laboratory support and information. provision of external quality assessment. this allows a better understanding of the diagnostic capacity of the surveillance system, by checking the provision of external quality assessment to the national reference laboratory and clinical local laboratories, and the use of international standards for diagnostics and ast. antimicrobial resistance data as stated in the glass reports, glass requires amr data to be collected through a surveillance system that gathers results from ast for common human bacterial pathogens in four infection sites, specifically bsis caused by acinetobacter spp., e. coli, k. pneumoniae, salmonella spp., s. aureus and s. pneumoniae, utis caused by e. coli and k. pneumonia, gastrointestinal infections caused by salmonella spp. and shigella spp., and genital infections caused by n. gonorrhoeae. data are generated by the collation of results from specimens that are routinely sent to laboratories for clinical testing and includes blood, urine, stool and cervical and urethral samples. the rationale for selection of these particular specimens is that the growth of a pathogen is a proxy of infection in the associated anatomical sites. the target population under surveillance is the national population of patients seeking care in hcfs. data to be collected includes: ‘numbers of patients with susceptible, non-susceptible, intermediate, and resistant isolates, as well as numbers of isolates with unknown susceptibility.8,9,10 two types of unknown results are recorded. the first, ‘unknown_no_ast’, is the number of isolates with ast results that were not reported (or not performed) for a specific antibiotic. the second, ‘unknown_no_breakpoints’, is the number of isolates for which ast was performed but which had no interpretation of results available for a specific antibiotic. additionally, countries are invited to report patients’ microbiological results (bacterial isolation and identification, ast), as well as demographic and epidemiological variables such as age, gender, and origin of infection in tested patients, in aggregated format.8 the distribution of infections and bacteria analysed and submitted to glass by countries during the three data calls (2017–2019) is summarised by year and shown in table format. antimicrobial resistance rates: proportions of patients with resistant infections reported by countries during three data calls are presented for: bloodstream infections caused by escherichia coli and klebsiella pneumoniae resistant to carbapenems and third-generation cephalosporins (3gc), and bsis caused by methicillin-resistant staphylococcus aureus (mrsa). urinary tract infection caused by e. coli and k. pneumoniae resistant to carbapenems and ciprofloxacin. as aligned to the method outlines in the glass report 2016–2017, ‘rates are shown only if [countries reported] results for > 10 patients, and for pathogen–antibiotic combinations with > 10 ast results and < 30% unknown ast results’11. box-and-whisker plots are used to summarise the reported median rate of resistance for specific specimen–pathogen–antibiotic combinations. the plots portray the distribution of the submitted data, outliers, and the median. the box within the chart displays where around 50% of the data points fall and it contains the lower quartile, the upper quartile, and the median in the centre. the median is the value separating the higher half from the lower half of the results. information gathered through countries’ and who regional offices’ feedback after the end of the first glass data call in august 2017 and the last data call in august 2019, enrolled african countries, the who regional office for africa and the who regional office for the eastern mediterranean were asked to provide feedback to a set of questions covering three themes: constraints: the difficulties encountered to participate in glass data calls. impact: the positive impact that the three data calls might have had to foster data generation. value: the added value of participating in glass. the qualitative data obtained from countries’ and regional offices’ feedback is summarised in the article using a set of codes identified for each theme and shown in pie chart format. coding was done by identifying a passage in the text, searching, and identifying concepts, and finding relationships between them. data analysis data on information on the implementation of the national surveillance system were exported from the glass information technology platform into microsoft excel (microsoft corporation, california, united states), and bar charts were used to visualise the proportion of each variable outcome for all reporting countries in order to interpret the data. antimicrobial resistance data were exported from the glass information technology platform and validated and analysed using stata (statacorp, college station, texas, united states) software. for each country, the number of patients with confirmed bacterial infection was calculated by collapsing all the laboratory results (susceptible, non-susceptible, intermediate, and resistant isolates, as well as numbers of isolates with unknown susceptibility) for a specific pathogen and choosing the antibiotic with the highest number of results reported. the number of patients with ast results by pathogen is calculated in the same way, but unknown susceptibility results are not included. the proportion of patients with resistant infection for a specific indicator (see ‘antimicrobial resistance rates’ mentioned earlier) is then calculated for each country using the following formula: all countries’ results were pulled together, and the median rate of resistance was calculated. tableau software (tableau, mountain view, california, united states) was used to visualise the data though box-and-whisker plots. the questionnaires’ contents, with countries’ and who regional offices’ feedback, were pulled together in microsoft excel and screened for key words to define codes for the feedback themes. the proportion of respondents for each code was then calculated, based on the total number of questionnaires received. results global antimicrobial resistance and use surveillance system data calls countries’ participation and reporting enrolment and reporting varied during the three data calls, both on submission on the information of the status of the national amr surveillance systems and amr data. by the end of the last data call in august 2019, 23 out of 54 (43%) african countries were formally enrolled in glass: cote d’ivoire, egypt, ethiopia, gabon, gambia, ghana, kenya, liberia, libya, madagascar, malawi, mali, mauritania, mauritius, mozambique, nigeria, south africa, sudan, tanzania, tunisia, uganda, zambia and zimbabwe. table 1 shows progress in country participation and reporting from 2017 to 2019, with almost a 100% increase in number of countries over the three years. table 1: number of countries on the african continent enrolled in glass and which reported information on their national surveillance systems and amr data during the three glass data calls (2017–2019). most countries have been able to report amr data with age and gender stratification (table 2). the reporting of the number of tested patients, the denominator used to calculate frequency of amr infection in patients with suspected bacterial infection, has increased from one (17%) reporting country in 2017, to 10 (100%) reporting countries in 2019. infection origin has proved to be the least reported variable throughout the three data calls. table 2: number of countries on the african continent enrolled in glass and reporting ast results during the three data glass calls (2017–2019), as well as availability of data stratification by age, gender and infection origin and data on the number of patients from which a diagnostic sample was taken (tested). information of the status of national antimicrobial resistance surveillance systems data show that the national coordinating centre is established, or is in the process of being established, and the national reference laboratories are nominated in around 80% of countries participating in the three data calls. the number of surveillance sites reporting to glass over the three years went from 52 hospital and five outpatient facilities in 2017, to 63 hospital and 62 outpatient facilities in 2019. in certain cases, the number of surveillance sites could not be retrieved so the number of laboratories supporting the surveillance systems was reported instead; this was done for five laboratories in 2018, and 12 laboratories in 2019. overall, the total number of surveillance sites increased from 57 in 2017 to 137 in 2019. almost 80% of countries reported in three data calls having the national reference laboratories participating in an external quality assessment scheme, while external quality assessment for clinical laboratories that contribute to the national amr surveillance programmes went from being performed by 27% of countries in 2017, to 61% of countries in 2018, and 48% of countries in 2019. in around 70% of the countries, clinical laboratories performed ast according to recognised standards, from either the clinical & laboratory standards institute or the european committee on antimicrobial susceptibility testing. antimicrobial resistance data distribution of infections and bacteria analysed: compared to the first data call, with six countries reporting ast results for 11 060 patients with confirmed bacterial infections, in 2019 glass received ast results from 10 countries for 32 117 patients, three times the number from 2017 (table 3). bloodstream infection is the most frequent infection reported for the three years, followed by uti, gastroenteric infection and gonorrhoea (table 3). bloodstream infections caused by s. aureus and k. pneumoniae appeared to be the most recurrent, while e. coli is the most frequent etiological agent of reported utis. rate of gastroenteric infections caused by shigella species and salmonella species were reported equally through the years. table 3: summary of confirmed bacterial infections and patient with ast results, by infection site and pathogen, reported by egypt, ethiopia, madagascar, malawi, mali, mozambique, nigeria, south africa, sudan, tunisia, uganda, and zambia during three in glass data calls (2017–2019). antimicrobial resistance rates: the patterns of resistance of e. coli and k. pneumoniae to different antibiotics by infections sites and specific organisms between 2016 and 2018, as reported in 2017–2019 data calls, are summarised in table 4. results show that resistance to 3gc in bsis is above 50% for e. coli and 81% for k. pneumoniae, while carbapenem resistance reaches a maximum of 8% for e. coli and 24% for k. pneumoniae; mrsa is found to be the cause of about 20% of bsis. similarly, resistance of e. coli and k. pneumoniae to carbapenems is generally found to be below 10% in utis, while resistance to ciprofloxacin is between 36% and 60%. table 4: proportion of resistance (median) for selected infection site, by antibiotics and pathogen, in egypt, ethiopia, madagascar, malawi, mali, mozambique, nigeria, south africa, sudan, tunisia, uganda and zambia between 2016 and 2018. boxplots for each pathogen–antibiotic combination in different infections sites (bsi and uti) are presented in figure 1. although the list and number of countries reporting on specific pathogen–antibiotic combination varied throughout the three data calls, data show a certain consistency in the reported rates. figure 1: boxplots showing proportion (median) of infection syndrome due to bacteria resistant to selected antibiotics in egypt, ethiopia, madagascar, malawi, mali, mozambique, nigeria, south africa, sudan, tunisia, uganda, and zambia between 2016 and 2018. the number of countries reporting for each year and the number of patients with antimicrobial susceptibility testing (ast) results are shown in the x-axis. the list and the number of countries reporting each year may vary. each red dot corresponds to a single country result. (a) boxplots showing proportion (median) of bloodstream infections due to escherichia coli resistance to third-generation cephalosporins in egypt, ethiopia, madagascar, malawi, mali, mozambique, nigeria, south africa, sudan, tunisia, uganda and zambia between 2016 and 2018. the number of countries reporting for each year and the number of patients with ast results are shown below the x-axis. the list and the number of countries reporting each year may vary. each red dot corresponds to a single country result. (b) boxplots showing proportion (median) of bloodstream infections due to escherichia coli resistance to carbapenems in egypt, ethiopia, madagascar, malawi, mali, mozambique, nigeria, south africa, sudan, tunisia, uganda and zambia between 2016 and 2018. the number of countries reporting for each year and the number of patients with ast results are shown below the x-axis. the list and the number of countries reporting each year may vary. each red dot corresponds to a single country result. (c) boxplots showing proportion (median) of bloodstream infections due to klebsiella pneumoniae resistance to third-generation cephalosporins, in egypt, ethiopia, madagascar, malawi, mali, mozambique, nigeria, south africa, sudan, tunisia, uganda and zambia, between 2016 and 2019. the number of countries reporting for each year and the number of patients with ast results are shown below the x-axis. the list and the number of countries reporting each year may vary. each red dot corresponds to a single country result. (d) boxplots showing proportion (median) of bloodstream infections due to klebsiella pneumoniae resistance to carbapenes in egypt, ethiopia, madagascar, malawi, mali, mozambique, nigeria, south africa, sudan, tunisia, uganda and zambia, between 2016 and 2019. the number of countries reporting for each year and the number of patients with ast results are shown below the x-axis. the list and the number of countries reporting each year may vary. each red dot corresponds to a single country result. figure 1 (continues...): boxplots showing proportion (median) of infection syndrome due to bacteria resistant to selected antibiotics in egypt, ethiopia, madagascar, malawi, mali, mozambique, nigeria, south africa, sudan, tunisia, uganda, and zambia between 2016 and 2018. the number of countries reporting for each year and the number of patients with antimicrobial susceptibility testing (ast) results are shown in the x-axis. the list and the number of countries reporting each year may vary. each red dot correspond to a single country result. (e) boxplots showing proportion (median) of bloodstream infections due to methicillin-resistant staphylococcus aureus (mrsa) in egypt, ethiopia, madagascar, malawi, mali, mozambique, nigeria, south africa, sudan, tunisia, uganda and zambia, between 2016 and 2019. the number of countries reporting for each year and the number of patients with ast results are shown below the x-axis. the list and the number of countries reporting each year may vary. each red dot corresponds to a single country result. (f) boxplots showing proportion (median) of urinary tract infections due to escherichia coli resistance to ciprofoloxacin in egypt, ethiopia, madagascar, malawi, mali, mozambique, nigeria, south africa, sudan, tunisia, uganda and zambia between 2016 and 2019. the number of countries reporting for each year and the number of patients with ast results are shown below the x-axis. the list and the number of countries reporting each year may vary. each red dot corresponds to a single country result. (g) boxplots showing proportion (median) of urinary tract infections due to escherichia coli resistance to carbapenems in egypt, ethiopia, madagascar, malawi, mali, mozambique, nigeria, south africa, sudan, tunisia, uganda and zambia between 2016 and 2019. the number of countries reporting for each year and the number of patients with ast results are shown below the x-axis. the list and the number of countries reporting each year may vary. each red dot corresponds to a single country result. (h) boxplots showing proportion (median) of urinary tract infections due to klebsiella pneumoniae resistance to ciprofloxacin in egypt, ethiopia, madagascar, malawi, mali, mozambique, nigeria, south africa, sudan, tunisia, uganda and zambia between 2016 and 2019. the number of countries reporting for each year and the number of patients with ast results are shown below the x-axis. the list and the number of countries reporting each year may vary. each red dot corresponds to a single country result. figure 1 (continues...): boxplots showing proportion (median) of infection syndrome due to bacteria resistant to selected antibiotics in egypt, ethiopia, madagascar, malawi, mali, mozambique, nigeria, south africa, sudan, tunisia, uganda, and zambia between 2016 and 2018. the number of countries reporting for each year and the number of patients with antimicrobial susceptibility testing (ast) results are shown in the x-axis. the list and the number of countries reporting each year may vary. each red dot correspond to a single country result. (i) boxplots showing proportion (median) of urinary tract infections due to klebsiella pneumoniae resistance to carbapenems in egypt, ethiopia, madagascar, malawi, mali, mozambique, nigeria, south africa, sudan, tunisia, uganda and zambia between 2016 and 2019. the number of countries reporting for each year and the number of patients with ast results are shown below the x-axis. the list and the number of countries reporting each year may vary. each red dot corresponds to a single country result. countries’ and who regional offices’ feedback eleven countries, as well as the who regional office for africa and the who regional office for the eastern mediterranean, provided feedback on the first three years of glass implementation. the feedback from nfps was provided either at the end of the first data call, the third data call, or both (table 5). responses by nfps and regional offices are presented in figure 2 by the three themes (constraint, perceived impact, and value) and identified codes. limited resources, economic issues, bureaucratic bottlenecks, and political instability were reported as having a major impact on the roll out of national amr surveillance systems. for example, the partial absence of quality assurance provision and tools to extract, clean and aggregate data were listed as important limitations to countries’ participation. scarce availability of trained professionals and resources to hire new staff was also indicated as major issue. both who regional offices noted that glass data generation, validation, and processing are centralised and headed by a single nfp. however, due to the shortage of manpower, nfps frequently oversee other activities, which results in competing priorities that might delay data reporting. lack of it tools, technical training for data management and data preparation, and complex it system interoperability, were also mentioned as important constraint for data reporting. in some countries, where a national laboratory-based surveillance programme has been in place for a long period of time, an enhanced surveillance system had to be adapted to meet glass’s capacity to include population data. table 5: list of countries and years for which countries national focal points provided feedback to glass. figure 2: pie charts summarising the proportion of countries’ national focal points and who regional offices’ responses related to constraints, perceived impact, and value associated to reporting data to global antimicrobial resistance and use surveillance system (glass). feedback was received by kenya, madagascar, malawi, nigeria, south africa, sudan, tanzania, tunisia, uganda, zambia, zimbabwe and the who regional office for africa and regional office for the east mediterranean, in relation to 2017 and 2019 glass data calls. (a) pie chart summarising the proportion of countries’ national focal points and who regional offices’ responses related to constraints associated to reporting data to glass. feedback was received by kenya, madagascar, malawi, nigeria, south africa, sudan, tanzania, tunisia, uganda, zambia, zimbabwe and the who regional office for africa and regional office for the east mediterranean, in relation to 2017 and 2019 glass data calls. (b) pie chart summarising the proportion of countries’ national focal points and who regional offices’ responses related to perceived impact associated to reporting data to glass. feedback was received by kenya, madagascar, malawi, nigeria, south africa, sudan, tanzania, tunisia, uganda, zambia, zimbabwe and the who regional office for africa and regional office for the east mediterranean, in relation to 2017 and 2019 glass data calls. figure 2 (continues...): pie charts summarising the proportion of countries’ national focal points and who regional offices’ responses related to constraints, perceived impact, and value associated to reporting data to global antimicrobial resistance and use surveillance system (glass). feedback was received by kenya, madagascar, malawi, nigeria, south africa, sudan, tanzania, tunisia, uganda, zambia, zimbabwe and the who regional office for africa and regional office for the east mediterranean, in relation to 2017 and 2019 glass data calls. (c) pie chart summarising the proportion of countries’ national focal points and who regional offices’ responses related to perceived value associated to reporting data to glass. feedback was received by kenya, madagascar, malawi, nigeria, south africa, sudan, tanzania, tunisia, uganda, zambia, zimbabwe and the who regional office for africa and regional office for the east mediterranean, in relation to 2017 and 2019 glass data calls. nevertheless, the glass surveillance methods proposed, and the it tools offered, were found to be well-defined and easy to use. countries were able to reform data sharing systems to suit the glass data reporting model. mostly, this was done by establishing local laboratories data systems, which were electronically linked to clinical data and to national network. according to one nfp, feedback was also sent to hospitals and outpatient clinics, resulting in treatment changes. the application of whonet, a laboratory information system software, to glass data preparation was found to be very useful and succeeded to strengthen data management capability.12 the national reports produced using data collected for glass were used by countries to develop and/or implement amr national plans and policies, to develop proposals, to orient partners, and to direct the necessary technical assistance. participation in glass has helped to launch strategies on data use and development of policies on antimicrobial use and amr. the experience gained during the data calls has been used to establish the mechanism of monitoring and evaluation of the amr surveillance system. the evidence has also collectively brought together one health partners and ministries to implement multisectoral projects and integrated surveillance. discussion african countries have responded to the first glass three data calls with a high level of interest and dedication, and the glass framework has proven to be a vital tool to the establishment and/or development of national amr surveillance systems, as reflected by the overall positive feedback of nfps to glass participation. the main constraints encountered by countries during the data calls were linked to lack of specific national activities to tackle amr, scarce laboratory capability, staffing and budget issues, and data management. however, through support from glass, countries were also able to revitalise their laboratory components and microbiological output, both for infectious diseases and amr diagnostics. in order to respond to the glass data call, countries improved the collection, analysis and presentation of standardised data generated from healthcare facilities, which in some cases also resulted in improved patient treatment. countries without an amr surveillance system in place used the glass manual for early implementation to model the structure of the new national system.13 the initial steps of the participation and the data call also pushed countries to assess the capacity of their national amr reporting system(s). this allowed the identification of gaps to address in the future, and it acted in participating countries as platform for the establishment of the national surveillance core components. this is key, considering that the data reported to all three glass data calls (2017–2019) might suggest the presence of high rates of amr in the continent. as expected, ast results for bsi were most frequently reported, followed by uti, gastroenteric infection and gonorrhoea. this is in line with the available evidence which shows that, in africa, bsi is a major cause of morbidity and mortality, and utis are some of the most frequent bacterial infections affecting people, both in the community and in hospitals.7 reported high resistance to 3gc is particularly worrying in some parts of africa, where diagnostic facilities are scarce and antibiotics such as carbapenems and semi-synthetic aminoglycosides (e.g. amikacin) are either unavailable or prohibitively expensive.7 in many sub-saharan africa hospitals, limited nursing capacity favours the use of broad-spectrum antimicrobials with a once-daily dosing regimen and this has led to the widespread adoption of 3gc for the empirical management of hospitalised patients with suspected sepsis.14 moreover, extended spectrum beta-lactamase-producing enterobacterales, for which resistance to 3gc is a marker, are also resistant to penicillins and therefore represent an important threat to the treatment of bsis in these settings.7 the reported rate of bsi caused by mrsa was also high (between 21% and 24%). methicillin-resistant s. aureus has been linked to significant morbidity and mortality and it carries an evident threat to african countries, since there might be limited access to antibiotics effective against hospital-associated mrsa, such as linezolid and daptomycin.15 furthermore, the scarce implementation of infection prevention and control measures and widespread hiv infection and tuberculosis can amplify the difficulty of dealing with the mrsa epidemic in africa. escherichia coliand k. pneumoniae-reported resistance to ciprofloxacin in utis was found to be consistently high (between 36% and 60%). this could potentially be linked to samples obtained from complicated and hospitalised patients, as in almost all countries reporting to the glass community, utis are not tested for and treated empirically.3,16 this is important, as fluoroquinolones have an significant role in treating of severe infections, such as septicaemia, and therefore increasing resistance can have severe health consequences.17 finally, reported resistance of e. coli and k. pneumoniae to carbapenems was high in both bsis and utis. this is worrying as, until recently, carbapenems were the last-resort antibiotics used for managing multidrug-resistant bacterial infections.18 moreover, the organisms that are resistant to carbapenems are frequently resistant to many other classes of commonly-used antimicrobial agents; thus, managing infections caused by them poses a substantial challenge in clinical practice and their public health impacts cannot be over-emphasised.19,20 both ciprofloxacin and carbapenems are on the ‘watch’ list of the who 2019 aware classification, that comprises antibiotics with higher potential to induce resistance; ciprofloxacin is also on the who model list of essential medicines, where it is listed as a firstor second-choice empirical treatment option for definite infectious syndromes.21 considering the reported amr data, the spread of all listed resistant patterns needs to be carefully monitored, and every country should apply measures for continuous data collection, by strengthening surveillance activities or implementing population-based studies (e.g. prevalence survey). limitations due to the quality of the data reported to glass, and associated potential bias, no trends analysis was performed with presented amr data, nor comparisons among infection types, or the identification of risk factors linked to age, gender or the source of infection. as stated in the glass report 20208: [d]ata aggregation is a major limitation, as it considerably limits options for epidemiological characterization, obviating the detection and validation of data from countries … with unusual antimicrobial patterns. furthermore, … [l]ack of a sampling strategy results in selection bias, which may affect the representativeness and precision of results. cases are found and tested only in the population that seeks medical care, … and most data are still generated in laboratories, with no epidemiological insight. antimicrobial susceptibility testing varied widely among countries for the specimen–pathogen–antibiotic combinations chosen. the numbers of patients screened for resistance were still very low, suggesting that most data come from complicated and hospitalised patients. unfortunately, it was also not possible to show the frequency of amr for these syndrome-pathogen–antibiotic combinations in the tested population, as the needed denominator – the population of the patient for which a diagnostic sample is taken – was not always available.8 finally, it was not possible to obtain feedback from all of the african countries participating in the glass data calls. however, responses showed a homogeneous consensus to the global system participation and the benefits associated to it. conclusion although some african countries listed in this article still face important constraints while building their national amr surveillance systems, and even if not all of them have provided amr data, countries have shown a willingness to share information with glass, particularly the status and the development of their surveillance systems. countries on the continent are working towards reaching a status that will enable them to report data in a complete and systematic manner, through the establishment of surveillance core components and by assuring the quality of amr diagnostics. although reported data are still not representative at a national level, and ast varied considerably among countries, the participation in glass is clearly linked to improved national surveillance systems, which will also result in better clinical care in prescribing the appropriate antibiotics, one of the most challenging objectives of the global action plan-amr. future improvements involve the expansion of routine surveillance capacity for several countries and the implementation of surveys that allow for effective definition of the magnitude of amr on the continent. meanwhile, the evidence generated is supporting the identification of areas for further research – amr burden in healthcare settings, improvement of diagnostic stewardship, and amr in the human animal environment interface – and it is advocating for the continuous support of actions directed to amr monitoring and control. together with both national and regional partners, african countries’ participation in glass is leading the way towards the further development of an efficient and reliable global surveillance system, which will be able to function in various economic and socio-political contexts, and provide vital and actionable data. addressing antimicrobial resistance through glass is part of the ongoing efforts of member states to strengthen health security, improve health systems and ensure universal health coverage.22 acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions b.t., s.e., r.a., e.o.a., i.b., a.e., w.f., l.g., s.g., w.k., c.l-m., f.a.m., s.m-z., g.n., o.p., b.z., y.a.a., m.t.i. and c.l.p.d.s. contributed to the generation of content and the development of the manuscript. sources of support this research received no specific grant from any funding agency in the public , commercial, or not-for-profit sectors. data availability the data that support the findings of this study are openly available at https://docs.google.com/spreadsheets/d/1lqcx6wno4pul4kre6tqehtrn0ultumdu/edit#gid=1028268226 (glass 2016 amr data). disclaimer the authors alone are responsible for the views expressed in this publication and they do not necessarily represent the views, decisions or policies of the institutions with which they are affiliated. references who. antimicrobial resistance. 2017 [cited 2022 july 25]. available from: https://www.who.int/news-room/questions-and-answers/item/antimicrobial-resistance murray cj, ikuta ks, sharara f, et al. global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. lancet. 2022;399(10325):629–655. https://doi.org/10.1016/s0140-6736(21)02724-0 horton r. gbd 2010: understanding disease, injury, and risk. lancet. 2012;380(9859):2053–2054. https://doi.org/10.1016/s0140-6736(12)62133-3 tadesse bt, ashley ea, ongarello s, et al. antimicrobial resistance in africa: a systematic review. bmc infect dis. 2017;17:616. https://doi.org/10.1186/s12879-017-2713-1 bernabé kj, langendorf c, ford n, ronat jb, murphy ra. antimicrobial resistance in west africa: a systematic review and meta-analysis. int j antimicrob agents. 2017;50(5):629–639. https://doi.org/10.1016/j.ijantimicag.2017.07.002 leopold sj, van leth f, tarekegn h, schultsz c. antimicrobial drug resistance among clinically relevant bacterial isolates in sub-saharan africa: a systematic review. j antimicrob chemother. 2014;69(9):2337–2353. https://doi.org/10.1093/jac/dku176 lester r, musicha p, van ginneken n, et al. prevalence and outcome of bloodstream infections due to third-generation cephalosporin-resistant enterobacteriaceae in sub-saharan africa: a systematic review. j antimicrob chemother. 2020;75(3):492–507. https://doi.org/10.1093/jac/dkz464 world health organization. global antimicrobial resistance and use surveillance system (glass) report early implementation 2020 [homepage on the internet]. 2020 world health organization. global antimicrobial resistance surveillance system (glass) report: early implementation 2017-2018. 2019. available from: https://www.who.int/glass/resources/publications/early-implementation-report-2017-2018/en/ world health organization. global antimicrobial resistance and use surveillance system (glass) report: 2021. 2021 [cited 2020 jan 01]. available from: https://www.who.int/publications/i/item/9789240027336 world health organization. global antimicrobial resistance surveillance system (glass) report: early implementation 2016-2017. 2018 [cited 2020 jan 01]. available from: http://www.who.int/glass/resources/publications/early-implementation-report/en/ whonet. who collaborating centre for surveillance of antimicrobial resistance [homepage on the internet]. no date [cited 2021 jun 01]. available from: http://www.whonet.org/ world health organization. glass manual for early implementation. 2015 [cited 2020 jan 01]. available from: http://apps.who.int/iris/bitstream/10665/188783/1/9789241549400_eng.pdf musicha p, cornick je, bar-zeev n, et al. trends in antimicrobial resistance in bloodstream infection isolates at a large urban hospital in malawi (1998–2016): a surveillance study. lancet infect dis. 2017;17(10):1042–1052. https://doi.org/10.1016/s1473-3099(17)30394-8 falagas me, karageorgopoulos de, leptidis j, korbila ip. mrsa in africa: filling the global map of antimicrobial resistance. plos one. 2013;8:e68024. https://doi.org/10.1371/journal.pone.0068024 seifu wd, gebissa ad. prevalence and antibiotic susceptibility of uropathogens from cases of urinary tract infections (uti) in shashemene referral hospital, ethiopia. bmc infect dis. 2018;18:30. https://doi.org/10.1186/s12879-017-2911-x fasugba o, gardner a, mitchell bg, mnatzaganian g. ciprofloxacin resistance in communityand hospital-acquired escherichia coli urinary tract infections: a systematic review and meta-analysis of observational studies. bmc infect dis. 2015;15:545. https://doi.org/10.1186/s12879-015-1282-4 elshamy aa, aboshanab km. a review on bacterial resistance to carbapenems: epidemiology, detection and treatment options. future sci oa. 2020;6:fso438. https://doi.org/10.2144/fsoa-2019-0098 doi y, paterson dl. carbapenemase-producing enterobacteriaceae. semin respir crit care med. 2015;36(1):74–84. https://doi.org/10.1055/s-0035-1544208 nordmann p, naas t, poirel l. global spread of carbapenemase-producing enterobacteriaceae. emerg infect dis. 2011;17(10):1791–1798. https://doi.org/10.3201/eid1710.110655 world health organization. who model list of essential medicines [homepage on the internet]. 2021 [cited 2020 jan 01]. available from: https://www.who.int/publications/i/item/who-mhp-hps-eml-2021.02 who, the world bank. tracking universal health coverage: 2017 global monitoring report [homepage on the internet]. 2017 [cited 2020 aug 20]. available from: http://documents1.worldbank.org/curated/en/640121513095868125/pdf/122029-wp-revised-public.pdf abstract introduction methods results discussion acknowledgements references about the author(s) adeyemi t. adeyemo department of medical microbiology and parasitology, obafemi awolowo university teaching hospitals, ile-ife, nigeria babatope kolawole department of medicine, faculty of clinical science, obafemi awolowo university, ile-ife, nigeria vincent o. rotimi department of microbiology, faculty of medicine, kuwait university, kuwait city, kuwait aaron o. aboderin department of medical microbiology and parasitology, obafemi awolowo university teaching hospitals, ile-ife, nigeria department of medical microbiology and parasitology, faculty of basic medical science, obafemi awolowo university, ile-ife, nigeria citation adeyemo at, kolawole b, rotimi vo, aboderin ao. multicentre study of the burden of multidrug-resistant bacteria in the aetiology of infected diabetic foot ulcers. afr j lab med. 2021;10(1), a1261. https://doi.org/10.4102/ajlm.v10i1.1261 original research multicentre study of the burden of multidrug-resistant bacteria in the aetiology of infected diabetic foot ulcers adeyemi t. adeyemo, babatope kolawole, vincent o. rotimi, aaron o. aboderin received: 03 may 2020; accepted: 22 oct. 2020; published: 23 mar. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: infected diabetic foot ulcer (idfu) is a public health issue and the leading cause of non-traumatic limb amputation. very few published data on idfu exist in most west african countries. objective: the study investigated the aetiology and antibacterial drug resistance burden of idfu in tertiary hospitals in osun state, nigeria, between july 2016 and april 2017. methods: isolates were cultured from tissue biopsies or aspirates collected from patients with idfu. bacterial identification, antibiotic susceptibility testing and phenotypic detection of extended-spectrum beta-lactamase and carbapenemase production were done by established protocols. specific resistance genes were detected by polymerase chain reaction. results: there were 218 microorganisms isolated from 93 idfus, comprising 129 (59.2%) gram-negative bacilli (gnb), 59 (27.1%) gram-positive cocci and 29 (13.3%) anaerobic bacteria. the top five facultative anaerobic bacteria isolated were: staphylococcus aureus (34; 15.6%), escherichia coli (23; 10.6%), pseudomonas aeruginosa (20; 9.2%), klebsiella pneumoniae (19; 8.7%) and citrobacter spp. (19; 8.7%). the most common anaerobes were bacteroides spp. (7; 3.2%) and peptostreptococcus anaerobius (6; 2.8%). seventy-four idfus (80%) were infected by multidrug-resistant bacteria, predominantly methicillin-resistant s. aureus and gnb producing extended-spectrum β-lactamases, mainly of the ctx-m variety. only 4 (3.1%) gnb produced carbapenemases encoded predominantly by blavim. factors associated with presence of multidrug-resistant bacteria were peripheral neuropathy (adjusted odds ratio [aor] = 4.05, p = 0.04) and duration of foot infection of more than 1 month (aor = 7.63, p = 0.02). conclusion: multidrug-resistant facultative anaerobic bacteria are overrepresented as agents of idfu. a relatively low proportion of the aetiological agents were anaerobic bacteria. keywords: infection; diabetic foot; ulcers; multidrug-resistance; bacteria; antibiotic; anaerobic culture; samples. introduction infected diabetic foot ulcer (idfu) is associated with inlammation or purulence occurring in sites below the ankle in persons with diabetes mellitus.1 it is a major global public health issue with a substantial medical, socio-economic and psychological burden. infected diabetic foot ulcer is one of the most common diabetes-related infections in clinical practice, and a common indication for hospital admission.1 ulceration often precedes foot infection in diabetic patients, with peripheral vascular disease, peripheral neuropathy and visual impairment and immunological disturbances also playing contributory roles. infection impairs the healing process and aggravates the condition of patients with diabetic foot ulcer (dfu) and could lead to great disability, septicaemia and death if not promptly and properly managed. at 7.2% (95% confidence interval [ci]: 5.1–9.3%) and higher than the global prevalence of 6.3% (95% ci: 5.4–7.3%), africa has the second highest global prevalence of dfu, a precursor of idfu.2 foot infections are more common and lethal in africa than elsewhere globally.3 between 25% and 60% of diabetic patients with a background foot ulcer will develop idfu which remains a major reason for non-traumatic amputation of the lower limbs.4 the foot infection can progress to irreversible septic gangrene which often necessitates life-saving amputation of the lower limb.5 patients with idfu have 15–46 times higher risk of limb amputation than those with non-diabetic related ulcers.6 more than 1 million diabetic patients may require limb amputation worldwide yearly, and a greater percentage of them are in developing countries.7 wide varieties of organisms, including anaerobic bacteria, have been implicated in the aetiology of idfu depending on the severity of infection and time from onset to presentation at healthcare facilities. advanced idfus with features of sepsis at admission usually harbour anaerobic pathogens.8 the emergence and current global threat of antimicrobial resistance in the face of dwindling antibiotics in the development pipeline has added a new twist to the burden of idfu.9 increasing involvement of multidrug-resistant organisms (organisms resistant to at least three different antimicrobial classes)10 in diabetic patients with infected foot ulcers has significantly reduced antibiotic treatment options, thus posing a serious challenge particularly in resource-constrained lowand middle-income countries where access to antimicrobial drugs is of grave concern.11 it has also increased the length of hospital stay and cost of treatment, and caused additional morbidity and mortality.12 these situations have assumed worrisome trends in which resistance is building up to antibiotics of last resort; pathogens showing considerable resistance to vancomycin and carbapenems are particularly becoming more common as agents of foot infection in diabetic patients.13 various studies have reported many independent risk factors and predictors of multidrug-resistant idfu including previous hospitalisation for the same wound, prolonged antibiotic therapy, ulcer type and increased ulcer size, presence of osteomyelitis, poor glycaemic control, prolonged duration of foot ulcer infection as well as proliferative retinopathy.14,15,16,17,18 according to bakele et al., predictors of lower limb amputation by multivariate logistic regression analysis were advanced ulcer grade, inappropriate antibiotic use, overweight, obesity, poor blood glucose control and neuropathy.19 furthermore, albuminuria, diabetic nephropathy and charcot arthropathy were noted as predictors of poor healing of diabetic foot ulcer.20 a recent systematic review and meta-analysis on the global burden of diabetic foot ulceration in cameroon, west africa, concluded that paucity of data impedes strategies for treatment and prevention of foot infections in diabetic patients.2 thus, our study was designed to determine the prevalent bacteria involved in idfus, assess the burden of multidrug-resistance (mdr) among the isolates and evaluate the associated risk factors. methods ethical considerations ethical approval for this study was granted by the ethics and research committees of the obafemi awolowo university teaching hospitals complex and ladoke akintola university college of technology with protocol numbers erc/2015/11/02 and lth/er/2016/01/254. information about the study and participant involvement was fully explained to patients, and properly signed and dated written informed consent forms were obtained from patients before their recruitment into the study. results of wound biopsy microscopy, culture and sensitivity were made available for patients’ management. study population the prospective, cross-sectional, multicentre, hospital-based study was carried out in osun state, southwest nigeria, between july 2016 and april 2017. it included three existing tertiary healthcare facilities in the state: obafemi awolowo university teaching hospitals complex, wesley guild hospital, ilesa, and ladoke akintola university of technology teaching hospital, osogbo. all consecutive diabetic patients (both hospitalised and outpatients) with foot infections meeting the criteria for diagnosis of idfu seen and managed at these hospitals were recruited into the study. they were clinically assessed and foot lesions graded according to the diabetic foot infection severity classification system issued by the infectious disease society of america.8 only non-duplicate patients and samples were included in the study. all inpatients were followed up with regular check-ups physically in the wards until they either died or were discharged. sample collection and bacterial identification aspirates were obtained from deep-seated abscesses, and tissue samples were collected after washing the wound vigorously with sterile saline and debridement of the slough to exclude mere colonisers. necrotic tissues were curetted into anaerobic basal broth (oxoid, basingstoke, hants, united kingdom) for anaerobic culture. the samples were immediately transported to the laboratory and processed within 2 h of sample collection by inoculating them onto a set of selective and non-selective media which were: 5% (volume/volume) sheep blood agar (ba; oxoid, basingstoke, hants, united kingdom), macconkey agar (oxoid, basingstoke, hants, united kingdom), chocolate agar and anaerobic basal agar (oxoid, basingstoke, hants, united kingdom) supplemented with 5% (abscesses) laked sheep blood, vitamin k1 (1 µg/ml), l-cysteine hydrochloride (5 µg/ml) and gentamicin (100 µg/ml) (gentamicin blood agar). inoculated plain ba and macconkey agar plates were incubated in air and chocolate agar plates in co2 at 37 °c for 24 h. inoculated plain gentamicin ba plates, as well as gentamicin ba with kanamycin (75 µg/l) and vancomycin (5 µg/l) supplements, were incubated under anaerobic conditions made up of 80% h2, 10% co2 and 10% n2 for 48 h and extended for 5 days if necessary; anaerobiosis was achieved using a bactron anaerobic chamber (sheldon manufacturing, inc., cornelius, oregon, united states). representative colonies were identified by colonial morphology, gram staining characteristics and conventional biochemical tests including catalase and oxidase tests. facultative anaerobic gram-negative bacilli (gnb) and streptococcus spp. were further identified with microbact™ gnb 24e (oxoid, basingstoke, hants, united kingdom) and rapid™ str (thermo fisher scientific, remel products, lexena, kansas, united states), while staphylococcus spp. were further identified with a coagulase test, characteristic growth appearance on mannitol salt agar and a dnase test. the obligate anaerobes were identified by rapid™ ana ii (thermo fisher scientific, remel products, lexena, kansas, united states). yeast isolates were identified by gram staining and germ tube tests. quality control strains, staphylococcus aureus atcc 25923, escherichia coli atcc 25922, bacteroides fragilis atcc 25285 and peptostreptococcus anaerobius atcc 27337, were used to assess the quality of the media and identification systems. the quality of our bacterial identification system and procedures (for aerobes, facultative anaerobes and obligate anaerobes) were assured by ensuring that the control bacterial strains were identified to their names. antibiotic susceptibility test antibiotic susceptibility testing for aerobes and facultative anaerobes was performed using the modified kirby-bauer disc diffusion technique as recommended by the clinical and laboratory standards institute (clsi).21 gram-positive isolates were tested with the following antibiotic discs (oxoid, basingstoke, hants, united kingdom): penicillin (10 µg), cefuroxime (30 µg), ceftriaxone (30 µg), cefepime (30 µg), co-amoxiclav (20/10 µg), ciprofloxacin (5 µg), chloramphenicol (30 µg), gentamicin (10 µg), amikacin (10 µg), trimethoprim-sulfamethoxazole (1.25 µg/23.75 µg), cefoxitin (30 µg), erythromycin (15 µg), ampicillin-sulbactam (10 µg/10 µg), piperacillin-tazobactam (100 µg/10 µg) and vancomycin (30 µg). vancomycin (256–0.015 µg/ml) minimum inhibitory concentration (mic) strip (oxoid, basingstoke, hants, united kingdom) was used to test for vancomycin resistance among methicillin-resistant s. aureus (mrsa). gram-negative isolates were tested with the following antibiotic discs (oxoid, basingstoke, hants, united kingdom): ceftriaxone (30 µg), ceftazidime (30 µg), cefotaxime (30 µg), cefepime (30 µg), co-amoxiclav (20/10 µg), ciprofloxacin (5 µg), gentamicin (10 µg), amikacin (10 µg), chloramphenicol (30 µg), aztreonam (30 µg), trimethoprim-sulfamethoxazole (1.25 µg/23.75 µg), ampicillin-sulbactam (10 µg/10 µg), piperacillin-tazobactam (100 µg/10 µg), cefoxitin (30 µg), meropenem (10 µg) and ertapenem (10 µg). discrete colonies were emulsified in sterile saline to match 0.5 mcfarland turbidity standards from where confluence inocula were made on mueller-hinton agar (mha) plates with sterile cotton swabs. the swabbed mha plates were allowed to dry at room temperature and a set of six antibiotic discs were placed evenly spaced on each of the plates. vancomycin resistance was tested in the methicillin-resistant s. aureus isolates by placing a mic evaluation strip (oxoid, basingstoke, hants, united kingdom) on inoculated mha. after 18–24 h of incubation, the diameter of the zone of inhibition around each antibiotic disc was measured and recorded. vancomycin mic values were also recorded for the s. aureus. zones of inhibition of each antibiotic as well as vancomycin mic values were interpreted as ‘sensitive’, ‘intermediate’ or ‘resistant’ in accordance with clsi guidelines.21 isolates with intermediate sensitivity were regarded as ‘resistant’. the quality of antibiotic susceptibility testing consumables (including antibiotic discs and mha) and procedures were assured with bacterial control strains (e. coli atcc 25922, s. aureus atcc 25923 and s. aureus atcc 43300). zones of inhibition of tested antibiotics on the bacterial control strains fell within their quality control ranges according to clsi.21 extended-spectrum β-lactamase production was confirmed among enterobacteriaceae and other gnb that showed reduced susceptibility to at least one of the tested third-generation cephalosporins (cefotaxime 30 µg, ceftazidime 30 µg and ceftriaxone 30 µg) or aztreonam (30 µg) by a combination disc diffusion test (cddt).21 cddt was done using both single discs of cefotaxime (30 µg) and ceftazidime (30 µg) and their respective clavulanic acid containing discs (cetotaxime-clavulanate 30/10 µg, ceftazidime-clavulanate 30/10 µg). a 5 mm or more increase in zone of inhibition of one or more combination discs as compared with their respective single discs was taken as confirmatory evidence of extended-spectrum beta-lactamase (esbl) production.21 ampc beta-lactamase production was detected by ampc disc test as described by anjali et al. on isolates which show resistance to at least one third-generation cephalosporin and a β-lactamase inhibitor.22 a broth culture of e. coli atcc 25922 was adjusted to 0.5 mcfarland turbidity standard and inoculated onto mha plates. sterile filter paper discs (6 mm) were moistened with distilled water (about 20 µl) and up to five colonies of the test organism was transferred onto the filter paper. afterwards, a cefoxitin (30 µg) disc and the inoculated filter paper disc were placed next to each other and almost touching on inoculated media. this setup was incubated overnight at 37 °c. a flattening or indentation of the zone of inhibition of cefoxitin in the vicinity of the test disc (inoculated filter paper) indicated a phenotypic confirmatory evidence of ampc β-lactamase production.22 gram-negative bacilli with intermediate sensitivity or resistance to one or more carbapenems were tested for production of carbapenemases by the modified hodge test and interpreted by clsi guidelines.21 organisms that were phenotypically mdr, including esbl-producing gnb, carbapenem-resistant gnb and mrsa, were further tested for resistance-determining genes using polymerase chain reaction (pcr)-based protocols with specific oligonucleotide primers23–27 (table 1); template bacterial dna was extracted by the boiling method.28 electrophoresis of each pcr product (5 µl) was carried out in 1.5% (weight/volume) agarose gel (biomatik, kitchener, ontario, canada) in 1x tris-acetate-edta (tae) buffer for 45 min. the size of amplified products was estimated using 100 base pairs molecular weight marker (100–1200 base pairs). table 1: oligonucleotides primers and amplification reactions for targeted resistance genes. statistical analysis data analysis was performed with statistical package for social sciences version 20 (spss inc., chicago, illinois, united states). comparison of mean values was done using the student’s t-test for continuous variables and the chi-square test for categorical variables. risk factors for infection of diabetic foot by mdr organisms were identified by logistic regression analysis. logistic regression was used to determine predictive associations of variables that showed statistical significance by bivariate analysis. a p-value of less than 0.05 was considered to be statistically significant. results sociodemographic and clinical characteristics of patients ninety patients (53 male and 37 female) presented with 93 idfus during the 11-month study period. the patients ranged between 18 and 85 years (mean 54.7 ± 12.8 years) of age. of the 93 cases of foot infections, 56 (60.2%) had lasted for at least 1 month, and 70 (75.3%) were in-hospital patients. sixty-six (71.0%) of the ulcers were categorised as wagner’s grade 3 and above. most (n = 74; 82.2%) of the patients used antibiotics in the month preceding presentation at the healthcare facilities, while 93.3% (n = 84) had commenced antibiotics before collection of wound samples (table 2). table 2: association between clinical and sociodemographic variables of infected diabetic foot ulcer patients from tertiary healthcare facilities in osun state, nigeria, july 2016 to april 2017. predictors and treatment outcomes of multidrug-resistant bacterial infections in infected diabetic foot ulcer significant factors associated with the presence of mdr organisms in diabetic foot infections included peripheral sensory neuropathy, a foot infection duration of more than a month and admission duration of more than a month (table 2). further analysis with logistic regression however identified only peripheral neuropathy (adjusted odds ratio = 4.05, 95% ci: 1.08–15.13) and foot infection duration of more than a month (adjusted odds ratio = 7.63, ci: 1.64–35.39) as the predisposing factors for acquisition of multidrug-resistant bacteria among patients with idfu. a substantial proportion (33/70; 47.1%) of the inpatients had poor treatment outcomes; poor outcomes noted in 53.4% (31/58) of patients with mdr infections included major limb amputation (below and above knee amputation) (18/58; 31.0%) and death (13/58; 22.4%). infections by these mdr bacteria have significant association with poor treatment outcomes (adjusted odds ratio = 5.11, 95% ci: 1.23–29.67). distribution of isolates among diabetic foot cases all 93 wound specimens obtained from the 90 patients (three patients had bilateral foot ulcers) in the three healthcare facilities were positive on bacterial culture with only 10 (10.8%) of them yielding a single organism each. among the polymicrobial cultures, 45 (48.4%) yielded two organisms per culture, 34 (36.6%) yielded three organisms per culture while 4 (4.3%) yielded four organisms per culture. results further showed that there was a total of 218 organisms isolated from the 93 specimens cultured with an average of 2.34 organisms per sample. of the organisms, 129 (59.2%) were gram-negative aerobic and facultative anaerobic bacilli, 59 (27.1%) were gram-positive aerobic cocci and 29 (13.3%) were anaerobic bacteria; only 1 (0.5%) was yeast. s. aureus (34; 15.6%) was the single most common organism followed by e. coli (23; 10.6%) and pseudomonas aeruginosa (20; 9.2%). others included klebsiella spp. (19; 8.7%), citrobacter spp. (19; 8.7%), enterococcus spp. (14; 6.4%), enterobacter spp. (11; 5.0%), proteus mirabilis (10; 4.6%) and acinetobacter spp (9; 4.1%). on the other hand, the predominant anaerobic bacteria were bacteroides spp. (7; 3.2%) and p. anaerobius (6; 2.8%) as shown in table 3. table 3: bacterial aetiological agents of infected diabetic foot ulcers in tertiary healthcare facilities in osun state, nigeria, july 2016 to april 2017. antibiotic resistance pattern of aerobic isolates gram-positive bacteria were highly resistant to trimethoprim-sulfamethoxazole (69.5%), penicillin g (66.1%) and gentamicin (40.1%) but demonstrated low-level resistance to piperacillin/tazobactam (6.8%) and amikacin (10.2%). on the other hand, gram-negative bacteria were highly resistant to the third-generation cephalosporins which included ceftriaxone (56%), cefotaxime (55%) and ceftazidime (48.1%), as well as trimethoprim-sulfamethoxazole (89%), gentamicin (54.3%) and ciprofloxacin (54.3%). low rates of resistance were however shown to ertapenem (6.4%), piperacillin/tazobactam (9.3%) and amikacin (12.4%). prevalence and pattern of multidrug resistance among bacterial isolates in infected diabetic foot ulcer further analysis of resistance profiles in the organisms showed that of the 188 aerobic isolates, 121 (64.4%) were mdr, being resistant to one or more agents in at least three antibiotic classes (table 4). the prevalence rates of mdr among gpc and gnb were 55.9% and 68.2%. multidrug resistance rates were generally high among the isolated bacteria especially acinetobacter species (88.9%), enterococcus species (84.6%), enterobacter species (81.8%) and citrobacter species (73.7%). overall prevalence of mdr bacteria among the idfu cases was 80% (n = 74) with rates among in-patient and outpatient cases being 82.9% (n = 58) and 69.6% (n = 16). twelve (35.3%) s. aureus were methicillin-resistant, while of the 129 gnb, 43 (33.3%) were esbl-producing and 10 (7.8%) were carbapenem-resistant (table 5). high esbl production rates were seen among enterobacter species (54.6%), klebsiella species (52.6%), citrobacter species (52.6%) and e. coli (43.5%). other esbl-producing species found were hafnia alvei (1; 20.0%), providencia spp. (1; 33.0%) and morganella morganii (2; 28.6%). ampc β-lactamase production was detected among citrobacter spp. (1; 5.3%), e. coli (2; 8.7%) and m. morganii (1; 14.3%). carbapenem resistance was seen among acinetobacter baumannii (4; 44.4%), h. alvei (2; 40.0%), p. aeruginosa (3; 15.0%) and m. morganii (1; 14.3%). further tests showed that among the 10 carbapenem-resistant isolates, only four, h. alvei (2/4) and a. baumannii (2/4), were carbapenemase-producing. table 4: prevalence of multidrug resistance among bacterial isolates from infected diabetic foot ulcer patients from tertiary healthcare facilities in osun state, nigeria, july 2016 to april 2017. table 5: types and mechanisms of antibiotic resistance among bacterial agents of infected diabetic foot ulcer from tertiary healthcare facilities in osun state, nigeria, july 2016 to april 2017. detection of resistance genes ten (83.3%) of the 12 mrsa isolates harboured the meca gene. at least one of the esbl genes investigated was detected in 86.0% (n = 37) of the 43 esbl-producing organisms. the most common esbl gene detected was blactx-m, harboured by 30 (69.8%) of the phenotypically confirmed esbl-producing isolates. others were blatem (27; 62.8%) and blashv (8; 18.6%) (table 6). thirty-one (72.1%) of the esbl-producing gnb had at least two esbl genes. four of the 10 carbapenem-resistant species were phenotypically confirmed to be carbapenemase-producers. carbapenemase and metallo-beta-lactamase genes were detected in all of the four phenotypically confirmed carbapenemase-producers; they were blavim (n = 3), blakpc (n = 2) and blandm (n = 1). these genes were detected in all of the carbapenemase-producing h. alvei (2/2) and a. baumannii (2/2). table 6: detection of extended-spectrum beta-lactamase genes among phenotypically confirmed extended-spectrum beta-lactamase producing isolates from tertiary healthcare facilities in osun state, nigeria, july 2016 to april 2017. discussion this study shows that a wide range of bacteria are agents of infection of diabetic foot ulcers; it also reveals the high level of antibiotic resistance among the aerobic and facultative anaerobic bacteria with a large proportion of patients having multidrug-resistant infection leading to poor treatment outcomes. infected diabetic ulcers continue to be polymicrobial infections involving aerobic as well as obligate anaerobic organisms. infected diabetic foot ulcers in this study have an average of two different bacteria implicated in the disease, and this is typical of diabetic foot infections across sub-saharan africa and asia.29 gram-negative aerobic bacteria including e. coli, p. aeruginosa, klebsiella species and enterobacter species predominate, reflecting the long-standing nature of these infections as a consequence of poor health-seeking behaviour in low-resourced developing countries.8,30 gram-negative bacteria are more commonly implicated in infected diabetic ulcers in developing countries where most patients present late to healthcare facilities with advanced diseases.29 furthermore, a wide range of anaerobic bacteria primarily bacteroides species and p. anaerobius are important agents of the infections and were isolated from a third of the cases in this study. this suggests infections that are chronic and below the superficial layers of the skin.31 anaerobic bacteria account for 13.3% of the organisms isolated in this study, a higher prevalence than previously reported in the institution32 and may be attributable to deployment of a better anaerobic culture method in which specimens were processed and incubated in a bactron anaerobic chamber. higher prevalence rates of obligate anaerobes were however reported by ikeh et al. in jos, nigeria (17%),33 and al-benwan in kuwait (15.3%).34 low rates noted by richard et al. (1%)35 in france and yates et al. (1%)36 in australia may be due to the fact that most patients tend to seek medical care early enough in countries with good health insurance coverage which will enable a higher proportion to present with low-grade foot infection.37 antibiotic resistance remains a huge problem among diabetic foot ulcer infections; it worsens prognosis and makes treatment outcomes poor.38 multidrug-resistant bacteria were common (74/93; 80%) among idfu cases in this study, and this is possibly due to inappropriate antibiotics use and unrestricted access to antimicrobial drugs in many lowand middle-income countries.39 this is similar to findings in studies conducted in other developing countries40,41 but contrasts findings of several studies in high-income countries including france which reported low prevalence of mdr bacteria among patients with idfu.38,42 a wide spectrum of aerobic bacteria isolated in this study were found to be multidrug-resistant, comparable to findings elsewhere in africa and asia with high mdr rates involving mainly s. aureus, enterobacteriaceae, and p. aeruginosa.41,43 one in every three isolates of s. aureus in this study was mrsa. although prevalence of mrsa appears to be rising in africa, most of the countries have rates lower than 50%.44 our study also revealed that meca, the most common determinant that confers methicillin resistance on s. aureus, was detected in 83.3% of the mrsa strains and this is similar to the observation of chaudhry et al. who detected the gene in 20 (84%) of the 25 phenotypically confirmed mrsa isolates.45 mrsa strains that lack the meca gene may demonstrate methicillin resistance on account of alternate mechanisms of penicillin resistance such as the possession of mecc, a variant of meca discovered in 2011, or other mutations in genes encoding penicillin-binding proteins.46 extended-spectrum β-lactamases, which confer resistance to expanded-spectrum cephalosporins, were produced by 33.3% of all gnb isolated; all but two of the esbl-producing gnb belonged to the family enterobacteriaceae and included e. coli, klebsiella and citrobacter species. the burden of esbl-producing gnb is enormous among patients with idfu especially in poor-resourced countries with prevalence rates being reported to range from 23% to 49% across africa and asia.22,43,45,47,48 the most prevalent esbl gene was the ctx-m which has been reported as the most predominant variant worldwide.49 in the present study, only 10 (7.8%) of the gram-negative bacteria were resistant to the carbapenems. carbapenem resistance-determining genes were present in a. baumannii, h. alvei and m. morganii. carbapenems as drugs of last resort in the treatment of resistant gnb infections have variable but increasing rates of resistance.13 independent risk factors for acquisition of mdr bacteria found in our study are peripheral sensory neuropathy and foot infection duration longer than a month. peripheral neuropathy does not only make diabetics susceptible to foot ulceration but also makes insensate (neuropathic) foot ulcers become more extensive due to continuous painless trauma. loss of protective pains could cause patients not to present to healthcare facilities early enough. in developing countries, such patients with more chronic infections (> 1 month duration) would have engaged in self-prescribed antibiotic use for a prolonged period of time leading to selective pressure and emergence of mdr foot infection.39,50 this is similar to reports among idfu cases from india.40,51,52 other authors also documented the prolonged duration of wound infection as a predictor of infection of diabetic foot ulcers with mdr bacteria.53,54 contrary findings have however been documented from other studies in china, iran and portugal.41,42,55 our finding is also discordant with the report of noor et al. which established that ulcer size is a risk factor for infection by mdr organisms.54 this study also observed a significant association between presence of mdr bacteria in idfu and long duration of hospitalisation (> 1 month) similar to previously documented reports by another author in turkey.14 we did not find any sociodemographic factors that were significantly associated with the occurrence of mdr idfu in our study in agreement with other reports.40,52,53 in contrast, trivedi et al. in the united states noted smoking as an independent risk factor for multidrug-resistant foot wound infection.56 furthermore, in this study, infection of diabetic foot ulcers by mdr pathogens was found to have a significant association with poor treatment outcomes including major limb amputation and mortality. in agreement with our findings, the adverse effects of mdr diabetic foot infection on treatment had been underscored in a systematic review and meta-analysis of data from 28 studies reporting a treatment failure rate of 22.7% and significant association between mdr foot infections and treatment failure.57 limitations the limitation of the study was that the number of patients recruited was limited to 90 and this was because the study was time-bound. also, outpatients could not be followed up because of the multicentre nature of the study. resistance profiles of obligate anaerobic bacteria could not be determined and whole genome sequencing (for strain relatedness) was also not done due to lack of financial support for the study. conclusion the spectrum of agents causing idfu is wide and includes numerous species of aerobic and anaerobic bacteria. there is a high prevalence of mdr aerobic bacteria among them which poses a great limitation to the effective treatment of cases. acknowledgements the authors are grateful to faith bankole and ayo isinkaye for assistance in data management and babatunde odetoyin for technical assistance in the central science laboratory, obafemi awolowo university, ile-ife, nigeria. competing interests the authors have declared that no competing interests exist. authors’ contributions a.t.a. and a.o.a. conceived and designed the study. b.k. and v.o.r. contributed to the design of the study. a.t.a. and a.o.a. conducted laboratory experiments. a.t.a. and a.o.a. analysed the data. a.t.a., a.o.a. and v.o.r. wrote the final report. all authors reviewed and approved the final report. sources of support this study was supported by the obafemi awolowo university teaching hospitals complex. data availability the data sets used and analysed during the current study are available from the corresponding author on reasonable request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references lipsky ba, senneville e, abbas zg, et al. guidelines on the diagnosis and treatment of foot infection in persons with diabetes (iwgdf 2019 update). diabetes metab res rev. 2020;36(s1):e3280. https://doi.org/10.1002/dmrr.3280 zhang p, lu j, jing y, tang s, zhu d, bi y. global epidemiology of diabetic foot ulceration: a systematic review and meta-analysis (?). ann med. 2017 mar;49(2):106–116. https://doi.org/10.1080/07853890.2016.1231932 atun r, gale ea. the challenge of diabetes in sub-saharan africa. lancet diabetes endocrinol. 2015 sep;3(9):675–677. https://doi.org/10.1016/s2213-8587(15)00236-3 jia l, parker cn, parker tj, et al. incidence and risk factors for developing infection in patients presenting with uninfected diabetic foot ulcers. jan y-k, ed. plos one. 2017;12(5):e0177916. https://doi.org/10.1371/journal.pone.0177916 adigun i, olarinoye j. foot complications in people with diabetes: experience with 105 nigerian africans. diab foot j. 2008;11(1):36–42. alavi sm, khosravi ad, sarami a, dashtebozorg a, montazeri ea. bacteriologic study of diabetic foot ulcer. pakistan j med sci. 2007;23(5):681–684. khanolkar mp, bain sc, stephens jw. the diabetic foot. quart j med. 2008;101(9):685–695. https://doi.org/10.1093/qjmed/hcn027 lipsky ba, berendt ar, cornia pb, et al. infectious diseases society of america clinical practice guideline for the diagnosis and treatment of diabetic foot infections. clin infect dis. 2012;54(12):132–173. https://doi.org/10.1093/cid/cis346 ventola cl. the antibiotic resistance crisis: part 1: causes and threats. pharm therap. 2015;40(4):277–283. magiorakos ap, srinivasan a, carey rb, et al. multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. clin microb inf. 2012;18(3):268–281. laxminarayan r, matsoso p, pant s, et al. access to effective antimicrobials: a worldwide challenge. lancet. 2016 jan 9;387(10014):168–175. https://doi.org/10.1016/s0140-6736(15)00474-2 serra-burriel m, keys m, campillo-artero c, et al. impact of multi-drug resistant bacteria on economic and clinical outcomes of healthcare-associated infections in adults: systematic review and meta-analysis. plos one. 2020;15(1):e0227139. https://doi.org/10.1371/journal.pone.0227139 pitocco d, spanu t, di leo m, et al. diabetic foot infections: a comprehensive overview. eur rev med pharmacol sci. 2019;23(2):26–37. kandemir ö, akbay e, şahin e, milcan a, gen r. risk factors for infection of the diabetic foot with multi-antibiotic resistant microorganisms. j infect. 2007;54(5):439–445. https://doi.org/10.1016/j.jinf.2006.08.013 shahi sk, kumar a, kumar s, singh sk, gupta sk, singh tb. prevalence of diabetic foot ulcer and associated risk factors in diabetic patients from north india. j diab foot compl. 2012;4(4):83–91. kaur n, kaur n, kumar r, gill ak. clinical and susceptibility profile from diabetic foot patients in tertiary care hospital. scholars j appl med sci. 2014;2(2d):865–869. vasnessa pds, denise rds, roberto ac. risk factor for primary major amputation in diabetic patients. sao paulo med j. 2006;124(2):66–70. https://doi.org/10.1590/s1516-31802006000200004 adeleye jo. diabetic foot disease: the perspective of a nigerian tertiary health care centre. pract diab int. 2005;22(6):211–214. https://doi.org/10.1002/pdi.819 bekele f, chelkeba l, fekadu g, bekele k. risk factors and outcomes of diabetic foot ulcer among diabetes mellitus patients admitted to nekemte referral hospital, western ethiopia: prospective observational study. ann med surg (lond). 2020 mar;51:17–23. https://doi.org/10.1016/j.amsu.2020.01.005 caruso p, longo m, gicchino m, scappaticcio l, caputo m, maiorino mi, et al. long-term diabetic complications as predictors of foot ulcers healing failure: a retrospective study in a tertiary-care center. diab res clin pract. 2020;163:108147. https://doi.org/10.1016/j.diabres.2020.108147 clsi. performance standards for antimicrobial susceptibility testing. twenty-fifth informational supplement. clsi document m100-s25. wayne, pa: clinical and laboratory standards institute; 2015. anjli sj, shadija pg, ghosh sj. detection of multidrug resistant gram negative bacilli in type ii diabetic foot infections. inter j med health sci. 2013;2(2):186–194. sidjabat he, paterson dl, adams-haduch jm, et al. molecular epidemiology of ctx-m-producing escherichia coli isolates at a tertiary medical center in western pennsylvania. antimicrob agents chemother. 2009;53(11):4733–4739. https://doi.org/10.1128/aac.00533-09 monstein hj, ostholm-balkhed a, nilsson mv, dornbusch nl. multiplex pcr amplification assay for rapid detection of shv, tem and ctx-m genes in enterobateriacea. apmis. 2007;115(12):1400–1408. https://doi.org/10.1111/j.1600-0463.2007.00722.x pasanen t, koskela s, mero s, et al. rapid molecular characterization of acinetobacter baumannii clones with rep-pcr and evaluation of carbapenemase genes by new multiplex pcr in hospital district of helsinki and uusimaa. plos one. 2014;9(1):e85854. https://doi.org/10.1371/journal.pone.0085854 zhao s, jang d, xu p, et al. an investigation of drug-resistant acinetobacter baumannii infection in a comprehensive hospital in east india. ann clin microbiol antimicrob. 2015;14:7. https://doi.org/10.1186/s12941-015-0066-4 khan aks, shetty pj, sarayu yl, chidambaram a, ranganathan r. detection of meca genes of methicillin-resistant staphylococcus aureus by polymerase chain reaction. int j health rehabil sci. 2012;1:64–68. https://doi.org/10.5455/ijhrs.000000011 dashti aa, jadaon mm, abdulsamad am, dashti hm. heat treatment of bacteria: a simple method of dna extraction for molecular techniques. kuwait med j. 2009;41(2):117–122. zubair m. prevalence and interrelationships of foot ulcer, risk-factors and antibiotic resistance in foot ulcers in diabetic populations: a systematic review and meta-analysis. world j diabetes. 2020;11(3):78–89. https://doi.org/10.4239/wjd.v11.i3.78 desalu oo, salawu fk, jimoh ak, adekoya ao, busari oa, olokoba ab. diabetic foot care: self reported knowledge and practice among patients attending three tertiary hospital in nigeria. ghana med j. 2011;45(2):60–65. https://doi.org/10.4314/gmj.v45i2.68930 mendes jj, nerves j. diabetic foot infections: current diagnosis and treatment. j diab foot compl. 2012;4(1):26–45. ako-nai ka, ikem ci, akinloye oo, aboderin oa, ikem tr, kassim oo. characterization of bacterial isolates from diabetic foot infections in ile-ife, southwestern nigeria. foot. 2006;16(3):158–164. https://doi.org/10.1016/j.foot.2006.05.001 ikeh ei, puepet f, nwadiaro c. studies on diabetic foot ulcers in patients at jos university teaching hospital, nigeria. afr j clin exp microb. 2003;4(2):52–61. https://doi.org/10.4314/ajcem.v4i1.7324 al-benwan ka, al mulla a, rotimi vo. a study of the microbiology of diabetic foot infections in a teaching hospital in kuwait. j infect public health. 2012;5(1):1–8. https://doi.org/10.1016/j.jiph.2011.07.004 richard jl, lavigne jp, got i, et al. management of patients hospitalized for diabetic foot infection: results of the french opidia study. diabetes metab. 2011;37(3):208–15. https://doi.org/10.1016/j.diabet.2010.10.003 yates c, may k, hale t, allard b, rowlings n, freeman a, et al. wound chronicity, inpatient care, and chronic kidney disease predispose to mrsa infection in diabetic foot ulcers. diabetes care. 2009;32(10):1907–1909. https://doi.org/10.2337/dc09-0295 oecd (june 27, 2013). oecd health data: social protection. oecd health statistics (database). paris: oecd. https://doi.org/10.1787/data-00544-en richard jl, sotto a, jourdan n, et al. risk factors and healing impact of multidrug-resistant bacteria in diabetic foot ulcers. diab metab. 2008;34(2):363–369. https://doi.org/10.1016/j.diabet.2008.02.005 omolase co, adeleke oe, afolabi ao, afolabi ot. self medication amongst general out-patients in a nigerian community hospital. ann ibadan postgrad med. 2007;5(2):64–67. https://doi.org/10.4314/aipm.v5i2.64032 gadepalli r, dhawan b, sreenivas v, kapil a, amini ac, chaudhry ra. clinico-microbiological study of diabetic foot ulcers in an indian tertiary care hospital. diabetes care. 2006;29(8):1727–1732. https://doi.org/10.2337/dc06-0116 amini m, davati a, piri m. determination of the resistance pattern of prevalent aerobic bacterial infections of diabetic foot ulcer. iranian j pathol. 2013;8(1):21–26. mendes jj, marques-costa a, vilela c, et al. clinical and bacteriological survey of diabetic foot infections in lisbon. diab res clin pract. 2012;95(1):153–161. https://doi.org/10.1016/j.diabres.2011.10.001 dwedar r, ismail dk, abdulbaky a. lecturer diabetic foot infection: microbiological causes with special reference to their antibiotic resistance pattern. egyptian j med microbiol. 2015;24(3):95–102. https://doi.org/10.12816/0024935 falagas me, karageorgopoulos de, leptidis j, korbila ip. mrsa in africa: filling the global map of antimicrobial resistance. plos one. 2013;8(7):e68024. https://doi.org/10.1371/journal.pone.0068024 chaudhry wn, badar r, jamal m, jeong j, zafar j, andleeb s. clinico-microbiological study and antibiotic resistance profile of meca and esbl gene prevalence in patients with diabetic foot infections. exp ther med. 2016;11(3):1031–1038. https://doi.org/10.3892/etm.2016.2996 shore ac, deasy ec, slickers p, et al. detection of staphylococcal cassette chromosomemec type xi carrying highly divergent meca, meci, mecr1, blaz, and ccr genes in human clinical isolates of clonal complex 130 methicillin-resistant staphylococcus aureus. antimicrob agents chemother. 2011;55:3765–3773. https://doi.org/10.1128/aac.00187-11 varaiya a, dogra j, kulkarni m, bhalekar p. extended spectrum beta lactamase (esbl) producing escherichia coli and klebsiella pneumoniae in diabetic foot infection. indian j med microbiol. 2008;26(3):281–282. https://doi.org/10.4103/0377-4929.42513 saltoglu n, ergonul o, tulek n, et al. influence of multidrug resistant organisms on the outcome of diabetic foot infection. int j infect dis. 2018;70:10–14. https://doi.org/10.1016/j.ijid.2018.02.013 shaikh s, fatima j, shakil s, rizvi smd, kamal ma. antibiotic resistance and extended spectrum beta-lactamases: types, epidemiology and treatment. saudi j biol sci. 2015;22(1):90–101. https://doi.org/10.1016/j.sjbs.2014.08.002 levin me. an overview of the diabetic foot: pathogenesis, management and prevention of lesions. inter j. diab dev countr. 1994;14:39–47. valappil rk, krishnan s, bai sdk. multidrug resistant organisms in diabetic foot ulcers-analysis of risk factors and clinical outcome. j med sci clin res. 2017;9(2):18138–18143. https://doi.org/10.18535/jmscr/v5i2.144 zubair m, abida m, jamal a. clinicobacteriology and risk factors for the diabetic foot infection with multidrug resistant microorganisms in north india. biol med. 2010;2(4):22–34. hartemann-heurtier a, robert j, jacqueminet s, et al. diabetic foot ulcer and multidrug-resistant organisms: risk factors and impact. diabet med. 2004;21(7):710–715. https://doi.org/10.1111/j.1464-5491.2004.01237.x noor s, borse ag, ozair m, raghav a, parwez i, ahmad j. inflammatory markers as risk factors for infection with multidrug-resistant microbes in diabetic foot subjects. foot (edinb). 2017;32:44–48. https://doi.org/10.1016/j.foot.2017.05.001 ji x, jin p, chu y, feng s, wang p. clinical characteristics and risk factors of diabetic foot ulcer with multidrug-resistant organism infection. inter j lower extrem wounds. 2014;13(1):64–71. https://doi.org/10.1177/1534734614521236 trivedi u, parameswaran s, armstrong a, et al. prevalence of multiple antibiotic resistant infections in diabetic versus nondiabetic wounds. j pathog. 2014;2014:173053. http://doi.org/10.1155/2014/173053 vardakas kz, horianopoulou m, falagas me. factors associated with treatment failure in patients with diabetic foot infections: an analysis of data from randomized controlled trials. diabetes res clin pract. 2008;80:344–351. https://doi.org/10.1016/j.diabres.2008.01.009 background observations acknowledgements references about the author(s) pascale ondoa african society for laboratory medicine, addis ababa, ethiopia amsterdam institute for global health and development, academic medical centre, department of global health, university of amsterdam, amsterdam, netherlands nqobile ndlovu african society for laboratory medicine, addis ababa, ethiopia mah-sere keita african society for laboratory medicine, addis ababa, ethiopia marguerite massinga-loembe africa centres for disease, control and prevention, addis ababa, ethiopia yenew kebede africa centres for disease, control and prevention, addis ababa, ethiopia collins odhiambo african society for laboratory medicine, addis ababa, ethiopia teferi mekonen african society for laboratory medicine, addis ababa, ethiopia aytenew ashenafi africa centres for disease, control and prevention, addis ababa, ethiopia amha kebede african society for laboratory medicine, addis ababa, ethiopia john nkengasong africa centres for disease, control and prevention, addis ababa, ethiopia citation ondoa p, ndlovu n, keita m-s, et al. preparing national tiered laboratory systems and networks to advance diagnostics in africa and meet the continent’s health agenda: insights into priority areas for improvement. afr j lab med. 2020;9(2), a1103 https://doi.org/10.4102/ajlm.v9i2.1103 opinion paper preparing national tiered laboratory systems and networks to advance diagnostics in africa and meet the continent’s health agenda: insights into priority areas for improvement pascale ondoa, nqobile ndlovu, mah-sere keita, marguerite massinga-loembe, yenew kebede, collins odhiambo, teferi mekonen, aytenew ashenafi, amha kebede, john nkengasong received: 20 sept. 2019; accepted: 29 may 2020; published: 21 sept. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. background access to diagnostics remains sub-optimal in africa due to limited human, financial and technical resources that affect various components of the health system.1,2 additionally, the lack of standardised systems for evaluation and registration of diagnostics3 cripples the introduction of better technologies, representing missed opportunities to address healthcare challenges. examples of poor access to diagnostics and its dramatic consequences are numerous. despite strong vertical control programmes, 40% of hiv-infected patients on antiretroviral therapy do not receive the recommended yearly hiv viral load monitoring test.4 in 2016, a dramatic 21% of children born to hiv-positive mothers were reported as not receiving early infant diagnosis testing before age 8 weeks in west and central africa.5 data from the world health organization (who) global tuberculosis database of 20166 also indicate that drug-resistant tuberculosis is largely missed in africa. in addition to 70% of patients not being notified, a rifampicin susceptibility test was available to less than 10% of patients in 23 of 47 countries and second-line resistance testing was available only in 60% of the countries on the continent.7 the picture is equally worrisome for diseases that are poorly or not supported through dedicated programmes. nine in 10 individuals carrying the hepatitis b or c virus have never been tested, while these infections are estimated to cause 60% of liver cancers and an epidemic larger than that caused by hiv.8 in a study conducted in senegal in 2015–2016, less than 30% of pregnant women attending antenatal care at the primary healthcare level had access to the minimum panel of screening tests for the most common clinical conditions threatening maternal and child health.9 almost half of the mortality cases associated with cervical cancer are due to late detection of the disease.10 more recently, it appeared that when the first coronavirus disease 2019 (covid-19) case was reported in egypt in february 2020, only 2 of the 55 countries on the continent were capable of detecting the severe acute respiratory syndrome coronavirus 2 (sars-cov-2). insufficient access to in vitro diagnostics (ivd) at all levels of healthcare delivery in low-income and middle-income countries reduces access to life-saving treatments, impairs the delivery of quality healthcare and compromises progress towards universal health coverage in africa. implementation of simplified, more robust and more affordable diagnostic options has been proposed to increase access to diagnostics in low-resource settings. rapid diagnostic tests that can be performed at the community level or through self-testing have transformed the management and prevention of several diseases such as hiv infection, malaria and diabetes.11,12,13 point-of-care molecular technologies, such as the genexpert® or the m-pima®, have been demonstrated to increase specificity and simplicity of testing, while reducing the turnaround time for test results14,15 for improved patient retention and management. despite addressing critical testing processes or delivery gaps, innovative or conventional diagnostic technologies frequently fail to translate into tangible public health outcomes.16,17,18 the failure of diagnostics to reach a large proportion of the population in need of it can partly be linked to implementation approaches that are designed for the site level, with oversight of the tiered laboratory network requirements and insufficient attention given to the underlying laboratory systems (e.g. supply chain, workforce, finance, etc.). complementing the maputo declaration of 2008,19 the freetown declaration of 201520 underscores that delivering diagnostic services in the context of functional, integrated national laboratory networks is the recommended strategy for providing maximum population coverage and cost-effective utilisation and delivery of diagnostic services in resource-limited settings. apart from selected disease-dedicated investments (e.g. hiv, tuberculosis), national tiered laboratory networks generally remain grossly underfunded with mild to critical dysfunctions in the underlying laboratory systems. in addition to falling short in the provision of optimal access to essential clinical diagnostics, the current sub-optimal capacity of tiered laboratory networks translates into weaknesses in the detection component of the ‘prevent, detect, respond’ framework of health security21,22 missed opportunities to stop onward transmission of infectious diseases and to detect and prevent non-communicable diseases hinder efforts to reduce the burden of illnesses in africa. the who estimates that the resulting loss of annual productivity due to the heavy disease burden in africa equalled $2.4 trillion and 630 million years of healthy lives in 2015.23 despite the call for more attention in building effective public health laboratory systems,24 which was made in the aftermath of the ebola epidemic of 2015, few data exist that allow quantification of the overall performance of national laboratory networks and systems across diseases. this lack of information prevents the design of impactful interventions and hinders the uptake of the otherwise large and relevant range of diagnostic technologies available. in this report, the african society for laboratory medicine (aslm) and the africa centres for disease control and prevention (africa cdc) share a unique insight into some of the most critical areas for improvement to bridge the gap between the capacity of laboratory networks and the promises of diagnostic technologies. observations root causes of dysfunction in laboratory networks are not sufficiently measured or addressed actionable data on integrated laboratory network functionality are scarce. the who joint external evaluation25 tool provides a high-level overview of the performance of the national laboratory network across four domains: (d1.1) laboratory testing for the detection of priority diseases, (d1.2) specimen referral and transport system, (d1.3) effective national diagnostic network and (d1.4) laboratory quality system, as part of the evaluation of entire national health systems to support compliance with international health regulations requirements. data from 54 countries21 indicate that quality management and sample referral systems are the most neglected areas across the continent with 68.1% and 50.0% of countries having none to only a basic capacity in place (figure 1), further aggravating the gaps in diagnostic networks with issues related to coverage and reliability of testing. more than 48.0% of countries have gaps in defining or providing access to tier-specific laboratory testing strategies, while more than 84.0% of the countries assessed demonstrated sustainable capacity for laboratory testing to support the surveillance of 10 priority diseases. the global health security agenda laboratory network (labnet26) scorecard was developed to complement the joint external evaluation tool and to provide more granularity on nine core capabilities of the laboratory network and systems, enabling the identification of specific gaps and related root causes. in eight countries where this evaluation was conducted, the average performance of laboratory networks ranged from none to basic capacity across the nine core capabilities assessed (figure 2), highlighting the presence of many common disabling factors (table 1), including: weak laboratory governance. in several countries, no directorate of laboratories exists or it is not directly placed under the authority of the ministry of health, preventing adequate coordination of laboratory services and limiting the sphere of operation of the laboratory vis-à-vis the other health sectors. unclear assignment of administrative versus technical tasks and mandates between directorates and national public health institutes and national reference laboratories also prevents the overall development and enforcement of regulations related to various aspects of diagnostics (such as ivd evaluation and registration, definition and updates of tier-specific minimal testing packages, laboratory staffing norms or definition and implementation of quality standards). figure 1: proportion of african countries (n = 54) at various levels of capacity in the area ‘laboratory network’ of the joint external evaluation tool. figure 2: overall level of laboratory network functionality in eight countries assessed. table 1: most critical gaps in laboratory network core capabilities were identified through the laboratory network assessment of eight sub-saharan african countries between 2017 and 2019, and their implications for diagnostic services. two countries with strong laboratory governance include ethiopia, through the ethiopian public health institute, and uganda, through the directorate of laboratory services of uganda. they offer good examples of best practices in the management of the laboratory systems and networks. these countries report updated national policies and strategic plans, budget lines earmarked for laboratory functions and regulations defining and enforcing laboratory clinical and public health functions from the reference level to the community level: missed opportunities to prioritise diseases for both surveillance and care. countries do not have access to up-to-date and fit-for-purpose epidemiological data to prioritise diseases of public health importance using a risk-based approach. often, the who list of 43 pathogens for surveillance serves as a de facto national list of ‘priority’ pathogens. the who recommends prioritising 10 pathogens27 to ensure that communicable diseases with the most severe morbidity and mortality are effectively screened, confirmed and the treatment monitored at the various tiers of the diagnostic network. countries implementing the global health security agenda prioritise 10 core tests covering international health regulations immediately notifiable diseases among the who top 10 causes of death in low-income and middle-income countries. the integrated disease surveillance and response framework of the who regional office for africa recommends prioritising epidemic-prone diseases targeted for eradication and elimination, and endemic diseases, resulting in a list of 19 diseases. in practice, these overlapping recommendations are difficult to interpret. the plethora of ‘prioritised’ diseases complicates the implementation of robust tier-specific packages and surveillance systems that can support both epidemiology reporting and clinical care. insufficient attention to evidence-based management of laboratory networks. none of the countries assessed had well-defined processes to routinely monitor the performance of the laboratory network without the support of external partners. this situation precludes the establishment of a quality assurance loop of laboratory networks and systems. ultimately, the lack of monitoring and evaluation systems (i.e. lack of key performance indicators for the laboratory sector and of responsible units to collect, analyse and act upon the data) for laboratory networks undermines the return on investment of most capacity strengthening interventions aimed at advancing diagnostic services. recommendations strengthening of integrated quality management and specimen referral systems are the most urgent interventions needed, the outcomes of which should be evaluated against the advancement of joint external evaluation scores. various initiatives funded by global stakeholders are ongoing (e.g. united states agency for international development, united states president’s emergency plan for aids relief, global fund) with the potential to be transitioned to and sustained by stronger and empowered national laboratory leadership with the political support of africa cdc. the aslm and their partners can work together at formulating clearer guidance on the respective mandates of directorates of laboratories and national public health institutes (including the monitoring and evaluation framework for laboratory network functions) as well as advocating to empower the laboratory sector. these efforts align with the recommendations of the maputo and the freetown declarations. assisting countries to define tier-specific testing packages that address the needs of clinical diagnostics and disease surveillance is another important intervention with the potential to guide the introduction of diagnostics at the levels where they are most needed and cost-effective. we recommend that every country conduct an external or self-evaluation of their laboratory systems once a year or once every two years as part of a continuous quality improvement cycle for their national tiered laboratory network. while the joint external evaluation tool provides high-level dashboards for a set of four indicators, the labnet scorecard is designed to guide countries in selecting specific and feasible interventions most likely to tackle the root causes of the identified dysfunctions across a comprehensive set of indicators. laboratory networks are not configured to support diagnostic services that are integrated, cost-effective and with maximum population coverage mutualising scarce resources for most cost-effective health services is (or should be) a constant concern in low-resource settings. a data-driven configuration of a national laboratory network can support the design of faster and more affordable sample transportation routes towards testing hubs. from the specimen referral landscape, assessments that were performed during 2015 and 2016 in eight countries by aslm under the global health security agenda laboratory strengthening effort revealed that a certain level of integration of the specimen transport system (sts) can exist, often between hiv and tuberculosis programmes. the stss are generally fragmented, working in parallel, using different transport mechanisms depending on the disease programme, are funded by various donors and have challenges in terms of cost-effectiveness, turnaround time and coverage. a few countries, such as ethiopia, rwanda and uganda, have highly integrated specimen referral networks that cater to any disease programme and span across diagnosis to surveillance, detection and response. ideally, this disease-agnostic approach should be developed such that individual stss are effectively and efficiently networked, and that any type of specimen can be easily and seamlessly moved from where it originates to the appropriate diagnostic equipment. addressing the common problem of fragmented and disease-specific stss, aslm coordinated the development of a standardised sts assessment and development toolkit,28 addressing multiple diseases, surveillance and clinical diagnostic needs, and factoring in different modes of transportation. one country, burkina faso, was able to use the findings of the assessment to establish a new specimen referral system for surveillance that is now being built upon, scaled up and integrated to cover any disease programme.29 the availability of up-to-date geo-localised information about laboratory network capacity provides evidence on which to base the process of defining optimal service configurations, including the shortest routes for sample transportation, maximum population coverage for testing services, cost-effective supply chains, or opportunities for testing integration. initiatives aimed at collecting geographical information system data on laboratory capacity are gaining momentum to improve the performance of hiv and tuberculosis control programmes.30,31 the aslm and africa cdc have implemented a continent-wide laboratory capacity mapping programme (labmap32) across diseases, based on open-source tools (ona [ona systems inc., nairobi, kenya], planwise [instedd, sunnyvale, california, united states]33) and fostering country ownership. this system allows the easy collection, curation and analysis of geospatial data to make informed decisions on national laboratory networks and is interoperable with the district health information system (dhis2, university of oslo, norway) 234. thirteen of the 54 countries on the continent (24%) are currently using the system under the coordination of africa cdc’s regional coordinating centres.35 among the 101 level 4 and level 3 laboratories assessed, only 40% had the capacity to conduct culture or polymerase chain reaction-based tuberculosis diagnosis, 11% and 34% could perform hiv drug resistance genotyping and early infant diagnosis, and 36% could run confirmatory testing for meningitis through culture or polymerase chain reaction (figure 3). using labmap data from 2018, we determined that in niger,36 eight facilities at level 3 are conducting meningitis testing, including serology, bacterial culture and nucleic acid testing, covering a total of 7.9 million people (40% of the nigerien population) within a 2-hour drive radius. only one facility (covering 2.8 million people, 14% of the population) is equipped to conduct polymerase chain reaction for the differential diagnosis of pathogens causing meningitis, which is critical for the swift adjustment of antibiotic therapy. planwise calculated that three strategically located level 3 laboratories involved in meningitis testing could be upgraded with a maximum impact reaching out to an additional 4.1 million (20%) inhabitants. planwise and other similar software like the supply chain guru from llamasoft37 offer the opportunity to calculate the best options to improve the laboratory network testing capacity and coverage, including upgrading existing facilities, building new laboratories or configuring specimen referral and supply chain routes. figure 3: gaps between actual diagnostic capacity and world health organization recommendations for minimal testing package in 101 level 4 and level 3 laboratories in 12 african countries (data from 2018 to 2019). recommendations data-driven optimisation of laboratory networks is an important management activity that can support essential public health and clinical functions. key considerations of any optimisation exercise should include at least: the structure of the tiered laboratory networks, (essential) testing needs for both clinical diagnosis and disease surveillance, and opportunities for test integration. specimen referrals are important systems underlying national laboratory networks. taking a network approach for developing integrated stss may provide greater cost savings, efficiencies, increased coverage and increased access. this approach can begin by mapping and optimising the overall referral network based on the existing diagnostics network (or any upcoming changes), reducing redundancies where possible and leveraging existing or new systems to create economies of scale, clearly planning and budgeting for the optimised network and implementing the new approach. any potential benefits should be clearly estimated and used to convince governments of the approach and gain buy-in from other stakeholders as well. regular collection and updating of (geo-located) laboratory capacity information is necessary to inform network optimisation exercises. such activities could be enforced as part of laboratory registration or licensing and re-licensing processes under the coordination of the directorate of laboratories. most diagnostic services delivered through laboratory networks are not quality assured unreliable diagnostic tests can compromise the quality of healthcare delivery. a laboratory quality management system (lqms) is a formalised system that documents processes, procedures and responsibilities for achieving an international standard of quality. the implementation of lqms was identified as a priority to strengthen laboratory services in africa38,39 a decade ago. as part of this momentum effect, various tools and frameworks have been developed and disseminated to guide laboratories towards lqms implementation and international organization for standardization 15189 accreditation. some of these resources have a regional or a disease-specific focus; these include the strengthening laboratory management toward accreditation, the stepwise laboratory quality improvement process towards accreditation (slipta), which also includes a tuberculosis-specific checklist, the lqms training tool kit and the laboratory quality services international group. to date, around 520 laboratories have received international accreditation on the continent. more than 370 of these accredited facilities are in south africa and most of them belong to the public sector, highlighting geographic differences and the lagging behind of private laboratories. the slipta is a who regional office for africa programme assessing laboratory progress towards the international organization for standardization 15189 standards and with aslm serving as the secretariat. since its launch in 2012, around 430 laboratories implementing lqms have been assessed across 17 countries (table 2), illustrating the increasing awareness of national stakeholders with regard to quality requirements, as well as the commitment of the laboratory community to advance diagnostic services. however, this number is below the original target of 2500 enrolled laboratories set at the programme onset40 and represents a modest outcome at both the continental and country scale. a couple of countries have demonstrated impressive coverage of the slipta programme (e.g. botswana and namibia, table 2). however, assuming that only 50% of the total number of government laboratories are in hospitals (the target facilities to implement the international organization for standardization 15189 norm), the current number of laboratories engaged in slipta represent only 0.3% of the total number of government laboratories in nigeria and 3% in kenya. some bottlenecks with lqms implementation using slipta are inherent to the programme itself. the handling of certification requests and processes through countries’ ministry of health prioritises government over private laboratories. the lack of core funding supporting the programme translates into insufficient capacity for aslm to cover the needs of an entire continent, not only to conduct audits but also to mentor laboratories towards quality improvement and accreditation. benefitting from more stable united states president’s emergency plan for aids relief funding, and despite reaching an impressive 1333 laboratories across various continents,41 the mentorship-focused strengthening laboratory management toward accreditation programme is equally unable to comprehensively cover the needs of entire national laboratory networks. advocacy efforts towards policymakers are also impaired by the lack of simple indicators linking lqms implementation with improved health outcomes. country-specific roadblocks have also been identified such as low political commitment and the lack of regulatory and policy frameworks guiding the expansion of lqms as national programmes, adapted for each tier of laboratory networks. recently, continuous quality improvement initiatives targeting disease-specific testing (such as hiv) have been implemented. although continuous quality improvement directly addresses the quality of diagnostic testing through a problem-solving approach linked to patient outcomes, it still needs to be embedded into larger quality management programmes in order to contribute to sustainable outcomes supporting universal healthcare coverage and international health regulations. table 2: stepwise laboratory quality improvement process towards accreditation coverage of facilities in the public and private sector in 17 african countries (as of august 2019). in addition to the poor coverage of lqms implementation, most countries do not have systems in place to ensure that all testing sites comply with the basic components of quality assurance such as external quality assessments, proficiency testing schemes42 and various quality checks linked to post-market surveillance of ivd. collectively, these observations suggest that dramatically high proportions of diagnostic tests delivered in african countries are not adequately quality assured. this inevitably results in laboratory test results that are incorrect or fraudulent, causing a lack of trust among clinicians and communities. recommendations to overcome these challenges, the aslm and who regional office for africa launched slipta version 2.0,43 which proposes to institutionalise slipta at the country level, harness partner resources for slipta funding and redefine the role of aslm as a coordinating rather than an implementing body. this strategic approach is currently being implemented through regional collaborations where the west african health organization and east, central and southern africa health community are providing leadership to advance lqms using the slipta programme in west, central east and southern africa, with aslm serving as a high-level coordinator. according to this model, aslm and its partners work at generating a sufficient pool of local slipta auditors and laboratory mentors who can be mobilised to advance lqms and conduct slipta audits upon regional or national request. the efforts of africa cdc to establish the regional integrated laboratory network (rislnet) is also contributing to the extension of slipta version 2.0, through the implementation of lqms in national public health institutes, with subsequent deployment of the system in lower tiers of the national laboratory networks and across the private sector. a couple of african countries like ethiopia have stepped forward to ‘franchise’ the slipta model as national programmes aiming to cover all laboratory facilities from the public and the private sector. the foundation of a country-led slipta programme is the definition of national quality standards for diagnostic services at each level of service delivery, clearly describing which laboratory has the vocation to be accredited or certified and against which set of minimum quality standards. critical shortage of pathologists compromises present and future benefits of laboratory diagnostics clinical pathologists are physicians trained in various disciplines of laboratory medicine, such as haematology, medical microbiology, transfusion medicine, clinical biochemistry or cytopathology.44 they provide highly specialised knowledge and leadership, ensuring the quality of the pre-analytical, analytical and post-analytical phases of testing, as well as critical information on the severity and prognosis of diseases based on test results. clinical pathologists make sure that patients are started and maintained on the correct treatment regimen. in the united states and united kingdom, the pathology workforce varies between 3 and 5 per 100 000 inhabitants.45 a quick desk survey conducted by aslm reveals critical gaps in the clinical pathology profession in 10 countries of sub-saharan africa. firstly, the mere definition of this profession is often not well understood, with most survey respondents only aware of anatomic pathologists but not clinical pathologists. data from the college of pathologists in east, central and southern central africa show a bias towards anatomical pathology (60%) compared to clinical pathology (18%) of the 119 registered pathologists (dr shahin sayed, personal communication). some countries also use pharmacists as ‘pathologists’,46 although this profession requires a background in medical studies, suggesting insufficient clinical interpretation of laboratory test results that might compromise patient outcomes. secondly, an up-to-date inventory of pathologists by national professional councils seems to be lacking in many countries, suggesting that these highly specialised professionals are not adequately registered or certified in their respective countries. thirdly, the ratio of pathologists (regardless of their specialty) is 5–350 times lower than ratios observed in the united states (table 3), translating into gaps of more than 4000 pathologists for a country like ethiopia or more than 6000 for a country like nigeria. in most countries sampled, the number of pathologists is lower than the number of tier 2 and tier 3 hospitals (where pathology services are required), suggesting that clinical pathologists would have to serve in more than one facility to reduce the gaps. for example, 61 pathologists in ethiopia have to support a total of 400 hospitals. the number of medical microbiologists (a sub-specialty of clinical pathology), at 0.5, is even lower in all countries sampled. this worrisome situation raises concerns about the sustainability and impact of current global and national efforts to establish diagnostic capacity for the control of antibiotic resistance on the african continent. the introduction of novel diagnostic technologies such as point-of-care testing at the community level or next generation sequencing at the reference level will only increase the need for specialised laboratory medicine professionals who are able to ensure the correct use and interpretation of diagnostic tests for improved patient and public health outcomes. collectively, these data highlight severe gaps in general and clinical pathology, in particular in sub-saharan african countries. table 3: overview of numbers, coverage and needs of pathologists in nine surveyed countries of africa (data from august 2019). recommendations in-country capacity for pathology training is commonly reported, with efforts by the college of pathologists in east, central and southern central africa and other organisations to advocate for training of more pathologists and the laboratory workforce. however, the magnitude of the gaps highlighted here and by others demands that many more resources be deployed to produce higher numbers of pathologists at a faster pace in the disciplines associated with the most severe disease burdens, and to provide acceptable solutions for task shifting at each tier of the laboratory network. key interventions to reduce the shortage of clinical pathologists include: the formulation of staffing norms in national laboratory strategic plans and healthcare human resources development strategies by defining the number and profile of pathologists at each relevant tier of the laboratory network and increasing opportunities for education, training and mentorship at the regional level, including innovative digital, remote training options. this could be done as part of the objective of the workforce development institute of africa cdc,47 with the establishment of certification and qualification programmes that ensure standard levels of competency, at least in national public health institutes. regulatory bottlenecks slow down introduction of useful diagnostics the past decade has seen the introduction of game-changing technologies for major public health diseases such as hiv, tuberculosis and malaria, which promise easier access, use and impact of diagnostics at the community level where most patients seek care. the who’s ivd prequalification process is a standardised procedure to determine whether products meet requirements for safety, quality and performance. the findings of this prequalification are used to provide guidance to countries in selecting laboratory diagnostics to be implemented at the programme level. the who prequalification represents the ‘ticket’ for any ivd to penetrate national markets, and is a process that takes 2–3 years. additionally, national regulatory frameworks often foresee additional evaluations to verify that the prequalified diagnostic is adapted to specific contexts. however, the relevance of multiple, in-country evaluations is not always clear, represents unnecessary repetition, and has no additional value compared to who prequalification results or to a well-designed single evaluation conducted in one centre of excellence located in a region with similar disease epidemiology. delayed registration of ivds in-country prevents access to reliable existing diagnostics for many priority diseases including those associated with outbreaks. regulatory bottlenecks for ivds during country registration compromise the implementation of essential diagnostics, and prevent universal health coverage and african health security. recommendations an innovative approach to facilitate the swift evaluation and registration of useful and performant ivd and support the advancement of the diagnostic agenda in african regions is needed. leveraging existing networks of excellence that can quickly conduct the standardised evaluation of ivds and support collaborative registration procedures on behalf of entire regions of africa can lead to critical benefits in access to ivd, while waiting for or to complement who prequalification. the africa collaborative to advance diagnostics, led by africa cdc, was launched in abuja during the biannual aslm conference in 201848 with the overall aim of advocating for appropriate investment in diagnostics as well as accelerating regulations to facilitate timely and wide access to essential diagnostics. one of the goals of the africa collaborative to advance diagnostics is to work towards speedier registration of diagnostic technologies, through a pan-african approach that complements who prequalification, leverages existing continental expertise and provides opportunities for manufacturers and other relevant stakeholders to support the process. conclusion in addition to advancing the development of increasingly relevant, reliable, specific and affordable ivd products, the future of diagnostics in africa also depends on our collective ability to comprehensively and swiftly address the systemic weaknesses in national laboratory networks. under the leadership of the africa cdc and who regional office for africa, african technical agencies at the continental or regional level (e.g. aslm, the west african health organization and east central and southern africa health community) or national level (e.g. nigeria cdc) have a critical role to play in providing direct technical assistance and vision for advancing national laboratory network diagnostic capacity and on setting adequate priorities for international cooperation. implementing the african union recommendations on domestic investment in healthcare49 is critical to ensure that laboratory systems and networks are sustainably prepared for the future of diagnostics in africa. acknowledgements the authors thank the african society for laboratory medicine and the africa centres for disease control and prevention team members samba diallo, edwin shumba and anafi mataka for their input in data collection and analysis. we thank dr kameko nichols for her contribution to the section on sample transportation systems. we thank dr ali elbireer for his contribution to the initial design of the manuscript. we thank professor oni emmanuel idigbe for his support in collecting data on african pathologists. we thank the ministries of health of niger, cameroon, congo, ethiopia, democratic republic of congo, chad, zambia, zimbabwe, sao tome and principe, gabon, central african republic and malawi for sharing data on laboratory mapping. we thank the ministries of health of ethiopia, uganda, burkina faso, senegal, cameroon, tanzania, democratic republic of congo and nigeria for sharing data on the labnet scorecard assessment. competing interests the authors declare that they have no conflicts of interest. authors’ contributions all authors contributed equally to the work. ethical considerations ethical approval is not required for this opinion article. sources of support this work would not have been possible without funding from the bill and melinda gates foundation, the president emergency plan for aids relief, the global health security agenda, unitaid and vital strategies. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references petti ca, polage cr, quinn tc, ronald ar, sande m. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377–382. https://doi.org/10.1086/499363 nkengasong jn, yao k, onyebujoh p. laboratory medicine in low-income and middle-income counrties: progress and challenges. lancet. 2018;391(10133):1873–1875. https://doi.org/10.1016/s0140-6736(18)30308-8 mcnerney r, sollis k, peeling rw. improving access to new diagnostics through harmonized regulation: priorities for action. afr j lab med. 2014;3(1), art. #123. https://doi.org/10.4102/ajlm.v3i1.123 world health organization hiv diagnostic test in low and middle income countries forecasts of global demand for 2014–2008. geneva: world health organization; 2015. the joint united nations programme on hiv/aids. unaids, unicef and who urge countries in western and central africa to step up the pace in the response to hiv for children and adolescent [homepage on the internet]. press release, dakar/geneva, 16 january 2019. [cited 2020 may 11]. available from: https://www.unaids.org/en world health organization. global tuberculosis report 2018 [homepage on the internet]. [cited 2019 july 29]. available from: https://www.who.int/tb/publications/global_report/en/ ismail n, ismael n, omar sv, et al. drug resistant tuberculosis in africa: current status, gaps and opportunities. afr j lab med. 2018;7(4):781. https://doi.org/10.4102/ajlm.v7i2.781 world health organization. global hepatitis report 2017 [homepage on the internet]. [cited 2019 july 29]. available from: https://www.who.int/hepatitis/publications/global-hepatitis-report2017/en/ van’t hoog a, sarr a, koster w, et al. a study to understand the under-utilization of laboratory tests for antenatal care in senegal. plos one. 2020;15(1):e0225710. world health organization regional office for africa. cervical cancer common amongst african women [homepage on the internet]. [cited 2019 july 29]. available from: https://www.afro.who.int/news/cervical-cancer-common-amongst-african-women angotti n, bula a, gaydosh l, kimchi ez, thornton rl, yeatman se. increasing the acceptability of hiv counseling and testing with three c’s: convenience, confidentiality and credibility. soc sci med. 1982;68(2009):2263–2270. https://doi.org/10.1016/j.socscimed.2009.02.041 visser t, daily j, hotte n, et al. rapid diagnostic test for malaria. bull world health organ 2015;93:862–866. https://doi.org/10.2471/blt.14.151167 schell o, crocker b, weng j. impact of hba1c testing at point of care on diabetes management. j diabetes sci technol. 2017;11(3):611. https://doi.org/10.1177/1932296816678263 world health organization. automated real-time nucleic acid amplification technology for rapid and simultaneous detection of tuberculosis and rifampicin resistance: xpert mtb/rif assay for the diagnosis of pulmonary and extrapulmonary tb in adults and children. geneva: who; 2013. bianchi f, cohn j, sacks e, et al. evaluation of a routine point-of-care intervention for early infant diagnosis of hiv: an observational study in eight african countries. lancet hiv. 2019;6(6):e373–e381. https://doi.org/10.1016/s2352-3018(19)30033-5 di tanna gl, khaki ar, theron g. effect of xpert mtb/rif on clinical outcomes in routine care settings: individual patient data meta-analysis. lancet glob health. 2019;7(2):e191–e199. https://doi.org/10.1016/s2214-109x(18)30458-3 burchett hed, leurent b, baiden f, et al. improving prescribing practices with rapid diagnostic tests (rdts): synthesis of 10 studies to explore reasons for variation in malaria rdt uptake and adherence. bmj open. 2017;7(3):e012973. https://doi.org/10.1136/bmjopen-2016-012973 rao vb, schellenberg d, ghani ac, rao vb, schellenberg d, ghani ac. overcoming health systems barriers to successful malaria treatment. trends parasitol. 2013;29(4):164–180. https://doi.org/10.1016/j.pt.2013.01.005 world health organization. maputo declaration [homepage on the internet]. [cited 2019 july 29]. available from: https://www.who.int/diagnostics_laboratory/maputo-declaration_2008.pdf african society for laboratory medicine. freetown declaration [homepage on the internet]. [cited 2019 july 29]. available from: http://www.aslm.org/what-we-do/global-health-security/freetown-declaration/ who african region: jee mission reports [cited 2019 july 29]. available from: https://www.who.int/ihr/procedures/mission-reports-africa/en/ nuche-berenguer b, kupfer lej. readiness of sub-saharan africa healthcare systems for the new pandemic, diabetes: a systematic review. diabetes res. 2018;18:9262395. https://doi.org/10.1155/2018/9262395 world health organization regional office for africa. a heavy burden: the productivity cost of illnesses in africa [homepage on the internet]. 2019 [cited 2020 july 25]. available from: https://www.afro.who.int/publications/heavy-burden-productivity-cost-illness-africa nkengasong jn, skaggs ba. are post ebola reconstruction efforts neglecting public health laboratory systems? lancet glob health. 2015;3(11):e678. https://doi.org/10.1016/s2214-109x(15)00159-x bell e, tappero jw, ijaz k. joint external evaluation – development and scale-up of global multisectoral health capacity evaluation process. emerg infect dis. 2017;23 (suppl 1):s33–s39. https://doi.org/10.3201/eid2313.170949 ondoa p, datema t, keita-sow ms, et al. a new matrix for scoring the functionality of national laboratory networks in africa: introducing the labnet scorecard. afr j lab med. 2016;5(3), a498. https://doi.org/10.4102/ajlm.v5i3.498 mehand ms, millet p, al-shorbaji f, rith c, kieny mp, murgue b. world health organization methodology to prioritize emerging infectious diseases in need of research and development. emerg infect dis. 2019;24(9):e171427. https://doi.org/10.3201/eid2409.171427 stop tb partnership. gli specimen referral toolkit. [homepage on the internet]. [cited 2020 july 25it]. available from: http://www.stoptb.org/wg/gli/srt.asp dama e, nikiema a, nichols k, et al. designing and piloting the first sample referral system in burkina faso using the national postal service. health security. 2020; 18(s1):s98–s104 nichols be, girdwood sj, cropton t, et al. monitoring viral load for the last mile: what will it cost? j int aids soc. 2019;22(9):e25337. https://doi.org/10.1002/jia2.25337 girdwood sj, nichol be, moyo c, crompton t, chimhamhiwa d, rosen s. optimizing viral load testing access for the last mile: geospatial cost model for point of care instrument placement. plos one. 2019;4(8):e0221586. https://doi.org/10.1371/journal.pone.0221586 african society of laboratory medicine. laboratory mapping program (labmap) [homepage on the internet]. [cited 2019 sep 18]. available from: http://www.aslm.org/what-we-do/laboratory-mapping/ instedd. planwise: geographic optimization of health system coverage [homepage on the internet]. [cited 2019 sep 18]. available from: https://instedd.org/project/planwise/ dhis2. covid-19 surveillance package [homepage on the internet]. [cited 2020 july 25]. available from: https://www.dhis2.org/ africa centres for disease control and prevention. strategic plan 2017–2021 [homepage on the internet]. [2020 july 25]. available from: http://www.ianphi.org/_includes/documents/strategy%20africa%20english%20cdc.pdf worldpop. open spatial demographic data and research [homepage on the internet]. [2019 may 19]. available from: www.worldpop.org.uk llamasoft inc. llamasoft [homepage on the internet]. [cited 2020 may 19]. available from: https://llamasoft.com/ world health organization. joint who-cdc conference on health laboratory quality systems, lyon, 9–11 april 2008. who/hse/ihr/lyo/2008.3. accessed at https://www.who.int/ihr/publications/who_hse_ihr_lyo_2008.3/en/ [cited 2020 july 25] world health organization regional committee for africa. resolution afr/rc58/r2 to strengthen public health laboratories in who african region at all levels of the healthcare system: a critical need for disease control. in: final report 58th session of who regional committee for africa, yaounde, september 2008. african society of laboratory medicine. aslm2020: strategies and vision to strengthen public health laboratory medicine in africa [homepage on the internet]. [cited 2019 dec 29]. available from: https://www.eiseverywhere.com/file_uploads/e46acabb2952cbecd2d610ca4cbfd45c_aslm2020.pdf strengthening laboratory management toward accreditation. slmta [homepage on the internet]. [cited 2019 aug 16]. available from: www.slmta.org carter jy. external quality assessment in resource-limited countries. biochem med. 2017;27(1):97–109. https://doi.org/10.11613/bm.2017.013 nqobile n. slipta 2.0 – the next ‘country driven’ frontier for laboratory quality improvement [homepage on the internet]. lab culture. 2018;18:12–13. [cited 2019 july 29]. available from: http://www.aslm.org/stay-informed/press-room/lab-culture-newsletter/ fleming ka, naidoo m, wilson m, et al. an essential pathology package for low and middle income countries. am j clin pathol. 2017;147(1):15–32. robboy si, weintraub s, horvath ae, et al. pathologist workforce in the united states. arch pathol lab med. 2013;213:1723–1732. https://doi.org/10.5858/arpa.2013-0200-oa hance p. repenser la formation des biologistes. le laboratoire en zone tropicale. colloque organisé par le spe, 7 juin 2006. bull soc pathol exot. 2006;99:409–417. africa cdc. africa cdc institute for workforce development [homepage on the internet]. [cited 2019 sep 18]. available from: https://africacdc.org/africa-cdc-institutes/africa-cdc-institute-for-workforce-development/ african union. africa centres for disease control and prevention and partners launch the africa collaborative initiative to advance diagnostics – afcad to address existing barriers towards advancing the diagnostic agenda in africa [homepage on the internet]. [cited 2020 july 25]. available from: https://au.int/en/pressreleases/20181116/africa-centres-disease-control-and-prevention-and-partners-launch-africa africa health business. africa leadership meeting. investing in health [homepage on the internet]. 2019 executive report [cited 2020 may 19]. available from: https://ahb.co.ke/wp-content/uploads/2019/03/alm-report.pdf introduction an important epidemiological reminder in this current context what we know what we should do acknowledgements references about the author(s) khadim diongue parasitology-mycology service, faculty of medicine, pharmacy and odontology, cheikh anta diop university, dakar, senegal laboratory of parasitology and mycology, aristide le dantec university hospital, dakar, senegal mamadou a. diallo laboratory of parasitology and mycology, aristide le dantec university hospital, dakar, senegal citation diongue k, diallo ma. covid-19 during malaria transmission season in africa and why we should be prepared: an example from senegal. afr j lab med. 2020;9(1), a1332. https://doi.org/10.4102/ajlm.v9i1.1332 opinion paper covid-19 during malaria transmission season in africa and why we should be prepared: an example from senegal khadim diongue, mamadou a. diallo received: 08 july 2020; accepted: 14 sept. 2020; published: 18 nov. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction coronavirus disease 2019 (covid-19) is not just a global health crisis; it is also a socio-economic crisis1 with a huge number of associated deaths.2 on 30 june 2020, the world health organization reported 10 185 374 cases and 503 862 deaths related to covid-19 globally, of which 297 290 cases and 6010 deaths were recorded in africa.1 in senegal, malaria transmission is seasonal with high vector populations observed during the rainy season – from mid-june to october. the heaviest rainfalls are observed in the months of july, august and september. on 30 march 2020, as effort to combat the spread of covid-19 in senegal, a state of emergency was declared banning religious gatherings, closing schools and universities and imposing a curfew between 22:00 and 06:00. as of 30 june 2020, the day the state of emergency was lifted, the authorities had reported 6698 confirmed covid-19 cases and 108 covid-19 related deaths1,3,4 thus the country was confronted with managing the expected rainy-season malaria epidemic and the on-going covid-19 pandemic. hence, this article aims to draw the attention of authorities to this current situation. an important epidemiological reminder in this current context before and after the ease of lockdown measures on 04 june 2020, senegal had been experiencing constantly increasing covid-19 cases, as evidenced by a 22% increase in cases during the month of june 2020. there were 1838 covid-19 cases on 01 june 2020, which had reached 2249 cases by 30 june 2020. however, the most detrimental event during the covid-19 pandemic is that of the general population deserting the health facilities that are hosting infected covid-19 individuals for fear of being tested or infected. since the decentralisation and proliferation of covid-19 care centres around the country, covid-19 hosting centres are being avoided by the populace; worse still, patients have now deserted the isolation centres. senegalese people avoid going to places where any test that could suggest covid-19 is done, even a body temperature measurement. however, experience has shown that in an outbreak such as the covid-19 pandemic, it is essential to not ignore other diseases such as malaria. the recent west africa ebola epidemic revealed the deleterious impact of increased health service demand on an already fragile health system, as well as on the control of other diseases. consequently, the ebola outbreak led to a substantial increase in morbidity and mortality of other diseases, including malaria.5 the world health organization has warned of a twofold devastation by the covid-19 pandemic: the first is its direct effect on health – covid-19 morbidity and mortality – and the second is the related increase in morbidity and mortality of other diseases due to lack of or diversion of adequate health response for other diseases.5 for example, during the ebola epidemic year in guinea, health facilities recorded a lower malaria patients’ visitation than was expected – an estimated deficit of 74 000 visitations. conversely and sadly, malaria caused 1067 deaths in 2014 – an ebola year – compared to 108 deaths reported in 2013 – a non-ebola year.6 more worryingly, in liberia, sierra leone and guinea, about 7000 other deaths related to malaria were recorded among children under age 5 years, which were attributed to the 2014–2016 ebola outbreak.6 what we know malaria illness presents some similar symptoms to the covid-19 illness: fever, respiratory distress, fatigue and an acute onset headache.5,7 the major sign between both illnesses is fever. thus, a malaria case or a covid-19 case may each be confused for the other, if symptoms alone are used to define a case during this current pandemic. also, since the beginning of the pandemic in senegal, fever is the major symptom of infected individuals8; febrile individuals are isolated before samples are taken. the sampling team is deployed after contact with the senegalese medical emergency assistance service; however, the emergency assistance centre has been overwhelmed (more than 726 000 emergency calls between 02 march 2020 and 29 june 20209), which could be the reason why, unfortunately, sampling teams can sometimes take more than 24 h to intervene. this delays the differential diagnosis of malaria and similar illnesses, as well as increases the risk of development of severe malaria, if malaria diagnosis is correct but delayed. therefore, clinicians should go back to the good old method of ‘any case of fever must be considered as malaria until proven otherwise’. confirming either malaria or severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection with a diagnostic test does not rule out the possibility of the other in a patient. indeed, both false-positive and false-negative results have been reported worldwide with rapid diagnostic tests.10 likewise, approximately 50% of infected cases are likely missed by current screening approaches for covid-19, even in countries with good healthcare systems and available diagnostic capacities. as covid-19 can be transmitted even by asymptomatic people, and personal social distancing cannot be effective when performing a malaria test, such as a rapid diagnostic test,11 other infection prevention and control measures must be carefully put in place.7 these measures include but are not limited to: proper donning and doffing of personnel protection equipment, especially face masks; provision and use of soap and water for handwashing; provision and use of alcohol-based hand sanitiser and the practice of good respiratory hygiene (i.e. covering of the mouth and nose with a disposable tissue when sneezing or coughing or sneezing into the crease of the elbow).7 thus, health providers should also be more careful. what we should do in senegal, according to the last national malaria control programme report published in 2018, malaria caused 395 706 cases with 284 deaths including 95 deaths (33.45%) among children younger than age 5 years.12 however, the malaria burden was more than 50% lower between 2009 and 2015, permitting the country to achieve the objectives of roll back malaria in 2015.3 therefore, to not lose this positive momentum in the fight against malaria, the senegalese national malaria control programme should scrupulously follow the recommendations of the world health organization, which include tailoring malaria interventions in the covid-19 response5 with particular regard to the following points: case management, chemoprevention (intermittent preventive treatment in pregnancy and seasonal malaria chemoprevention); extraordinary interventions, including presumptive treatment of fever and mass drug administration, as well as the assurance of continued access to and use of recommended insecticide-treated mosquito nets; and implementation of core vector-control activities to the greatest extent possible (current and planned insecticide-treated nets campaigns should go ahead, if at all possible). malaria prevention and treatment is even more important during the covid-19 pandemic than under normal circumstances. rapid diagnosis plays an important role in the outcome of both malaria and covid-19; thus, in addition to these recommendations, quick, easy, reliable and cheap diagnostics requiring minimal invasion must be performed, especially in cases of likely overlap with malaria and covid-19. this is the time for malaria-endemic countries challenged with the covid-19 pandemic to apply modern techniques including point-of-care tests such as the loop-mediated isothermal amplification assay for rapid detection of both plasmodium and sars-cov-2.13,14 meanwhile, health authorities should also centralise the management of covid-19 infected individuals so that populations suffering from other diseases (diabetes, high blood pressure, cardiovascular disorders, arthritis, etc.) could return to health facilities without fear of being infected. however, number of tests as well as the test centers for covid-19 detection should be multiplied in order to attend benefits of early case identification. acknowledgements we are thankful to dr mamane nassirou garba for his english review and all the staff of the parasitology and mycology laboratory at le dantec university of dakar, especially to prof. daouda ndiaye. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions k.d. was responsible for the conception and design of the study and drafting the article. m.a.d. reviewed the article. all authors approved the final version to be submitted. ethical considerations the work did not involve the use of human subjects or animal experiments. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references world health organization (who). coronavirus disease (covid-19): situation report – 162. who; 2020 [cited 2020 jun 30]. available from: www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports gangneux j-p, bougnoux m-e, dannaoui e, cornet m, zahar jr. invasive fungal diseases during covid-19: we should be prepared. j mycol méd. 2020;30(2):100971. https://doi.org/10.1016/j.mycmed.2020.100971 programme national de lutte contre le paludisme (pnlp). sénégal – plan stratégique de lutte contre le paludisme 2016–2020 [homepage on the internet]. pnlp; 2015 [cited 2020 jun 30]. http://www.pnlp.sn/wp-content/uploads/2016/08/pnlp_psn_vff_03-02-2016.pdf agence nationale de la statistique et de la démographie/service régional de la statistique et de la démographie de dakar (ansd/srsd). situation économique et sociale régionale – 2012 [homepage on the internet]. ansd/srsd; 2015 [cited 2020 jun 30]. available from: http://www.ansd.sn/ressources/ses/ses_dakar-2012.pdf world health organization (who). tailoring malaria interventions in the covid-19 response [homepage on the internet]. who; 2020 [cited 2020 jun 30]. available from: http://www.who.int/malaria wang j, xu c, wong yk, et al. preparedness is essential for malaria-endemic regions during the covid-19 pandemic. lancet. 2020;395(10230):1094–1096. https://doi.org/10.1016/s0140-6736(20)30561-4 chanda-kapata p, kapata n. covid-19 and malaria: a symptom screening challenge for malaria endemic countries. int j infect dis. 2020;94:151–153. https://doi.org/10.1016/j.ijid.2020.04.007 seydi m, lakhe na. profil épidémiologique, clinique et évolution des cas de covid-19 au sénégal. ministère de la santé et de l’action sociale [homepage on the internet]. 2020 [cited 2020 jun 30]. available from: http://familyplanning2020.org/sites/default/files/resources/covid/covid-19%20afrehealth%20webinar%20talk_cmit_dakar_senegal_pr.%20seydi_dr.%20lakhe_march19_2020_french%20version.pdf ministère de la santé et de l’action sociale (msas). message à la nation de sem le président sall levée de l’état d’urgence instauré dans le cadre de la lutte contre la maladie à coronavirus covid-19 [homepage on the internet]. msas; 2020 [cited 2020 jun 30]. available from: http://www.sante.gouv.sn/sites/default/files/discours%20du%20pr.pdf kafai nm, odom john ar. malaria in children. infect dis clin n am. 2018;32(1):189–200. https://doi.org/10.1016/j.idc.2017.10.008 diallo ma, diongue k, ndiaye m, et al. evaluation of carestart™ malaria hrp2/pldh (pf/pan) combo test in a malaria low transmission region of senegal. malar j. 2017;16:328. https://doi.org/10.1186/s12936-017-1980-z programme national de lutte contre le paludisme (pnlp). bulletin épidémiologique annuel 2017 du paludisme au sénégal [homepage on the internet]. pnlp; 2018 [cited 2020 jun 30]. available from: https://fr.africacheck.org/wp-content/uploads/2018/04/senegal-paludisme-bulletin-annuel-2017-pnlp.pdf lucchi nw, gaye m, diallo ma, et al. evaluation of the illumigene malaria lamp: a robust molecular diagnostic tool for malaria parasites. sci rep. 2016;6:36808. https://doi.org/10.1038/srep36808 l’helgouach n, champigneux p, schneider fs, et al. easycov: lamp based rapid detection of sars-cov-2 in saliva [homepage on the internet]. 2020 [cited 2020 jun 30]. https://www.skillcell-alcen.com/sites/skillcell-alcen/files/pdf/press/clinical-assessment-of-easycov-v10_1.pdf abstract introduction methods results discussion acknowledgements references about the author(s) nireshni mitchev department of medical microbiology, school of laboratory medicine and medical sciences, university of kwazulu-natal, durban, south africa ravesh singh department of medical microbiology, school of laboratory medicine and medical sciences, university of kwazulu-natal, durban, south africadepartment of medical microbiology, national health laboratory service, durban, south africa nigel garrett centre for the aids programme of research in south africa, durban, south africa discipline of public health medicine, school of nursing and public health, university of kwazulu-natal, durban, south africa veron ramsuran kwazulu-natal research innovation and sequencing platform, school of laboratory medicine and medical sciences, university of kwazulu-natal, durban, south africa abraham j. niehaus department of medical microbiology, school of laboratory medicine and medical sciences, university of kwazulu-natal, durban, south africadepartment of medical microbiology, national health laboratory service, durban, south africa koleka p. mlisana department of medical microbiology, school of laboratory medicine and medical sciences, university of kwazulu-natal, durban, south africacentre for the aids programme of research in south africa, durban, south africadepartment of academic affairs, research and quality assurance, national health laboratory service, durban, south africa citation mitchev n, singh r, garrett n, ramsuran v, niehaus aj, mlisana kp. performance of taqman probes for the detection of sexually transmitted infections in south african women. afr j lab med. 2021;10(1), a1124. https://doi.org/10.4102/ajlm.v10i1.1124 brief report performance of taqman probes for the detection of sexually transmitted infections in south african women nireshni mitchev, ravesh singh, nigel garrett, veron ramsuran, abraham j. niehaus, koleka p. mlisana received: 07 nov. 2019; accepted: 08 jan. 2021; published: 31 mar. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract neisseria gonorrhoeae, chlamydia trachomatis, trichomonas vaginalis and mycoplasma genitalium are the four main aetiologies of sexually transmitted infections responsible for vaginal discharge syndrome (vds). commercially available multiplex polymerase chain reaction (pcr) assays are expensive and generally not customisable. we evaluated a highly customisable singleplex pcr approach by testing it in parallel with the anyplex™ ii sti-7 detection assay in a cohort of south african women that presented with vds between may 2016 and january 2017. our multiple singleplex pcr strategy proved to be a simple, accurate, rapid, affordable and scalable option for diagnosing vds. keywords: sexually transmitted infections; vaginal discharge syndrome; molecular diagnostics; validation; taqman. introduction the world health organization estimates that approximately one million curable sexually transmitted infections (stis) occur globally each day.1 neisseria gonorrhoeae, chlamydia trachomatis and trichomonas vaginalis are the three most common pathogens causing stis worldwide, with co-infection also common.1,2 globally, as of 2016, there were approximately 86 million cases of gonorrhoea, 127 million cases of chlamydia and 156 million cases of trichomoniasis among 15–49 year-olds, with estimated prevalence rates of 0.9%, 3.8% and 5.3% in women specifically.1,3 such estimates are important for the effective prevention and control of stis but are generally lacking for most lowand middle-income countries.4 torrone et al. recently published a meta-analysis of data from african countries that highlights this point.4 prevalence figures varied widely between studies and ranged from 1.4% to 15.2% for n. gonorrhoeae, 1.2% to 20.6% for c. trachomatis and 6.6% to 29.7% for t. vaginalis.4 the prevalence of most stis is generally greater in certain high-risk populations such as those with a high prevalence of hiv co-infection and women aged 15–24 years.4 sexually transmitted infections and their complications are one of the top five reasons why females in lowand middle-income countries attend healthcare facilities.1,2 although the majority of stis are asymptomatic, asymptomatic stis can increase the probability of transmission of hiv and other stis, as well as have adverse effects on reproduction potential and maternal and newborn health.1,2,5,6 syndromic management of stis is widely practised and was introduced in south africa in 1995.7,8 unfortunately, due to its inability to effectively identify and treat a large number of individuals with asymptomatic infections, syndromic management has failed to decrease the prevalence of gonorrhoea and chlamydia in south africa.7,8 diagnosing the aetiology of most stis using culture methods is notoriously difficult and can take several days to complete. multiplex polymerase chain reaction (pcr) assays are currently the preferred diagnostic method used for identifying and subsequently managing both symptomatic and asymptomatic stis in many well-resourced countries.9 since pcr assays are not dependent on organism viability, they are often up to 20% – 30% more sensitive than conventional phenotypic methods and they can simultaneously detect multiple pathogens and also serve as point-of-care tests.9,10,11,12,13 polymerase chain reaction also performs well on noninvasively obtained specimens like urine and self-collected vaginal swabs.10 our study aimed to evaluate the suitability of a highly customisable singleplex real-time pcr approach using commercially available taqman® probes (thermo fisher scientific, waltham, massachusetts, united states) for the identification of n. gonorrhoeae, c. trachomatis, t. vaginalis and myocoplasma genitalium in vaginal samples from our local population. the anyplex™ ii sti-7 detection assay (seegene, seoul, south korea) was used as the comparator method. this widely used multiplex real-time pcr assay displays excellent sensitivity and specificity characteristics compared with other diagnostic tools.10 methods ethical considerations women attending the prince cyril zulu communicable disease centre (durban, south africa) for sti care were approached and, if willing to participate, provided written informed consent for the study. study data were collected, anonymised and managed using password-protected redcap electronic data capture tools (vanderbilt university, nashville, tennessee, united states), and stored on a secure server. this study was approved by the biomedical research ethics committee of the university of kwazulu-natal (study approval number: be534/16). samples as part of the centre for the aids programme of research in south africa 083 cohort study between may 2016 and january 2017, which was previously described in detail, women aged 18–40 presenting for sti care at a clinic in durban were assessed for enrolment and participation.7,14 hiv-positive women, pregnant women and those engaging in sex work were excluded due to predetermined eligibility criteria.15 participants consented to vaginal swab specimen collection for molecular testing. eswab™ collection and transport kits (copan diagnostics, brescia, italy) were used as per the manufacturer’s instructions. dna extraction and polymerase chain reaction collection swabs were vortexed for 30 s while inside their transport tubes, whereafter 500 µl of the suspension was added to a 1.5 ml eppendorf tube. this was followed by centrifugation and resuspension of the sediment in 200 µl distilled water. after heating and sonication steps, 5 µl of the resultant sample was added to 15 µl pre-aliquoted master mix. for the taqman® probe assays, master mixes were prepared separately for each single target reaction in a 96-well plate. the anyplex™ ii sti-7 detection assay was used according to the manufacturer’s specifications and was performed using a cfx96 real-time pcr system (biorad, hercules, california, united states).10 taqman® probes were used in a singleplex format on the abi® 7500 real-time pcr instrument from applied biosystems (thermo fisher scientific, waltham, massachusetts, united states). data management and analysis anyplex™ ii sti-7 detection assay results were analysed and interpreted with seegene viewer software (seegene, seoul, south korea). contingency 2 × 2 tables were used to determine the sensitivity, specificity, positive predictive value and negative predictive value for each of the taqman® probes with anyplex™ ii sti-7 detection assay as the gold standard.10,16,17 results a total of 267 women were screened for stis at an urban clinic in durban, south africa. vaginal discharge (n = 106; 39.7%) was the most common symptom reported.15 vaginal swabs were available for molecular investigation from 250 (93.6%) women. two molecular assays were used in parallel to detect the presence of four sexually transmitted microorganisms implicated in vaginal discharge syndrome. the yield obtained from 250 samples by each of the two methods ranged between 3.6% and 13.6% (table 1). at least one of n. gonorrhoeae, c. trachomatis, t. vaginalis or m. genitalium was identified in 22.8% (57/250) of the study population. c. trachomatis was the most common organism found in co-infections and was present in 42% (5/12) of the n. gonorrhoeae, 25% (3/12) of the m. genitalium and 11% (1/9) of the t. vaginalis infections. table 1: yield obtained with anyplex™ ii sti-7 detection assay and taqman® probes, from 250 vaginal swab specimens collected from patients attending prince cyril zulu communicable disease centre (durban, south africa) between may 2016 and january 2017. taqman® probe sensitivity ranged from 91.67% to 100%, and specificity ranged from 98.74% to 100.00% (table 2). negative predictive values ranged from 99.08% to 100.00% and positive predictive values ranged from 90.00% to 100.00%, except for m. genitalium (78.57%). table 2: performance characteristics of taqman® probes for 250 vaginal swab specimens collected from patients attending prince cyril zulu communicable disease centre (durban, south africa) between may 2016 and january 2017. final results were available after 130 min and 180 min with the taqman® probes and anyplex™ ii sti-7 detection assay, respectively. this includes 45 min for dna extraction and 15 min for results analysis. discussion the prevalence of any of the main curable stis, including syphilis and those caused by n. gonorrhoeae, c. trachomatis and t. vaginalis, in women in kwazulu-natal, south africa was previously reported to be as high as 13%.18 in our study, we observed the overall prevalence of stis to be 22.8%. our prevalence data is not a true representation of the general population, because we recruited participants from a sexually active cohort of women that presented with symptoms to an sti clinic. the xpert® ct/ng assay (cepheid, sunnyvale, california, united states) was previously used to investigate this cohort of samples.7 for the present study, we employed the anyplex™ ii sti-7 detection assay as the comparator, because the xpert® ct/ng assay can identify c. trachomatis and n. gonorrhoeae, but not t. vaginalis and m. genitalium. sensitivity, specificity, positive predictive value and negative predictive value characteristics of the taqman® probes for the four investigated organisms compared well to the chosen reference method. the only exception was the low positive predictive value observed for m. genitalium. this deviation may be due to the small sample size and requires further investigation. except for certain antibiotic-resistant organisms, the stis detected in our study can usually be treated with a course of antibiotics; however, if they remain undiagnosed and subsequently untreated, a range of serious health issues may follow. complications include elevated hiv transmission rates, infertility as well as life-threatening ectopic pregnancy and stillbirths.19 a rapid, accurate and affordable sti diagnostic tool is therefore essential to identify and treat affected individuals.13 molecular testing methods are advantageous over phenotypic testing methods due to the rapid availability of results. polymerase chain reaction methodologies can also detect various predefined targets at the same time, including drug-resistance determinants. singleplex pcr methods offer some important advantages over multiplex methods, principally in terms of ease of optimisation, customisability and target quantification. new gene targets of interest can be added in a flexible way without the requirement of a full revalidation process of the existing targets. our multiple singleplex pcr system can easily be adapted to diagnose various other clinical syndromes caused by bacterial, viral, fungal and parasitic microorganisms, including meningitis, blood stream infections, respiratory infections, diarrhoea, and urinary tract infections. singleplex pcr systems allow target quantification, which has potential diagnostic, prognostic and clinical monitoring functions. turnaround times with our multiple singleplex pcr were 50 min shorter than with the multiplex method. the clinical impact of this time difference, when non-immediately life-threatening infections are concerned, is probably insignificant. multiplex pcr methodologies are generally significantly more cost-effective than singleplex pcrs. however, this does not hold true when small numbers of molecular targets are considered.20 costing of the four targets used in our testing system indicates a considerable cost saving of $15.27 (united states dollars [usd]) per test, over the comparator method that is priced at $24.67 usd per test. affordability can be increased even further by using a 384-well reaction plate, making it an attractive option for high-throughput environments. with our system, we can easily adopt a workflow that is suitable for both research and clinical laboratories. limitations limitations of this study include the relatively small sample size and the low positivity rate obtained by both methodologies. multiple other microorganisms and dual infections may also have been responsible for vaginal discharge syndrome in some women, but were not considered in this investigation. conclusion the performance of four taqman® probes (thermo scientific) was assessed by comparison with the anyplex ii sti-7 detection assay. our multiple singleplex real-time taqman® pcr approach proved to be highly sensitive and specific for the detection of c. trachomatis, n. gonorrhoeae, t. vaginalis and m. genitalium. this approach offers an accurate, cost-effective and scalable option for identifying these pathogens in our patient population. acknowledgements the authors would like to acknowledge the aids programme of research in south africa 083 sti study participants and staff at the national health laboratory services (inkosi albert luthuli central hospital, durban, south africa) and centre for the aids programme of research in south africa. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions n.m., r.s. and k.p.m. conceptualised the study. k.p.m. was responsible for the overall supervision of the project and the acquisition of funding. n.m. and n.g. were responsible for project administration. n.m., r.s. and v.r. decided on the methodology and performed the laboratory investigations. n.m. curated all the data and the data analysis was done together with a.j.n. all authors participated in the writing of the manuscript. sources of support this project was funded through a grant by the department of science and technology and the national research foundation centre of excellence in hiv prevention as well as a grant from the united states – south african program for collaborative biomedical research, through the south african medical research council and the united states national institutes of health (grant number: ai116759). data availability the authors confirm that the data supporting the findings of this study are available within the article or its supplementary materials. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references world health organization. report on global sexually transmitted infection surveillance 2018 [homepage on the internet]. 2019 [cited 2019 jul 13]. available from: https://www.who.int/reproductivehealth/publications/stis-surveillance-2018/en/ tayoun ana, burchard pr, caliendo am, scherer a, tsongalis gj. a multiplex pcr assay for the simultaneous detection of chlamydia trachomatis, neisseria gonorrhoeae, and trichomonas vaginalis. exp mol pathol. 2015;98(2):214–218. https://doi.org/10.1016/j.yexmp.2015.01.011 rowley j, van der hoorn s, korenromp e, et al. chlamydia, gonorrhoea, trichomoniasis and syphilis: global prevalence and incidence estimates, 2016. bull world health organ. 2019;97:548–562. torrone ea, et al. prevalence of sexually transmitted infections and bacterial vaginosis among women in sub-saharan africa: an individual participant data meta-analysis of 18 hiv prevention studies. plos med. 2018;15(6):e1002511. https://doi.org/10.1371/journal.pmed.1002511 kaida a, dietrich jj, laher f, et al. a high burden of asymptomatic genital tract infections undermines the syndromic management approach among adolescents and young adults in south africa: implications for hiv prevention efforts. bmc infect dis. 2018;18:499. https://doi.org/10.1186/s12879-018-3380-6 workowski ka, bolan ga, centers for disease control and prevention. sexually transmitted diseases treatment guidelines, 2015. mmwr recomm rep. 2015;64(rr-03):1–137. garrett nj, osman f, maharaj b, et al. beyond syndromic management: opportunities for diagnosis-based treatment of sexually transmitted infections in lowand middle-income countries. plos one. 2018;13(4):e0196209. https://doi.org/10.1371/journal.pone.0196209 garrett nj, mcgrath n, mindel a. advancing sti care in low/middle-income countries: has sti syndromic management reached its use-by date? sex transm infect. 2016;93(1):4–5. https://doi.org/10.1136/sextrans-2016-052581 lee sj, park dc, lee ds, choe hs, cho yh. evaluation of seeplex std6 ace detection kit for the diagnosis of six bacterial sexually transmitted infections. j infect chemother. 2012;18(4):494–500. https://doi.org/10.1007/s10156-011-0362-7 choe hs, lee ds, lee sj, et al. performance of anyplex ii multiplex real-time pcr for the diagnosis of seven sexually transmitted infections: comparison with currently available methods. int j infect dis. 2013;17(12):e1134–e1140. https://doi.org/10.1016/j.ijid.2013.07.011 trembizki e, costa amg, tabrizi sn, whiley dm, twin j. opportunities and pitfalls of molecular testing for detecting sexually transmitted pathogens. pathology. 2015;47(3):219–226. https://doi.org/10.1097/pat.0000000000000239 papp jr, schachter j, gaydos ca, van der pol b. recommendations for the laboratory-based detection of chlamydia trachomatis and neisseria gonorrhoeae – 2014. mmwr recomm rep. 2014;63(rr-02):1–19. unemo m. current and future antimicrobial treatment of gonorrhoea – the rapidly evolving neisseria gonorrhoeae continues to challenge. bmc infect dis. 2015;15:364. https://doi.org/10.1186/s12879-015-1029-2 garrett n, mitchev n, osman f, et al. diagnostic accuracy of the xpert ct/ng and osom trichomonas rapid assays for point-of-care sti testing among young women in south africa: a cross-sectional study. bmj open. 2019;9(2):e026888. https://doi.org/10.1136/bmjopen-2018-026888 garrett n, maharaj b, osman f, et al. p4.115 high uptake of effective expedited partner therapy among young women with stis and their partners in south africa. sex transm infect. 2017;93(suppl 2):a233. https://doi.org/10.1136/sextrans-2017-053264.610 naaktgeboren ca, bertens lcm, van smeden m, et al. value of composite reference standards in diagnostic research. bmj. 2013;347:f5605. https://doi.org/10.1136/bmj.f5605 alonzo ta, pepe ms. using a combination of reference tests to assess the accuracy of a new diagnostic test. stat med. 1999;18(22):2987–3003. https://doi.org/10.1002/(sici)1097-0258(19991130)18:22%3c2987::aid-sim205%3e3.0.co;2-b naidoo s, wand h, abbai ns, ramjee g. high prevalence and incidence of sexually transmitted infections among women living in kwazulu-natal, south africa. aids res ther. 2014;11:1. https://doi.org/10.1186/1742-6405-11-31 centers for disease control and prevention. sexually transmitted disease surveillance report [homepage on the internet]. 2016 [cited 2019 may 28]. available from: https://www.cdc.gov/std/stats16/cdc_2016_stds_report-for508websep21_2017_1644.pdf deshpande a, white ps. multiplexed nucleic acid-based assays for molecular diagnostics of human disease. expert rev mol diagn. 2012;12(6):645–659. https://doi.org/10.1586/erm.12.60 acknowledgements references about the author(s) collins o. odhiambo african society for laboratory medicine, addis ababa, ethiopia anafi mataka african society for laboratory medicine, addis ababa, ethiopia marguerite massinga loembe african society for laboratory medicine, addis ababa, ethiopialaboratory division, africa centres for disease control and prevention, addis ababa, ethiopia pascale ondoa african society for laboratory medicine, addis ababa, ethiopiaamsterdam institute for global health and development, department of global health, university of amsterdam, amsterdam, the netherlands citation odhiambo co, mataka a, massinga loembe m, ondoa p. maintaining routine hiv and tuberculosis testing services in sub-saharan african countries in the context of covid-19: lessons learnt and opportunities for improvement. afr j lab med. 2021;10(1), a1413. https://doi.org/10.4102/ajlm.v10i1.1413 opinion paper maintaining routine hiv and tuberculosis testing services in sub-saharan african countries in the context of covid-19: lessons learnt and opportunities for improvement collins o. odhiambo, anafi mataka, marguerite massinga loembe, pascale ondoa received: 29 sept. 2020; accepted: 24 mar. 2021; published: 17 june 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. since being declared a public health emergency of international concern on 30 january 2020, the coronavirus disease 2019 (covid-19) has spread internationally, reaching the stage of a global pandemic.1 african countries quickly put in place social and public health measures to limit the spread of the disease, with some of the most ‘visible’ measures being lockdowns, physical distancing and the overall surge of healthcare services to support the covid-19 response. the director-general of the world health organization, calling for increased testing, recommended ‘test, test, test’ as a critical step to contain the spread of the disease.2 when the first african case was reported in egypt in february 2020, only two centres of excellence laboratories on the continent were capable of conducting severe acute respiratory syndrome coronavirus 2 polymerase chain reaction testing, the gold standard assay recommended by the world health organization. one key strategy to swiftly scale-up the testing capacity for severe acute respiratory syndrome coronavirus 2 has been the re-tooling of existing nucleic acid amplification testing platforms toward the control of covid-19. nucleic acid amplification testing platforms available from hiv and tuberculosis control programmes offered a unique opportunity given their large footprint across several countries and the availability of in-service training programmes for the operators. at least three covid-19 molecular diagnostic assays listed under the emergency use authorisations of the united states food and drug administration or the world health organization can be used on platforms commonly available within the hiv and tuberculosis programmes – the abbott m2000 realtime system, cepheid’s genexpert® system, and the roche cobas 6800/8800 system.3 although covid-19 testing volumes in most african countries remain below or at the lower threshold required for relaxing the containment measures (10–30 tests per confirmed case),4 have we gone a step too far in refocusing most of the laboratory capacity to covid-19 testing? mounting evidence suggests that the ‘covidisation’ of the healthcare system and, more specifically, diagnostics poses a risk to maintaining other routine testing services for the control and prevention of endemic diseases like hiv, malaria, syphilis and tuberculosis.5 additionally, lockdowns imposed in most countries prevent patients from accessing healthcare and this may result in increased treatment failure among those on medication, and increased maternal and child mortality in the context of poorly attended childbirth and reduced antenatal care. without intervention, there is a risk of reversing many of the gains achieved towards meeting the targets of the joint united nations programme on hiv/aids 95:95:95, the end tb strategy, and the united nations sustainable development goals, including ending the ‘big three’ diseases – hiv, tuberculosis and malaria. hiv, tuberculosis and malaria programmes have made substantial progress towards achieving global targets due to massive donor funding. however, the joint united nations programme on hiv/aids estimates that there could be hundreds of thousands of extra deaths from hiv if routine services, including hiv screening, viral load and early infant diagnosis, are disrupted.6 eighty-five percent of national-level respondents from 61 countries participating in a world health organization, united nations children’s fund and global aids vaccine initiative poll reported lower vaccination proportions in may 2020 compared to the level in january 2020 – february 2020.7 additionally, disruption of maternal and child health services is estimated to contribute to at least 8% more deaths per month.8 a survey of the global fund’s supported programmes across 106 countries revealed that at least 85%, 80% and 76% of hiv, tuberculosis and malaria control services, respectively have been disrupted by the covid-19 pandemic.9 a recent modelling analysis from the stop tb partnership further illustrated how an additional lockdown period of three months could lead to 6.3 million more cases of tuberculosis globally by 2025, causing a setback of at least five years on the progress achieved thus far.10 more specifically, the survey revealed that at least 20% of hiv and tuberculosis laboratory services are facing high disruption with much of the advanced diagnostic equipment repurposed and the workforce reassigned for covid-19 testing. a world health organization survey of essential services revealed that hiv testing (n = 38) and viral load monitoring (n = 23) were the two most frequently interrupted testing services in the 61 countries that responded.11 it is important to note that laboratory testing is key to identifying disease cases, breaking transmission chains and monitoring patients on treatment. in outbreak situations, healthcare stakeholders at the national level must be able to quickly address testing needs and, at the same time, identify the testing threshold needed to ensure that routine testing services are maintained and health targets for the control of essential diseases remain on track. however, data about the overall function of health services are usually fragmented and rarely realtime on the continent. furthermore, movement restrictions have further hampered the collection of information on the continuation of essential healthcare services, particularly diagnostics. the african society for laboratory medicine through the laboratory systems strengthening community of practice (labcop)12 conducted an online survey (june 2020) among labcop member countries to seek a deeper understanding of the disruptions in hiv and tuberculosis testing services during covid-19 diagnosis scale-up. the labcop is funded by the bill and melinda gates foundation and is composed of multidisciplinary teams (clinicians, laboratorians and civil society) with strong collaboration with the ministries of health from 14 sub-saharan african countries.12 consensus responses to the survey questionnaire were received from the multidisciplinary teams of 10 of the 14 countries. among those, nine countries reported a decline in viral load testing volumes, eight reported a decline in tuberculosis genexpert testing volumes, while seven reported a decline in early infant diagnosis volumes during the covid-19 response, thereby confirming the global fund survey findings.9 in uganda, one of the few labcop participant countries with a publicly available viral load dashboard for testing services, there was a 28% reduction in viral load test volume (from 104 474 to 74 841 tests) between march 2020, when the first covid-19 case was reported in uganda, and may 2020.13 the main reason provided by most countries (9 of 10 countries) for the decline in testing volumes was either the inability of patients to visit the health facilities to access hiv and tuberculosis services due to movement restrictions aimed at limiting covid-19 transmission (at least 42 african countries had imposed partial or complete lockdowns as of may 2020) or patient fear of contracting the disease while visiting health facilities. five countries reported stock-outs of testing kits, reagents and laboratory consumables as a result of border closures. four countries indicated that the surge in covid-19 testing volumes caused or exacerbated the shortage of healthcare workers, as they were reassigned to support the covid-19 response (especially personnel skilled in molecular testing), thereby affecting the provision of other essential diagnostics based on polymerase chain reaction technology. whereas 8 of 10 countries reported re-purposing between 25% to 83% of the total hiv and tuberculosis testing equipment capacity in-country for covid-19 testing, only two countries identified insufficient molecular testing capacity as a barrier to maintaining hiv and tuberculosis diagnostic services. two separate analyses indicated that most hiv and tuberculosis instruments are often operated below their full capacity,14,15 indicating that available instruments in most countries may be sufficient to support both covid-19 and hiv and/or tuberculosis testing. moreover, under the impulse of strong hiv and tuberculosis disease control programmes funded by the united states president’s emergency plan for aids relief and the global fund, the equipment is used almost exclusively for one disease area due to vertical programming, despite the instruments’ multiplexing capability and the recommendation to ‘integrate’ testing.16 additionally, many of the countries who either repurposed hiv and/or tuberculosis equipment for covid-19 testing or refocused testing still experience challenges in long turn-around times for results, quality assurance and procurement issues, among others, indicating systemic weaknesses that need attention. the difficulty in scaling the covid-19 response while maintaining routine testing services for other essential diseases indicates that many countries are still struggling to achieve a multipurpose, resilient and effective laboratory network to address the needs of clinical and surveillance testing services and meet the prevention and control targets for all essential diseases.17 with more than a decade of large initiatives aimed at laboratory system capacity building in africa, what are we still missing to ensure that laboratory networks can more effectively forecast and organise laboratory services in both routine and emergency situations? through the labcop country annual self-assessment reports, we have been able to identify some of the inherent weaknesses in the viral load testing cascade, including laboratory network optimisation and monitoring and evaluation (m&e). laboratory stakeholders at the central level often do not have a comprehensive overview of available resources and capacities across the network, or how these can be leveraged to strengthen the various functions of the laboratory system; for example, how higher-tier laboratories can organise external quality assessment for those in lower tiers. further, at the laboratory level, managers and directors often lack the managerial skills and leadership needed to organise and implement comprehensive laboratory management systems that enable laboratories to conduct the tiered functions of the network for multiple diseases; these include knowledge in determining staffing levels, testing capacity, and roles and responsibilities, as well as skills in the use of geographic information system tools like labmap18 and labready to map, analyse and optimise the laboratory network capacity, functions and services. an inclusive approach that fosters strong collaboration across sectors (i.e., public and private, academic, civilian and military, human and animal health, vertical disease programmes, etc.), as well as an evidence-based decision-making process based on available laboratory data, should be used to inform scale-up of testing for covid-19 and any emerging disease. our survey has highlighted that evidence was seldom leveraged to inform scale-up of covid-19 diagnostic services; only one of three countries that reported the procurement of additional molecular testing equipment to support the surge in covid-19 testing identified a shortage of testing platforms as the reason for the reduction in hiv/tuberculosis testing, whereas only two of the four countries that reported hiring additional laboratory staff identified insufficient staff capacity as a gap. to address some of these gaps, the african society for laboratory medicine is working with various stakeholders, including the association of public health laboratories, the clinton health access initiative, the foundation for innovative new diagnostics, the world health organization, and the united states centers for disease control and prevention, among others, to develop a leadership and mentorship programme focused on leading and managing tiered laboratory networks to offer clinical care and public health services during routine and emergency situations. a guidance document for scaling up covid-19 diagnosis within the laboratory network while maintaining essential testing has been developed.19 additionally, the african society for laboratory medicine is convening an m&e sub-community of practice of the labcop articulated around the delivery of a fit-for-purpose m&e training and mentoring curriculum with the opportunity for direct technical assistance to labcop country teams. the key outcome of this initiative is the development or improvement of national dashboards to monitor and evaluate the implementation and performance of the hiv viral load testing cascade and diagnosis of other diseases, including covid-19. additionally, it is anticipated that the multi-country discussion will highlight best practices and identify actual needs that will inform the update of the current guidance document for establishing an m&e framework.20 the difficulty of scaling up covid-19 diagnostics while maintaining routine testing of hiv, tuberculosis and other essential diseases relates to pre-existing laboratory systemic weaknesses. solutions to durably tackle the gaps crippling diagnostic services include better knowledge, management and optimisation of national tiered networks to become more resilient in the face of health emergencies. attaining skills in the management of laboratory networks and strengthening of m&e systems in-country will contribute to better and faster identification of strengths and bottlenecks and mutualisation of existing resources across the laboratory network for routine and emergency testing needs. additionally, robust political commitment and sufficient provision of domestic funding are needed to ensure that all local health needs are adequately covered and are not compromised when addressing global priorities. acknowledgements the authors thank members of the labcop country teams for their responses to the survey questionnaire. we also thank dr george alemnji, dr clement zeh and jason williams for the insightful discussion on barriers to maintaining routine hiv and tuberculosis testing in the context of covid-19. we are grateful to mr michael waweru and mr getachew kassa for supporting the design of the questionnaire and for reviewing this manuscript. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions c.o.o. and p.o. conceptualised the idea, c.o.o. drafted the original manuscript, and p.o., a.m. and m.m.l. made substantial revisions to the manuscript for intellectual content. all authors reviewed and approved the manuscript. ethical considerations ethical clearance was not required for the study. this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support this work would not have been possible without funding from the bill and melinda gates foundation (investment number inv-003603) and resolve to save lives through the project ‘strengthening national capacity for covid 19 surge testing in selected countries across africa’ (surge cov19 testing, grant code: 4406-20-1-vs-covid-19-response). data availability statement data sharing is not applicable to this article. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references cucinotta d, vanelli m. who declares covid-19 a pandemic. acta biomed. 2020;91(1):157–160. who calls for more coronavirus testing: ‘test, test, test’ [homepage on the internet]. [cited 2020 sept 28]. available from: https://www.youtube.com/watch?v=3gqhapwmfta who. list of eul products eligible for procurement [homepage on the internet]. [cited 2020 jul 03]. available from: https://www.who.int/diagnostics_laboratory/eual/listing/en/index1.html who. covid-19 – virtual press conference – 30 march 2020 [homepage on the internet]. 2021 [cited 2020 sept 28]. available from: https://www.who.int/docs/default-source/coronaviruse/transcripts/who-audio-emergencies-coronavirus-press-conference-full-30mar2020.pdf?sfvrsn=6b68bc4a_2 madhukar p. covidization of research: what are the risks? nat med. 2020;26:1159. https://doi.org/10.1038/s41591-020-1015-0 hogan ab, jewell bl, sherrard-smith e, et al. potential impact of the covid-19 pandemic on hiv, tuberculosis, and malaria in low-income and middle-income countries: a modelling study [published correction appears in lancet glob health. 2021 jan;9(1):e23]. lancet glob health. 2020;8(9):e1132–e1141. https://doi.org/10.1016/s2214-109x(20)30288-6 who. special feature: immunization and covid-19 [homepage on the internet]. 2020 [cited 2020 sept 28]. available from: https://www.who.int/immunization/monitoring_surveillance/immunization-and-covid-19/en/ roberton t, carter ed. early estimates of the indirect effects of the covid-19 pandemic on maternal and child mortality in low-income and middle-income countries: a modelling study. lancet glob health. 2020;8(7):e901–e908. https://doi.org/10.1016/s2214-109x(20)30229-1 the global fund. mitigating the impact of covid-19 on countries affected by hiv, tuberculosis and malaria [homepage on the internet]. [cited 2020 jul 24]. available from: https://www.theglobalfund.org/media/9819/covid19_mitigatingimpact_report_en.pdf?u=637293077390000000 stop tb partnership. the potential impact of the covid-19 response on tuberculosis in high-burden countries: a modelling analysis. 2020 [cited 2020 sept 28]. available from: http://www.stoptb.org/assets/documents/news/modeling%20report_1%20may%202020_final.pdf who. disruption in hiv, hepatitis and sti services due to covid-19 [homepage on the internet]. 2020 [cited 2020 sept 28]. available from: https://www.who.int/docs/default-source/hiv-hq/disruption-hiv-hepatitis-sti-services-due-to-covid19.pdf?sfvrsn=5f78b742_6 aslm. labcop [homepage on the internet]. [cited 28 sept 2020]. available from: https://aslm.org/what-we-do/labcop/ uganda viral load dashboard [homepage on the internet]. [cited 22 jun 2020]. available from: https://vldash.cphluganda.org lecher s, williams j, fonjungo pn, et al. progress with scale-up of hiv viral load monitoring – seven sub-saharan african countries, january 2015–june 2016. mmwr morb mortal wkly rep. 2016;65(47):1332–1335. https://doi.org/10.15585/mmwr.mm6547a2 cazabon d, pande t, kik s, et al. market penetration of xpert mtb/rif in high tuberculosis burden countries: a trend analysis from 2014–2016. gates open res. 2018;2:35. https://doi.org/10.12688/gatesopenres.12842.1 who. considerations for adoption and use of multi-disease testing devices in integrated laboratory networks [homepage on the internet]. 2017 [cited 2020 sept 28]. available from: https://apps.who.int/iris/bitstream/handle/10665/255693/who-htm-tb-2017.06-eng.pdf?sequence=1 who regional office for africa. the maputo declaration on strengthening of laboratory systems [homepage on the internet]. 2008 [cited 2011 aug 18]. available from: http://www.who.int/diagnostics_laboratory/maputo-declaration_2008.pdf african society of laboratory medicine. laboratory mapping program (labmap) [homepage on the internet]. [cited 2021 feb 22]. available from: http://www.aslm.org/what-we-do/laboratory-mapping/ labcop cookbook of best practices. decentralizing covid-19 pcr diagnostic capacity to sub-national level [homepage on the internet]. [cited 28 sept 2020]. available from: https://aslm.org/wp-content/uploads/2020/09/bookletlabcopcookbook4-2020-09-02-webquality.pdf?x64533 considerations for developing a monitoring and evaluation framework for viral load testing [homepage on the internet]. geneva: world health organization; 2019 [cited 2020 sept 28] (who/cds/hiv/19.5). license: cc by-nc-sa 3.0 igo. available from: https://apps.who.int/iris/bitstream/handle/10665/324745/who-cds-hiv-19.5-eng.pdf abstract introduction methodology results discussion acknowledgements references about the author(s) winny koster amsterdam institute for global health and development, amsterdam, the netherlands department of public health, faculty of social and behavioural sciences, amsterdam institute for social science research, university of amsterdam, amsterdam, the netherlands albert g. ndione institut de recherche pour le développement (ird), transvihmi, crcf-hôpital fann, dakar, senegal mourfou adama centre national de lutte anti tuberculeuse (cnlat), laboratoire national de référence des mycobactéries, ouagadougou, burkina faso ibrehima guindo institut national de recherche en santé publique, bamako, mali iyane sow directorate of laboratory services, ministry of public health and social welfare, dakar, senegal souleymane diallo centre d’infectiologie charles mérieux, bamako, mali jean sakandé fondation mérieux, ouagadougou, burkina faso pascale ondoa amsterdam institute for global health and development, amsterdam, the netherlands department of global health, amsterdam university medical centers, amsterdam, the netherlands african society for laboratory medicine, addis ababa, ethiopia citation koster w, ndione ag, adama m, et al. an oral history of medical laboratory development in francophone west african countries. afr j lab med. 2021;10(1), a1157. https://doi.org/10.4102/ajlm.v10i1.1157 note: additional supporting information may be found in the online version of this article as supplementary document 1. original research an oral history of medical laboratory development in francophone west african countries winny koster, albert g. ndione, mourfou adama, ibrehima guindo, iyane sow, souleymane diallo, jean sakandé, pascale ondoa received: 20 dec. 2019; accepted: 20 oct. 2020; published: 16 mar. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: underdeveloped and underused medical laboratories in sub-saharan africa negatively affect the diagnosis and appropriate treatment of ailments. objective: we identified political, disease-related and socio-economic factors that have shaped the laboratory sector in senegal, mali and burkina faso to inform laboratory-strengthening programmes. methods: we searched peer-reviewed and grey literature from february 2015 to december 2018 on laboratory and health systems development from colonial times to the present and conducted in-depth interviews with 73 key informants involved in (inter)national health or laboratory policy, organisation, practice or training. this article depended on the key informants’ accounts due to the paucity of literature on laboratory development in francophone west african countries. literature and interview findings were triangulated and are presented chronologically. results: until around 1990 there were a few disease-specific research laboratories; only the larger hospitals and district health facilities housed a rudimentary laboratory. the 1990s brought the advent of donor-dictated, vertical, endemic and epidemic disease programmes and laboratories. despite decentralising from the national level to the regional and district levels, these vertical laboratory programmes biased national health resource allocation deleteriously neglecting the development of the horizontal, general-health laboratory. after the year 2000, the general-health laboratory system received more attention when, influenced by the world health organization, national networks and (sub-)directorates of laboratories were installed. conclusion: to advance national general healthcare, as opposed to disease-specific healthcare, national laboratory directors and experts in general laboratory development should be consulted when national policies are made with potential laboratory donors. keywords: oral history; medical laboratory; senegal; burkina faso; mali. introduction underdeveloped and underused medical laboratories in sub-saharan africa hamper the diagnosis and management of potentially epidemic infectious diseases as well as general public health conditions.1,2,3,4 recent national surveys of laboratory capacity conducted in senegal, mali and burkina faso – francophone west africa for short, and the countries this study focuses on – found that many laboratories still have insufficient personnel, a lack of or dysfunctional basic equipment, and operate in inappropriate rooms.5,6,7,8 studies in senegal on barriers to the uptake of the seven recommended routine maternal diagnostic tests found that laboratory problems were part of the reason why only around one-third of pregnant women received the complete set of tests.9,10 identifying the historical developments that have shaped the laboratory sector could help to understand the gaps and provide lessons for laboratory-strengthening programmes. as far as we know, this is the first study on this topic. the guiding question of our study was: what political, socio-economic, and disease-related factors have influenced the current status of medical laboratories in senegal, mali and burkina faso? the development of medical laboratories in africa cannot be dissociated from the development of the healthcare sector at large.11,12,13,14 the healthcare sector in colonial times was mainly directed controlling infectious disease control and keeping the colonial workforce healthy to ensure economic productivity.15 the years after independence saw optimism towards national economic growth that focused on improved healthcare for the general population.15 the global economic recession of the early 1980s and the structural adjustment programmes of the late 1980s–1990s had a significant negative impact on national economies and forced governments to cut spending and lower their development goals of early post-independence.15,16,17 the ensuing cuts in the health sector thus created institutional, technological and personnel capacity problems.13,17,18 the late 1990s saw the emergence of global health initiatives in response to worldwide health-related problems, mainly epidemic diseases, that could pose global security threats. our article progresses chronologically: it begins in late colonial times, goes on to the post-independence period, followed by the time of structural adjustment, and ends with the emergence of global health. in each period, we describe the developments in the laboratory sector and the influencing factors. methodology ethical considerations the objectives of the study were explained to all key informants; all gave verbal consent to be interviewed and recorded, as can be proven by the audio recordings. study design the data for this qualitative descriptive study are derived primarily from in-depth key informant interviews and a literature review. the study proposal identified themes in the interrelated factors influencing laboratory development. themes explored were: organisation of services, regulations, health policies and plans; economic conditions and by extension personnel, equipment, training and infrastructure development; endemic and epidemic disease contexts; and lastly, funding by national governments and (inter)national donors. data collection methods and study participants literature review between february 2015 and february 2016, three country teams of three to six social science researchers and laboratory professionals searched for peer-reviewed and grey literature on the study’s themes and identified factors in local libraries, organisations and government departments (online supplementary document – table 1), and freely accessible websites (online supplementary document – list 1). key informant interviews key informants, selected by the country team members, were in 2015 interviewed for 30 min to 3 h, mainly at their workplace. selection criteria were having an active role in the (inter)national health or laboratory policy, organisation, practice or training (past and present). for every key informant, a personalised question guide was prepared, considering their professional background, the period of their professional work and their involvement with laboratories. a summary of the 73 key informants’ background characteristics is presented in table 1, details are provided in the online supplementary document – table 2. key informants were trained in clinical or laboratory medicine (or both) and included retired (n = 8) and current (n = 65) staff members of various departments in the ministry of health (moh), international organisations, private and public health facilities, professional associations, training institutions, and universities; many had worked in several functions. one key informant’s professional career commenced during the colonial period, nine commenced in the two decades after the three countries’ independence in 1960, while the remainder commenced from the 1980s. table 1: background of the 73 key informants, francophone africa, february 2015 – february 2016. data analysis and presentation the audio-recorded key informant interviews (in french) were transcribed verbatim. nvivo qualitative data analysis software, version 10 (qsr international, melbourne, australia), was used for the thematic analysis of the interviews and a timeline was constructed for each country. each country team summarised their literature findings thematically and chronologically. in this article, the findings derived from the two data collection methods have been triangulated and are presented for the three countries combined. occasionally, differences and specificities across countries are pointed out. it should be noted that when the article refers to literature, the same information was usually corroborated by key informants. all quotations in the text are english translations of the french citations. results the study’s primary finding and challenge was the scarcity of written sources on laboratories; consequently, this history of laboratory development mainly hinges on the accounts of key informants, all of whom willingly recounted their experiences. notably, those involved in laboratory practice or policy welcomed the attention; as a senegalese biologist involved in laboratories since 1982 expressed: ‘the laboratory has habitually been the forgotten part of medicine, meaning one thinks of everything, and of the laboratory only after that’ (key informant 62, male, interview date 11 march 2015, dakar). descriptions of developments for colonial times and during the first decades after independence are briefer than for the periods afterwards because most of the key informants commenced their laboratory experience after these two periods. colonial times in colonial times, all three countries housed renowned research laboratories, including centre muraz in burkina faso, institut pasteur in senegal and laboratoire central de biologie in mali. these laboratories were involved in the research and the control of endemic and epidemic diseases – including trypanosomiasis, cholera, onchocerciasis, meningitis, malaria and syphilis – to preserve the labour and military force,15 and were funded by fides (fonds d’investissements pour le développement economique et social des territoires d’outre-mer).19 the staff were french doctors and local assistants – mainly nurses – trained on-the-job.19,20 a retired burkinabé pharmacist stated that everything related to laboratories in french west africa started with colonial doctors interested in tropical diseases; for instance, gaston muraz, a french military doctor, created centre muraz in bobo dioulasso in 1939. the few existing national and regional hospitals had small laboratories attached.19 in dakar, the capital of french west africa – which included present-day burkina faso and mali – principal, dantec and fann hospitals were among the first modern healthcare facilities in sub-saharan africa.21 mali had kayes, mopti, markala and point g hospitals. in burkina faso, the first hospital was a military facility at bobo dioulasso (now the university hospital sanou sourô). a retired director of institut national de recherche en santé publique (inrsp) in mali, who replaced the french director of the central laboratory in bamako in 1956, remembered that some pharmacies also had a small laboratory attached. post-independence (≈1960–1979) after independence, the colonial research laboratories remained – although some under another name (the malian laboratoire central de biologie became institut national de biologie humaine) – as did those attached to the few public national and regional hospitals.22 a few medical laboratories were attached to private pharmacies in burkina faso, while mali had 16 public stand-alone medical laboratories, mainly for technical support of disease control programmes.19 laboratory diagnostic tests were limited and aimed mainly at detecting parasites and measuring glycosuria. even in larger public hospitals, laboratory services were embryonic and only a few laboratory training programmes existed. in burkina faso, nurses could specialise in laboratory technology at ecole jamot and centre muraz. in mali, schools were opened for medical doctors and pharmacists, two of which offered biology: l’école des assistants médicaux (founded 1969) and l’école nationale de médecine et de pharmacie (founded 1974). in mali, a microbiology research laboratory was set up in 1973, where many of the malian key informants received training. the head of this laboratory had a wide network of international partners who helped provide the necessary resources to make the laboratory internationally renowned. laboratory assistants were trained in the secondary school of health (founded 1963) and the institute national de formation en sciences de la santé. in senegal, the faculty of medicine at cheikh anta diop university (founded 1962) trained pharmacists who worked all over west africa.21 in the three countries, assistants trained on-the-job represented a large part of the laboratory workforce. the national moh in senegal and burkina faso did not prioritise laboratories, resulting in a lack of equipment and supplies for the few existing public facilities. in mali, however, the minister of health acknowledged the importance of laboratories relatively early; he created the national research laboratory inrsp in 197323 and aimed to strengthen public hospital laboratories and establish a national coordinating body and policy. the former inrsp director, recounted that in 1974 this health minister created the division of laboratories and appointed him head, telling him to assess the status of laboratories in the country. based on his situation analysis, he recommended that laboratory services be decentralised to the regional level. disease outbreaks, in particular the 1974–1975 meningitis and cholera outbreaks, added to the health minister’s motivation to decentralise laboratory services. the former inrsp director remembered how cumbersome the control of these outbreaks had been, notably the transportation of suspected cases’ stool samples from the regional laboratories to the central laboratories for analysis. primary healthcare and structural adjustment (≈1980–the late 1990s) this period saw the implementation of two global strategies for health systems development: the 1978 alma ata primary health care declaration and the 1988 bamako initiative. these called for the decentralisation of basic health services, a focus on clinical diagnosis and the supply of essential drugs and equipment. however, structural adjustment programmes, with their efficiency-driven economic reforms, implied less state involvement, public spending cuts and privatisation of healthcare services.15,17 decentralisation of laboratories in the three countries, health centres at (sub-)district level were built, usually with a supporting laboratory to provide the stipulated minimum of primary healthcare diagnostic tests.24,25 the revolutionary sankara regime (1983–1987) aimed to bring healthcare, including rudimentary laboratories, to all corners of burkina faso, as the then minister of health and sports (1985–1987) explained. a pharmacist and current health inspector recounted that the reorganisation of the burkinabé national health system into districts became a fact in 1992–1993, with service norms set by level; laboratories were designated to the district level. medical doctors and laboratory personnel working at district level narrated that at this time if there was a laboratory at all, it was rudimentary and very few types of tests were done, usually in a room not designed to house a laboratory. a burkinabé laboratory technician depicted how, in the 1980s, a typical health centre laboratory was just a small room with a microscope, a manual centrifuge and a fridge with some reagents. ‘community people called us “stool doctors” because they only saw us examining stools’ (key informant 35, male, interview date 06 may 2015, ouagadougou), recounted the lecturer for laboratory technicians at ecole nationale de santé publique in ouagadougou. a challenge for peripheral laboratories was the absence or unreliability of electricity. a medical doctor, currently at conseil national de lute contre le sida, shared his experiences in rural burkina faso: ‘during the years 1984–1990, it was challenging. it was apparent that when you were in a place without electricity you could use very little equipment. only those working in private laboratories had solar microscopes. thus, we had to seek a window and work with sunlight; in that way, we could only do parasitology tests’. (key informant 46, male, interview date 04 may 2015, ouagadougou) he concluded: ‘sophisticated laboratory development followed electrification’. his midwife colleague at conseil national de lute contre le sida added that most health problems were diagnosed at this time through physical patient examination. two senegalese medical doctors, both working in district health centres, remembered that in the 1980s and 1990s, they only sporadically referred patients to regional hospital laboratories for diagnostic tests if the patients could afford the travel costs. key informants in mali and burkina faso reported that structural adjustment programmes had stimulated private practice; this was supported by government laws authorising private medical practice.26 consequently, some private laboratories were created, either stand-alone, like rive droite in bamako, or attached to private hospitals (six in burkina faso). this legal opening prompted some pharmacists to leave the public sector and open private pharmacies with a laboratory attached. the malian and burkinabé key informants generally applauded the development of private laboratories because it increased access to laboratory services, though they acknowledged quality control challenges. training opportunities and insufficiencies training opportunities at this time increased. the university of mali (founded in 1993) offered pharmacists and medical doctors additional training options in medical biology. cheikh anta diop university in dakar offered training for senior laboratory technicians. in burkina faso, a three-year training programme for laboratory technicians started in 1985 at the ecole nationale de santé publique, while during the sankara regime some nurses, including one key informant, were sent for laboratory medicine training in cuba. before 1985, most burkinabé laboratory technicians were trained in dakar, and some in canada and france. the year 1999 saw the start of the licence professionnelle option analyses at the university of ouagadougou. insufficient local training opportunities persisted for assistant and specialisation levels. also, no formal training existed for laboratory assistants and nurses trained on-the-job, who still formed a large portion of laboratory personnel.27 the saying went: ‘you are a laboratory worker and you die a laboratory worker’. due to the lack of laboratory training and career opportunities, many nurses left laboratory work to pursue further nursing training, although some nurses among our key informants stayed because they liked the work. a clinical biology university professor explained that because there was a lack of advanced specialisation training in medical biology or biochemistry in senegal, medical doctors such as himself had to go for further studies abroad, mainly to france. (in)visibility of laboratories in national policies and programmes the first national demographic and health surveys were conducted in the mid-1980s. national health plans focused on equity in service access through primary healthcare, the reduction of infant, child and maternal mortality, and family planning.28 these demographic and health survey reports and national plans made little reference to laboratories. only in 1999 did the malian public health sector set up a referral system for laboratory tests: from first-level centre de santé de référence to second-level regional hospitals to third-level university hospitals.24 no national funds were dedicated to laboratories, and therefore laboratory operations relied on budgetary allocations from health facility management. in those periods of economic austerity, the management of health facilities struggled to maintain all services, including laboratory services. limited budgets and the consequently erratic reagent availability made it difficult for laboratories to function well.28 biological pharmacists among the key informants explained that it was demotivating and boring to work in laboratories with so little support and rudimentary equipment when from their training they knew that the technology was more advanced in europe. however, some doctors in charge of health facilities took the initiative to strengthen their laboratories. a director at the reproductive health directorate senegal recounted how when he became the medical director of a district health centre (1995–1998), the laboratory gained reference in the area because he asked a french friend to help him equip the laboratory with full blood count and glycaemia machines. laboratories became more visible in senegal in 1990 as part of the direction de la pharmacie et des medicaments avec volet laboratoires,29 and in burkina faso in 1993, when they gained a place in the direction générale de la pharmacies, des medicaments traditionels et des laboratoires. a current health inspector reasoned that the burkinabé moh realised the need to coordinate and supervise laboratory activities because the number of laboratories had increased. disease and donors many informants pointed out that the rapid development of laboratories from the 1990s onwards was mainly linked to aids control programmes which, compared to tuberculosis and malaria control programmes, required more than reagents and microscopes. once hiv cases were discovered in the mid-1980s, national aids programmes were set up with large international donor support to gather epidemiological data. until the mid-1990s, hiv testing was centralised – either the suspected hiv-positive individuals had to go to central laboratories or the laboratory staff went to the regions. a director of the senegalese direction des laboratoires remembered that in cases of suspected hiv, laboratory staff had to travel to the regions to collect the blood samples and bring them to dakar for analysis. hiv serology and immunology tests were gradually decentralised to the regional hospital level. donors supported regional hospitals and selected district health centre laboratories with equipment and supplies and by training laboratory staff – often assistants – to execute specific tests. the president of the burkinabé association of technicians explained that in 1999 the entrance qualifications for laboratory technician training at the ecole nationale de santé publique were raised from a middle-school exam to baccalauréat-level (final exam of secondary school) because the sophisticated equipment and more complicated techniques required more highly trained laboratory personnel. two important drivers for laboratory development were the 1996 world health organization (who) meeting in ouagadougou and the 1998 meeting in bamako on epidemic preparedness and the vital role of laboratories. ministry of health representatives from 16 west african countries participated. key informants who attended these meetings remembered that the who stressed laboratory development, training of laboratory staff for early diagnosis of epidemic diseases, and setting up of national laboratory networks. some few years later, all mohs agreed to support medical laboratories to prevent and fight epidemic diseases. emergence of global health (late 1990s–present) increasing but suboptimal laboratory services in all three countries, more public regional hospitals and health centres were built from the late 1990s onwards, which included buildings or rooms dedicated to laboratory services.30 referral laboratories were connected to university hospitals and research laboratories were established for specific diseases. compared to mali and burkina faso, senegal had fewer private stand-alone laboratories. in 2012, while senegal had only 6, burkina faso had 80.31,32,33 with the arrival of more equipment and machines, the array of tests that laboratories could process increased.34,35 the head of the senegalese aids control programme and the head of the who hiv programme in ouagadougou explained that technological developments enabled further roll-out of the prevention of mother to child (hiv) transmission (pmtct) programme. around 2006, all health centre laboratories could perform hiv confirmation diagnostic tests. a burkinabé pharmacist noted doctors began to increasingly use laboratory services when they observed a significant increase in the number of laboratories, tests and more qualified laboratory personnel. other key informants observed that some younger doctors even overused the laboratory, with one expressing: if the person [clinician] knows he can have a haemoglobin count, he does not bother to check the eyes or tongue of the patient, because that would take him more time than to write a test request. (key informant 47, female, interview date 04 may 2015, ouagadougou) medical laboratories exist from the health centre level. however, many (public) laboratories are often substandard. they have inadequate qualified human resources, often face stock-outs of rapid tests, encounter recurrent machine maintenance problems and non-availability of supplies and reagents, and have inadequate laboratory space. the latter was particularly true in mali. a malian biological pharmacist described how a health centre would find a laboratory space: ‘here there is a spare storeroom that only has to be transformed to be a laboratory’ (key informant 28, male, interview date 14 august 2015, bamako). laboratory staff worried that working in a non-dedicated room compromises safety and biosecurity. for instance, a laboratory technician in a malian health centre complained that laboratory training was not practicable in these settings. for instance little space means that equipment is sometimes placed too close to the wall, or on top of each other. some health centre laboratories are still headed by inadequately trained staff. mali has a shortage of clinical biologists due to the lack of training opportunities. in burkina faso, biological pharmacists are available, but many opt to work in private pharmacies, so they are ‘lost for the [public] laboratory’. in senegal, the bottleneck is the result of an insufficient budget to recruit the many required trained laboratory technicians. improved organisation the three countries’ mohs realised the need to prepare and enable health facility laboratories to quickly respond to epidemic threats and to be less dependent on specialised research laboratories. national plans began to increasingly include laboratory services: national laboratory networks, directorates and sub-directorates. interestingly, an outbreak of meningitis in burkina faso in 2002 led directly to the installation of the direction des laboratoires as one of the four technical sub-directorates of the direction générale de la pharmacie, du médicament et des laboratoires (décret n°2011/pres/pm/ms). two key informants who were involved in this recounted what had made the moh realise the importance of laboratory service coordination. during the outbreak, the country’s meningitis research laboratory had identified the outbreak virus to be a different strain (w135) previously unknown in the country, brought by pilgrims from mecca. however, this discovery was not communicated to the moh, leading to a wasteful high-cost countrywide meningitis a vaccination campaign. in mali, the national network of laboratories was created in 2004, consisting of specialised laboratories, hospital and centre de santé de référence (district) laboratories, a few cscom (sub-district) laboratories and private laboratories. in 2011, the division des laboratoires was added to the directorate of pharmacy and medicines. in senegal, the laboratory was in 2002 included in the direction de la pharmacie et des laboratoires and in february 2005 the national network of laboratories was officialised. this network extended to laboratories at the health centre level.36 senegal created a direction des laboratories in 2012.31 its director narrated the 10-year process of its birth: he advocated for its creation in 2002, and the then minister of health had been in favour; however, she was replaced and the new minister was less interested. when the former health minister was reinstated in 2012, the directorate was created. a public health medical doctor at who dakar explained that the advantage of being a stand-alone directorate is visibility at the ministry level, which goes with resource allocations. the problem of not having a budget for the sub-directorate in burkina faso was explicated by its former director: ‘i was appointed director without materials, without anything. it is with my car that i visited all the laboratories in the country [for quality assessment]’ (key informant 30, male, interview date 04 may 2015, ouagadougou). the director of the division of laboratories in mali noted that the division still struggles to function well because of a lack of resources for personnel. the national laboratory networks, the (sub-)directorate and division depend heavily on donor funds for their outreach activities such as general supervision and quality control. however, these donors mainly support disease-focused programmes and outreach stops when the programme ends. for example, laboratory supervision activities in mali no longer take place since money from the global fund to fight aids, tuberculosis and malaria stopped. to improve organisation and monitoring, the réseau d’afrique de l’ouest des laboratoire was created in 2009. resaolab is a regional laboratory network that includes senegal, burkina faso and mali, domiciled in bamako funded by agence française de développement and fondation mérieux. national laboratory policies and plans the who guided the three mohs in developing national laboratory policies and strategic plans. the first step was to conduct a countrywide inventory and evaluation of the status of laboratories. the united states centres for disease control and prevention (cdc) funded this exercise in mali in 2012, and the malian national laboratory policy was accepted in the same year. in burkina faso, the national laboratory policy was endorsed by the government in 2007, including the first five-year strategic plan.37 senegal has no approved plan yet. the laboratory became more integrated into the health system through the specific mention of laboratory testing in management guidelines for certain conditions. for example, in senegal and mali, the antenatal care guidelines identify laboratory tests that all pregnant women should receive.38,39,40 increased training opportunities in-country training opportunities for all levels of laboratory personnel have increased since the late 1990s, and medical staff could specialise in laboratory sciences. public and some private schools (the latter more prevalent in senegal) were opened and existing schools or universities offered new curricula. since 2007, laboratory technicians can upgrade their qualification through the bachelor in applied medical biology (biologie médicale appliquée – bams) in bamako, organised in collaboration with the centre d’infectiologie charles mérieux, fondation mérieux, and université catholique de lyon. in 2010, a nine-month training programme started at ecole nationale de santé publique, burkina faso, for lower-trained technicians to upgrade to the same level as the later-trained baccalauréat-level technicians. the association of laboratory technicians had pushed for this training, and as of 2015 nearly all had been trained. specialised training in laboratory sciences for medical doctors and pharmacists (diplôme d’études spécialisées de biologie clinique [des bc]) is offered in ouagadougou (since 2004), dakar (since 2008) and bamako (since 2013). a diplôme d’études spécialisées lecturer in dakar explained that these regional training opportunities are cost-efficient because students no longer need to go overseas for diplôme d’études spécialisées training, as his generation had to do. since 2010, réseau d’afrique de l’ouest des laboratoires organises refresher training in dedicated institutes in all three countries for biologists and senior technicians working in laboratories, including modules on quality control, biosecurity and biosafety, and health information systems. a general problem reported for many laboratory training programmes is the scarcity of equipment and supplies for hands-on practice. a lecturer in dakar explained how lecturers have to buy equipment and reagents for the students’ practical work because since 2008 or 2009 the university does not receive government funding for research: ‘we do not even have a bottle of reagent for the practical work. the state has no money’ (key informant 60, male, interview date 10 march 2015, dakar). to tackle this problem, senegalese university and training school laboratories have been allowed to generate money by offering paid services to the public. donor support focusing on specific diseases donors have continued to support specific laboratories with training and equipment for the disease they focus on. hiv programmes have greatly increased the availability and level of laboratory services for diagnosis and treatment follow-up. international donors, including the united states cdc, the bill and melinda gates foundation, the clinton health access initiative, the world bank and the global fund, have financially supported or provided equipment, supplies and staff training. the global fund and world bank financially supported infrastructure, surveillance, and supervision of hiv programmes. the united states cdc gave technical and financial support for sentinel site surveillance of hiv and syphilis among pregnant women, while providing quality controls for the testing services. the who provided financial and technical support for laboratory services to fight (other) epidemic-threat diseases, focusing on bacterial meningitis, bacterial diarrhoeal diseases, yellow fever, measles and rubella. in 2004, the who conducted training on the stratégie de surveillance integrée de la maladie et la riposte in ouagadougou, which was attended by several key informants. the training covered, among other things, epidemiology, security, quality assurance and laboratory systems development. in burkina faso, this training led to the national external quality assessment programme in 2006, supervision of laboratory systems and several training programmes in biosecurity.41 the who published a document on laboratory development42 that all countries ratified – the key informant at the who burkina faso office was one of the authors. the west african ebola epidemic of 2013–2016 was a wake-up call for donors to support laboratories and for governments to establish national research laboratories. the inrsp already existed in mali, and the institute’s ex-director and a current lecturer both recounted how donors had supported the inrsp with equipment that enabled it to perform ebola diagnostic tests on regional samples. these key informants believed that this timely and efficient action contributed to the containment of ebola in mali. many key informants criticised donor support for its focus on specific diseases. the former head of several senegalese moh directorates (2004–2012) complained that donors for aids and malaria programmes only support selected laboratories with equipment and training. two biotechnologists active in the malian national association of laboratory technicians highlighted the lack of long-term vision and soundness of donor support during the ebola outbreak when just a few technicians were trained and general biosecurity was only maintained as long as ebola was a threat. a burkinabé pharmacist recounted how a donor had given 10 microscopes – even though the (vertical) programme only needed three – but had not allowed the extra seven microscopes to be used for other services. informants also criticised the fact that some donors imported their machines, not considering what was already available. for example, the head of the directorate for sexual and reproductive health in burkina faso recounted how, as a medical doctor in the region, she experienced that the laboratory equipment given by donors differed from what is normally used in the country. for instance, they received cd4 count machines that used different reagents and could be maintained or repaired by only one person in west africa when the machines were out of commission for three months. réseau d’afrique de l’ouest des laboratoires is one of the few donor-funded programmes focusing on the laboratory system. it supports the training of different levels of laboratory staff, the renovation and equipment of laboratories, and the equipment of training centres.43 involved key informants were very positive about the contributions of réseau d’afrique de l’ouest des laboratoires. some other programmes and funds also address laboratory systems, including the global health security agenda (launched 2014), the regional disease surveillance systems enhancement ii project and the west african health organisation which conducts large laboratory system strengthening through the world bank funding. discussion until the late 1990s, there were few investments in the expansion of laboratory capacity, partly as a result of economic austerity that affected the overall health sector, but also because laboratories were not considered a priority. from the 1990s onwards, the development of laboratories was mainly influenced by the emergence of potential pandemic diseases that require laboratory confirmation for treatment and control. new technology has led to the expansion of laboratory services. however, operating a laboratory (including machines and equipment) depends on the availability of amenities such as electricity and running water. at the time of this report, unavailable and erratic public amenities challenge proper laboratory functioning. not only do power cuts hinder testing, power fluctuations also cause equipment breakdown. the mohs in these economically constrained countries have had and still do have to operate with limited public finances, relying heavily on external financial aid for the running of public health services. donors have therefore played the most important role in how laboratories have been developmentally geared towards specific diseases; national policymakers did not take a leading role. dependence on donors puts the mohs in a weak position in terms of setting priorities for laboratory development and research. donor support for specific diseases did and might not strengthen the laboratory system for the diagnosis and follow-up of all health conditions. an unexpected finding was the big influence of ‘laboratory champions’: persons committed to laboratories, who have a ‘vision’ and ‘fight’ for the development of the national laboratory system. they are individuals who got ‘the laboratory virus’, as the director of the senegalese directorate of laboratories aptly described them. these champions included biologists and pharmacists who lobbied national ministers of health and donors to set up research laboratories, national health ministers and laboratory technicians active in their associations. these champions were often constrained in putting their vision into practice by the larger national context of unsupportive political leadership, general poverty and the lack of basic infrastructure and amenities. recommendations drawing lessons from the study findings, the following recommendations for laboratory-strengthening programmes are directed at political leaders, mohs, health facilities and donors: have a stand-alone directorate of laboratories with a dedicated national budget, as per the recommendation of the 2008 maputo declaration.44 the who regional office for africa, the african society for laboratory medicine and the africa cdc should continue to remind and support national policymakers who signed the declaration. national policymakers should dedicate more national budget to laboratory development and give the national directorate the mandate to coordinate and guide donor involvement in public and private laboratories at all levels. prioritise continuous professional development opportunities for laboratory personnel at all levels, including laboratory assistants trained on-the-job. professional councils and associations, and the african society for laboratory medicine, should play a role in regulating the profession while the national leadership supports with sufficient funding. national public health laboratories should be established – this is one of the goals of the africa cdc, supported by the african society for laboratory medicine – and should have political influence at the moh to set national research priorities. health facility management committees should establish a dedicated budget for the laboratory, reasoning that the laboratory is a source of direct income and needs to function optimally. as the director of a senegalese regional hospital expressed: ‘that will make the facility function. the laboratory and the radiology are the “lungs” of the health facility’ (key informant 65, male, interview date 16 march 2015, kaolack). limitations the authors acknowledge that personal accounts do not constitute hard, objective data, as is the norm in the medical sector, including laboratory medicine. the authors are also aware that respondents may have constructed this history of, and recommendations for, laboratory development to suit their own or their organisations’ interests. nevertheless, by triangulating literature and accounts of key informants from three countries and different health professions and levels, the authors opine that this oral history presents a true reflection of the development of medical laboratories in francophone west africa. conclusion this unique article on the long-term historical developments of the laboratory sector in francophone west africa demonstrates that by collecting and recording the experiences of people who lived through and have been actors in laboratory developments, a history that would have been otherwise forgotten has been constructed. many of the historical laboratory sector challenges still exist and will not be surmounted without national leaders prioritising the alleviation of national poverty and the development of basic infrastructure. as exemplified by the champions described in this article, national policymakers must be abreast of their population’s disease burdens and needs and play the important leadership role to donors ensuring donor programmes match the populace’s healthcare or laboratory needs, consequently advancing their population’s healthcare. ministries of health should see laboratories as an integral part of the health system, and not simply as part of vertical disease programmes. the retired director of inrsp in mali aptly reiterated: ‘the laboratory is the brain of the health system’ (key informant 24, male, interview date 13 august 2015, bamako). acknowledgements we thank the francophone west africa country team members – other than the authors of this article – who participated in the literature studies. in senegal, they are mouhamed ahmed badji and papa ngore sarr sadio; in burkina faso, dr nikiéma abdoulaye, dr paul somda, naby alphonse and zélé issa; in mali, prof. bourèma kouriba, dr seydou diarra and mamadou fadiala sissoko. we also thank the west african and dutch socialab members who contributed to the overall socialab study that this study is part of: dr aicha marceline sarr, prof. robert pool, prof. constance schultz. special thanks go to prof. robert pool for critically reading the final draft manuscript and oumou badji and sylvain yaméogo for transcribing the key informant interviews. we are most grateful to the key informants who gave their time and enthusiasm to share their experiences with us; without them this history could not have been written. we apologise for having had to sacrifice their often very rich contributions for the general history. competing interests the authors have declared that no competing interests exist. authors’ contributions w.k. was responsible for the study proposal and design, interviews (senegal and burkina faso), coordination, analysis of key informant interviews and drafting of manuscripts. a.g.n. drafted the literature study and report. m.a. and i.g. conducted interviews (mali) and drafted the literature study and report. i.s., s.d. and j.s. were involved in the study proposal and design, selecting key informants and the literature study. p.o. worked on the study proposal and design and was the principal investigator at socialab. all authors critically commented on the draft manuscripts and gave their approval of the final article. sources of support the study was funded by the netherlands organization for scientific research, science for global development (nwo/wotro) under w07.4.203.00. data availability statement upon request, transcripts of interviews can be provided. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references okeke in. divining without seeds. the case for strengthening laboratory medicine in africa. ithaca and london: cornell university press; 2011. petti ca, polage cr, quinn tc, ronald ar, sande a. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377–382. https://doi.org/10.1086/499363 bates i, maitland k. are laboratory services coming of age in sub-saharan africa? clin infect dis. 2006;42(3):383–384. https://doi.org/10.1086/499368 nkengasong jn, skagg ba. are post-ebola reconstruction efforts neglecting public health laboratory systems? lancet glob health. 2015;3(11):e678. https://doi.org/10.1016/s2214-109x(15)00159-x centre national de lutte contre le sida (cnls). plan stratégique national de lutte contre le sida 2011–2015 [national aids strategic plan] [homepage on the internet] [cited 2018 dec 15]. available from: https://apf.francophonie.org/img/pdf/2013_10_vih_dakar_senegalstrategie.pdf ministère de la santé et de l’hygiène publique (mshp). plan stratégique national de renforcement des laboratoires de biologie médicale au mali 2017–2021 [national strategic plan for the strengthening of medical biology laboratories in mali 2017–2021]. bamako: ministère de la santé et de l’hygiène publique; 2016 (unpublished). ministère de la santé et de l’hygiène publique (mshp). la politique nationale des laboratoires de biologie médicale au mali [the national policy of medical biology laboratories in mali]. bamako: ministère de la santé et de l’hygiène publique; 2016 (unpublished). direction des laboratoires. rapport d’activités 2004 [activity report 2004]. ouagadougou: direction des laboratoires; 2004 (unpublished). koster w, ondoa p, sarr am, et al. barriers to uptake of antenatal maternal screening tests in senegal. ssm population health. 2016;2:784–792. https://doi.org/10.1016/j.ssmph.2016.10.003 van’t hoog a, sarr am, koster w, et al. a study to better understand under-utilization of laboratory tests for antenatal care in senegal. plos one. 2020;15(1):e0225710. https://doi.org/10.1371/journal.pone.0225710 becker c, collingnon r. epidémies et médecine coloniale en afrique de l’ouest [epidemics and colonial medicine in west africa]. santé : cahiers d’etudes et de recherches francophones. 1998;8(6):411–416. bado jp. médecine coloniale et grandes endémies en afrique 1900–1960: lèpre, trypanosomiase humaine et onchocercose [colonial medicine and great endemics in africa 1900–1960: leprosy, human trypanosomiasis and onchocerciasis]. paris: éditions karthala (collections hommes et sociétés); 1996. bado jp. histoire, maladies et médecines en afrique occidentale xixe-xxe siècles [history, diseases and medicine in west africa 19th-20th centuries]. outre-mers revue d’histoire. 1999;86:237–268. https://doi.org/10.3406/outre.1999.3727 jacquemot p. les systèmes de santé en afrique et l’inégalité face aux soins [health systems in africa and inequality of care]. afrique contemporaine. 2012;243(3):95–97. https://doi.org/10.3917/afco.243.0095 prince rj. introduction. situating health and the public in africa. historical and anthropological perspectives. in: prince rj, marsland r, editors. making and unmaking public health in africa: ethnographic and historical perspectives. athens: ohio university press (cambridge centre of african studies series), 2014; pp 1–51. jaffré y, olivier de sardan jp. une médecine inhospitalière: les difficiles relations entre soignants et soignés dans cinq capitales d’afrique de l’ouest [inhospitable medicine: the difficult relations between carers and patients in five west african capitals]. paris: karthala; 2003. foley ee. your pocket is what cures you: the politics of health in senegal. new brunswick: rutgers university press; 2010. tousignant n. edges of exposure: toxicology and the problem of capacity in postcolonial senegal. durham: duke university press; 2018. dembélé m. système de santé publique au mali, d’hier à aujourd’hui [mali’s public health system, from yesterday to today]. bamako: ministère de la santé; 2004 (unpublished conference paper). centre muraz. rapport d’activité 1963 [activity report 1963]. bobo dioulasso: centre muraz; 1963. garenne m, cantrelle p, diop i. le cas du sénégal (1960–1980) [the case of senegal (1960-1980)]. in: vallin j, lopez a, editors. la lutte contre la mort. l’influence des politiques sociales et des politiques de santé sur l’évolution de la mortalité [the struggle against death. the influence of social and health policies on the evolution of mortality]. présentation d’un cahier de l’ined, in: population, 40:2:pp 307-330. 1985 [homepage on the internet] [cited 2018 oct 18]. available from: https://www.researchgate.net/publication/32986832_les_cas_du_senegal ministère de la santé et de la prévention (msp). politique de santé du sénégal 1989 [health policy of senegal 1989]. dakar: ministère de la santé et de la prévention; 1989 (unpublished). ministère de la santé. carte sanitaire du mali, mise à jour 2011 (rapport de synthèse) [health map of mali, update 2011 (synthesis report)] [document on the internet]. bamako: ministère de la santé; 2012. available from: http://www.mail.cnom.sante.gov.ml samaké s. l’approche sectorielle dans le domaine de la santé au mali [the sector-wide approach in the health sector in mali]. bamako: dossche printing; 2009. djiba d. bilan d’activités du laboratoire d’analyses de biologie médicale du centre de santé dominique de pikine [report on the activities of the medical biology analysis laboratory at the health centre dominique in pikine]. dakar: université cheikh anta diop, faculté de médecine de pharmacie et d’odonto-stomatologie; 2000 (unpublished). présidence de la république du mali. loi n°85-41/an-rm du 22 juin 1985 portant autorisation de l’exercice privée des professions sanitaires [law no. 85-41/an-rm of 22 june 1985 authorising the private practice of health professions] [document on the internet]. 1985 [cited 2016 jan 15]. available from: http://cnop.sante.gov.ml oninga j. les laboratoires de santé au sénégal: bilan et perspectives [health laboratories in senegal: review and prospects] [unpublished thesis]. dakar: université cheikh anta diop, faculté de médecine de pharmacie et d’odonto-stomatologie; 1990. mané pyb. performance des centres de santé publics au sénégal [performance of public health centres in senegal]. santé publique. 2012;24(6):497–509. https://doi.org/10.3917/spub.126.0497 de roodenbeke e, néné m, loock p. rapport analytique santé et pauvreté: senegal [analytical report on health and poverty: senegal] [document on the internet]. africa region human development working paper series; no. 55. washington, d.c.: world bank group; 2006 [cited 2015 june 16]. available from: http://documents.worldbank.org/curated/en/724991468304516826/rapport-analytique-sante-et-pauvrete-senegal zurn p, codja l, sall fl. la fidélisation des personnels de santé dans les zones difficiles d’accès au sénégal [health worker retention in hard-to-reach areas in senegal] [document on the internet]. rapport intermédiaire national. geneva: oms; 2008 [cited 2016 july 10]. available from: https://www.who.int/hrh/migration/case_study_senegal_2008.pdf direction des laboratoires. rapport d’activités 2012 [activity report 2012] [document on the internet] [cited 2015 may 20]. available from: http://dirlabosn.com institut bioforce développement. la professionnalisation de la chaine d’approvisionnement des produits de santé en afrique de l’ouest, rapport préliminaire [professionalisation of the health commodity supply chain in west africa, preliminary report] [document on the internet]. 2012 [cited 2015 may 20]. available from: https://peoplethatdeliver.org institut national de la statistique et de la démographie (insd). annuaire statistique 2015 [statistical yearbook 2015] [homepage on the internet]. ouagadougou: institut national de la statistique et de la démographie; 2016 [cited 2016 july 16]. available from: http://insd.bf ministère de la santé. arrêté n°2007/202/ms/cab portant contrôle national de qualité des analyses de biologie médicale [order n°2007/202/ms/cab on national quality control of medical biology analyses]. ouagadougou: ministère de la santé; 2007. ministère de la santé, direction des laboratoires. document cadre des politiques nationales en matières d’analyses de biologie médicales [framework document for national policies on medical biology analysis]. ouagadougou: ministère de la santé; 2007. ministère de la santé et de la prévention. arrêté n° 00275 du 03 février 2005 mettant en place un réseau national des laboratoires au sénégal [order n° 00275 of february 3, 2005 setting up a national network of laboratories in senegal]. 2005. direction des laboratoires. plan directeur national 2006–2010 pour le développement du secteur des laboratoires d’analyses de biologie médicale [national master plan 2006–2010 for the development of the medical biology laboratory sector]. ouagadougou: direction des laboratoires; 2005. division santé de la reproduction (dsr), ministère de la santé. politiques et normes des services de santé de la reproduction [policies and standards for reproductive health services. national policy document]. document de politique nationale. bamako: politique du ministère de la santé; 2005. ministère de la santé. protocole des services de la santé de la reproduction, sénégal [reproductive health services protocol, senegal]. dakar: ministère de la santé; 2000. présidence de la république. loi n°02–044 /du 24 juin 2002 relative à la santé de la reproduction [law n°02–044 /of 24 june 2002 relating to reproductive health]. 2002. sakandé j, niekiema a, kabré e, et al. national external quality assessment for medical biology laboratories in burkina faso: an overview of three years of activity. ann biol clin. 2010;68(6):637–642. oms. capacités requises des laboratoires en vertu du règlement sanitaire international et leur mise en place dans la région africaine de l’oms [laboratory capacity requirements under the international health regulations and their implementation in the who african region]. brazzaville: bureau régional de l’oms pour l’afrique; 2013. fondation mérieux. rapport annuel 2011. lyon: fondation mérieux; 2011. world health organisation, regional office for africa. the maputo declaration on strengthening of laboratory systems. 2008 [homepage on the internet] [cited 2015 dec 18]. available from: https://www.who.int/diagnostics_laboratory/maputo-declaration_2008.pdf introduction covid-19 and cytokine storm covid-19 and t cell impairment covid-19 and immunological investigations acknowledgements references about the author(s) brahim admou center of clinical research, faculty of medicine and pharmacy, mohamed vi university hospital, cadi ayyad university, marrakech, morocco abdelhamid hachimi department of intensive care, faculty of medicine and pharmacy, mohamed vi university hospital, cadi ayyad university, marrakech, morocco mohamed abdenasser samkaoui department of anesthesiology and intensive care, faculty of medicine and pharmacy, mohamed vi university hospital, cadi ayyad university, marrakech, morocco citation admou b, hachimi a, samkaoui ma. how can clinical immunology laboratories contribute to the management of severe covid-19 cases in limited resource contexts? afr j lab med. 2020;9(1), a1282. https://doi.org/10.4102/ajlm.v9i1.1282 opinion paper how can clinical immunology laboratories contribute to the management of severe covid-19 cases in limited resource contexts? brahim admou, abdelhamid hachimi, mohamed abdenasser samkaoui received: 27 may 2020; accepted: 03 sept. 2020; published: 02 nov. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction since its appearance, the coronavirus disease 2019 (covid-19) that is caused by severe acute respiratory disease coronavirus 2 (sars-cov-2), has been responsible for severe respiratory disease similar to diseases commonly associated with the coronaviruses such as severe acute respiratory syndrome and middle east respiratory syndrome; covid-19 frequently requires hospitalisation in the intensive care unit and has had a high mortality rate.1 the severity of covid-19 is generally stratified as asymptomatic, mild (without signs of severe or critical illness), severe (with signs of breathing difficulties) or critical (with signs of respiratory failure, shock and multiple organ failure).2 the contribution of the medical laboratory to the categorisation of covid-19 clinical forms is not yet well defined.3 after confirmation of infection by real-time polymerase chain reaction, biochemical and haematological analyses provide fundamental biological parameters for determining disease severity. similarly, the clinical immunology laboratory could play an important role in elucidating diverse immunological abnormalities associated with the disease. in particular, immunological testing could better categorise the severe and critical forms of covid-19, and subsequently assist treating physicians during the entire course of therapy. the purpose of this opinion article is to highlight the position of the immunology laboratory in the management of severe and critical covid-19 cases, specifically in countries with limited resources. covid-19 and cytokine storm covid-19 is marked by an overproduction of pro-inflammatory cytokines and chemokines, mainly interleukin (il)-6, il-10, tumor necrosis factor (tnf)-α, il-1β, il-2, il-10, interferon-γ and monocyte chemoattractant protein-1.4 other biological abnormalities may predict the severity or progression of the disease, such as abnormal coagulation activation, leukopenia, high levels of c-reactive protein, ferritin, d-dimers, aminotransferase, lactate dehydrogenase and creatin kinase.3,5 elevated levels of pro-inflammatory cytokines have been shown to characterise severe lung infection, which manifests as respiratory distress, multiple organ failure and adverse outcomes in sars-cov-2 infection.6,7 in addition, il-6, il-10, and tnf-α levels significantly increase during infection and drop during recovery,8 suggesting that the intensity of the cytokine release correlates with disease activity. interestingly, il-6 may be a marker for monitoring serious covid-19 cases.9 moreover, simultaneous high levels of il-6 and d-dimers have been shown to be narrowly associated with severe forms of the disease in adult patients. therefore, measuring them in combination allows for greater specificity and sensitivity for predicting severe cases of covid-19.3 covid-19 and t cell impairment deregulation of the immune response, particularly t lymphocytes, appears to be strongly linked to the pathology of covid-1910; direct infection of lymphocytes by sars-cov-2 has been proposed as a cause for acute lymphocyte decline.3,11 lymphocytes express the receptor angiotensin-converting enzyme 2, which has been suggested to be the primary receptor targeted by sars-cov-2.12 moreover, altered t lymphocytes could be an important factor in worsening symptoms in patients, which makes lymphopenia a relevant marker of disease severity, hence the need for intensive care unit admission.11 it has been demonstrated that the severe respiratory syndrome that manifests due to covid-19 is characterised by cluster of differentiation (cd)4 and cd8 t lymphopenia, correlating with disease severity.4,13 on the other hand, covid-19 patients who require intensive care unit hospitalisation have significantly higher levels of il-6, il-10 and tnf-α, with lower levels of cd4 and cd8 t cell counts,8 with which levels of cytokines and t cells are inversely correlated.6,8 moreover, because t lymphocytes are generally crucial for amortising exaggerated natural immune reactions against viral infection, t cell defects can lead to worsening inflammatory responses during covid-19, whereas restoring the number of these lymphocytes can improve them.6 in accordance with this hypothesis, it has been shown that 4–6 days following the onset of infection, t lymphocyte counts drop to their lowest level, whereas cytokine levels reach their maximum. conversely, the restoration of the number of t lymphocytes is associated with a decrease in serum levels of various cytokines, such as il-6, il-10 and tnf-α.6 owing to the cytokine storm, and other immunological predictors of severity of sars-cov2 infection, these may also be helpful in choosing anti-inflammatory drugs, especially corticosteroids for their potential benefit in the reduction of inflammation-induced lung damage in severe covid-19 patients.1 covid-19 and immunological investigations having shown that the most relevant immunological parameters include inflammatory cytokines and t lymphocyte subpopulations, the clinical immunology laboratory is positioned to be important for assessing covid-19 patients, especially for categorising, or even predicting severe cases.2,10 cytokine levels, especially that of il-6, can be individually measured on immunoassay analysers, whereas other cytokine profiles can be explored using either enzyme-linked immunosorbent assays or other specific biotechnologies like luminex®, 200™ (or multiplex) (luminex®, austin, texas, united states) and flow cytometry systems, which allow for a large-scale quantitative measurement.14 the assessment of the main t cell subsets, such as cd3+, cd4+ and cd8+, requires only a simple phenotyping procedure conductible on a mini-cytometer, which is largely available worldwide even in limited-resource context laboratories, thanks to hiv management programs. more developed flow cytometry platforms allow for comprehensive phenotyping assays, enabling the investigation of naive, memory and regulatory t cells; in severe covid-19 patients, early data on t cell subpopulation abnormalities show increased naive helper t cells and decreased memory helper t cells, associated with a lower number of regulatory t cells.10 using a receiver operating characteristic curve to compare fatal and recovered covid-19 cases, xu et al.2 considered 559 cells/µl, 235 cells/µl, and 104 cells/µl of cd3+, cd4+t, and cd8+ t cell subsets as warning values, below which there was a significantly higher risk of in-hospital death.2 indeed, there is a need for regional data analyses before determining cut-off values of these t cell subsets. however, t cell lymphopenia might be accentuated by possible primary or acquired immune deficiency conditions, potentially revealed by covid-19, and must then be considered first when interpreting cell phenotyping values and when managing patients as well.15 conclusion for sars-cov-2 infection, alongside other clinical and biological parameters, the measurement of inflammatory cytokines, mainly il-6, as well as cd4+ and cd8+ t cell assessment, should be systematically planned during management of the disease. these markers could be useful for identifying severe cases requiring prompt admission to intensive care units, and for monitoring patient disease progression. these investigations are within the reach of almost all clinical immunology laboratories in the world. close collaboration between immunologists and physicians is essential for effective global efforts against this highly threatening pandemic. acknowledgements we would like to express our sincere gratitude and deepest appreciation to yacine berka for his highly valuable contribution in the editing of this manuscript. competing interests the authors have declared that no competing interests exist. authors’ contributions all authors contributed equally to this work. b.a. conceptualised and wrote the major parts of the manuscript. a.h. co-wrote the clinical aspects of the manuscript. m.a.s. contributed to the conceptualisation and validated a writing review of the manuscript. ethical considerations ethical clearance was not required for this study. sources of support this research received no specific grant from any funding agency in the public commercial or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references huang c, wang y, li x, et al. clinical features of patients infected with 2019 novel coronavirus in wuhan, china. lancet. 2020;395(10223):497–506. https://doi.org/10.1016/s0140-6736(20)30183-5 xu b, fan c-y, wang a-l, et al. miao. suppressed t cell-mediated immunity in patients with covid-19: a clinical retrospective study in wuhan, china. j infect. 81(1):e51–e60. gao y, li t, han m, et al. diagnostic utility of clinical laboratory data determinations for patients with the severe covid-19. j med virol. 2020;92(7):791–796. https://doi.org/10.1002/jmv.25770 pedersen s-f, ho y-c. sars-cov-2: a storm is raging. j clin invest. 2020;130(5):2202–2205. https://doi.org/10.1172/jci137647 henry b-m, de oliveira m-h-s, benoit s, et al. hematologic, biochemical and immune biomarker abnormalities associated with severe illness and mortality in coronavirus disease 2019 (covid-19): a meta-analysis. clin chem lab med. 2020;58(7):1021–1028. https://doi.org/10.1515/cclm-2020-0369 liu j, li s, liu j, et al. longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of sars-cov-2 infected patients. ebio med. 2020;55:102763. https://doi.org/10.1016/j.ebiom.2020.102763 zhou j, chu h., li c, et al. active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis. j infect dis 2014;209(9):1331–1342. https://doi.org/10.1093/infdis/jit504 diao b, wang c, tan y, et al. reduction and functional exhaustion of t cells in patients with coronavirus disease. front. immunol. 2020;11:827. https://doi.org/10.3389/fimmu.2020.00827 zhu z, cai t, fan l, et al. clinical value of immune-inflammatory parameters to assess the severity of coronavirus disease 2019. int j infect dis. 2020;95:332–339. https://doi.org/10.1016/j.ijid.2020.04.041 qin c, zhou l, hu z, et al. dysregulation of immune response in patients with covid-19 in wuhan, china. clin infect dis. 2020. https://doi.org/10.1093/cid/ciaa248 tan l, wang q, zhang d, et al. lymphopenia predicts disease severity of covid-19: a descriptive and predictive study. signal transduct target ther. 2020;5(1):33. https://doi.org/10.1038/s41392-020-0159-1 xu h, zhong l, deng j, et al. high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa. int j oral sci. 2020;12:8. https://doi.org/10.1038/s41368-020-0074-x chen g, wu d, guo w, et al. clinical and immunologic features in severe and moderate coronavirus disease 2019. j clin invest. 2020;130(5):2620–2629. https://doi.org/10.1172/jci137244 medeiros ni, gomes jas. cytometric bead array [cba] for measuring cytokine levels in chagas disease patients. methods mol biol. 2019;1955:309–314. https://doi.org/10.1007/978-1-4939-9148-8_23 brough h-a, kalayci o, sediva a, et al. managing childhood allergies and immunodeficiencies during respiratory virus epidemics – the 2020 covid-19 pandemic. pediatr allergy immunol. 2020;31(5):442–448. https://doi.org/10.1111/pai.13262 acknowledgements references about the author(s) awadia a. ahmeidi department hematology, faculty of medical laboratory science, university of science and technology, khartoum, sudan ashraf musa abeer hospital, muscat, oman hend s. ahmed department hematology and blood transfusion, faculty of medical laboratory science, omdurman ahlia university, khartoum, sudan adel a. elahmar communicable disease center, harmad medical corporation, doha, qatar ryhana b. goota ministry of health, khartoum, sudan ibtihal a. ahmed faculty of medical laboratory science, ibn sina university, khartoum, sudan abdelhakam h. ali department microbiology, faculty of medical laboratory science, university of al butana, rufaa, sudan mushal allam national institute for communicable diseases, national health laboratory service, johannesburg, south africa mozan o. hassan department hematology and blood transfusion, faculty of medical laboratory science, omdurman ahlia university, khartoum, sudan citation ahmeidi a.a, musa a, ahmed h.s, et al. inflammatory markers as predictors of mortality in covid-19 infection. afr j lab med. 2020;9(1), a1298. https://doi.org/10.4102/ajlm.v9i1.1298 scientific letter inflammatory markers as predictors of mortality in covid-19 infection awadia a. ahmeidi, ashraf musa, hend s. ahmed, adel a. elahmar, ryhana b. goota, ibtihal a. ahmed, abdelhakam h. ali, mushal allam, mozan o. hassan received: 12 june 2020; accepted: 07 oct. 2020; published: 21 dec. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. since the origination of the severe acute respiratory syndrome coronavirus-2 (sars-cov-2) in china in december 2019 and its subsequent global spread, it became critical to understand the inflammatory process associated with this virus and the coronavirus disease 2019 (covid-19) and to understand the changes associated with the different inflammatory parameters. the clinical presentation of covid-19 cases varies greatly from mild symptoms among the majority of patients, to severe disease leading to death in some patients. many covid-19 cases had increased levels of inflammatory cytokines and other infection-related biomarkers.1 the inflammatory markers, including interleukin-6, d-dimer, neutrophil-to-lymphocyte ratio and high-sensitivity c-reactive protein (hs-crp) levels, were found to be indicative of severe covid-19 in reports that emerged from china.2 however, their association with mortality among covid-19 patients has not been reviewed. in this article, we identify inflammatory markers (interleukin-6, d-dimer, neutrophil-to-lymphocyte ratio and hs-crp) as predictors of covid-19 mortality. laboratory tests for these markers are simple, cheap and available and can be performed in outbreak areas with limited resources. according to zhou et al., d-dimer levels exceeding 1.0 µg/ml at hospital admission correlated significantly with death among hospitalised covid-19 patients in china, with a p-value of less than 0.001.3 another study in wuhan, china, which included 343 in-patients with confirmed covid-19, showed that elevated d-dimer levels over 2 µg/ml at an early stage of hospitalisation correlated significantly with a high risk of death.4 also, according to another study from china that included 1099 confirmed covid-19 patients, results revealed that median d-dimer and c-reactive protein levels were higher among severe cases compared to non-severe cases, demonstrating that high d-dimer and c-reactive protein levels were significantly associated with covid-19 severity.5 additionally, in wuhan jinyintan hospital, china, a study showed that high levels of interleukin-6 were significantly associated with a high mortality rate.6 this finding was corroborated by silberstein in this study.7 liu y et al. in zhongnan hospital of wuhan university identified an elevation in the neutrophil-to-lymphocyte ratio as an independent and significant predictor of mortality among 245 hospitalised covid-19 patients, with an 8% increase in mortality with each unit increase in neutrophil-to-lymphocyte ratio.8 many studies reported a correlation between high levels of hs-crp and mortality rate in covid-19 patients. a study done among 375 patients with confirmed sars-cov-2 infection revealed that elevated hs-crp levels were significantly associated with a high mortality risk.9 in another study conducted to evaluate fatal outcomes among covid-19 patients, 187 patients from china were included among whom 43 died, and results revealed that high levels of hs-crp was significantly associated with mortality.10 we conclude that elevation in levels of these four inflammatory markers may be indicative of covid-19 infection severity and mortality. we suggest that these parameters may be helpful predictors of covid-19 severity and could be used as early predictors for case management before deterioration. on the other hand, these parameters cannot be used independently for initial diagnosis and physicians need to monitor the presence of other infections that may interfere with the elevation of these markers. finally, we recommend that similar studies on inflammatory markers should be conducted in african populations to demonstrate the levels of these markers among african covid-19 patients and their association with covid-19 severity and mortality. acknowledgements competing interests the authors have declared that no competing interests exist. authors’ contributions a.a.a., a.m., a.a.e. and i.a.a. developed the concepts and performed the literature search. m.o.h. and m.a. helped to supervise the project. a.h.a and r.b.g. edited the manuscript. a.a.a., a.h.a. and a.a.e. prepared and reviewed the manuscript. h.s.a. helped in writing the literature review. m.a. and m.o.h. were responsible for final approval of the version to be published. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references qin c, zhou l, hu z, et al. dysregulation of immune response in patients with covid-19 in wuhan, china. clin infect dis. 2020;71(15):762–768. https://doi.org/10.1093/cid/ciaa248 chen n, zhou m, dong x, et al. epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study. lancet. 2020 feb 15;395(10223):507–513. https://doi.org/10.1016/s0140-6736(20)30211-7 zhou f, yu t, du r, et al. clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study. lancet. 2020;395(10229):1054–1062. https://doi.org/10.1016/s0140-6736(20)30566-3 zhang l, yan x, fan q, et al. d-dimer levels on admission to predict in hospital mortality in patients with covid-19. j thromb haemost. 2020 jun;18(6):1324–1329. https://doi.org/10.1111/jth.14859 guan wj, ni zy, hu y, et al. clinical characteristics of coronavirus disease 2019 in china. n engl j med. 2020 apr 30;382(18):1708–1720. wu c, chen x, cai y, et al. risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease 2019 pneumonia in wuhan, china. jama intern med. 2020;180(7):934–943. https://doi.org/10.1001/jamainternmed.2020.0994 silberstein m. correlation between premorbid il-6 levels and covid-19 mortality: potential role for vitamin d. int immunopharmacol. 2020 nov;88:106995. https://doi.org/10.1016/j.intimp.2020.106995 liu y, du x, chen j, et al. neutrophil-to-lymphocyte ratio as an independent risk factor for mortality in hospitalized patients with covid-19. j infect. 2020;81(1):e6–e12. https://doi.org/10.1016/j.jinf.2020.04.002 yan l, zhang ht, goncalves j, et al. an interpretable mortality prediction model for covid-19 patients. nat mach intell. 2020;2(5):283–288. https://doi.org/10.1038/s42256-020-0180-7 guo t, fan y, chen m, et al. cardiovascular implications of fatal outcomes of patients with coronavirus disease 2019 (covid-19). jama cardiol. 2020;5(7):811–818. https://doi.org/10.1001/jamacardio.2020.1017 abstract introduction the tuberculosis data fellowship programme training structure focus areas of the training key insights from the programme discussion acknowledgements references about the author(s) natasha gous global health, systemone, llc, johannesburg, south africa alaine u. nyaruhirira management sciences for health, pretoria, south africa bradford cunningham strategic initiatives, systemone, llc, johannesburg, south africa chris macek business development, systemone, llc, northampton, massachusetts, united states citation gous n, nyaruhirira au, cunningham b, macek c. driving the usage of tuberculosis diagnostic data through capacity building in lowand middle-income countries. afr j lab med. 2020;9(2), a1092. https://doi.org/10.4102/ajlm.v9i2.1092 lessons from the field driving the usage of tuberculosis diagnostic data through capacity building in lowand middle-income countries natasha gous, alaine u. nyaruhirira, bradford cunningham, chris macek received: 04 sept. 2019; accepted: 12 aug. 2020; published: 18 nov. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: connectivity platforms collect a wealth of data from connected genexpert instruments, with the potential to provide valuable insights into the burden of disease and effectiveness of tuberculosis programmes. the challenge faced by many countries is a lack of training, analytical skills, and resources required to understand and translate this data into patient management and programme improvement. objective: we describe a novel training programme, the tuberculosis data fellowship, designed to build capacity in lowand middleincome countries for tuberculosis data analytics. methods: the programme consisted of classroom and remote training plus mentorship over a 12-month period. the focus was on skills development in tableau software, followed by training in exploration, analysis, and interpretation of genexpert tuberculosis data across five key programme areas: patient services, programme monitoring, quality of testing, inventory management, and disease burden. results: the programme was piloted in six countries (bangladesh, ethiopia, ghana, malawi, mozambique) in july 2018 and nigeria in september 2018; 20 participants completed the training. a number of key outputs have been achieved, such as improved instrument utilisation rates, decreased error rates, and improved instrument management. conclusion: the training programme empowers local tuberculosis programme staff to discover and fix critical inefficiencies, provides high-level technical and operational support to the tuberculosis programme, and provides a platform for continued sharing of insights and best practices between countries. it supports the notion that connectivity can increase efficiencies and clinical benefits with better data for decision making, if coupled with commensurate capacity building in data analysis and interpretation. keywords: tuberculosis; genexpert; diagnostic data; monitoring and evaluation; data analysis; programmatic. introduction tuberculosis has been declared a global public health emergency. an estimated one-third of the world’s population is infected with tuberculosis; 10 million people developed tuberculosis disease in 2017 alone,1 a number that may be underestimated due to under-reporting and lack of reliable data. the widespread implementation of the xpert® mtb/rif assay (cepheid, sunnyvale, california, united states) for detection of mycobacterium tuberculosis and rifampicin resistance as a first-line tuberculosis diagnostic, has been hailed as the most significant advancement in decades, becoming the first molecular assay to provide a tuberculosis and first-line drug resistance diagnosis in just 2 hours. following widespread adoption of this technology, the world health organization’s agenda for action on digital health for the end tuberculosis strategy called for 100% of all sites using rapid tuberculosis diagnostic instruments to be connected by 2020,2 becoming the first to recognise the role of digital health in the fight against tuberculosis. over the past 2 to 3 years, numerous countries have begun adopting connectivity platforms to help monitor and manage their genexpert fleet by collecting the vast amounts of rich clinical diagnostic and operational data produced by the instrument. rarely before has such a rich data resource been both produced by a diagnostic instrument and been made available via connectivity platforms, at scale, for analysis. as yet, it remains largely untapped.3 if these data can be analysed, interpreted and translated into appropriate recommendations and actions, they have the potential to provide significant transformative impact on the management and effectiveness of infectious disease programmes worldwide. gxalert® (systemone, llc, northampton, massachusetts, united states) is currently collecting data from genexpert platforms in 43 countries running cepheid’s xpert mtb/rif assay. gxalert is a connectivity platform that integrates directly with diagnostic instruments to collect and send a digital copy of test results and associated instrument metadata to an in-country or gxalert server. from there, results can be sent and accessed via short message service and email alerts, microsoft excel (microsoft corp, redmond, washington, united states) reports and web dashboards. the type of data being collected includes not only the diagnostic result, but also information on when and where the test was run and by whom, demographic information about the patient (through an application called gxconnect), reagent lot numbers, probe data, cycle thresholds as well as instrument operational data such as instrument failures, inventory consumption and instrument downtime. from these data, critical insights can be gained or inferred about the tuberculosis programme and can help shed light on both clinical and operational return on investment. data can also provide useful information on testing coverage, disease status and trends, circulating strains and drug resistance profiles, instrument utilisation rates, training needs, supply chain, inventory, and quality of the testing programme.4 but there is a challenge: even though countries now collect this type of data in large volumes, it is a new arena for them. most high-disease burden countries lack the tools, resources and expertise required to analyse, understand and translate these data into improved programme and patient outcomes. a recent study by the foundation for innovative diagnostics (find), found that despite large investments by donors to implement electronic data management systems, there is limited usage of the data to improve service delivery, mainly due to a lack of understanding and awareness of what data means.5 as a result, tuberculosis programmes are accumulating but not using the data being collected to drive decision making. this becomes apparent when one considers the various challenges still hindering tuberculosis programmes today, including gross under-utilisation of instruments,3,6,7,8 high unsuccessful test and error rates (loss of tests),9,10 cartridge stock-outs, instrument breakdowns, and lack of adequate module replacements and maintenance of instruments.11 there is a dire need to build capacity in health data analytical skills amongst staff within national tuberculosis programmes (ntps) in order to bolster the usage of data. to address this need, we designed a novel training programme to develop the expertise and skills required for the analysis and understanding of connected diagnostic data. the tuberculosis data fellowship programme the tb data fellowship (tdf) programme was initiated in 2018 through a joint collaboration between systemone and management sciences for health, with the support of the tableau foundation. designed to build the foundation for sustainable in-country capacity, the programme aimed to enhance the understanding of tuberculosis data and its translation into actionable outputs. achieving these goals required a new cadre of healthcare worker to be trained, one with the ability to understand and interpret the vast amounts of diagnostic and operational data being collected through connected diagnostic systems. for the pilot programme, staff from the ntps, national tuberculosis reference laboratories and ministries of health from several countries using the gxalert connectivity platform were invited to apply. countries invited included bangladesh, ethiopia, ghana, malawi, mozambique and nigeria. the selection criteria for the programme included a minimum of 2 years’ work experience in the field of tuberculosis, and more specifically the genexpert tuberculosis programme, and at least 6 months of work experience with gxalert software. participants also had to have experience working in excel. informatics infrastructure the programme leveraged the existing connectivity infrastructure, gxalert, to gain access to live xpert mtb/rif data. in addition, each participant was provided with a tableau desktop and tableau online licence (tableau, seattle, washington, united states). tableau is a powerful data visualisation and analytics software package that is specifically aimed at helping people understand large amounts of data through the creation of structured storyboards, dashboards and visual representations. systemone developed the server architecture to enable participants to extract live country data from gxalert and to import it into tableau (figure 1). this allowed participants to safely interact with data, enforce the necessary patient privacy and country-specific data permissions, and generate visualisations to allow them to share this with the ntp, neighbouring disease programmes, the ministry of health or donors, via tableau online (figure 1). figure 1: general informatics infrastructure and data flow for tb data fellowship programme. live xpert mtb/rif data from each country is collected via gxalert and stored on a in-country or private cloud hosted gxalert server (depending on country preference). each data fellow is able to download an extract of their own country data to tableau desktop in order to create various graph-like visualisations and basic analytics. once analysis is complete, data fellows could choose to publish a subset of these visualisations, unlinked to the data source, via a community folder on tableau online, allowing them to share insights, ideas and graphics with other data fellows, the ministry of health or ntp. training structure two pilot training programmes were undertaken: the first accepted participants from five countries supported by the global challenge tb project, funded by the united states agency for international development. the first countries were bangladesh (n = 2), ethiopia (n = 2), mozambique (n = 2), ghana (n = 1) and malawi (n = 1). the second programme accepted participants from nigeria only (n = 12). the training lasted 12 months and was structured in two parts: a 1-week in-person classroom training followed by an 11-month remote training and mentorship. classroom training the in-person classroom training consisted of a 1-week intensive centralised training, the first of which was held in johannesburg, south africa from 9–13 july 2018, and the second in abuja, nigeria from 24–28 september 2018. during the first three days of each session, participants were trained extensively on tableau desktop v2018.1 software (tableau, seattle, washington, united states). this was followed by two days of training on how to integrate and interpret gxalert tuberculosis data in tableau, perform basic data analysis, and prepare visualisations to improve the interpretation and reporting of tuberculosis data. remote training and mentorship for 11 months following the classroom training, participants received monthly training and mentorship remotely via 2-hour skype sessions as two separate groups, depending on which classroom training session they attended. systemone designed these sessions to allow in-depth data analysis and development of visualisations to improve understanding, with a focus on the development of data-driven recommendations for programmatic improvement. to promote a platform for sharing of data, insights and best practices, each month participants were required to complete assignments and publish their developed visualisations and insights on the tableau online server for the entire group to see. this encouraged collaboration among members of the groups. focus areas of the training through the 12-month programme, participants were taught how to understand genexpert tuberculosis diagnostic and operational data being collected by the gxalert platform and to translate this data into insights about their respective tuberculosis programmes (table 1). they identified programme gaps and appropriate intervention needs in different programme areas (table 1), while learning how to lower operating costs (by reducing supervision frequency and troubleshooting services), improve the quality of the programme and manage the programme more effectively. table 1: key topics covered during the 2018 tb data fellowship training. key insights from the programme the training has yielded new country-level insights and programme improvements due to improved data use and decision making as demonstrated by numerous technical reports, conference abstracts and presentations. for example, in bangladesh, participants have used data about instrument utilisation rates to influence the ntp to improve referral mechanisms for underperforming sites and further optimise genexpert placement within their ntp to better meet testing demand.12 these insights have also helped the ntp plan for future placement of additional genexpert machines. in ethiopia, analysis of instrument utilisation and subsequent proactive monitoring have led to an improvement in utilisation, from 28% to 75%.13 such dramatic improvements can translate immediately into programme return on investment – whereby tuberculosis programmes can make existing resources go much farther than expected and deploy resources more effectively when receiving future grants or allocating domestic budgets. by teaching participants how to monitor unreportable test rates or the number of tests resulting in errors, no results and invalid results, programme efficiency and response speed can be improved. unreportable or unsuccessful tests do not provide a clinically valid result to the patient and thus need to be repeated. besides the cost in ‘lost’ cartridges, when one considers that the actual cost per test performed has been estimated at $23.00 (united states dollors [usd]) and the cost per diagnosis at $99.00 usd,14 unsuccessful tests represent a significant cost to the health system. the majority of unsuccessful tests are due to error results and can, to a large extent, be corrected. unfortunately, countries seldom know how to interpret error codes to inform appropriate corrective actions. the tdf helped participants categorise error codes according to their suspected sources and, through doing this, identify the most frequent types of errors to troubleshoot while pinpointing the sites needing supervision and follow-up. this real-time support is less costly compared to conventional monitoring, which requires a person to visit sites to troubleshoot issues, without any understanding of which issues pertain to which sites. across all participating countries, the majority of errors were user or technical errors. these errors are associated with incorrect specimen processing or volumes added to the cartridge.15 for example, in ghana, up to 67% of error results were identified as user related, and this insight has led to the introduction of refresher training and targeted supervision for sites.16 the same issue was identified in nigeria and bangladesh, where both programmes have managed to reduce their national error rates due to targeted supervision, refresher training for laboratory staff and regular feedback to laboratories aimed at addressing the high incidence of these user related errors.12,17 another focus area of the tdf training that has led to significant programmatic improvement is the monitoring of testing fleet and instrument downtime. a challenge faced by many tuberculosis programmes is that genexpert instruments are often located at remote facilities, leading to delays in maintenance and replacement of broken modules. by monitoring trends in how instruments report in real-time through connectivity tools, the bangladesh ntp are now identifying directly when modules are down or instruments require calibration. through this real-time monitoring of instrument performance, bangladesh has managed to reduce instrument maintenance turn-around time from anywhere between 5 and 14 months to just 2 weeks, and is now also maintaining 90% module functionality.12 ethical considerations ethical clearance was not required for this study. discussion various connectivity solutions exist to collect diagnostic data, some of which have already been adopted widely. connectivity tools can play a major role in addressing many of the challenges that tuberculosis programmes face by facilitating the central collection and aggregation of diagnostic instrument data so that it can be analysed. however, the introduction of connectivity tools is not sufficient to ensure improved programme management. well-functioning health systems need to utilise this data at all levels in order to drive evidence-based decisions and interventions to improve the quality of care provided.18 to our knowledge, the tdf programme is the first of its kind to build capacity and resources in lowand middle-income countries for the analysis of tuberculosis data collected from connected diagnostics. to address sustainability, the programme provided participants with the much-needed tools required to drive data analytics, counting on participants to lead the ongoing analysis of tuberculosis-related health data from the national genexpert programme in their respective countries. we chose tableau software as a data analytics and visualisation tool, because it enables users to explore, manipulate and create visual representations of large amounts of data in order to produce insights as well as to communicate those insights to a broader audience. we leveraged an existing connectivity footprint, namely the gxalert system (systemone, llc, northampton, massachusetts, united states), to gain access to genexpert tuberculosis data within each respective country, but the programme is translatable to any connectivity platform collecting tuberculosis genexpert data. while the initial pilot programme focused on tuberculosis, the intent is to expand into related disease streams and diagnostics within the ministry of health, such as hiv. by providing an online tool (tableau online) where participants could post their visualisations and insights, the programme also provides a platform whereby countries can share best practices and help to create value. the tdf programme has already seen rapid development and analysis of country key performance indicators leading to immediate publications, programme engagements and strengthening.12,13,16,17 by using the data to recognise programme gaps and identify needs, issues and priorities, participants have been equipped to help develop their national strategies, address challenges and inform data-driven decision making. conclusion the programme empowers local ministry of health, ntp and national tuberculosis reference laboratory staff to lead the analysis of tuberculosis-related data, discover and fix critical inefficiencies, and provide high-level technical and operational support to tuberculosis programmes. through data-driven, actionable recommendations, the tdf helps to strengthen, improve and complement ntps in lowand middle-income countries and, ultimately, improve healthcare delivery. acknowledgements the authors wish to acknowledge and thank neal myrick and jason schumacher from the tableau foundation for their technical and financial support of the programme, sarah hinrichsen from tableau and iwan rÿnders from moyo business advisory, south africa for providing the tableau training. barbara k. timmons edited the article. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions n.g. was the project leader, and n.g., a.u.n. and b.c. were responsible for project design. n.g. wrote the article, and a.u.n., b.c. and c.m. contributed to the conceptualisation, design, development and editing of this article. sources of support the tableau foundation funded this study under award number r007 tableau foundation. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the offifical policy or position of any affiliated agency of the authors. references world health organization. tuberculosis. key facts [homepage on the internet]. c2018 [updated 2018 sept 18; cited 2019 aug 14]. available from: https://www.who.int/news-room/fact-sheets/detail/tuberculosis world health organization. digital health for the end tb strategy: an agenda for action. geneva: who press; 2015. albert h, nathavitharana rr, isaacs c, et al. development, roll-out and impact of xpert mtb/rif for tuberculosis: what lessons have we learnt and how can we do better? eur respir j. 2016;48(2):516–525. https://doi.org/10.1183/13993003.00543-2016 gous n, boeras di, cheng b, et al. the impact of digital technologies on point-of-care diagnostics in resource-limited settings. expert rev mol diagn. 2018;18(4):385–397. https://doi.org/10.1080/14737159.2018.1460205 foundation for innovative diagnostics (find). case study: be data driven: find’s actionable diagnostics data for improved tb care (add for tb) initiative [homepage on the internet]. c2018 [cited 2019 july 10]. available from: https://digitalprinciples.org/wp-content/uploads/find-case-study.pdf creswell j, codlin aj, andre e, et al. results from early programmatic implementation of xpert mtb/rif testing in nine countries. bmc infect dis. 2014;14:2. https://doi.org/10.1186/1471-2334-14-2 karamagi e, nturo j, donggo p, et al. using quality improvement to improve the utilisation of genexpert testing at five lab hubs in northern uganda. bmj open qual. 2017;6(2):e000201. ndlovu z, fajardo e, mbofana e, et al. multidisease testing for hiv and tb using the genexpert platform: a feasibility study in rural zimbabwe. plos one. 2018;13(3):e0193577. gounder a, gounder s, reid sa. evaluation of the implementation of the xpert® mtb/rif assay in fiji. public health action. 2014;4(3):179–183. https://doi.org/10.5588/pha.14.0025 gidado m, nwokoye n, nwadike p, et al. unsuccessful xpert® mtb/rif results: the nigerian experience. public health action. 2018;8(1):2–6. https://doi.org/10.5588/pha.17.0080 joshi b, lestari t, graham sm, et al. the implementation of xpert mtb/rif assay for diagnosis of tuberculosis in nepal: a mixed-methods analysis. plos one. 2018;13(8):e0201731. hossain st, imtiaz es, modak pk, et al. gxalert for real-time monitoring management and strengthening of remote genexpert network in bangladesh [homepage on the internet]. technical brief. c2018 [updated 2018 sept 21; cited 2019 july 10]. available from: https://www.msh.org/resources/gxalert-for-real-time-management-and-strengthening-of-remote-genexpert-network-in mengesha e, nyaruhirira a, scholten j, et al. the experience of innovative specimen transportation and genexpert expansion in ethiopia. presented at: 49th union world conference on lung health; 2018 oct 24–27; the hague. unitaid. unitaid end-of-project evaluation: tb genexpert: scaling up access to contempory diagnostics for tb. geneva: dalberg; 2017. cepheid. improving your experience with xpert mtb/rif [homepage on the internet]. c2012 [updated may 2012; cited 2019 aug 12]. available from: https://www.ghdonline.org/uploads/improving_your_experience_of_xpert_mtb_rif.pdf kudzawu s. genexpert error codes: an evaluation of their definitions and its implications on program strenghtening efforts. accepted to:50th union world conference on lung health; 2019 oct 30–nov 2; hyderabad. agbaiyero kj, emeka e, kuye o. benefits of using technology supported gxalert in managing genexpert high error rates in nigeria. presented at: 49th union world conference on lung health; 2018 oct 24–27; the hague. wagenaar bh, hirschhorn lr, henley c, et al. data-driven quality improvement in low-and middle-income country health systems: lessons from seven years of implementation experience across mozambique, rwanda, and zambia. bmc health serv res. 2017;17(suppl 3):830. https://doi.org/10.1186/s12913-017-2661-x abstract introduction ethical considerations case presentation management and outcome discussion acknowledgements references about the author(s) fatima b. jiya department of paediatrics, usmanu danfodiyo university teaching hospital, sokoto, nigeria paul k. ibitoye department of paediatrics, usmanu danfodiyo university teaching hospital, sokoto, nigeria nma m. jiya department of paediatrics, usmanu danfodiyo university teaching hospital, sokoto, nigeria maryam amodu-sanni department of paediatrics, usmanu danfodiyo university teaching hospital, sokoto, nigeria yahaya mohammed department of medical microbiology and parasitology, usmanu danfodiyo university teaching hospital, sokoto, nigeria dada m. aquib department of radiology, usmanu danfodiyo university teaching hospital, sokoto, nigeria lukman k. coker department of paediatrics, usmanu danfodiyo university teaching hospital, sokoto, nigeria citation jiya fb, ibitoye pk, jiya nm, et al. emphysematous pyelonephritis in an infant from sokoto, north-western nigeria. afr j lab med. 2021;10(1), a1181. https://doi.org/10.4102/ajlm.v10i1.1181 case study emphysematous pyelonephritis in an infant from sokoto, north-western nigeria fatima b. jiya, paul k. ibitoye, nma m. jiya, maryam amodu-sanni, yahaya mohammed, dada m. aquib, lukman k. coker received: 27 jan. 2020; accepted: 03 dec. 2020; published: 26 apr. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract introduction: emphysematous pyelonephritis is a life-threatening necrotising bacterial infection of the kidneys. it is rare among children and can be fatal if not promptly identified and treated. case presentation: a 7-month-old male infant presented to the emergency paediatric unit of usmanu danfodiyo university teaching hospital, sokoto, nigeria, on 12 november 2019 with a 5-day history of fever and vomiting, and a 3-day history of a progressively enlarging, left-side abdominal mass. there was associated excessive crying on micturition, refusal to feed and weight loss. he looked ill and was in respiratory distress, irritable, febrile (38.8 °c), moderately dehydrated and pale. his weight and length were 5.5 kg and 64 cm. he had a tender, firm and ballotable abdominal mass on the left flank measuring 8 cm × 10 cm. his pulse rate was 140 beats/min, blood pressure 60/40 millimetres of mercury and respiratory rate was 65 cycles/min. he had widespread coarse crepitations and normal heart sounds on chest auscultation. management and outcome: an initial diagnosis of sepsis was made. other considerations were nephroblastoma and neuroblastoma. ceftriaxone and blood transfusion were commenced with subsequent administration of intravenous fluids. further radiologic investigations revealed emphysematous pyelonephritis. the patient had percutaneous drainage and extended spectrum β-lactamase-producing escherichia coli (sensitive to meropenem) which was isolated from the aspirate culture after 48 h of incubation. meropenem could not be commenced because of non-availability and high cost. the patient subsequently deteriorated and died from septic shock. conclusion: emphysematous pyelonephritis has a fulminant course when not diagnosed promptly and treated adequately. keywords: emphysematous pyelonephritis; infection; kidney; infant; sokoto. introduction emphysematous pyelonephritis (epn) is a severe necrotising and progressive infection of the kidneys which is characterised by the formation of gas within the renal parenchyma, collecting system, or the perinephric tissue.1 it is rare in children, with the majority of cases occurring among adults with diabetes mellitus.2 gas-forming organisms are said to be the causative organisms of which escherichia coli is the most common.3 the pathogenesis is unclear but factors thought to increase predisposition to developing epn include decreased host immunity, increased levels of glucose in the tissue, impaired tissue perfusion, obstruction of the urinary system and presence of gas-forming organisms in the host tissue.1 the diagnosis of epn requires clinical features supported by radiologic investigations and isolation of the offending organisms. depending on the stage of the disease, treatment could be medical alone or a combination of medical and surgical interventions.1 ethical considerations ethical approval to conduct the study was obtained from uduth health research ethics committee with registration number nhrec/30/012/2019. authors obtained permission from the caregivers to publish the clinical details of the patient. case presentation we report the case of y.b (initials of infant used to retain anonymity), a 7-month-old male infant that was referred from a secondary health facility in sokoto, nigeria, to usmanu danfodiyo university teaching hospital (uduth), sokoto in november 2019. he was brought by his parents to the emergency paediatric unit of uduth on account of a 5-day history of fever and vomiting, and a 3-day history of progressively enlarging left-side abdominal mass which was noticed incidentally and said to be tender to touch. there was no preceding history of trauma and there were no masses on other body parts. there was associated excessive crying, crying on micturition, refusal to feed and weight loss. he was admitted at the referring hospital at the onset of illness where he had anti-malaria (artesunate), and antibiotic (cefuroxime) treatment, as well as a blood transfusion with no significant improvement, necessitating referral to uduth sokoto 72 h later. operating within a resource-constrained health system, uduth is the highest tertiary level healthcare facility within the state. both of his parents have no formal education and are ‘petty traders’ with a combined average earning of 40 000.00 nigerian naira ($132.00 united states dollars [usd]) per month. physical examination revealed an ill-looking child in respiratory distress, irritable, febrile (axillary temperature = 38.8 °c), moderately dehydrated, pale, anicteric acyanosed with no significant peripheral lymphadenopathy. his weight and length were 5.5 kg and 64 cm, while oxygen saturation (spo2) was 89% in room air. he had a tender, firm and ballotable abdominal mass extending from the left lumbar region to the left iliac region, measuring 8 cm × 10 cm. the right kidney, liver and spleen were not palpable. he had normal male external genitalia and was not circumcised. his pulse rate was 140 beats/min, blood pressure 60/40 millimetres of mercury and respiratory rate was 65 cycles/min. chest auscultation revealed vesicular breath sounds with widespread coarse crepitations and normal heart sounds. the neurologic examination was also normal. a clinical diagnosis of sepsis with focus on the chest and urinary tract with malaria was made. other considerations were nephroblastoma and neuroblastoma. management and outcome broad spectrum empirical antibiotic (intravenous ceftriaxone) treatment was commenced empirically, and the patient was transfused with blood (haematocrit was 22%) and subsequently placed on intravenous fluids. blood film for malaria parasite was negative, and metabolic panel and complete blood count were normal except for anaemia (table 1). urinalysis showed proteinuria, glycosuria and leucocyturia but urine microscopy and culture yielded no significant growth (table 1). human immunodeficiency virus dna polymerase chain reaction was negative. chest radiograph revealed multiple patchy perihilar opacities. abdominal ultrasound demonstrated relative renomegaly of the left kidney with a bipolar length of 99 mm, turbid collection with multiple pockets of air within it and marked thinning of the parenchyma. the right kidney was normal in outline, position and size (72 mm in bipolar length) and other organs were normal in appearance (figure 1). computerised tomography (ct) scan of the abdomen revealed an enlarged left kidney with bipolar length and transverse diameters of 97 mm and 68 mm, reduced renal parenchyma enhancement (hounsfield units = 36–42), while multiple oval and tubular negative density areas (hounsfield units = 543–723) were noted centrally and in subcapsular regions suggestive of air in the collecting system and renal parenchyma with overall features in keeping with epn. the right kidney was normal in position, outline and size (bipolar diameter 63.8 mm and transverse diameter 40.8 mm), no mass lesions or calculus were seen, and other organs were normal (figure 2 and figure 3). the patient’s diagnosis was changed to septicaemia complicated by unilateral (left) emphysematous pyelonephritis class ii. ceftriaxone treatment was continued while awaiting the culture result, and an ultrasound guided percutaneous drain was inserted, which drained about 60 ml of purulent, blood-stained material containing air bubbles. the result of aspirate microscopy, culture and sensitivity revealed numerous pus cells on microscopy, and growth of e. coli confirmed to be extended spectrum β-lactamase-positive via phenotypic method, which was sensitive only to meropenem. antibiotic therapy was changed to intravenous meropenem but could not be commenced due to unavailability, as well as the high cost of the medication where available. although there was reduction in his temperature to 37.9 °c, he subsequently developed shock and deteriorated rapidly. he did not respond to resuscitation and died 24 h later. although not the recommended treatment for this case with stage ii epn, nephrectomy was considered but the rapidity in deterioration in the patient’s condition (shock and death within few hours) did not allow for adequate counselling and preparation for the procedure. figure 1: sonogram of the left kidney of an emphysematous pyelonephritis patient at the usmanu danfodiyo university teaching hospital, sokoto, nigeria, november 2019. figure shows renomegaly, reduced renal parenchyma echogenicity and multiple irregular mixed echoes with dirty shadowing (blue arrow). figure 2: contrast enhanced computed tomogram of an emphysematous pyelonephritis patient at the usmanu danfodiyo university teaching hospital, sokoto, nigeria, november 2019. figure shows left renomegaly, contrast enhancement and multiple air densities within the calyceal system (orange arrow). figure 3: non–contrast-enhanced computed tomogram of an emphysematous pyelonephritis patient at the usmanu danfodiyo university teaching hospital, sokoto, nigeria, november 2019. figure shows left renomegaly with multiple air density area within the parenchyma and subcapsular region (orange arrows). table 1: laboratory data of the emphysematous pyelonephritis patient on the second day of admission at the usmanu danfodiyo university teaching hospital, sokoto, nigeria, november 2019. discussion the 7-month-old male infant in this study had acute onset of fever, vomiting and a left-side ballotable abdominal mass with radiologic features of stage ii epn confirmed to be caused by extended spectrum β-lactamase e. coli on aspirate microscopy, culture and sensitivity. emphysematous pyelonephritis is a severe necrotising infection of the renal parenchyma that causes gas accumulation within the renal tissues, with or without the involvement of the peri-renal spaces.4,5 reports of epn has been documented in adults, especially among diabetics, hypertensives and renal transplant recipients following end-stage renal disease.6 women are said to be more at risk than men.1,5 emphysematous pyelonephritis is rare among children with the first known paediatric case reported in a 10-year-old female in 1985.7 there are few reports of epn among children in studies from south africa,8 texas9 and saudi arabia,10 with none of the patients having diabetes mellitus nor was there significant gender preponderance in the occurrence of epn. however, one of the reported cases was a transplant recipient.10 the index patient is, to the best of our knowledge, the first documented case in a child in sokoto state, and possibly in nigeria. unlike in previous reports9,10 in which cases had underlying medical conditions (chromosomal abnormality with ectopic right ureter, and end-stage renal disease from neurogenic bladder), our case was apparently healthy with no identifiable risk factors prior to the development of epn. possibly, our case is an indication that epn can in very rare instances occur in previously apparently healthy children. the most common causative bacterial organism is e. coli, which is also the organism that was isolated from the aspirate specimen of our patient. other pathogenic agents of epn include klebsiella, proteus, pseudomonas, citrobacter, enterococcus and streptococcus species. rare organisms such as coagulase negative staphylococcus, clostridium, candida species and aspergillus fumigates have also been reported.1,3,9,10 unlike in our case, the study by siddique et al.9 reported enterobacter cloacae as the causative agents of epn in their patient. the pathogenesis of epn is not yet known; however, the formation of carbon dioxide from the fermentation of glucose in urine and kidney tissues is thought to be the main mechanism.1 the predisposing factors that have been implicated include the presence of a gas-forming bacterial organisms, high glucose levels in tissues, immunosuppression (e.g. diabetes and immunosuppressive therapy), urinary tract obstruction, and poor tissue perfusion.1,11,12 local tissue ischaemia in the presence of a gas-producing pathogen is thought to exacerbate tissue destruction, encourage the production of pus, and inhibit the removal of locally produced gas, leading to epn.11 other speculated mechanisms are that the increased levels of glucose in the tissues together with decreased blood supply to the kidneys contributes to the anaerobic metabolism of glucose and lactate by the organisms and thereafter the production of gases like carbon dioxide, hydrogen, nitrogen, oxygen and methane by the gas-forming organisms.13 the clinical manifestation of epn in children is similar to that of adults with the main features being fever, anorexia, nausea, vomiting, flank pain with or without palpable mass, and dysuria.7,13 our patient presented with some of the aforementioned symptoms. the classification of epn is via radiologic imaging, and the most commonly used classification system is by huang and seng using ct scan to classify epn into five (1–4b) classes.1 our patient’s ct scan findings (figure 2 and figure 3) placed him at epn class ii because the demonstrated air went beyond the left collecting system to involve the renal parenchyma. although earlier studies9,10 reported renal ultrasonograms suggesting air collection in the renal parenchyma of their patients, epn was not staged using a impossible ct scan in the studies making it impossible to compare the stage of epn in our study with theirs. the clinical presentation, radiologic imaging and isolation of causative organisms confirms the diagnosis of epn.1 modalities of treating patients with epn are said to have changed over time, with intensive conservative management assuming a prominent position, depending on the class of epn and other co-morbidities. broad spectrum antibiotic therapy with percutaneous drainage is the standard recommended treatment protocol for epn classes i and ii. the treatment options for patients with epn classes iii and iv depend on the presence and number of risk factors such as shock, acute kidney injury, thrombocytopaenia and coma. the choice of treatment for patients with fewer than two risk factors is antibiotics and percutaneous drainage. in the presence of two or more risk factors, nephrectomy is the recommended treatment.1 our patient had percutaneous drainage but could not commence the appropriate antibiotic (meropenem) therapy due to non-availability and high cost. although uduth is the highest tertiary level option within the state, meropenem is not indigenously produced to the best of our knowledge. although available, his parents could not afford the supply of ten 500 mg powder for the injection, which cost 18 000.00 nigerian naira ($59.00 usd).14 unlike in our patient, other studies have reported good response to antibiotics like third-generation cephalosporin, fluoroquinolones and vancomycin.6,8,9 good outcomes for epn have been reported with prompt initiation of recommended treatment.9 however, epn may run a fatal course if not identified and treated early.4,5,9 there was a delay in diagnosing the index case due to late presentation. additionally, the isolated organism (extended spectrum β-lactamase e. coli) was resistant to the empirical antibiotic (ceftriaxone) therapy. the recommended antibiotic (meropenem) could not be commenced because of the aforementioned reasons. it is, therefore, not surprising that the patient succumbed to the illness. conclusion our experience brings to the fore the occurrence of epn in an infant and the fulminant course it can take when not diagnosed promptly and treated adequately. our case highlights the importance of a high index of suspicion, the resistance of extended spectrum β-lactamase e. coli to the empirical antibiotic (ceftriaxone), and the need to ensure availability as well as cost effectiveness of recommended antibiotics in the study location. these measures will go a long way in the timely identification of cases and will improve the outcome of management of initial classes (1–3) of epn. acknowledgements competing interests the authors have declared that no competing interests exist. authors’ contributions f.b.j. conceptualised, designed and wrote the original draft. p.k.i., n.m.j. and y.m. critically revised and supervised the manuscript. m.a.-s., d.m.a. and l.k.c. acquired the data. all authors approved the final version to be published. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references huang jj, tseng cc. emphysematous pyelonephritis: clinicoradiological classification, management, prognosis, and pathogenesis. arch intern med. 2000;160(6):797–805. https://doi.org/10.1001/archinte.160.6.797 pontin ar, barnes rd, joffe j, kahn d. emphysematous pyelonephritis in diabetic patients. br j urol. 1995;75(1):71–74. https://doi.org/10.1111/j.1464-410x.1995.tb07237.x mohsin n, budruddin m, lala s, al-taie s. emphysematous pyelonephritis: a case report series of four patients with review of literature. ren fail. 2009;31(7):597–601. https://doi.org/10.1080/08860220903003396 lu yc, chiang bj, pong yh, et al. emphysematous pyelonephritis: clinical characteristics and prognostic factors. int j urol. 2014;21(3):277–282. https://doi.org/10.1111/iju.12244 schicho a, stroszczynski c, wiggermann p. emphysematous cystitis: mortality, risk factors, and pathogens of a rare disease. clin pract. 2017;7(2):930. https://doi.org/10.4081/cp.2017.930 camelia a, abhijit s, shankar a, et al. a case series of emphysematous pyelonephritis. case rep med. 2014;2014:587926. https://doi.org/10.1155/2014/587926 pode d, perlberg s, fine h. emphysematous renal and perirenal infection in nondiabetic patient. urology. 1985;26:313–315. https://doi.org/10.1016/0090-4295(85)90139-6 ambaram pr, petersen kl. emphysematous pyelonephritis in children. pediatr infect dis j. 2016;35(10):1159–1161. https://doi.org/10.1097/inf.0000000000001254 siddique k, seikaly mg. emphysematous pyelonephritis in an infant. pediatr infect dis j. 2013;32(10):1157–1158. https://doi.org/10.1097/inf.0b013e31829aaae3 al-makadma as, al-akash si. an unusual case of pyelonephritis in a pediatric renal transplant recipient. pediatr transplant. 2005;9(2):258–260. https://doi.org/10.1111/j.1399-3046.2004.00276.x turney jh. renal conservation for gas-forming infections. lancet. 2000;355(9206):770–771. https://doi.org/10.1016/s0140-6736(99)00351-7 yang wh, shen nc. gas-forming infection of the urinary tract: an investigation of fermentation as a mechanism. j urol. 1990;143(5):960–964. https://doi.org/10.1016/s0022-5347(17)40151-0 singh su, mcglynn l, fordham m. emphysematous pyelonephritis. bju int. 2010;107(9):1474–1478. https://doi.org/10.1111/j.1464-410x.2010.09660.x drugs.com. meropenem prices, coupons and patient assistance programs [homepage on the internet]. [cited 2019 nov 10]. available from: http://www.drugs.com about the author(s) rajiha a. ibrahim ethiopian public health institute, addis ababa, ethiopia amete m. teshale ethiopian public health institute, addis ababa, ethiopia surafel f. dinku ethiopian public health institute, addis ababa, ethiopia negga a. abera ethiopian public health institute, addis ababa, ethiopia abebe a. negeri ethiopian public health institute, addis ababa, ethiopia feven g. desta ethiopian public health institute, addis ababa, ethiopia eyasu t. seyum ethiopian public health institute, addis ababa, ethiopia adugna w. gemeda ethiopian public health institute, addis ababa, ethiopia wubshet m. keficho american society for microbiology, addis ababa, ethiopia citation ibrahim ra, teshale am, dinku sf, et al. corrigendum: antimicrobial resistance surveillance in ethiopia: implementation experiences and lessons learned. afr j lab med. 2019;8(1), a1109. https://doi.org/10.4102/ajlm.v8i1.1109 note: doi of original article: https://doi.org/10.4102/ajlm.v7i2.770 corrigendum corrigendum: antimicrobial resistance surveillance in ethiopia: implementation experiences and lessons learned rajiha a. ibrahim, amete m. teshale, surafel f. dinku, negga a. abera, abebe a. negeri, feven g. desta, eyasu t. seyum, adugna w. gemeda, wubshet m. keficho published: 06 dec. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. in the version of this article published earlier, the surname of the second author, amete m. teshale, was inadvertently misspelt as ‘teshal’. the author’s surname should have appeared as ‘teshale’ throughout the author list and ‘how to cite’ information section. this correction does not alter the study’s findings of significance or overall interpretation of the study results. the authors apologise for any inconvenience caused. http://www.ajlmonline.org open access page 1 of 1 reviewer acknowledgement acknowledgement to reviewers in an effort to facilitate the selection of appropriate peer reviewers for the african journal of laboratory medicine, we ask that you take a moment to update your electronic portfolio on https://ajlmonline.org for our files, allowing us better access to your areas of interest and expertise, in order to match reviewers with submitted manuscripts. if you would like to become a reviewer, please visit the journal website and register as a reviewer. to access your details on the website, you will need to follow these steps: 1. log into the online journal at https:// ajlmonline.org 2. in your ‘user home’ [https://ajlmonline.org/ index.php/ajlm/user] select ‘edit my profile’ under the heading ‘my account’ and insert all relevant details, bio statement and reviewing interest(s). 3. it is good practice as a reviewer to update your personal details regularly to ensure contact with you throughout your professional term as reviewer to the african journal of laboratory medicine. please do not hesitate to contact us if you require assistance in performing this task. publisher: publishing@aosis.co.za tel: +27 21 975 2602 tel: 086 1000 381 the editorial team of the african journal of laboratory medicine recognises the value and importance of the peer reviewer in the overall publication process – not only in shaping the individual manuscript, but also in shaping the credibility and reputation of our journal. we are committed to the timely publication of all original, innovative contributions submitted for publication. as such, the identification and selection of reviewers who have expertise and interest in the topics appropriate to each manuscript are essential elements in ensuring a timely, productive peer review process. we would like to take this opportunity to thank all reviewers who participated in shaping this volume of the african journal of laboratory medicine. special thanks go to those reviewers who assessed fast-tracked covid-19 and other epidemic papers, who completed reviews on very short timelines. we appreciate the time taken by all of you to perform your review(s) thoroughly. ibrahim abdelazim molla abebe teferi m. abera aaron o. aboderin olusola a. adejumo ayorinde o. afolayan ali akhtar heidi albert george alemnji r. joseph alexis getnet ali linda e. amoah e. andres aje j. anejo-okopi john i. anetor enoch aninagyei kingsley anukam eunice aroyewun sola aruna george aryee george awungafac funmilola ayeni oluwatoyin a. babalola colleen bamford fay betsou debrah i. boeras seye bolaji yap j.b. boum ii robert butcher jane y. carter naseem cassim jennifer coetzee tjeerd datema angel n. desai samba diallo alpha a. diallo kwabena duedu adetoun ejilemele stephen g. emerson patrick erdman uchenna ezenkwa catherine falade emmanuel j. favaloro glen fine onikepe a. folarin adeola fowotade willy m. frança tiago s. garcia maria gazouli lisa gerwing-adima mark a. giffen jr chetna govind xu-xiao guo tomáš hanke dominic j. harrington ekua houphouet noah hull hiroshi ichimura joseph igietseme zeynep o. inal emmanuel o. irek farzana ismail harparkash kaur luc kestens daniel kimani eric s. klug stephania k. deme kekoura kourouma joseph larmarange kim lewis ako-egbe louis robert luo robert n. maina david j. marchant puleng marokane talkmore maruta patrick mateta genevieve mezoh james miller a.h. mohamed meade morgan fausta s. mosha jacob s. mwebi prisha naidoo kameko nichols patrick njukeng johannes nossent ifeanyi nsofor jacinta nwogu roseangela nwuba stephen obaro celestina obiekea michael ofori olusegun s. ojo jude n. okoyeh ayobami olaniyi gisele oliveira geoffrey omuse cyprian onyeji jose ortiz de la rosa philip o. oshun judith owen maria pardos de la gándara dimitri poddighe nira pollock raymond pranata jackson a. roberts vincent rotimi celal satici nadeem shaikh andre st. aubyn bowers yogesh kumar swami vu thi thom ralph timperi oyewale tomori cristina touriño noemi r. tousignant lara vojnov larry westerman p. yagupsky http://www.ajlmonline.org� https://ajlmonline.org https://ajlmonline.org https://ajlmonline.org https://ajlmonline.org/index.php/ajlm/user https://ajlmonline.org/index.php/ajlm/user mailto:publishing@aosis.co.za abstract introduction ethical considerations case presentation management and outcomes discussion acknowledgements references about the author(s) christoffel j. opperman division of medical microbiology, national health laboratory service, university of cape town, cape town, south africa gert j.k. marais division of medical virology, national health laboratory service, university of cape town and groote schuur hospital, cape town, south africa michelle naidoo division of medical virology, national health laboratory service, university of cape town and groote schuur hospital, cape town, south africa marvin hsiao division of medical virology, national health laboratory service, university of cape town and groote schuur hospital, cape town, south africa nazlee samodien division of medical microbiology, national health laboratory service, university of cape town, cape town, south africa citation opperman cj, marais gjk, naidoo m, hsiao m, samodien n,. response to a cluster of severe acute respiratory syndrome coronavirus 2 cases at a diagnostic laboratory. afr j lab med. 2020;9(1), a1307. https://doi.org/10.4102/ajlm.v9i1.1307 case study response to a cluster of severe acute respiratory syndrome coronavirus 2 cases at a diagnostic laboratory christoffel j. opperman, gert j.k. marais, michelle naidoo, marvin hsiao, nazlee samodien received: 18 june 2020; accepted: 06 july 2020; published: 25 aug. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract introduction: we report on the first documented cluster of coronavirus disease 2019 cases amongst diagnostic laboratory staff and outline some of the initial and ongoing steps that are being implemented to manage and prevent the spread of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in our laboratory. case presentation: on 24 april 2020, three staff members of a tertiary diagnostic laboratory in groote schuur hospital, cape town, south africa, tested positive for sars-cov-2. within seven days, a further nine cases were identified, which suggested an outbreak and prompted a full investigation. management and outcome: a multifaceted strategic approach was adopted to halt the spread of sars-cov-2 in our laboratory. interventions focused on simultaneously establishing appropriate risk mitigation and stratification strategies through the upscaling of infection prevention and control measures, whilst minimising disruption to service delivery. conclusion: laboratory coronavirus disease 2019 outbreaks have the potential to cripple a laboratory’s testing capacity. contingency planning and risk assessments should occur early, and interventions should be modified according to each laboratory’s available resources and infrastructure. keywords: sars-cov-2; covid-19; diagnostic laboratory; outbreak; occupational exposure. introduction to our knowledge this is the first reported cluster of coronavirus disease 2019 (covid-19) cases in a laboratory in south africa. with transmission of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) firmly established in our communities, infection amongst laboratory staff and laboratory outbreaks pose a serious threat to both private and public pathology laboratories and have the potential to derail the already-strained pathology services. we outline some of the initial and ongoing steps that are being implemented in our laboratory to manage and prevent the spread of sars-cov-2. ethical considerations this work was done in accordance with all ethical standards for carrying out research, in an emergency situation, without direct contact with human or animal subjects. institutional (national health laboratory service) and departmental (department pathology, division microbiology,) approval was obtained. case presentation on 24 april 2020, three staff members at the national health laboratory service situated within groote schuur hospital, tested positive for sars-cov-2. within seven days, a further nine cases were identified, which suggested an outbreak and prompted a full investigation. this resulted in a temporary closure of affected sections of the laboratory and a significant disruption to laboratory services in the subsequent week. management and outcomes outbreak investigation strategy once the initial cluster was identified, a task team comprising pathologists and managers from multiple pathology disciplines was established to lead an outbreak investigation, whilst simultaneously upscaling infection prevention and control measures. the completion of a line list and analysis of the descriptive epidemiology allowed for the identification of laboratory-specific risk factors and transmission ‘hotspots’. screening for severe acute respiratory syndrome coronavirus 2 infection amongst staff members it is well described that asymptomatic and pre-symptomatic people can transmit sars-cov-2 effiently.1 thus, three days after the three initial confirmed cases, samples were collected from all staff members over a one-week period, regardless of exposure. a designated swabbing station was set up in the laboratory and staff were informed to collect their own swabs for reverse transcriptase polymerase chain reaction (rt-pcr) testing. a video on the correct technique for self-sampling was circulated prior to sample collection. this further limited potential exposure of laboratory staff, whilst reducing strain on hospital testing sites, and facilitated a shorter time to result, allowing for the resumption of clinical diagnostic services. currently, all laboratory staff are being screened daily with a temperature check and a simple symptom-based questionnaire to help identify cases early.2 response interventions and contact tracing all staff that tested positive for sars-cov-2 by rt-pcr were instructed to self-isolate at home and to return to work only after two weeks, if there was a resolution of clinical symptoms. this was in accordance with the south african covid-19 national guidelines.3 contact tracing was carried out immediately for all laboratory personnel who tested positive, to contain the spread of the virus. the contact tracing was limited to laboratory workers. expanded contact tracing, including family members, was conducted independently by the western cape department of health. asymptomatic staff with confirmed exposure to colleagues who tested positive by rt-pcr, were categorised as high-risk for covid-19 according to national guidelines.4 these asymptomatic employees were given special leave to quarantine for seven days and were required to submit a nasopharyngeal or mid-turbinate swab on day 8 for rt-pcr testing. they were to return to work if their rt-pcr test was negative but had to continue daily symptom self-checks until day 14 post exposure. in our laboratory, one staff member with known risk factors for severe covid-19 passed away, whilst the remaining 11 (this includes the first three cases) qualified to return to work after 14 days; 10 of the 11 were mildly symptomatic and one was asymptomatic.3,4 positive specimens have been stored with a view to conduct genomic sequencing and analysis. the resources to perform sequencing in real time were not available, although we did recognise that it would have been extremely helpful in deducing transmission dynamics.5 infection prevention and control measures reinforced in the laboratory laboratory infection prevention and control measures were communicated swiftly to all staff. the local pathology management team recommended several measures to combat intra-laboratory transmission of sars-cov-2. these included: wearing of appropriate masks for all staff as opposed to virology and specimen reception personnel only, frequent and thorough hand-washing and cleaning of surfaces, frequent sanitisation of hands with 70% alcohol-based solutions, correct usage of personal protective equipment in addition to masks in appropriate circumstances and practice of social distancing. to aid the implementation of the above-mentioned measures, masks and personal protective equipment were made available, and extra bottles of alcohol-based solutions were placed at the entry and exit points of the laboratory.6 risk mitigation strategies a shift system was implemented where amenable, to increase social distancing and to avoid having to isolate or quarantine the whole workforce if virus transmission continued. the need to acutely reduce the size of the on-site workforce in the sars-cov-2 diagnostic laboratory had to be weighed carefully against the inevitable increase in test turn-around time this would cause and the resultant public health implications for the community it serves. flexible solutions included staggering shifts and encouraging staff who could work from home to do so. in some departments, where feasible, senior staff over the age of 60 with risk factors7 for covid-19 were advised not to return to work during the initial phase of the outbreak. it took much deliberation to determine which of the high-risk employees could stay at home, because it had to be balanced against the consequences on service delivery in the context of a pandemic. extra personnel from other pathology departments within the national health laboratory service, as well as from affiliated departments at the university of cape town, were trained as backup sars-cov-2 testing staff. additional automated kit-based platforms requiring minimal molecular training to operate were also introduced. the purpose was to increase the operator pool and to avoid key-person dependency, which could potentially disrupt service delivery if any or all of the key persons were isolated or quarantined. testing platforms included the seegene allplex™ 2019-ncov assay (seegene, seoul, republic of korea) after in-house or nuclisens® easymag® nucleic acid extraction (biomérieux, marcy l’étoile, france) and the abbott realtime sars-cov-2 assay (abbott laboratories, abbott park, illinois, united states). assay data were analysed according to the manufacturers’ instructions. daily laboratory activities all academic activities were suspended and only essential on-site meetings that did not violate social distancing measures were held. areas where staff were less vigilant about infection prevention and control precautions, such as the tearoom, were identified and risks mitigated as far as possible. for the tea room, ipc measures included frequent reminders to practise social distancing, request that staff bring and clean their own crockery and cutlery, and limit on the number of staff members in the room at any given time to six people. in addition to the tearoom, high-touch areas, for example, communal telephones, scanners, keyboards, light switches and door handles, were also identified as high-risk areas. therefore, more frequent surface cleaning and disinfection was instituted. furthermore, carpooling by staff, especially from covid-19 ‘hot spots’ in the cape metropole, was discouraged and management provided masks for those commuting to and from work via public transport. maintaining service delivery sections of the laboratory (haematology and chemical pathology) needed to be closed briefly for decontamination during the outbreak. these samples were diverted to a neighbouring national health laboratory service facility to avoid a breakdown in service delivery. however, the sudden transfer of large volume of samples to an understaffed and unprepared laboratory did present challenges that delayed result turn-around times. in another attempt for the laboratory to meet with the increased sars-cov-2 testing demands, non-priority tests were suspended unless suitably justified by the clinician. communication staff were kept abreast of new covid-19-related laboratory information through regular small group or zoom (zoom video communications, san jose, california, united states) meetings. laboratory section-specific communication strategies, such as whatsapp (whatsapp, menlo park, california, united states) groups, were implemented. pathologists were also in regular telephonic contact with affected staff members who were in isolation to ensure that they had the necessary support during recovery. transparency and collaboration were a prominent feature of our response. in particular, the laboratory worked with groote schuur hospital and the provincial outbreak response team to minimise the service disruption and to ensure that the laboratory could be reopened both safely and timeously. discussion challenges and lessons learned the lack of a guideline specific for management of a sars-cov-2 laboratory outbreak posed a significant challenge in mobilising a quick outbreak response plan. although international guidelines were available and consulted, they were not applicable to our setting. the dynamic nature of the outbreak necessitated frequent revisions to staff scheduling rosters when certain employees had to be isolated or quarantined. this was not an easy task, especially because the skill levels amongst all staff were not equally matched. another difficulty was ensuring the safety of staff from sars-cov-2, whilst travelling to and from work. many relied on public transport and carpooling and had no alternative means of travel. lessons learned include the importance of early implementation of a symptom screening tool to detect cases earlier and to prevent spread within the laboratory. we realised the importance of developing contingency plans for any crisis causing laboratory closure in the future. had there been an existing plan, referral of large numbers of samples would probably have occurred faster and more smoothly, without impacting test turn-around time negatively. this outbreak emphasised the need for skills transfer amongst staff to avoid key person dependency issues. we need to institute training programmes that will allow staff to fulfil multiple roles, thereby making it less likely that we jeopardise testing. conclusion a sars-cov-2 outbreak in a diagnostic laboratory can cripple its testing capacity, particularly one that is already under strain. a multifaceted strategic approach was adopted to halt the spread of sars-cov-2 in our laboratory, whilst minimising service delivery disruptions. hopefully, our experiences serve to help other laboratories that find themselves in a similar situation. it is necessary for interventions to be modified based on each facility’s infrastructure and available resources. these recommendations should also be adjunctive to good laboratory practice principles. acknowledgements we would like to acknowledge all the staff in our laboratory who have worked tirelessly at the frontlines of this pandemic. competing interests the authors have no competing conflict of interest to declare with regards to the material discussed in the article. authors’ contributions all authors contributed equally to this work. sources of support the research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement data sharing is not applicable to this article, as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect an official policy or position of any affiliated agency of the authors. references gandhi m, yokoe ds, havlir dv. asymptomatic transmission, the achilles’ heel of current strategies to control covid-19. n engl j med. 2020;382(22):2158–2160. https://doi.org/10.1056/nejme2009758 world health organization. risk assessment and management of exposure of health care workers in the context of covid-19 interim guidance [homepage on the internet]. c2020 [cited 2020 may 14]. available from: https://apps.who.int/iris/bitstream/handle/10665/331496/who-2019-ncovhw_risk_assessment-2020.2-eng.pdf nicd clinical management of suspected or confirmed covid-19 disease version 3 [homepage on the internet]. c2020 [cited 2020 may 14]. available from: https://www.nicd.ac.za/wp-content/uploads/2020/03/clinical-management-of-suspected-or-acute-covid-19-version-3.pdf department of health, south africa. guidelines for quarantine and isolation in relation to covid-19 exposure and infection [homepage on the internet]. c2020 [cited 2020 may 14]. available from: https://www.nicd.ac.za/wp-content/uploads/2020/05/guidelines-for-quarantine-and-isolation-in-relation-to-covid-19.pdf nutman a, marchaim d. how to: molecular investigation of a hospital outbreak. clin microbiol infect. 2019;25(6):688–695. https://doi.org/10.1016/j.cmi.2018.09.017 department of health, south africa. covid-19 disease: infection prevention and control guidelines version 1 [homepage on the internet]. c2020 [cited 2020 may 14]. available from: http://www.health.gov.za/index.php/component/phocadownload/category/626 zhou f, yu t, du r, et al. clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study. lancet. 2020;395(10229):1054–1062. https://doi.org/10.1016/s0140-6736(20)30566-3 article information authors: maxwell waema1 naomi maina2 simon karanja1 beatrice gachie2 maina ngotho3 john kagira4 affiliations: 1institute of tropical medicine and infectious diseases, jomo kenyatta university of agriculture and technology, kenya2biochemistry department, jomo kenyatta university of agriculture and technology, kenya 3animal science department, institute of primate research, kenya 4department of land resources planning management, jomo kenyatta university of agriculture and technology, kenya correspondence to: john kagira postal address: jomo kenyatta university of agriculture and technology, po box 62000 – 00200 nairobi, kenya dates: received: 01 mar. 2013 accepted: 23 oct. 2013 published: 29 oct. 2014 how to cite this article: waema m, maina m, karanja s, gachie b, ngotho m, kagira j. development of a safer laboratory vervet monkey model for the study of human african trypanosomiasis. afr j lab med. 2013;3(1), art. #100, 6 pages. http://dx.doi.org/10.4102/ ajlm.v3i1.100 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. development of a safer laboratory vervet monkey model for the study of human african trypanosomiasis in this original research... open access • abstract • introduction • research methods and design    • trypanosomes    • drugs    • experimental animals    • experimental design    • clinical examination and sample collection    • laboratory analysis    • disease staging criteria    • statistical analysis    • ethical consideration • results    • clinical signs    • parasitaemia and cerebrospinal fluid parasitosis    • haematology profiles       • erythrocyte changes       • platelets       • leucocytes • trustworthiness    • reliability and validity of the research • discussion • limitations of the study    • recommendations • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: there are three subspecies of trypanosoma brucei: t. b. gambiense, t. b. rhodesiense and t. b. brucei. the first two are infectious to humans, whilst t. b. brucei is not. identifying an animal model of t. b. brucei that mimics human african trypanosomiasis (hat) would enable researchers to study hat without subjecting themselves to undue risks such as accidental infection.objectives: this study assessed the sequential clinical, parasitological and haematological changes in vervet monkeys infected with t. b. brucei. methods: three vervet monkeys were infected with a 104 inoculum of t. b. brucei (isolate gutat 1). late-stage disease was induced by subcurative treatment with diminazene aceturate 28 days post-infection. the animals were treated curatively with melarsoprol upon relapse. parasitaemia and clinical signs were monitored daily and, at weekly intervals, the monkeys’ blood and cerebrospinal fluid (csf) were sampled for haematology and parasitosis assessments, respectively. results: the first-peak parasitaemia was observed between seven and nine days post-infection. clinical signs associated with the disease included fever, dullness, pallor of mucous membranes, lymphadenopathy, splenomegaly and oedema. late-stage signs included stiffness of joints and lethargy. the monkeys developed a disease associated with microcytic hypochromic anaemia. there was an initial decline, followed by an increase, in total white blood cell counts from earlyto late-stage disease. trypanosomes were detected in the csf and there was a significant increase in white cell counts in the csf during late-stage disease. infected vervet monkeys displayed classical clinical symptoms, parasitological and haematological trends that were similar to monkeys infected with t.b. rhodesiense. conclusion: the t. b. brucei vervet monkey model can be used for studying hat without putting laboratory technicians and researchers at high risk of accidental infection. introduction top ↑ trypanosoma brucei (t. brucei) is a protozoan parasite, of which there are three subspecies: t. b. gambiense, t. b. rhodesiense and t. b. brucei. the first two subspecies are infectious to humans, causing human african trypanosomiasis (hat) (also known as sleeping sickness),1 whilst t. b. brucei only causes disease in animals. the disease has two recognised stages: the early (haemolymphatic) stage when parasites appear in the blood and the late (encephalitic) stage when the central nervous system is involved.t. b. rhodesiense is found in eastern and southern africa and causes an acute infection. the first clinical symptoms are observed a few weeks or months after initial infection – a result of the parasite invading the central nervous system (cns). t. b. gambiense is found in west and central africa and causes a chronic infection. the parasite can be present in the body for months or even years without causing severe symptoms. for the majority of patients infected with t. b. gambiense, the disease is already in an advanced stage by the time symptoms emerge, with parasites having affected the cns.1 hat remains a neglected disease of major health and socioeconomic consequence in sub-saharan africa,1 with an estimated 60 million people in 36 countries at risk.2 the management of the disease remains a major challenge due to poor diagnostics and treatment regimens, most of which have been used for decades. research and development of diagnostics and new drugs normally rely upon the use of laboratory animal models, principally mice and monkeys, which mimic human disease. six accidental, laboratory-acquired cases of t. b. rhodesiense have been reported.3,4,5 rather than identifying an animal model of a dangerous human pathogen, a safer option would be to identify a non-human pathogen that mimics hat. unlike t. b. rhodesiense, t. b. brucei is unable to infect humans, but mimics the pathogenesis of human-infective t. b. rhodesiense in infected animals.6 thus, an animal model of t. b. brucei that mimics hat would enable researchers to study the disease without subjecting themselves to undue risks. given that the vervet monkey disease model of t. b. rhodesiense bears a close relationship to hat,7 the authors hypothesised that infection with t. b. brucei could provide similar disease progression. these monkeys may provide an excellent opportunity to investigate controlled laboratory studies on serum and cerebrospinal fluid (csf) samples, which would also allow for identification of potential biomarkers of the disease stages. this study was designed to determine the clinical, parasitological and haematological profile of vervet monkeys infected with t. b. brucei and to determine whether a t. b. brucei model would be a suitable and safer alternative to the vervet monkey model of t. b. rhodesiense. research methods and design top ↑ trypanosomes the t. b. brucei gutat 1 isolate was used. the isolate was obtained from the international livestock research institute biobank, passaged three times in mice and preserved at the institute of primate research’s trypanosomes cryobank. the cryopreserved isolate was thawed and injected into donor swiss white mice for expansion. at peak parasitaemia, the mice were euthanased with co2 and their blood was harvested by cardiac puncture. the blood was serially diluted in phosphate saline glucose to a final concentration of 104 trypanosomes/ml. drugs the trypanocidal drugs used in this study were diminazene aceturate (pharma links, india) and melarsoprol (arsobal®, specia, france).diminazene aceturate is an aromatic diamidine used as treatment for livestock trypanosomiasis and is also effective against early-stage t. b. gambiense and t. b. rhodesiense infection.7 in monkey models of hat, the drug clears bloodstream parasites but is unable to clear parasites in the cns since it does not cross the blood-brain barrier. its mode of action involves interference with rna editing and trans-splicing and it inhibits adomet decarboxylase in trypanosomes, resulting in the reduction of spermidine content and the elevation of putrescine levels in the parasite. melarsoprol is a trivalent arsenical compound used for the treatment of late-stage sleeping sickness. the mode of action involves inhibition of trypanothione reductase in trypanosomes. melarsoprol can cross the blood-brain barrier and can thus clear trypanosomes that have crossed into the brain parenchyma. in hat monkey models, melarsoprol is used for curative treatment in late-stage disease.8 experimental animals five wild-caught vervet monkeys, each weighing 2–4 kg, were obtained from the animal unit colony of the institute of primate research. prior to experimentation, the monkeys underwent quarantine for 90 days, during which time they were screened for zoonotic diseases as well as ectoand endoparasites. because of the evidence of minor strongyle infections and mange infestations, they were treated with subcutaneous injections of ivermectin at dosages of 300 μg/kg for three days. the monkeys were housed in single 90 x 60 x 60 cm stainless-steel cages in a room maintained at temperatures in the range of 23–25 °c. they were fed twice daily with monkey cubes (goldstar feeds® ltd., nairobi, kenya), vegetables, green maize and bananas; water was provided ad libitum. experimental design three monkeys were infected intravenously with 1 ml of the suspension containing 104 trypanosomes of t. b. brucei (gutat 1), whilst the remaining two monkeys were kept as non-infected controls. the infected monkeys were monitored daily for parasitaemia as described in previous studies.7 at 28 days post-infection (dpi) the monkeys were treated subcuratively for three consecutive days using intramuscular diminazene aceturate at a dose rate of 5 mg/kg body weight. the monkeys were then monitored for parasitaemia and, on relapse (114 dpi), were treated curatively for four consecutive days with intravenous melarsoprol at a dose rate of 3.6 mg/kg body weight. after 180 days of monitoring, the monkeys were euthanased using euthatal (20% sodium pentobarbitone). clinical examination and sample collection the clinical status of the monkeys was monitored daily. at weekly intervals, the monkeys were anaesthetised with intramuscular injections of ketamine hydrochloride (ketamine®, rotexmedica, trittau, germany) at dosages of 10 mg/kg body weight and full physical examinations were conducted. furthermore, 2 ml of blood were collected weekly by venepuncture of the femoral vein and placed in tubes containing ethylenediaminetetraacetic acid (edta). csf was collected weekly by lumbar puncture. laboratory analysis immediately after blood collection, the haematological assays were performed using an ac3diff t coulter counter (miami, florida, usa). parasitaemia was scored using the method described by herbert and lumsden,8 which included the daily use of wet smear detection of microscopic parasites using the rapid matching method. to determine pathological effects, baseline biochemical values were compared to those post-infection. normal ranges are not generally used because of the variation found amongst vervet monkeys.10 disease staging criteria the stage of trypanosome infection was determined in accordance with world health organization criteria and as previously described.9,11 csf was collected by lumbar puncture and examined for the presence of trypanosomes and the number of white blood cells (wbcs). the wbcs were estimated by counting in a neubauer cell chamber. when cells were < 5 cells/µl, infection was classified as first stage. late-stage infection was diagnosed when trypanosomes were detected in the csf and the wbc count of the csf was > 5 cells/µl.12 statistical analysis data were managed using microsoft excel (microsoft us, version 2007). descriptive statistics and summary tables were employed for the initial description of the data and the results were displayed in excel charts. differences between the means were compared using the student’s t-test and were deemed to have statistical significance at p < 0.05. ethical consideration prior to commencement of the study, all protocols and procedures used were reviewed and approved by the institute of primate research institutional animal care and use committee (irc/19/10). results top ↑ clinical signs all the infected animals developed acute symptoms characteristic of rhodesian hat, including fever with a mean temperature of 40 °c, dullness, increased pulse and respiratory rates, pallor of the mucous membranes, enlarged superficial lymph nodes and spleen, raised hair coat, peri-orbital oedema and stiffness of joints. upon subcurative treatment with diminazene aceturate, most of the clinical signs of disease disappeared within 14 days. however, starting at 42 dpi, one monkey exhibited a general body weakness, sleepiness, ataxia and an arched back when sitting on the cage floor and was euthanased for humane reasons. the other two infected monkeys had relapses of parasitaemia from 114–119 dpi with clinical signs that included stiffness of joints and hind leg paralyses. these signs disappeared within 14 days after curative treatment with melarsoprol. parasitaemia and cerebrospinal fluid parasitosis all experimentally-infected animals had a prepatent period ranging from two to four days. the first parasitaemia peak of 107 trypanosomes per ml of blood occurred between 7–9 dpi (figure 1). treatment with diminazene aceturate resulted in clearance of the trypanosomes in the blood. in all three infected monkeys, the parasites relapsed, starting at 114 dpi. trypanosomes were detected in the csf of two monkeys on days 28 and 105 post-infection. treatment with melarsoprol at 119 dpi led to clearance of both the parasitaemia and csf parasitosis by 123 dpi. there was an increase in csf wbc counts during the late stage (figure 2). figure 1: mean daily parasitaemia of vervet monkeys infected with t. b. brucei gutat 1, indicating the point of subcurative treatment with diminazene aceturate (da) and curative treatment with melarsoprol (mel b). figure 2: white blood cell (wbc) count numbers in the cerebrospinal fluid of t. b. brucei-infected vervet monkeys, showing the point of subcurative treatment with diminazene aceturate (da) (28 dpi) and curative treatment with melarsoprol (mel b) (119 dpi). haematology profiles erythrocyte changes in the control animals, the mean total red blood cell (rbc) count was 5.4 x 106 cells/μl, packed cell volume (pcv) was 42% and haemoglobin (hb) was 12 g/dl. blood count parameters did not change significantly throughout the experimental period. however, by 28 dpi, the infected vervet monkeys had a decrease (p < 0.05) in the mean values for the rbc count, pcv and hb values to 4.7 x 106 ± 0.72 cells/μl, 27 ± 2.05% and 8.2 ± 0.8 g/dl, respectively (figure 3). after subcurative treatment, the levels increased gradually to those of pre-infection within 14 days post-diminazene aceturate treatment. blood count parameters decreased again at 98 dpi but recovered and attained the pre-infection levels within seven days of curative treatment with melarsoprol. figure 3: mean changes in red blood cell (rbc) counts, packed cell volume (pcv) and haemoglobin (hb) of vervet monkeys infected with t. b. brucei gutat 1, showing the point of subcurative treatment with diminazene aceturate (da) (28 dpi) and curative treatment with melarsoprol (mel b) (119 dpi). red cell distribution width (rdw) increased during early-stage infection and peaked (14.2 ± 1.5%) at 42 dpi. thereafter, rdw decreased during late-stage disease to normal levels (10.5 ± 0.6%) at 140 dpi. a decrease in mean corpuscular volume (mcv) was observed from the onset of infection until 7 dpi, after subcurative treatment with diminazene aceturate, thereafter returning to pre-infection levels (figure 4). mean corpuscular haemoglobin (mch) decreased 14 dpi from 23.5 to 22.5 pg, levelling off by 28 dpi. levels appeared stable after melarsoprol treatment (119 dpi), returning to pre-infection levels by 140 dpi (figure 5). figure 4: mean changes in red cell distribution width (rdw) and mean cell volume (mcv) of vervet monkeys infected with t. b. brucei gutat 1, showing the point of subcurative treatment with diminazene aceturate (da) (28 dpi) and curative treatment with melarsoprol (mel b) (119 dpi). figure 5: mean changes in mean corpuscular haemoglobin (mch) and mean corpuscular haemoglobin concentration (mchc) in vervet monkeys infected with t. b. brucei gutat 1, showing the point of subcurative treatment with diminazene aceturate (da) (28 dpi) and curative treatment with melarsoprol (mel b) (119 dpi). platelets platelet numbers declined after infection, reaching their lowest levels (58.5 × 10³ ± 0.15 cells/µl) at 21 dpi. levels increased after subcurative treatment with diminazene aceturate, returning to those of pre-infection by 56 dpi (figure 6). platelet counts decreased steadily for three weeks before trypanosome relapse in the blood (84 dpi), but stabilised to pre-infection levels (550 × 10³ cells/µl) after melarsoprol treatment (119 dpi). figure 6: changes in mean platelet numbers in vervet monkeys infected with t. b. brucei gutat 1, showing the point of subcurative treatment with diminazene aceturate (da) (28 dpi) and curative treatment with melarsoprol (mel b) (119 dpi). leucocytes in uninfected animals, the mean total wbc count ranged from 4.3 x 103 cells/μl to 5.7 x 103 cells/μl and did not change significantly throughout the experimental period (p < 0.05). . mean neutrophil counts ranged from 1.72 x 103 cells/µl to 2.12 x103 cells/µl, lymphocyte counts from 3.6 x103 cells/µl to 4.48 x 103 cells/ul and monocyte counts from 0.3 x 103 cells/µl to 0.26 x 103 cells/µl. none of these counts changed significantly throughout the experimental period (p < 0.05). in infected animals, total wbc counts declined during early stage infection (4.7 x 103 cells/µl to 2.5 x 103 cells/µl). after subcurative treatment with diminazene aceturate, wbc counts increased and were significantly higher at 84 dpi (7.7 x 103 ± 1.75 cell/µl, p < 0.05) than pre-infection levels (4.3 x 103 ± 1.18 cells/µl). thereafter, wbc counts declined to 3 x 103 cells/µl by 154 dpi. both lymphocyte and neutrophil counts followed a similar pattern (figure 7). changes in eosinophil and basophil counts were not significant during the course of the disease. monocyte counts dropped significantly from 0.2 x 103 cells/µl to 0.07 x 103 cells/µl between 0 and 14 dpi. they then peaked at 42 dpi (0.38 x 103 ± 0.04 cells/µl), after which there was a decline. a further increase in monocyte counts was noted, starting at 112 dpi with a peak at 126 dpi (0.3 x 103 ± 0.05 cells/µl) (figure 8). figure 7: mean changes in total white blood cells (wbc), lymphocyte and neutrophil counts of t. b. brucei gutat 1-infected vervet monkeys showing the point of subcurative treatment with diminazene aceturate (da) (28 dpi) and curative treatment with melarsoprol (mel b) (119 dpi). figure 8: mean changes in monocyte counts of t. b. brucei gutat 1-infected vervet monkeys, showing the point of subcurative treatment with diminazene aceturate (da) (28 dpi) and curative treatment with melarsoprol (mel b) (119 dpi). trustworthiness top ↑ to judge from the outcome of t. b. brucei infection in vervet monkeys, the observations obtained here appeared to compare well with those arising from t. b. rhodesiense and other related studies. reliability and validity of the research the experimental design of this study is reliable and valid. the procedures used in this research have been tested in other studies as cited in this article. discussion top ↑ this is the first study reporting the infection of vervet monkeys with t. b. brucei. human beings and some non-human primates have a trypanolytic factor, which prevents them from being infected with t. b. brucei and other livestock trypanosomes.13,14 results from this study suggest that vervet monkeys may lack trypanolytic factors, which have been reported to be present in other non-human primates, such as baboons and gorillas. the haptoglobin-related protein which has been demonstrated to have trypanolytic activity might be absent in the sera of vervet monkeys. in spite of the lack of human infection, t. b. brucei infections in rodents follow a similar pathogenesis to that of t. b. rhodesiense, thus rodents have been used as models for hat.8 some of the disadvantages of rodent hat models include phylogenetic distance and an inability to perform sequential monitoring of csf changes. these limitations can be addressed by using monkey models.in the current study, infected vervet monkeys displayed classical clinical symptoms, parasitological and haematological trends that were similar to monkeys infected with t. b. rhodesiense.15in addition, upon subcurative treatment with diminazene aceturate, disease relapse and re-emergence of blood parasitaemia took 85 days, which is similar to t. b. rhodesiense ketri 2537 infection.16 however, the duration was longer than that reported for t. b. rhodesiense ipr 001 infections.15 the differences noted may be due to variation in parasite virulence. anaemia gives a reliable indication of the disease status and productive performance of trypanosome-infected animals.17 disease in vervet monkeys tended to manifest with microcytic hypochromic anaemia during the early stages. this type of anaemia has been associated with iron deficiency that, in hat, might arise from lack of incorporation into red cell precursors – despite the presence of adequate iron storage – and inefficient recovery of iron from the phagocytosed erythrocytes.18,19 microcytic hypochromic anaemia has been described in vervet monkeys infected with t. b. rhodesiense.20 in this study, the severity of anaemia was greater in the acute stage of the disease (day 4 to 28 pi) than during the late stage (day 28 to 119 pi) and appeared to be associated with the level of parasitaemia. after curative treatment (119 dpi), all of the erythrocyte values recovered by 140 dpi because melarsoprol can clear parasites in all bodily compartments.21 there was a rapid decline in platelet counts in early-stage disease. low platelet counts could be indicative of toxic products, originating from the trypanosomes, which cause platelet destruction.18 splenic pooling of platelets and phagocytic removal of platelets by mononuclear cells has also been associated with thrombocytopaenia.19 severe progressive thrombocytopaenia has been reported in t. b. rhodesiense vervet monkey models and human cases of sleeping sickness.23,24 similarly to t. b. rhodesiense infections in vervet monkeys, lymphocytopoenia was noted in the early stage of the disease and lymphocytosis in the late stage.17 limitations of the study top ↑ only three animals were used in this study; however an extensive amount of data was obtained. parasitaemia was limited to the wet smear microscopic observation method only and, at very low parasitaemia, the method could have missed out early relapses. the characterisation of the model was also limited to haematological profiles and clinical examination. recommendations more extensive studies are needed to establish baseline levels of various biochemical parameters in vervet monkeys to aid in the determination of significant variations during infection/disease. haematological profiles need to be studied in shorter sampling intervals (e.g. daily sampling for the first week of infection) in order to discern properly any changes during the acute disease phase. conclusion top ↑ the clinical, parasitological and haematological observations obtained in this study compare well with those arising from t. b. rhodesiense; therefore, the vervet monkey t. b. brucei model may be used as substitute. this animal model may enable researchers and laboratory technicians to study hat without the high risk of accidental infection. acknowledgements top ↑ this project was funded jointly by the institute of primate research (ipr), jomo kenyatta university of agriculture and technology-research production and extension and foundation for innovative new diagnostics (find). we are grateful to the technical assistance provided by mr. ken waititu and mr tom adino of animal science department in institute of primate research. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions n.m. (jomo kenyatta university of agriculture and technology) was the project leader and in charge of animal welfare. j.k. (jomo kenyatta university of agriculture and technology) and n.m. were responsible for the experimental and project design. m.w. (jomo kenyatta university of agriculture and technology), k.j., n.m., s.k. (jomo kenyatta university of agriculture and technology) and b.g. (institute of primate research) performed the experiments, whilst m.w. and s.k. performed the data analysis. all the authors participated in the writing of the manuscript. references top ↑ 1. world health organization. trypanosomiasis, human african (sleeping sickness). fact sheet 259. geneva, switzerland: world health organization; 2006. 2. world health organization. african trypanosomiasis. world health organization fact sheet no.259; 2000, revised august 2006. http://www.who.int/mediacentre/factsheets/fs259/en/ 3. herwaldt bl. laboratory-acquired parasitic infections from accidental exposures. clin microbiol rev. 2001;14(4):659–688. http://dx.doi.org/10.1128/cmr.14.3.659-688.2001 4. robertson dhh, pickens s, lawson jh, et al. an accidental laboratory infection with african trypanosomes of a defined stock i. the clinical course of the infection. j infect. 1980;2(2):105–112. http://dx.doi.org/10.1016/s0163-4453(80)91084-1 5. herbert wj, parratt d, van meirvenne n, et al. an accidental laboratory infection with trypanosomes of a defined stock ii. studies on the serological response of the patient and the identity of the infecting organism. j infect. 1980;2(2):113–124. http://dx.doi.org/10.1016/s0163-4453(80)91109-3 6.6. keita m, bouteille b, enanga b, et al. trypanosoma brucei brucei: a long-term model of human african trypanosomiasis in mice, meningo-encephalitis, astrocytosis, and neurological disorders. exp parasitol. 1997;85(2):183–192. http://dx.doi.org/10.1006/expr.1996.4136 7. farah io, ngotho m, kariuki m, et al. animal models of tropical human diseases. in: j hau, g hoosier, editors. handbook of laboratory animal science, vol. iii. new york: crc press, 2005; pp. 169–224. 8. herbert wj, lumsden wh. trypanosoma brucei: a rapid ‘matching’ method for estimating the host’s parasitemia. exp parasitol. 1976;40(3):427–431. http://dx.doi.org/10.1016/0014-4894(76)90110-7 9. gould ss, sayer pd. a simple concentration technique for direct detection of trypanosomes in the cerebrospinal fluid of vervet monkeys, 17th isctrc meeting. in: proceedings of the international scientific council for trypanosomiasis research and control) (oau) arusha, tanzania, 1983; p. 259–260. 10. kagira jm, ngotho m, thuita jk, et al. hematological changes in vervet monkeys (chlorocebus aethiops) during eight months’ adaptation to captivity. amer j primatol. 2007;69(9):1053–1063. http://dx.doi.org/10.1002/ajp.20422 11. ngotho m, kagira jm, gaithuma ak, et al. a robust and improved monkey model of human african trypanosomiasis. in: proceedings of the xii international congress of parasitology melbourne (australia)august 15–20, 2010; p. 39–46. 12. clerinx j, vlieghe e, asselman v, et al. human african trypanosomiasis in a belgian traveller returning from the masai mara area, kenya, february 2012. euro surveill. 2012;17(10):pii=20111. available online: http://www.eurosurveillance.org/viewarticle.aspx?articleid=20111 13. gillett mp, owen js. comparison of the cytolytic effects in vitro on trypanosoma brucei brucei of plasma, high density lipoproteins, and apolipoprotein a-i from hosts both susceptible (cattle and sheep) and resistant (human and baboon) to infection. j lipid res. 1992;33(4):513–523. 14. pays e, vanhollebeke b. mutual self-defence: the trypanolytic factor story. microbes infect. 2008;10(9):985–989. http://dx.doi.org/10.1016/j.micinf.2008.07.020 15. gaithuma ak, karanja sm, ngotho m, et al. lipid metabolism and other metabolic changes in vervet monkeys experimentally infected with trypanosoma brucei rhodesiense. j med primatol. 2011;41(2):75–81. http://dx.doi.org/10.1111/j.1600-0684.2011.00523.x 16. ngotho m, maina n, kagira j, et al. il-10 is up regulated in early and transitional stage in vervet monkeys experimentally infected with trypanosoma brucei rhodesiense. parasitol int. 2006;55(4) 243–248. http://dx.doi.org/10.1016/j.parint.2006.06.004 17. ngotho m, kagira jm, kariuki c, et al. influence of trypanocidal therapy on the haematology of vervet monkeys experimentally infected with trypanosoma brucei rhodesiense. acta trop. 2011;119(1):14–18. http://dx.doi.org/10.1016/j.actatropica.2011.02.013 18. dow rb. the clinical and laboratory utility of platelet volume parameters. australia journal of medical science.1994;15:1–8. 19. robins-browne rm, schneider j, metz j. thrombocytopenia in trypanosomiasis. am j trop med hyg.1975;24(2):226–231. 20. kagira jm, thuita jk, ngotho m, et al. haematology of experimental trypanosoma brucei rhodesiense infection in vervet monkeys. afr j health sci. 2006;13(3–4):59–65. 21. egbe-nwiyi tn, igbokwe io, onyeiyili pa. the pathogenicity of diminazene aceturate-resistant trypanosoma brucei in rats after treatment with the drug. j comp pathol. 2003;128(2–3):188–191. http://dx.doi.org/10.1053/jcpa.2002.0599 22. umar ia, ogenyi e, okodaso d, et al. amelioration of anaemia and organ damage by combined intraperitoneal administration of vitamins a and c to trypanosoma brucei brucei-infected rats. afr j biotechnol. 2007;6(18):2083–2086. 23. thuita jk, kagira jm, mwangangi d, et al. trypanosoma brucei rhodesiense transmitted by a single tsetse fly bite in vervet monkeys as a model of human african trypanosomiasis. plos negl trop dis. 2008;2(5):e238. http://dx.doi.org/10.1371/journal.pntd.0000238 24. chisi je, misiri h, zverev y, et al. anaemia in human african trypanosomiasis caused by trypanosoma brucei rhodesiense. east afr med j. 2004;81(10):505–508. http://dx.doi.org/10.4314/eamj.v81i10.9232 article information authors: dianna edgil1 jason williams2 peter smith2 joel kuritsky1 affiliations: 1united states agency for international development (usaid), washington, united states 2the partnership for supply chain management (pfscm), arlington, united states correspondence to: dianna edgil postal address: 1300 pennsylvania ave. nw, washington dc, united states dates: received: 14 mar. 2013 accepted: 03 apr. 2014 published: 05 sept. 2014 how to cite this article: edgil d, williams j, smith p, kuritsky j. optimising the laboratory supply chain: the key to effective laboratory services. afr j lab med. 2014;3(1), art. #101, 7 pages. http://dx.doi.org/10.4102/ ajlm.v3i1.101 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. optimising the laboratory supply chain: the key to effective laboratory services in this original research... open access • abstract • introduction • research method and design    • pepfar procurement    • cd4 cost-per-test analysis    • country-level cost analysis • results    • pepfar procurement    • calculated cd4 cost-per-test analysis    • country-level calculated cost analysis       • countries a and b       • country c • discussion • limitations of the study    • recommendations • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: the supply chain management system (scms) is a contract managed under the partnership for supply chain management (pfscm) consortium by the united states agency for international development (usaid). scms procures commodities for programmes supported by the us president’s emergency plan for aids relief (pepfar). from 2005 to mid-2012, pepfar, through scms, spent approximately $384 million on non-pharmaceutical commodities. of this, an estimated $90m was used to purchase flow cytometry technology, largely for flow cytometry platforms and reagents.objectives: the purpose of this paper is to highlight the cost differences between low, medium and high utilisation rates of common cd4 testing instruments that have been procured though pepfar funding. method: a scale of costs per test as a function of test volume through the machine was calculated for the two most common cd4 testing machines used in hiv programmes: becton dickinson (bd) facscount™ and bd facscalibur™. instrument utilisation data collected at the facility level in three selected countries were then used to calculate the onsite cost-per-test experienced in each country. results: cost analyses indicated that a target of at least 40% utilisation for facscount™ and 15% utilisation for facscalibur™, respectively, closely approach maximal per-test cost efficiency. the average utilisation rate for cd4 testing instruments varies widely by country, level of laboratory and partner (0% − 68%). conclusion: our analysis indicates that, because cost-per-test is related inversely to sample throughput, the underutilisation of flow cytometry machines is resulting in an increase in average cost-per-test for many instruments. introduction top ↑ the us president’s emergency plan for aids relief (pepfar) has directed significant resources toward diagnosing and treating hiv in selected developing countries. in support of this programme, the united states government established a supply chain contract mechanism that is implemented by the partnership for supply chain management (pfscm) and managed by the united states agency for international development (usaid). the purpose of this contract is to provide technical assistance to host countries with procurement of pharmaceuticals, laboratory products and other items for country programmes.the global hiv community has recognised that increasing the efficiency of current programmes through better management, data-driven decision making and appropriate resource allocation are key to the continued scale-up of hiv programmes necessary for universal access to care and treatment.1,2 laboratory diagnostic services are a critical component of hiv programmes and are key to programmes’ ability to scale up treatment for hiv.3 building laboratory capacity requires resources and is accompanied by supply chain challenges.4,5 optimising laboratory procurement by using an evidence-based strategy to inform the procurement of laboratory instruments that are appropriate to the throughput (i.e., tier) of the laboratory in which they are to be placed, offers an opportunity to increase value for money6 and ensures that programmes can maximise the number of patients with access to reliable laboratory diagnostic services. the purpose of this article is to analyse costs associated with flow cytometry platforms by studying utilisation rates in three countries and comparing them to the maximum throughput recommended by manufacturers. the data will help inform country purchases and direct optimal deployment strategies of this technology. research method and design top ↑ pepfar procurement procurement data were collected from the pfscm orion procurement system. the orion system is pfscm’s integrated procurement system that controls, monitors and records all procurement activity for scms/pepfar. all figures represent the value of delivered commodities in us dollars ($) from september 2005 to june 2012. data are categorised into pharmaceuticals (pharma; 66%), rapid test kits (9%), analysers and/or reagents (13%), laboratory consumables (lab; 8%) and ‘other’ (4%). cd4 cost-per-test analysis we established a unit cost-per-test for the benton dickinson (bd) facscount™ and bd facscalibur™ instruments based on current prices paid by pfscm and manufacturer-established consumption amounts.7,8 consumption ratios were calculated for each product defined by the instrument manufacturer and end-user experiences (commodity list, consumption ratios and pricing in table 1). third-party control samples were not included in the overall pricing because of inconsistency in use. total costs were then calculated based on the product requirements needed to conduct cd4 testing over a one-day period. per-test costs were calculated using the following formula: table 1: cd4 count platform reagents and usage rates. cost-per-test = [(σ (unit quantity commodity cost/usage rate per test)a-e * tests per day) + daily control costf]/tests per day {eqn 1} estimated costs do not include product wastage, start-up and shutdown product consumption, or any additional equipment maintenance costs, which can vary considerably by end users and across countries. country-level cost analysis demographic, morbidity and service-statistics data were collected to inform multi-year (threeto five-year) laboratory instrumentation commodity requirements for three national quantification workshops in 2011 and 2012. instrument test numbers were gathered through data collection exercises at all laboratory sites in countries a and b and at a subset of sites in country c. data were provided by national laboratory leadership in each country, as well as pepfar implementing partners and united states government missions (usaid/us centers for disease control and prevention). where information on instrument service interruptions as a result of commodity stock-outs was available, diagnostic consumption was adjusted in order to account for a reduction in the number of days of operation. demographic and morbidity data, adjustments to test numbers associated with programme scale-up and general assumptions were documented. final forecast estimates were then validated through consensus at each quantification workshop. quantification outputs were used to develop first-year supply plans; to determine overall laboratory network commodity resource requirements, hiv diagnostics, care and treatment monitoring tests; and to prioritise laboratory spending when funding gaps were identified.to determine the overall efficiency of cd4 testing instruments within the laboratory system, cd4 facscount™ and facscalibur™ testing data were extracted from the quantification and forecast data gathered during the country’s first-year forecast period. for each country, the number of individual instruments and where they were located within each national laboratory network were determined. we compared the number of tests by instrument with the manufacturer-recommended average instrument throughput capacity (50 sample tests per day for facscount™ and 350 per day for facscalibur™) to determine instrument utilisation rates.7,8 the rates were not adjusted for instrument breakdowns because of a consistent lack of data in each represented country at the time of data review. diagnostic contribution was calculated by comparing individual machine throughput with total service provision estimates (service statistics), disaggregated by instrument type and level within the laboratory networks. for country c, we calculated product use to diagnostic contribution by the seven implementing partners using bd instruments (five partners using other brands were removed from the analysis). the total cd4 commodity cost was established for 2011 based on actual testing services provided during that year. the 2012 unit prices were calculated by projecting programmatic growth, planned instrument procurements and testing demand increases based on pepfar care and treatment targets. results top ↑ pepfar procurement as of june 30, 2012, pfscm assisted in procuring pharmaceuticals (antiretrovirals, treatment for opportunistic infections) and other products with a value of about $1.1 billion (figure 1). thirty-four per cent of pfscm’s overall commodity procurement, $384m, was spent on non-pharmaceutical products to supply pepfar-supported laboratories and testing sites in 53 countries. $150m of the non-pharmaceutical commodity amount was spent on reagents for analytical testing, with the majority ($90m) going to cd4 testing. procurement of analytical testing products, specifically for cd4 testing, has increased by almost 20% annually for the past five years and in 2011 accounted for 8% of all procurement (figure 2). figure 1: partnership for supply chain management (pfscm) total spending as of june 30, 2012. pfscm historical procurement data were used to determine the total expenditures for the supply chain management systems life of project (lop). all figures represent the value of delivered commodities in us dollars from september 2005 to june 2012. data are categorised into pharmaceuticals (pharma [66%], hiv rdts [9%], analysers and/or reagents [13%], lab/clinical supplies [8%] and other [4%]). figure 2: partnership for supply chain management (pfscm) spending on laboratory commodities delivered through june 2012. pfscm historical procurement data are displayed as expenditures per year from 2007 through june 2012. pepfar flow-cytometry expenditures show continual increases on a per-year basis. pepfar’s largest procurement of laboratory reagents during the period of september 2005 to june 2012 was for flow cytometry (64% of total reagent costs). bd products ($81m) represented 54% of the overall reagent costs and 90% of flow cytometry costs. for this reason, the two most commonly used bd flow cytometry platforms, facscount™ and facscalibur™, were chosen for further analysis in order to identify areas of cost savings or cost efficiency. calculated cd4 cost-per-test analysis we hypothesised that the cost for a test performed using these platforms would depend on volume, as was seen in a previous analysis.9 the cd4 cost-per-test analysis was limited to pfscm prices paid for the reagents required for the bd facscount™ and facscalibur™ platforms (table 1).using pfscm pricing for necessary testing commodities, we found that a higher throughput did result in a lower cost-per-test, with the rate of cost savings decreasing as the volume approached the manufacturer-recommended maximum throughput of the instrument (figure 3). the costs per test in this analysis were found to range from $14.64 to $7.29 for the facscount™ systems and $14.06 to $8.67 for the facscalibur™ system. we chose a rate of return of less than $0.01 per additional test per day as the point at which further investment in scale-up does not gain significant returns. whilst in both cases the rate of return diminishes to less than $0.01 per additional test added per day after a critical volume is achieved, for facscount™ the volume must exceed 40% (n > 20 tests) of the maximum daily throughput (50 tests), whereas for facscalibur™, the critical volume is approximately 15% (n > 45 tests) of maximum throughput (350 tests). figure 3: facscount™ versus faccalibur™ utilisation price per test in us dollars. the reagent unit cost-per-test for the bd facscount™ and bd facscalibur™ instruments was based on historical partnership for supply chain management (pfscm) pricing and manufacturer-established consumption amounts,7,8 using the reagents and consumption ratios in table 1. total costs were then calculated based on the product requirements needed to conduct cd4 testing over a one-day period. the rate of return diminishes to less than $0.01 per additional test added per day after a critical volume is achieved: facscount™, n > 20 tests or 40% throughput; facscalibur™, n > 45 tests or 15% throughput. several variable costs drive the cost-per-test of the bd systems, especially at low throughput volumes. in this analysis, the bd control and/or calibration kit is an important cost driver for both instruments in that unit pricing reductions are based on maximising use of the control kit by increasing the volume of tests processed per day. in comparison with the facscount™, the facscalibur™ has an initially lower baseline cost-per-test cd4 reagent (table 2). in fact, although the cost-per-test for both machines is similar at very low throughput, because of the lower baseline cost-per-test for facscalibur™ cd4 reagents, at extremely low volumes (n < 2) the facscalibur™ is slightly less expensive than the facscount™. however, as shown in figure 3, for volumes of more than two tests per day, the cost-per-test of the facscount™ system drops below that of the facscalibur™ system. the facscalibur™ system maintains a unit pricing per test at about $1.40 higher than that of the facscount™ as efficiency is gained. these results indicate that for all bd platforms, cost savings can be achieved by maximising daily testing volumes per machine with optimal targets of 40% throughput for facscount™ and 15% throughput for facscalibur™ systems. additionally and significantly, for volumes of tests of n > 2, the facscount™ system is the more cost efficient testing platform. table 2: facscount™ and facscalibur™ reagent unit price per test. country-level calculated cost analysis countries a and b to estimate potential cost savings that might be achieved by maximising throughput volumes, data from two countries were used to compare existing and recommended targeted throughput in each country. the cost-per-test as a function of throughput, as described above, was compared with actual utilisation rates collected from testing facilities in two pepfar countries in order to calculate the average cost-per-test of a cd4 test performed on facscount™ machines. for facscount™, country a (table 3) is shown to maintain an aggregated average instrument utilisation rate of 47%, which translates to an average cost-per-test of $7.46. country b (table 4) is shown to have an average facscount™ instrument utilisation rate of 10%, resulting in a calculated average cost of $8.64 per test. between countries a and b, the average cost-per-test difference for tests performed using a facscount™ system amounts to an estimated $1.18 in unit pricing overall. table 3: country a cd4 testing equipment utilisation. to identify areas in which the greatest cost efficiencies could be gained through increased utilisation of cd4 testing equipment within the tiered laboratory system, we performed a more targeted analysis of country b. in our review of the data for country b, we observed that laboratories in regional and provincial laboratories had 51 facscount™ machines. these sites had an utilisation rate of 9% (n < 5 tests per day), but contributed 30% of the total cd4 tests conducted for the country (table 4). costs in these laboratories averaged $8.83 per unit test. according to our analysis of cost-per-test as a function of instrument utilisation, country b would achieve a cost-per-test of $7.89 were it to increase its utilisation of these machines to 20%; a per-test savings of $0.94. moreover, were country b to increase utilisation to 40%, the target for maximising efficiency, the cost-per-test would be $7.51, a per-test savings of $1.32. extrapolating the savings accrued by country b for increasing cd4 equipment utilisation to 20% or 40% in regional and/or provincial laboratories as a percentage of the total budget spent on cd4 testing would reduce expenditures by 14% and 17%, respectively. overall, small increases in utilisation rates, when targeted to facilities with large diagnostic contribution to the total number of tests performed, can result in significant cost savings. table 4: country b cd4 testing equipment utilisation. country c in country c we calculated cd4 test costs by seven different implementing partners. targeting implementing partners with high cd4 testing contribution and seeking ways to increase utilisation rates may be one way to reduce testing costs and establish further commodity consumption efficiencies. the analysis of implementing partners in country c provided an opportunity to investigate implementing partner cd4 testing contributions for pepfar in 2011 and to examine projected growth into 2012. partners 2, 4 and 5 use facscount™ machines and contribute significantly to cd4 testing services within the pepfar country c programme for 2011 and 2012 (table 5). instrument utilisation rates for partners 2, 4 and 5 are, respectively, 30%, 4% and 43% in 2011 and 5%, 4% and 57% in 2012. the model for optimal instrument utilisation is partner 5, with a cost-per-test average of $7.49, which further gained efficiency in 2012 by increasing instrument utilisation to 57% with an average per-test cost of $7.40. in contrast, partner 4 had a throughput of 4% capacity in 2011 and 2012, with a calculated cost of $10.89 per test. table 5 indicates that the projected number of tests performed has increased substantially (by 13%), yet the percent utilisation remains unchanged. this indicates that, rather than increasing throughput on existing machines, additional machines have been procured by this partner such that an exceedingly high cost-per-test is maintained over time (10 new facscount™ instruments planned for 2012). given the extremely low throughput observed for partner 4, even a 2% increase in utilisation (an average of one more test per day) could reduce per-test costs by over $1 ($10.89 − $9.64). here, consolidating testing into existing machines to increase efficiency rather than procuring new equipment would result in significant cost savings over time. table 5: country c comparison of cd4 testing equipment utilisation in 2011 and 2012. for country c, product use and diagnostic contribution were disaggregated according to those implementing partners using bd instruments (five partners using other brands were removed from the analysis). total cd4 commodity costs for 2011 were based on actual testing services provided. 2012 unit prices were based on projected programmatic growth and planned instrument procurements. instrument utilisation rates for partners 2, 4 and 5 are, respectively, 30%, 4% and 43% in 2011 and 5%, 4% and 57% in 2012, indicating appropriate growth for partner 5, but a decrease or no increase in efficiency (reduced cost-per-test) for partners 2 and 4. similarly, in 2011 partner 2 operated with an average instrument utilisation of 30% and a cost-per-test of $7.64 (table 5). ideally, for 2012, partner 2 would increase instrument utilisation to 40% to gain efficiency and lower the cost-per-test for its cd4 testing programme. however, in this case, despite a significant projected increase in tests performed, instrument utilisation decreases to 5%, giving an average cost-per-test in 2012 of $10.27. this is an increase of $2.63 per test over 2011 costs. once again, the decrease in utilisation likely indicates the procurement of additional equipment (43 new facscount™ instruments planned for 2012) within a small testing pool that reduces the average volumes for all machines operated by partner 2. using the 2011 per-test cost ($7.64) to calculate the budget needed for partner 2 to perform the 55 798 tests projected for 2012 results in an estimated budget of $426 297 for reagent procurement. in this case, the decrease in utilisation and subsequent increase in per-test cost in 2012 (to $10.27) results in an actual budgetary requirement of $573 045. the underutilisation of cd4 testing equipment results in an additional $146 748 in reagent procurement needed to perform the same number of tests. these results indicate that efficient systems seeking to expand coverage must consider the cost implications of reducing volumes through existing equipment. discussion top ↑ our work in reviewing procurement and participation in laboratory commodity quantification exercises indicates that cost savings can be realised with better utilisation of cd4 testing instruments. in this analysis of national quantification exercises in three countries, we identify one area in which greater efficiency may be established by maximising cd4 instrument utilisation rates to reduce the cost-per-test of cd4 testing (represented by bd facscount™ and facscalibur™). these results indicate that for all bd platforms, cost savings can be achieved by maximising daily testing volumes per machine with optimal targets of 40% throughput for facscount™ and 15% throughput for facscalibur™ systems. this is particularly important for testing sites with large diagnostic contribution where small increases in utilisation rates can result in significant cost savings. for programmes seeking to expand coverage, acquisition of additional cd4 machines should consider the need to increase utilisation by consolidating testing into existing machines where existing referral networks are adequate.considering instrument placement before procurement, including accurate estimation of the appropriate demand at deployment locations, ultimately increases consumption efficiencies and reduces overall costs. to that end, it is particularly important to understand the cost implications of decentralising services to sites that underutilise instruments that contribute little to overall diagnostics. underutilising instruments that contribute less to the overall testing uptake has less of an impact on overall commodity costs, whereas underutilising instruments that have higher overall testing contributions has a higher impact on cost. for example, a facscount™ instrument operating at $9.00 per test that contributes to only 4% of the national testing target will have a lower impact on overall programme costs than if that same machine were contributing to 35% of the national testing targets. strategically relocating existing instruments could improve utilisation, as could replacing underutilised equipment with lower capacity point-of-care (poc) tests. it is important to either match site-level demand to the instrument (placing smaller instruments in low-volume clinics) or to place underutilised instruments into higher volume sites as back-up instruments in order to add redundancy in the event of equipment breakdown. planned expansion should first seek to efficiently utilise existing equipment and minimise the appropriation of tests from currently functioning sites. throughput of a selected cd4 testing machine should match testing consumption at the service delivery point. for certain facilities, diagnostics consumption falls below the optimal throughput volume for the more commonly-used cd4 platforms into the range of the low-throughput poc cd4 testing platforms, for which the cost-per-test is a flat rate from a commodity consumption perspective and does not vary with volume. in these cases, it may be more cost efficient to use a poc cd4 testing platform. this decision would require selecting a single supported poc instrument that is included in the national health laboratory strategic plan and strategically integrated into the tiered laboratory network to optimise existing instrumentation, balancing access to service. it should be noted that this analysis shows that for nearly all levels of throughput, the facscount™ platform is less expensive on a per-test basis than the facscalibur™ system. choosing to implement the facscalibur™, however, does have some benefit in very high-throughput facilities because it is fully automated and processes samples more quickly than the facscount™. thus, use of the facscalibur™ has the potential to reduce overall laboratory costs by allowing for higher testing volumes at service provision sites with less dependence upon laboratory staff time. whilst an acceptable level of instrument utilisation may appear to be achieved at the national level, for some countries disaggregating instrument utilisation for cd4 testing equipment by tiered level, region, or possibly by implementing partner, can assist in developing a more targeted intervention. for example, at the level of an individual implementing partner, diagnostic throughput may be very low, resulting in a high cost-per-test for testing sites managed by that implementing partner. for these partners, it may be useful to consolidate testing to existing equipment or to consider the suitability of new equipment for proposed expansion sites. further comparative analysis could potentially guide appropriate instrument deployment based on the site-specific demands, maximising partner cost efficiencies and further reducing the overall cd4 testing unit pricing scheme. limitations of the study top ↑ the study presented here has several limitations. the first is that the cost-per-test analysis considers only the cost of reagents. this likely results in an underestimation of the true cost-per-test, which would also include the cost of the equipment, human resources, service maintenance contracts and of expired and wasted product because of low equipment throughput and instrument breakdown. the second limitation to the analysis is a lack of information on efficiencies gained at very-low-volume sites by batching test samples collected over several days. very-low-volume sites using batch testing would at some level increase throughput and decrease the daily cost-per-test, perhaps significantly, at very low volume. finally, the analysis centres on bd cd4 testing platforms; whilst these are the most commonly-used cd4 testing platforms in pepfar-supported hiv testing programmes, other platforms with considerably lower reagent costs are used, albeit far less commonly, throughout africa. recommendations the 2008 maputo declaration on strengthening of laboratory systems6 called for harmonisation and standardisation of tests, reagents, consumables and instruments at each level within a tiered laboratory system. since that time, harmonisation has proven to be a challenge for many reasons, including evolving diagnostic coverage during scale-up, system maturation, existing procurement policies and changes in demographic and morbidity demands. nonetheless, in support of the maputo declaration, we must pursue a strategy of optimising laboratory procurement through informed decision making in order to advance harmonisation and maximise consumption efficiencies. such a strategy can advance harmonisation at all levels within the tiered system by using site-level data that informs the selection of equipment based on need or consumption at the point of use (consumption efficiencies); including the platform within the national testing algorithm, if one exists (hiv, tuberculosis and malaria); and understanding the sustainability of the platform within the regional setting (adequate infrastructure, reagent supply, maintenance and training curricula). the overall objective of optimising laboratory procurement is to develop instrument placement strategies that will increase appropriate coverage, increase overall commodity consumption efficiencies and ensure that instruments are operational, accounted for and, ultimately, maximise return on investment.as donor support and financial resources must stretch further to meet mounting global health needs, it is critical that potential savings be sought wherever possible. recognising the commitment and financial obligation required to support instrument life cycles amongst many competing priorities requires a broad perspective and regular re-evaluation of needs. in this way, translating historical laboratory procurement and quantification data into service performance indicators across all laboratory instrumentation and across different platforms can provide visibility into the critical operational aspects and create opportunities to establish further efficiencies of a laboratory network over time. product consumption and testing numbers can identify commodity wastage, supply chain management inefficiencies and the underutilisation of machines based on their potential testing capacity. armed with this information, those directing and managing laboratory networks are better equipped to develop responsive laboratory optimisation strategies that enable the best use of every donor dollar. conclusion top ↑ donors, implementing partners and host-country governments must make deliberate, transparent and coordinated efforts to advance evidence-based laboratory optimisation processes. countries must be positioned to take into account how best to balance costs, increase consumption efficiencies and ensure overall access to services. to realise further cost savings and continue to increase access to patient testing services, the development of laboratory optimisation strategies should serve as a critical step in further advancing overall healthcare delivery. fundamental to this strategy is ensuring a clear understanding of how effectively laboratory-related commodities are consumed in support of healthcare programmes. acknowledgements top ↑ we thank vincent wong for his thoughtful comments and sue carrington for her technical assistance.this research has been supported by the president’s emergency plan for aids relief (pepfar) through usaid under the terms of project number gpo-i-00-05-00032-00. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions the opinions of the authors of this article do not represent the opinions of usaid. the authors’ contributions are as follows: j.k. (usaid) was the project leader and d.e. (usaid) wrote the manuscript and was responsible for project design. j.w. and p.s. (both pfscm) collected and analysed the data (at country level and global expenditures, respectively) and made conceptual contributions regarding the optimisation of cd4 diagnostics in order to maximise return on investment on equipment procurement. references top ↑ 1.el-sadr wm, holmes cb, mugyenyi p, et al. scale-up of hiv treatment through pepfar: a historic public health achievement. j acquir immune defic syndr. 2012;60 suppl 3:s96–104. http://dx.doi.org/10.1097/qai.0b013e31825eb27b2.larson hj, bertozzi s, piot p. redesigning the aids response for long-term impact. bull world health organ. 2011;89(11):846–852. http://dx.doi.org/10.2471/blt.11.087114 3.nkengasong jn, nsubuga p, nwanyanwu o, et al. laboratory systems and services are critical in global health: time to end the neglect? am j clin pathol. 2010;134(3):368–373. http://dx.doi.org/10.1309/ajcpmpsinq9brmu6 4.thairu l, katzenstein d, israelski d. operational challenges in delivering cd4 diagnostics in sub-saharan africa. aids care. 2011;23(7):814–821. http://dx.doi.org/10.1080/09540121.2010.541416 5.birx d, de souza m, nkengasong jn. laboratory challenges in the scaling up of hiv, tb, and malaria programs: the interaction of health and laboratory systems, clinical research, and service delivery. am j clin pathol. 2009;131(6):849–851. http://dx.doi.org/10.1309/ajcpgh89qdswfons 6.the world health organization. the maputo declaration on strengthening of laboratory systems [document on the internet]. c2008 [cited 2013 mar 13]. available from: http://www.who.int/diagnostics_laboratory/maputo-declaration_2008.pdf 7.bd biosciences. bd facscount system: technical specifications [document on the internet]. c2009 [cited 2013 mar 13]. available from: http://www.bdbiosciences.com/external_files/is/doc/tds/instruments/live/web_enabled/23-6575-02.pdf 8.bd biosciences. bd facscalibur flow cytometry system: technical specifications [document on the internet]. c2010 [cited 2013 mar 13]. available from: http://www.bdbiosciences.com/documents/facscalibur_flowcytometry_techspec.pdf 9.jani iv. cost comparison of point-of-care and laboratory cd4 testing in resource-limited settings. presented at the 6th ias conference on hiv pathogenesis and treatment: 2011 july 17–20; rome, italy. abstract introduction methods results discussion acknowledgements references about the author(s) kafui c. kouassi unity of external quality assessement, division of laboratories, ministry of health and public hygiene, lomé, togo medical and biological analysis-biochemistry, higher school of biological and food techniques, university of lomé, lomé, togo améyo m. dorkenoo division of laboratories, ministry of health and public hygiene, lomé, togo department of health sciences, faculty of health sciences, university of lomé, lomé, togo komivi gbada division of laboratories, ministry of health and public hygiene, lomé, togo lomé commune regional hospital, ministry of health and public hygiene, lomé, togo yaovi-gameli afanyibo division of laboratories, ministry of health and public hygiene, lomé, togo national institute of hygene, ministry of health and public hygiene, lomé, togo minogblon têko division of laboratories, ministry of health and public hygiene, lomé, togo bè secondary hospital, ministry of health and public hygiene, lomé, togo adjane koura division of laboratories – resaolab, ministry of health and public hygiene, lomé, togo citation kouassi kc, dorkenoo am, gbada k, afanyibo y-g, têko m, koura a. the togo national proficiency test pilot programme for basic clinical chemistry tests. afr j lab med. 2022;11(1), a1565. https://doi.org/10.4102/ajlm.v11i1.1565 original research the togo national proficiency test pilot programme for basic clinical chemistry tests kafui c. kouassi, améyo m. dorkenoo, komivi gbada, yaovi-gameli afanyibo, minogblon têko, adjane koura received: 24 feb. 2021; accepted: 09 mar. 2022; published: 24 june 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: a national proficiency test (pt) programme is not currently implemented in most low-income countries. however, participation in such pt programmes assists improves test performance and result accuracy. objective: this study assessed how well 11 government hospital laboratories performed 18 basic clinical chemistry tests and identified areas needing improvement. methods: a cross-sectional study was carried out by the division of laboratories of the ministry of health of togo from 01 july 2016 to 31 december 2016. the test performance was evaluated using panels provided by one world accuracy, canada (vancouver). the clinical laboratory improvement amendments criteria were used in evaluating the laboratories, and their success rates were compared with the world health organization regional office for africa’s target of 80%. results: the overall rate of acceptable results at the laboratories was over 80% for glucose, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, alkaline phosphatase and triglycerides tests. the laboratories using fully automated spectrophotometers had an acceptable results rate of 89% (p = 0.001). the overall performance of the laboratories by cycles varied from 71% to 82%. conclusion: this national pt programme identified the tests, which laboratories must improve their performance (urea, creatinine, uric acid, bilirubin, cholesterol, total protein, calcium, magnesium, phosphorus). it demonstrated the need for the use of routine appropriate internal quality control in all laboratories. the proficiency test programme should be extended to all clinical laboratories and target all biology disciplines. keywords: quality control; biochemistry; laboratory; performance; togo. introduction clinical laboratory test results are for screening, diagnosis, prognosis, therapeutic monitoring of chronic pathologies and epidemiology surveillance.1 these results are more reliable when internal quality control (iqc) and external quality assessment (eqa) are implemented in the clinical laboratories. a proficiency test (pt) is one form of eqa that uses pre-established criteria to evaluate the performance of a participating laboratory (pl) compared with other laboratories.2 the inter-laboratory comparison, audit, and accreditation culture has not yet taken root in low-income countries, such as togo, with a gross national income per capita of $1035 or less. a 2013 survey reported that more than 90% of african countries had no accredited laboratories meeting the international organization for standardization standard 15189 (iso 15189), the international quality standard for clinical laboratories.3,4 clinical biochemistry tests remain predominant in the management of pathologies, whether resulting from transmissible or non-transmissible diseases.5,6 it is therefore essential that the results of these clinical chemistry tests are accurate. the participation of government hospital laboratories in a national pt programme as required by the iso 15189 standard could help to ensure accurate test results.7 in togo, the clinical diagnostics laboratories face many challenges, the most important of which are: obtaining market authorisation from distributors of in vitro diagnostic medical devices; implementing appropriate quality assurance processes, including iqc, and participating in a national or private eqa (pt) programme; and getting the iso 15189 accreditation (particularly for the national reference laboratories). since 2012, only three clinical laboratories in togo have been iso 15189:2012 accredited, two within the public health institute by the west african accreditation system and one private laboratory by the french accreditation committee.8,9 after a successful pt feasibility assessment in 2012 involving 11 clinical laboratories across the lomé municipalities, in 2016, the division of laboratories, ministry of health, togo, implemented a national pt programme.10 the resaolab project (west african network of clinical laboratories) implemented the pt programme, supported by fondation mérieux.11 in total, the health system of togo is composed of 179 government hospital laboratories organised in a tiered system, depending on their capabilities. a pilot phase of this programme was initiated the same year and involved 11 government hospital laboratories representing the central, intermediate and peripheral laboratory levels. the study aimed to assess how well these 11 government hospital laboratories performed 18 basic clinical chemistry tests and to identify areas where improvement may be required. methods ethical considerations this study did not involve human subjects or animal research. this pt programme is part of regular assessment activities of the ministry of health of togo and does not require any particular ethical consideration. a unique pt biological material was obtained from the canadian company one world accuracy and sent to all participating laboratories. the pt programme is part of the ministry of health’s regular assessment activities and does not require a particular ethical consideration. more so, the pt activity did not violate the current ethical considerations of the declaration of helsinki. study design a cross-sectional assessment was carried out from 01 july 2016 to 31 december 2016. the programme comprised four cycles at the rate of one cycle per month from august to november 2016. the study was conducted in three steps: (1) identification and training of participating laboratories (pls); (2) selection of tests, reception of samples at the central level, and dispatch of samples to pls; (3) collection of pt results and data evaluation. participating laboratories and personnel training eleven government hospital laboratories (representing 32% of 34 laboratories that routinely perform the 18 basic clinical chemistry tests), were enrolled in the pt programme. these 34 government hospital laboratories represent 19% of all the government hospital laboratories spread across the six health regions in togo. the pls were purposefully selected to ensure that all three levels of the health system were represented: three university hospital laboratories for the central level; six regional hospital laboratories for the intermediate level; and two district hospital laboratories for the peripheral level. these pls were randomly anonymised by numbering them 1–11. none of these pls had iso/international electrotechnical commission (iec) 15189 accreditation. a unique pt panel was obtained from oneworld accuracy® (owa) located in vancouver, british columbia, canada, and shipped by fedex® expedition services. following arrival and customs clearance, the package was immediately sent to the institut national d’hygiène, the national public health reference laboratory in togo, which is iso 15189 accredited. at institut national d’hygiène, sample integrity and adherence to temperature requirements (2 °c – 8 °c) were confirmed using a calibrated thermometer. these samples were stored for up to 24 h at 2 °c – 8 °c at the institut national d’hygiène and then sent to the pls for immediate testing upon reception. samples were transported refrigerated in individual insulated containers to each pl. the maximum transit time was 12 hours for the furthest pl. two laboratory technicians from each pl were trained by two members of the coordinating team of the national pt programme of the division of laboratories, ministry of health of togo. the training was provided by members with experience in the areas of quality management, statistical analysis, and biochemical analysis. the pl technicians were trained on pt principles under iso/iec 17043, iqc and the inter-laboratory comparison requirements in iso/iec 15189, and owa® pt pl guidelines for demonstration.2,7,12 training on the use of the oneworld accuracy system software platform (collaboration secretariat of oneworld accuracy group, vancouver, british columbia, canada), for setting measurement units, methods, equipment and test result submission, was also provided. laboratory tests and samples the 18 basic clinical chemistry tests and serum analytes selected by the ministry of health division of laboratories for this pt programme were: urea, blood glucose, creatinine, uric acid, alanine aminotransferase (enzyme commission [ec] number: 2.6.1.2 [international union of biochemistry and molecular biology, https://iubmb.qmul.ac.uk]), aspartate aminotransferase (ec 2.6.1.1), gamma-glutamyl transferase (ec 2.3.2.2), alkaline phosphatase (ec 3.1.3.1), total bilirubin, direct bilirubin, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, total protein, calcium, magnesium, and phosphorus. these tests were performed by the technicians of the 11 pls using commercially purchased reagents and iqc was used where available. analytical methods used for each test were recorded. the 11 pls used the same methods but not the same vendor kits for the following tests: urea (urease), blood glucose (glucose oxidase-hydrogen peroxide), creatinine (alkaline picrate), uric acid (uricase-hydrogen peroxide), alanine aminotransferase (international federation of clinical chemistry and laboratory medicine method without pyridoxal-5-phosphate cofactor), aspartate aminotransferase (international federation of clinical chemistry and laboratory medicine method without pyridoxal-5-phosphate cofactor), gamma-glutamyl transferase (carboxy–gamma-glutamyl-p-nitroanilide), alkaline phosphatase (p-nitrophenyl phosphate-diethanolamine), cholesterol (cholesterol oxidase-hydrogen peroxide), high-density lipoprotein cholesterol (phosphotungstic acid), low-density lipoprotein cholesterol (calculated by friedewald’s formula), triglycerides (glycerol phosphate oxidase-hydrogen peroxide), total protein (biuret), phosphorus (phosphomolybdic by ultraviolet spectrophotometry) and direct bilirubin (diazosulfanilic acid). for the three remaining tests (total bilirubin, calcium and magnesium), two different methods (a and b) were used by the pls (table 1). table 1: clinical chemistry tests with two different methods used in togo from july 2016 to december 2016. calibration traceability information was stated in the package inserts and all assays were traceable to an appropriate international reference standard or method. tests were performed by a fully automated or semi-automated spectrophotometer, depending on the analyser available in each pl (table 2). table 2: spectrophotometers and brands of analysers and reagents used by the participating laboratories in togo from july 2016 to december 2016. the sample analysed by each pl consisted of a lyophilised multiparametric serum supplied by owa. in the four cycles of this pilot programme, the pls received pt samples of different ranges in each cycle. five millilitre vials of philco water 5® brand distilled water (philco pharma carsten, grosshansdorf, germany) was provided for the reconstitution of samples using a volumetric micropipette. proficiency test data collection participating laboratories were instructed to test each sample in the same manner as patient samples. results were documented and sent to owa for analysis, with the final pt reports typically being received from owa within 15 days of sample receipt in togo. a whatsapp group (whatsapp llc, menlo park, california, united states) including all 11 pls and staff from the coordinating team was created to monitor the timely submission of results and pt reports. this whatsapp group was also used to discuss the implementation of corrective actions when a result was unacceptable or when a pl had issues with continuing the programme. the corrective actions were tracked using a ‘non-conformity management sheet’. data evaluation and performances criteria the pls were divided into two main groups to determine and compare their pt performance: one group of pls used fully automated spectrophotometers and the second group used semi-automated spectrophotometers and volumetric micropipettes. in addition, the performance of labs that used iqc was compared with that of labs that did not, evaluating the impact of iqc. the performance measurements included the overall performance of pls by cycle, the performance of the pls in each test across the four cycles, the performance of labs using automated or semi-automated spectrophotometers, and the adherence to the iqc process as identified through the whatsapp group discussions. the evaluation criteria were based on the clinical laboratory improvement amendments (clia) acceptable limits. this acceptable limit corresponded to the owa peer group mean ± (allowable total error) defined by clia.13 the total error of all the 18 tests was given in plus or minus percentage (± %) except for urea and calcium, which were expressed as an absolute value.2,14 the pt reports were qualified as acceptable when the results of the test provided by the pl were within the acceptability limits. for tests where there were no clia criteria, such as gamma-glutamyl transferase and direct bilirubin, owa used the peer group mean ± 2 s.d. (standard deviation) to determine the acceptability limits. allowable error rates were determined by owa for each test and stated on the pt reports for each pl during each cycle. statistical data analysis results of all pls were sent both to pls and directly to the ministry of health division of laboratories by owa in a microsoft excel file (microsoft, redmond, washington, united states), with the quantitative results of the pls identified as compliant or non-compliant. the pt results of each pl, with the assigned values of each test, were also checked by the pt coordinating team as recommended in the iso 13528 guideline.15 the data were collated and analysed using epi-info software version 3.5.3 (2011, centers for disease control and prevention, atlanta, georgia, united states). the calculation of the number of compliant results for a test or an identified group was used to determine the compliance rates in percentage (%). the performance rates of identified groups were compared using the uncorrected chi-square test or fisher’s exact test where appropriate. the same statistical method was used in comparing the number and percentage of acceptable results between two successive cycles. the target score of acceptable results was 80% as recommended by the world health organization regional office for africa.16 a cycle participation rate of 100% was expected from all pls. the p-value significance level was < 0.05. results laboratory participation rate each pl submitted results after performing all 18 tests. a cycle participation rate of 100% was obtained by nine pls (82%). the participation rate for the two remaining pls was 50% for pl4 and 25% for pl9. overall analytical performance of participating laboratories in performing tests seventy-six per cent of 775 results were acceptable. the performance scores for urea and direct bilirubin tests were less than 60%. the pls had a performance score above 80% for blood glucose, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase, alkaline phosphatase and triglycerides (figure 1). figure 1: analytical performance score of participating laboratories for each test performed in togo from july 2016 to december 2016. comparing the overall analytical performance between cycles assessing the overall performance level by cycle provides a measurement for the rate of improvement. the performance score increased from 71% to 81% (table 3). eight corrective actions were implemented at least once during the four cycles: providing a refrigerator thermometer to monitor the cold chain for better preservation of reagents and samples; planning a daily temperature measurement and performing root cause analysis for any observed deviations relating to out-of-range temperatures; purchasing new refrigerators suitable for laboratory use; calibrating micropipettes; performing periodic preventive maintenance on instruments and corrective maintenance when required; running iqc after each maintenance and re-calibrating instruments when necessary; implementing the use of iqc periodically; and analysing the results of the iqc using a levey-jennings chart. table 3: comparison of participating laboratories’ performance between cycles in togo from july 2016 to december 2016. comparing participating laboratories’ performance based on the use of internal quality control out of the 11 pls, four (36%) used control sera before performing each test, but none of the sites calculated uncertainty. during cycles 2, 3 and 4, the acceptable results rate of pls using iqc serum was significantly higher than for those not using iqc (table 4). table 4: comparison of participating laboratories’ performance based on the use of internal quality control in togo from july 2016 to december 2016. rate of acceptable results based on the spectrophotometer category five out of 11 pls used a fully automated spectrophotometer. three hundred and thirteen (89%) out of 352 results produced were acceptable, against 292 (69%) acceptable results (p = 0.001) in the six pls that used semi-automated spectrophotometers. discussion this study assessed how well 11 government hospital laboratories performed 18 basic clinical chemistry tests and identified areas needing improvement may be required. this assessment focused on the most common clinical chemistry laboratory tests and therefore a logical starting point for national pt activities.17 in togo, eqa initiatives have only been possible with external funding, and are currently available for tuberculosis, malaria and hiv testing.18 on 12 august 2015, the togo ministry of health issued ministerial decree n°115/2015/msps/cab/sg, which formally adopted iso/iec 15189:2012 as the quality management system standards to be met by every clinical laboratory in the country. article 4 of this decree stipulates that: ‘directors of government hospitals and heads of clinical laboratories are required to comply with the standard requirements laid down in iso 15189’. consequently, they must start planning and budgeting for pt activities as part of the national funding priorities to ensure a sustainable pt programme. planning and budgetting is important and should be improved for a sustainable national pt programme.5 the low participation rate of pl9 in this study was caused by the breakdown of their spectrophotometer. three months was insufficient for pl9 to perform the appropriate corrective action and to participate in subsequent cycles. in addition, pl4 suffered reagentstock-outs for six tests, resulting in low participation. the scenario demonstrates the challenge of pls in low-income countries to implement corrective actions in a timely fashion, even when root causes are identified.17,19 this challenge in the present pilot study could in part be mitigated by including at least a three months delay between pt cycles. insufficient delay between cycles could also explain the lack of significant improvement between cycles. implementing corrective maintenance and servicing actions for the spectrophotometers and other equipment needs a high level of commitment from the hospital authorities in charge of clinical laboratories, as recommended by iso/iec 15189 standards.7 this commitment should be manifested through the provision of resources to ensure the availability of reagents and equipment with efficient maintenance services.19,20 in addition, a quality management system to monitor overall laboratory quality, including equipment repair, maintenance and calibration, should be implemented as required by the iso/iec 15189 regulations.16,19 the routine use of iqc by pls will also enhance laboratory performance, as shown in this study. the use of iqc material at concentrations equal to or close to clinical decision values results in improved test performance and validates reported results.7 the laboratories should consider using independent third-party control materials in place of or in addition to reagent or instrument manufacturer iqc materials. the regular review of iqc data to identify acceptable and unacceptable results as well as trends is a good indicator for measuring laboratory performance, and helps to detect performance trends that may indicate problems in the analytical system.7,20 the study results showed that pls using semi-automated spectrophotometers obtained a significantly lower rate of acceptable results than those using fully automated systems. one likely source of this additional error with semi-automated spectrophotometers is the use of micropipettes to measure sera and reagents. these volumetric micropipettes were not calibrated by an iso/iec 17025 accredited laboratory and so may not be dispensing accurate volumes. measurement instruments should be subject to initial reference calibration and regular recalibrations to monitor performance.21 also, pls using semi-automated spectrophotometers performed poorly because they failed to maintain correct assay temperature or assay incubation period and used test tubes that may not have been washed properly.22 the lower performance in urea testing was also documented in a similar study in ethiopia that used the same owa pt panel as used in this study. the performance rate for urea testing in 12 ethiopian laboratories over six cycles was 21% lower than those obtained in the present study.23 the suboptimal performance of the pls for the urea test in this assessment could be attributed to the infrequent urea calibration when using different batches of reagents. also, the failure to maintain consistent assay temperature might have contributed to the poor urea testing performance.22 generally, proficiency testing evaluation is often done by a specific instrument group or analytical method used. the pls studied utilised multiple small instruments and multiple reagent kits that are not likely to fit into a specific peer group. this could lead to poorer pt performance for urea and other tests, because the mean method utilised for comparison may not be optimal.2,24 the low-performance scores for both total and direct bilirubin can be attributed to multiple factors, such as failure to maintain consistent assay temperature to which the diazo reaction is sensitive, failure to maintain reagent cold chain, and failure to protect calibrators and specimens from exposure to light, as bilirubin is light sensitive.25 limitations the major limitations of this study are the use of multiple instruments and reagent kits by pls. other limitations of the study include the few numbers of pls because of limited funds, and the insufficiently spaced pt cycles that did not allow for corrective actions to be implemented before the subsequent pt cycle. in a future study, the impact of the implementing iso/iec 15189 requirements on pls’ performance will be evaluated with the possibility of benchmarking between the central, intermediate and peripheral health levels. conclusion this study identified areas for improvement for a national pt programme and also demonstrated the value of such work in togo. it also identified some tests (urea, creatinine, uric acid, bilirubins, cholesterols, otal protein, calcium, magnesium, phosphorus) for which laboratories must improve their performance. it showed that the use of fully automated spectrophotometers is more likely to lead to reliable test results and demonstrated the need for the use of routine appropriate iqc in all laboratories. it emphasised the necessity to plan cycles with sufficient delay for implementing sustainable corrective actions. the national pt programme should be extended to all clinical laboratories in togo with three cycles per year and should also target all clinical laboratory disciplines. acknowledgements the authors would like to thank fondation mérieux and their partners for funding the resaolab project, togo’s ministry of health, dr katawa gnatoulma, and the members of participating laboratories. competing interests the authors declare that they have no financial or personal relationship that may have inappropriately influenced them in writing this article. authors’ contributions k.c.k. and a.m.d. conceived of the presented idea; k.c.k. developed methods and followed up the field activities; k.g. and m.t. verified the analytical methods and helped participating laboratories with results submission; y-g.a. wrote the manuscript with support from a.m.d. and k.c.k.; a.k. helped supervise the project under a.m.d.’s supervision. all authors discussed the results and contributed to the final manuscript. sources of support this work was supported by fondation mérieux and partners, who funded resaolab project (phase 2). data availability the authors confirm that the data supporting the findings of this study are available within the article. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references wians fh. clinical laboratory tests: which, why, and what do the results mean? lab med. 2009;40(2):105–113. https://doi.org/10.1309/lm4o4l0hhutwwudd international standardization organization. iso 17043:2010 – conformity assessment – general requirements for proficiency testing. genève: iso/iec; 2010. schroeder lf, amukele t. medical laboratories in sub-saharan africa that meet international quality standards. am j clin pathol. 2014;141:791–795. https://doi.org/10.1309/ajcpq5ktkagsscfn world bank country and lending groups [homepage on the internet]. [cited 2021 jun 29]. available from: https://datahelpdesk.worldbank.org/knowledgebase/articles/906519 fleming ka, naidoo m, wilson m, et al. essential pathology package for lowand middle-income countries. am j clin pathol. 2017;147(1):15–32. https://doi.org/10.1093/ajcp/aqw143 murray rk, kennelly pj, bender da, rodwell vw, botham km, weil pa. biochimie et medecine. 4th ed. bruxelles: de boeck: biochimie de harper; 2011 international standardization organization. iso 15189:2012 – medical laboratories – requirements for quality and competence. genève: iso/iec; 2012. cofrac. compétence le magazine de l’accréditation n°71 de 2017 [homepage on the internet]. [cited 2020 jul 13]. available from: https://www.cofrac.fr/fileadmin/user_upload/competences_71_0117.pdf waas. accredited conformity assessment bodies [homepage on the internet]. [cited 2020 jul 29]. available from: https://www.soacwaas.org/internal-documents.html kouassi k, fétéké l, assignon s, dorkenoo a, napo-koura g. external quality assessment in clinical biochemistry laboratories: pilot study in 11 laboratories of lomé. ann biol clin. 2015;73(2):165–175. https://doi.org/10.1684/abc.2015.1026 fondation mérieux. report of 4th steering commitee of resaolab at dakar [homepage on the internet]. 2017. [cited 2020 feb 04]. available from: https://www.resaolab.org/wp-content/uploads/2018/05/4e-resaolab-comite-de-pilotage-senegal-2017-compte-rendu.pdf one worldaccuracy. one world accuracy® pt participating laboratory guidelines for demonstration [homepage on the internet]. [cited 2018 oct 24]. available from: https://demo.oneworldaccuracy.com/healthmetrx/secure/home.do;jsessionid=cadb0995a1e394ec622cdc7d4186fe8a.node7?method=prepare westgard j. clia requirements for analytical quality [homepage on the internet]. westgard. [cited 2018 oct 25]. available from: https://www.westgard.com/clia.htm westgard jows. new clia proposed rules for acceptance limits for proficiency testing. [cited 2020 mar 02]. available from: https://www.westgard.com/2019-clia-changes.htm international standardization organization. iso 13528:2015 – statistical methods for use in proficiency testing by inter-laboratory comparison. geneva; 2015. world health organization regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region (slipta) [homepage on the internet]. brazzaville: who-afro; 2015 [cited 2020 mar 02]. available from: https://www.afro.who.int/sites/default/files/2017-06/slipta-checkist0711.pdf krleza jl, celap i, tanaskovic jv. external quality assessment in laboratory medicine: external quality assessment in croatia: problems, challenges, and specific circumstances. biochem med. 2017;27(1):86–92. https://doi.org/10.11613/bm.2017.011 carter jy. external quality assessment in laboratory medicine: external quality assessment in resource–limited countries. biochem med. 2017;27(1):97–109. https://doi.org/10.11613/bm.2017.013 john nn, katy y, philip o. laboratory medicine in low-income and middle-income countries: progress and challenges. lancet. 2018 391(10133):1873–1875. https://doi.org/10.1016/s0140-6736(18)30308-8 kaseha wm, manyilizu wb. variations on external quality assessment performance of public medical laboratories: the effect of strengthening laboratory management towards. acta sci med sci. 2019;33:1–10. https://doi.org/10.31080/asms.2019.03.0419 lothar s, robert iw, craig mj, linda t, stephane m. outline of the calibration and measurement hierarchy in laboratory medecine – quality policy and definitions wg2-p-00 [homepage on the internet]. [cited 2020 feb 13]. available from: https://www.bipm.org/en/committees/jc/jctlm-wg2/wg2_quality-manual.html bertrand s. biochimie clinique. instruments et techniques de laboratoire. diagnostics médico-chirugicaux. 2nd ed. paris: maloine; 1985. ashebir g, abay s. proficiency test feedback utilization at government hospitals laboratory, addis ababa, ethiopia. j med diagn methods. 2017;6:1–8. https://doi.org/10.4172/2168-9784.1000258 kristensen gbb, meijer p. external quality assessment in laboratory medicine: interpretation of eqa results and eqa-based trouble shooting. biochem med. 2017;27(1):49–62. https://doi.org/10.11613/bm.2017.007 westwood a. the analysis of bilirubin in serum. ann clin biochem. 1991;28(2):119–130. https://doi.org/10.1177/000456329102800202 conclusion acknowledgements references about the author(s) olayinka s. ilesanmi department of community medicine, college of medicine, university of ibadan, oyo state, nigeria department of community medicine, university college hospital, ibadan, oyo state, nigeria aanuoluwapo a. afolabi department of community medicine, college of medicine, university of ibadan, oyo state, nigeria citation ilesanmi os, afolabi aa. biobanking of covid-19 specimens during the pandemic: the need for enhanced biosafety. afr j lab med. 2021;10(1), a1379 https://doi.org/10.4102/ajlm.v10i1.1379 scientific letter biobanking of covid-19 specimens during the pandemic: the need for enhanced biosafety olayinka s. ilesanmi, aanuoluwapo a. afolabi received: 10 sept. 2020; accepted: 18 june 2021; published: 21 oct. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. dear editor, biobanking holds promising benefits particularly for improving the understanding of specific diseases and illnesses,1 as evidenced for the zika virus disease. research using biobanked blood samples helped resolve the ‘dengue-like syndrome’ misunderstanding associated with the zika virus. secondly, it provided comprehensive knowledge on the possibility for vertical transmission of the zika virus between mother and child, as well as transmission via sexual relationships and blood transfusions.2 biobanked biological samples could be kept for indefinite periods, allowing for long-term retrospective research in the future. the disease control opportunities that abound in biobanking can only be activated through efficient biobank structures. biobanks receive, process, store, and use available biological samples for research purposes to improve healthcare and reduce the incidence of disease occurrence. biobanks are essential elements in the epidemiological surveillance of diseases and are often integrated into normal laboratory practices. biobank staff are often trained on the rudiments of ethical guidelines for sample collection and handling and infection prevention and control measures. the efficiency of biobanks is influenced by both internal and external factors. internal factors could include the choice of specimen storage, information management, and communication technologies.4 external factors include ethical issues such as informed consent to use provided samples, conflict of interests of stakeholders (e.g. scientists, biobank administrators, sample donors, and commercial organisations), as well as commercialisation and profit distribution issues. inefficient handling and storage of specimens, information management systems, and communication modalities between sample handling and storage units of biobanks, as well as the commercialisation of samples, will reduce the efficiency of biobanks. this letter aimed to describe strategies for maintaining efficiency and safety in the coronavirus disease 2019 (covid-19) biobanks. safety guidelines have been developed by the governments of many countries for covid-19 biobanks.5,6 these guidelines mandate general laboratory practices and standard biosafety precautions but barely dwell on the sustenance of safety protocols.7 laboratory safety could be maintained by regular training of biobank staff on covid-19 guidelines and standard operating procedures, particularly specimen handling, risk assessments, emergency operating procedures, and overall safety precautions.8 this will ensure that laboratory managers and staff remain updated and maintain good laboratory practice. the use of personal protective equipment, such as disposable latex gloves, face shields, and laboratory gowns, is required for the safety of covid-19 biobank staff.8 while preventing staff infection, personal protective equipment also reduces the risk of specimen contamination. donning and doffing of personal protective equipment by biobank staff should be done in restricted areas, and proper handwashing when exiting the laboratory should be practised. individual covid-19 samples must be handled as biohazardous material to prevent the infection of biobank staff. when covid-19 samples are collected in designated laboratories, they are to be packaged and transferred to a biobank at 2 °c to –8 °c or frozen at –70 °c in a viral medium from the source laboratory8 and samples are to be transferred within 5 days of collection to a biobank. affiliation with the nearest biobank is advised to allow for prompt transfer of collected covid-19 samples. laboratory infrastructure may need to be reviewed to promote the efficiency of covid-19 biobanks. the decontamination of specimen containers should be done before removing each specimen from the safety cabinet. in biosafety level-2 laboratories, routine testing should be automated and limited to inactivated specimens.8 tasks involving the culture of severe acute respiratory syndrome coronavirus 2 must be carried out in biosafety level-3 laboratories.9 all examinations of covid-19 specimens should be conducted in a certified class ii biological safety cabinet with a high-efficiency particulate air filter, which should be used alongside a high-efficiency particulate air-filtered incubator for culture samples obtained from suspected covid-19 cases.9 inactivated specimens should be stored in biosafety level-2 or higher safety laboratories and sample integrity must be ensured. periodic internal and external quality assurance assessment of laboratories handling covid-19 biospecimens should be conducted to validate the generated results by biobanks. to achieve this, partnerships with public or private organisations with an excellent external quality assurance track record should be instituted. internal quality assurance could be commenced and promoted through the establishment of a quality assurance unit within each biobank. similarly, regular quality assurance training, reporting, and appraisal should be instituted. the implementation of biosecurity measures should be implemented at all stages from sampling to labelling, tracking, and handling of covid-19 specimens and results.7 due to the highly infectious nature of covid-19 samples, they could be targeted as weapons of bioterrorism, and biobank staff could be used to gain access to these samples. thus, regulations such as a material transfer agreement similar to those used in middle east respiratory syndrome coronavirus biobanks should be developed. this will guard against the use of collected covid-19 samples for personal, self-motivated purposes. also, ethical considerations such as privacy, fairness, and beneficence must be considered when sharing covid-19 specimen-related data among donors, researchers, and institutions. biobanked specimens could be used to conduct population-wide genetic sequencing research, which can identify severe acute respiratory syndrome coronavirus 2 mutation patterns among the population. presently, covid-19 vaccines such as moderna, astrazeneca, pfizer, and johnson & johnson are being rolled out globally.4 however, many countries have reported that these vaccines are inadequate to cover the variants circulating within their population.10 hence, prioritisation of certain populations may be needed during this period. therefore, biobanking could provide essential information to influence the distribution of covid-19 vaccines. conclusion biobanks provide an opportunity through which insights can be gained into the diagnosis of diseases. they are also important for providing long-term storage of biological samples. while carrying out their roles, biobank staff could be at risk of covid-19 infection. to prevent such occurrences, adherence to infection prevention and control measures should be promoted in biobanks. in addition, regular internal and external quality assurance should be ensured in all biobanks. to sustain biobanks, measures such as multisectoral collaboration are needed to ensure that the financial needs of biobanks are met. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions o.s.i. conceptualised the study. a.a.a. drafted the manuscript. all authors provided critical feedback and helped shape the manuscript. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support the authors received no financial support for the research, authorship, or publication of this article. data availability data sharing does not apply to this article, as no new data were created or analysed in this study. disclaimer the views expressed in this manuscript are those of the authors and are not an official position of any institution. references amin l, hashim h, mahadi z, ismail k. determinants of the willingness to participate in biobanking among malaysian stakeholders in the klang valley. mc med res methodol. 2018;18:163. https://doi.org/10.1186/s12874-018-0619-2 madhav n, oppenheim b, gallivan m, et al. pandemics: risks, impacts, and mitigation. in: jamison dt, gelband h, horton s, et al., editors. disease control priorities: improving health and reducing poverty. 3rd ed. [homepage on the internet]. washington, dc: the international bank for reconstruction and development/the world bank; 2017. chapter 17. available from: https://www.ncbi.nlm.nih.gov/books/nbk525302/ henderson ge, cadigan rj, edwards tp, et al. characterizing biobank organizations in the u.s.: results from a national survey. genome med. 2013;5(1):3. https://doi.org/10.1186/gm407 sargsyan k, jaksa b, hartl g, et al. risk management in biobanks. in: j. rocha, s. oliveira 7 c. capinha, editors, risk management and assessment. london: intechopen. https://doi.org/10.5772/intechopen.91463 peeling rw, boeras d, wilder-smith a, sall a, nkengasong j. need for sustainable biobanking networks for covid-19 and other diseases of epidemic potential. lancet infect dis. 2020;20(10):e268–e273. https://doi.org/10.1016/s1473-3099(20)30461-8 guidance covid-19: a safe handling and processing for samples in laboratories [homepage on the internet]. gov.uk (online). [cited 2020 sept 09]. available from: https://www.gov.uk/government/publications/wuhan-novel-coronavirus-guidance-for-clinical-diagnostic-laboratories/wuhan-novel-coronavirus-handling-and-processing-of-laboratory-specimens cloudlims. covid-19 clinical data management using lims [homepage on the internet]. 2020. [cited 2020 sept 10]. available from: https://www.cloudlims.com/blog/covid-19-clinical-data-management-using-lims-html who. laboratory testing for coronavirus disease (covid-19) in suspected human cases [homepage on the internet]. world health organization; 2020. [cited 2020 sept 09]. available from: https://apps.who.int/iris/handle/10665/331501 yang j-r, liu m-t, huang h-i, teng h-j, chen j-h, li s-y. building the national sars-cov-2 laboratory diagnostic capacity in taiwan. health secur. 2020;18(5):383–391. https://doi.org/10.1089/hs.2020.0056 scroll. in. coronavirus crises: covid-19: vaccine production and distribution has exposed – and intensified – global inequality [homepage on the internet]. [cited 2021 may 04]. available from: https://scroll.in/article/992328/covid-19-vaccine-production-and-distribution-has-exposed-and-intensified-global-inequality abstract introduction methods results discussion acknowledgements references about the author(s) bonifasius s. singu school of pharmacy, faculty of health sciences, university of namibia, windhoek, namibia helen morrison school of pharmacy, faculty of health sciences, university of namibia, windhoek, namibia lydia irengeya school of pharmacy, faculty of health sciences, university of namibia, windhoek, namibia roger k. verbeeck school of pharmacy, faculty of health sciences, university of namibia, windhoek, namibia citation singu bs, morrison h, irengeya l, verbeeck rk. therapeutic drug monitoring of phenytoin and valproic acid in critically ill patients at windhoek central hospital, namibia. afr j lab med. 2022;11(1), a1628. https://doi.org/10.4102/ajlm.v11i1.1628 original research therapeutic drug monitoring of phenytoin and valproic acid in critically ill patients at windhoek central hospital, namibia bonifasius s. singu, helen morrison, lydia irengeya, roger k. verbeeck received: 22 may 2021; accepted: 14 apr. 2022; published: 21 july 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: phenytoin and valproic acid, anticonvulsants, have a low therapeutic index and are highly plasma protein bound, mainly to albumin. hypoalbuminaemia is common in critically ill patients and increases the unbound drug concentration. thus, monitoring unbound rather than total plasma drug concentrations is recommended to optimise the dosing of these drugs. objective: this retrospective study determined unbound plasma concentrations of phenytoin and valproic as a more accurate value of drug levels than total plasma drug concentrations. methods: total plasma concentrations were retrieved for 56 intensive care unit patients for phenytoin and 93 for valproic acid. total drug concentrations were converted to unbound concentrations using a serum albumin-based normalising equation. results: total phenytoin plasma concentration was below (41.1% of patients), within (46.4%) or above (12.5%) the therapeutic range (10 μg/ml – 20 μg/ml). however, the predicted unbound plasma concentration of phenytoin was above the therapeutic range (1 μg/ml – 2 μg/ml) in the majority of patients (57.1%). for valproic acid, the total plasma concentration of most patients (87.1%) was below the therapeutic range (50 μg/ml – 100 μg/ml); among remaining patients (12.9%), it was within the therapeutic range. in the majority of patients (91.4%), the predicted unbound plasma concentration of valproic acid was between 2.5 μg/ml and 20 μg/ml. conclusion: the usefulness of monitoring the total phenytoin or valproic acid levels for dose optimisation is limited as it is an inaccurate indicator of a patient’s drug therapeutic state. thus, the unbound plasma drug concentrations should be quantified experimentally or predicted in resource-limited settings. keywords: phenytoin; valproic acid; critically ill patients; therapeutic drug monitoring; unbound concentration. introduction the incidence of seizures in north american general intensive care units (icus) was reported in 2013 to range from 3.3% to 34.0%.1 risk factors of seizures include head trauma, stroke, brain tumour, infections, hypoglycaemia, electrolyte abnormalities, and drug overdose. phenytoin and valproic acid are anticonvulsants frequently used to prophylax or treat seizures in critically ill patients.2,3 however, both drugs have a low therapeutic index; thus, drug monitoring is required to minimise the risk of toxicity and optimise therapeutic efficacy.4,5,6 following oral administration, both drugs are well-absorbed, highly bound to plasma albumin and eliminated by metabolism.2,4,5 the cytochrome p450 2c9 and 2c19 enzymes metabolise phenytoin to the inactive hydroxy-phenytoin, following first-order kinetics. however, at therapeutic concentrations, the rate of phenytoin metabolism approaches saturation and hence shows capacity-limited elimination (zero-order kinetics).5 metabolism of valproic acid proceeds mainly by glucuronidation (50.0%) and βand ω-oxidation (40.0%) and, to a much lesser extent, by cytochrome p450-catalysed oxidation (10.0%).6 the β-oxidation of valproic acid has been reported to be saturable and subject to autoinduction.6 the pharmacokinetic behaviour of phenytoin and valproic acid is complicated because their elimination kinetics are nonlinear. in addition, the pharmacokinetics of both drugs show a high degree of interindividual variability as a result of pharmacogenetic differences in enzyme and transporter activities, as well as high interpatient variability in plasma protein binding, in the case of valproic acid.7,8,9 as a result of this high interpatient variability, there is a poor correlation between the dose of these drugs and patient plasma concentrations. hence, monitoring patients’ phenytoin or valproic acid plasma concentrations is common, even in critically ill patients.2,7 phenytoin and valproic acid are highly bound to serum albumin (≥ 90%). the binding of these drugs depends on the drug plasma and albumin concentrations and the presence of other albumin-binding substances that may compete for binding sites with these drugs.10 thus, hypoalbuminaemia, a condition in which concentrations of the plasma protein albumin are lower than 35 g/l, increases the unbound proportion of these drugs because of the decreased albumin (binding sites) concentrations.11,12,13 hence, monitoring total plasma concentrations of phenytoin and valproic acid hypoalbuminaemia patients, such as critically ill patients, as the clinical indicator for therapeutic efficacy and safety, is not as reliable as utilising the unbound concentrations.14,15 since it is only the unbound drug that can cross biological membranes and cause pharmacological action through interaction with the drug target, the therapeutic range and dosage individualisation in hypoalbuminaemia patients should be informed by the unbound plasma drug concentrations to prevent toxicity or therapeutic failure.10,12,16,17 unbound plasma concentrations can be determined by equilibrium dialysis and ultrafiltration. however, the use of unbound plasma concentrations in clinical practice is limited because it is time consuming and costly. therefore, equations have been formulated to estimate the unbound concentrations of phenytoin and valproic acid using a patient’s measured total drug plasma concentration and serum albumin level.5,18,19,20,21,22 the therapeutic range for the total phenytoin plasma concentration is 10 μg/ml to 20 μg/ml, and 1 μg/ml to 2 μg/ml for the unbound phenytoin plasma concentration.4,5 for valproic acid, the therapeutic range for the total plasma drug concentration is 50 μg/ml to 100 μg/ml; for the unbound plasma concentration, a therapeutic range of 2.5 μg/ml to 20 μg/ml has been suggested.4,5 the windhoek central hospital in windhoek (namibia) conducts therapeutic phenytoin and valproic acid monitoring in critically ill patients in the icu facility by measuring only total plasma concentrations of the drugs. this study aimed to retrospectively estimate unbound plasma concentrations as a more accurate measure of the therapeutic plasma levels of two drugs. methods ethical considerations the research and ethics committees of the ministry of health and social services, the republic of namibia, approved this study; reference numbers hm2019 and lni2019. data were retrieved from patient record archives; no patients were required to participate in the data collection procedure of this study, and the ethics committee waived the requirement to obtain consent from patients before accessing their records. furthermore, names were excluded from the data collected for this study to protect patients’ privacy. patient population and data collection this retrospective analysis utilised data from the icu of windhoek central hospital, namibia. records of patients admitted between january 2013 and july 2019 were reviewed. patients who received phenytoin or valproic acid while at the icu and whose anticonvulsant plasma concentrations and serum albumin levels had been recorded were included in the study. the following information was extracted from the records: age, gender, diagnosis, total plasma concentrations of phenytoin or valproic acid, serum albumin concentration, and concomitant medication. serum albumin concentrations were measured at the namibia institute of pathology using the bromocresol purple method (cobas® 6000 analyser, roche diagnostics international, rotkreuz, switzerland). the total plasma concentrations of phenytoin and valproic acid were measured by fluorescence polarisation immunoassay (abbott architect i2000, abbott laboratories, chicago, illinois, united states). data analysis for each patient, the unbound plasma concentration of phenytoin (cu pht) was calculated based on the measured total plasma concentration of phenytoin (cp pht) and the serum albumin concentration (alb), in grams/decilitre, by using the original winter-tozer equation5 (equation 1). the therapeutic range for the total phenytoin plasma concentration is 10 μg/ml to 20 μg/ml, and 1 μg/ml to 2 μg/ml for the unbound phenytoin plasma concentration:4,5 the unbound plasma fraction of valproic acid (α vpa) was calculated based on the serum albumin concentration (alb) in μmol/l, by use of the hyperbolic equation proposed by parent et al.16,17 (equation 2): the unbound plasma concentration of valproic acid (cu vpa) was then estimated for each patient based on the measured total valproic acid plasma concentration (cp vpa) (equation 3). the therapeutic ranges were 50 μg/ml to 100 μg/ml for total valproic acid plasma concentration and 2.5 μg/ml to 20.0 μg/ml for unbound valproic acid plasma concentration:4,5 descriptive statistics were used to summarise the results. all statistical calculations were carried out using the excel software package of windows 10 (microsoft corporation, redmond, washington, united states). results in total, 1661 files of patients hospitalised between january 2013 and july 2019 at the icu of windhoek central hospital were reviewed. fifty-six patients, given phenytoin and 93 patients given valproic acid, who had data recorded for total plasma concentrations and serum albumin levels, were included in the study. patients were admitted to the icu for the following conditions: head trauma, hypoxic brain injury, infectious diseases such as meningitis and acute gastroenteritis, renal failure, and hepatic failure. the age of the patients ranged from two months to 66 years. the mean serum albumin levels were below 35 g/l in both sets of patients (mean ± s.d.: 23.5 ± 5.00 g/l for phenytoin, mean ± s.d.: 23.8 ± 5.9 g/l for valproic acid) (table 1). forty-five percent of patients on phenytoin were also receiving drugs known to interfere with its plasma binding or metabolism. for valproic acid, 28% of patients were concomitantly treated with drugs interfering with its plasma binding or metabolism. table 1: patient characteristics and total and predicted plasma concentrations of phenytoin and valproic acid in critically ill patients in the intensive care unit at windhoek central hospital (windhoek, namibia) from 2013 to 2019. the total plasma concentration of phenytoin recorded for 56 patients ranged from 0.1 μg/ml to 43.0 μg/ml, with an average of 12.6 ± 8.20 μg/ml (table 1). in 23 patients (41.1%), the phenytoin total plasma concentration was within the therapeutic range (10 μg/ml to 20 μg/ml) (figure 1). in 26 patients (46.4%), the plasma concentrations were subtherapeutic, that is, below 10 μg/ml, and in seven patients (12.5%) they were above 20 μg/ml. in 16 patients (28.6%), the predicted unbound plasma concentration of phenytoin was within the therapeutic range (1 μg/ml to 2 μg/ml). the unbound plasma concentration of phenytoin was subtherapeutic in eight patients (14.3%) and supratherapeutic in 32 patients (57.1%). figure 1: plasma concentrations of (a) total and (b) unbound phenytoin plasma concentration in 56 critically ill patients in the intensive care unit at windhoek central hospital (windhoek, namibia) from january 2013 to july 2019. the therapeutic range is indicated by dashed lines. for valproic acid, in 81 patients (87.1%), the total plasma concentration was below the therapeutic range of 50 μg/ml to 100 μg/ml (figure 2). binding of valproic acid to albumin in individual patients ranged from 9.0% to 39.0%. in 12 patients (12.9%), the total plasma concentration was within the therapeutic window, and no patients had a total plasma concentration of valproic acid above the therapeutic range. the average estimated unbound valproic acid plasma concentration was 24.0% (7.0% to 67.0%). in the majority of patients (n = 85, 91.4%), the unbound plasma concentration of valproic acid was within the therapeutic range (2.5 μg/ml to 20 μg/ml). in seven patients (7.5%), the unbound plasma concentration was below the therapeutic range, and in only one patient (1.1%) was it above the therapeutic range. figure 2: plasma concentrations of (a) total and (b) unbound valproic acid plasma concentration in 93 critically ill patients in the intensive care unit at windhoek central hospital (windhoek, namibia) from january 2013 to july 2019. the therapeutic range is indicated by dashed lines. the suggested therapeutic range for unbound valproic acid concentrations is quite wide because the lower and upper limits of therapeutic ranges reported in the literature differ substantially. discussion this retrospective study determined the plasma concentrations of phenytoin and valproic acid in critically ill patients at the icu of windhoek central hospital and found that the estimated unbound plasma concentration is a more accurate measure of the therapeutic plasma levels for the two drugs compared to total plasma concentration. using the total plasma concentration, most patients were within or below the phenytoin therapeutic range. in contrast, unbound plasma concentration showed that less than one-third of the patients were within the therapeutic range and over half were above. for valproic acid, total plasma concentrations placed most patients were below the therapeutic range. however, unbound plasma concentrations placed majority within the therapeutic range. noval et al. reported a 2-fold increase in critical care patients having phenytoin concentrations falling within the therapeutic range when unbound concentrations instead of total phenytoin concentrations were considered for dose adjustment.23 in our study, the percentage of patients within therapeutic concentrations using total plasma concentrations reduced from 41.1% to 28.6% when an unbound phenytoin concentration was used. thus, the use of total phenytoin concentrations suggests that dosing in these patients was at target therapeutic concentrations, suggesting no optimal patient dosage. the high variability in the binding of valproic acid to albumin observed in this study (9.0% – 39.0%) reflects reports in the literature, such as the 10.0% – 60.0% range reported in icu patients by lagneau et al.13 and the 15.0% – 89.0% range reported by riker et al.24 this high variability in plasma protein binding explains the marked difference in the percentage of patients having concentrations that were within the therapeutic range when total valproic acid was considered (12.9%) compared to when the unbound valproic acid concentrations were taken into account (91.4%). total valproate concentration is a poor predictor of the unbound drug concentration, even when correction is made for albumin.13 therapeutic drug monitoring of phenytoin and valproic acid by measuring unbound plasma concentrations is not widespread because of the additional time and cost implications. thus, some therapeutic drug monitoring services measure only the total drug plasma concentrations or estimate the unbound plasma drug concentrations. these equations, including those used in this study, estimate the unbound concentrations of phenytoin and valproic acid based on the patient’s measured total drug plasma concentration and serum albumin level.5,18,19,20,21,22 unfortunately, these equations are not very accurate and may underestimate the unbound plasma concentrations of phenytoin and valproic acid in critically ill patients.25,26,27,28,29,30 in addition, although the therapeutic plasma range for unbound phenytoin is relatively established (1 μg/ml – 2 μg/ml), various therapeutic ranges for the unbound plasma concentration of valproic acid have been proposed in the literature. however, more research is needed to determine the optimal unbound plasma concentration range for this anticonvulsant.31 furthermore, recent investigations on the predictive performance of these equations led to the conclusion that more complex, multivariate predictive equations may be required, which, in addition to the total drug plasma concentration and serum albumin level, considering the icu status of the patient, age, and blood urea nitrogen level.30,28 therapeutic drug monitoring of phenytoin and valproic acid should be based on unbound drug concentrations. the best way to monitor the unbound drug concentration would be by measuring it directly in ultrafiltrate obtained from serum to ensure that the accurate concentration of the free drug is determined and used to inform dosage adjustment. limitations the main limitation of this retrospective study is that the unbound plasma concentrations of phenytoin and valproic acid were not determined experimentally. however, because the albumin-based normalising equations that were used underestimate the patient’s unbound plasma concentrations of phenytoin and valproic acid, the discordance between total and unbound concentrations is less pronounced than it would be in reality. in addition, there was no follow-up to adjust the dosage regimen of these two drugs in the individual patients based on the observed total plasma concentrations. conclusion in conclusion, for therapeutic drug monitoring of phenytoin and valproic acid in critically ill patients, direct measurement of the unbound phenytoin and valproic acid concentration in plasma would be the best approach. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions b.s.s. supervised the study and reviewed the write-up of the manuscript. h.m. and l.i. collected the data and provided the draft manuscript. r.k.v. was the overall supervisor of the project and provided the final manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability data supporting the findings of this study are available from the corresponding author, b.s.s., on request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policiy or position of any affiliated agency of the authors. references varelas pn, spanaki mv, mirski ma. seizures and the neurosurgical intensive care unit. neurosurg clin n am. 2013;24(3):393–406. https://doi.org/10.1016/j.nec.2013.03.005 farrokh s, tahsili-fahadan p, ritzl ek, lewin iii jj, mirski ma. antiepileptic drugs in clinically ill patients. crit care. 2018;22(1):153. https://doi.org/10.1186/s13054-018-2066-1 allen b, vespa pm. antiseizure medications in critical care: an update. curr opin crit care. 2019;25(2):117–125. https://doi.org/10.1097/mcc.0000000000000587 garnet wr, anderson gd, collins rj. antiepileptic drugs. in: burton me, shaw lm, schentag jj, evans we, editors. applied pharmacokinetics & pharmacodynamics: principles of therapeutic drug monitoring. 4th ed. philadelphia, pa: lippincott williams & wilkins; 2006, pp. 491–511. winter me, tozer tn. phenytoin. in: burton me, shaw lm, schentag jj, evans we, editors. applied pharmacokinetics & pharmacodynamics: principles of therapeutic drug monitoring. 4th ed. philadelphia, pa: lippincott williams & wilkins; 2006. pp. 463–490. ghodke-puranic y, thorn cf, lamba jk, et al. valproic acid pathways: pharmacokinetics and pharmacodynamics. pharmacogenet genomics. 2013;23(4):236–241. https://doi.org/10.1097/fpc.0b013e32835ea0b2 patsalos pn, berry dj, bourgeois bf, et al. antiepileptic drugs – best practice guidelines for therapeutic drug monitoring: a position paper by the subcommission on therapeutic drug monitoring, ilae commission on therapeutic strategies. epilepsia. 2008;49(7):1239–1276. https://doi.org/10.1111/j.1528-1167.2008.01561.x thorn cf, whirl-carillo m, leeder js, klein te, altman rb. pharmgkb summary: phenytoin pathway. pharmacogenet genomics. 2012;22(6):466–470. https://doi.org/10.1097/fpc.0b013e32834aeedb gomez bellver mj, garcia sanchez mj, alonso gonzales ac, santos buelga c, dominguez-gil a. plasma protein binding kinetics of valproic acid over a broad dosage range: therapeutic implications. j clin pharm ther. 1993;18(3):191–197. https://doi.org/10.1111/j.1365-2710.1993.tb00612.x rowland m. plasma protein binding and therapeutic drug monitoring. ther drug monit. 1980; 2(1):29–37. https://doi.org/10.1097/00007691-198001000-00005 wiedermann cj. hypoalbuminemia as surrogate and culprit of infections. int j mol sci. 2021;22(9):4496. https://doi.org/10.3390/ijms22094496 von winckelmann sl, spriet i, willems l. therapeutic drug monitoring of phenytoin in critically ill patients. pharmacotherapy. 2008;28(11):1391–1400. https://doi.org/10.1592/phco.28.11.1391 riker rr, gagnon dj, hatton c, et al. valproate protein binding is highly variable in icu patients and not predicted by total serum concentrations: a case series and literature review. pharmacotherapy. 2017;37(4):500–508. https://doi.org/10.1002/phar.1912 perucca e. plasma protein binding of phenytoin in health and disease: relevance to therapeutic drug monitoring. ther drug monit. 1980;2(4):331344. https://doi.org/10.1097/00007691-198010000-00005 hatton c, riker rr, gagnon dj, may t, seder db, fraser gl. free serum valproate concentration more reliable than total concentration in critically ill patients. resuscitation. 2016;10:e15e16. https://doi.org/10.1016/j.resuscitation.2016.05.027 heuberger j, schmidt s, derendorf h. when is protein binding important? j pharm sci. 2013;102(9):3458–3467. https://doi.org/10.1002/jps.23559 lenn nj, robertson m. clinical utility of unbound antiepileptic drug blood levels in the management of epilepsy. neurology. 1992;42(5):988–990. https://doi.org/10.1212/wnl.42.5.988 parent x, marzullo c, gutbub am. acide valproique: estimation simple de la concentration sérique libre. ann biol clin (paris). 1993;51(6):649–650. hermida j, tutor jc. a theoretical method for normalising total serum valproic acid concentration in hypoalbuminemic patients. j pharmacol sci. 2005;97(4):489–493. https://doi.org/10.1254/jphs.fpe04007x sheiner lb, tozer tn. clinical pharmacokinetics: the use of plasma concentrations. in: melmon kl, morelli hf, editors. clinical pharmacology: basic principles in therapeutics. new york, ny: macmillan; 1978, p. 1978. anderson gd, pak c, doane kw, et al. revised winter-tozer equation for normalised phenytoin concentrations in trauma and elderly patients with hypoalbuminemia. ann pharmacother. 1997;31(3):279–284. https://doi.org/10.1177/106002809703100301 kane sp, bress ap, tesoro ep. characterisation of unbound phenytoin concentrations in neurointensive care unit patients using a revised winter-tozer equation. ann pharmacother. 2013;47(5):628–636. https://doi.org/10.1345/aph.1r651 noval m, seung h, armahizer m. evaluation of fosphenytoin therapeutic drug monitoring in the neurocritical care unit. drugs r d. 2020;20(1):17–22. https://doi.org/10.1007/s40268-019-00292-1 lagneau f, perbet s, delefosse d, et al. drugs pharmacokinetics in icu patients: consequences of hypoalbuminemia upon drugs monitoring and dosing scheme. intensive care med 2004;30:1247. https://doi.org/10.1007/s00134-004-2313-6 smetana ks, cook am, thompson bastin ml, oyler dr. antiepileptic dosing for critically ill adult patients receiving renal replacement therapy. j crit care. 2016;36:116–124. https://doi.org/10.1016/j.jcrc.2016.06.023 wolf gh, mcclain cd, zurakowski d, dodson b, mcmanus ml. total phenytoin concentrations do not accurately predict free phenytoin concentrations in critically ill children. pediatr crit care med. 2006;7(5):434–439. https://doi.org/10.1097/01.pcc.0000235252.43921.de cheng w, kiang tkl, bring p, ensom mhh. predictive performance of the winter-tozer and derivative equations for estimating free phenytoin concentration. can j hosp pharm. 2016;69(4):269–279. https://doi.org/10.4212/cjhp.v69i4.1573 kiang tkl, ensom mhh. a comprehensive review of the predictive performance of the sheiner-tozer and derivative equations for the correction of phenytoin concentrations. ann pharmacother. 2016;50(4):311–325. https://doi.org/10.1177/1060028016628166 javadi s-s, mahjub r, taher a, mohammadi y, mehrpooya m. correlation between measured and calculated free phenytoin serum concentration in neurointensive care patients with hypoalbuminemia. clin pharmacol. 2018;10:183–190. https://doi.org/10.2147/cpaa.s186322 barra me, phillips km, chung dy, rosenthal es. a novel correction equation avoids high-magnitude errors in interpreting therapeutic drug monitoring of phenytoin in critically ill patients. ther drug monit. 2020;42(4):6-17–25. https://doi.org/10.1097/ftd.0000000000000739 tseng y-j, huang s-y, kuo c-h, wang c-y, wang k-c, wu c-c. safety range of free valproic acid serum concentration in adult patients. plos one. 2020;15(9):e0238201. https://doi.org/10.1371/journal.pone.0238201. abstract introduction methods results discussion acknowledgements references about the author(s) victor n. fondoh bamenda regional hospital laboratory, regional hospital bamenda, cameroon department of medical laboratory sciences, school of health and medical sciences, catholic university of cameroon, bamenda, cameroon department of health economics policy and management, faculty of business management, catholic university of cameroon, bamenda, cameroon nobert ndzenjempuh department of medical laboratory sciences, school of health and medical sciences, catholic university of cameroon, bamenda, cameroon tamunjoh stella department of medical laboratory sciences, school of health and medical sciences, catholic university of cameroon, bamenda, cameroon richard m. fondoh north-west regional fund for health promotion, bamenda, cameroon charles n. awasom department of anatomy, school of health and medical science, catholic university of cameroon, bamenda, cameroon rebecca enow-tanjong department of medical laboratory sciences, school of health and medical sciences, catholic university of cameroon, bamenda, cameroon egbe p. egbengu department of medicine and surgery, school of health and medical science, catholic university of cameroon, bamenda, cameroon robert leke department of medicine and surgery, school of health and medical science, catholic university of cameroon, bamenda, cameroon njini f.n. rose regional hospital bamenda, bamenda, cameroon denis nsame regional hospital bamenda, bamenda, cameroon citation fondoh vn, ndzenjempuh n, stella t, et al. prevalence of alpha and beta haemolysin among blood group o donors in bamenda, cameroon. afr j lab med. 2022;11(1), a1432. https://doi.org/10.4102/ajlm.v11i1.1432 original research prevalence of alpha and beta haemolysin among blood group o donors in bamenda, cameroon victor n. fondoh, nobert ndzenjempuh, tamunjoh stella, richard m. fondoh, charles n. awasom, rebecca enow-tanjong, egbe p. egbengu, robert leke, njini f.n. rose, denis nsame received: 25 oct. 2020; accepted: 03 feb. 2022; published: 19 apr. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the occurrence of high titres of alpha (anti-a) and beta (anti-b) haemolysin immunoglobulin g antibodies in blood causes haemolysis during blood transfusion from a group o donor, commonly and inappropriately known as the ‘universal blood donor’, to a group a, b or ab recipient. surprisingly, haemolysin testing is not routinely done during blood transfusion services in bamenda, cameroon. objective: this study aimed to determine the prevalence of haemolysin among blood group ‘o’ donors at the regional hospital bamenda blood bank, bamenda, cameroon. methods: this was a cross-sectional descriptive study carried out between june and september 2020 at the regional hospital bamenda blood bank, bamenda, cameroon. blood group o donors who were free from transfusion-transmissible infections were selected systematically and serially and their serum tested for the presence of haemolysin. haemolysin titres were determined, and titres ≥ 8 were considered significant. the associations between haemolysin prevalence and age group, gender and rhesus d blood group were determined using the chi-square test. results: the prevalence of haemolysin among the 480 study participants was 52.1% and significant haemolysin titres were detected in 18.5%. there was no association between haemolysin and gender, age group or the rhesus d blood group. conclusion: the prevalence of significant titres of haemolysin among participants in this study was high. there is the need to test for haemolysin in blood group o donors to prevent the potential risk to blood group a, b, and ab recipients and to provide safer blood for transfusion. keywords: prevalence; haemolysin; immunoglobulin; blood group o; donors; bamenda; cameroon. introduction the occurrence of alpha (anti-a) and beta (anti-b) immunoglobulin m antibodies in the absence of corresponding red blood cell antigens is a significant feature of the abo blood group system in individuals.1 when blood transfusion is done without consideration for abo compatibility, these naturally occurring antibodies are a potential cause of dangerous haemolysis in recipients.2 although these antibodies react optimally at 4 °c, they may also cause haemolysis at 37 °c. also, some blood group o, and sometimes blood group a2, individuals may develop alpha and beta antibodies of the immunoglobulin g class that react optimally at 37 °c. these antibodies are commonly referred to as haemolysins and are more dangerous compared to naturally occurring haemolysins.2 in cases of non-iso group-compatible abo transfusion, these antibodies can trigger the complete complement cascade, leading to haemolytic reactions.2 several studies have reported complications in patients who were transfused with non-identical blood groups, including disseminated intravascular coagulation,3,4 hepatic and renal failure leading to death,3,5 paleness, jaundice, fever, ecchymosis and generalised exfoliation of the skin,6 significant reduction in packed cell volume,7 hyperbilirubinaemia,8 varying degrees of haemoglobinaemia, and intravascular agglutination.9 several studies have advocated that the transfusion of group o blood to a, b, or ab recipients be discontinued due to the high prevalence of alpha and beta haemolysins among blood group o donors.10,11,12 despite this concern, the practice is yet to be discontinued. several studies have postulated that these alpha and beta antibodies originated as products of immunisation from allogenic stimulation (due to transfer of antigen by red cells from pregnancy with an abo-incompatible foetus, incompatible blood transfusion, tissue transplant, etc.) and heterogeneous stimulation (due to vaccination, serotherapy, and inoculation from vectors such as blood-sucking insects and certain pharmaceutical preparations contaminated with alphaand beta-like antigens, etc.).13,14,15 due to the high demand for and shortage of allogenic blood in most developing countries, including cameroon, as well as the relative difficulty in getting abo group-compatible blood during emergencies, blood group o, which is often inappropriately referred to as the ‘universal blood donor’, has been increasingly transfused to non-group o recipients.16 besides, there is evidence that blood group o is the most abundant group in the abo blood system, with a prevalence of 51.1% reported in a study in cameroon.17 blood transfusion is an essential medical practice that replenishes lost blood or blood products in the recipient. the transfused blood should be as safe as possible to ensure maximum benefit to the recipients. thus, there is the need to strictly adhere to standard screening protocols to achieve safe blood transfusion. unfortunately, this practice is limited in the developing world due to inefficient blood banking systems and scarcity of screening facilities. besides, the standard protocol for compatibility testing to ensure safe blood has been omitted or abbreviated by many screening services. this standard compatibility testing protocol18 requires that all group o blood intended for transfusion to group a, b, or ab recipients should be screened for the presence of high titres of alpha and beta haemolysins and that only haemolysin-free blood should be reserved for blood group a, b, or ab recipients, while haemolysin-positive blood should be reserved for group o recipients only.13 several countries, including côte d’ivoire, have included a haemolysin test as part of the standard protocol in their national blood transfusion programmes.18 surprisingly, this practice is yet to be implemented in blood bank facilities in bamenda or cameroon in general. hence, this study aimed to determine and demonstrate the presence of significant titres of alpha and beta haemolysins among blood group o donors at the regional hospital bamenda blood bank (rhbbb), bamenda, cameroon, to guide the implementation of a policy to include haemolysin testing in the protocol for compatibility testing in cameroon. methods ethical considerations administrative authorisation to carry out this work was provided by the catholic university of cameroon, bamenda, north-west regional delegation of public health, bamenda, and regional hospital bamenda. ethical clearance was provided by the institutional review board of the regional hospital bamenda (irb number: 211/app/rdph/rhb/irb). participants provided written informed consent and were free to withdraw from the study at any time. the participants’ data were coded by assigning numbers to identify the participants instead of names. the anonymity of participants and their data were ensured by storing the data on password-protected computers and in locked file cabinets accessible only to the study staff and researchers. study area this research was carried out in bamenda at the rhbbb, bamenda, a unit at the regional hospital bamenda. the rhbbb receives approximately 5400 blood donors and issues about 4200 safe pints of blood yearly. it has a standard blood transfusion service and is enrolled in a certification programme with the safe blood for africa foundation. it is also the largest blood transfusion centre in the north-west region and provides transfusion services to the region and beyond. research design this was a descriptive, cross-sectional study conducted between june 2020 and september 2020 at the rhbbb. the sample size was calculated based on a previous study conducted in 2011 in eastern nigeria16 that reported an overall haemolysin prevalence of 55.4%. a minimum of 385 participants was required for this study. blood donors arriving at the reception area of the rhbbb undergo routine screening to determine physical fitness to donate blood using a standard questionnaire validated and provided by the rhbbb quality team. this routine screening selects individuals who had not donated blood and had no history of sexually transmitted diseases in the three months preceding the blood donation, were free from non-communicable diseases such as diabetes and hypertension, had not been vaccinated in the last four months, had not taken medication for at least one week, and had not smoked on the day of the donation or taken alcohol in the last 24 h. women who were pregnant, breastfeeding, or menstruating or expecting their menses within one week were excluded. in addition, only donors who weighed greater than 50 kg, were between the ages of 18 and 60 years (women) or 18 and 65 years (men) and had blood pressures between 100 mmhg and 140 mmhg over 60 mmhg – 100 mmhg and temperatures between 36 °c and 37.5 °c were endorsed as fit for blood donation. as part of the routine protocol for screening donors to obtain safe blood in the blood bank, abo and rhesus d blood group tests and transfusion transmittable infection (tti) tests were done on samples from all donors. abo and rhesus blood groups were determined using the procedure described by dacie and lewis19 using the blood in the ethylenediaminetetraacetic acid tube. the tti test was done using the blood in the plain tube. tti testing included the following: hiv test using the hiv-1/2 ag/ab combo determine (alere medical co., ltd, matsuhidai, matsudo-shi, chiba-ken, japan) as the first-line test and oraquick (orasure technologies, inc., bethlehem, pennsylvania, united states) as the second-line test; hepatitis b and hepatitis c virus tests using the diaspot diagnostic kit (diaspot diagnostics, jawa barat, indonesia); syphilis test using the rapid plasma reagin (rpr)-carbon slide agglutination assay (cypress diagnostics, langdorp, belgium) and treponema pallidum haemagglutination assay (omega diagnostic, alva, scotland, united kingdom); and malaria test using the carestartth malaria pf/pan (hrp2/pldh)ag combo rdt (accessbio, somerset, new jersey, united states). as part of the donation process, blood samples were collected into two tubes – one in a plain tube and the other in an ethylenediaminetetraacetic acid tube from donors already screened using the questionnaire as fit for blood donation. the screened donors were systematically and serially contacted to participate in the study. only donors who were blood group o, free from all the ttis, and who consented to be part of the study were included. a standard data collection format was used to collect information on the age, blood groups and gender of the study participants. haemolysin test and titration blood specimens collected in the plain tubes (used for tti screening) were used to determine haemolysin titres within 24 h of specimen collection. briefly, the sample was allowed to clot for about 45 min and then centrifuged to separate the serum. the serum was then tested for the presence of haemolysins.11,16 zero point 5 mililetres of the serum was placed in three test tubes labelled ‘a’, ‘b’ and ‘o’, and 0.5 ml of 5% blood group a, b, or o washed red cells suspended in physiological saline was added to each tube. the blood group o red cells were used as a negative control. the setup was incubated at 37 °c for 2 h and centrifuged afterwards. the supernatant was then examined macroscopically (in bright light) and microscopically for the presence of haemolysins. the degree of haemolysis was graded as 1+ for traces of haemolysis, 2+ for partial (greater than 50% but not complete) haemolysis, 3+ for complete haemolysis, and negative when no haemolysis was observed.11,20,21,22 all sera positive for haemolysis were titrated to quantify the degree of haemolysis.11,20,21 0.5 ml of the sera was diluted twofold with physiological saline to a titre of 526, and 0.5 ml of 5% washed red cells of the respective positive sera was added. the setup was incubated for 2 h and observed for haemolysis macroscopically and microscopically. the reciprocal of the serum dilution in the last tube with haemolysis was considered as the titre.11,20,21 statistical analysis collected data were entered into microsoft excel 2010 (microsoft corporation, redmond, washington, united states) and double-checked for errors by a second person. all analyses were done using statistical package for social sciences version 20 (ibm corp., chicago, illinois, united states). haemolysin prevalence was determined as the proportion of participants whose blood samples were positive for haemolysin (alpha, beta or both alpha and beta haemolysin). haemolysin titres equal to or greater than 8 were considered significant. the prevalence of significant titres was also determined as the proportion of participants with significant haemolysin titres. associations between haemolysin prevalence and age group, gender and rhesus d blood group were determined using chi-square test, and p-values < 0.05 were considered as statistically significant. results in total, 1161 blood donors presented to the rhbbb for blood donation between june 2020 and september 2020, 820 of whom were screened for physical fitness. of the 820 donors, 812 were classified as physically fit based on the applied standard questionnaire prepared by the rhbbb, and all the 812 donors consented to participate in the study. out of the screened 812 donors, 493 were blood group o, 480 of whom were free from ttis and were included to participate in the study (figure 1). figure 1: selection of study participants among blood group o donors at the rhbbb, cameroon, june 2020 – september 2020. participants were aged between 18 years and 55 years and comprised 383 (79.8%) men and 97 (20.2%) women (table 1). four hundred and sixty-three (96.5%) of the participants were rhesus d positive and 17 (3.5%) were rhesus d negative. of the 480 blood group o donors tested for haemolysins, 230 were negative while 250 were positive, giving a haemolysin prevalence of 52.1%. haemolysins were detected in 204 (53.3%) men and 46 (47.4%) women. the single participant aged ≥ 55 years was positive for haemolysin. in the other age groups, haemolysin prevalence was highest among participants aged between 45 and 54 years (28/48; 58.3%), followed by participants aged 18–24 years (86/156; 55.1%), 35–44 years (44/87; 50.6%) and 25–34 years (91/188; 48.4%). there was no association between haemolysin production and gender (p = 0.304), age group (p = 0.501) or rhesus d positivity (p = 0.628). two hundred and forty (240) of the 463 rhesus d-positive participants (51.8%) were positive for haemolysin while 10 (58.8%) of the 17 rhesus d-negative participants were positive for haemolysin. table 1: association between haemolysin production and gender, age group or rhesus d positivity of blood group o donors at the rhbbb, cameroon, june 2020 – september 2020. of the 250 participants positive for haemolysin (alpha, beta or both), 105 (42.0%) were positive for only alpha haemolysin, 69 (27.6%) were positive for only beta haemolysin, and 76 (30.4%) were positive for both alpha and beta haemolysins (figure 2). haemolysins from the 181 participants positive for alpha haemolysin showed trace haemolysis (47; 26.0%), partial haemolysis (95; 52.5%) and complete haemolysis (39; 21.5%) (table 2). of the haemolysins from the 145 participants positive for beta haemolysin, 58 (40.0%) showed trace haemolysis, 58 (40.0%) showed partial haemolysis, and 29 (20.0%) showed complete haemolysis. the highest observed haemolysin titre was 32 and was detected in five (2.8%) alpha haemolysin-positive participants and three (2.1%) beta haemolysin-positive participants. eighty-nine (89; 18.5%) participants presented with significant haemolysin titres (table 3), of which 45 (50.6%) were alpha haemolysin-positive only, 16 (18.0%) were beta haemolysin-positive only, and 14 (15.7%) were positive for both alpha and beta haemolysin (figure 3). figure 2: the prevalence of alpha and beta haemolysins among haemolysin-positive blood group o donors at the rhbbb, cameroon, june 2020 – september 2020. figure 3: prevalence of significant titres of alpha and beta haemolysins among blood group o donors at the rhbbb, cameroon, june 2020 – september 2020. table 2: degree of haemolysis of alpha and beta haemolysins from blood group o donors at the rhbbb, cameroon, june 2020–september 2020. table 3: titres of alpha and beta haemolysins among blood group o donors at the rhbbb, cameroon, june 2020–september 2020. discussion this study was carried out to determine the prevalence and titres of haemolysin among blood group o donors at the rhbbb. our study found a high haemolysin prevalence of 52.1% among the study population. significant haemolysin titres (defined as titres ≥ 8) were also detected in a high proportion (18.5%) of participants. this high prevalence may be attributed to immunisation arising from exposure to mosquito bites and parasitic infections of the gastrointestinal system.22 high prevalence rates of malaria23 and gastrointestinal parasites24 have been reported in bamenda, north-west region, cameroon.25 the high prevalence of haemolysin in this study is comparable to that (52.8%) reported by a study in abakaliki, nigeria, in 2014.25 lower haemolysin prevalence rates have been reported by studies among healthy blood donors in south india (10.8% in 2019),26 abidjan, côte d’lvoire18 (35.1% in 2016), lagos, nigeria10 (30.3% in 2015), bauchi, nigeria27 (22.2% in 2015), anambra, nigeria28 (16.06% in 2015), tunisia14 (6.6% in 2008), ilorin, nigeria11 (23.2% in 2001), and nigeria12 (30.6% in 1990). a higher prevalence of 69.0% was reported in a 2012 study on healthy blood donors in bangkok, thailand.29 these differences in prevalence rates may be due to the admixture of blood of immigrants as a result of intermarriages,20 variations in serum-cell ratios,30 or differences in geographical location,26 particularly due to the differences in the degree of exposure to gastrointestinal parasites22 and mosquitoes.13,14,15 it has been reported that higher serum-cell ratios increase the tendency for red cell lysis.30 the alpha haemolysin prevalence in this study (53.6%) was higher than that reported in a study conducted in 2010 in southeast nigeria that reported a prevalence of 10.3% for alpha haemolysin.20 in contrast, the observed prevalence rates of beta haemolysin, and both alpha and beta haemolysins in this study were lower than that reported in the same study (8.3% vs 12.6% for beta haemolysin, and 15.8% vs 32.5% for both alpha and beta haemolysins).20 alpha haemolysins were more prevalent in our study compared to beta haemolysin, which is consistent with the findings of a study conducted in 201511 in lagos, nigeria, but different from the findings of another study conducted in 2001 in ilorin, nigeria, that observed a higher prevalence of beta haemolysin compared to alpha haemolysin.11 the reasons for these variations may either be genetic or environment-induced.31 the absence of associations between haemolysin production and gender, age group or rhesus d blood group of blood group o donors in our study is consistent with the findings from previous studies.4,10,11,14,18,31,32 the prevalence of significant haemolysin titres (titres ≥ 8) in our study is noteworthy, considering the evidence that titres above this threshold can significantly cause haemolysis in vivo.33 this may be because parasitic infections such as malaria are endemic in the study area. there is evidence that the malaria parasite can stimulate the production of haemolysin.22 our observation is lower than those reported by studies conducted among blood group o donors in lagos, nigeria, in 2015 (18.6%)10 and ilorin, nigeria, in 2021 (31.7%).11 this may be due to differences in geographical location26 and the degree of exposure to gastrointestinal parasites22 and mosquitoes.13,14,15 group o blood should not be transfused to blood group a, b, or ab recipients except when such blood is tested and determined to be free of haemolysins. the haemolysin test should be included in the protocol for screening and compatibility testing of blood donors in the national blood transfusion programme of cameroon. this can be achieved through the collaborative efforts of the government, the ministry of public health of cameroon, the national blood transfusion programme of cameroon, as well as staff of the rhb and rhbbb. training for haemolysin testing should be conducted at the national level. furthermore, more studies should be carried out in other localities in cameroon to determine the haemolysin prevalence or presence of significant titres of haemolysin among blood group o donors. limitations due to limited resources, we only used the visual titration method and could not carry out the spectrophotometric or gel methods for the quantification of red blood cell lysis; this could have influenced the detection of lysis. however, our findings were compared only with studies that used the visual method. conclusion alpha and beta haemolysins are prevalent and exist in significant titres among blood group o donors in bamenda, cameroon. thus, there is an urgent need for public health intervention. considering the frequent practice of non-iso group-compatible abo transfusion, there is a need to routinely test for the presence of haemolysins in blood donors to prevent the potential risk to recipients and to provide safer blood for maximum benefits to the recipient. acknowledgements we acknowledge all the participants who gave their blood for this study. thanks to the management of the department of medical laboratory science, faculty of health and medical science, and the department of health economics, policy and management, faculty of business management – catholic university of cameroon-bamenda for academic support during this study. lastly, we appreciate the management and staff of the rhbbb for their wonderful support during the study. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions v.n.f. (study lead) designed the model and the computational framework, conceived, and planned the experiments and finalised the writing of the manuscript. t.s. and v.n.f. were responsible for supervision of the findings. v.n.f., r.m.f and c.n.a. were responsible for statistical analysis. v.n.f. and n.n. were responsible for specimen collection, performing the experiments and data collection. t.s., n.n., r.m.f. c.n.a., r.e-t., e.p.e., r.l, n.f.n.r. and d.n. reviewed and edited the manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability the data supporting the findings of this study are available within the article. data are also available on request from the corresponding author, v.n.f. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references boorman ke, dodd be, lincoln pj. blood group serology: theory, techniques, practical applications. london: churchill livingstone; 1977. waters a, lloyd e. laboratory aspects of blood transfusion. practical haematology. 7th ed. edinburgh: churchill livingstone; 1991, p. 115–124. berséus o, boman o, nessen sc, et al. risks of hemolysis due to anti-a and anti-b caused by the transfusion of blood or blood components containing abo-incompatible plasma. transfusion. 2013;53(s1):114s–123s. https://doi.org/10.1111/trf.12045 angiolillo, anne et luban, naomi lc. hemolysis following an out-of-group platelet transfusion in an 8-month-old with langerhans cell histiocytosis. j pediatr hematol oncol. 2004;26(4):267–269. https://doi.org/10.1097/00043426-200404000-00012 sapatnekar s, girish s, downes ka, et al. acute hemolytic transfusion reaction in a pediatric patient following transfusion of apheresis platelets. j clin apheresis. 2005;20(4):225–229. https://doi.org/10.1002/jca.20072 suleiman am, aisha i, mamman et, akanmu as. transfusion of the dangerous universal donor blood leading to maternal mortality: a case report. afr sanguine. 2016;18(2):1–3. shittu ao. incidence of acute haemolytic transfusion reactions in abo group compatible compared with group identical adult blood recipients in ilorin. fmcpath dissertation. ibadan: faculty of laboratory medicine, medical college of nigeria. 2005. available from: https://www.dissertation.npmcn.edu.ng/index.php/fmcpath/article/download/1349/1242 aubert ef, boorman ke, dodd be, et al. the universal donor with high titre iso-agglutinins. br med j. 1942;1(4247): 659. https://doi.org/10.1136/bmj.1.4247.659 tisdall ml, garland cd, szanto lb, et al. the effects of the transfusion of group o blood of high iso-agglutinin titer into recipients of other blood groups. am j clin pathol. 1946;16(3):193–206. https://doi.org/10.1093/ajcp/16.3.193 oyedeyi o, adeyemo t, ogbenna a, et al. prevalence of anti‑a and anti‑b hemolysis among blood group o donors in lagos. niger j clin pract. 2015;18(3):328–332. https://doi.org/10.4103/1119-3077.151760 olawumi h, olatunji p. prevalence and titre of alpha and beta haemolysins in blood group ‘o’ donors in ilorin. afr j med med sci. 2001;30(4):319–321. emeribe a. the status of alpha and beta haemolysins in nigerian blood donors. east afr med j. 1990;67(3):205–208. uko e, erhabor o, ahmed h, et al. prevalence of high titre alpha and beta haemolysins among blood donors in sokoto, north western nigeria. int j med sci health care. 2013;1:1–8. louati n, cherif j, ben amor i, rekik h, gargouri j. recherche des hémolysines chez les donneurs de sang. tinisia. j inform méd sfax. 2008;15(16):17–19. ukaejiofor e. the blood groups antigen-antibody reactions. blood transfus trop. 1996;1:13–23. kagu m, ahmed s, askira b. utilisation of blood transfusion service in north eastern nigeria. highland med res j. 2007;5(2):27–30. bamou r, sevidzem sl. abo/rhesus blood group systems and malaria prevalence among students of the university of dschang, cameroon. microwave j. 2016;7(4). victorine g-kap, liliane sk, honoré aa, et al. prevalence of anti-a and anti-b haemolysins in group o blood donors at the national blood transfusion center of abidjan, côte d’ivoire. int j immunol. 2016;4(6):68–72. https://doi.org/10.11648/j.iji.20160406.14 cheesbrough m. district laboratory practice in tropical countries, part 2. cambridge: cambridge university press; 2006. kagu m, ahmed sg, mohammed aa, moshood wk, malah mb, kehinde jm. anti-a and anti-b haemolysins amongst group ‘o’ voluntary blood donors in northeastern nigeria. j blood transfus. 2011;2011:302406. https://doi.org/10.4061/2011/302406 edinoton g, gilles hm. pathology in the tropics, 2nd edition. london: edward arnold (publishers) ltd; 1979. shanbhag s, joshi s, bhatia h. evaluation of the two screening techniques in the detection of high titre anti-a and anti-b. ind j med res. 1973;61(12):1824–1830. ntonifor nh, veyufambom s. assessing the effective use of mosquito nets in the prevention of malaria in some parts of mezam division, northwest region cameroon. malar j. 2016;15(1):390. https://doi.org/10.1186/s12936-016-1419-y bissong m, nguemain n, ng’awono t, kamga f. burden of intestinal parasites amongst hiv/aids patients attending bamenda regional hospital in cameroon. afr j clin exp microbiol. 2015;16(3):97–103. https://doi.org/10.4314/ajcem.v16i3.3 ugah u, ibekailo s, mbamagu d. rate of haemolysins among blood donors in abakaliki, ebonyi state, nigeria. gjmr stud. 2014;1(3):61–65. amita r, vijayalakshmi k. prevalence and haemolytic significance of red cell antibodies among dangerous universal donors in a tertiary care hospital in south india. int j med lab. 2019;6(4):234–240. https://doi.org/10.18502/ijml.v6i4.1998 obisesan oa, ogundeko to, iheanacho cu, et al. evaluation of alpha (α) and beta (β) haemolysin antibodies incidence among blood group ‘o’ donors in. am j clin med res. 2015;3(3):42–44. https://doi.org/10.12691/ajcmr-3-3-2 ibeh n, aneke j, okocha c. prevalence of haemolysins in blood donors in nnamdi azikiwe university teaching hospital, nnewi, anambra state, nigeria. orient j med. 2015;27(1–2):34–39. khampanon k, chanprakop t, sriwanitchrak p, setthakarn m, oota s, nathalang o. the characteristics of abo antibodies in group ot hai blood donors. j clin lab anal. 2012;26(4):223–226. https://doi.org/10.1002/jcla.21499 polley mj, adinolfi m, mollison p. serological characteristics of anti-a related to type of antibody protein (7s γ or 19s γ). vox sanguinis. 1963;8(4):385–409. https://doi.org/10.1111/j.1423-0410.1963.tb04159.x anyanwu r, emeribe a, igwe c, et al. occurrence of haemolysin antibodies among sickle cell anaemia patients within calabar metropolis of nigeria. afr j biotechnol. 2007;6(10):1217–1220. adewuyi j, gwanzura c. racial difference between white and black zimbabweans in the haemolytic activity of a, b, o antibodies. afr j med med sci. 2001;30(1–2):71. saphire d, rudolph n, hackleman s, stone w. the effect of age on the level of human abo blood group antibodies. aging clin exp res. 1993;5(3):177–184. https://doi.org/10.1007/bf03324152 abstract background description of the intervention lessons learnt recommendations acknowledgements references about the author(s) timothy amukele icon clinical laboratories, icon clinical research plc, farmingdale, new york, united states ryland n. spence department of pathology, johns hopkins school of medicine, johns hopkins bayview medical center, baltimore, maryland, united states citation amukele t, spence rn. african countries established covid-19 testing in one month: here’s how they did it. afr j lab med. 2021;10(1), a1457. https://doi.org/10.4102/ajlm.v10i1.1457 lessons from the field african countries established covid-19 testing in one month: here’s how they did it timothy amukele, ryland n. spence received: 17 nov. 2020; accepted: 18 aug. 2021; published: 15 dec. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: as a novel and deadly acute respiratory syndrome, which later became known as coronavirus disease 2019 (covid-19), spread beyond china in late january 2020, there were no laboratories in africa that could test for the disease. however, in early march, just over a month later, 42 african countries had developed the expertise and resources to perform covid-19 testing. our goal was to document this public health success story, learn from it, and use it to inform future public health action. intervention: three groups were primarily responsible for establishing covid-19 testing capacity in africa. the first group comprised early test manufacturers who reacted with incredible speed and ingenuity early in the pandemic, such as the german company tib molbiol that developed a molecular test for covid-19 before the sars-cov-2 genome sequence was available. the second group included private and public donors such as the jack ma foundation, and the last were the coordinators of the rollout, such as the world health organization and the africa centres for disease control and prevention (cdc). lessons learnt: the first lesson was that speed is critical, especially during a crisis. it was also demonstrated that being a predictable and transparent trusted institution opens doors and improves effectiveness. africa cdc, which was only three years old, was able to secure significant resources from external partners and rapidly build substantial testing capacity within africa because it is a trusted institution. recommendations: lowand middle-income countries must build local trusted institutions to better prepare for public health challenges. keywords: testing capacity; covid-19; pandemic; coronavirus; africa; laboratory. background when côte d’ivoire had its first suspected coronavirus disease 2019 (covid-19) case in late january 2020, the nearest laboratory capable of performing covid-19 testing was in paris, france.1 yet just over a month later, by early march, 42 sub-saharan african countries had acquired the instruments, reagents and know-how to perform laboratory diagnostic testing for covid-19.2 broadly speaking, ongoing covid-19 infection can be diagnosed using two types of tests: antigen-based and nucleic acid-based tests. antigen-based tests detect the virus’s coronal spike proteins from which the virus derives its name. nucleic acid-based tests (also called molecular or polymerase chain reaction [pcr] tests) work by directly detecting severe acute respiratory syndrome coronavirus 2 (sars-cov-2) rna. these molecular tests are more specific and sensitive than antigen-based tests but are 3–10 times more expensive and require more complex instrumentation.3 nevertheless, in early 2020, the overwhelming majority of covid-19 tests available globally were molecular. in addition, although sub-saharan africa had less than 1% of the molecular testing capacity of that in the united states, thanks to hiv control efforts, every country had at least one location that could perform molecular testing once the sars-cov-2 molecular reagents were available.4 this article tells the story of how 42 african countries were able to acquire covid-19 pcr testing capacity in two months in early 2020. description of the intervention ethical considerations this study followed all ethical standards for research without direct contact with human or animal subjects. data collection we started by independently corroborating the ‘42 countries’ figure announced by the director of the africa centres for disease control and prevention (cdc) in his interview on 11 march 2020.2 specifically, we searched the internet and social media platforms such as twitter for announcements by individual countries that they could now test for covid-19. next, we used google searches to identify relevant sources (including those in the grey literature and popular press) that detailed how covid-19 testing capacity was established in africa in the first two months of 2020. data analysis raw data were collected as described above, entered into excel (microsoft corporation, redmond, washington, united states), and presented in this study without any transformation. no additional statistical analysis was performed. the figure displaying these data (figure 1) was also created using excel (microsoft corporation, redmond, washington, united states). figure 1: the total number of sub-saharan african countries reporting coronavirus testing results or laboratory testing capacity, january 2020 – may 2020. additional country-level data is available on the covid-19 testing dashboard of pathologists overseas (https://www.pathologistsoverseas.com/). lessons learnt as of march 2020, the total number of countries reporting coronavirus testing results or laboratory testing capacity in sub-saharan africa was 42 (figure 1). this public health success story involved three principal actors: early manufacturers of covid-19 tests, private and government donors, and coordinating bodies, including the world health organization (who) and africa cdc, that coordinated efforts within and across the african countries. the first lesson learnt from this experience was that speed is critical during a crisis, as with the much-criticised motto of facebook’s founder: ‘move fast and break things’. while this may not be good advice for every situation, it was the approach successfully adopted by all the actors in this story. the activities of these major players are discussed in greater detail below. covid-19 test manufacturers test manufacturers reacted with incredible speed and ingenuity in the early days of the pandemic. to illustrate, the announcement of the isolation of the sars-cov-2 virus on 07 january 2020 and the determination of its genome sequence on 10 january 2020, occurred less than a week after china first shared information about a new and deadly flu-like respiratory illness with the who and other countries on 03 january 2020.5,6 while the speed of virus isolation and genome sequencing was remarkable in itself, something even more prodigious was accomplished by an early covid-19 test developer. the german company tib molbiol (tib molbiol syntheselabor gmbh, berlin, germany), in collaboration with the charite hospital in berlin, announced a molecular test for covid-19 on 10 january, the same day the sars-cov-2 genome sequence became available.7,8 they accomplished this feat by creating their molecular sars-cov-2 test based on the sequences of other coronaviruses.9 this is akin to sewing custom clothing for a stranger based on pictures of their parents and siblings. tib molbiol made several versions of this blind test and vetted them using actual covid-19 patient samples before selecting the one that worked best. it was a gamble, but it paid off. the protocol for the best version was published by the who on 17 january 2020, and tib molbiol had sold four million tests by early march 2020.10,11 coordinating bodies many of the aforementioned tib molbiol tests were purchased by the who with funding from the bill and melinda gates foundation and sent to countries around the world to support the scale-up of the novel coronavirus diagnostic efforts.12 in particular, reagent kits were shipped to more than 20 countries in the african region by the who before 31 january 2020.13 this was to expand diagnostic capacity beyond the two referral laboratories in senegal and south africa that had it at the time. of note, the who had a much more expansive role in responding to the covid-19 pandemic.13 for example, on 31 january 2020, the who identified 13 top-priority countries that had either direct links with or a high volume of travel to china (algeria, angola, côte d’ivoire, the democratic republic of the congo, ethiopia, ghana, kenya, mauritius, nigeria, south africa, tanzania, uganda and zambia), and subsequent active screening was established in a majority of the airports in these countries.13 however, these initiatives that are not directly about the establishment of covid-19 diagnostic capacity in africa in the first month of 2020 will not be covered in this focused report. in africa, the who-sourced kits were primarily distributed by africa cdc, a technical institution of the african union that was established in 2016 but officially launched in january 2017.14 like other actors in this story, africa cdc acted swiftly. on 22 january 2020, five days after the publication of the tib molbiol protocol on the who website, africa cdc announced that it was working with member states to identify laboratories that were capable of receiving and testing specimens.10,15 on 05 february 2020, they created the africa task force for coronavirus preparedness and response,16 a multi-country multi-agency group designed to collaborate, communicate and coordinate efforts in response to the coronavirus pandemic. a day later and exactly a week after announcing that they were identifying laboratories in member countries, africa cdc, in collaboration with the pasteur institute in dakar, organised a workshop and training on covid-19 diagnosis for medical teams from 16 african countries, where each trainee received a kit that could run 100 tests (figure 2).17 the countries were côte d’ivoire, cameroon, the democratic republic of the congo, egypt, ethiopia, the gambia, gabon, ghana, kenya, nigeria, morocco, senegal, south africa, tunisia, uganda, and zambia. figure 2: first training on laboratory diagnosis of covid-19 in dakar, senegal, 06–08 february 2020. in partnership with the national institute for communicable disease, a second training on laboratory diagnosis of covid-19 was organised by africa cdc in south africa on 20–22 february 2020 (figure 3). each of the 12 countries that took part in the training received kits for the testing of 192 specimens, while egypt was provided with 700 additional kits, and nigeria and rwanda each received 1000 additional kits.18 figure 3: second training on laboratory diagnosis of covid-19, south africa, 21 february 2020. as many of these groups that were trained lacked the equipment to perform the testing in their home countries, the procurement and placement of equipment and biosafety protective gear were also addressed by the who and africa cdc. in addition, as stated in a media briefing on 04 february 2020, africa cdc planned to set up a referral system where the 16 laboratories that would be set up to perform covid-19 testing could receive samples and support other countries who were unable to test.19 it is not clear if this referral system was ever established but it likely was not needed for long because by 11 march 2020, john nkengasong, the director of africa cdc, had announced in an interview that there were 42 african countries with covid-19 testing capacity at the central level.2 like the who, the efforts of africa cdc transcended laboratory testing and included significant efforts in five other high-priority areas for coronavirus control, namely surveillance, infection prevention and control, clinical care, risk communication, and the distribution of donations by other groups and individuals.20 pre-existing disease surveillance networks also helped in the response to covid-19 in africa. for example, the who has 27 who collaborating centers (defined as ‘institutions such as research institutes, parts of universities or academies, which are designated by the director-general to carry out activities in support of the organization’s programmes’) in 14 african countries.21 other laboratory networks in africa include the regional integrated surveillance laboratory network, which is run by africa cdc and based in zambia, kenya, gabon, nigeria, and senegal; the west african network of biomedical analysis laboratories, which is a network of laboratories in francophone west africa supported by the mérieux foundation22,23; and many other disease-based laboratory networks. these laboratories were already in close contact and sharing surveillance and other information with the who or cdc before covid-19. thus, they were logical sites for situating covid-19 testing once the kits became available. private and government donors one of the most significant donations to help counter the impact of the coronavirus in africa was that made by the chinese billionaire and founder of alibaba, jack ma.24 jack ma’s massive operation had, by september 2020, shipped over 200 million units of personal protective equipment, testing kits and ventilators to over 150 countries and regions, including the united states and over 30 countries in africa. although this was not the largest covid-mitigating financial donation by a wealthy donor, it was arguably the most impactful as alibaba’s logistic muscle allowed the rapid delivery of life-saving face masks and ventilators to many countries that were outcompeted during the global jostle for life-saving equipment.25 on 16 march 2020, jack ma announced a donation of 100 000 masks, 20 000 test kits and 1000 sets of protective clothing and face shields to each of the 54 nations on the african continent.26 on 06 april 2020, an additional donation of medical supplies to all 54 countries of africa was announced.27 this second donation included 500 ventilators, 200 000 sets of protective clothing and face shields, 2000 thermometers, one million swabs and extraction kits, and 500 000 gloves. this brief article does not capture all the activities of all the partners that helped establish laboratory capacity for covid-19 diagnosis in the early days of the pandemic in africa. for example, many government and private partners, including the ethiopian government, the africa cdc, the united nations world food programme, the who, and ethiopian airlines, helped distribute jack ma’s large donation. there were many other groups involved in the early days of the coronavirus laboratory response by countries in africa, including wellcome, the department for international development, the mérieux foundation, oxford nanopore technologies, the united states cdc, and others.20,28,29 the denominating factor across the activities by all the players in this story was the speed with which they responded to build capacity. having a pandemic nipping at one’s heels is a strong incentive to move fast and stay focused. although the lightning-fast response by all partners to the covid-19 pandemic described in this article may not be possible under normal operating conditions, it does demonstrate what is feasible and gives us something to aspire to in terms of speed and efficiency. trusted institutions are crucial for moving africa forward another lesson from this experience is that trusted institutions are the key to effective public health interventions. much of the credit for the rapid establishment of testing capacity should go to the africa cdc as the main coordinating body for external partners (the who, donors, etc.) and countries within africa. but how was a three-year-old institution so effective at such a scale? the africa cdc had obvious advantages such as the resources to hire and retain world-class staff, deep institutional connections to the who and the united states cdc, as well as the pressure of a pandemic. however, our opinion is that the africa cdc was so effective because it is a trusted institution. being a trusted institution does not mean that an institution is perfect or even efficient. rather, a trusted institution has ‘policies and mechanisms that showcase their commitment to transparency, high standards, fiscal management, measurable results, and zero tolerance for corruption and mismanagement of funds’ (brough r, april 2018, personal communication). the establishment of covid-19 testing capacity in africa, while remarkable, was not an unmitigated success. african countries are still behind high-income countries in terms of the number of tests done per capita.30 nevertheless, it was a remarkable achievement and provides a clear example of the positive impact of trusted institutions in public health. recommendations we need to build more local trusted institutions. trusted institutions are characterised by predictability and transparency, which makes them more effective than other institutions because partners can engage without concerns that their donations may be diverted. more so, the influence of trusted institutions goes beyond donors and philanthropists as it also makes it easier for the recipients to engage. when invited by the africa cdc for training or capacity building, countries are much more likely to respond positively because they know that such offers are usually without any hidden motives or expectations. naturally, this begs the question, the answers to which are beyond the scope of this article but have been comprehensively addressed elsewhere31,32: how do we build trusted institutions? acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions t.a. conceived of the presented idea. t.a. and r.n.s. performed the research and found the original sources. both authors discussed the results and contributed to the final manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the data that support the findings of this study are available on request from the corresponding author, t.a. the data are not publicly available due to it containing information that could compromise the privacy of research participants. disclaimer the views expressed in this article are those of the authors and do not necessarily reflect the views of the institutions the authors represent. references washington post. africa has 1.2 billion people and only six labs that can test for coronavirus. how quickly can they ramp up? [homepage on the internet]. https://www.washingtonpost.com. c2020 [updated 2020 feb 6; cited 2020 may 12]. available from: https://www.washingtonpost.com/world/africa/africa-has-12-billion-people-and-only-six-labs-that-can-test-for-coronavirus-how-quickly-can-they-ramp-up/2020/02/05/68af96de-4758-11ea-91ab-ce439aa5c7c1_story.html pandemics explained. building testing capacity in africa – 42 countries and counting [homepage on the internet]. https://www.globalpandemics.org. c2020 [updated 2020 mar 13; cited 2020 mar 29] available from: https://globalepidemics.org/2020/03/13/42-countries-and-counting-how-africas-cdc-is-testing-for-sars-cov-2/ the conversation. african countries need cheaper covid-19 tests: here’s how to get them [homepage on the internet]. https://www.theconversation.com. c2020 [updated 2020 jul 12; cited 2021 aug 1] available from: https://theconversation.com/african-countries-need-cheaper-covid-19-tests-heres-how-to-get-them-141315 schroeder lf, elbireer a, jackson jb, amukele tk. laboratory diagnostics market in east africa: a survey of test types, test availability, and test prices in kampala, uganda. plos one. 2015;10(7):e0134578. science. chinese researchers reveal draft genome of virus implicated in wuhan pneumonia outbreak [homepage on the internet]. https://www.sciencemag.org. c2020 [updated 2020 jan 11; cited 2020 apr 24] available from: https://www.sciencemag.org/news/2020/01/chinese-researchers-reveal-draft-genome-virus-implicated-wuhan-pneumonia-outbreak international antiviral society – usa. croi aud d3b [homepage on the internet]. https://www.capitalreach.com. c2020 [updated 2020 mar 10; cited 2020 may 18]. available from: https://special.croi.capitalreach.com/arc/audd3b/o1 tib molbiol. home [homepage on the internet]. https://www.tib-molbiol.de. c2018. [cited 2020 mar 5]. available from: https://www.tib-molbiol.de/es/index.html corman vm. detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr. euro surveill. 2020;25(3):2000045. world health organization. diagnostic detection of wuhan coronavirus 2019 by real-time rtpcr [homepage on the internet]. c2020 [updated 2020 jan 13; cited 2020 jun 12]. available from: https://www.who.int/docs/default-source/coronaviruse/wuhan-virus-assay-v1991527e5122341d99287a1b17c111902.pdf?sfvrsn=d381fc88_2 world health organization. laboratory testing of 2019 novel coronavirus (2019-ncov) in suspected human cases: interim guidance [homepage on the internet]. c2020 [updated 2020 jan 17; cited 2020 mar 8]. available from: https://apps.who.int/iris/handle/10665/330676 bloomberg. a berlin biotech company got a head start on coronavirus tests [homepage on the internet]. c2020 [updated 2020 mar 11; cited 2020 mar 29]. available from: https://www.bloomberg.com/news/articles/2020-03-12/a-berlin-biotech-company-got-a-head-start-on-coronavirus-tests twitter. africa center for disease control [homepage on the internet]. https://twitter.com/. c2020 [updated 2020 feb 7; cited 2020 feb 25]. available from: https://twitter.com/africacdc/status/1225707932656054272 world health organization. who ramps up preparedness for novel coronavirus in the african region [homepage on the internet]. https://afro.who.int/. c2020 [updated 2020 jan 31; cited 2020 apr 11] available from: https://www.afro.who.int/news/who-ramps-preparedness-novel-coronavirus-african-region africa center for disease control. about us [homepage on the internet]. https://africacdc.org. c2020 [cited 2020 feb 26] available from: https://africacdc.org/about-us/ africa center for disease control. novel coronavirus (2019-ncov) global epidemic – 22 january 2020 [homepage on the internet]. https://africacdc.org. c2020 [updated 2020 jan 22; cited 2020 apr 18]. available from: https://africacdc.org/disease-outbreak/novel-coronavirus-2019-ncov-global-epidemic-22-january-2020/ africa center for disease control. africa cdc establishes continent-wide task force to respond to global coronavirus epidemic [homepage on the internet]. https://www.africacdc.org. c2020 [updated 2020 feb 5; cited 2020 mar 15]. available from: https://africacdc.org/news-item/africa-cdc-establishes-continent-wide-task-force-to-respond-to-global-coronavirus-epidemic/ twitter. [https://www.twitter.com/] africa center for disease control [homepage on the internet]. c2020 [updated 2020 feb 7; cited 2020 apr 20]. available from: https://twitter.com/africacdc/status/1225707932656054272 africa center for disease control. novel coronavirus (2019-ncov) global epidemic – 3 march 2020 [homepage on the internet]. https://www.africacdc.org. c2020 [updated 2020 mar 3; cited 2020 apr 16]. available from: https://africacdc.org/disease-outbreak/novel-coronavirus-2019-ncov-global-epidemic-3-march-2020/ africa center for disease control. novel coronavirus (2019-ncov) global epidemic – 4 february 2020 [homepage on the internet]. https://www.africacdc.org. c2020 [updated 2020 feb 4; cited 2020 apr 24]. available from: https://africacdc.org/disease-outbreak/novel-coronavirus-2019-ncov-global-epidemic-4-february-2020/ africa center for disease control. wellcome and dfid support africa covid-19 continental response with € 2.26 million [homepage on the internet]. https://africacdc.org. c2020 [updated 2020 apr 21; cited 2020 apr 29]. available from: https://africacdc.org/news-item/wellcome-and-dfid-support-africa-covid-19-continental-response-with-e-2-26-million/ world health organization. who collaborating centres database and portal [homepage on the internet]. https://who.int. c2020 [cited 2020 jun 1]. available from: https://apps.who.int/whocc/ africa center for disease control. rislnet laboratory [homepage on the internet]. https://africacdc.org. c2020 [cited 2020 jun 4]. available from: https://africacdc.org/rislnet/rislnet-laboratory/ merieux foundation. resaolab [homepage on the internet]. https://https://www.fondation-merieux.org/. c2020 [cited 2020 jun 11]. available from: https://www.fondation-merieux.org/en/projects/resaolab/ alizila. factsheet: jack ma foundation and alibaba foundation’s global donations and efforts to combat covid-19 [homepage on the internet]. https://alizila.com. c2020 [updated 2020 apr 15; cited 2020 may 15]. available from: https://www.alizila.com/factsheet-jack-ma-foundation-alibaba-foundations-coronavirus-donations-and-efforts/ british broadcasting corporation. jack ma: the billionaire trying to stop coronavirus (and fix china’s reputation) [homepage on the internet]. https://bbc.com. c2020 [updated 2020 apr 26; cited 2020 jun 10]. available from: https://www.bbc.com/news/world-asia-china-52325269 twitter. [https://twitter.com] jack ma [homepage on the internet]. c2020 [updated 2020 apr 6; cited 2020 may 12]. available from: https://twitter.com/jackma/status/1247014237303537664?s=20 twitter. [https://twitter.com] jack ma [homepage on the internet]. c2020 [updated 2020 mar 16; cited 2020 may 12]. available from: https://twitter.com/jackma/status/1239581509125726210 merieux foundation. covid-19: the merieux foundation deploys diagnostic and research expertise on the ground [homepage on the internet]. https://www.fondation-merieux.org/. c2020 [cited 2020 jun 11]. available from: https://www.fondation-merieux.org/en/the-merieux-foundation-deploys-diagnostic-and-research-expertise-on-the-ground/ africa center for disease control. outbreak brief number 11: covid-19 pandemic – 31 march 2020 [homepage on the internet]. https://africacdc.org. c2020 [updated 2020 mar 31; cited 2020 jun 19]. available from: https://africacdc.org/download/outbreak-brief-11-covid-19-pandemic-31-march-2020/ our world in data. research and data to make progress towards the world’s largest problems [homepage on the internet]. [https://ourworldindata.org]. [cited 15 oct 2020]. available from: https://ourworldindata.org faith and leadership. dave odom: what makes an institution trustworthy? [homepage on the internet]. https://faithandleadership.com. c2019 [updated 2019 aug 6; cited 2020 sep 12]. available from: https://faithandleadership.com/dave-odom-what-makes-institution-trustworthy world bank blogs. who is responsible for building trust in institutions? [homepage on the internet]. https://www.blogs.worldbank.org. c2014 [updated 2014 nov 13; cited 10 oct 2020]. available from: https://blogs.worldbank.org/governance/who-responsible-building-trust-institutions acknowledgements references about the author(s) iruka n. okeke department of pharmaceutical microbiology, university of ibadan, ibadan, nigeria citation okeke in. extending the breadth of african laboratory medicine. afr j lab med. 2019;8(1), a1128. https://doi.org/10.4102/ajlm.v8i1.1128 editorial extending the breadth of african laboratory medicine iruka n. okeke copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the african journal of laboratory medicine’s (ajlm) eighth volume contains almost 30 articles, our largest issue yet. a few areas of laboratory medicine are not represented, but the impressively expansive scope of the volume extends from bacteriology (including mycobacteriology), clinical chemistry, clinical pharmacology, haematology, histopathology and virology to informatics, laboratory development and quality management. while appreciating that growth has come with good subject area representation, we have to be just as intentionally concerned with the diversity of matters other than content. if nothing is done, some estimates suggest that it will be over 200 years before gender parity is reached throughout society1 and other forms of imbalance may be even more entrenched. ajlm is therefore not alone in its current introspection and quest for diversity. in a bid to accelerate the quality improvements that will come with inclusiveness, a number of editors, journals and independent researchers have published their recent observations and reflections on the gender and geographic balance of editors, reviewers and authors.2,3,4 almost universally, they find that representation in scientific journals is slanted towards the male northern bias that permeates most of science, even if the tilt angles differ. as a journal owned by an african scientific society, with both editorial and publishing offices based on the continent, ajlm has an uncommon position in this discourse. firstly, women are severely underrepresented in african science5 and globally women play a prominent role in healthcare delivery but are much less visible in its leadership.6 both of these deficits could well influence representation in a laboratory medicine journal with a lens on africa, the continent that is home to the fewest scientists per capita. secondly, as a regional journal with global reach, we receive and publish a large number of scientific articles from african authors. we simultaneously provide an owned perspective and a global view of african science, a dual role that is rare in scientific publishing.2,4,7 this vantage-point notwithstanding, ajlm also represents a powerful mouthpiece for speaking to african scientists and therefore we cannot take for granted that our editorial processes and authorship will reflect our readership. the vision and content of a journal is shaped by its editors. as of october 2019, ajlm’s editorial team comprised a female editor-in-chief, five section editors (two female) and a 22-person editorial board that included only three women. this team had affiliations on four continents with a third of us working in africa. with three of our six editors being women (not counting our managing editor, who is also female), we merit only 60% by bhaumik and jagnoor’s8 composite editorial board diversity score. but, given our focus on africa, we have a greater proportion of africa-based editors than any of the 27 global health journals that were assessed in that study. we lag most noticeably in geographic diversity. scientists from all over the world work with laboratory medicine experts on our continent to better health, and findings from studies in or on africa will inform the practice of laboratory medicine anywhere. so, our editorial board is deliberately global even though we do seek editors who are familiar with science in africa. we, therefore, would benefit from representation from australasia, latin america and the caribbean, which is currently absent from our editorial board. even though this is not the case with respect to african biomedical research overall,5 the vast majority of our articles are wholly or predominantly authored by africans. in spite of the well-documented male skew in african science,5 ajlm vol. 7(1) 2018 comprised articles with nine female and five male first authors (with one indeterminate). first authors on articles in vol. 8 include 11 men and nine women (4 indeterminate based on first name and online searches). therefore, ajlm is an important venue for publishing female-led work, particularly from africa. we are also pleased to find that ajlm continues to feature articles from a range of african countries and from beyond them and that it addresses health issues of different populations, including ethnic minorities or those living in rural or remote areas. unfortunately, while we have good representation from africa, we find that there is less diversity in the geographic origin of ajlm articles within the continent. five 2018 corresponding authors listed south african addresses and one each from rwanda, ethiopia, the netherlands, kenya, cameroon, barbados, haiti, namibia, nigeria and canada. in 2019, we published six articles with corresponding authors from south africa, four each from nigeria and the united states (us), one with a double nigeria-us affiliation, two from kenya and saudi arabia and one each from belgium, botswana, cameroon, ethiopia, ghana and zimbabwe. while preponderance of anglophone corresponding authors is expected for an english-language journal, the underrepresentation of submissions from western, central, north and east africa in our recent volumes is obvious and concerning. if ajlm has uncommon success in publishing high-quality african research, it should do so for those parts of the continent least represented in science. what could ajlm do to enhance diverse representation in our journal going forward? firstly, we need to maintain the diversity that we do have and to ensure that with the growth in our size and reputation, minority contributors do not become overshadowed or, worse, replaced. ajlm will continue to encourage female scientists to submit to, review for and edit for the journal as well as to support those that work with us. we will also continue to offer a mentored review process and copy-editing support for accepted articles, which allows us to feature work by new authors and non-anglophone scientists. however, we will have to do much more to bring in manuscripts from across the continent and explicitly wish to call for papers from countries not featured in our last two volumes. from time to time, ajlm offers heavily subscribed manuscript writing training workshops and in future, we will prioritise early-career participants from african countries who are underrepresented in the journal or who are female. we will also endeavour to invite more female scientists to review for the journal and take places on our editorial board. finally, with this editorial, ajlm is in the process of keeping track of the demographics of our authors, reviewers and board because, unless we do, we will have no idea how far we are from our goal of being the primary outlet for truly balanced scientific work in laboratory medicine across the continent. acknowledgements i am grateful to el-shama monu-nwoko for collating data. competing interests i declare that i have no financial or personal relationships that may have inappropriately influenced me in writing this article. references gates m. gender equality is within our reach. harv bus rev. 2019. briggs rc, weathers s. gender and location in african politics scholarship: the other white man’s burden? afr aff. 2016;115(460):466–489. https://doi.org/10.1093/afraf/adw009 espin j, palmas s, carrasco-rueda f, et al. a persistent lack of international representation on editorial boards in environmental biology. plos biol. 2017;15(12):e2002760. https://doi.org/10.1371/journal.pbio.2002760 chersich mf, blaauw d, dumbaugh m, et al. local and foreign authorship of maternal health interventional research in low-and middle-income countries: systematic mapping of publications 2000–2012. glob health. 2016;12(1):35. https://doi.org/10.1186/s12992-016-0172-x okeke in, babalola cp, byarugaba dk, djimde a, osoniyi or. broadening participation in the sciences within and from africa: purpose, challenges, and prospects. cbe life sci educ. 2017;16(2):es2. https://doi.org/10.1187/cbe.15-12-0265 world health organization. delivered by women, led by men: a gender and equity analysis of the global health and social workforce. who: geneva;2019. abimbola s. the foreign gaze: authorship in academic global health. bmj glob health. 2019;4(5):e002068. https://doi.org/10.1136/bmjgh-2019-002068 bhaumik s, jagnoor j. diversity in the editorial boards of global health journals. bmj glob health. 2019;4(5):e001909. https://doi.org/10.1136/bmjgh-2019-002068 abstract introduction methods results discussion acknowledgements references about the author(s) vuyolwethu fadana school of pathology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa department pathology, national health laboratory services, johannesburg, south africa teena thomas school of pathology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa infectious control services laboratory, national health laboratory services, johannesburg, south africa nina von knorring school of pathology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa mycobacteriology referral laboratory, national health laboratory service, johannesburg, south africa citation fadana v, thomas t, von knorring n. retrospective analysis of vitek®2 performance compared to manual broth micro-dilution for colistin susceptibility testing of acinetobacter baumannii complex isolates in south africa. afr j lab med. 2022;11(1), a1597. https://doi.org/10.4102/ajlm.v11i1.1597 brief report retrospective analysis of vitek®2 performance compared to manual broth micro-dilution for colistin susceptibility testing of acinetobacter baumanniicomplex isolates in south africa vuyolwethu fadana, teena thomas, nina von knorring received: 29 mar. 2021; accepted: 27 oct. 2021; published: 28 feb. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the manual broth micro-dilution (mbmd) is the recommended reference method for colistin minimum inhibitory concentration determination; however, it is not as readily available in south africa as the vitek®2. this retrospective study compared the performance of vitek®2 against mbmd in determining the colistin minimum inhibitory concentration of 337 extensively drug-resistant acinetobacter baumannii complex isolates. vitek®2 yielded a categorical agreement of 89%, an essential agreement of 56%, a major error rate of 8% and a very major error rate of 55%. the vitek®2 is not an alternative to mbmd for colistin susceptibility testing. keywords: acinetobacter; colistin; broth micro-dilution; vitek®2; antimicrobial susceptibility testing. introduction the increasing antimicrobial resistance in the acinetobacter baumannii complex, and the recently observed resistance to the polymyxin antibiotic (colistin), demands timeous identification and antimicrobial susceptibility profiling of causal pathogens to ensure timely and appropriate antimicrobial therapy decisions.1,2 different laboratory methods for the assessment of colistin susceptibility testing among a. baumannii isolates have been assessed.3 since 2016, the reference colistin susceptibility testing method recommended by the clinical and laboratory standards institute and the european committee on antimicrobial susceptibility testing is the international organization for standardization (iso) broth micro-dilution (bmd) method (iso-20776).4 implementing manual bmd (mbmd) is currently not feasible in the routine microbiology laboratory due to its laborious nature.5 the vitek®2 (biomerieux inc., marcy l’étoile, france) is an automated microbial identification and antimicrobial susceptibility testing (ast) platform. most public and private south african laboratories use the vitek®2, making it an attractive alternative to the mbmd. however, studies comparing vitek®2 and the mbmd method have reported discordant colistin susceptibility results. dafopoulou et al. compared the mbmd with polysorbate-80, vitek®2, etest, agar dilution, and the minimum inhibitory concentration (mic) methods for the colistin susceptibility testing of 51 klebsiella pneumoniae and 20 a. baumannii clinical isolates.6 eighteen of the a. baumannii isolates were colistin-resistant by mbmd, and vitek®2 categorically agreed in 85% and essentially agreed in 90% of the a. baumannii isolates. there were no ‘very major errors’ (vme) reported. these findings were similar to those obtained by lo-ten-foe et al.7 piewngam and kiratisin also observed a low vme rate of 0.7% when testing 290 a. baumannii isolates.8 these findings suggest that vitek®2 could be a viable alternative to the mbmd. in contrast to these results, vourli et al. reported unacceptably high vme rates for phoenix100 and vitek®2 against the mbmd (41.4% and 37.9%).9 additionally, in 2017 biomerieux retracted the use of vitek®2 for colistin testing owing to the clinical and laboratory standards institute-european committee on antimicrobial susceptibility testing recommendations and an in-house observed performance issue: high vme rate against agar dilution and mbmd methods.10 due to these contradictory findings in the literature, further research into this area was warranted. this study aimed to compare the performance of vitek®2 colistin susceptibility testing to mbmd for clinical extensively drug-resistant (xdr) a. baumannii complex isolates at charlotte maxeke johannesburg academic hospital in johannesburg, south africa. methods ethical considerations ethical approval was obtained from the university of the witwatersrand human research ethics committee (clearance certificate number m191048 med 19-10-043). data were anonymised before analysis to maintain patient confidentiality. patient consent was not required. approval to utilise patient data was obtained from the chief executive officer of charlotte maxeke johannesburg academic hospital. data collection this was a descriptive, retrospective analysis of the a. baumannii complex isolated from charlotte maxeke johannesburg academic hospital inpatients between 01 january 2017 and 30 june 2019. the xdr a. baumannii complex isolates were resistant to one or more agents in all but one or two categories of antibiotics. microbiological data, including the sample type, vitek®2 and mbmd colistin mic results, were extracted from the corporate data warehouse, a division of the national health laboratory service (nhls). isolate identification and ast microbiology services within the hospital are provided by the nhls. the identification of isolates within the institution was performed using either the vitek®2 (biomerieux inc., marcy l’étoile, france) gram-negative identification (gn id) card or matrix-assisted laser desorption ionisation-time of flight mass spectrometry. these methods are unable to differentiate species within the a. baumannii complex. routine ast was performed using the kirby-bauer disc diffusion susceptibility method or vitek®2 ast-n256 card. isolates that had ast by the former method were excluded from the study. quality control strains and pure cultures of test isolates were used for the vitek®2 ast. isolates were further tested using mbmd when colistin therapy was considered, that is, for clinically significant xdr a. baumannii complex isolates. manual bmd was performed by trained personnel with appropriate controls according to the iso-20776 recommendation. isolates that were colistin-resistant by mbmd were then sent to a reference laboratory for mcr 1-5 testing (data not shown). data analysis all xdr a. baumannii complex isolates cultured between 01 january 2017 and 30 june 2019 were analysed. no patient admission data was available to discriminate between community-acquired and hospital-acquired infections. isolates that were obtained from outpatient departments or without the ward specified were excluded. duplicate patient samples, such as blood cultures collected within two weeks and other sample types collected within one month of the initial sample, were excluded. intravenous central venous catheter tips were not included as they were processed in a separate laboratory using a different automated ast platform. only isolates with both vitek®2 and mbmd colistin susceptibility results were included. the performance of vitek®2 colistin susceptibility testing was determined by evaluating the categorical and essential agreements and the major and vme rates in comparison to the mbmd colistin susceptibility testing method, according to the food and drug administration (fda) recommendations.11 microsoft® excel 2016 (microsoft corporation, redmond, washington, united states) was used for data analysis. the following equations were employed to assess agreement and error rates: categorical agreement = (number of isolates correctly classified by vitek®2 as either colistin susceptible or resistant in comparison to mbmd ÷ total number of isolates tested) × 100     [eqn 1] essential agreement = (number of isolates within one doubling dilutions of the mbmd mic on vitek®2 ÷ total number of isolates tested) × 100     [eqn 2] major error rate = (number of falsely resistant isolates on vitek®2 ÷ number of susceptible isolates by mbmd) × 100     [eqn 3] very major error rate = (number of falsely susceptible isolates by vitek®2 ÷ number of resistant isolates by mbmd) × 100     [eqn 4] results data for 523 isolates were obtained from all specimen types that harboured xdr a. baumannii complex and were submitted for colistin mbmd. after appropriate exclusions, 337 (64%) isolates had both vitek®2 and mbmd mic results (figure 1). figure 1: isolate selection and inclusion for vitek®2 colistin susceptibility assessment. of the 337 isolates with both vitek®2 and mbmd colistin susceptibility results, 20 (6%) were resistant to colistin by mbmd. the highest proportion of colistin-resistant acinetobacter was isolated from tracheal aspirates and swabs (figure 2). figure 2: distribution of colistin-resistant and susceptible xdr a. baumannii complex isolates by sample type (n = 337). vitek®2 was found to have a categorical agreement of 89% (300/337) and an essential agreement of 56% (190/337) with mbmd. the vme rate was 55% (11/20) and the major error rate was 8% (26/317) (figure 3). figure 3: colistin minimum inhibitory concentration (µg/ml) results for xdr a. baumannii complex isolates by both vitek®2 and manual broth micro-dilution. discussion colistin susceptibility testing by mbmd according to iso-20776 is difficult to implement in routine microbiology laboratories. due to financial constraints and the technical competencies required, only one nhls laboratory can offer mbmd in south africa. this is likely to impair patient care due to delays in turnaround times of results. in contrast, vitek®2 is available in most nhls microbiology laboratories and serves as an alternative. however, our study demonstrates unacceptable performance, with 11 of 20 (55%) colistin-resistant isolates being falsely susceptible by vitek®2. the vitek®2 also falsely reported some isolates with mbmd colistin mic of > 64 µg/ml as susceptible. this has the potential to result in inappropriate antimicrobial therapy and adverse patient outcomes. this extremely high vme contrast with previous studies mentioned earlier. however, those studies included fewer a. baumannii isolates compared to our study. in addition to the unacceptable vme rate, the categorical agreement, essential agreement and the major error rates with vitek®2 were also unacceptable according to the fda requirements for an ast testing platform.11 attempts to make mbmd more readily available and easier to implement until other testing options become available are required. matuschek et al. evaluated five recently developed commercial bmd systems – sempa1 (sensititre™ custom plate [thermo fisher scientific, east grinstead, united kingdom], micronaut-s and micronaut mic-strip [merlin diagnostika gmbh, bornheim, germany], sensitest™ [liofilchem, roseto degli abruzzi, italy] and umic [biocentric, bandol, france]).12 these were evaluated against mbmd using various gram-negative organisms including 22 acinetobacter spp isolates.12 they demonstrated overall better performance compared to our findings with vitek®2. the majority of the platforms had acceptable categorical agreement and essential agreement (> 90%), with significantly lower error rates than obtained in this study. interestingly, there were no vmes detected in the a. baumannii isolates. however, this study tested fewer isolates than our study. these methods may offer an alternative to mbmd and further research on their performance is required. limitations only a small number of colistin-resistant isolates were obtained for the study. analysis of larger numbers of resistant isolates with wider mic distribution is required to confirm our finding of high vmes. conclusion based on the results of this study, vitek®2 is not an alternative for mbmd for colistin ast in our setting. further studies are required to determine if the commercially available colistin bmd methods are a cost-effective option with acceptable analytical performance. additionally, the semi-automated platforms such as vitek®2 should be better optimised for colistin ast. acknowledgements the co-authors played an invaluable role in completion of this work. gratitude also goes to the employees of the nhls who ensured that the data used for this study were of high quality. competing interests the authors declare that there were no competing interests, financial or otherwise, that may have affected the writing of this article. authors’ contributions v.f. conceptualised the idea of the manuscript, was the primary author and collated co-authors’ feedback. t.t. contributed to the write-up of the article and provided critical feedback. n.v.k. contributed to the write-up of the article and provided critical feedback. sources of support the authors received no funding for the research or write-up of this study. data availability the data that support the findings made in this study can be made available from the corresponding author, v.f., on request. disclaimer the views expressed in this study are those of the authors and are not an official position of the university of the witwatersrand. references almasaudi sb. acinetobacter spp. as nosocomial pathogens: epidemiology and resistance features. saudi j biol sci. 2018 mar 1;25(3):586–596. https://doi.org/10.1016/j.sjbs.2016.009 ahmed ss, alp e, hopman j, voss a. global epidemiology on colistin-resistant acinetobacter baumannii. j infect dis ther. 2016;4(4):287. https://doi.org/10.4172/2332-0877.1000287 bakthavatchalam y, pragasam a, biswas i, veeraraghavan b. polymyxin susceptibility testing, interpretative breakpoints and resistance mechanisms: an update. j glob antimicrob resist. 2018;12:124–136. https://doi.org/10.1016/j.jgar.2017.09.011 clsi-eucast polymyxin breakpoints working group. recommendations for mic determination of colistin (polymyxin e) [homepage on the internet]. eucast; 2016 [cited 2020 feb 03]. available from: http://www.eucast.org/fileadmin/src/media/pdfs/eucast_files/general_documents/recommendations_for_mic_determination_of_colistin_march_2016.pdf poirel l, jayol a, nordmann p. polymyxins: antibacterial activity, susceptibility testing, and resistance mechanisms encoded by plasmids or chromosomes. clin microbiol rev. 2017 apr 1;30(2):557–596. https://doi.org/10.1128/cmr.00064-16 dafopoulou k, zarkotou o, dimitroulia e, et al. comparative evaluation of colistin susceptibility testing methods among carbapenem-nonsusceptible klebsiella pneumoniae and acinetobacter baumannii clinical isolates. antimicrob agents chemother. 2015 aug 1;59(8):4625–4630. https://doi.org/10.1128/aac.00868-15 lo-ten-foe jr, de smet am, diederen bm, kluytmans ja, van keulen ph. comparative evaluation of the vitek 2, disk diffusion, etest, broth microdilution, and agar dilution susceptibility testing methods for colistin in clinical isolates, including heteroresistant enterobacter cloacae and acinetobacter baumannii strains. antimicrob agents chemother. 2007 jul 23;51(10):3726–3730. https://doi.org/10.1128/aac.01406-06 piewngam p, kiratisin p. comparative assessment of antimicrobial susceptibility testing for tigecycline and colistin against acinetobacter baumannii clinical isolates, including multidrug-resistant isolates. int j antimicrob agents. 2014 nov 1;44(5):396–401. https://doi.org/10.1016/j.ijantimicag.2014.06.014 vourli s, dafopoulou k, vrioni g, tsakris a, pournaras s. evaluation of two automated systems for colistin susceptibility testing of carbapenem-resistant acinetobacter baumannii clinical isolates. j antimicrob chemother. 2017 jun 12;72(9):2528–2530. https://doi.org/10.1093/jac/dkx186 biomerieux. urgent product correction notice [homepage on the internet]. [cited 2020 mar 26]. available from: https://www.bfarm.de/shareddocs/kundeninfos/en/08/2017/04963-17_kundeninfo_en.pdf?__blob=publicationfile&v=1 us food and drug administration. class ii special controls guidance document: antimicrobial susceptibility test (ast) systems; guidance for industry and fda. rockville, md: us food and drug administration; 2003. matuschek e, åhman j, webster c, kahlmeter g. antimicrobial susceptibility testing of colistin–evaluation of seven commercial mic products against standard broth microdilution for escherichia coli, klebsiella pneumoniae, pseudomonas aeruginosa, and acinetobacter spp. clin microbiol infect. 2018 aug 1;24(8):865–870. https://doi.org/10.1016/j.cmi.2017.11.020 abstract introduction methods results discussion acknowledgements references about the author(s) naseem cassim department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa national health laboratory service, johannesburg, south africa neeshan ramdin department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa sadhaseevan moodly department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa national health laboratory service, johannesburg, south africa deborah k. glencross department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa national health laboratory service, johannesburg, south africa citation cassim n, ramdin n, moodly s, glencross dk. cost of running a full-service receiving office at a centralised testing laboratory in south africa. afr j lab med. 2022;11(1), a1504. https://doi.org/10.4102/ajlm.v11i1.1504 original research cost of running a full-service receiving office at a centralised testing laboratory in south africa naseem cassim, neeshan ramdin, sadhaseevan moodly, deborah k. glencross received: 29 dec. 2020; accepted: 19 apr. 2022; published: 13 july 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the national health laboratory service operates a platform of 226 laboratories across south africa, ranging from highly sophisticated central academic hospitals to distant rural hospitals. the core function of the national health laboratory service is to provide cost-effective and efficient health laboratory services in the public healthcare sector. objective: this study aimed to assess the comprehensive cost of running a full-service receiving office (ro) at the charlotte maxeke johannesburg academic hospital (cmjah) laboratory. methods: top-down costing was conducted, with the cost per registration as the main outcome of interest. the annual equivalent costs (aec) for the following categories were determined: registration materials, collection materials, staffing, laboratory equipment, building and electricity, and other operating costs. data for the period from 01 april 2019 to 31 march 2020 were included in the analyses. results: the aec was $1 657 483.00 united states dollars (usd) and the cost per registration was $0.766 usd. staff contributed 59.9% of the total cost per registration, while collection materials contributed 21.4%. the ro core staff (data clerks) contributed 50.8% of the total staffing costs, while messengers and drivers contributed 31.2%. the introduction of order entry at the cmjah and other primary healthcare facilities reduced the total aec by 20%. a single order entry application would serve both the cmjah and primary healthcare facilities hence we would prefer to not refer to order entries. conclusion: providing a comprehensive ro service costs approximately $1.00 usd per registration. the implementation of order entry at the cmjah would reduce aecs substantially and improve efficiency. keywords: receiving office; cost per registration; order entry; costing; pre-analytical. introduction the national health laboratory service (nhls) operates a platform of 226 laboratories across south africa, ranging from central academic to distant rural laboratories, with a mandate to provide cost-effective and efficient services in the public healthcare sector.1 to carry out this mandate, the nhls is premised on three pillars, namely diagnostics, research, and teaching and training.1 the charlotte maxeke johannesburg academic hospital (cmjah) is the largest nhls reference laboratory in south africa. the laboratory is international standards organization 15189-accredited and provides quality results that contribute to patient care.2 this laboratory houses the largest automated laboratory track system within the nhls network that allows third-party instrument connectivity, thus providing a wide repertoire of clinical pathology services.3 the automated track creates a harmonious flow of samples through the pre-analytical, analytical and post-analytical phases and ensures that the laboratory can provide state-of-the-art diagnostic services with efficiency, improved turn-around times, and minimal wastage.3 an estimated 4.8 million tests are performed annually at the cmjah laboratory, with steady year-on-year increases recorded. the receiving office (ro) plays a pivotal role in the correct and timeous capturing of samples into the laboratory information system (lis) to facilitate the delivery of patients’ reports and expenditure reporting. unfortunately, unlike the analytical phase at the cmjah laboratory, the pre-analytical processes at the ro are not automated. pre-analytics refer to the procedures carried out before actual sample testing and include patient identification, preparation, sample collection, sample packaging, sample transportation, and sample preparation for analysis and storage.4 currently, the ro receives completed paper-based request forms to start the data capture process, which is time-consuming as it requires the entry of details of the health facility, healthcare worker, patient, and requested test into the lis. details such as the time of sample receipt, sample sorting, data capture, and delivery to the laboratory are also manually entered into the lis. furthermore, in the present system, samples are handled multiple times in the ro in contrast to the analytical phase where the automated track uses a one-touch approach.3 in other settings, many of these manual processes can be automated through order entry (oe). for example, public sector laboratories such as the inkosi albert luthuli central hospital have adopted a paperless approach that uses oe to replace multiple manual ro tasks.5 order entry is an application that enables healthcare workers to create electronic orders within the facility,6 replacing traditional pen-and-paper methods for ordering laboratory investigations using request forms. a simplified linear workflow for oe would be as follows: at source, the healthcare worker identifies the need for a laboratory test to be performed and uses the oe application to select the tests required, with the patient and healthcare worker information populated by an electronic patient record system (eprc). the order is then electronically transcribed and sent to the national lis where it is accepted and the testing process commences. after results authorisation, data are translated back to the eprc and patient care is provided based on the results.7 with the oe workflow, all the required information is transmitted electronically and testing can commence with minimal ro processing, thus minimising transcription errors and improving order response,8 turn-around time, test ordering efficiency, laboratory utilisation, and, ultimately, patient care.9,10 as oe is part of the clinical workflow, it should ideally be implemented as a collaborative effort with the entire healthcare team.7 order entry can influence and control test ordering patterns through structured order screens, manipulation of order sets and the analysis of real-time data to assess the impact of such changes.10 this is especially important in the context of national treatment guidelines such as for the care of hiv-positive patients, in which tests are performed based on standardised patient workup and care.11 electronic gatekeeping, which is used to reduce unnecessary test requests, can also be programmed into the oe application to alert clinicians at the time of placing an order that their samples will likely not be processed.11 this study aimed to assess the comprehensive cost of running a full-service ro at a busy centralised academic laboratory that processes local samples and receives referred samples for specialist pathology testing and to assess the impact of the implementation of oe on ro costs. methods ethical considerations ethical clearance (approval number: m160978) was obtained from the university of the witwatersrand human research ethics committee (medical). no patient identifiers were collected and thus patient consent was not required. study setting this study was conducted at the full-service ro based at the cmjah laboratory, johannesburg, south africa. data are reported for the april 2019 to march 2020 financial period. workflow at the charlotte maxeke johannesburg academic hospital laboratory receiving office forty-two primary healthcare (phc) facilities and two national central hospitals are directly served by the cmjah laboratory; the laboratory offers all routine haematology, chemistry and microbiology testing, as well as specialist pathology testing (e.g. hiv viral load or cd4 testing, flow cytometry, cytogenetics, etc.). in addition, similar specialised tests that are referred from other phc facilities and hospitals outside the immediate precinct of the cmjah laboratory (n = 354) are also processed at the cmjah laboratory, including those based in the west rand, ekurhuleni, city of johannesburg, and sedibeng districts. samples collected at distant health facilities are delivered to the closest local nhls source laboratory using a hub-and-spoke courier network with multiple health facilities located along designated routes (figure 1). these source laboratories serve their respective surrounding health facilities and perform routine clinical pathology testing. all specialised tests such as hiv viral load are subsequently referred within the nhls network to academic testing facilities such as cmjah. an inter-laboratory referral courier network is used to transport the referred samples to the larger academic laboratories. locally sourced specimens are collected from hospital wards at cmjah (n = 64; samples transported every 2 h) and the nelson mandela children’s hospital (n = 6; samples transported every 4 h). the final source of specimens is the surrounding health facilities (e.g. alexandria community health centre) that deliver samples directly to the cmjah ro using a courier network. figure 1: sources of specimens and activities performed at the charlotte maxeke johannesburg academic hospital laboratory receiving office, south africa, april 2019 – march 2020. pre-analytical activities include sample receipt, sample sorting to identify where testing will take place, and registration on the lis. these activities take place at both the cmjah ro as well as at surrounding source laboratories. the registration process involves entering the details provided on the request form (patient demographics and health facility and healthcare worker details) into the lis and attaching barcodes with the episode numbers on all samples. samples not tested at the source laboratory are referred on the lis; this involves preparing a shipping list and packaging samples for courier pick-up. the pre-analytical activities performed at the cmjah ro are the same for surrounding health facilities. referral samples are accepted on the lis before testing. after testing, patient reports are printed and delivered to each health facility. costing methodology the costing analysis was done using microsoft excel (microsoft corporation, redmond, washington, united states). costs were obtained using historical cmjah expenditure data for the april 2019 to march 2020 financial period. the accounting stance was as a provider of the ro service, that is, all costs were obtained as the provider of laboratory services (costs incurred by the health facility were excluded). a top-down costing approach was used, with the main outcomes of interest being the annual equivalent cost (aec) and cost per registration. the cost per registration refers to the costs of all the activities conducted in the ro for each patient visit, during which one or more tests may be requested. therefore, it is not the cost per sample received as multi-disciplinary registration is performed. multi-disciplinary registration refers to a single registration on a single episode number of multiple tests requested for multiple pathology disciplines. costs were collected in south african rand (zar) and reported in united states dollars (usd) (using the monthly average exchange rate of r14.7835 zar to $1.00 usd for november 2019).12 the consolidated health economic evaluation reporting standards checklist was used in the preparation of this manuscript.13 costs were reported for the following categories: registration materials, collection materials, staff, laboratory equipment, building and electricity, and other operating costs. the consumer price index ranged from 3.6% (november 2019) to 4.6% (february 2020).14 furthermore, the world bank reported an ‘inflation, consumer prices (annual %)’ value of 4.12% for 2019.15 therefore, we assumed a 4% discount rate. organisational overhead costs were excluded; these included all corporate services such as human resources, finance, and information technology offered by the nhls corporate offices. registration materials included items such as disposable gloves, n95 masks, thermal barcode printer paper, paper (to print worklists for the laboratory), disposal boxes, etc. specimen collection materials included laboratory request forms, sharps disposal boxes, specimen collection kits and biohazard bags, which are all issued by the nhls to healthcare facilities for sample collection. receiving office staff included the business manager (grade d5), the ro manager (d1), supervisors (c3), and operational staff, consisting of data clerks (b2/4), messengers (a3), administrative officers (c1), drivers (b4) and cleaners (a1). for staff costs, we used the annual salary data that include medical aid, pension and other allowances.16 staff were categorised as ro core staff, messengers or drivers, ro management, cleaning, and overall management. the percentage of time spent on sample registration by each staff category was determined (table 1). table 1: percentage of time spent on registrations by staff categories and types at the charlotte maxeke johannesburg academic hospital laboratory receiving office, south africa, april 2019 – march 2020. the costs of the following laboratory equipment were included: a messenger monitoring system (used to track and log the times when messengers arrive and collect samples at each ward and deliver samples back to the ro), date and time stamp devices, multi-function printers, barcode scanners, operator chairs, workbenches, computers, network points, barcode printers, specimen tube racks, shopping baskets (used for sample delivery to each section), air conditioners, fridges, freezers (–20 °c), document scanners, and filing cabinets. to calculate the aec for laboratory equipment, working life was set at five years with a discount rate of 4%. working life is defined as the period that an asset is likely to remain in service. we used the microsoft excel ‘sum’ and ‘pmt’ functions (equation 1): studies have reported that the ‘pmt’ function in microsoft excel is easy to use for calculating the aec for equipment using assumptions of the discount rate, working life, and purchase price.17,18 to determine the total building costs, we multiplied the total area of the ro in square metres (m2) by the average building cost of r8163 zar per square metre.19 this average building cost is based on the statistics south africa estimate for building office spaces.19 to calculate the aec for building costs, we used a working life of 50 years and a discount rate of 4% and applied this to the total costs using the pmt formula. we determined the monthly cost of electricity at the cmjah laboratory and attributed 2% of the cost to the ro based on its size. we used the ro income statement to assign other operating costs. these included the aec for items such as cellular phones, computer consumables, printing, telephones, couriers, freight, postage, laundry, accreditation, waste disposal, uniforms, etc. the cost for lis licenses and other costs were also obtained from the income statement. costing analysis we reported the aec and the cost per registration. data were reported for each cost category. the percentage contribution of each cost category to the total cost per registration was reported. we assessed the impact of implementing oe at surrounding phc facilities only versus implementing oe at all surrounding phc facilities and all cmjah wards. to calculate costs for each oe scenario, the percentage reduction in manual ro activities was applied to the staff costs of ro clerks. a rejection rate of 6% was used to determine the reduction in data collection materials and registration materials (based on lis reports). samples are rejected because of electronic gatekeeping rules (such as minimum re-testing intervals) or for failing to meet essential criteria stipulated in the laboratory handbook (such as missing mandatory information or using the incorrect specimen type).20,21,22,23 it was assumed that oe would integrate electronic gatekeeping and rejection rules to reduce wastage.22,23 by integrating these rules, the oe application would alert the healthcare worker that mandatory information was missing and prevent the order from being placed. this would reduce costs by preventing the use of specimen collection materials, as well as other time-consuming activities, including sample transport, sample sorting, data capture, and sample rejection. currently, ro staff capture only 6% of samples that are rejected on the lis. the aec for the base case (as is) was compared to the two oe scenarios for each cost category. results data from a total of 2 163 421 registrations at the cmjah ro were included in the analyses. the cmjah ro consists of 68 employees (excluding the business manager and administrative assistant), including 34 data capturers, 22 messengers, four drivers, four cleaners, three managers or supervisors, and one administrative officer. assuming a 24/7 service for 365 days, this equates to a daily workload of 5927 registrations. on average, it takes 12 min per sample to complete sample receipt, sorting, registration, barcoding, and addition of test tubes to the track for delivery to each laboratory section. annual equivalent cost an aec of $1 657 482 usd was reported for the cmjah ro. staffing contributed $992 664 usd (59.9%), collection materials contributed $355 450 usd (21.4%) and other operating costs contributed $222 653 usd (13.4%) to the aec (table 2). registration materials ($54 622 usd), equipment ($20 444), and building and electricity ($11 650) collectively contributed $86 716 usd (5.2%) to the aec. table 2: annual equivalent costs and the cost per registration for each cost category at the charlotte maxeke johannesburg academic hospital laboratory receiving office, south africa, april 2019 – march 2020. cost per registration the total cost per registration was $0.766 usd. staffing contributed $0.459 usd, collection materials contributed $0.164 usd, other operating costs contributed $0.103 usd, registration materials contributed $0.025 usd, equipment contributed $0.009 usd, and building and electricity contributed $0.005 usd to the cost per registration. staffing costs the ro core staff (50.8%) and messengers and drivers (31.2%) contributed 82.0% of the total staffing costs, while the ro supervisor and ro manager contributed 14.4% of the total staffing costs. impact of order entry introducing oe at the surrounding phc facilities, which contribute 50% of registrations at cmjah, would reduce costs by $285 081 usd. by implementing oe at the surrounding phc facilities and the cmjah wards, the estimated cost savings increased to $335 514 usd (figure 2). this is a 20.2% cost reduction from the current costs of running the ro service. figure 2: impact of order entry implementation on the annual equivalent cost at the charlotte maxeke johannesburg academic hospital (cmjah) laboratory receiving office, south africa, april 2019 – march 2020. discussion the cost to register one sample at the cmjah ro was less than $1.00 usd. staffing was the major contributor to the overall cost, highlighting the manual nature of activities performed by ro staff. collection materials and other operating costs, on the other hand, contributed only one-third of the total ro costs. one of the current data challenges in healthcare services in south africa is that patient demographics and personal identifiers are captured on multiple data systems in different formats. this highlights the inefficiencies of the current paper-based system. at the cmjah, patients are assigned a unique patient identifier and their details are captured on the hospital information system when they first present for care. when they present to the pharmacy or radiology units, for example, their details are re-captured. also, when a laboratory test is requested, this information is transcribed onto the laboratory request form and then manually captured in the lis by data clerks. aside from the multiplied workload, this multipronged, multi-layered approach to patient data capture and sample tracking can lead to transcription errors and data field omissions. these manual transcription errors have been confirmed during random data audits of patient request forms where differences were observed between lis-contained information and information on the request forms (data not shown). inefficient use of existing information within the healthcare system thus increases the workflow complexity and workload in the ro, thereby increasing the need for a large staffing component to process and capture information on the lis. for the implementation of oe across the public health sector in south africa, the use of a national unique patient identifier is imperative to ensure consistency of information irrespective of the site of presentation. currently, no unique patient identifier is used within the public health system.24 in the present system, duplicate patient records are inadvertently created on the hospital information system or eprc systems as most phc facilities use alphanumeric codes that are not based on the national health identifier (nhid) to identify patients.25 the development of an electronic system that can generate a master patient index or nhid would prevent unnecessary data duplication.25 this nhid can be implemented using a centralised, semi-distributed or highly distributed model.25 the different models determine where the nhid is assigned, that is, at the national, regional or facility level.25 the aim would be to develop an nhid system that contains a master record of all patients that have accessed public healthcare services across south africa. a new nhid would only be assigned after searching the national master patient index list to prevent the duplication of patient information. this will facilitate the seamless data transfer of patient information to the oe application, enable longitudinal tracking of patients, and reduce healthcare costs by not repeating laboratory investigations that were already requested by another health facility. the cost to implement oe (capital expenditure and maintenance) and an electronic health record system is a function of bed size.26 the one-time capital cost to implement oe for a 720-bed facility was estimated at $16 026 676 usd (2012 equivalent),26,27 and the annual maintenance costs were estimated to be $2 015 807 usd.26,27 as this data were reported for four hospitals with an existing electronic health record system, we calculated the cost for a single hospital and used the interest rates reported by the international monetary fund to determine the equivalent value in 2019.28 between 2013 and 2019, the south african central bank policy rate reported a cumulative inflation rate of 6.63%.28 this equates to an annual cost of $4 809 449 usd per hospital in 2019.28 in contrast, a district hospital in kenya reported a cost of $2.1 million usd for the implementation of oe and $435 000 usd for annual maintenance.29 given that this hospital has 320 beds, this is not a reasonable cost estimate for oe implementation at cmjah (1088 beds).30 therefore, a local costing study is required to assess the implementation and maintenance costs of oe for all wards at cmjah, nelson mandela children’s hospital, and surrounding phc facilities. given the connectivity and information technology infrastructure challenges in the public health sector in south africa, extensive investments would have to be made to make oe accessible and to develop the necessary interfaces with the different hospital information system and eprc systems used by hospitals and phc facilities. to implement oe at phc facilities, some minimum infrastructures such as tablets, computers, internet connection and an eprc interface are required.9 these are required for the electronic transfer of orders to the lis and the return of results.9 for this electronic transfer, logical observation identifier names and codes could be used.31 implementing oe at the surrounding phc facilities and cmjah wards would result in an estimated annual cost saving of around 20%. should the cost of oe installation be similar to the cost of the cmjah ro, it would be possible to pay the capital cost of the system with the savings generated over a five-year period. there may also be additional cost savings generated by better adherence to treatment and pathology testing guidelines, reduced rejections and more appropriate ordering. another study reported that the implementation of oe at a large academic hospital resulted in an annual saving of $2.2 million usd compared to an investment of $11.8 million usd, albeit it took over five years for these savings to be realised.32 as indicated by birkmeyer et al., the primary motivation for introducing oe should be to improve the quality of care provided and not to reduce healthcare expenditure.33 it has been shown that oe has the potential to increase efficiency and effectiveness, thereby enhancing the quality of patient care.32 when implemented with clinical decision support systems, oe has the potential to reduce rejections and unnecessary test requests and alert clinicians of the required mandatory data fields and specimen criteria.34 in addition, as an electronic platform, oe could streamline the workflow in the health facility. the development of eprc systems in the united states has been challenging due to closed, proprietary and incompatible systems.35 likewise, for developing countries, purchasing proprietary oe systems from developed countries leads to higher implementation costs due to travel costs and exchange rate fluctuation. as a result, the use of locally developed or open-source oe could result in lower implementation costs and minimal expenditure required to extend the application across the public health sector. a good example of a locally developed information system is tier.net, which is used to collect data on patients receiving antiretroviral therapy.34 after being piloted in the western cape province, tier.net is now used across south africa. the implementation of oe across the public health system beyond cmjah would dramatically streamline the process of placing laboratory orders and receiving results. the major benefit of oe would be a move from paper-based to electronic systems, removing the requirement to re-capture information that exists in the eprc. in addition, the ro at laboratories would be dramatically scaled down given the electronic transfer of data. this would change the visibility of orders at both the health facility and laboratory, with better monitoring of samples and results that are outstanding. overall, the interface with the laboratory would become efficient and streamlined by bypassing multiple manual steps in the current workflow (figure 3). figure 3: typical workflow with order entry implemented at the charlotte maxeke johannesburg academic hospital laboratory receiving office, south africa, april 2019 – march 2020. once oe is implemented, the cmjah ro data clerks will not be retrenched. staff may be re-allocated, deployed to other duties, or transferred to other laboratories with staff shortages. in addition, clerks could be encouraged to consider training as laboratory technicians and migrating to the analytical phase where shortages exist. limitations this study excluded the cost of organisational overheads. should the organisational overheads be included, it would result in a minor change to the cost per registration as the organisational overheads would be divided by the number of registrations across the nhls. the costs reported would also vary depending on the size and complexity of the ro, as well as the distance of the ro from source laboratories. in a small province such as gauteng, health facilities are close (≤ 50 km) to testing laboratories. however, in more rural provinces such as the northern cape, facilities could be located over 300 km from their local laboratory, thus leading to higher logistics costs. another limitation is that this study did not consider the costs of training required to introduce oe. although the coronavirus disease 2019 pandemic has demonstrated that it is possible to conduct clinical training remotely,36 health facilities may not have access to the necessary bandwidth and computers present in a university environment.36 therefore, the cost of introducing oe should be assessed given the challenges with remote learning, especially in rural settings. conclusion providing a comprehensive ro service at a large referral laboratory in south africa costs less than $1.00 usd per registration; however, most of this expenditure can be attributed to the high ro staffing costs due to the manual nature of ro activities. the implementation of oe has the potential to reduce ro costs by as much as 20% while also improving efficiency, reducing turn-around times, and improving patient outcomes. acknowledgements the authors would like to thank the various suppliers for providing quotations. we would also like to thank the national health laboratory service for providing costs from the oracle erp system. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions n.c. developed and executed the research, conducted the costing analysis, prepared the first draft and made editorial input. s.m. and n.r. provided costing information, reviewed the data and reviewed the article. d.k.g. was the project leader and provided editorial input. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the data are not publicly available due to the authors not having permission to share the costing data. disclaimer the views expressed in this manuscript are those of the authors and not those of the university of witwatersrand or the nhls. references national health laboratory service (nhls). annual report 2018/19 [homepage on the internet]. johannesburg: national health laboratory service (nhls); 2019 [cited 2020 feb 19]. available from: https://www.nhls.ac.za/key-documents/annual-reports/ schneider f, maurer c, friedberg rc. international organization for standardization (iso) 15189. ann lab med. 2017;37(5):365–370. https://doi.org.10.3343/alm.2017.37.5.365 south african government. gauteng health launches automated pathology laboratory at charlotte maxeke [homepage on the internet]. johannesburg: south african government; 2017 [cited 2020 nov 03]. available from: https://www.gov.za/speeches/official-launch-nhls-charlotte-maxeke-johannesburg-academic-hospital%e2%80%99s-automated-pathology plebani m. quality indicators to detect pre-analytical errors in laboratory testing. clin biochem rev. 2012;33(3):85–8. seemela vpp. public-private partnership as a tool for developmental state. j public admin. 2008;43(si-2):483–491. https://doi.org.doi:10.10520/ejc51650 dixon be, zafar a. inpatient computerized provider order entry (cpoe): findings from the ahrq health it portfolio [homepage on the internet]. agency for healthcare research and quality (ahrq), national resource center for health information technology; 2009 [cited 2021 nov 05]. available from: https://digital.ahrq.gov/sites/default/files/docs/page/09-0031-ef_cpoe.pdf aarts j, ash j, berg m. extending the understanding of computerized physician order entry: implications for professional collaboration, workflow and quality of care. int j med inform. 2007;76:s4–s13. https://doi.org/10.1016/j.ijmedinf.2006.05.009 teich jm, hurley jf, beckley rf, aranow m. design of an easy-to-use physician order entry system with support for nursing and ancillary departments. proc annu symp comput appl med care. 1992:99–103. baron jm, dighe as. computerized provider order entry in the clinical laboratory. j pathol inform. 2011;2(1):35. https://doi.org.10.4103/2153-3539.83740 westbrook ji, georgiou a, dimos a, germanos t. computerised pathology test order entry reduces laboratory turnaround times and influences tests ordered by hospital clinicians: a controlled before and after study. j clin pathol. 2006;59(5):533–536. https://doi.org.10.1136/jcp.2005.029983 cassim n, coetzee lm, schnippel k, glencross dk. compliance to hiv treatment monitoring guidelines can reduce laboratory costs. s afr j hiv med. 2016;17(1):449. https://doi.org.10.4102/sajhivmed.v17i1.449 standard bank. rand/dollar exchange rate: monthly average for november 2019 [homepage on the internet]. standard bank; 2020 [cited 2020 oct 20] available from: https://ws15.standardbank.co.za/cdp_bluecurve/publishedresearchdocument?id=650c6031-0c3c-4a49-b0eb-7d1450c782ca husereau d, drummond m, petrou s, et al. consolidated health economic evaluation reporting standards (cheers) statement. eur j health econ. 2013;14(3):367–372. https://doi.org.10.1007/s10198-013-0471-6 south african reserve bank (sarb). selected statistics [homepage on the internet]. 2021 [cited 2021 nov 03]. available from: https://www.resbank.co.za/en/home/what-we-do/statistics/releases/selected-statistics world bank. inflation, consumer prices (annual %) – south africa [homepage on the internet]. 2021 [cited 2021 nov 03]. available from: https://data.worldbank.org/indicator/fp.cpi.totl.zg?locations=za payscale. average salary for nhls employees in south africa [homepage on the internet]. 2021 [cited 2021 nov 04]. available from: https://www.payscale.com/research/za/employer=nhls/salary larson b, schnippel k, ndibongo b, long l, fox mp, rosen s. how to estimate the cost of point-of-care cd4 testing in program settings: an example using the alere pima™ analyzer in south africa. plos one. 2012;7(4):e35444. https://doi.org.10.1371/journal.pone.0035444 george g, chetty t, strauss m, et al. costing analysis of an sms-based intervention to promote hiv self-testing amongst truckers and sex workers in kenya. plos one. 2018;13(7):e0197305. https://doi.org.10.1371/journal.pone.0197305 statistics south africa (stats sa). construction: what are the costs per square metre? [homepage on the internet]. statistics south africa (stats sa); 2019 [cited 2020 oct 20]. available from: http://www.statssa.gov.za/?p=7974 national department of health (ndoh), national health laboratory service (nhls). primary health care laboratory handbook: a step by step guide [homepage on the internet]. 2018 [cited 2021 nov 03]. available from: https://www.idealhealthfacility.org.za/app/document/download/20 pema ak, kiabilua o, pillay ts. demand management by electronic gatekeeping of test requests does not influence requesting behaviour or save costs dramatically. ann clin biochem. 2018;55(2):244–253. https://doi.org.10.1177/0004563217707980 elnenaei mo, campbell sg, thoni aj, lou a, crocker bd, nassar ba. an effective utilization management strategy by dual approach of influencing physician ordering and gate keeping. clin biochem. 2016;49(3):208–212. https://doi.org/10.1016/j.clinbiochem.2015.11.005 sripa p, hayhoe b, garg p, majeed a, greenfield g. impact of gp gatekeeping on quality of care, and health outcomes, use, and expenditure: a systematic review. br j gen pract. 2019;69(682):e294. https://doi.org.10.3399/bjgp19x702209 haeri mazanderani a, sherman gg, moyo f, goga ae, feucht u. leveraging the road to health booklet as a unique patient identifier to monitor the prevention of mother-to-child transmission programme. s afr med j. 2018;108(9):729–733. https://doi.org.10.7196/samj.2018.v108i9.13093 beck ej, shields jm, tanna g, et al. developing and implementing national health identifiers in resource limited countries: why, what, who, when and how? glob health action. 2018;11(1):1440782. https://doi.org.10.1080/16549716.2018.1440782 nuckols tk, asch sm, patel v, et al. implementing computerized provider order entry in acute care hospitals in the united states could generate substantial savings to society. jt comm j qual patient saf. 2015;41(8):341–350. https://doi.org.10.1016/s1553-7250(15)41045-1 zimlichman e, keohane c, franz c, et al. return on investment for vendor computerized physician order entry in four community hospitals: the importance of decision support. jt comm j qual patient saf. 2013;39(7):312–318. https://doi.org.10.1016/s1553-7250(13)39044-8 international monetary fund (imf). interest rates selected indicators: united states [homepage on the internet]. 2021 [cited 2021 nov 08]. available from: https://data.imf.org/regular.aspx?key=61545855 kathini e, kiongo jg, editors. utilization levels of computerized physician order entry (cpoe) by health care workers in mbagathi district hospital nairobi, kenya. nurs health sci. 2020;9(2):57–64. https://doi.org.10.9790/1959-0902025764 nganga sw, otieno na, adero m, et al. patient and provider perspectives on how trust influences maternal vaccine acceptance among pregnant women in kenya. bmc health serv res. 2019;19(1):747. https://doi.org.10.1186/s12913-019-4537-8 campbell ws, karlsson d, vreeman dj, lazenby aj, talmon ga, campbell jr. a computable pathology report for precision medicine: extending an observables ontology unifying snomed ct and loinc. j am med inform assoc. 2018;25(3):259–266. https://doi.org.10.1093/jamia/ocx097 kaushal r, jha ak, franz c, et al. return on investment for a computerized physician order entry system. j am med inform assoc. 2006;13(3):261–266. https://doi.org.10.1197/jamia.m1984 birkmeyer cm, lee j, bates dw, birkmeyer jd. will electronic order entry reduce health care costs? eff clin pract. 2002;5(2):67–74. osler m, hilderbrand k, hennessey c, et al. a three-tier framework for monitoring antiretroviral therapy in high hiv burden settings. j int aids society. 2014;17(1):18908. https://doi.org.https://doi.org/10.7448/ias.17.1.18908 fraser h, biondich p, moodley d, choi s, mamlin b, szolovits p. implementing electronic medical record systems in developing countries. j innov health inform. 2005;13(2):83–95. https://doi.org.10.14236/jhi.v13i2.585 schmutz ams, jenkins ls, coetzee f, et al. re-imagining health professions education in the coronavirus disease 2019 era: perspectives from south africa. afr j prim health care fam med. 2021;13(1):e1–e5. https://doi.org.10.4102/phcfm.v13i1.2948 abstract introduction methods results discussion acknowledgements references about the author(s) leonard mutema department of haematological pathology, tygerberg hospital, national health laboratory service and stellenbosch university, cape town, south africa department of internal medicine, university of kwazulu-natal, durban, south africa zivanai chapanduka department of haematological pathology, tygerberg hospital, national health laboratory service and stellenbosch university, cape town, south africa fungai musaigwa department of haematological pathology, tygerberg hospital, national health laboratory service and stellenbosch university, cape town, south africa nomusa mashigo department of haematological pathology, tygerberg hospital, national health laboratory service and stellenbosch university, cape town, south africa citation mutema l, chapanduka z, musaigwa f, mashigo n. in-depth investigation of turn-around time of full blood count tests requested from a clinical haematology outpatient department in cape town, south africa. afr j lab med. 2021;10(1), a1318. https://doi.org/10.4102/ajlm.v10i1.1318 original research in-depth investigation of turn-around time of full blood count tests requested from a clinical haematology outpatient department in cape town, south africa leonard mutema, zivanai chapanduka, fungai musaigwa, nomusa mashigo received: 27 june 2020; accepted: 06 jan. 2021; published: 29 apr. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the performance of laboratories can be objectively assessed using the overall turn-around time (tat). however, tat is defined differently by the laboratory and clinicians; therefore, it is important to determine the contribution of all the different components making up the laboratory test cycle. objective: we carried out a retrospective analysis of the tat of full blood count tests requested from the haematology outpatient department at tygerberg academic hospital in cape town, south africa, with an aim to assess laboratory performance and to identify critical steps influencing tat. methods: a retrospective audit was carried out, focused on the full blood count tests from the haematology outpatient department within a period of 3 months between 01 february and 30 april 2018. data was extracted from the national health laboratory service laboratory information system. the time intervals of all the phases of the test cycle were determined and total tat and within-laboratory (intra-lab) tat were calculated. results: a total of 1176 tests were analysed. the total tat median was 275 (interquartile range [iqr] 200.0–1537.7) min with the most prolonged phase being from authorisation to review by clinicians (median 114 min; iqr: 37.0–1338.5 min). the median intra-lab tat was 55 (iqr 40–81) min and 90% of the samples were processed in the laboratory within 134 min of registration. conclusion: our findings showed that the intra-lab tat was within the set internal benchmark of 3 h. operational phases that were independent of the laboratory processes contributed the most to total tat. keywords: audit; turn-around time; full blood count; haematology clinic. introduction the increase in the number of patients accessing the public health system and the disproportionate patient to healthcare provider ratio in developing countries necessitates more efficient and cost-effective approaches to healthcare provision.1,2 the efficiency of healthcare providers is evaluated in part by how rapidly diagnoses are made and patients are prioritised for treatment.3 in addition, the rate at which laboratory results are made accessible to clinicians impacts both the patient outcome and the overall performance of a diagnostic laboratory.4 a prolonged turn-around time (tat) results in delayed diagnosis and impacts the management of patients. this may lead to prolonged hospital stay and increased costs.5,6 the consequences of prolonged tat are more pronounced in an emergency service setting and in outpatient departments, where a narrow window of opportunity exists to make key management decisions.7 therefore, constant monitoring and improvement of the tat in this setting is important to ensure the efficiency of laboratory services.8 the tat is crucial in clinical practice and is used as a measure of the performance of laboratories.5 however, the tat definition often varies between laboratories and clinicians, often resulting in unrealistic expectations.5,6,9,10 total tat is categorised into preanalytical, analytical and post-analytical stages and some define it as time from collection of the sample to when results are available for review by clinicians.9 clinicians tend to view tat as the time between requisition of a test and when they view a result.11 laboratories are inclined to exclude components of the test cycle that they have no control over, such as phlebotomy and specimen transport12,13 because it is difficult for laboratories to address delays in these phases.5 therefore, in laboratories, the tat is usually defined as a measure of the time taken from sample registration to authorisation of results, also known as intra-lab tat.5 although this approach is not all inclusive of the phases of total tat, it is generally accepted as the best representation of those elements of tat that the laboratory controls.12 to assess the hypothesis that our laboratory performance was within set local benchmarks and comparable with the widely accepted international benchmark of completion of 90% of the sample processing within 60 min, we performed a retrospective analysis to evaluate the respective contribution of the various phases of the test cycle to the total tat at tygerberg academic hospital (tbh) in cape town, south africa.14 methods ethical considerations this retrospective study was approved by the stellenbosch university research ethics committee (s18/10/232) and performed according to the declaration of helsinki. a waiver of consent was obtained, and patient confidentiality was maintained. moreover, patient-level data was not accessed. study design and setting a retrospective audit was conducted over a 3-month period between 01 february and 30 april 2018 at the haematological pathology laboratory of the tertiary referral tbh in cape town, south africa. the laboratory and its reception operate on a 24-h service and have three shifts: regular shift (08:00 to 16:30), evening shift (16:30 to 20:00) and night shift (20:00 to 08:00). the laboratory provides diagnostic pathology services to regional hospitals and clinics in the western cape, south africa. at tbh laboratory reception, samples go through the processes of sorting, registration and labelling before being transported to the haematology laboratory for analysis where samples are loaded into a siemens advia 2120i haematology analyser (siemens healthcare diagnostics, erlangen, germany). this analyser has the capacity of performing 120 full blood counts (fbcs) and differentials per hour. from the haematology analyser the preliminary results are entered into the laboratory information system (lis) at which point the results are available to clinicians as provisional results. the laboratory fast-tracks all urgent samples and aims to release the results within 3 h. samples from the haematology clinic are treated as urgent and are colour coded and given preference over other routine samples. the lis (intersystems trakcare® lab enterprise, cambridge, massachusetts, united states) tracks the processing of samples at various time points (preanalytical, analytical, and post-analytical). the haematology clinic is housed in a separate building from the main building that houses the haematology laboratory. at this clinic, tests are requested through paper forms which are then placed alongside the samples into transporting bags and transported to the laboratory reception by porters or, rarely, by doctors or nurses. the clinic operates monday to friday from 07:00 to 16:00. clinicians start seeing patients at 08:00 and most of the patient consultations are usually complete by 14:00, after which time the clinicians leave for the ward. patients who arrive late are seen by one clinician until 16:00 when the clinic closes. data collection we retrieved and extracted data from the lis to determine the tat reflecting all the operational phases. reports with two or more missing entries of the date or time points on the lis were excluded. full blood counts done as part of bone marrow examination requests were also excluded as they are not reported separately but as part of the full bone marrow report. four main phases of the test cycles were identified and evaluated. the first phase is between collection of samples (phlebotomy) and registration of the same in the laboratory. this is followed by the phase between registration of samples, processing by the haematology analyser, and loading of preliminary results into the lis. the third phase is when preliminary results are reviewed by laboratory staff and then authorised, and the final phase is from authorisation of results to when clinicians view these results. in this study, the total tat was defined as the time taken from specimen collection to the review of the results by the requesting clinician, while the intra-laboratory tat was defined as the time taken from specimen registration to authorisation of results after review of preliminary results by the laboratory staff. total tat was therefore calculated, for each sample, by adding the time taken in the four phases which are: collection to registration, registration to acquisition into lis, acquisition into lis to authorisation and, finally, authorisation to review by clinicians. intra-lab tat was calculated by adding the time taken in the two phases which are: registration to acquisition into lis and acquisition into lis to authorisation of results. we also carried out a sub-analysis of samples collected in the month of april. the operation phase from the collection of samples to registration was divided into two phases: transportation time (collection to receipt in the laboratory) and sorting time (from receipt in the laboratory to registration of samples). the relative contribution of these two phases were determined and compared to that of a similar study.12 statistical analysis data items which included sample collection, registration, acquisition to lis, authorisation and review by clinicians were extracted from the lis onto microsoft® office excel (redmond, washington, united states) 2016 (version 16.0). assessment of normality was done using the kolmogorov-smirnov test at p < 0.05 significance level.15 descriptive statistics, including median, percentages and interquartile range (iqr) were used. intra-lab and total tat were expressed as median with iqr and time to complete 90% of tests (90% completion time).16 all time intervals were calculated in minutes. the chi-square test was used to compare the expected total tat with the observed total tat and to compare the sorting time of our laboratory with that of a study carried out at new york presbyterian hospital12; values of p < 0.05 were considered statistically significant. using the percentage contribution of each phase for all 1176 samples, median percentages were calculated. the same was done for the intra-lab tat for each of the 1176 samples. all data analyses were performed using statistica software version 8 (tibco software inc., palo alto, california, united states). results a total of 1505 fbcs were requested between 01 february and 30 april 2018 from the haematology clinic, tbh, cape town. we performed an analysis on a subset of the retrieved fbc reports over the study period. a total of 329 (22%) patient reports were excluded either due to missing data (2 or more missing data points) or being reports for fbcs requested as part of a bone marrow biopsy (figure 1). in all, this retrospective study included a total of 1176 fbc reports. delays in registration of samples after receipt by the laboratory would not be reflected on the intra-laboratory tat but on the total tat. therefore, a sub-analysis of the phase between collection and registration was performed for the month of april 2018. this comprised 412 samples for the phase between collection of samples to receipt in the laboratory and 420 samples for the phase from receipt to registration. seventy percent of the 1176 samples were collected by 9:00 and 90% by 11.00. considering the closing time of the clinic, the clinicians had a window of 7 h and 5 h to receive results and make clinical decisions for 70% and 90% of patients. figure 1: screening and selection of reports of full blood counts requested from the haematology outpatient department at tygerberg academic hospital in cape town, south africa, over the period february 2018 to april 2018. intra-laboratory turn-around time and total turn-around time the majority of the fbc samples (93.3%) had an intra-lab tat of less than 3 h and most of them (80.1%) were processed within 90 min (table 1). the median intra-lab tat was 55 (iqr 40–81) min and had a median percentage of 17.1% of the total tat (table 2). ninety percent of the results were ready for viewing by clinicians within 134 min of registration. the median total tat was 275 (iqr 200.0–1537.7) min. the phases within the preanalytical and post-analytical phases contribute the most to the total tat, with collection time to registration and time to review results by clinicians representing a median of 32.5% (iqr 7.8–48.7) and 44.3% (iqr 18.1–87.0). the total tat was significantly delayed compared to the calculated expected tat (p < 0.05). table 1: contribution of the different phases of sample processing to total turn-around time and intra-lab turn-around time. table 2: summary of turn-around times across phases of workflow for 1176 full blood count requests from the haematology outpatient department at tygerberg academic hospital in cape town, south africa, over the period february 2018 to april 2018. preanalytical phase the preanalytical phase, defined in this study as the time from collection of samples to registration, had a median of 96 min (table 2). ninety percent of the samples completed this phase within 218 min. a sub-analysis of this phase during the month of april revealed a median of 60 (iqr 45–85) min for the period between collection of the sample to receipt in the laboratory (transportation) and a median of 39 (iqr 23.35–58.00) min from receipt to registration (sorting) (table 3). most of the samples (90%) spent less than 95 min in the sorting area. our laboratory performed significantly worse when we compared the time spent in the sorting area for each sample in april with the median of 15 min at new york presbyterian hospital, another tertiary hospital described in literature, using the chi-square test.12 table 3: summary of phases between collection to registration of full blood count samples at the haematology outpatient department for the month of april 2018 at tygerberg academic hospital in cape town, south africa. analytical phases we categorised the analytical phase into two independent intervals (interval 2 and interval 3) (table 1). our analysis showed that most samples (73.3%) spent less than 60 min in interval 2 while 85.2% of samples were authorised within 30 min in interval 3. notably, a few samples, 26.6 % and 6.3%, took more than 1 h in interval 2 and interval 3. the authorisation of reports is also impacted by the battery of laboratory tests requested and this may explain the minority of samples authorised after an hour. post-analytical phase we further evaluated the time taken by clinicians to review the authorised reports (interval 4). we observed a significant number of reports (48.9%) that were accessed after 2 h, while only 22.7% of the reports were viewed within 30 min (table 1). notably, 38.6% of reports were viewed after 4 h. the median time for this phase was 114 (iqr 37.0–1338.5) min and the time to view 90% of the reports was 10.78 days (table 2). discussion the constant increase in medical care cost and the growing demand for improved quality of care has necessitated the monitoring of determinants of the quality of healthcare services.17,18 the tat is one of the determinants that is commonly measured, as it is both objective and easily measured.19 however, different views exist regarding the definition of what the composition of the tat should be. these are often compounded by the lack of an interface for dialogue between the laboratory staff and clinicians. howanitz et al. showed that there was also no agreement among clinicians as to the definition of tat, with 56% using test requisition as the first step of tat, while 44% used the time of phlebotomy as the initial step. although most studies use reporting of results as the endpoint of tat,17 this study defined total tat as the time period from the time of phlebotomy to the time the results are reviewed by clinicians, whereas we considered the intra-laboratory tat as the time from registration of the sample to reporting of results (authorisation). this was done in efforts to determine the relative contribution of different phases of the laboratory process to tat and to identify phases requiring the most attention in alleviating delays. this study showed that the phases outside of the laboratory environment needed the most attention and that the laboratory was meeting its predetermined mandate. samples from the haematology outpatient department at tbh are regarded as urgent because for most patients, fbcs are required to make decisions on chemotherapy, transfusion and other interventions before leaving the clinic. the results of this study were therefore compared to findings of studies on tat of emergency departments. our findings were similar to those previously reported.5,20 the preanalytical and post-analytical phases accounted for a greater proportion of the total tat, with a median of 60 min transportation time (preanalytical phase) and 48.9% of the post-analytical phase taking more than 120 min. in contrast, most of the samples were processed in the laboratory within 90 min. the median intra-lab tat was 55 (iqr 40–81) min and 90% of samples were authorised within 134 min. therefore, our findings suggest that the laboratory is performing within the set threshold of 3 h as stated by the tbh laboratory standard operating procedure hae1813 for tests from the haematology clinic. this highlights the need to further interrogate the phases that the laboratory has no control over, that is, the preanalytical and post-analytical phases. in addition to the lack of consensus on the definition of tat, there is also no specific benchmark for tat parameters.21 however, databases used to benchmark tat thresholds can be obtained from the college of american pathologists q-probes and q-tracks programmes.16 we therefore used these databases and experiences from other international hospitals to assess our performance. the 1998 college of american pathologists q-probes study of emergency department tats showed a 90% completion time for haemoglobin testing of 55 min or less.22 in this study 90% completion time of 60 min or less is recommended,22 as opposed to that of tbh which is 134 min. in a survey carried out in nigeria involving 109 doctors, 91.3% of the respondents considered a tat of less than 2 h as ideal for their laboratory, although their median tat in emergency rooms was 5.12 h.23 in a chinese national survey the median intra-lab tat for white blood cells was 44.7 min.24 although our laboratory performance was within the local threshold, our laboratory performed below the expectations when compared to other laboratories in a similar setting.5,7,23,24 the april sub-analysis exposed the sorting area as a problematic area that is responsible for prolongation of the intra-lab tat and ultimately total tat. few studies focus on the analysis of this phase as the time of receipt of samples in the laboratory is often missing; therefore, the study at new york presbyterian hospital, which analysed total tat with emphasis on quantification of sorting time, was best suited for comparison with this study.12 as with this study, sorting time was also prolonged at new york presbyterian hospital. opening of sample bags, generation and printing of barcodes and decoding handwritten requests were responsible for the delays in the sorting area, which is also partly the case at tbh.12 electronic requesting of tests as well as the generation of barcodes at the time of requesting tests were suggested as solutions to this delay.12 total tat was significantly prolonged compared to the expected tat, with phases outside the laboratory causing the most delay. the time taken to view results by clinicians was the longest with a median time of 114 (iqr 37.0–1338.5) min. this could be due to the limited period during which clinicians can view results. once this window has elapsed the results can only be viewed on the patient’s next appointment which can vary from a day to several months later. this is a limitation of total tat in assessing laboratory performance, which explains in part why laboratories resort to the use of intra-lab tat.12 these delays can be improved by ensuring patients’ punctuality and having the laboratory contact the clinicians with results as soon as they are authorised. the other cause of delay for total tat was the time taken to transport samples from the clinic to the laboratory as well as sorting time. porters tend to wait for samples to pile up before transporting them. having a set time for phlebotomy and ensuring that patients avail themselves at this time may mitigate this problem. in addition, the use of pneumatic tube systems results in faster and reliable transportation of samples.25,26 as it is noted that a pragmatic approach is to set tat goals locally, informed by both the published literature and by local expectations,5 we made the following recommendations: establishment of point-of-care testing at the haematology clinic, employment of additional staff for the sorting area or the use of an automated barcode system, and the use of pneumatic tube systems. we also recommend the use of an interactive tat dashboard, a recently described system that offers information to enable the review of performance in real time.27 this may help in ensuring timely response to changes in performance. however, solutions requiring extra funding may take time to implement due to economic challenges facing the health sector in south africa.2,28 as noted earlier, some results are only reviewed on the patient’s next visit and this may pose serious challenges to patient care. therefore, the use of other communication avenues such as short message services, whatsapp, or emails to send authorised results to clinicians is recommended. not only will this improve the total tat, but it will also allow clinicians to act on results immediately rather than waiting for the patient’s next appointment. another suggestion is for clinicians to employ a clerk to review results even after the clinicians have left the clinic. for the laboratory, we recommend that considerations be made to incorporate phlebotomy and specimen transportation services into its armamentarium of service delivery. this will increase laboratory influence on preanalytical processes and subsequently improve total tat. limitations this study was limited by the absence of an interventional process, and we therefore recommend a follow-up audit after implementing some of the proposed solutions. the results from this study cannot be extrapolated to all hospitals and departments but only to those with a similar setting, as they are solely based on the experience of the haematology clinic at tbh. the collection times were based on what was reported by the nurses at the clinic; therefore, the accuracy of the recorded time following each phlebotomy cannot be ascertained. this is not the case for other time points retrieved from the lis as they are computer generated in real time. in addition, the laboratory receipt time for samples was not available for the entire study period. as noted earlier, 22% of the data had 2 or more missing data points which were critical in the calculation of intra-lab tat and total tat. this has the potential of introducing bias, which is likely if the proportion of missing data is greater than 10%.29 these missing data points were random occurrences on the extracted lis data and tats could not be computed; therefore, the samples were removed in a non-biased manner by listwise deletion. due to the random nature of these missing points, a case is therefore made that the generalisability of the study is not affected. the statistical method of handling missing data used in this case was complete-case analysis, which only includes participants or variables that are complete on all waves of data collection.30 conclusion this study demonstrated that tbh laboratory was compliant with its intra-lab tat benchmark and that the 90% completion time target was achieved for samples from the haematology outpatient clinic. the phases outside the control of the laboratory were primarily responsible for prolonged total tats. the monitoring of tat is a powerful tool for assessing a laboratory service’s performance and contributes to patient care. however, as demonstrated in this study, monitoring and process improvement requires measurement of tats for individual phases of the test cycle. acknowledgements the authors would like to thank ms fazlin kolia, the laboratory manager, for providing us with permission to use lis data and standard operation procedures. we also would like to extend our gratitude to mr wessel kleinhans for his critical role in data mining from the lis. lastly, we would like to thank prof. bongani nkambule for assistance in analysis of the data. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions n.m. and z.c. supervised the study by providing leadership and oversight. l.m. conceptualised the study, conducted the research and wrote the initial manuscript. f.m. and l.m. conducted data analysis. all authors contributed to final manuscript development. sources of support the authors received no financial support for the research, authorship or publication of this article. data availability the data that support the findings of this study are available from the corresponding author, l.m., upon reasonable request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references veld m, van de voorde k. how to take care of nurses in your organization: two types of exchange relationships compared. j adv nurs. 2013;70(4):855–865. https://doi.org/10.1111/jan.12247 van rensburg h. south africa’s protracted struggle for equal distribution and equitable access – still not there. 2014;12(26):1–16. https://doi.org/10.1186/1478-4491-12-26 mishra v. measuring technical efficiency in healthcare service: a case study. pacific bus rev int. 2019;11(6):36–43. valenstein p. laboratory turnaround time. am j clin pathol. 1996;105(6):676–688. https://doi.org/10.1093/ajcp/105.6.676 hawkins rc. laboratory turnaround time. clin biochem rev. 2007;28(4):179–194. jalili m, shalileh k, mojtahed m, moradi-lakesh m. identifying causes of laboratory turnaround time delay in the emergency department. arch iran med. 2012;15(12):759–763. kaushik n, khangulov vs, o’hara m, arnaout r. reduction in laboratory turnaround time decreases emergency room length of stay. open access emerg med. 2018;10:37–45. https://doi.org/10.2147/oaem.s155988 imoh lc, mutale m, parker ct, erasmus rt, zemlin ae. laboratory-based clinical audit as a tool for continual improvement: an example from csf chemistry turnaround time audit in a south-african teaching hospital. biochem medica. 2016;26(2):194–201. https://doi.org/10.11613/bm.2016.021 schimke i. quality and timeliness in medical laboratory testing. anal bioanal chem. 2009;393(5):1499–1504. https://doi.org/10.1007/s00216-008-2349-5 groenewald aj, potgieter hd. physician satisfaction and emergency (stat) laboratory turnaround time during various developmental stages. med technol sa. 2014;28(1):20–25. weinstein s. quality in pathology laboratory practice. j qual clin pr. 1995;15(2):121–126. stotler ba, kratz a. determination of turnaround time in the clinical laboratory. am j clin pathol. 2012;138(5):724–729. https://doi.org/10.1309/ajcpyhbt9oqrm8dx chung hj, lee w, chun s, park h-i, min wk. analysis of turnaround time by subdividing three phases for outpatient chemistry specimens. ann clin lab sci. 2009;39(2):144–149. howanitz pj. errors in laboratory medicine: practical lessons to improve patient safety. arch pathol lab med. 2005;129(10):1252–1261. mishra p, pandey c, singh u, gupta a, sahu c, keshri a. descriptive statistics and normality tests for statistical data. ann card anaesth. 2019;22(1):67–72. https://doi.org/10.4103/aca.aca_157_18 howanitz jh, howanitz pj. timeliness as a quality attribute and strategy. am j clin pathol. 2001;116(3):311–315. https://doi.org/10.1309/h0dy-6vtw-nb36-u3l6 howanitz p, cembrowski g, steindel sj, long t. physician goals and laboratory test turnaround times. a college of american pathologists q-probes study of 2763 clinicians and 722 institutions. arch pathol lab med. 1993;117(1):22–28. breil b, fritz f, thiemann v, dugas m. mapping turnaround times (tat) to a generic timeline: a systematic review of tat definitions in clinical domains. bmc med inform decis mak. 2011;11(34):1–12. https://doi.org/10.1186/1472-6947-11-34 bloch dm. computer-generated management tools for the clinical pathology laboratory – ii. computer-generated graphic work flow. j med syst. 1982;6(3):305–310. https://doi.org/10.1007/bf00992807 mahdaviazad h, javidialesaadi f, hosseinzadeh m, masoompour sm. turnaround times for hematology and chemistry tests in the emergency department: experience of a teaching hospital in iran. shiraz e med j. 2016;17(4–5):e37101. https://doi.org/10.17795/semj37101 dey b, bharti j, chakraborty m. laboratory turnaround time. int j heal sci res. 2013;3(5):82–84. steindel sj, howanitz pj. physician satisfaction and emergency department laboratory test turnaround time. arch pathol lab med. 2001;125(7):863–871. bolodeoku j, ogbeiwi o, kuti ma, adebisi sa. laboratory tests turnaround time in outpatient and emergency patients in nigeria: results of a physician survey on point of care testing. int j med res heal sci. 2017;6(5):76–81. fei y, zeng r, wang w, he f, zhong k, wang z. national survey on intra-laboratory turnaround time for some most common routine and stat laboratory analyses in 479 laboratories in china. biochem medica. 2015;25(2):213–221. https://doi.org/10.11613/bm.2015.021 fernandes c, worster a, eva k, hill s, mccallum c. pneumatic tube delivery system for blood samples reduces turnaround times without affecting sample quality. j emerg nurs. 2006;32(2):139–143. https://doi.org/10.1016/j.jen.2005.11.013 guss d, chan t, killeen j. the impact of a pneumatic tube and computerized physician order management on laboratory turnaround time. ann emerg med. 2008;51(2):181–185. https://doi.org/10.1016/j.annemergmed.2007.03.010 cassim n, coetzee lm, tepper me, perelson l, glencross dk. timely delivery of laboratory efficiency information, part i: developing an interactive turnaround time dashboard at a high-volume laboratory. afr j lab med. 2020;9(2):a948. https://doi.org/10.4102/ajlm.v9i2.948 malakoane b, heunis jc, chikobvu p, kigozi ng, kruger wh. public health system challenges in the free state, south africa: a situation appraisal to inform health system strengthening. bmc health serv res. 2020;20(1):1–14. https://doi.org/10.1186/s12913-019-4862-y bennett da. how can i deal with missing data in my study? aust n z j public health. 2001;25(5):464–469. https://doi.org/10.1111/j.1467-842x.2001.tb00294.x shortreed sm, forbes ab. missing data in the exposure of interest and marginal structural models: a simulation study based on the framingham heart study. stat med. 2010;29(4):431–443. abstract background description of the intervention lessons learnt recommendations acknowledgements references about the author(s) barbara s. van deventer department of forensic medicine, faculty of health sciences, university of pretoria, pretoria, south africa lorraine du toit-prinsloo department of forensic medicine, faculty of health sciences, university of pretoria, pretoria, south africa chantal van niekerk department of chemical pathology, faculty of health sciences, university of pretoria, pretoria, south africa citation van deventer bs, du toit-prinsloo l, van niekerk c. practical tips to using formalin-fixed paraffin-embedded tissue archives for molecular diagnostics in a south african setting. afr j lab med. 2022;11(1), a1587. https://doi.org/10.4102/ajlm.v11i1.1587 note: additional supporting information may be found in the online version of this article as online supplementary documents 1, 2 ,3 and 4. lessons from the field practical tips to using formalin-fixed paraffin-embedded tissue archives for molecular diagnostics in a south african setting barbara s. van deventer, lorraine du toit-prinsloo, chantal van niekerk received: 16 mar. 2021; accepted: 21 mar. 2022; published: 23 june 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: formalin-fixed paraffin-embedded (ffpe) tissue archives in hospitals, biobanks, and others offer a vast collection of extensive, readily available specimens for molecular testing. unfortunately, the use of tissue samples for molecular diagnostic applications is challenging; thus, the forensic pathology ffpe tissue archives in africa have been a largely unexploited genetic resource, with the usability of dna obtainable from these samples being unknown. intervention: the study, conducted from january 2015 to august 2016, determined the usefulness of ffpe tissue as a reliable source of genetic material for successful post-mortem molecular applications and diagnostics. formalin-fixed paraffin-embedded tissue samples were collected and archived from autopsies conducted over 13 years in the forensic medicine department of the university of pretoria (pretoria, south africa). deoxyribonucleic acid from ffpe tissue samples and control blood samples was amplified by high-resolution melt real-time polymerase chain reaction before sequencing. the procurement parameters and fixation times were compared with the quantity and quality of the extracted dna and the efficiency of its subsequent molecular applications. lessons learnt: this study has shown that ffpe samples are still usable in molecular forensics, despite inadequate sample preparation, and offer immense value to forensic molecular diagnostics. recommendations: ffpe samples fixed in formalin for more than 24 h should still be used in molecular diagnostics or research, as long as the primer design targets amplicons not exceeding 300 base pairs. keywords: autopsy; deoxyribonucleic acid; formalin-fixed paraffin-embedded tissue; formalin-fixed paraffin-embedded tissue archive; high-resolution melt analysis; molecular diagnostics; polymerase chain reaction; post-mortem genetic testing; sequencing. background advances in molecular biology have enabled the detection of preventable and treatable diseases at a genetic level and it is a field of study receiving growing attention over the years.1,2,3 globally, formalin-fixed paraffin-embedded (ffpe) tissue samples are the largest available and most used source of biological material for molecular applications – diagnostic purposes or research.3,4,5 formalin-fixed paraffin-embedded tissue archives in hospitals, private pathology institutions, biobanks, and tertiary academic pathology departments offer a vast collection of extensive, readily available specimens for molecular testing.6,7,8 the critical information these specimens contain and their undeniable usefulness to necessary health-related investigations are considered by many researchers to be invaluable for molecular diagnostics.4,8,9 unfortunately, the low quality and efficiency of dna extracted from ffpe samples in molecular diagnostic applications limits the use of archived ffpe tissue samples.10,11,12 potential factors influencing the limited molecular utility include different parameters used in the procurement, fixation and processing of these samples.1,6,11 particularly, formalin fixation causes dna fragmentation, formaldehyde exposure causes dna-protein cross-linking, and paraffin in dna samples inhibits polymerase chain reaction (pcr) amplifications. these factors impair the dna quality and efficiency for molecular applications, such as pcr and sequencing.9,11,12,13 although blood collected in ethylenediaminetetraacetic acid or fresh frozen tissue samples are the best source of dna and the preferred material used for genetic testing, numerous studies have reported on the successful use of ffpe tissue samples.2,14,15 the successful use of ffpe tissue samples is particularly encouraging because african forensic pathology still faces the dire reality of inadequate funding and resource allocation required to maintain freezer preservation systems for routine collection and storage of blood or tissue samples.15,16,17 forensic molecular pathology is an emerging field with significant clinical impact; its techniques are used to diagnose preventable and inherited causes of death.18,19,20 the role of the forensic pathologist in the medico-legal investigation of death includes determining the cause and manner of death. despite the implementation of forensic molecular investigations and research in many high-income countries, there is still a significant lack of it in lowand middle-income countries, mainly due to financial and resource constraints.9,15,21 several south african universities’ forensic medicine departments conduct valuable research, mainly focusing on possible causes of sudden deaths; however, there is still a remarkable paucity of publications on molecular forensic studies and their applications in africa.22,23,24 social and medico-legal issues challenge molecular forensic research, including personal and public concerns.25 fortunately, in most large forensic pathology centres, which are often linked to tertiary academic institutions, forensic pathologists have established extensive ffpe tissue archives through routine histology casework. these archives are excellent and sometimes the only archives for conducting large retrospective genetic epidemiological studies.9,15,20 until now, these forensic pathology ffpe tissue archives have mainly been unexploited; thus, the quality of dna obtainable from these samples is unknown.9,15,24 african countries, particularly south africa, have some of the highest numbers of unnatural deaths per year, compared to other countries in the world.26,27 unfortunately, this increases the burden on the already-scarce africa-practising forensic pathologists. consequently, the increased caseload delays case investigation, prolonging formalin fixation times by days, weeks, and even months.26,27,28 according to recommended guidelines for optimised molecular testing, tissue samples should be fixed in 10% buffered formalin for 14 h – 24 h.9,14 hence, most forensic pathology departments doubt the value and efficacy of their decades worth of ffpe tissue archives in the context of molecular diagnostic applications or research.9,15,20 this study aimed to determine the utility of ffpe tissue samples as a reliable source of genetic material for post-mortem molecular applications and diagnostics. firstly, the study evaluated the influence of the procurement parameters, fixation time, and storage periods of the archived ffpe tissue samples on the quantity and quality of the dna extracted and its efficiency for subsequent molecular applications. secondly, the study aimed to determine which dna extraction kits yield the best quality and quantity of ffpe dna. description of the intervention ethical considerations ethics approval was obtained from the faculty of health sciences research ethics committee university of pretoria (reference number 142/2014) to use the retrospective and prospective ffpe tissue samples and the control blood samples. the retention and use of tissues obtained at medico-legal post-mortem examinations were guided by the south african legislation (inquests act 58 of 1995, national health act 61 of 2003 and the regulations regarding the rendering of forensic pathology service r636). thus, patient and family consent was not required. healthy volunteers provided written informed consent before blood sample collection. all tissue and blood samples were assigned a case number, with no identifying features linked to any of these samples. study location and design this retrospective observational study was conducted in the department of forensic medicine, the university of pretoria, pretoria, south africa, from january 2015 to august 2016 using ffpe myocardial tissue samples obtained over 13 years from post-mortem examination of sudden unexplained infant deaths in the pretoria medico-legal laboratory. the samples were stored for further ancillary investigations to determine the cause of death, including genetic testing for genes linked to inherited cardiac arrhythmogenic disorders and sudden unexplained infant deaths, such as scn5a. specimen description the study included a total of 58 ffpe myocardial tissue samples. these ffpe tissue samples were obtained from sudden unexplained infant death complete post-mortem investigations between january 2002 and january 2015 and identified from the department of forensic medicine electronic repository. additionally, in january 2015, two prospective myocardial tissue samples (samples 59 and 60) were also obtained from autopsy and, starting in march 2015, venous blood from nine healthy volunteers was drawn into two 5 ml ethylenediaminetetraacetic acid tubes. specimen processing when retained, the archived ffpe myocardial tissue samples were immediately fixed in 10% neutral buffered formalin solution per routine and processed using the shandon pathcentre from thermo scientific (waltham, massachusetts, united states) and the tissue-tek® tec from sakura finetek (torrance, california, united states). the ffpe processing included dehydration in ethanol, clearing in xylene, and embedding in paraffin blocks. when obtained, the prospective myocardial tissue samples (samples 59 and 60) were immediately fixed in formalin for 14 h – 24 h, per qiagen protocol. subsequently, they were cleared in xylene, embedded in paraffin blocks (following the same procedure as the archived samples), and stored for a month. all ffpe tissue samples were cut into 20 μm thick sections using a microtome (leica biosystems, wetzlar, germany). the first three to four cuts of each ffpe specimen were discarded because the sample surface had been exposed to air. then, to ensure sufficiency, six to eight sections of each sample were put into a well-labelled sterile 1.5 ml microcentrifuge tube for dna extraction. during this specimen preparation process, the microtome’s blade and the cutting surface were cleaned with 100% ethanol between each specimen to prevent cross-contamination. deoxyribonucleic acid extraction deoxyribonucleic acid was extracted from all ffpe samples (archived and prospective) using two different extraction kits; the qiaamp dna ffpe tissue kit (qiagen, hilden, germany) and the isolate п ffpe dna kit (bioline, london, united kingdom). thus, two dna extraction procedures were performed on every ffpe tissue sample. the two kits differed in the type of deparaffinisation solution and incubation time at 90 °c for the reversal of dna cross-linking. the qiaamp dna ffpe tissue kit used standard qiagen-provided deparaffinisation solution and required an incubation time of 2 h at 90 °c, while the isolate п ffpe dna kit used xylene and incubation of 1 h at 90 °c.29 for the blood samples drawn into two 5 ml ethylenediaminetetraacetic acid tubes, the buffy coat was isolated (200 μl) and stored at – 80 °c until dna extraction using the qiaamp dna blood mini kit (qiagen, hilden, germany) per manufacturer’s instructions. further on, the concentration and purity of all dna samples (archived ffpe, prospective ffpe and blood) were determined spectrophotometrically (nanodrop, thermo scientific [waltham, massachusetts, united states]). for this study, a dna concentration range of 40 ng/μl – 75 ng/μl and a purity ratio above 1.75 were deemed sufficient. high-resolution melt real-time pcr and sequencing the dnas were diluted to concentrations between 40 ng/μl and 75 ng/μl. thirty-four amplicon primer pairs (online supplementary document table 1) for the scn5a gene30 generating 152 base pairs (bp) to 514 bp amplicon sizes amplified all extracted dna (60 ffpe tissue samples: two prospective and 58 archived; and nine blood samples). amplification was by high-resolution melt real-time pcr using sensifast high resolution melt master mix (bioline, london, united kingdom) on the rotorgene q (qiagen, hilden, germany). successful real-time pcr results had sigmoidal amplification and single melt curves. afterwards, the pcr amplicons were sanger sequenced by inqaba biotec, pretoria, south africa. sequencing chromatograms were analysed using clc main workbench 5 software (clc bio®, aarhus, denmark). a successful sanger sequencing had evenly spaced peaks and a minimal noise chromatogram. analysis the tissue retention date and the 10% formalin fixation duration for each ffpe tissue sample (online supplementary document table 2) were used for student’s t-test in microsoft excel 2013 (redmond, washington, united states) to determine a possible correlation with the quality of pcr amplifiability. lessons learnt deoxyribonucleic acid yield deoxyribonucleic acid extracted from all nine control blood samples yielded concentrations ranging from 15 ng/μl to 56 ng/μl, with sufficient 260/280 ratios between 1.75 and 2.0. comparison of dna yield obtained from two different extraction kits the maximum period of tissue sample storage (embedded in paraffin blocks) was 13 years, while the minimum was one month. the minimum period of tissue fixation in formalin wax was two days and the maximum, 271 days. the average value obtained for the fixation period of all 58 tissue samples was 26.1 days, with a median of 11.5 days. our findings showed an extensive delay in the department’s processing of ffpe tissue samples, particularly long tissue fixation in formalin (online supplementary document table 2). these findings are in keeping with similar ffpe archive conditions reported by other forensic pathology departments practising in resourceand fund-limited settings.9,15,16,24 the qiagen ffpe kit yielded much higher dna concentrations, 50 ng/μl – 900 ng/μl. after extraction, an elution volume of 50 μl of dna of each sample was obtained, with purity ratios between 1.7 and 2.1. the bioline ffpe kit yielded lower dna concentrations, 33 ng/μl – 137 ng/μl, with 260/280 ratios between 2.0 and 2.1 and an elution volume of 50 μl. for this study, a dna concentration range of 40 ng/μl – 75 ng/μl and a purity ratio above 1.75 were deemed sufficient. although the two different kits yielded dna concentrations of quite different ranges, no difference in pcr amplification results was observed. furthermore, both kits’ pcr amplification quality correlated; thus, successful or failed/unsuccessful amplification was recorded for each tissue sample. therefore, we concluded that the two kits yielded the same quality of extracted dna and pcr amplification and, by extension, the exact molecular analysis results. does size matter? amplification of all 34 amplicons, high resolution melt analysis, gel electrophoresis, and sequencing were successful for all dna extracted from blood. polymerase chain reaction amplified several amplicons from the ffpe dna; however, there was a significant reduction in the amplification of amplicons with a length greater than 300 bp (figure 1 and online supplementary document table 3). in contrast to vitosevic et al.,31 our study (as indicated in online supplementary document table 2) did not find a correlation between longer sample storage time and pcr amplification failure, as successful amplification was observed for samples stored between 1 month and 13 years. instead, we found that prolonged periods of formalin fixation, which causes dna fragmentation, correlated with failed pcr amplification (figure 2). figure 1: effect of amplicon size on pcr amplification success rate of dna extracted from ffpe tissue samples, pretoria, south africa, june 2015 – may 2016 (online supplementary document table 3). figure 2: laboratory results showing the association between a prolonged formalin fixation period and a decline in pcr amplification success rate, pretoria, south africa, march 2015 – july 2016 (online supplementary document table 2). formalin fixation time approximately half of the ffpe dna samples produced large amplicons; an association existed between successful amplification and a shorter (approximately four days) formalin fixation duration (figure 2, online supplementary document table 2 and online supplementary document table 4). converse to archived ffpe dna, which had been fixed in formalin for a period exceeding 24 h, the two prospective ffpe samples (fixed in formalin for only 24 h) yielded all 34 targeted amplicons, including a 514 bp amplicon (the largest amplicon). only four retrospective ffpe dna samples yielded the 514 bp amplicon. on analysis, these four ffpe dna samples, all stored for six years, were subjected to shorter periods of formalin fixation (figure 1 and online supplementary document table 2). this bar graph shows the significant decrease in successful pcr amplification by using dna extracted from ffpe tissue samples that have been fixated in formalin for periods longer than the prescribed 24 h. dna samples extracted from those ffpe tissue samples which adhered to the prescribed 24-h formalin fixation illustrates a 100% success rate in pcr amplification. samples were grouped according to days in formalin (see online supplementary document table 4). downstream applications sanger sequencing of amplicons was successful for both blood and ffpe tissue samples. formalin-fixed paraffin-embedded tissue sequences that showed variations upon alignment to the reference sequence were re-amplified and re-sequenced to validate these variations and exclude the possibility of dna cross-linking. results showed no aberrant variations in ffpe dna samples compared to amplicons resulting from blood dna, indicating that the fixation method does not seem to increase the chance of interpreting these as legitimate variations. recommendations cut your losses? this study showed that dna extracted from ffpe tissue samples could successfully be both amplified and sequenced. however, it is essential to note that dna measurements (concentration and purity ratio) obtained using a spectrophotometer do not assure successful pcr amplification and sequencing because it does not measure the extent of dna fragmentation. commercially available kits, such as the quantifiler® trio dna quantification kit, are designed to detect and help overcome external factors affecting pcr amplifiable quality. however, in a resource-poor setting, a spectrophotometer, which most laboratories have, will indicate the ‘usability’ of extracted dna.32 polymerase chain reaction success was limited to amplicons smaller than 300 bp in cases of prolonged formalin fixation. thus, ffpe samples could, and should, still be used in molecular diagnostics or research, as long as the primers are designed to generate pcr amplicons not exceeding 300 bp. our study confirmed that ffpe samples are still useful for molecular forensics and diagnostics despite inadequate sample preparation. thus far, ffpe tissue archives in most african forensic pathology departments have been an underexploited resource for conducting large retrospective and prospective genetic epidemiological studies.14,15,16 it is time to utilise these resources at our disposal to the advantage of the african population. the most significant benefit of post-mortem molecular testing is the high disease-specific diagnostic, therapeutic, and prognostic benefits derivable from subsequent genetic analysis.1,2,3 understanding the genetics of inherited diseases specific to african populations informs the development of more meaningful and relevant risk stratification techniques.3,15,33 this includes the development of more targeted molecular tests for various challenges in the forensic setting, including population genetics, human identification, and post-mortem interval estimation.9,15 acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions b.s.v.d., l.d.t.-p. and c.v.n. conceived and planned the experiments/study. b.s.v.d. carried out the experiments. b.s.v.d., l.d.t.-p. and c.v.n. contributed to the interpretation of the results. b.s.v.d. took the lead in writing the manuscript. all three authors provided critical feedback and helped shape the research, analysis, and manuscript. sources of support the authors would like to thank the genomics research institute (university of pretoria) for funding this project. we would also like to thank the south african national research foundation for funding part of this project. data availability the data that support the findings of this study are available from the corresponding author, b.s.v.d., upon request. disclaimer the views expressed in this article are those of the authors and do not necessarily reflect the official position of any affiliated agency to the authors. references bhagwate av, liu1 y, stacey j, et al. bioinformatics and dna-extraction strategies to reliably detect genetic variants from ffpe breast tissue samples. bmc genom. 2019;20:1–10. https://doi.org/10.1186/s12864-019-6056-8 gao xh, li j, gong hf, et al. comparison of fresh frozen tissue with formalin-fixed paraffin-embedded tissue for mutation analysis using a multi-gene panel in patients with colorectal cancer. front oncol. 2020;10:1–8. https://doi.org/10.3389/fonc.2020.00310 sarnecka ak, nawrat d, piwowar m, et al. dna extraction from ffpe tissue samples – a comparison of three procedures. contemp oncol. 2019;23(1):52–58. https://doi.org/10.5114/wo.2019.83875 gaffney ef, riegman ph, grizzle we, et al. factors that drive the increasing use of ffpe tissue in basic and translational cancer research. biotech histochem. 2018;93(5):373–386. https://doi.org/10.1080/10520295.2018.1446101 senguven b, baris e, oygur t, et al. comparison of methods for the extraction of dna from formalin-fixed, paraffin-embedded archival tissues. int j med sci. 2014;11(5):494–499. https://doi.org/10.7150/ijms.8842 atanesyan l, steenkamer mj, horstman a, et al. optimal fixation conditions and dna extraction methods for mlpa analysis on ffpe tissue-derived dna. am j clin pathol. 2017;147(1):60–68. https://doi.org/10.1093/ajcp/aqw205 steinau m, patel ss, unger er. efficient dna extraction for hpv genotyping in formalin-fixed, paraffin-embedded tissues. j mol diagn. 2011;13(4):377–381. https://doi.org/10.1016/j.jmoldx.2011.03.007 doncza b, guttman a. biomedical analysis of formalin-fixed, paraffin-embedded tissue samples: the holy grail for molecular diagnostics. j pharm biomed. 2018;155:125–134. https://doi.org/10.1016/j.jpba.2018.03.065 lin y, gryazeva t, wang d, et al. using post-mortem formalin-fixed paraffin-embedded tissues for molecular testing of sudden cardiac death: a cautionary tale of utility and limitations. forensic sci int. 2020;308:1–8. https://doi.org/10.1016/j.forsciint.2020.110177 linton km, hey y, saunders e, et al. acquisition of biological relevant gene expression data by affymetrix microarray analysis of arhival – fixed paraffin-embedded tumours. br j canc. 2008;98:1403–1414. https://doi.org/10.1038/sj.bjc.6604316 huijsmans cjj, damen j, van der linden jc, et al. comparative analysis of four methods to extract dna from paraffin-embedded tissues: effect on downstream molecular applications. bmc res notes. 2010;3:1–9. https://doi.org/10.1186/1756-0500-3-239 gilbert mtp, haselkorn t, bunce m, et al. the isolation of nucleic acid from fixed, paraffin-embedded tissues – which methods are useful when? plos one. 2007;2(6):1–12. https://doi.org/10.1371/journal.pone.0000537 snow an, stence aa, pruessner ja, et al. a simple and cost-effective method of dna extraction from small formalin-fixed paraffin-embedded tissue for molecular oncologic testing. bmc clin pathol. 2014;14:1–10. https://doi.org/10.1186/1472-6890-14-30 yil q, yang r, shi j, et al. effect of preservation time of formalin-fixed paraffin-embedded tissues on extractable dna and rna quantity. j int med res. 2020;48(6):1–10. https://doi.org/10.1177/0300060520931259 reid km, maistry s, ramesar r, et al. a review of the optimisation of the use of formalin-fixed paraffin-embedded tissue for molecular analysis in a forensic post-mortem setting. forensic sci int. 2017;280:181–187. https://doi.org/10.1016/j.forsciint.2017.09.020 naidoo d, ihekweazu c. nigeria’s efforts to strengthen laboratory diagnostics – why access to reliable and affordable diagnostics is key to building resilient laboratory systems. afr j lab med. 2020:9(2):a1019. https://doi.org/10.4102/ajlm.v9i2.1019 shaboodien g, spracklen tf, kamuli s, et al. genetics of inherited cardiomyopathies in africa. cardiovasc diagn ther. 2020;10:262–278. wang d, shah kr, yon um s, et al. cardiac channelopathy testing in 274 ethnically diverse sudden unexplained deaths. forensic sci int. 2014;237:90–99. https://doi.org/10.1016/j.forsciint.2014.01.014 semsarian c, ingles j, wilde aam. sudden cardiac death in the young: the molecular autopsy and a practical approach to surviving relatives. eur heart j. 2015;36(21):1290–1296. https://doi.org/10.1093/eurheartj/ehv063 bulzan o, precup c, tamas la, et al. the genetic investigation of old tissue samples paraffin-embedded as a source for molecular autopsies in sudden cardiac death cases. life. 2014;24:391–397. heathfield lj, martin lj, ramesar r. a systematic review of molecular autopsy studies in sudden infant death cases. j pediatr genet. 2018;7(4):143–149. https://doi.org/10.1055/s-0038-1668079 heathfield lj, martin lj, ramesar r. massively parallel sequencing in sudden unexpected death in infants: a case report in south africa. forensic sci int genet suppl ser. 2019;7(1):459–461. https://doi.org/10.1016/j.fsigss.2019.10.051 heathfield lj, bhengu w, louw s, et al. assessment of candidate variants causative of inborn metabolic diseases in sudi cases in south africa, and a case report. int j leg med. 2020;134:1639–1645. https://doi.org/10.1007/s00414-020-02337-6 van deventer bs, du toit-prinsloo l, van niekerk c. feasibility of analysis of the scn5a gene in paraffin-embedded samples in sudden infant death cases at the pretoria medico-legal laboratory, south africa. forensic sci med pathol. 2018;14:276284. https://doi.org/10.1007/s12024-018-9995-5 maeda h, ishikawa t, michiue t. forensic molecular pathology: its impact on routine work, education and training. leg med. 2014;16(2):61–69. https://doi.org/10.1016/j.legalmed.2014.01.002 du toit-prinsloo l, saayman g. performance of autopsies in south africa: selected legal and ethical perspectives. cme. 2012;30:53–55. saayman g. south africa: vulnerable persons and groups in a vulnerable democracy – can forensic medical services help to ensure justice in critical times? acad forensic pathol. 2017;7(3):434–443. https://doi.org/10.23907/2017.036 du toit-prinsloo l, pickworth g, saayman g. the forensic autopsy as a teaching tool: attitudes and perceptions of undergraduate medical students at the university of pretoria, south africa. afr j health professions educ. 2016;8(1):77–80. https://doi.org/10.7196/ajhpe.2016.v8i1.589 qiagen. qiagen supplementary protocol qa50 – purification of genomic dna from ffpe tissue using the qiaamp® dna ffpe tissue kit and deparaffinisation solution. hilden: qiagen. 2011; p. 1–3. millat g, chevalier p, restier-milon l, et al. spectrum of pathogenic mutations and associated polymorphisms in a cohort of 44 unrelated patients with long qt syndrome. clin genet. 2006;70(3):214–227. https://doi.org/10.1111/j.1399-0004.2006.00671.x vitosevic k, todorovic m, slovic z, et al. dna isolated from formalin-fixed paraffin-embedded healthy tissue after 30 years of storage can be used for forensic studies. forensic sci med pathol. 2021;17:47–57. https://doi.org/10.1007/s12024-020-00327-z gouveia n, brito p, serra a, et al. validation of quantifiler® trio dna quantification kit in forensic samples. forensic sci int genet. 2015;5:24–25. https://doi.org/10.1016/j.fsigss.2015.09.010 laing m, kraus sm, shaboodien g, et al. an overview of the genetic basis of cardiovascular disease. samj. 2019;109(6):364–370. https://doi.org/10.7196/samj.2019.v109i6.14069 abstract introduction methods results discussion acknowledgements references about the author(s) tjeerd a.m. datema datos b.v., leiden, the netherlands linda oskam datos b.v., leiden, the netherlands jacqueline e.w. broerse department of science communication, faculty of science, vrije universiteit amsterdam, amsterdam, the netherlands paul r. klatser athena institute, faculty of science, vrije universiteit amsterdam, amsterdam, the netherlands citation datema tam, oskam l, broerse jew, klatser pr. review of the stepwise laboratory quality improvement process towards accreditation (slipta) version 2:2015. afr j lab med. 2020;9(1), a1068. https://doi.org/10.4102/ajlm.v9i1.1068 original research review of the stepwise laboratory quality improvement process towards accreditation (slipta) version 2:2015 tjeerd a.m. datema, linda oskam, jacqueline e.w. broerse, paul r. klatser received: 18 july 2019; accepted: 19 aug. 2020; published: 28 oct. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: in 2011 the stepwise laboratory quality improvement process towards accreditation (slipta) was launched, aimed at strengthening the quality and competence of african clinical, public health and reference laboratories. we reviewed the first version of the slipta checklist in 2011. the continued development and publication of a new version of the international organization for standardization (iso) 15189 standard demands a renewed review. objective: this study aimed to determine the suitability of slipta in guiding laboratories towards iso 15189:2012 compliance and accreditation and provide recommendations for further slipta improvement. methods: the study was conducted between september 2018 and april 2019. coverage of iso 15189:2012 by slipta checklist version 2:2015 was determined and the point distribution of the scoring system over the different sections of the slipta checklist was re-investigated. these findings were compared with the review of the first version of the slipta checklist (based on iso 15189:2007) and with findings published on slipta implementation and roll-out. results: the coverage of iso 15189 by the slipta checklist has increased, even though iso 15189:012 is more extensive than iso 15189:2007. the point distribution is still skewed towards sections related to quality planning rather than quality control and improvement. although to date 314 laboratories have been assessed, barriers for laboratories to participate in slipta are high. sustainability of slipta results is insufficiently studied. conclusion: slipta checklist version 2:2015 has improved compared to earlier versions. we recommend increasing accessibility for laboratories to participate and increasing guidance for iso-based quality management system implementation. keywords: accreditation; iso 15189; laboratory; slipta; slmta; quality assurance; total quality management. introduction in 2011 the stepwise laboratory quality improvement process towards accreditation (slipta) was launched, aimed at strengthening quality and competence of clinical, public health and reference laboratories in the african region.1,2,3,4 the launch of slipta was the result of a series of events. in 2008 the world health organization (who) and the united states centers for disease control and prevention (cdc) organised a conference on laboratory quality systems. one of the recommendations stated that laboratories in resource-limited settings should consider taking a staged approach towards implementation of a quality management system (qms).5,6 in the following years, several resolutions on laboratory strengthening were drafted for the african region.7,8 in 2009, the who regional office for africa (who-afro), in collaboration with cdc and other partners, launched the who-afro laboratory accreditation process and the strengthening laboratory management towards accreditation (slmta) training and mentoring programme.9,10,11,12 in 2011, the who-afro accreditation process was renamed slipta.4,10,13 in 2012, who-afro designated the african society for laboratory medicine (aslm) as the slipta secretariat.4,12,14 the aslm established slipta’s implementation structure consisting of4,14: who-afro slipta focal point, responsible for mobilisation of resources, providing guidance on content and implementation, and reviewing and updating slipta. ministry of health slipta focal point, responsible for -in-country promotion of laboratory improvement through slipta. the ministry of health develops an implementation plan and prioritises slipta applicant laboratories and allocates financial and human resources for slipta implementation. aslm-certified slipta auditors, responsible for conducting audits and providing advice to auditees. slipta independent advisory group, enrolling laboratories into the slipta programme, organising audits and making a final decision on laboratory recognition through awarding a star rating based on audit reports (varying from zero to five stars) (table 1). star ratings are valid for a two-year period.12,14 table 1: stepwise laboratory quality improvement process towards accreditation checklist compliance levels versus star ratings. the aslm aims to enrol 2500 laboratories in slipta and have 250 public laboratories accredited to international standards by 2020.12 not all laboratories can voluntarily participate in slipta because ministries of health are encouraged to select laboratories in phases, considering tiered laboratory networks and giving precedence to laboratories that have already completed laboratory quality improvement training. eligibility criteria include a slipta self-audit score of 55% or higher, participation in proficiency testing schemes or alternative methods in the past 6 months and having conducted internal audits and a management review in the past 12 months. the laboratory should also have documented its qms.14 upon enrolment a laboratory is audited to determine its initial star rating. laboratories are expected to work towards the next star. laboratories that achieve a five-star rating are encouraged to apply for iso 15189 accreditation.14 key in the slipta programme is the slipta checklist, which is primarily based on iso 15189 and, to a lesser extent, clinical and laboratory standards institute (clsi) guideline qms01-a4.3,15 because laboratories with a five-star rating are encouraged to apply for accreditation, it is important to obtain insight into the coverage of iso 15189 requirements by the slipta checklist. this determines how much of the iso 15189 requirements still must be addressed before full compliance with iso 15189 is achieved. in 2011, the authors reviewed the first version of the slipta checklist (published in 2009), referred to as the alpha version,16 and determined coverage of iso 15189:2007.1 in that same year slipta was revised and the slipta alpha version became slipta checklist v1.0.11,12 because a new version of the iso 15189 standard was published in 2012, slipta checklist v2, based on iso 15189:2012, was published in 2015.3,12 the current study determines coverage of iso 15189:2012 by slipta checklist v2:2015 and re-investigates the point division over the different sections of the slipta checklist to determine the relative weight of qms elements in slipta. we also reviewed published slipta implementation data. this article informs potential users about slipta’s suitability to guide laboratories towards iso 15189:2012 compliance and accreditation and provides recommendations for further improvement of slipta. methods ethical considerations ethical clearance was not required for this study. study design the study was conducted between september 2018 and april 2019. the methodology used in this study was adapted from datema et al. 2011.1 the first analysis determined the slipta checklist’s coverage of iso 15189:2012 by linking each question of slipta checklist v2:2015 to iso 15189:2012 clauses. the second analysis provides insight into the point distribution of the scoring system over the different slipta checklist sections. the slipta checklist is divided into 12 sections corresponding with 12 qms elements. for each section, points can be scored, the total of which determines the number of stars awarded. excel 2016 (microsoft, redmond, washington, united states) was used to analyse and compare the number of points that can be scored per section. results were compared with results of the review of the slipta checklist alpha version.1 in datema et al. 2011, the 12 sections of the slipta checklist were divided over the categories ‘resource management’, ‘process management’ and ‘improvement management’. in this article we renamed these categories to ‘quality planning’, ‘quality control’ and ‘quality improvement’, in line with the juran trilogy (table 2).17 the overall intention of each slipta checklist section led the categorisation process, which was identical to the review of the alpha version of the slipta checklist.1 hence, the overall aim of the sections assigned to the juran category quality planning is to ensure quality of work before it is started, that is, before work can be conducted in a quality-assured way, proper organisation and functioning of equipment, purchasing and inventory management processes, good facilities and competent personnel are needed. as such, with the implementation of these elements the laboratory is ‘planning for quality’, justifying the decision for assigning these sections to the juran category quality planning. similarly, the primary, shared objective of the sections on process control, information management, documents and records, and client management is to control quality of work while it is being conducted. therefore, these sections were assigned to the juran category quality control. the sections on management reviews, evaluation and audits, occurrence or incident management and process improvement, and identification of non-conformities, corrective and preventive actions all share the common goal of continuously improving the quality of laboratory work. therefore, these sections were assigned to the juran category quality improvement. table 2: distribution of stepwise laboratory quality improvement process towards accreditation checklist v2:2015 sections over the different categories of the juran trilogy. a pubmed search was conducted on 06 march 2019 to gather literature on outcomes of slipta implementation and roll-out. the search terms were ‘slipta’ or ‘stepwise laboratory improvement process towards accreditation’ and the search yielded 29 hits. after primary and secondary selection based on title and abstract, and identification of additional reports through snowballing, a total of 23 articles were identified. upon further scrutiny 12 papers were excluded because they did not report findings on slipta implementation, roll-out, effectiveness or sustainability per se. finally, 11 papers were included. results changes to stepwise laboratory quality improvement process towards accreditation checklist v2:2015 compared with the alpha version structurally, slipta checklist v2:2015 is very similar to the alpha version. the checklist is still divided over 12 sections based on the quality system essential structure developed by the clsi.15 a notable change is the addition of one very detailed question on the presence and content of 36 specific standard operating procedures (sops). iso 15189:2012 coverage the slipta checklist v2:2015 addresses 82% of iso 15189:2012 clauses, of which 35% are fully addressed, 47% addressed partially and 18% are not addressed at all. this is an improvement compared to the alpha version, which wholly or partially covered 52% of iso 15189:2007 clauses. in some areas slipta checklist v2:2015 is more detailed and prescriptive compared to iso 15189 requirements, whereas in other areas iso 15189 requirements are more extensive than the slipta checklist. point distribution and relative weight (importance) of quality management system elements the total number of points in the slipta checklist v2:2015 has increased from 250 to 275. in most sections a higher number of points can be scored compared to the alpha version. however, the relative weight of each section has remained similar due to the increase in the total number of points (see figure 1). figure 1: relative point distribution of the scoring system over the different sections of stepwise laboratory quality improvement process towards accreditation checklist alpha version and version 2:2015. when the point distribution of the slipta checklist v2:2015 scoring system was analysed using the juran trilogy model, points were still heavily skewed towards quality planning (45% of the weight) and quality control (33%). quality improvement received the lowest number of points (22%) (figure 2). figure 2: relative point distribution of the scoring system of stepwise laboratory quality improvement process towards accreditation checklist alpha version and version 2:2015 over the different categories of the juran trilogy. outcomes of stepwise laboratory quality improvement process towards accreditation roll-out up to 24 april 2019, 314 laboratories had been audited in 20 countries, which is still far below the ambition to enrol 2500 laboratories by 2020 (table 3).18 the percentage of laboratories per star rating is shown in figure 3. the distribution is still in line with data published by ndihokubwayo et al. in 2016, and andiric et al. in 2018.12,19 figure 3: percentage of laboratories per star rating, of a total of 314 laboratories, retrieved from stepwise laboratory quality improvement process towards accreditation database on 24 april 2019.18 table 3: number of laboratories per star level per country on 24 april 2019.18 although many papers have been published on the slmta training and mentoring programme, in which the slipta checklist was used to measure progress, papers evaluating the slipta initiative per se are scarce. the paper by ndihokubwayo et al. (2016) is the only paper summarising slipta implementation and lessons learned.12 two additional papers were found that present slipta implementation findings, but these also combined slipta with additional assistance.20,21 both studies indicated that combining slipta with mentoring has a positive effect on qms implementation, although neither control laboratories nor findings on sustainability of this model were included.20,21 ndihokubwayo et al. found that slipta laboratories performed most poorly on the internal audit and corrective action sections.12 this finding is corroborated by other studies.22,23,24,25,26,27,28 they further state that advocacy for laboratory strengthening is key to the slipta process (as it is owned by the ministry of health) and that particularly francophone and lusophone countries are not well covered. an explanation for the latter finding might be that slipta was initially implemented through the united states president’s emergency plan for aids relief, which is oriented towards anglophone countries.12 discussion slipta checklist v2:2015 has improved compared to the alpha version. even though iso 15189:2012 is more extensive than iso 15189:2007, iso 15189 coverage has increased, decreasing the gap that still needs to be bridged by 5-star laboratories aiming for iso 15189 accreditation. however, the gap is still considerable: only 35% of iso 15189 clauses are fully covered, leaving 47% partially covered and 18% not covered at all. compliance with some of these requirements may be reached as part of the continuous improvement process which 5-star laboratories may have already partially implemented. the main point for slipta improvement remains the absence of prioritisation of qms implementation activities. there are no conditions for the different star ratings (other than the star rating thresholds) that encourage laboratories to implement the qms in a specific, rational manner, indicating that qms implementation guidance remains limited. most slipta checklist v2:2015 questions are supplemented with short notes including examples, but neither a stepwise plan nor a detailed explanation of implementation of requirements is provided, showing that slipta remains primarily an assessment checklist. slipta auditors may provide advice on implementation during assessments4,12 but this might come late, for laboratories may first try to implement a qms before being assessed as laboratories must score at least 55% in self-assessment to meet eligibility criteria for enrolment.14 currently, most points can be scored in slipta checklist sections related to quality planning, followed by quality control. the least number of points can be scored on quality improvement. this creates an imbalance. implementing a qms is a ‘systems approach’: all qms elements work together to create a sustainable system that can deliver quality-assured laboratory services and continuously improve itself. when one qms element is not (correctly) functioning, quality assurance of overall laboratory services and continuous quality improvement may be compromised. therefore, one could argue that slipta should award an equal number of points for each section. on the other hand, the skewed point distribution may point laboratories in the right direction by encouraging them to address sections related to quality planning first because of the high number of points that can be scored in this category, as was also argued by datema et al. in 2011 (although no evidence was found in literature supporting this hypothesis).1 however, this is counterbalanced by the higher amount of work required for implementing the sections related to quality planning. another improvement opportunity for the slipta checklist is the imbalance in the effort required to earn points per question. for example, for question 1.5 one needs to develop 36 sops to earn five points, whereas by developing a list of documents used in the laboratory (question 1.4), making sure that sops are accessible to staff (question 1.6), and indicating date of authorisation, location and date of discontinuation on each sop (question 1.8) one can score a total of six points. writing 36 sops obviously requires considerably more effort. stepwise laboratory quality improvement process towards accreditation implementation and roll-out a strong point is that slipta requires the ministry of health to play an active role; government commitment has been shown to be key to success in both slipta21 and slmta.11,12,20,24,25,27,29,30,31,32,33,34,35 also, slipta can be well combined with other guidance methods for laboratory accreditation as is evident from many studies on slmta implementation.20,22,27,29,30,33,34,35,36 a major downside is the indirect accessibility of the slipta programme: laboratories are selected by the ministry of health for participation. only 27 of the 47 who-afro member states have established a slipta focal point within their ministry of health. moreover, the programme has a language bias towards anglophone countries.12,18,28 also, up to april 2019, 314 laboratories had been assessed, which is low considering that kampala, the capital of uganda, alone counts 954 laboratories18,37 and that aslm aimed to include 2500 laboratories by 2020.12 recommendations for improvement the level of guidance provided by slipta for qms implementation could be increased by making better use of the star rating system. currently, stars are simply awarded based on the number of points scored, regardless of the section these points are scored in. setting certain benchmarks and conditions for the different star ratings may improve guidance. an example could be the definition of key questions that have to be implemented for each star rating. this may help laboratories in using a rational approach towards qms implementation. it may also assist laboratories in lower tiers of laboratory networks, for which iso 15189:2012 is not (yet) feasible, to implement a basic yet functional qms. the phased approach incorporated in the who laboratory quality stepwise implementation (lqsi) tool as well as the tiered approach of the laboratory quality management system stepwise improvement process (lqms-sip) used in the caribbean region could serve as models in assigning key questions to the different star ratings, which would also contribute to harmonisation of slipta with these laboratory strengthening tools and initiatives.38,39,40 sustainability is a challenge for laboratory strengthening efforts. literature on slipta implementation does not provide sufficient clarity on sustainability. slipta assessments, like accreditation assessments in general, are snapshot measurements of laboratory compliance, creating the risk that the efforts may weaken after an assessment, as was also witnessed in slmta evaluations.11,24,32 a possible measure to decrease this risk is announcing assessment visits only shortly before they are scheduled, leaving just enough time for a laboratory to prepare logistics but not for correction of elements that would otherwise not have been corrected, yielding a more representative view of the daily practice. another measure is the adoption of a point scoring system that awards negative points for questions that are not in place anymore compared to the previous assessment. this emphasises the importance of quality assurance and may be an extra driver for the laboratory to ensure continued compliance. in the case of slipta, both measures could be adopted without requiring major revisions as it would primarily require amendment of the audit scoring section of the slipta checklist. it should be noted that slipta checklist v2:2015 already indicates that audit scores should be based on laboratory performance during the 12 months preceding the slipta audit, which is an encouragement for laboratories to maintain compliance.3 the last recommendations relate to implementation and roll-out of slipta: increasing the accessibility of slipta by increasing the capacity, among others through ensuring the presence of slipta focal points at each ministry of health and training of more slipta auditors. this should include training of more slipta auditors fluent in portuguese and french to increase accessibility for laboratories in lusophone and francophone countries. limitations the analysis using the juran trilogy model was limited to categorisation of overall slipta sections. although the authors are aware that the same analysis can be conducted at the individual question level, the decision was made to categorise based on the overall intention of each slipta section and, therefore, categorise each slipta checklist section as a whole as described in the methods section. this was also required to enable comparison with the review of the alpha version of the checklist. this study is a desk-based review. ideally, the findings of this study should be triangulated through an observational study monitoring slipta implementation with a sufficiently large sample size. this would enable substantiation of the findings and may reveal additional opportunities for improvement of the slipta checklist and programme. conclusion slipta checklist v2:2015 has improved compared to the alpha version. suggestions for improvement are mainly related to the point scoring system, including the designation of key questions to specific star ratings to improve the level of guidance for implementation of a qms. the lqsi tool and lqms-sip could serve as examples, leading to harmonisation of slipta with these tools. recommendations for enhanced slipta roll-out include increasing accessibility by translation into french and portuguese, training more auditors, and increasing capacity for participation. acknowledgements competing interests the authors have declared that no competing interests exist. authors’ contributions t.a.m.d. designed and performed the review and wrote the manuscript. l.o., j.e.w.b. and p.r.k. critically reviewed the analysis and the manuscript. all authors approved the present version of the manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability statement data are available upon request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references datema tam, oskam l, van beers sm, klatser pr. critical review of the stepwise laboratory improvement process towards accreditation (slipta): suggestions for harmonization, implementation and improvement. trop med int health. 2012;17(3):361–367. https://doi.org/10.1111/j.1365-3156.2011.02917.x world health organization regional office for africa. laboratory accreditation checklist. brazzaville: who-afro; 2009. world health organization regional office for africa. stepwise laboratory quailty improvement process towards accreditation (slipta) checklist. brazzaville: who-afro; 2015. [cited 2019 march 06]. available from: https://apps.who.int/iris/bitstream/handle/10665/204423/slipta-checkist0711.pdf?sequence=1&isallowed=y world health organization regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region (slipta). brazzaville: who-afro; 2015 [cited 2019 march 06]. available from: http://www.afro.who.int/en/who-guide-for-the-stepwise-laboratory-improvement-process-towards-accreditation-in-the-african-region-with-checklist.html world health organisation. joint who – cdc conference on health laboratory quality systems. lyon: who; 2008. available from: https://www.who.int/csr/ihr/lyon/report20080409.pdf?ua=1 world health organization. joint who-cdc conference on laboratory quality systems, lyon, april 2008 – joint statement and recommendations. wkly epidemiol rec. 2008;83(32):285–292. [cited 2019 march 06]. available from: http://www.who.int/wer/2008/wer8332/en/ world health organization regional office for africa. the maputo declaration on strengthening of laboratory systems. maputo: who-afro; 2008. available from: http://www.who.int/diagnostics_laboratory/maputo-declaration_2008.pdf regional committee for africa. strengthening public health laboratories in the who african region: a critical need for disease control (resolution afr/rc58/6). maputo: who-afro; 2008. [cited 2019 march 06]. available from: https://www.afro.who.int/sites/default/files/2017-06/afr-rc58-6.pdf world health organization. press release: kigali host the launch of a program to accelerate national laboratory service capcity building towards accreditation in the african region. kigali: who; 2009 [cited 2019 march 06]. available from: https://www.who.int/hiv/amds/diagnostics/amds_kigali_pr_lab.pdf nordling l. african disease labs to get health check. nature. 2009. https://doi.org/10.1038/news.2009.735 andiric lr, massambu cg. laboratory quality improvement in tanzania. am j clin pathol. 2015;143(4):566–572. https://doi.org/10.1309/ajcpab4a6wwpyien ndihokubwayo jb, maruta t, ndlovu n, et al. implementation of the world health organization regional office for africa stepwise laboratory quality improvement process towards accreditation. afr j lab med. 2016;5(1):a280. https://doi.org/10.4102/ajlm.v5i1.280 gershy-damet g-m, rotz p, cross d, et al. the world health organization african region laboratory accreditation process. am j clin pathol. 2010;134(3):393–400. https://doi.org/10.1309/ajcptuuc2v1wjqbm world health organization regional office for africa. who/afro guide for the stepwise laboratory quality improvement process towards accreditation (slipta) in the african region – revision 2 (draft). brazzaville: who-afro; 2019. clinical and laboratory standards institute. quality management system : a model for laboratory services; approved guideline – 4th ed. wayne, pa: clsi; 2011. yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3):401–409. https://doi.org/10.1309/ajcpnbbl53fwuiqj juran jm. juran on leadership for quality – an executive handbook. new york, ny: collier macmillan; 1989. aslm laboratory accreditation/slipta – aslm [homepage on the internet]. [cited 2019 apr 24]. available from: http://www.aslm.org/slipta-map/ andiric lr, chavez la, johnson m, landgraf k, milner jr da. strengthening laboratory management toward accreditation, a model program for pathology laboratory improvement. clin lab med. 2018;38(1):131–140. https://doi.org/10.1016/j.cll.2017.10.010 viegas so, azam k, madeira c, et al. mozambique’s journey toward accreditation of the national tuberculosis reference laboratory. afr j lab med. 2017;6(2):a491. https://doi.org/10.4102/ajlm.v6i2.491 maruta t, motebang d, mathabo l, rotz pj, wanyoike j, peter t. impact of mentorship on who-afro strengthening laboratory quality improvement process towards accreditation (slipta). afr j lab med. 2012;1(1):6. https://doi.org/10.4102/ajlm.v1i1.6 taremwa im, ampaire l, iramiot j, et al. assessment of three medical and research laboratories using who afro_slipta quality standards in southwestern uganda: long way to go. pan afr med j. 2017;28(1):129. https://doi.org/10.11604/pamj.2017.28.129.10995 mokobela ko, moatshe mt, modukanele m. accelerating the spread of laboratory quality improvement efforts in botswana. afr j lab med. 2014;3(2):207. https://doi.org/10.4102/ajlm.v3i2.207 mbah h, ojo e, ameh j, et al. piloting laboratory quality system management in six health facilities in nigeria. plos one. 2014;9(12):e116185. https://doi.org/10.1371/journal.pone.0116185 guevara g, gordon f, irving y, et al. the impact of slmta in improving laboratory quality systems in the caribbean region. afr j lab med. 2014;3(2):199. https://doi.org/10.4102/ajlm.v3i2.199 maina rn, mengo dm, mohamud ad, et al. progressing beyond slmta: are internal audits and corrective action the key drivers of quality improvement? afr j lab med. 2014;3(2):222. https://doi.org/10.4102/ajlm.v3i2.222 mothabeng d, maruta t, lebina m, lewis k, wanyoike j, mengstu y. strengthening laboratory management towards accreditation: the lesotho experience. afr j lab med. 2012;1(1):9. https://doi.org/10.4102/ajlm.v1i1.9 world health organization regional office for africa. who/afro slipta update [homepage on the internet]. slipta/slmta symposium 2016; 2016 [cited 2019 jul 17]. available from: https://slmta.org/uploads/category_file/29/1.4-sliptaupdates.pdf nzabahimana i, sebasirimu s, gatabazi jb, et al. innovative strategies for a successful slmta country programme: the rwanda story. afr j lab med. 2014;3(2):a217. https://doi.org/10.4102/ajlm.v3i2.217 skaggs b, pinto i, masamha j, turgeon d, gudo es. implementing laboratory quality management systems in mozambique: the becton dickinson-us president’s emergency plan for aids relief public-private partnership initiative. j infect dis. 2016;213(suppl 2):s47–s52. https://doi.org/10.1093/infdis/jiv544 yao k, maruta t, luman et, nkengasong jn. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(3):a194. https://doi.org/10.4102/ajlm.v3i2.194 andiric lr, massambu cg. one laboratory’s progress toward accreditation in tanzania. afr j lab med. 2014;3(2):202. https://doi.org/10.4102/ajlm.v3i2.202 nkrumah b, van der puije b, bekoe v, et al. building local human resources to implement slmta with limited donor funding: the ghana experience. afr j lab med. 2014;3(2):a214. https://doi.org/10.4102/ajlm.v3i2.214 nkwawir sc, batumani nn, maruta t, awasom cn. from grass to grace: how slmta revolutionised the bamenda regional hospital laboratory in cameroon. afr j lab med. 2014;3(2):a203. https://doi.org/10.4102/ajlm.v3i2.203 ntshambiwa k, ntabe-jagwer w, kefilwe c, samuel f, moyo s. translating a national laboratory strategic plan into action through slmta in a district hospital laboratory in botswana. afr j lab med. 2014;3(2):a209. https://doi.org/10.4102/ajlm.v3i2.209 masamha j, skaggs b, pinto i, et al. working toward a sustainable laboratory quality improvement programme through country ownership: mozambique’s slmta story. afr j lab med. 2014;3(2):a253. https://doi.org/10.4102/ajlm.v3i2.253 elbireer am, jackson jb, sendagire h, opio a, bagenda d, amukele tk. the good, the bad, and the unknown: quality of clinical laboratories in kampala, uganda. plos one. 2013;8(5):e64661. https://doi.org/10.1371/journal.pone.0064661 caricom regional organization for standards and quality [homepage on the internet]. lqms-sip. [cited 2019 jul 17]. available from: https://www.crosq.org/index.php/projects/lqms-sip alemnji g, edghill l, guevara g, et al. development and implementation of the caribbean laboratory quality management systems stepwise improvement process (lqms-sip) towards accreditation. afr j lab med. 2017;6(1):a496. https://doi.org/10.4102/ajlm.v6i1.496 world health organization. laboratory quality stepwise implementation tool [homepage on the internet]. 2014. [cited 2019 jul 17]. available from: https://extranet.who.int/lqsi abstract introduction methods results discussion acknowledgements references about the author(s) cheikh fall department of virology, institut pasteur de dakar, dakar, senegal aurélie cappuyns praesens foundation, brussels, belgium oumar faye department of virology, institut pasteur de dakar, dakar, senegal steven pauwels praesens foundation, brussels, belgium gamou fall department of virology, institut pasteur de dakar, dakar, senegal ndongo dia department of virology, institut pasteur de dakar, dakar, senegal moussa m. diagne department of virology, institut pasteur de dakar, dakar, senegal cheikh t. diagne department of virology, institut pasteur de dakar, dakar, senegal makhtar niang department of virology, institut pasteur de dakar, dakar, senegal alassane mbengue department of virology, institut pasteur de dakar, dakar, senegal martin faye department of virology, institut pasteur de dakar, dakar, senegal idrissa dieng department of virology, institut pasteur de dakar, dakar, senegal babacar gningue quality department, institut pasteur de dakar, dakar, senegal abdoulaye bousso senegalese health emergency operation center, ministry of health, dakar, senegal ousmane faye department of virology, institut pasteur de dakar, dakar, senegal rudi pauwels praesens foundation, brussels, belgium amadou a. sall department of virology, institut pasteur de dakar, dakar, senegal citation fall c, cappuyns a, faye o, et al. field evaluation of a mobile biosafety laboratory in senegal to strengthen rapid disease outbreak response and monitoring. afr j lab med. 2020;9(2), a1041 https://doi.org/10.4102/ajlm.v9i2.1041 original research field evaluation of a mobile biosafety laboratory in senegal to strengthen rapid disease outbreak response and monitoring cheikh fall, aurélie cappuyns, oumar faye, steven pauwels, gamou fall, ndongo dia, moussa m. diagne, cheikh t. diagne, makhtar niang, alassane mbengue, martin faye, idrissa dieng, babacar gningue, abdoulaye bousso, ousmane faye, rudi pauwels, amadou a. sall received: 25 apr. 2019; accepted: 29 may 2020; published: 20 aug. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: past and recent outbreaks have highlighted the vulnerability of humans to infectious diseases, which represent serious economic and health security threats. a paradigm shift in the management of sanitary crises is urgently needed. based on lessons from the 2014 ebola outbreak, the praesens foundation has developed an all-terrain mobile biosafety laboratory (mbs-lab) for effective field diagnostics capabilities. objective: the aim of the study was to train african teams and run a field evaluation of the mbs-lab, including robustness, technical and operational sustainability, biosafety, connectivity, turn-around times for testing and result delivery. methods: the mbs-lab was deployed in senegal in october 2017 for a six-month field assessment under various ecological conditions and was mobilised during the dengue outbreaks in 2017 and 2018. results: the mbs-lab can be considered an off-grid solution that addresses field challenges with regard to working conditions, mobility, deployment, environment and personnel safety. blood (n = 398) and nasal swab (n = 113) samples were collected from 460 study participants for molecular screening for acute febrile illnesses and respiratory infections. the results showed that malaria (particularly in kédougou) and upper respiratory tract infections remain problematic. suspected dengue samples were tested on board during the dengue outbreaks in 2017 (882 tests; 128 confirmed cases) and 2018 (1736 tests; 202 confirmed cases). conclusion: the mbs-lab is an innovative solution for outbreak response, even in remote areas. the study demonstrated successful local ownership and community engagement. the mbs-lab can also be considered an open mobile healthcare platform that offers various opportunities for field-deployable, point-of-care technologies for surveillance programmes. keywords: mobile biosafety laboratory; mbs-lab; field deployment; outbreak; senegal; point-of-care. introduction past and recent disease outbreaks (e.g. severe acute respiratory syndrome, middle east respiratory syndrome-related coronavirus, severe acute respiratory syndrome coronavirus, ebola, zika, dengue) have shown that infectious diseases continue to affect the lives of people, while also representing social, economic and national security threats that can quickly evolve into global health crises.1 for example, the 2014–2016 ebola outbreak in west africa cost $32.6 billion and resulted in the loss of 11 000 lives.2 furthermore, dengue fever has become endemic in africa with recurrent outbreaks in different countries.3,4,5 a parallel threat is the rise of antimicrobial resistance with an estimated 4.1 million deaths per year, expected to lead to a $42 trillion loss to the african economy by 2050.2 it is therefore urgent to further strengthen infectious disease surveillance and outbreak preparedness. compared to the resources devoted to dealing with other global threats such as terrorism, climate change or war, little investment is dedicated to infectious disease outbreak preparedness.6,7,8 in recent years, health professionals and decision-makers have identified several strategies needed to mitigate risk.9 however, up to now, preventive initiatives have underestimated threats. in view of this, there is an increasing international community alliance, not only to raise funding, but more importantly to develop adequate and rapid deployment of task forces to prevent or contain declared outbreaks.10 in fact, the typical pattern of infectious disease preparedness today can be characterised by international mobilisation during outbreaks, followed by relaxation and diminishing investments. the dependence on crisis response is both costly and ineffective (especially in preventing future outbreaks).8 whereas ignorance and lack of technology may have been an excuse in the past, more can and should be done for the development of strategies to achieve global health security, including a commitment from public authorities, the availability of appropriate healthcare infrastructure and qualified staff, and international operating ‘disease-fighting forces’ that can be deployed rapidly when needed.11,12 according to the world bank, africa needs between $2 billion and $3.5 billion a year for epidemic preparedness. in addition, this requires political leadership, financial commitment, partnerships and innovation.11 based on their field observations in west africa during the 2014–2016 ebola epidemic and driven by the ambition to bring advanced technologies to communities that need it most, the praesens foundation developed a truck-based mobile biosafety laboratory (mbs-lab) that included an isolator for safe handling of samples and a fully automated molecular diagnostic platform. the goal was to develop an additional tool for better preparedness and faster response to outbreaks and epidemics. the purpose was to fill the gap between the so-called suitcase-based or boxed field mobile laboratories and the much larger container-based laboratories. this combines rapidity, biosafety and advanced diagnostic technology for the rapid detection and identification of pathogens, even in hard-to-reach regions with limited to non-existent healthcare infrastructure. the key objective of this study was to evaluate the mbs-lab in an african context and to prepare local staff for its use, especially during outbreaks. the mbs-lab was challenged under various field conditions to test its operational readiness regarding the following indicators: robustness, technical and operational sustainability, autonomy, connectivity, maintenance, biosafety procedures, logistics, turn-around times and communication of test results. with its current ecosystem of fixed laboratories, field stations, national surveillance network and experience in responding to various outbreaks, the institut pasteur de dakar (ipd), which hosts a world health organization collaborating center for arboviruses and haemorrhagic fever viruses, was chosen as a partner to evaluate this mbs-lab. methods ethical considerations ethical approval to conduct the study was provided by the ministry of health, republic of senegal (0154/msas/dprs/cners; protocol sen17/48). biosafety considerations a closed biosafety isolator equipped with a high-efficiency particulate air filter was integrated in the mbs-lab. a negative pressure cascade (−25 pascal [pa], −50 pa and −25 pa inside the isolator) provided a safe environment for handling different classes of pathogen, thereby eliminating the need for personal protective equipment required for high-containment laboratory facilities. in addition, regular decontaminations were conducted by fumigation with hydrogen peroxide (h2o2) both within the isolator and the workspace of the mbs-lab (nocospray, oxy’pharm, paris, france). to prevent external contamination and ensure cleanliness inside the mbs-lab, operators were required to wear disposable shoe covers. study setting the mbs-lab was built in belgium and shipped to dakar, senegal, in september 2017. this prospective study was carried out in five different localities in senegal based on their climatological and ecological differences (from the dry northern to the humid southern regions) for six months (from october 2017 to march 2018). work was conducted in various healthcare settings ranging from district hospitals and laboratories to primary healthcare settings and outreach initiatives in remote areas. after a week of training of ipd staff, the mbs-lab was first deployed in the kédougou area (south-eastern senegal) from 08 to 23 october 2017, before being mobilised unexpectedly for dengue outbreak management in louga city (north-western senegal) from 25 october to 23 november 2017. following this event, extensive field evaluation continued in the following sites: (1) barkedji, linguère and dahra localities, which are not far from louga (03–29 december 2017), (2) bandafassi and angoussaka villages near kédougou (13–27 january 2018), (3) saint-louis city and debi-tiguette village next to the bird sanctuary of djoudj in the north (11–23 february 2018) and (4) sokone and karang in the fatick region in the central part of country, near the gambia (11–24 march 2018) (figure 1). the mbs-lab travelled over 7000 kilometres during the pilot study. figure 1: map indicating the study areas in senegal, 2017–2018. prior to deployment, ipd laboratory staff received appropriate training to work in the mbs-lab, which covered (1) necessary precautions to prevent exposures, (2) biosafety practices and procedures and (3) data and material management. in addition, the training covered realistic scientific, medical, technical and operational challenges that could be encountered in a field situation. drivers and maintenance technicians were also trained extensively for their jobs. overall, a team of more than 15 people have been trained and have participated in one or more field deployments. study population and sampling the study population included people visiting local healthcare facilities and presenting acute febrile illness or respiratory symptoms. they consisted of male and female patients of all ages who fully consented to participate in the study. for children and patients under 18 years of age, the parents or legal guardians signed the consent forms. a syndromic approach was adopted for diagnostics. therefore, blood (n = 398) and nasopharyngeal swab (n = 113) samples were collected and analysed using a multiplex strategy for the detection of plasmodium genus, arboviruses (including main flavivirus species, chikungunya and rift valley fever virus), salmonella genus and respiratory viruses (such as influenza, respiratory syncytial virus and human metapneumovirus). staff composition one scientist was in charge of coordination between local healthcare settings and the laboratory, such as specimen reception and the release of analysis results. two laboratory technicians were in charge of operations, including molecular diagnostic activities, cleaning of the laboratory and daily reporting. the dedicated driver of the mbs-lab assisted in setting up the laboratory and stabilising the vehicle. during operations, he ensured security around the laboratory and monitored its energy supply. he was also trained to resolve any minor technical issues related to the vehicle and equipment. maintenance and biosafety personnel were on call in dakar and assisted in the field on request. mobile biosafety laboratory workflow sample reception samples and clinical information forms were collected daily on-site and from neighbouring healthcare facilities for analysis in the mbs-lab, respecting cold-chain protocols during transportation. samples were then introduced from the outside directly into the isolator using the secured exterior sample hatch. the access to the sample hatch and entrance to the mbs-lab were for authorised personnel only, using a magnetic badge or key. handling inside the isolator for the protection of laboratory staff and the environment, all samples were unpacked only inside the isolator, in order to minimise the risk of exposure (figure 2). the surfaces of packaging and sample tubes were decontaminated with aniospray disinfectant (laboratoires anios, lille, france). easy and safe handling was the main objective for the design of the isolator with a negative internal pressure of up to −50 pa. the entrance and exit pass boxes flanking the isolator were set at −25 pa. therefore, specimens containing any class of infectious pathogen can be handled within the mbs-lab. figure 2: features of the mobile biosafety laboratory and platforms, senegal, 2017–2018. (1) closed under-pressured biosafety isolator; (2) idylla platform; (3) idylla cartridge; (4) smartcycler instrument; (5) telecommunication system; (6) external view of the mbs-lab; (7) biohazard waste container; (8) laboratory refrigerator. molecular platforms the modularity and flexibility of the mbs-lab enable rapid detection and identification of various pathogens using the on-board multiplexing molecular platforms. the idylla™ system (biocartis, mechelen, belgium) is a fully automated, real-time polymerase chain reaction (real-time transcriptase–polymerase chain reaction) molecular testing system designed to offer results in the minimum amount of time. all components required for nucleic acid extraction, purification, real-time transcriptase–polymerase chain reaction amplification and detection are integrated in a single cartridge that was further loaded into the idyllatm system. handling time was less than five min per sample and the liquid-tight, disposable cartridges greatly reduce the risk of contamination.13 the idylla™ ebola virus triage test, which was approved by the united states food and drug administration for emergency use authorisation, and tropical fever panel cartridges (prototype assay) were tested and compared with reference methods (manuscript in progress).14 in addition, real-time multiplex transcriptase–polymerase chain reaction tests were performed on the smartcycler device (cepheid, sunnyvale, california, united states), using a set of lightmix kits containing pre-mixed primers and probes (tib molbiol, berlin, germany) for the simultaneous detection and differentiation of up to three different pathogens in less than 1 h. results were analysed according to the manufacturer’s recommendations. runs were valid if results generated for all controls (positive and negative) were correct. samples were considered positive if there was an amplification curve with a crossing point value within the defined cut-off (crossing point < 39), equivocal if the crossing point value was higher than the cut-off and negative if there was no amplification. specimen and reagents storage the mbs-lab was equipped with a 4 °c refrigerator for short-term storage of inactivated products and reagents (figure 2). in addition, there was a portable ultra-low mini −80 °c freezer (shuttle™ ult-25ne, stirling, athens, ohio, united states) with capacity to store up to 1000 specimens, which were first stowed in cryoboxes. testing report, data processing and analysis prior to testing, samples were codified with unique anonymous numbers for traceability purposes, linking the sample number, patient identification, case definition and consent form. data were reported in a microsoft excel database (microsoft corporation, redmond, washington, united states), cleaned, and analysed using r software (r foundation, vienna, austria). the diagnostic results were delivered to physicians as early as possible for improved patient management. results overall, 460 participants, consisting of 224 women and 236 men (sex ratio = 1.05), were recruited. the median age was 18 years (range: 2 months to 70 years). a total of 398 blood samples and 113 nasal swabs were screened using the commercial lightmix kits (tib molbiol, berlin, germany) based on a syndromic approach for the diagnosis of febrile infections, including malaria, those caused by salmonella or arboviruses (chikungunya and rift valley fever virus, and flaviviruses such as dengue, zika, west nile and yellow fever virus) and respiratory diseases associated with influenza, parainfluenza, human metapneumovirus, adenovirus and respiratory syncytial viruses. the results showed that malaria remains problematic in senegal, particularly in the southern areas, where 60% (98/162) of the blood samples collected in kédougou were positive (table 1). the other malaria cases (15%, 29/195) were reported during the deployment in the north-western areas (barkedji and dahra-linguère) in december 2017. table 1: distribution of blood samples collected and pathogens identified in kédougou, barkedji, dahra-linguère, saint-louis and karang-sokone areas, october 2017–march 2018. the other major health problem was respiratory tract infections, particularly among young children. indeed, 21 metapneumovirus, 15 influenza, 3 parainfluenza and 4 picornavirus cases were diagnosed from nasal swab samples. co-infection was found in two patients with metapneumovirus and influenza virus, one with malaria and metapneumovirus, one with malaria and picornavirus and one with metapneumovirus and picornavirus (tables 1 and 2). table 2: distribution of nasopharyngeal swab samples collected and pathogens identified in kédougou, barkedji, dahra-linguère, saint-louis and karang-sokone areas, october 2017–march 2018. otherwise, the mbs-lab was mobilised during the 2017 and 2018 dengue outbreaks at the request of the ministry of health. consequently, 882 and 1736 suspected samples have been tested on-board, with 128 and 202 confirmed cases (manuscript in progress). performance of the mbs-lab the features and performance of the mbs-lab are detailed below. all-terrain truck a mercedes sprinter was converted with a six-wheel driveline and increased gross vehicle mass to 7 tons. a fixed laboratory cabin was then installed on the chassis. this combination offers a good balance between optimised mobility, size and robustness. the mbs-lab fills the gap between the extreme flexibility and mobility of suitcase-based mobile laboratories and fixed container-based solutions. the all-terrain vehicle can cover most of africa’s diverse terrain and poor roads with a driving range of about 550 km. a support vehicle (toyota hilux) accompanied the mbs-lab during deployments for personnel transportation and for carrying additional laboratory materials (figure 3). figure 3: deployment and implementation of the mobile biosafety laboratory in the field, kédougou, senegal, october 2017. power system power for the mbs-lab was supplied by lithium-ion batteries, which can be charged by the on-board diesel generator, the local electrical grid (with an inline voltage regulator) or by the vehicle’s alternator. this system guaranteed energy autonomy for the proper functioning of the fully equipped laboratory, including the air conditioning, refrigerator, lighting, telecommunication system and diagnostic equipment (idylla™ and smartcycler™ instruments) for at least one working day relying on batteries alone. the whole laboratory was self-reliant for at least a week; the air conditioning system had the largest impact on energy consumption. the vehicle was equipped with a 100-litre fuel tank and as long as diesel fuel is available, the laboratory can be operational. in the event of a total power cut-off, the isolator had a dedicated uninterruptible power supply to ensure safe shut down and decontamination. the entire power management system can be remotely monitored and controlled using the on-board communication capabilities. geolocation system a real-time satellite geo-positioning system was installed that allows for remote tracking of the vehicle. the system sent alerts when the mbs-lab left or entered a predefined geofenced zone. the cabin was equipped with an emergency distress signal and badge identification of drivers for security and traceability. communication system the mbs-lab was equipped with 3g/4g cellular routers and worldwide-secured satellite networks, which guaranteed permanent connectivity and secure communication channels between the on-field mobile laboratory and the external world. both systems also had the ability to create a local wi-fi network. these allowed for the remote monitoring of connected instruments and control computers that together form a data acquisition and monitoring system. the status and data of the mbs-lab, including the isolator pressures, local time, temperature, humidity, power system monitoring and control, test reports and error logs, were displayed on several dedicated screens in the mbs-lab. laboratory air conditioning the mbs-lab interior was kept at a controlled temperature and humidity even in hot (up to 54 °c) and humid environments. this not only created a comfortable working environment for personnel during long shifts in the laboratory, but also ensured that all equipment could function within optimal operating conditions. laboratory refrigerator a laboratory refrigerator (4 °c) provided a cold chain for the short storage of reagents as well as (inactivated) samples for further confirmation testing. the safe storage of vaccines and medicines and their transportation to and from faraway health clinics until the point of administration is another potential use. hydraulic levelling system this system automatically stabilises and levels the mbs-lab in less than five minutes on uneven terrain. once deployed, the mbs-lab is no longer susceptible to any motion induced by people entering the laboratory or by wind shear, and complies with the requirements of certain equipment to only be operated when levelled. maintenance because the base vehicle was kept standard and is based on a widely used light commercial vehicle, the manufacturer’s recommended engine maintenance intervals are not affected and regular spare parts can be used. a selection of spare parts for both the vehicle and the power supply equipment is always on board for field repairs. special spare parts were shipped to dakar when needed and installed when the mbs-lab was between deployments. in one instance, a maintenance technician was sent to the field to perform an urgent repair. the power supply system can be monitored and controlled remotely, which allows the system to send out alerts in case of issues that can also be resolved remotely, sometimes in combination with the installation of spare parts carried on board such as fuses. systematic technical checks were performed in dakar after each mission to keep the mbs-lab ready for its next (unexpected) deployment. turn-around times prior to the availability of the mbs-lab, samples had to be sent to ipd for specific diagnostic testing, causing slow turn-around times, which have a considerable effect on the quality of samples, the reliability of tests and the reporting of results. the decentralisation of testing with the mbs-lab has dramatically cut turn-around times for samples processing and results delivery from at least a week to hours (average: 4 h). biosafety risk management the mbs-lab was designed for the safe handling of all types of pathogens and reduced human and environmental exposure to potentially harmful agents. the three key elements of biological containment are laboratory practices, safety equipment and facility design. some characteristics are detailed below. personnel flow usually the mbs-lab’s occupancy does not exceed two laboratory technicians. before and after manipulation, the surface and materials used were decontaminated with aniospray disinfectant (laboratoires anios, lille, france). samples and materials flow samples and materials were brought into the isolator through the first pass box with a negative pressure of −25 pa. this pass box has two entry doors, one inside the laboratory for materials and one connected to the external sample hatch. accordingly, samples were deposited from outside into the mbs-lab via this sample hatch and from there were handled safely within the isolator, avoiding any contact between the sample and the laboratory technician. a unidirectional workflow was respected. waste management all potentially infectious waste materials were disposed of in a leak-proof biohazard waste container (jce biotechnology, za bioparc, lyon, france) (compliant with the normative requirements nf x 30-511, un 3291 and un 3249). this was safely connected to the inside of the isolator via a leak-proof hatch system (according to the recommendations of the international organisation for standardization [iso] 10648-2), preventing contamination risks.15,16 any materials which had to be taken out of the isolator, including rna samples and test cartridges, were disinfected with aniospray (laboratoires anios, lille, france) and placed through the sterile pass box before being carried out for further operations. another trash bin was dedicated to non-infectious materials. full containers were securely closed and awaited transport in the accompanying vehicle before being sent to the ipd for incineration. discussion the rising frequency of emerging infectious diseases, their increasing geographic spread and their expanding impact should make overcoming pandemic diseases an international priority. it is widely recognised that infectious diseases constitute social, economic and global security threats. outbreak control that relies exclusively on international response, mostly mounted in crisis mode, leaves little time or opportunity to train local teams and is not sustainable, as after the crisis response very little infrastructure or expertise is left behind. if time is of the essence, it seems logical to have local, regional and continental rapid response teams that are well trained and equipped with the appropriate knowledge and tools. diagnostic needs for pathogens with epidemic potential need to be addressed ahead of the next epidemic. by doing so, we can create a preparedness ecosystem that will allow the shift from a cumbersome, costly emergency response to rapid, cost-effective action for both known and unknown pathogens. key issues seem to be support for public health emergency preparedness, surveillance and response management, workforce development and knowledge at the regional and country level to withstand emerging diseases and other unexpected health events. different models of truck-based laboratories have already been developed for outbreak management or bioterrorism response17,18,19; some were deployed during the 2014−2016 ebola outbreak in west africa.19 however, mobility and flexibility to move from one hotspot to another and energy management, especially in remote areas, turned out to be the biggest challenges. in this study, the performance of the mbs-lab was evaluated against field conditions over a six-month period. the mbs-lab can be considered an off-grid solution that addresses field challenges with regard to mobility, deployment, environment and personnel safety, and operator working conditions. permanent connectivity using cellular and satellite network connections makes the transmission of data and provision of real-time information possible. the design of the mbs-lab offers safe and comfortable working conditions for operators. designed for and tested by african experts, it has demonstrated successful local ownership and management. placing patients, their healthcare providers and local communities at the centre of these activities has contributed to local support from both political and medical authorities, which proved to be key factors for a successful initiative. acting under the auspices of the senegalese ministry of health and local health authorities, the mbs-lab was fully integrated and embedded into the local health system to reinforce capacity building. the mbs-lab can be seen as an extension of a strong local reference laboratory that serves as a base station, providing trained operators with logistical support in terms of laboratory consumables and supplies, waste management and storage of biological samples collected during missions. the pilot study met the overall objective of cutting down turn-around times for diagnostic testing from (at least) a week to hours, through the decentralisation of testing at the most remote level and on-site multiplex testing using molecular platforms at the sentinel sites that served as satellite health posts. no extensive set-up or installation time is required. once on-site, the mbs-lab can be operational within the hour and moves easily between sites, which is useful during an epidemic investigation. by avoiding the need for the transportation of infectious clinical samples to centralised laboratories, the time to generate actionable results and logistical burdens are dramatically reduced. this approach led to better-informed decision-making and improved case management, even of highly mobile populations. because they relied on accurate diagnosis, treatments were more targeted and not based on clinical symptoms only. more than 1300 samples have been safely handled inside the mbs-lab, including blood samples and nasopharyngeal swabs for the detection of pathogens associated with the main tropical infectious diseases. overall, tests were correctly conducted and the results reported on average within 4 h upon receipt. prior to the deployment, no local laboratory capacity was available in selected sites and samples had to be sent to the ipd for analysis, resulting in a turn-around time of at least a week. in other words, a functional mechanism was in place but might be improved by mobile testing capacity. during the six-month period (from october 2017 to march 2018), acute respiratory infections were the common causes of febrile illnesses in practically all the settings in which the mbs-lab operated. human metapneumovirus influenza viruses, parainfluenza virus and picornavirus were the most commonly detected pathogens and were found mostly in children under 5 years, as previously reported.20 these results highlighted once again the pivotal role of respiratory viruses in acute respiratory infections. similar results were found in other countries.20,21,22 co-infection cases have become more apparent since the introduction of multiplex molecular assays; however, the impact on disease severity with arboviruses seems less well defined.23 besides the acute respiratory infections, malaria was also observed in a significant proportion. malaria is endemic in senegal, with a stratified transmission pattern characterised by a low incidence in the dry and northern regions and a relatively high incidence in the southern and humid areas. the high transmission season starts from july and continues until october, corresponding to the rainy season.24 the majority of malaria cases were found in kédougou with a peak of transmission in october during the rainy season. these findings are in agreement with other malaria reports, which shows the accuracy of the results provided by the mbs-lab and indicates that this platform could also be used to monitor the most deadly human parasite.24,25 however, no arbovirus cases were observed during the investigation at kédougou, an area where arbovirus infection is reputed to be endemic.25 whereas in asia and the americas human-to-human transmission by mosquitoes is the current form of arbovirus circulation, in west africa sylvatic circulation is predominant.26 with entomological and virological surveillance programmes, several epidemic events have been observed in kédougou after sylvatic amplification in mosquitoes.26 with climate change, urbanisation and population mobility, sporadic cases or small outbreaks are at risk of turning into large epidemics.27,28 indeed, ancestral sylvatic dengue transmission, initially carried by non-human primates and aedes mosquitoes in the forests of west africa, is now characterised by larger outbreaks, as exemplified by the recent epidemics in senegal in 2017 and 2018.29,30 in that framework, the mbs-lab was deployed, and 882 (in 2017) and 1736 (in 2018) sera samples were handled (unpublished data). the mbs-lab played a key role in managing the outbreaks with proximity, rapid response and early patient management. the response to the dengue virus outbreak has shown that the surveillance network (senegalese syndromic sentinel surveillance network) can mobilise targeted diagnostic efforts to assist in controlling disease outbreaks. as such, the mbs-lab complements this system by adding mobile testing capacity. on top of acute disease surveillance with a direct impact on public health, the mbs-lab can act in a rapid response capacity to address epidemics, ensure preventive disease surveillance and serve as a health monitoring platform and a provider of primary healthcare. as an open mobile healthcare platform, it offers various opportunities for field-deployable point-of-care technologies (e.g. molecular detection platform with multiplexing or syndromic panel testing, lateral flow, sequencing, etc.). moreover, this platform generates high-quality data that can be turned into new insights and knowledge (disease intelligence) and offers great potential for the development of disease surveillance software and epidemiological tools for integrated public health surveillance. conclusion in summary, we describe the set-up and operations of the mbs-lab, first deployed in senegal for extensive field evaluation resulting from a strong partnership between the praesens foundation and the ipd. the trained teams and mbs-lab stationed in dakar now act as a standby epidemic task force. overall, the mbs-lab is a forward-looking solution for outbreak response in remote areas with a high risk for emerging infectious diseases. however, it can also be considered an open mobile healthcare platform that offers various opportunities for field-deployable point-of-care technologies for surveillance programmes. it offers rapidly deployable, connected and state-of-the-art technology for effective field diagnostics capabilities. with extensive training and knowledge sharing, this experience perfectly illustrates that by investing in local capacity building efforts that engage communities and establish the necessary trust before a crisis hits, a country will be able to take local ownership of potential future outbreak responses and to address regional laboratory testing needs autonomously. this innovative solution has the potential to be scaled across the african continent. other domains such as public health emergencies, research, refugee camps, armies, clinical trials and vaccination campaigns also need to be explored for intervention. acknowledgements we thank the hospital staff of all partner healthcare settings and local populations for their involvement and efforts in the project. we thank the drivers, and any other staff involved in the project at the institut pasteur de dakar for their help in logistics maintenance, quality management and laboratory activities. many thanks to our partners and donors such as the praesens foundation, the senegalese ministry of health (via the prevention branch) and the global health security agenda programme for their continuous support and efforts in the project. competing interests the authors have declared that no competing interests exist. authors’ contributions r.p., s.p., a.c., ousmane f. and a.a.s. conceived the project. oumar f., b.g., a.c., c.f. and c.t.d. conceived and planned the experiments. a.b. coordinated activities of dengue outbreak responses. c.f., c.t.d., oumar f., b.g., a.c., s.p., m.f., m.m.d., i.d., m.n., g.f., a.m. and n.d. were involved in the field study. c.f. and a.c. took the lead in writing the manuscript in consultation with all the authors. sources of support the praesens foundation supported the mobile laboratory development and part of the field missions. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references nii-trebi ni. emerging and neglected infectious diseases: insights, advances, and challenges. biomed res int. 2017;2017:5245021. https://doi.org/10.1155/2017/5245021 african union. peace and security council discuss public health threats to the continent urge integration of effective public health strategies in the african union peace and security architecture, addis ababa: african union. sanou as, dirlikov e, sondo ka, et al. building laboratory-based arbovirus sentinel surveillance capacity during an ongoing dengue outbreak, burkina faso, 2017. health secur. 2018;16(s1):s103–s110. https://doi.org/10.1089/hs.2018.0048 faye o, ba y, faye o, et al. urban epidemic of dengue virus serotype 3 infection, senegal, 2009. emerg infect dis. 2014;20(3):456–459. https://doi.org/10.3201/eid2003.121885 franco l, di caro a, carletti f, et al. recent expansion of dengue virus serotype 3 in west africa. euro surveill bull eur sur mal transm eur commun dis bull. 2010;15(7). commission on a global health risk framework for the future; national academy of medicine, secretariat. strengthening the global and regional system for outbreak preparedness, alert, and response. washington, dc: national academic press (us); 2016. fatiregun aa, isere ee. epidemic preparedness and management: a guide on lassa fever outbreak preparedness plan. niger med j j niger med assoc. 2017;58(1):1–6. https://doi.org/10.4103/0300-1652.218414 global monitoring of disease outbreak preparedness: preventing the next pandemic – a shared framework – world. cambridge, ma: harvard global health institute; 2018. world health organization. risk reduction and emergency preparedness: who six-year strategy for the health sector and community capacity development. geneva: world health organization; 2007. rull m, kickbusch i, lauer h. policy debate | international responses to global epidemics: ebola and beyond. int dev policy rev int polit dév. 2015;6(6.2). https://doi.org/10.4000/poldev.2178 nkengasong jn. how africa can quell the next disease outbreaks. nature. 2019;567:147. https://doi.org/10.1038/d41586-019-00789-4 heymann dl, chen l, takemi k, et al. global health security: the wider lessons from the west african ebola virus disease epidemic. lancet. 2015;385(9980):1884–1901. https://doi.org/10.1016/s0140-6736(15)60858-3 huang h, springborn s, haug k, et al. evaluation, validation, and implementation of the idylla system as rapid molecular testing for precision medicine. j mol diagn. 2019;21(5):862–872. https://doi.org/10.1016/j.jmoldx.2019.05.007 cnops l, de smet b, faye o, et al. evaluation of a multi-pathogen tropical fever prototype test for the diagnosis of acute arboviral and malarial infections. 1st international conference on (re-)emerging infectious diseases (icreid) meeting in addis ababa, ethiopia, march 12–14, 2018. afnor, ed. nf x30-511 [homepage on the internet]. 2015 [cited 2020 may 18]. available from: https://www.boutique.afnor.org/standard/nf-x30-511/packaging-for-medical-care-waste-additional-and-or-alternative-characteristics-and-requirements-for-sharps-containers/article/818735/fa185194 who/whe/cpi/2019.20. guidance on regulations for the transport of infectious substances 2019–2020, geneva: world health organization; 2019. nisii c, castilletti c, raoul h, et al. biosafety level-4 laboratories in europe: opportunities for public health, diagnostics, and research. plos pathog. 2013;9(1). https://doi.org/10.1371/journal.ppat.1003105 paweska jt, jansen van vuren p, meier gh, et al. south african ebola diagnostic response in sierra leone: a modular high biosafety field laboratory. plos negl trop dis. 2017;11(6):e0005665. https://doi.org/10.1371/journal.pntd.0005665 zhang y, gong y, wang c, et al. rapid deployment of a mobile biosafety level-3 laboratory in sierra leone during the 2014 ebola virus epidemic. plos negl trop dis. 2017;11(5):e0005622. https://doi.org/10.1371/journal.pntd.005622 assane d, makhtar c, abdoulaye d, et al. viral and bacterial etiologies of acute respiratory infections among children under 5 years in senegal. microbiol insights. 2018;11:1178636118758651. https://doi.org/10.1177/1178636118758651 simusika p, bateman ac, theo a, et al. identification of viral and bacterial pathogens from hospitalized children with severe acute respiratory illness in lusaka, zambia, 2011–2012: a cross-sectional study. bmc infect dis. 2015;15:52. https://doi.org/10.1186/s12879-015-0779-1 ouédraogo s, traoré b, nene bi zab, et al. viral etiology of respiratory tract infections in children at the pediatric hospital in ouagadougou (burkina faso). plos one. 2014;9(10):e110435. https://doi.org/10.1371/journal.pone.0110435 asner sa, science me, tran d, smieja m, merglen a, mertz d. clinical disease severity of respiratory viral co-infection versus single viral infection: a systematic review and meta-analysis. plos one. 2014;9(6):e99392. https://doi.org/10.1371/journal.pone.0099392 seck mc, thwing j, fall fb, et al. malaria prevalence, prevention and treatment seeking practices among nomadic pastoralists in northern senegal. malar j. 2017;16:413. https://doi.org/10.1186/s12936-017-2055-x sow a, loucoubar c, diallo d, et al. concurrent malaria and arbovirus infections in kedougou, southeastern senegal. malar j. 2016;15:47. https://doi.org/10.1186/s12936-016-1100-5 diallo m, ba y, sall aa, et al. amplification of the sylvatic cycle of dengue virus type 2, senegal, 1999–2000: entomologic findings and epidemiologic considerations. emerg infect dis. 2003;9(3):362–367. https://doi.org/10.3201/eid0903.020219 rezza g. dengue and chikungunya: long-distance spread and outbreaks in naïve areas. pathog glob health. 2014;108(8):349–355. https://doi.org/10.1179/2047773214y.0000000163 murthy s, keystone j, kissoon n. infections of the developing world. crit care clin. 2013;29(3):485–507. https://doi.org/10.1016/j.ccc.2013.03.005 diagne ct, barry ma, ba y, faye o, sall aa. dengue epidemic in touba, senegal: implications for the grand magal pilgrimage for travellers. j travel med. 2019;26(7):tay123. https://doi.org/10.1093/jtm/tay123 sokhna c, goumballa n, gautret p. the grand magal of touba in the time of a dengue outbreak in senegal. travel med infect dis. 2019;28:107–108. https://doi.org/10.1016/j.tmaid.2018.11.002 abstract introduction methods results discussion acknowledgements references about the author(s) teena s.m. thomas infection control services laboratory, national health laboratory services, johannesburg, south africa department of clinical microbiology and infectious disease, school of pathology, university of the witwatersrand, johannesburg, south africa juno thomas centre for enteric diseases, national institute of communicable diseases, johannesburg, south africa karren le roux infection control services laboratory, national health laboratory services, johannesburg, south africa department of clinical microbiology and infectious disease, school of pathology, university of the witwatersrand, johannesburg, south africa sanelisiwe t. duze department of clinical microbiology and infectious disease, school of pathology, university of the witwatersrand, johannesburg, south africa faith mkhwanazi infection control services laboratory, national health laboratory services, johannesburg, south africa adriano duse infection control services laboratory, national health laboratory services, johannesburg, south africa department of clinical microbiology and infectious disease, school of pathology, university of the witwatersrand, johannesburg, south africa citation thomas tsm, thomas j, le roux k, duze st, mkhwanazi f, duse a. diagnostic challenges with accurate identification of listeria monocytogenes isolates from food and environmental samples in south africa. afr j lab med. 2022;11(1), a1482. https://doi.org/10.4102/ajlm.v11i1.1482 note: additional supporting information may be found in the online version of this article as supplementary documents 1, 2 and 3. original research diagnostic challenges with accurate identification of listeria monocytogenes isolates from food and environmental samples in south africa teena s.m. thomas, juno thomas, karren le roux, sanelisiwe t. duze, faith mkhwanazi, adriano duse received: 02 dec. 2020; accepted: 11 feb. 2022; published: 23 may 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the 2017–2018 listeriosis outbreak in south africa warranted testing for listeria monocytogenes in food products and processing environments. diagnostic tests are needed to accurately differentiate l. monocytogenes from other listeria species. objective: the study assessed the performance of the commonly used tests in our setting to accurately identify l. monocytogenes. methods: the study was conducted in a public health laboratory in south africa. cultured isolates from food and environmental samples were tested both prospectively and retrospectively between august 2018 and december 2018. isolates were phenotypically identified using tests for detecting β-haemolysis, christie-atkins-munch-peterson, alanine arylamidase (alaa), mannosidase, and xylose fermentation. listeria monocytogenes isolates were identified using automated systems, microscan walkaway plus 96, vitek® ms, vitek® 2 and surefast listeria monocytogenes plus pcr. all results were compared to whole-genome sequencing results. results: β-haemolysis and christie-atkins-munch-peterson tests gave delayed positivity or were negative for l. monocytogenes and falsely positive for one strain of listeria innocua. the alaa enzyme and colorex listeria agar lacked specificity for l. monocytogenes identification. based on a few phenotypic test results, an aberrant l. monocytogenes strain and listeria seeligeri strain were reported. all automated platforms overcalled l. monocytogenes in place of other listeria species. conclusion: no test was ideal in differentiating listeria species. this is an issue in resource-limited settings where these tests are currently used. newer technologies based on enzyme-linked immunosorbent assay and other molecular techniques specific to l. monocytogenes detection need to be investigated. keywords: listeria monocytogenes; food; environmental samples; diagnostic challenges; africa. introduction an extensive outbreak of listeriosis occurred in south africa in 2017–2018. to date, it is the largest laboratory-confirmed listeria monocytogenes foodborne outbreak described globally.1 the l. monocytogenes strain responsible for the outbreak was characterised on whole-genome sequencing (wgs) analysis as multi-locus sequence type 6.2 consequently, wide-scale testing for l. monocytogenes in various food products and processing environments commenced. for appropriate food safety and public health interventions, the utilised diagnostic tests should reliably differentiate l. monocytogenes, the outbreak pathogen, from other listeria species. listeria species and l. monocytogenes share the same growth requirements and often coexist in the same environment; therefore, l. monocytogenes should be accurately discriminated from other co-occurring listeria species.3 six listeria species (listeria marthii, listeria ivanovii, listeria seeligeri, listeria innocua, listeria grayi and listeria welshimeri) are closely related to l. monocytogenes. this close-relatedness challenges species differentiation.4 although uncommon, ‘atypical’ strains, which do not exhibit typical phenotypic characteristics, of l. monocytogenes and other listeria species have also been described.5 several test methodologies are utilised to discriminate between the listeria species; however, each has its pitfalls. l. monocytogenes is positive for the christie-atkins-munch-peterson (camp) test on sheep blood agar within 24 h of incubation.4 however, weakly haemolytic (showing haemolysis beyond 24 h of incubation) or non-haemolytic strains are frequently detected. weak or no haemolysis is due to deletion of the hyl gene or its regulatory protein prfa, which regulates the expression of virulence factors required for l. monocytogenes pathogenesis.4 other listeria species, such as l. ivanovii (particularly with the camp test utilising rhodococcus equi), l. seeligeri, and some l. innocua strains also show haemolytic capabilities which can make the utility of this test pointless.5,6,7 listeria agar by ottaviani and agosti (agar listeria ottaviani & agosti medium, biorad, berkeley, california, united states) is recommended in the international organization for standardization 11290–1, 2017 standard for the isolation and differentiation of l. monocytogenes from other listeria species.8 all listeria species are selected for growth on the medium and produce blue-green colonies due to substrate degradation by β-d-glucosidase activity. l. monocytogenes and l. ivanovii can be differentiated from the other species due to the production of an opaque halo around the colonies as a result of phosphatidylinositol-specific phospholipase (pi-plc) activity.9 the timing of the appearance of the opaque halo is also indicative of the species type. the halo is produced after 24 h incubation by l. monocytogenes and after 48 h of incubation by l. ivanovii.3,10 other strains, such as l. seeligeri, l. welshimeri, and a few strains of l. innocua, may also possess the plca gene, which codes for phospholipase activity that is responsible for creating the opaque halo around its colonies.4,5 in addition, other bacterial species, like bacillus species, cellulosimicrobium funkei, enterococci, kochuria kristinae, marinilactibacillus psychrotolerans, rothia terrae, and coagulase-negative staphylococci, may also grow as blue-green colonies on agar listeria ottaviani & agosti medium.11 bacillus circulans, bacillus licheniformis, enterococcus faecalis, enterococcus faecium/durans, and staphylococcus sciuri can produce a halo as well, which can make differentiation of l. monocytogenes from l. ivanovii difficult.11 the analytical profile index listeria test (biomerieux, marcy d’etoile, france) fails in 10% – 15% of identification cases. the main reason for this failure is due to the weak colour determinations. this is particularly applicable to the arylamidase test. the arylamidase enzyme, tested for in the popular differentiation innocua monocytogenes (dim) test, is supposed to be negative in l. monocytogenes and positive in other listeria species.12 often a weak positive dim result was considered a negative result, increasing the false positive l. monocytogenes determinations.4 this might be due to the doubtfulness of the colour determinations by the reader of the test. furthermore, false negative identification in atypical l. monocytogenes strains is also frequent.4 matrix-assisted laser desorption time of flight (maldi-tof) mass spectrometry is a quick and easy methodology gaining popularity in several microbiology laboratories. however, maldi-tof reportedly misidentifies l. innocua as l. monocytogenes american type culture collection (atcc) strain and the l. seeligeri atcc strain as l. monocytogenes or l. innocua.4 rychert et al. reported that vitek® mass spectrometer (ms) version 2.0 system (biomerieux, marcy d’etoile, france) correctly identified only 76% (34/45) of l. monocytogenes to the species level and 9% (4/45) to the genus level, while in 15% (7/45) identification could not be finalised because split identification and re-testing were not performed.13 the vitek® 2 system has also been reported to misidentify l. monocytogenes as l. innocua based on a negative reaction for phospholipase c in 1.4% (4/288) of a collection of isolates tested.14 the instrument could not identify an l. monocytogenes strain and gave a species error in another study.15 in a previous evaluation of the vitek system, when genus level identification of various listeria species was sought, the instrument had a sensitivity of 97.5%.16 the microscan walkaway si system (siemens healthcare diagnostics, west sacramento, california, united states) could not identify one l. monocytogenes atcc strain baa–751 during a comparative study with the vitek® 2 compact system.17 the reason for this was potentially attributed to the limited number of listeria species strains on the database. during the investigation of the south african listeriosis outbreak in 2017–2018, four listeria species (l. monocytogenes, l. innocua, l. welshimeri, and l. seeligeri) were detected from food samples and environmental swabs tested at the infection control services public health laboratory in johannesburg, south africa. this is similar to what has been described elsewhere in outbreak settings.5 as a result, accurate discrimination of l. monocytogenes from other species is of critical importance. whole-genome sequencing is a useful tool for confirmatory identification of l. monocytogenes and can be used as the reference standard test for comparing other tests.18 subsequent to the reported limitations of listeria tests commonly utilised in most public health laboratories, particularly in lowand middle-income countries, the infection control services public health laboratory evaluated the performance of the commonly utilised phenotypic tests (conventional phenotypic tests and chromogenic media) for the identification of listeria species in comparison to wgs results. the infection control services public health laboratory also compared the performance of the different automated diagnostic systems available in the institution for the identification of l. monocytogenes utilising known listeria isolates characterised by wgs. the study results will inform whether current tests are acceptable for future use and, if not, it will justify the evaluation of other technologies for accurate identification of l. monocytogenes. methods ethical considerations only cultured isolates from food and environmental samples were utilised in this research. no isolates from animals or animal-derived samples were used. therefore, no ethical clearance was required. study design and samples used data for this analysis were collected prospectively from august 2018 to december 2018 at the infection control services public health laboratory in johannesburg. isolates were cultured from food and environmental swabs of several food processing facilities across all of the provinces in south africa during the listeriosis outbreak period. all listeria isolates were identified on vitek® 2 (biomerieux, marcy-i’etoile, france). the phenotypic tests were performed either to (1) confirm the initial identification from vitek® 2 or (2) to discriminate between listeria species if two species identifications were given by vitek® 2. as a result, not all phenotypic tests were performed on all isolates. the accuracy of the conventional phenotypic tests to discriminate the four listeria species (l. monocytogenes, l. innocua, l. welshimeri, and l. seeligeri) was assessed (table 1).3 the isolate identity (vitek® 2 and phenotypic testing) was confirmed by wgs. table 1: phenotypic tests used to discriminate listeria monocytogenes from l. innocua, l. welshimeri and l. seeligeri. these isolates included 39 l. monocytogenes and 36 listeria non-monocytogenes species, including 28 l. innocua, seven l. welshimeri, and one l. seeligeri. laboratory analyses beta (β)-haemolysis was performed on sheep blood agar. plates were checked daily for up to 72 h. the camp test was performed using the staphylococcus aureus atcc strain 25923. the positive controls used for this test were streptococcus agalactiae atcc 13813 and l. monocytogenes atcc 19115. the plates were examined daily for up to 72 h. the presence of arylamidase, mannosidase enzymes and acid production from d-xylose (dxyl) fermentation was assessed on the vitek® 2 gram-positive card based on the results of alanine arylamidase (alaa), α-mannosidase (aman), and dxyl. the vitek® 2 gram-positive card was inoculated with the isolates as per the vitek® 2 instrument training manual.19 the colorex listeria agar (e&o laboratories ltd, bonnybridge, united kingdom) has the same constituents as the agar listeria ottaviani & agosti medium. this medium was assessed and analysis was performed retrospectively using 59 of the 75 banked listeria isolates from the analysis of the phenotypic tests. the isolates included 36 l. monocytogenes, 16 l. innocua, and seven l. welshimeri. the laboratory also verified the performance of the automated platforms available. analysis was performed retrospectively in december 2018 using 50 known listeria isolates from the laboratory repository. the listeria species included 20 l. monocytogenes strains and 30 listeria species. the 30 listeria species included 27 l. innocua, two l. seeligeri, and one l. welshimeri. these isolates were tested on four platforms, namely (1) microscan walkaway plus 96 (beckman coulter life sciences, indianapolis, indiana, united states), (2) vitek® ms version 3.0 system (biomerieux, marcy-i’etoile, france), (3) vitek® 2, and (4) surefast listeria monocytogenes plus polymerase chain reaction kit (congen, berlin, germany), run on the roche light cycler 2.0 (roche, basel, switzerland) instrument. the surefast kit identifies l. monocytogenes by amplifying a fragment of prfa and the detection limit of this assay is 10 cfu/ml as per our laboratory verification. staff were blinded to the confirmatory wgs results of the isolates during the colorex listeria agar and automated systems assessments. data analysis the sensitivity, specificity, positive predictive value, and negative predictive value of the test methods for detecting l. monocytogenes were calculated. data were collected on excel spreadsheets (microsoft, redmond, washington, united states) and analysis was performed using two-by-two tables.20 calculations were done as follows: sensitivity = true positive l. monocytogenes isolates (test and wgs positive) / total wgs-confirmed l. monocytogenes isolates (true positive + false negative) × 100 specificity = true negative l. monocytogenes isolates (test and wgs negative) / total wgs-confirmed non-l. monocytogenes isolates (true negative + false positive) × 100 positive predictive value = true positive l. monocytogenes isolates (test and wgs positive) / total positive test results (true positive + false positive) × 100 negative predictive value = true negative l. monocytogenes isolates (test and wgs negative) / total negative test results (true negative + false negative) × 100 results the phenotypic results of the listeria species in comparison to the wgs results are summarised in the online supplementary table 1. performance of the phenotypic tests for l. monocytogenes identification the three phenotypic tests used to confirm the identification of l. monocytogenes by vitek® 2 and discriminate it from the other listeria species were β-haemolysis, the camp test, and alaa activity (table 2). table 2: performance characteristics of β-haemolysis, christie, atkins and munch-peterson test and differentiation innocua monocytogenes test in comparison to whole-genome sequencing in the identification of listeria monocytogenes, infection control services public health laboratory, south africa, august 2018 – december 2018. of the 39 l. monocytogenes isolates identified by wgs, all three phenotypic tests corroborated the wgs findings in 82% (32/39) of the isolates. β-haemolysis and the camp test were absent in 18% (7/39) of the isolates. delayed positivity to both of these tests occurred at 72 h in 5.1% (2/39) of isolates. one l. innocua isolate was falsely positive to both of these tests. of the l. monocytogenes isolates, 18% (7/39) were falsely positive for alaa on vitek® 2. all l. innocua isolates and the one l. seeligeri isolate were falsely negative for alaa. performance of the phenotypic tests for l. innocua identification of note, one out of the 28 l. innocua isolates was positive for both β-haemolysis and the camp test. performance of the phenotypic tests for l. welshimeri identification the phenotypic tests used to identify l. welshimeri identified all seven of the isolates correctly. however, there was one isolate that had two identification options on vitek® 2, namely l. monocytogenes and l. welshimeri. to discriminate between the two listeria species, β-haemolysis, camp, and dxyl fermentation tests were assessed. the isolate was negative for β-haemolysis and the camp test but positive for dxyl fermentation, which suggested that the isolate is l. welshimeri. however, wgs results identified the isolate as l. monocytogenes. performance indicators, such as sensitivity, specificity, positive predictive value, and negative predictive value, for the phenotypic tests (β-haemolysis, camp test and dxyl fermentation) differentiating l. welshimeri from l. monocytogenes were not done due to the low number of l. welshimeri isolates identified during the study period. performance of the phenotypic tests for l. seeligeri identification one isolate was previously identified as l. monocytogenes based on vitek® 2 identification (99% probability), positive β-haemolysis (at 24 h incubation), positive camp test (at 24 h incubation), negative alaa enzyme activity, negative dxyl fermentation, and positive aman activity. however, wgs identified this isolate as l. seeligeri. since there was only one l. seeligeri isolate, the performance of the tests (alaa activity, aman activity, and dxyl fermentation) to discriminate this species from l. monocytogenes was not done. performance of the colorex listeria agar in the identification of l. monocytogenes all 59 isolates representing the three listeria species produced blue-green colonies on colorex listeria agar and 73% (43/59) of these isolates produced an opaque halo around the colonies (figure 1).21 of the 43 isolates that produced a halo, 84% (36/43) were identified as l. monocytogenes on wgs, while the remaining 16% (7/43) of isolates were identified as l. innocua (n = 5) and l. welshimeri (n = 2) on wgs (online supplementary table 2). the sensitivity and specificity of the medium for accurate l. monocytogenes identification were 100% and 69%. the positive predictive value was 83.7% and negative predictive value was 100%. figure 1: listeria monocytogenes colonies on colorex listeria agar. performance of the automated systems in the identification of l. monocytogenes all the systems overcalled l. monocytogenes in place of other species (table 3). all listeria species results on the various automated systems are provided in online supplementary table 3. table 3: performance characteristics of the automated systems in comparison to whole-genome sequencing in the identification of listeria monocytogenes, infection control services public health laboratory, south africa, december 2018. discussion from the evaluation, there was no ideal test for differentiating the listeria species: all had limitations. β-haemolysis and the camp test are recommended to differentiate l. monocytogenes from l. innocua. however, these tests can give delayed positivity (up to three days later) or be negative for l. monocytogenes. in addition, they may be falsely positive for certain l. innocua strains. the dim test for alaa enzyme activity lacks specificity for l. monocytogenes detection. all of the other listeria species also tested negative for this enzymatic activity, disproving its utility. we report the first aberrant l. monocytogenes strain that fermented dxyl and an aberrant l. seeligeri strain that was negative for alaa activity and dxyl fermentation and positive for aman activity. this further illustrates atypical strains that may potentially exist in our setting, complicating identification. unfortunately, wgs could not be repeated on both of these isolates again to reconfirm the results and rule out the possibility of isolate mix-up. the colorex listeria agar was able to correctly identify all l. monocytogenes isolates; however, it lacked specificity in discriminating other listeria species from l. monocytogenes. limitations there were also several shortcomings associated with the four automated diagnostic platforms tested. all platforms overcalled l. monocytogenes in place of the other listeria species. this could be because these instruments were validated for clinical samples in which l. monocytogenes is the predominant species isolated. the vitek® ms misidentified l. monocytogenes for other listeria species and the vitek® 2 gave both l. monocytogenes and l. innocua options for a few l. innocua isolates. the worst-performing platform for l. monocytogenes identification was microscan and the best performer was the surefast polymerase chain reaction kit. based on the above results, alternate testing platforms for l. monocytogenes identification need to be investigated. several other diagnostic methodologies are available to detect l. monocytogenes. these include (1) detection of l. monocytogenes by antibody-based assays, (2) molecular test methods such as loop-mediated isothermal amplification (lamp), dna hybridisation or polymerase chain reaction utilising l. monocytogenes–specific gene targets that have been identified, and (3) the use of genetically engineered bacteriophages.3,7,9,12 many of these assays are available as commercial automated kits approved by regulatory authorities. these technologies have been reported to perform well in the detection of l. monocytogenes. in addition, the automated systems are high throughput and significantly shortens the time to results of the traditional methods. the possible disadvantages of the above technologies would be: cost, staff expertise to perform the tests, and inhibition of tests (antibody-based and molecular) by the sample matrix. the molecular assays may also detect non-viable organisms. conclusion we have demonstrated that the commonly used methodologies in most public health laboratories, particularly in lowand middle-income settings, are limited in differentiating l. monocytogenes from the other listeria species. the accurate identification of l. monocytogenes is critical since it is the most predominant listeria species causing human disease and, therefore, must not be missed by diagnostic tests. the large scale of this outbreak required upscaling laboratory support for public health sample testing. however, if the available systems in routine microbiology laboratories cannot discriminate between l. monocytogenes and the other listeria species, overcalling or underreporting of l. monocytogenes can occur. underreporting l. monocytogenes will prevent or delay identifying an outbreak source and promote its continuity with huge public health impact. overcalling l. monocytogenes leads to the unnecessary closure of food production lines, which has huge financial implications for the company involved. depending on the company’s distribution level, halting production can also impact the community. in the outbreak setting, where l. monocytogenes prevalence in samples was comparatively higher, the positive predictive value of most of the tests assessed was unacceptable. hence, in a non-outbreak setting, the performance of these tests will be worse. therefore, other technologies must be investigated for their discriminatory capabilities and accurate identification of l. monocytogenes in microbiology laboratories in lowand middle-income countries. acknowledgements the authors would like to acknowledge the following for their role in the study: (1) the molecular and public health staff of the infection control services laboratory, national health, laboratory services, for conducting the tests that were required, (2) the microbiology laboratory at charlotte maxeke johannesburg academic hospital for granting permission to use the vitek® 2 and vitek® ms instruments, and (3) the national institute of communicable diseases for providing whole-genome sequencing results for all the isolates used for this research. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions t.s.m.t. and a.d. conceived the original idea for the study. t.s.m.t. supervised the study. f.m., s.t.d. and k.l.r. carried out the laboratory work and data collection. t.s.m.t. analysed the data. j.t. assisted with the whole-genome sequencing data and provided critical input to the article. all authors provided critical inputs to the manuscript. sources of support this work was supported by the national health laboratory services, south africa, by providing all consumables used for this research. data availability derived data supporting the findings of this study are available from the corresponding author, t.s.m.t., on request. disclaimer the views expressed in this publication are those of the authors and do not directly represent the views of any support agencies. references smith am, tau np, shannon l, et al. outbreak of listeria monocytogenes in south africa, 2017–2018: laboratory activities and experiences associated with whole-genome sequencing analysis of isolates. foodborne pathog dis. 2019;16(7):524–530. https://doi.org/10.1089/fpd.2018.2586 thomas j, govender n, mccarthy km, et al. outbreak of listeriosis in south africa associated with processed meat. nejm. 2020;382(7):632–643. https://doi.org/10.1056/nejmoa1907462 chen j-q, regan p, laksanalamai p, healey s, hu z. prevalence and methodologies for detection, characterization and subtyping of listeria monocytogenes and l. ivanovii in foods and environmental sources. food sci hum wellness. 2017;6(3):97–120. https://doi.org/10.1016/j.fshw.2017.06.002 pusztahelyi t, szabó j, dombrádi z, kovács s, pócsi i. foodborne listeria monocytogenes: a real challenge in quality control. scientifica. 2016;5768526:1–6. https://doi.org/10.1155/2016/5768526 orsi rh, wiedmann m. characteristics and distribution of listeria species including listeria species newly described since 2009. appl microbiol biotechnol. 2016;100(12):5273–5287. https://doi.org/10.1007/s00253-016-7552-2 johnson j, jinneman k, stelma g, et al. natural atypical listeria innocua strains with listeria monocytogenes pathogenicity island 1 genes. appl environ microbiol. 2004;70(7):4256–4266. https://doi.org/10.1128/aem.70.7.4256-4266.2004 gasanov u, hughes d, hansbro pm. methods for the isolation and identification of listeria species and listeria monocytogenes: a review. fems microbiol rev. 2005;29(5):851–875. https://doi.org/10.1016/j.femsre.2004.12.002 iso 11290-1. microbiology of the food chainhorizontal method for the detection and enumeration of listeria monocytogenes and of listeria spppart 1: detection method. 2nd ed. geneva: internation standards organization; 2017. law jw-f, ab mutalib n-s, chan k-g, lee l-h. an insight into the isolation, enumeration, and molecular detection of listeria monocytogenes in food. front microbiol. 2015;6 (1227):1–15. https://doi.org/10.3389/fmicb.2015.01227 beumer rr, hazeleger wc. listeria monocytogenes: diagnostic problems. fems immunol med microbiol. 2003;35(3):191–197. https://doi.org/10.1016/s0928-8244(02)00444-3 angelidis as, kalamaki ms, georgiadou ss. identification of non-listeria species bacterial isolates yielding a β-d-glucosidase-positive phenotype on agar listeria according to ottaviani and agosti (aloa). int j food microbiol. 2015;193:114–129. https://doi.org/10.1016/j.ijfoodmicro.2014.10.022 janzten mm, navas j, corujo a, moreno r, lópez v, martínez-suárez jv. specific detection of listeria monocytogenes in foods using commercial methods: from chromogenic media to real-time pcr. span j agric res. 2006;4(3):235–247. https://doi.org/10.5424/sjar/2006043-198 rychert j, burnham c-ad, bythrow m, et al. multicenter evaluation of the vitek ms matrix-assisted laser desorption ionization–time of flight mass spectrometry system for identification of gram-positive aerobic bacteria. j clin microbiol. 2013;51(7):2225–2231. https://doi.org/10.1128/jcm.00682-13 de lappe n, lee c, o’connor j, cormican m. misidentification of listeria monocytogenes by the vitek 2 system. j clin microbiol. 2014;52(9):3494–3495. https://doi.org/10.1128%2fjcm.01725-14 guo l, ye l, zhao q, ma y, yang j, luo y. comparative study of maldi-tof ms and vitek 2 in bacteria identification. j thorac dis. 2014;6(5):534–538. https://doi.org/10.3978%2fj.issn.2072-1439.2014.02.18 odumeru ja, steele m, fruhner l, et al. evaluation of accuracy and repeatability of identification of food-borne pathogens by automated bacterial identification systems. j clin microbiol. 1999;37(4):944–949. https://doi.org/10.1128/jcm.37.4.944-949.1999 rhoads s, marinelli l, imperatrice ca, nachamkin i. comparison of microscan walkaway system and vitek system for identification of gram-negative bacteria. j clin microbiol. 1995;33(11):3044–3046. https://doi.org/10.1128/jcm.33.11.3044-3046.1995 stasiewicz mj, oliver hf, wiedmann m, et al. whole genome sequencing allows for improved identification of persistent listeria monocytogenes in food associated environments. appl environ microbiol. 2015;81(17):6024–6037. https://doi.org/10.1128/aem.01049-15 biomerieux. vitek 2 systems: vitek 2, xl, compact training manual. biomerieux-south africa local training manual, vt2c/ vt2/vt2 xl, v1-01/06/2014. marcy-i’etoile: biomerieux; 2014. trevethan r. sensitivity, specificity and predictive values: foundations, pliabilities and pitfalls in research and practice. front public health. 2017;307(5):1–7. https://doi.org/10.3389/fpubh.2017.00307 e&o laboratories ltd webpage. pp7021 – colorex™ listeria (iso). https://www.eolabs.com/product/pp7021-colorex-listeria-iso/. abstract introduction methods results discussion acknowledgements references about the author(s) ashandree reddy department of chemical pathology, faculty of health sciences, university of kwazulu-natal, durban, south africanational health laboratory service, durban, south africa nadine rapiti national health laboratory service, durban, south africadepartment of haematology, faculty of health sciences, university of kwazulu-natal, durban, south africa verena gounden department of chemical pathology, faculty of health sciences, university of kwazulu-natal, durban, south africanational health laboratory service, durban, south africa citation reddy a, rapiti n, gounden, v. comparison of 24-hour versus random urine samples for determination and quantification of bence jones protein in a south african population. afr j lab med. 2021;10(1), a1228. https://doi.org/10.4102/ajlm.v10i1.1228 original research comparison of 24-hour versus random urine samples for determination and quantification of bence jones protein in a south african population ashandree reddy, nadine rapiti, verena gounden received: 20 mar. 2020; accepted: 04 mar. 2021; published: 04 aug. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the international myeloma working group and college of american pathologists recommend a 24-h urine collection to determine the bence jones protein (bjp) excretion level for monitoring treatment response in patients with multiple myeloma (mm). there are several issues related to sample collection and the method is prone to inaccuracy. objective: this study compared measured 24-h to random urine collections for the quantitation of bjp in a south african population. methods: sixty-six patients with mm submitted random urine samples with their routine 24-h urine collection from april 2016 – march 2018. measured 24-h urine bjp was compared to two estimated 24-h bjp excretions calculated as follows: estimation 1 (e1): estimated 24-h bjp (mg/24 h) = urine bjp/creatinine ratio (mg/mmol) × 10. estimation 2 (e2): estimated 24-h bjp (mg/24 h) = urine bjp/creatinine ratio (mg/mmol) × 15 mg/kg for women or × 20 mg/kg for men. results: correlation of estimation equations e1 and e2 to the measured 24-h urine bjp was 0.893. patients showed no difference in classification of treatment response using either the e1 or e2 estimation equations when compared to the measured 24-h urine bjp results. conclusion: this study demonstrates that the estimated 24-h bjp shows a high degree of correlation with the measured 24-h bjp and can likely be used to monitor treatment response in south african patients with mm. keywords: multiple myeloma; bence jones protein; random urine; estimated 24-h bence jones protein. introduction plasma cell neoplasms are a group of disorders which include multiple myeloma (mm), where a clone of plasma cells can secrete a homogenous immunoglobulin or its components. these may be identified as a monoclonal peak on analysis by serum or urine protein electrophoresis (upep).1 the presence, level and type of monoclonal immunoglobulin have important implications in diagnosis, staging and treatment of these disease states.2 the core diagnostic features of mm include the presence of neoplastic plasma cells on bone marrow aspirate, radiological evidence of osteolytic lesions and detection of monoclonal immunoglobulin in serum or urine.1 it is the second most common haematological cancer accounting for 1% of all malignancies worldwide. multiple myeloma was responsible for 0.43% of newly diagnosed cases of malignancies in south africa in 1999 with the incidence being reported at approximately 0.00054%.3 while the incidence is highly variable among countries, studies indicate that the incidence of mm has increased uniformly since 1990 with the largest increase in middle and low-middle income countries.4,5 the prevalence of mm is higher in hiv-positive compared to uninfected individuals.6 this increases the disease burden of mm in south africa, a country that has a high hiv epidemic prevalence with 7.5 million people living with hiv.7 monoclonal free light chains (flcs) appearing in the urine are referred to as bence jones proteins (bjps). detection and measurement of bjps are utilised to diagnose and monitor monoclonal gammopathies.8,9 once renal tubular reabsorption is saturated, bjp is present in urine. in approximately 20% of mm cases, bjps may occur in the absence of a monoclonal band in the serum, making their detection a valuable test for this malignancy.8,10 levels of bjps may be quantified by upep. following electrophoresis of the urine specimen and staining of the gel, the size of the bjp peak is measured using a densitometry scan of the peak. the percentage area of the peak is then multiplied by the total urine protein concentration of the sample to provide a semi-quantitative value for the bjp. confirmation of the presence of bjp following upep is performed via immunofixation. the international myeloma working group (imwg) and the college of american pathology recommend a 24-h urine collection for quantification of urine bjp.9,11 although 24-h urine is a definitive means to determine renal protein excretion, it has several issues especially those related to sample collection (table 1)12,13,14,15. in particular, the impracticality of a 24-h collection together with the high likelihood of incomplete collections hinder the accuracy of the test. hence the use of random or early morning urine collections has been suggested to avoid the problems associated with 24-h collections. the clinical utility of measured urine protein is improved when expressed as a ratio to urine creatinine.12,13,14 as creatinine excretion in urine is fairly constant throughout the 24 h, measurement of protein creatinine ratios allows correction for variations in urine concentration. the use of protein creatinine ratios has become widespread for routine urine protein analysis and several studies have demonstrated good correlation with the 24-h collection.11,12,13 table 1: comparison of the advantages and disadvantages of random and 24-h urine collection for bence jones protein. while the use of bjp to creatinine ratio has emerged as an alternative to the 24-h collection, few studies have examined its correlation with the 24-h collection and no reported study to the authors’ knowledge has reviewed its utility in an african population.14,15,16,17 the haematology clinic at king edward viii hospital is the referral centre for the management of patients with mm and other plasma cell neoplasms from the entire province of kwazulu-natal, south africa. many of these patients carry their 24-h collections, travelling several hundred kilometres using public transport to reach the haematology clinic. this is not ideal for maintaining sample stability while also being inconvenient and embarrassing for the patient.18 despite new therapy options, mm is largely incurable, and most patients relapse and require a change in management. laboratory testing plays a vital role in monitoring response to treatment as well as detecting a relapsed inpatient on treatment.9 this, together with the previously described issues related to 24-h urine collections, prompted us to examine the utility and validity of measured 24-h urine compared to random urine collections for the quantitation of bjp in a south african population. methods ethical considerations ethical approval to conduct the study was acquired from the biomedical research ethics committee, university of kwazulu-natal (ref. no. be509/15). written informed consent was taken from each participant in english or isizulu depending on their requirement. the raw data were securely stored by the principal investigator and the compiled electronic data were anonymised and password protected for use only by those involved in the study. participants study participants were recruited from the haematology clinic at king edward viii hospital, durban, south africa. samples were collected over a period of two years (april 2016 – march 2018). all participants had the diagnosis of mm (per imwg criteria) and were at different stages of the disease and treatment. sample collection each participant collected a 24-h urine sample for bjp following a standard protocol as part of the routine clinical assessment. the 24-h collection was started the day before the clinic visit. on submission of the 24-h collection, the participants immediately collected a random urine sample as per instructions provided. thymol was used as the preservative for the 24-h urine sample and no preservative was utilised for the random sample. both samples were submitted to the laboratory immediately. the 24-h collections were analysed as per routine by the chemical pathology laboratory. the random urine samples were analysed for urine total protein (utp) and creatinine. aliquots of the random urine samples were then frozen at –70 °c and stored for a maximum of one month (stability as per manufacturer) until the upep was performed.18 laboratory analysis for both random and 24-h urine collections, upep was performed using the sebia hydragel 7 high resolution kit run on the sebia hydrasys (sebia, norcross, georgia, united states). quantitation of upep fractions was performed using the sebia hydrasys densitometer system and phoresis software (sebia, norcross, georgia, united states). acid violet staining was used, and the sensitivity of this method allows bjp to be detected at concentrations of 15 mg/l – 20 mg/l of the original urine. urine samples for immunofixation electrophoresis (ife) analysis were concentrated using bjp concentrators from the sebia hydrasys kit for all utp samples measuring less than 0.7 g/l.18 urine total protein and urine creatinine were measured using standard spectrophotometric methods on the siemens advia 1800 chemistry analyser (siemens diagnostics, tarrytown, new york, united states). a dye-binding method using pyrogallol red was used to quantify utp.19 urine creatinine was measured using the modified kinetic jaffe method.20 only those 24-h urine samples that were positive for bjp had their respective random samples analysed to determine comparability. the measured 24–h bjp excretion was calculated as follows: %bjp peak on densitometer × utp (g/l) × 24-h urine volume (litre [l]) and multiplied by 1000 mg/24 h. the estimated 24 bjp using the random urine values were calculated as per the two formulae below: for e1, a factor of 10 was utilised because while daily excretion of creatinine is dependent on muscle mass, an average daily loss of 10 mmol of creatinine can be expected.13 e2 was based on the average urinary creatinine excretion which is higher in men (14 mg/kg/day – 26 mg/kg/day) than women (11 mg/kg/day – 20 mg/kg/day).13 of the included participants, three had paired before and after treatment samples. for the purpose of this study, they were classified according to imwg response criteria21 using only the urine and serum electrophoresis data. a complete response is a negative serum and urine m-protein immunofixation; a very good partial response is serum and urine m-protein detectable by immunofixation but not by electrophoresis or a greater than 90% reduction in serum m-protein plus urine m-protein level under 100 mg/24 h; a partial response is a greater than 50% reduction in serum m-protein and a greater than 90% or to under 200 mg/24 h reduction in 24 h urine m-protein; progressive disease is described as an increase of greater than 25% from the lowest response value in any one or more of the following: (1) serum m-component or the absolute increase must be over 0.5 g/dl, (2) urine m-component and/or the absolute increase must be > 200 mg/24 h. stable disease is not meeting criteria for complete response, very good partial response, partial response or progressive disease.21 demographic details and clinical histories were collected from the patients’ clinical records. the body mass index was calculated as weight/height2 (kg/m2) and categorised according to the world health organization. statistical analysis statistical analyses were performed using microsoft® excel (microsoft® office 2016, microsoft, redmond, washington, united states) and medcalc for windows, version 10.0 (medcalc software, ostend, belgium). the shapiro-wilk test was used to assess normality. for non-parametric data, the spearman rank correlation and passing-bablock regression analysis was utilised for comparison of different estimated 24-h bjp equations to the measured 24-h bjp. categorical data were compared using the kruskal wallis test. wilcoxon paired-sample analysis was used to compare continuous variables. a p-value of less than 0.05 was deemed to be statistically significant. results a total of 66 paired 24-h and random urine samples were collected. twenty-two samples (33%) had detectable bjp on 24-h upep and 19 (29%) had a quantifiable bjp in g/24 h. three patients had faint bands below the detectable limit (< 15 mg/l) on the measured 24-h urine with percentage bjp calculated, but did have a quantifiable bjp peak on their paired random urine sample. the urine total protein (tp) for those three 24-h samples were 0.1 g/l, 0.07 g/l and 0.05 g/l which was much lower than their random paired samples of 1.6 g/l, 1.1 g/l and 1.8 g/l. one sample had a quantifiable 24-h bjp peak (tp 0.1 g/l) with no peak on the random urine sample (tp 4.2 g/l) but the monoclonal band was present on urine immunofixation. the 22 samples with detectable bjp were from 19 patients as three patients had repeat collections within the study period. for the three patients, only their first collection was included when analysing data except when categorising the responses. there were 10 women and 9 men with 18 of the 19 patients being black african. the remaining patient was of indian descent. only 10 patients were tested for hiv with only 1 being positive. serum free light chains (sflcs) were also only measured in seven of these patients. of the 19 patients, two had no urine immunofixation request by a pathologist or analysis despite having detectable bjp on upep (table 2). the mean age was 55.8 years old (standard deviation ± 6.6) and the mean body mass index was 27.5 m2/kg (standard deviation ± 5.4) (table 3). table 2: patient characteristics including demographics, immunotyping and relevant laboratory results obtained from king edward hospital, durban, south africa, april 2016 – march 2018. table 3: summary data of the patient characteristics, the mean or median for the measured 24-h bence jones protein and the estimated bence jones protein for the 19 patients, between april 2016 and march 2018, durban, south africa. the correlation between e2 and the 24-h bjp is 0.88 for women and 0.988 for men. the e2 equation was based on the average urinary creatinine excretion which is higher in men (14 mg/kg – 26 mg/kg per day) than women (11 mg/kg – 20 mg/kg per day). the correlation between e1 which is based on creatinine excretion and 24-h bjp is 0.82 for women and 0.975 for men (table 4). table 4: estimated equations (e1 and e2) and 24-h bence jones protein categorised according to gender between april 2016 and march 2018, durban, south africa. analysis following categorisation of the three patients with paired samples per imwg bjp response criteria (table 5) showed no significant difference in classification of treatment response using either the e1 or e2 estimation equations when compared to the measured 24-h urine bjp results. absolute or percentage difference from sample a (before treatment) and sample b (after treatment) did not show pr which is characterised by a greater than 50% reduction of serum m-protein and reduction in 24-h urinary m-protein by over 90% or to under 200 mg/24 h. nor did it show progressive disease with an increase of more than 25% from the lowest response value urine m-component (the absolute increase must be > 200 mg/24 h). the average period between sample a and sample b was nine months. the treatment comprised supportive and specific individualised therapy. the specific therapy included the use of alkylating agents such as cyclophosphamide or melphalan, immunomodulatory agents like thalidomide and steroids. all three patients were classified as stable disease as per the response criteria. the revised international staging system for mm is not affected by the difference between the measured and estimate equations for these three patients as bjp is not included in its criteria. table 5: comparison of 24-h bence jones protein with the estimate equations (e1 and e2) in three paired patient samples before and after treatment over the april 2016 – march 2018 period in durban, south africa, together with their response classification. the spearman rank correlation for both estimation equations e1 and e2 was 0.893 when compared to the measured 24-h bjp. passing-bablock regression analysis showed that the e2 estimation equation had a smaller proportional bias with a slope of 0.968 compared to the e1 estimation equation slope of 0.671 when compared to the measured 24-h bjp (figure 1 and figure 2). figure 1: regression analysis of measured 24hr bence jones protein excretion versus e1 estimation for 24-h bence jones protein during the period april 2016 – march 2018 in durban, south africa. figure 2: regression analysis of measured 24-h bence jones protein excretion versus e2 estimation for 24-h bence jones protein, during the period april 2016 – march 2018 in durban, south africa. discussion the spearman rank correlation of 0.893 signifies a high degree of correlation between the estimate equations and 24-h bjp. this indicates that although the estimate equations do not precisely approximate the 24-h bjp, they are comparable and hence can be useful. the estimate equations also did not consistently overestimate or underestimate the 24-h measurement. the e2 estimation demonstrates a closer correlation and smaller proportional bias with the measured 24-h bjp compared to the e1 estimation equation and may be preferred (table 4, figure 1 and figure 2). importantly, when the three paired samples’ estimated bjps were used to classify patients according to imwg treatment response, there was no significant difference with the performance of the measured 24-h bjp and the estimated bjp using the e1 and e2 equations (table 5). this is key with regard to being able to use the random specimens for monitoring of disease. although there was a limited number of samples, this study indicates the potential use of both 24-h bjp estimates to monitor response in patients with mm and this is in keeping with prior findings in other studies performed in different population groups.16,17 the average age of mm patients in this study was 55.8 years old, which is much younger when compared to that reported in developed countries with the average age at diagnosis ranging from 65 to 70 years.22 due to the high prevalence of hiv infection in the 15–49-year-old age group (19%) in south africa, further research with the appropriate clinical and laboratory information will be key in determining if hiv is a significant risk factor for mm in the younger population.7 unfortunately this information was not available to us at the time. other findings like translocation (11;14) have been reported to be more prevalent in younger myeloma patients.23 a previous study demonstrated that it may be possible to use the protein/creatinine ratio from random urine samples to estimate the 24-h bjp excretion.16 another study concluded that protein concentrations in the same individual are relatively constant. this group also demonstrated that early morning spot specimens had a linear relation with measured 24-h bjp collections and were preferred over random urine collection.17 because patients travelled long distances and arrived at the haematology clinic at varying times, early morning specimens were a challenge to collect. despite this, our study was still able to demonstrate that a random sample can be comparable to a 24-h bjp and can be used to monitor disease response to treatment. light chains are more challenging to detect than complete igs.24 the sflc assay has increasingly been used and tracks well with proteinuria in individual patients.25,26 the greater sensitivity when compared to urine analysis has brought forth the widespread use and incorporation of sflc measurements into multiple guidelines for the management of myeloma; most recently it is a myeloma defining event in asymptomatic patients.9,27 all the study participants had a serum protein electrophoresis and a upep but surprisingly only a few had sflcs. we found only seven patients had sflcs and one of the seven samples had leaked during transit. the sflc assay is not readily available in our province (kwazulu-natal). additionally, due to inter-patient variation in the renal metabolism of light chains, quantification of proteinuria cannot be predicted by the sflc concentration.28,29,30 fifteen of the 19 patients had glomerular infiltration rates under 60 ml/min per 1.73 m2 which may affect the renal metabolism of sfl’s. the imwg states that once a diagnosis of mm is made, a 24-h upep and immunofixation should be done for patient monitoring and these measures are not replaceable with sflc.21,30 multiple myeloma is associated with significant mortality and morbidity and is considered largely incurable and fatal without treatment. with the introduction of new classes of effective drugs for the treatment of mm, improved frequencies and the degree of patient response have been observed. many treatments have been shown to significantly prolong survival and simultaneously improve the quality of life. unfortunately, all patients will ultimately relapse after treatment and will require a change to a more responsive therapy. this necessitates regular periodic monitoring of disease to detect relapse. laboratory testing plays a vital role in monitoring response to treatment as well as detecting relapse in a patient on treatment.9,21 to further elucidate the findings in this study, future studies should include a larger cohort of patients that have measurements before and after treatment enabling a more robust comparison for response criteria. this study is the first to use and to demonstrate the potential utility of the estimated 24-h urine bjp in an african population group. both the e1 and e2 calculations are simple to perform while utp and urine creatinine measurements are easily available on routine chemistry analysers. together with other studies, this study adds to the body of evidence available for use of random urine in estimating 24-h bjp for patient monitoring.16,17 limitations as a result of only including patients with densitometrically quantifiable bjp on the measured 24-h bjp, the small sample size was a limitation. however, this was also a limitation noted in other studies reviewing the use of random urine collections for bjp estimation.16,17 measuring the creatinine on the 24-h urine collections to verify the accuracy of the collection would have been beneficial.17 another limitation is the challenges associated with the method to quantify bjp. different proteins have varying affinities for the dyes used to stain electrophoretic gels and thus a lack of linearity of the densitometry response may be seen. bence jones protein may also co-migrate with other proteins or present with several bands making it complex to define the bjp peak correctly by densitometry. the measurement of bjp is not standardised and to minimise the mentioned analytical variability, it is suggested that patients should be followed up at the same laboratory, which was adhered to in this study.31 we suggest using the random bjp to monitor known patients with mm who already have confirmed bjp on immunofixation to minimise the above-mentioned limitations associated with measuring bjp on electrophoresis. conclusion the random urine bjp estimates e1 and e2 are simple, rapid, easily available and an inexpensive method that is comparable to a 24-h bjp. it can potentially be used for monitoring known patients with mm including light-chain disease. we have demonstrated that when using the imwg response classification, both e1 and e2 equations did not differ from the measured 24-h bjp. although we had a few paired samples, their encouraging comparison and utility will promote further studies with larger cohorts. acknowledgements the authors are grateful to sunitha sathabridg, a technologist at the national health laboratory service based at inkosi albert luthuli central hospital, for assistance in analysis of the urine protein electrophoresis samples. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions v.g. conceived the research idea and assisted with literature review, research protocol development, statistical analysis, interpretation of data and review of the first draft write-up. a.r. compiled the literature review and assisted in research protocol development, ethical and hospital management approval, data collection and processing, statistical analysis, interpretation of data and the journal article first draft write-up. n.r. contributed by reviewing the research protocol and coordinating data collection. sources of support the authors would like to acknowledge the south african national health laboratory service for the funding of this study. data availability raw and derived data supporting the findings of this study are available from the corresponding author, a.r., on request. disclaimer the views expressed in the submitted article are those of the authors and not an official position of the institution. references burtis ca, ashwood er, bruns de. tietz textbook of clinical chemistry and molecular diagnostics. volume 25. london: elsevier health sciences, 2012; p. 547–548. tate j, caldwell g, daly j, et al. recommendations for standardized reporting of protein electrophoresis in australia and new zealand. ann clin biochem. 2012;49(3):242–256. https://doi.org/10.1258/acb.2011.011158 visser hf, visser a, snyckers ch, goller r, pool r, myburgh jg. retrospective review of multiple myeloma and immunosecretory disorder cases diagnosed in a tertiary setting. sa orthop j. 2008;8(4):38–43. ludwig h, miguel js, dimopoulos ma, et al. international myeloma working group recommendations for global myeloma care. leukemia. 2014;28(5):981–992. https://doi.org/10.1038/leu.2013.293 cowan aj, allen c, barac a, et al. global burden of multiple myeloma: a systematic analysis for the global burden of disease study 2016. jama oncol. 2018;4(9):1221–1227. https://doi.org/10.1001/jamaoncol.2018.2128 de groot jjb, webb mj, raubenheimer je, struwig mc, louw vj. concomitant hiv infection in newly diagnosed multiple myeloma patients is hard to recognise and should be tested for routinely in areas of high endemicity. s afr med j. 2017;107(9):781–787. https://doi.org/10.7196/samj.2017.v107i9.12360 unaids. unaids data 2019. geneva: unaids; 2019. beetham r. detection of bence-jones protein in practice. ann clin biochem. 2000;37(5):563–570. https://doi.org/10.1258/0004563001899690 rajkumar sv, dimopoulos ma, palumbo a, et al. international myeloma working group updated criteria for the diagnosis of multiple myeloma. lancet oncol. 2014;15(12):e538–e548. https://doi.org/10.1016/s1470-2045(14)70442-5 dispenzieri a, kyle r, merlini g, et al. international myeloma working group (imwg) guidelines for serum free light chain analysis in multiple myeloma and related disorders. leukemia. 2009;23(2):215–224. https://doi.org/10.1038/leu.2008.307 keren df, alexanian r, goeken ja, gorevic pd, kyle ra, tomar rh. guidelines for clinical and laboratory evaluation of patients with monoclonal gammopathies. arch pathol lab med. 1999;123(2):106–107. https://doi.org/10.5858/1999-123-0106-gfcale beetham r, cattell wr. proteinuria: pathophysiology, significance and recommendation for measurement in clinical practice. ann clin biochem. 1993;30(5):425–434. https://doi.org/10.1177/000456329303000502 burtis ca, ashwood er, bruns de. tietz textbook of clinical chemistry and molecular diagnostics. volume 25. london: elsevier health sciences, 2012; p. 675–676. inker la, astor bc, fox ch, et al. kdoqi us commentary on the 2012 kdigo clinical practice guideline for the evaluation and management of ckd. am j kidney dis. 2014;63(5):713–735. https://doi.org/10.1053/j.ajkd.2014.01.416 citalia vc, kothari j, wells ej, et al. cost-benefit analysis and prediction of 24-hour proteinuria from the spot urine protein-creatinine ratio. clin nephrol. 2001;55(6):436–447. kaplan js, horowitz gl. twenty-four-hour bence jones protein determinations: can we ensure accuracy? arch pathol lab med. 2011;135(8):1048–1051. https://doi.org/10.5858/2010-0547-oar brigden ml, neal ed, mcneely md, hoag gn. the optimum urine collections for the detection and monitoring of bence jones proteinuria. am j clin pathol. 1990;93(5):689–693. https://doi.org/10.1093/ajcp/93.5.689 sebia© hydragel 7.15 & 30 β1β2 package insert. 2015-06. siemens advia chemistry systems. advia 1800 reagent package insert for total protein (urine) (upro). 2007-05. siemens advia chemistry systems. advia 1800 reagent package insert for creatinine (creatinine_2). 2014-06. kumar s, paiva b, anderson kc, et al. international myeloma working group consensus criteria for response and minimal residual disease assessment in multiple myeloma. lancet oncol. 2016;17(8):e328–e346. surveillance, epidemiology, and end results program. seer stat fact sheets: myeloma [homepage on the internet]. national cancer institute. [cited 2018 jan 11]. available from: http://seer.cancer.gov/statfacts/html/mulmy.html duek a, trakhtenbrot l, avigdor a, nagler a, leiba m. multiple myeloma presenting in patients younger than 50 years of age: a single institution experience. acta haematol. 2020;144(1):58–65. https://doi.org/10.1159/000507414 abraham rs, clark rj, bryant sc, et al. correlation of serum immunoglobulin free light chain quantification with urinary bence jones protein in light chain myeloma. clin chem. 2002;48(4):655–657. https://doi.org/10.1093/clinchem/48.4.655 alyanakian ma, abbas a, delarue r, arnulf b, aucouturier p. free immunoglobulin light-chain serum levels in the follow-up of patients with monoclonal gammopathies: correlation with 24-hour urinary light-chain excretion. am j hematol. 2004;75(4):246–248. https://doi.org/10.1002/ajh.20007 bradwell ar, carr-smith hd, mead gp, harvey tc, drayson mt. serum test for assessment of patients with bence jones myeloma. lancet. 2003;361(9356):489–491. https://doi.org/10.1016/s0140-6736(03)12457-9 jenner el, evans jar, harding sj. serum free light chain (flc) analysis: a guiding light in monoclonal gammopathy management. j appl lab med. 2017;2(1):98–106. https://doi.org/10.1373/jalm.2016.021352 dispenzieri a, zhang l, katzmann ja, et al. appraisal of immunoglobulin free light chain as a marker of response. blood. 2008;111(10):4908–4915. https://doi.org/10.1182/blood-2008-02-138602 nowrousian mr, brandhorst d, sammet c, et al. serum free light chain analysis and urine immunofixation electrophoresis in patients with multiple myeloma. clin cancer res. 2005;11(24 pt 1):8706–8714. https://doi.org/10.1158/1078-0432.ccr-05-0486 singhal s, stein r, vickrey e, mehta j. the serum-free light chain assay cannot replace 24-hour urine protein estimation in patients with plasma cell dyscrasias. blood. 2007;109(8):3611–3612. https://doi.org/10.1182/blood-2006-11-060368 graziani m, merlini g, petrini c. guidelines for the analysis of bence jones protein. clin chem lab med. 2005;41(3):338–346. https://doi.org/10.1515/cclm.2003.054 abstract background description of the intervention lessons learnt recommendations acknowledgements references about the author(s) anafi mataka african society for laboratory medicine, addis ababa, ethiopia esther a.j. tumbare elizabeth glaser pediatric aids foundation (egpaf), maseru, lesotho tsietso motsoane laboratory services, ministry of health, maseru, lesotho david holtzman partners in health, maseru, lesotho monkoe leqheka laboratory services, ministry of health, maseru, lesotho kolisang phatsoane laboratory services, ministry of health, maseru, lesotho emma sacks school of public health, george washington university, washington, district of columbia, united states anthony isavwa elizabeth glaser pediatric aids foundation (egpaf), nairobi, kenya appolinaire tiam elizabeth glaser pediatric aids foundation (egpaf), washington, district of columbia, united states how to cite this article: mataka a, tumbare eaj, motsoane t, et al. strategic site selection for placement of hiv early infant diagnosis point-of-care technology within a national diagnostic network in lesotho. afr j lab med. 2021;10(1), a1156. https://doi.org/10.4102/ajlm.v10i1.1156 lessons from the field strategic site selection for placement of hiv early infant diagnosis point-of-care technology within a national diagnostic network in lesotho anafi mataka, esther a.j. tumbare, tsietso motsoane, david holtzman, monkoe leqheka, kolisang phatsoane, emma sacks, anthony isavwa, appolinaire tiam received: 23 dec. 2019; accepted: 28 may. 2021; published: 24 aug. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: new technologies for rapid point-of-care (poc) diagnostic tests hold great potential for improving the health outcomes of hiv-exposed infants. poc testing for hiv early infant diagnosis (eid) was introduced in lesotho in late 2016. here we highlight critical requirements for selecting routine poc eid sites to ensure a sustainable and optimised eid diagnostic network. intervention: lesotho introduced poc eid in a phased approach that included assessments of national databases to identify sites with high test volumes, the creation of local networks of sites to potentially increase access to poc eid, and a standardised capacity assessment to determine site readiness. potential site networks comprising ‘hub’ testing sites and ‘spoke’ specimen referring sites were created. lessons learnt: after determining optimal placement, a total of 29 testing facilities were selected for placement of poc eid to potentially increase access to 189 facilities through the use of a hub-and-spoke model. site capacity assessments identified vital human resources and infrastructure capacity gaps that needed to be addressed before introducing poc eid and informed appropriate poc platform selection. recommendations: poc placement involves more than just purchasing the testing platforms. considering the relatively small proportion of sites that can be eligible for placement of a poc platform, utilising a hub-and-spoke model can maximise the number of health facilities served by a poc platform while reducing the necessary capacity building and infrastructure investments to fewer sites. keywords: hiv early infant diagnosis; point-of-care; increased health access; site selection. background in lesotho, a country with a high burden of hiv infection of nearly one in every four pregnant women and adults aged between 15 years and 49 years,1 antenatal care and hiv treatment for women and children are delivered at three levels: primary, secondary and tertiary. at the primary healthcare level are health centres, health posts and all community-level outreaches. district hospitals serve as next level referral facilities for all health centres in the district and refer cases to a national referral hospital at the tertiary level. laboratory services are structured similarly. however, in 2016 only one laboratory, the national reference laboratory (nrl) in the capital city, processed dried blood spot (dbs) samples for hiv early infant diagnosis (eid) testing, and this was done using conventional complex laboratory equipment. the dbs samples were transported from various health facilities all over the country to the nrl using a motorcycle-based national sample transport system. one of the main challenges with this centralised eid system was long turn-around times (tats) – typically between 30 days and 90 days – for returning results to local health centres and caregivers.2,3 reasons for the long tats include delays at the clinics before specimens are dispatched to the district hospital laboratory and delays in sending samples from the district hospital laboratory to the nrl. also, at the nrl, waiting to batch samples before analysis to allow sufficient numbers for full test runs on the instruments added to the long tat. further delays occur during the return of paper-based results to clinics through the same sample transport system before finally reaching caregivers, who may not have telephone access to facilitate quicker result communication or live far from facilities. to strengthen this centralised system, the ministry of health has implemented mechanisms such as routine training on dbs collection to ensure the quality of specimens and reduce rejection rates and provision of more motorcycles to increase the pick-up frequency of dbs and other clinical samples from facilities; however, many challenges remain. new technologies for rapid, point-of-care (poc) molecular diagnostic tests hold great potential for improving timely management of infants with hiv infection by eliminating delays in the return of results and enabling rapid initiation of antiretroviral therapy.4,5 lesotho embraced poc eid testing in 2016, making it one of the first countries to introduce the technology into routine clinical settings. the poc platforms were initially thought to be particularly useful for hard-to-reach areas, where delays in result return were often the longest. however, given the large numbers of primary and secondary health facilities (255 such facilities in lesotho) and the limited number of platforms that were available due to funding constraints, not all health facilities or service delivery points could receive poc eid platforms. hence, strategic placement of the poc eid platforms was necessary. before 2016, poc eid testing was primarily introduced in controlled environments such as research and pilot projects in sub-saharan africa.5,6,7 many poc platforms for various diagnostic tests have been deployed in healthcare settings. however, few implementers have tried to describe and explain the actual real-life process for introducing and integrating a new poc into a national health system for routine use. a better understanding of how the introduction of new poc technologies is implemented could help us anticipate possible challenges and identify elements needed for successful implementation in other limited-resource settings. this article aims to share lesotho’s experiences of a systematic approach to identifying and assessing the readiness of potential poc eid sites and to highlight the critical requirements for selecting sites to integrate routine poc eid into a sustainable and optimised eid diagnostic network. description of the intervention ethical considerations the approval to conduct site selection and capacity assessments was obtained from the lesotho ministry of health research and ethics committee (approval number id 29-2016, dated 13 january 2016). setting the processes of selecting sites eligible for poc eid testing, assessing site capacity and needs, and designing the poc eid network were carried out for all the ten districts of lesotho in the year 2016. the geography of lesotho’s highly mountainous kingdom poses many challenges to providing high-quality healthcare because of the difficulties to reach the remote facilities, especially those high up the mountains. the site selection was carried out in two main phases: an initial desk analysis to determine potential sites that could access poc eid and capacity assessments to determine the readiness for implementation of poc testing. desk analysis to determine potential health facilities eligible for poc eid two data sources were used in the desk analysis, namely the 2014 national population projections (unpublished) for catchment areas of health facilities (n = 158) in rural, peri-urban and urban settings and the ministry of health’s laboratory information system. data on the expected volume of eid tests in each site were extracted; these data were based on the highest number of expected pregnancies from hiv-positive women, the expected number of hiv-exposed infants, or historical dna polymerase chain reaction tests. data was also collected on the availability of onsite antiretroviral therapy or paediatric antiretroviral therapy services, facilitating the ability to use results immediately in a real poc setup. the ministry of health’s laboratory information system was queried in march 2016 for all eid test samples collected from hiv-exposed infants in the 255 facilities nationwide in the previous year (january 2015 to december 2015). the data were disaggregated to total samples per site in a year. to get testing rates per day, the annual test volume was divided by 12 months, following which the result was divided by 22, which is the number of working days per month. a threshold of 0.5 tests per day (at least one test every two days) was set as the minimum test volume for sites that would receive a poc platform. this threshold was regarded as sufficient to maintain operator competency and provided a throughput of tests high enough to justify the cost of the poc platforms. using a hub-and-spoke model, several health facilities with low test volumes were combined with other low-volume sites or with sites with higher test volumes to form local testing networks that reached or exceeded the threshold of 0.5 tests per day. in this model, the sites were grouped around a central location (hub) with a testing platform that would serve the surrounding health facilities (spokes). criteria for eligibility of a spoke site included proximity to the nearest potential poc hub site and the existence of a sample transport system to a nearby possible hub site. proximity was defined as facilities that were, on average, no further than 60 km from the possible testing site. spoke sites are required to send samples using sample transport carriers to a nearby hub site that provides onsite poc eid testing service, thus eligibility was also based on the ability to send samples in ethylenediaminetetraacetic acid-treated capillary tubes according to laboratory and manufacturers’ standards. the latter recommends specimens to be carried within 24 h before testing if kept at ambient temperature and within three days if kept and transported between 2 °c and 8 °c. these hubs and spokes already existed as part of the national sample transport system; hence, no additional transport or new routes were created. eligible sites were then scheduled for site capacity assessments to determine their readiness for poc eid introduction. the first 15 sites assessed were located in rural, peri-urban and urban settings across all the 10 districts. the remaining 14 testing sites were also scheduled to undergo capacity assessments later and were placed in a ‘scale-up’ phase of the implementation. site capacity assessments of selected poc eid sites capacity assessments were conducted using a standardised checklist adapted from the stepwise process for improving the quality of hiv-related point-of-care testing checklist version 2.0, 16 september 2014.8 the stepwise process for improving the quality of hiv-related point-of-care testing checklist attempts to harmonise with international regulations to evaluate sites consistently. the adapted tool has eight domains and 49 questions. each domain had questions on how well the facility performs specific tasks. based on the findings, each of the eight domains was given a weighted score. the total for all the domains was computed to arrive at a numerical percentage score. site readiness level, ranging from level 0 to level 4, was determined based on these scores. level 0 (0% – 40%) sites were those that needed improvement in all areas, level 1 (40% – 59%) sites required improvement in specific areas, while level 2 (60% – 79%) sites were partially eligible. level 3 (80% – 89%) sites were close to pilot site capacity but needed some upgrades or improvements, while level 4 (90% or higher) sites were fully eligible for selection as pilot sites. data management and analysis descriptive analyses (percentage scores by checklist domain for each health facility and median scores per checklist domain) were carried out using microsoft excel (microsoft corporation, redmond, washington, united states). statistical comparisons between facilities that underwent capacity assessments were not conducted due to the small sample size (n = 15). data on historical laboratory eid test volumes were exported into an excel database to calculate the test rate per site and aggregated test volumes for each potential local testing network. lessons learnt this article presents the steps taken before the introduction of poc platforms in lesotho, including health facility pre-selection, data analysis aimed at maximising the use of available poc platforms, and the determination of site readiness for poc eid. desk analysis to increase access to poc eid testing important considerations for setting up poc eid testing at a health facility include sufficient test volumes of at least one test every two days and the availability of hiv treatment services or referral treatment, including paediatrics, at the health facility or within a set distance to allow quick referrals. the geographic locations are also an important consideration as tats for delivery of eid results from the centralised laboratory may be prolonged in hard-to-reach areas and affect the ability to monitor the performance of eid testing. most of the sites assessed had test volumes that were too low to justify the placement of an instrument. creating local testing networks to increase testing volumes can improve access in underserved areas that are very far from the nrl. we considered all facilities with records of having collected samples for polymerase chain reaction from infants in the previous year. it has been demonstrated that some areas in hard-to-reach regions have challenges sending samples to the national laboratories and often have very long tats from sample collection to return of results.3 thus, in developing an optimised network map for both conventional and poc eid testing, poc testing should be considered not only as a tool for hard-to-reach areas but also to reduce tat from sample collection to return of results. the availability of systems for sample transport to the hubs with poc platforms would mean that although these sites would not get the eid result on the same day, they would get them in a few days since most of the sites were serviced by the sample transport system at least twice a week. this would be a potentially significant improvement compared to conventional eid when specimens are processed in the conventional laboratory, where results often took quite long (usually months) to reach the caregiver for several reasons.3 these poc sites would also take some of the burden off conventional laboratories that are overloaded with viral load (vl) testing and improve tat. following the desk analysis, in which a total of 255 sites were analysed, poc eid was determined to be potentially suitable for introduction into 29 testing sites. through the use of the hub-and-spoke model, it was found that access to poc eid could be increased 6.5-fold from 29 to 189 sites; five of the 29 sites were stand-alone testing sites with no associated spoke sites and 24 were testing hubs that were designated to receive samples from an additional 160 spoke sites (table 1). thus, in total, 189 sites were selected for possible access to poc eid based on the hub-and-spoke model, most (88%; 166/189) of which had fewer than one test every two days (< 0.5 eid/day). the remaining 66 of the 255 sites would continue to be served by the conventional system because of their proximity to the nrl and the sample transport network. table 1: outcomes of the desk analysis for site selection and mapping for placement of hiv point-of-care early infant diagnosis using a hub-and-spoke model in lesotho, 2016. during the assessment of each spoke site, it was essential to assess distance and accessibility to the nearby potential testing site in terms of how samples could be transported in ethylenediaminetetraacetic acid-treated capillary tubes according to laboratory and manufacturers’ standards. for the optimal quality of samples, the facility should be within 60 min of the hub site by normal transport modes. where this is not possible, a dbs sample becomes ideal; this is in place in many countries. however, the challenge of long tats for results delivery still exists. therefore, governments need to strengthen systems to improve the value of using alternative sample types for eid and vl.9 creating a local hub-and-spoke network contributes to strengthening the overall network by potentially reducing tat and placing less burden on the conventional system that could use the space for vl testing. due to the high workload at the nrl, which had a backlog, eid was run only 1–2 days per week. the platforms selected for potential poc facilities, namely the cepheid genexpert (gx-vi) four-module instrument (cepheid, sunnyvale, california, united states) and the m-pima (abbott laboratories, chicago, illinois, united states), formerly the alere q, are capable of conducting other tests beyond hiv eid. for example, both can do hiv vl testing, while the cepheid genexpert can also test for other infections such as tuberculosis, hepatitis, human papillomavirus and more.10 however, at the time of selection, there was no evidence of the feasibility of poc testing for hiv vl monitoring. since these were new, it was decided to start testing for eid and not perform integrated testing. point-of-care site capacity assessments a total of 15 sites with the highest test volumes were assessed for capacity to conduct poc eid testing. of the 15 facilities assessed, only one facility was fully eligible (level 4) for implementation of poc eid (table 2). the majority (n = 14) were not immediately ready to implement new poc eid testing and required structural upgrades, including process-related improvements. the required infrastructural improvements included providing room and tables for poc platforms and shelves to store poc commodities, providing fridge thermometers, and installing air conditioners and power inverters or backup for some sites. required process-related improvements included improving site-level stock management through training, integrating standardised forms to document patient and specimen information, as well as printing and distributing eid testing algorithms, job aids, logbooks, and poc eid testing forms. documentation of standard operating procedures, safety practices and data management mechanisms also needed to be improved in all facilities (table 3). also, competent poc platform operators were required to be present or recruited in all facilities. across all health facilities, the highest median scores were obtained in integrating poc services into hiv care (100%), which evaluated the prior experience in poc testing for any disease, such as rapid testing for hiv and malaria. this was followed by the domains on quality control (92%) and safety practices (92%). the lowest median scores were obtained in the supplies, reagents and equipment management domain (57%) and the personnel training, competency and certification domain (67%) (figure 1). figure 1: median performance scores per stepwise process for improving the quality of hiv-related point-of-care testing checklist domain of potential point-of-care early infant diagnosis health facilities (n = 15) in lesotho, february 2016. table 2: readiness of selected health facilities in lesotho (n = 15) in 2016 to introduce hiv point-of-care early infant diagnosis based on a standardised tool adapted from the stepwise process for improving the quality of hiv-related point-of-care testing checklist version 2.0. 9/16/2014. table 3: common gaps identified during the site capacity assessments of health facilities (n = 15) in lesotho, 2016. there was adequate space in all the 15 facilities where poc testing equipment could be placed, although not all were designated for poc. all facilities had reliable paediatric hiv counselling and treatment services at the facility or within a reasonable distance. however, despite the prior experience of conducting poc tests for rapid hiv or any other disease, there were several common gaps across all health facilities. several sites had inadequate security or lacked room temperature monitoring at the poc testing or storage areas for commodities. the facilities also had poor documentation, did not sufficiently manage the supplies of already existing poc tests and lacked storage capacity for poc commodities (table 3). before the introduction of poc eid in these sites, room temperature monitoring thermometers, security padlocks, tables, and cabinets to store poc commodities had to be procured. in addition to equipment-related training, all poc testing personnel were trained on supplies and commodities management, safety, pre-testing, and post-testing procedures, including result management and poc eid use in the existing national eid algorithm. since poc eid was new in the country, a testing algorithm for use at poc sites was developed and provided to facilities as a reference guide. the algorithm followed the same testing interval as the existing national algorithm. still, it specified how to handle test results, given that tests are available on the same day, which was not the case with the current centralised system. another critical consideration was the duration of the steps taken to introduce poc testing. the site selection process took three months, while the procurement, including site upgrades, took nearly four months to complete. it is therefore essential to take into account the time lag, including procurement lead times for commodities. where possible, it is best to consider procuring materials for upgrades locally to reduce lead times associated with external procurement. the procurement of poc eid platforms was planned to suit each site’s needs-based infrastructure, availability of reliable electricity, dynamics of daily patient testing volumes, and other characteristics as determined on the findings of the physical capacity assessments. the selection was informed by a side-by-side analysis, using a tool12 that compared three platforms based on various characteristics such as maximum throughput, storage temperature of reagents, power requirements, type of sample (whole blood or capillary), time to result, waste management needs and regulatory approvals. many of these elements were assessed during the capacity assessment. the cepheid genexpert four-module instrument and a laptop computer were placed in 16 sites, and the m-pima, formerly the alere q, was found to be suitable for 13 of the sites. a phased rollout plan was developed, which involved the implementation of five sites (three hubs and two stand-alone sites) in the first phase, the implementation of a further ten sites after 6 months with the modification of appropriate steps based on lessons learned in the first phase, and the implementation of 14 additional sites in a final scale-up phase after 12 months. at the time of writing, a total of 23 hubs with 131 spokes and six stand-alone sites had been implemented. we used historical testing rates to estimate expected test volumes. one limitation of this method is that we could not fully guarantee the same test volumes in future because other variables such as population movements and birth rates could change. while the results of the site selection and network mapping process showed that poc eid could potentially increase access to testing, a post evaluation is needed to verify this potential impact in this setting. recent data from the multi-country evaluation showed that poc eid resulted in better outcomes for hiv-exposed and hiv-positive infants. the data, which included lesotho, showed that compared to conventional laboratory testing,12 there was a five-fold increase in the proportion of infants receiving eid results within 30 days of the eid test and the number of infants initiated on antiretroviral therapy within 2 months more than doubled. given the many steps involved in introducing poc testing in a country and the data requirements to make evidence-based decisions, all key stakeholders, must be identified and consulted. stakeholders’ participation should be coordinated, preferably by the directorate of laboratories or equivalent leadership for diagnostics in a country. such stakeholders include development partners who may also have plans to roll out poc in specific areas, other ministries such as the information ministry to advise on data connectivity, and environmental units to guide waste management. clinicians and key technical working groups such as the prevention of mother to child transmission and the laboratory technical working groups are also important stakeholders in their advisory roles for the ministry of health. supply chain and procurement units need to be looped in early to avoid unnecessary delays for procured materials. recommendations our site selection process suggests that placement of poc eid platforms at well-prepared sites within diagnostic networks in limited-resource settings can improve access and that optimal introduction of poc eid transcends mere poc platform procurement. for pre-selected potential sites meeting all preliminary criteria, more detailed site capacity assessments of human resources and infrastructure capacity are required. in the case of many sites being ineligible for a poc platform based on insufficient testing volumes, and considering that the vast majority of them require some level of capacity building or infrastructure upgrade, a hub-and-spoke model can be adopted to potentially increase access to poc eid. acknowledgements the authors wish to thank the poc eid teams in lesotho and the elizabeth glaser pediatric aids foundation headquarters, the ministry of health in lesotho, and the laboratory and health worker staff at all participating facilities. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.m. drafted the manuscript, contributed to the data analysis and revised the manuscript. e.a.j.t. and d.h. contributed to the study design and data analysis and revised the manuscript, and collected data. m.l. and k.p. collected data and revised the manuscript. t.m., e.s. and a.i. analysed and interpreted the data and revised the manuscript. a.t. contributed to the design of the study and revised the manuscript. all authors gave final approval of the version to be published and agree to be accountable for the accuracy and integrity of the work. sources of support the introduction of poc eid in lesotho was made possible through funding from unitaid, ‘introduction of point-of-care early infant diagnosis in decentralized settings: creating a market for affordable, effective, and equitable hiv testing of exposed infants’. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references ministry of health/lesotho and icf international. lesotho demographic and health survey 2014 [internet]. maseru, lesotho: ministry of health/lesotho and icf international; 2016 [cited 2018 aug 27]. available from: http://dhsprogram.com/pubs/pdf/fr309/fr309.pdf gill mm, hoffman hj, mokone m, et al. assessing very early infant diagnosis turnaround times: findings from a birth testing pilot in lesotho. aids res treat. 2017;2017:8. https://doi.org/10.1155/2017/2572594 tiam a, gill mm, hoffman hj, et al. conventional early infant diagnosis in lesotho from specimen collection to results usage to manage patients: where are the bottlenecks? plos one. 2017;12(10):e0184769. https://doi.org/10.1371/journal.pone.0184769 mwenda r, fong y, magombo t, et al. significant patient impact observed upon implementation of point-of-care early infant diagnosis technologies in an observational study in malawi. clin infect dis. 2018;67(5):701–707. https://doi.org/10.1093/cid/ciy169 jani iv, meggi b, mabunda n, et al. accurate early infant hiv diagnosis in primary health clinics using a point-of-care nucleic acid test. j acquir immune defic syndr. 2014;67(1):e1. https://doi.org/10.1097/qai.0000000000000250 dube q, dow a, chirambo c, et al. implementing early infant diagnosis of hiv infection at the primary care level: experiences and challenges in malawi. bull world health organ. 2012;90(9):699–704. https://doi.org/10.2471/blt.11.100776 technau k-g, kuhn l, coovadia a, carmona s, sherman g. improving early identification of hiv-infected neonates with birth pcr testing in a large urban hospital in johannesburg, south africa: successes and challenges. j int aids soc. 2017;20(1):21436. https://doi.org/10.7448/ias.20.01/21436 world health organization. improving the quality of hiv-related point-of-care testing: ensuring the reliability and accuracy of test results [homepage on the internet]. who; 2015 [cited 2018 aug 27]. available from: http://apps.who.int/iris/bitstream/10665/199799/1/9789241508179_eng.pdf egpaf. side-by-side analysis of poc eid products | children & aids [homepage on the internet]. [cited 2021 feb 16]. available from: http://childrenandaids.org/node/982 world health organization. toolkit: hiv molecular diagnostics toolkit to improve access to viral load testing and infant diagnosis: hiv treatment and care [homepage on the internet]. world health organization; 2019 [cited 2020 jun 26]. available from: https://apps.who.int/iris/handle/10665/325961 cepheid | molecular testing [homepage on the internet]. [cited 2021 feb 16]. available from: https://www.cepheid.com/en/tests bianchi f, cohn j, sacks e, et al. evaluation of a routine point-of-care intervention for early infant diagnosis of hiv: an observational study in eight african countries. lancet hiv. 2019;6(6):e373–e381. https://doi.org/10.1016/s2352-3018(19)30033-5 article information authors: rosemary a. audu1 catherine c. onubogu2 rosemary n. okoye3 nkiru n. nwokoye2 chika k. onwuamah1 adesola z. musa4 toyosi y. raheem2 maureen n. aniedobe1 samuel j. nduaga3 ini-obong essien3 emmanuel o. idigbe2 affiliations: 1human virology laboratory, nigerian institute of medical research, nigeria2national tuberculosis reference laboratory, nigerian institute of medical research, nigeria 3clinical diagnostic laboratory, nigerian institute of medical research, nigeria 4monitoring and evaluation unit, nigerian institute of medical research, nigeria correspondence to: rosemary audu postal address: nigerian institute of medical research, pmb 2013, yaba, nigeria. dates: received: 25 mar. 2013 accepted: 09 july 2014 published: 24 oct. 2014 how to cite this article: audu ra, onubogu cc, okoye rn, et al. proficiency testing for hiv, tuberculosis and malaria diagnosis in clinical laboratories in nigeria. afr j lab med. 2014;3(1), art. #102, 9 pages. http://dx.doi.org/10.4102/ ajlm.v3i1.102 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. proficiency testing for hiv, tuberculosis and malaria diagnosis in clinical laboratories in nigeria in this original research... open access • abstract • introduction • research method and design    • phase 1: questionnaire survey    • phase 2: provision of proficiency testing service    • characterisation of panels       • hiv       • tuberculosis       • malaria    • panel distribution    • mentoring component    • data analysis    • ethical considerations • results    • questionnaire survey    • participation and response rate of laboratories    • proficiency testing panel testing results    • feedback from participating laboratories • discussion    • limitations of the study    • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: proficiency testing (pt) is a means of verifying the reliability of laboratory results, but such programmes are not readily available to laboratories in developing countries. this project provided pt to laboratories in nigeria.objectives: to assess the proficiency of laboratories in the diagnosis of hiv, tuberculosis and malaria. methods: this was a prospective study carried out between 2009 and 2011. a structured questionnaire was administered to 106 randomly-selected laboratories. forty-four indicated their interest in participation and were enrolled. four rounds of pre-characterised plasma panels for hiv, sputum films for tuberculosis and blood films for malaria were distributed quarterly by courier over the course of one year. the results were returned within two weeks and scores of ≥ 80% were reported as satisfactory. mentoring was offered after the first and second pt rounds. results: average hiv pt scores increased from 74% to 95% from the first round to the third round, but decreased in the fourth round. for diagnosis of tuberculosis, average scores increased from 42% in the first round to 78% in the second round; but a decrease to 34% was observed in the fourth round. malaria pt performance was 2% at first, but average scores increased between the second and fourth rounds, culminating in a fourth-round score of 39%. many participants requested training and mentoring. conclusions: there were gross deficiencies in the quality of laboratory services rendered across nigeria. in-country pt programmes, implemented in conjunction with mentoring, will improve coverage and diagnosis of hiv, tuberculosis and malaria. introduction top ↑ the importance of quality services in healthcare laboratories in developing countries has been recognised universally. 1, 2, 3 at present, the laboratory infrastructure and test quality for all types of clinical laboratories remains weak in most countries in africa. 4,5 laboratories applying the principles of a quality management system (qms) generate reliable and cost-effective results; moreover, quality management is one of the major building blocks of the accreditation process in the african region. 6,7 it has become necessary to strengthen the capacity of clinical laboratories in order to ensure the generation of quality results that are suitable for use by clinicians and which benefit patients.proficiency testing (pt) is an external quality assessment (eqa) programme involving sending a panel of samples to a group of participating laboratories.8 although the organisers issuing the panels know the result, the participating laboratory personnel do not. pt verifies that laboratories are proficient in their testing process and can obtain accurate and reliable results. 6 comparison of results between groups of laboratories may also be used to validate a particular measurement process. as beneficial as the pt programmes may be, they are not readily available to many laboratories in developing countries. some of the limitations to local laboratories' enrolment in foreign programmes are the high cost, challenges of transportation with respect to country regulations, suitable means of transport of specimens to sites, difficulty in interpretation of pt results and the absence of technical support with regard to identifying and correcting causes of poor performance. 6 since laboratory-confirmed diagnosis of hiv, tuberculosis (tb) and malaria is essential to public health prevention and support services, accurate and reliable laboratory results are critical.9 in addition, the world health organization's regional office for africa (who afro) has recommended that national public health reference laboratories develop and implement a qms,2 including participation in an eqa scheme.7 these reference laboratories, according to who afro, should, in turn, provide national eqa programmes to other laboratories within the country.7 as a result of the challenges encountered by laboratories, a national pt feasibility study for hiv, tb and malaria was undertaken at both public and private health laboratories in nigeria. this study was conducted to evaluate the level of implementation of qms in nigeria and to assess the proficiency of laboratories in the diagnosis of hiv, tb and malaria. research method and design top ↑ this was a prospective study carried out in two phases between 2009 and 2011. the first phase was a questionnaire survey to provide baseline information on quality practices in laboratories, whilst the second phase was the provision of pt services. phase 1: questionnaire survey in the first phase, six states were selected at random from each of the six geopolitical zones of nigeria. six focal persons who were laboratory state coordinators were then identified and recruited to serve as zonal coordinators. zonal coordinators identified 20 laboratories, each from different local government areas within the six states. structured questionnaires, adapted from the who template, 10 were developed and field tested prior to distribution of the survey (box 1). the questionnaire had five sections, including general questions about the laboratory, specific questions about hiv, tb and malaria units and questions on general quality issues. the laboratory head and respective heads of each unit completed the questionnaires. the questionnaires were sent to the zonal coordinators to administer to the 120 laboratories, 106 of which consented to participate and returned completed questionnaires. data entry and analysis were performed using the filemaker pro v10 (2009) database and microsoft® excel, respectively. phase 2: provision of proficiency testing service of the 106 laboratories that completed the questionnaires, 44 indicated their interest regarding participation in the joint pt programme for the three major diseases of public health importance, namely, hiv, tb and malaria. these laboratories were enrolled in the second phase of the study. by september 2010, pre-characterised panels were prepared for hiv, tb and malaria in the nigerian institute of medical research (nimr)’s reference laboratories and were distributed to the participating laboratories by courier. four rounds of panels each of hiv, tb and malaria were sent to each laboratory on a quarterly basis for a year. characterisation of panels hiv blood samples obtained from blood banks were characterised at the national reference laboratory by testing on the determine™ hiv-1/2 rapid test (abbott, usa), genscreen™ ultra hiv ag-ab enzyme immunoassay (bio-rad, france) and new lav-blot i and new lav-blot ii western blotting (bio-rad, france). the tests were all carried out according to their manufacturers’ instructions. 11,12,13 five panels, each comprising three positive and two negative samples, were sent to the participating laboratories for each round. the pt panels were scored based on the hiv-positive or -negative status assigned by the reference laboratory. the correct use of the national testing algorithm was also assessed. box 1: needs assessment questionnaire for the national external quality assessment programme for hiv, tuberculosis and malaria. box 1 (continues): needs assessment questionnaire for the national external quality assessment programme for hiv, tuberculosis and malaria. box 1 (continues): needs assessment questionnaire for the national external quality assessment programme for hiv, tuberculosis and malaria. box 1 (continues): needs assessment questionnaire for the national external quality assessment programme for hiv, tuberculosis and malaria. tuberculosis following informed consent, fresh sputum specimens were collected from patients who attended the directly observed treatment short-course (dots) clinic at nimr. panel slides were prepared as described by martinez-guarneros et al. 14 and each slide reading was carried out by two independent microscopists who arrived at a consensus. a total of five unstained slides per panel was sent to each laboratory (20 slides in total), with instructions to stain panels using the laboratory’s routine procedure to identify and quantify the acid-fast bacilli (afb) using the who or international union against tuberculosis and lung disease (iuatld) grading system. the ratio of positive to negative was varied randomly in each round of panel distribution. the participating laboratories were assessed on correct identification of the slide status and quantification of the afb, as compared with the assigned characteristics from the reference laboratory. malaria following informed consent, blood was collected in edta tubes from malaria parasite-positive patients with varying degrees of parasitaemia, as well as from malaria parasite-negative subjects. before samples were sent out, thick and thin films were made on the same slide and stained with giemsa stain at the reference laboratory, according to the standard method.15 the films were screened to ensure a good staining reaction. the slides were examined by two independent microscopists for cell distribution, parasite density count, species and stage identification. a limit of 30% was set to reach a consensus; however, where consensus was not reached, a third microscopist read the slide as a tiebreaker. the consensus information was recorded for each slide. for each round, the slides were packaged in the slide boxes in sets of three negatives and two positives. laboratories were expected to determine the parasite status and density as well as analyse each slide for species identification. results were assessed based on these parameters and the errors identified. reports were returned showing error types and suggestions for improvement. panel distribution for each shipment, the panels were parcelled in a triple packaging format, including instructions and reporting forms. the panels were sent in cold boxes through a courier agent to each zonal coordinator in order to save cost. each report form contained sections for results and comments, enabling participants to provide feedback. the zonal coordinators were responsible for distributing the panels to the participating laboratories within their zone. participants were instructed to return results within two weeks of receipt of the panels and the same channel was then used for returning the results and feedback forms to nimr. returned results were assessed and scored based on performance as compared with the expected results and individual performance scores were then returned to the participants through the same zonal coordinators. mentoring component after the first round of panel distribution, job aids (i.e., brief procedural instructions) for diagnosis of each disease type were prepared and sent to all the laboratories to help improve performance. at the end of the second round of panel distribution, because of cost constraints, 13 laboratories were recommended for personnel retraining as a result of poor performance. these laboratories were invited for a fully-sponsored training course at nimr. personnel from 11 of the 13 laboratories attended the week-long training, during which participants spent two days each on practical sessions on the laboratory diagnosis of tb and malaria and one day on hiv diagnosis. the training also consisted of didactic sessions on qms. panels were sent out to the laboratories for the third round immediately after the training. there was no mentoring session before the fourth round. data analysis results were scored based on the assigned pt provider ratings and characteristics. discordant results were assigned zero points, whilst concordant results were assigned 20 points per sample, for a total of 100 points per panel. scores of 80% and above were reported as satisfactory, which is the generally-accepted standard. 16 an unassigned score for a particular distribution indicated that a laboratory did not return the result. all scored results were entered into a filemaker pro v10 database where individual reports were generated for each laboratory. the data were then exported and analysed in a microsoft® excel spreadsheet. the feedback from the laboratories was analysed and suggested improvements for the pt services were implemented where possible. ethical considerations ethical approval was obtained from the institutional review board of the nimr (11 may 2009). only those sites that indicated an interest in participating were enrolled in the study, at no cost. laboratories were free to decline participation in the study at any point in time. results top ↑ questionnaire survey table 1 shows the results of the surveyed laboratories from the first phase of the study. this study used an abridged grading system of the who stepwise laboratory quality improvement process towards accreditation (slipta) to assess laboratories' adherence to the international organization for standardization (iso) 15189 standard, measuring laboratory quality on a scale of zero to five stars. 17 the laboratories attained, based on self-reporting, an average score of 65%, which is equivalent to two stars out of five. it was also found that of the 106 laboratories that completed the questionnaire, 68 (64%) reported having a system of result validation and only 34% (n = 36/106) provided scheduled maintenance of equipment. most of the laboratories did not have preventive maintenance policies, thereby possibly undermining the quality of results generated by the equipment. internet access was found to be uncommon (n = 36/106; 34%) amongst the respondents. the survey also showed that very few laboratories were registered for pt for hiv (n = 35/106; 33%), tb (n = 33/106; 31%) and malaria (n = 19/106; 18%). forty-four (42%) of the 106 laboratories indicated interest in participating in the pt for the three diseases offered in this study as they had not been registered for pt previously. table 1: survey results of the 106 surveyed laboratories. participation and response rate of laboratories of the 44 laboratories that indicated interest in participating in the joint pt programme, 10 (23%) were publicly-owned laboratories within hospital settings whilst 34 (77%) were private, stand-alone laboratories. in the second phase of the study, two laboratories opted out, one at the first round of panel distribution and the other after the second round of distribution. a few laboratories did not return results and were thus not assigned scores. laboratories that did not receive scores included 3/44 (7%) in the first round, 5/43 (12%) in the second, 2/42 (5%) in the third and 7/42 (17%) in the fourth round. on average, 10% failed to return results on one or more pt samples. proficiency testing panel testing results most laboratories returned the pt panel results within the two weeks stipulated, but the time had to be extended for some laboratories because of political insecurity. all the laboratories tested the hiv panel using rapid test kits, either serially or in parallel algorithms. some did not confirm a positive result with a second test kit. the pt results showed improvement in hiv pt scores from an average of 74% in the first round to 95% in the third round, but this was not sustained in the fourth round (figure 1). the major issue observed with hiv pt was the incorrect use of the national testing algorithm, which requires consistent results from two rapid test kits before confirming a positive hiv status; some laboratories used only a single reactive result (figure 2). figure 1: average performance of laboratories at different rounds of panel testing. figure 2: use of national testing algorithm in hiv diagnosis. for tb pt, all laboratories stained the slides using the ziehl-neelson staining technique. there was an improvement after the first round from an average score of 42% to 78% in the second round; however, the average dropped consistently from that point to 34% in the fourth round (figure 1). the issues observed with tb pt included quantification errors and a high level of false negative results (figure 3). an unusually high level of false positives was observed in the third round of the pt. figure 3: comparison of errors in tuberculosis diagnosis. although performance in malaria pt appeared poor, there was a steady increase in average scores from 2% in the first round to 39% in the fourth round (figure 1). participants appeared to continue to have difficulties with parasite detection and count throughout the testing period (figure 4). one laboratory confirmed the use of rapid test kits for malaria; its results were not included in the analysis. figure 4: frequency and types of errors identified in malaria diagnosis. feedback from participating laboratories a total of 81 persons provided feedback throughout the duration of the pt. the respondents were at liberty to express their concerns to nimr on any issues whatsoever. figure 5 shows the categorised comments from the participating laboratories. figure 5: categorised comments by participating laboratories. there was a great demand for training expressed by 25 (31%) of those who gave feedback. some requested on-site mentoring visits, whilst others wanted practical training sessions organised by the pt provider. some stated that training would enhance their competence and serve as a motivating factor for participation. twenty (25%) of the feedback responses provided useful suggestions to the provider for improvement. some requested that the time to return results be extended, whilst others requested that the quarterly exercise be replaced by samples being distributed every 2 months. fifteen (19%) respondents also requested the continuation of the programme and expansion of the scope to include more analytes for laboratory investigations of other diseases, aside from the hiv, tb and malaria, as well as inclusion of more laboratories. whilst 12 (15%) of the respondents requested the provision of reagents for pt, four (5%) complained about bad microscopes and needed some assistance, either financially or through direct provision of better microscopes and supervisory visits. a number of comments were actionable immediately and helped the provider to improve the quality of pt samples. the most prominent example was a series of complaints of leakage of plasma samples, which the provider responded to by changing the sample tubes. equally important complaints after the first round were broken slides and the quality of some of the malaria parasite slides. the provider improved on the packaging by ensuring proper sealing and positioning of the slide boxes so as to prevent damage during transportation. to address the slide quality, three or four readers reviewed the stained slides for subsequent panels for the second, third and fourth rounds and the best slides with the lowest inter-reader variability were selected and sent. an expert on malaria panel preparation from the national research centre, burkina faso, where the staff had previously acquired malaria panel preparation skills, visited the provider to ensure quality practices. discussion top ↑ the initial phase of the study indicates the prevalence of a poor culture of qms in nigerian laboratories. from the questionnaire survey, the laboratories earned an average of two stars, despite the fact that the grade was attained by self-reporting and not by an external audit. the lack of qms culture creates concern regarding the accuracy, reliability and timeliness of clinical results generated in laboratories. this finding supports previous observations that, in sub-saharan africa, laboratory infrastructure and personnel are affected adversely by the lack of resources and prioritisation, hampering laboratory system efforts in the fight against infectious and chronic diseases. 4,5 as a result, the accessibility of laboratory testing and the quality of available services remain a serious challenge.7 there is a dire need to create a culture of quality management in nigerian laboratories, which will help practitioners appreciate the necessity of results validation and participation in pt and other programmes. nimr plans to address this need with its newly-awarded training grant to build the culture of qms in both private and government-owned laboratories. this grant utilises the strengthening laboratory management toward accreditation (slmta) programme to develop capacity for laboratories not supported by the us president’s emergency plan for aids relief (pepfar) fundsa lack of resources as well as a poor understanding of the benefits of participation in an external pt programme may have been responsible for the low enrolment in this study. of the laboratories that did enrol, two private laboratories opted out of the pt programme after commencement. without any formal communication, the first laboratory was closed down at the point of delivery of a set of panels. the closure may have been related to the political crises in that region. the second laboratory communicated to the provider that the staff would no longer be able to participate in the study because the laboratory owner had gone back to school for full-time study. investing in qms is expensive and time consuming. as such, care should be taken in the selection of the laboratories enrolled in such pt projects in order to ensure their ability and willingness to provide the needed services. the rate of failure to return results varied despite the fact that the due dates for the results were extended at the request of some of the participating laboratories. one reason for this variation could possibly be a poor understanding of the importance of pt programmes. some of the laboratories’ staff members reported that they had to wait for the most senior laboratory scientist, often the owner of the laboratory, to be present during sample analysis. training will help improve understanding and increase participation. the incorrect use of the national hiv testing algorithm was common at the outset of this study. the nationally recommended algorithm includes serial testing, which requires a second test for an initial reactive sample. some laboratories used test kits outside the nationally-approved kits; some used a single test to confirm hiv infection; and others used parallel testing algorithms. correct use of the standardised hiv testing algorithm would reduce the risk of issuing false reactive results. when so much time and so many resources are spent on developing hiv testing algorithms, it is essential that countries ensure their proper dissemination to all levels where hiv testing is carried out. in this study, provision of a job aid with step-by-step instructions for hiv testing after the first round of panel distribution resulted in marked improvements. however, some laboratory scientists complained that their job aids were kept in the office of the head of the laboratory and not at the point of use in the laboratory. lack of available job aids in the laboratory may have affected the laboratories' performance in this study. provision of job aids may also have contributed to the improvements observed in the diagnosis of tb in the second round of panel distributions; however, this improvement was not sustained and quantification errors were common. this finding underscores the challenges of managing tb patients on therapy, as the efficacy of therapy would be difficult to monitor. other error types that could have a serious impact on the community were the high rate of false positive results observed in the third round and the persistent false-negative results observed throughout the study. these errors have serious implications: individuals diagnosed incorrectly as positive will most likely be placed on unnecessary therapy, whilst individuals who are diagnosed incorrectly as negative will be released into the community and will spread the infection. all measures must be put in place to halt this trend, especially as nigeria has been ranked 13th on a list of 22 countries with the highest burden of tb. 16 unlike the positive performances reported in an eight-year eqa of public reference laboratories, which recorded 82% acceptable results for malaria species identification,18 this study observed a comparatively low rate of acceptable malaria results. the poor performance observed for both tb and malaria diagnoses may be connected to the report in phase 1 of the study, in which only 34% of the laboratories reported having preventive maintenance for their equipment. it is important that preventive maintenance programmes be established in laboratories in order to ensure proper functioning of equipment so as to guarantee the accuracy and reliability of test results. confirming the need for better equipment maintenance, 5% of respondents complained about the quality of their microscopes. inadequate equipment maintenance, as observed in this study, is not limited to nigeria; poor maintenance culture is one of the major challenges in strengthening health systems in sub-saharan africa. 19,20 in spite of equipment limitations, there was continual improvement in performance on malaria panels, particularly with regard to parasite count and staging. this progression gives hope for improvement in the diagnostic proficiency of malaria microscopists if more efforts can be devoted to their training. to bridge this gap, nimr now provides annual training for malaria microscopists from all over the country. study participants demonstrated that they recognise that there are gaps in their knowledge and are willing to be trained for improvement. thirty-one per cent of the participants who gave feedback from phase 2 of the study requested further professional training. moreover, some of the participants who attended the resulting training commended the organisers, as they had not undertaken any prior in-service training. efforts directed at in-service training should be increased and extended to private laboratories that contribute a great deal to the health system, particularly in nigeria. those who had not participated in a pt programme previously also requested that the programme be sustained and expanded with regard to the scope of analytes and the number of participating laboratories. this request came despite the varying acceptable result rates obtained from the laboratories. such feedback is indeed a clarion call for more donor investment in eqa. it is essential that individual african countries be strengthened in order to take up the challenge of providing eqa in their respective countries, in order to extend pt programmes to these laboratories. currently available programmes are accessible to national public health or reference laboratories in africa, but do not benefit peripheral or private laboratories. access to pt programmes will also motivate the drive toward accreditation, as observed in south africa.7 the quest for accreditation can help laboratories address most other concerns. preparing for accreditation helps to strengthen laboratory management in and application of best practices throughout the laboratory system. awareness of laboratory accreditation is gathering momentum at present in nigeria, as is evidenced by the enrolment of 30 laboratories for accreditation preparedness training, the preparation for enrolment by others and the high demand for training by still more laboratories. there are currently 30 personnel who have been trained to roll-out the slmta programme in nigeria, three of whom qualified as master trainers;21 several other in-country training courses to prepare laboratories for accreditation are on-going. in summary, this study identified gross deficiency in the quality of laboratory services rendered across the country, indicating a poor state of qms. the pt study was well received, with demands to extend its scope. although most participants requested training and on-site mentoring, the training provided by this exercise was too short and did not have the desired impact for hiv and tb diagnoses. nevertheless, just as it has been reported from regional eqas that public health and reference laboratories in the african region are capable of the accurate determination of disease status,18,22 so it is believed that the laboratories that participated in this study also can perform satisfactorily if given the necessary support. for example, similar laboratories in uganda23 and other resource-constrained countries 24 have been supported in their endeavours to improve the quality of their services and have yielded remarkable improvements. there is, therefore, a need to strengthen laboratory systems in individual countries by providing pt programmes to clinical laboratories, including those that are privately owned with high volumes of work. the implementation of pt programmes will enhance the drive toward laboratory accreditation in the region. limitations of the study differences may have arisen from the self-administered questionnaires that could have influenced the findings reported in this study. also, there may have been varied readings by microscopists for the blood and sputum films, affecting study findings. conclusion there was gross deficiency in the implementation of qms which inadvertently affected the proficiency of the laboratories in the diagnosis of hiv, tb and malaria. concerted efforts are therefore required to train nigerian laboratories on qms, which would yield the desired outcome as observed from this study. acknowledgements top ↑ this project was supported by funds from the international association of national public health institutes (ianphi) under the sub-award no. 6-38223-g1. we appreciate ianphi for this support. we are also grateful to the zonal coordinators who assisted us in identifying the laboratories and who served as a bridge between us and the participating laboratories. we thank all the laboratories that made time to be part of this project. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions r.a.a. (human virology laboratory) developed the proposal, coordinated the project and wrote the manuscript. c.c.o. (national tuberculosis reference laboratory) led the tb team in preparing the tb panel. r.n.o. (clinical diagnostic laboratory) led the malaria team in preparing the malaria panels and evaluating the reports. n.n.n. (national tuberculosis reference laboratory) prepared the tb panel and evaluated the reports. c.k.o. (human virology laboratory) was responsible for all paperwork and prepared reports for the participating laboratories. a.z.m (monitoring and evaluation unit) provided bio-statistical expertise for the team. t.y.r. (national tuberculosis reference laboratory) prepared the tb panel and maintained the link with the zonal coordinators. m.n.a. (human virology laboratory) prepared the hiv panel and evaluated the reports. s.j.n. (clinical diagnostic laboratory) prepared the malaria panel and also helped to evaluate the reports. i.-o.e. (clinical diagnostic laboratory) prepared the malaria panel and e.o.i. (national tuberculosis reference laboratory) was involved in designing the project. references top ↑ 1. maher d, harries ad. quality care: a link between clinical and public health approaches to hiv infection in developing countries. trop med int health. 2010;15(4):391–395. 2. maher d, raviglione m. the history of the dots strategy: achievements and perspectives. in: schaaf hs, zumla a, editors. tuberculosis – a comprehensive clinical reference, 2nd ed. london: elsevier, 2009; pp. 270–273. 3. colebunders r, decock r, mbeba mj. improving the quality of care for persons with hiv infection in sub-saharan africa. trop geogr med. 1995;47(2):78–81. 4. derua ya, ishengoma dr, rwegoshora rt, et al. users' and health service providers' perception on quality of laboratory malaria diagnosis in tanzania. malar j. 2011;10:78. http://dx.doi.org/10.1186/1475-2875-10-78 5. hailegiorgis b, girma s, melaku z, et al. laboratory malaria diagnostic capacity in health facilities in five administrative zones of oromia regional state, ethiopia. trop med int health. 2010;15(12):1449–1457. http://dx.doi.org/10.1111/j.1365-3156.2010.02646.x 6. constantine nt, saville rd, dax em. retroviral testing and quality assurance: essentials for laboratory diagnosis. ann arbor, mi: malloy printers; 2005; pp. 578–590. 7. gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm 8. hertzberg ms, mammen j, mccraw a, et al. achieving and maintaining quality in the laboratory. haemophilia. 2006;12 suppl 3:61–67. http://dx.doi.org/10.1111/j.1365-2516.2006.01278.x 9. birx d, de souza m, nkengasong jn. laboratory challenges in the scaling up of hiv, tb, and malaria programs: the interaction of health and laboratory systems, clinical research, and service delivery. am j clin pathol. 2009;131(6):849–851. http://dx.doi.org/10.1309/ajcpgh89qdswfons 10. world health organization’s regional office for africa. hiv and aids laboratory capacity: where are we? overview of laboratory capacity in africa 2005–2007 [document on the internet]. c2010 [cited 2014 aug 18]. available from: http://www.afro.who.int/en/clusters-a-programmes/dpc/acquired-immune-deficiency-syndrome/features/2736-hiv-and-aids-laboratory-capacity.html 11. arai h, petchclai b, khupulsup k, et al. evaluation of a rapid immunochromatographic test for detection of antibodies to human immunodeficiency virus. j clin microbiol. 1999;37(2):367–370. 12. bio-rad. genscreen™ ultra hiv ag-ab: screening kit for the detection of hiv p24 antigen and antibodies to hiv-1 and hiv-2 in human serum/plasma by enzyme immunoassay [document on the internet]. c2010 [cited 2014 mar 06]. available from: http://www.bio-rad.com/webroot/web/pdf/inserts/cdg/en/883605_en.pdf 13. bio-rad. new lav blot ii: confirmation kit for anti-hiv2 antibodies detection in serum/plasma by immunoblotting [document on the internet]. c2007 [cited 2014 mar 06]. available from: http://www.bio-rad.com/webroot/web/pdf/inserts/cdg/en/883521_en.pdf 14. martinez-guarneros a, balandrano-campos s, solano-ceh ma, et al. implementation of proficiency testing in conjunction with a rechecking system for external quality assurance in tuberculosis laboratories in mexico. int j tuberc lung dis. 2003;7(6):516–521. 15. world health organization. tuberculosis profiles by country [homepage on the internet]. c2014 [cited 2014 mar 07]. available from: http://www.stoptb.org/countries/tbdata.asp 16. forbes ba, sahm df, weissfeld as. bailey & scott's diagnostic microbiology. 12th ed. st. louis, mo: mosby, 2007; p 625–626. 17. world health organization regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region (with checklist) [document on the internet]. c2011 [cited 2014 mar 06]. available from: http://www.afro.who.int/index.php?option=com_docman&task=doc_download&gid=8642&itemid=2593 18. frean j, perovic o, fensham v, et al. external quality assessment of national public health laboratories in africa, 2002–2009. bull world health organ. 2012;90:191–199a. http://dx.doi.org/10.2471/blt.11.091876 19. penfold s, shamba d, hanson c, et al. staff experiences of providing maternity services in rural southern tanzania – a focus on equipment, drug and supply issues. bmc health serv res. 2013;13:61. http://dx.doi.org/10.1186/1472-6963-13-61 20. fonjungo pn, kebede y, messele t, et al. laboratory equipment maintenance: a critical bottleneck for strengthening health systems in sub-saharan africa? j public health policy. 2012; 33(1):34–45. http://dx.doi.org/10.1057/jphp.2011.57 21. ianphi public health institutes of the world. slmta training of trainers workshop: creating a culture of quality for nigerian laboratories [homepage on the internet]. c2013 [cited 2014 mar 06]. available from: www.ianphi.org/whatwedo/projects/list/nigeria5.html 22. cham f, maleka m, masango m, et al. the world health organization african region external quality assessment scheme for anti-hiv serology. afr j lab med. 2012;1(1), art. #39, 6 pages. 23. yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3):401−409. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj 24. gaydos ca, rizzo-price p, balakrishnan p, et al. impact of international laboratory partnerships on the performance of hiv/sexually transmitted infection testing in five resource-constrained countries. int j std aids. 2011;22(11):645–652. http://dx.doi.org/10.1258/ijsa.2011.010527 next-generation sequencing where short reads come short long-read sequencing ‘nano-promises, giga-prospects’ situation report lessons from the past the illumina-pacbio merger outlook acknowledgements references about the author(s) boluwatife a. adewale medicine and surgery, faculty of clinical sciences, college of medicine, university of ibadan, ibadan, nigeria college research and innovation hub (crih), university of ibadan, ibadan, nigeria university college hospital, ibadan, nigeria citation adewale b.a. will long-read sequencing technologies replace short-read sequencing technologies in the next 10 years? afr j lab med. 2020;9(1), a1340 https://doi.org/10.4102/ajlm.v9i1.1340 opinion paper will long-read sequencing technologies replace short-read sequencing technologies in the next 10 years? boluwatife a. adewale received: 18 july 2020; accepted: 07 sept. 2020; published: 26 nov. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the year 1977 is quite remarkable in the history of genomics. it was the first time that the complete genome of an organism (phage φx174) would be sequenced – the advent of the first generation of sequencing technologies. of the two sequencing methods published that year, fred sanger’s ‘chain-termination’ method would become the mainstay of sequencing technology for the next three decades. because of its better usability compared to the maxam-gilbert method, it was widely preferred and became commercialised1 by applied biosystems inc. thanks to the collaborative efforts of scientists across the world,2 sanger sequencing eventually produced a reference human genome, courtesy of the us$2.7-billion (united states dollar) human genome project completed in 2003.3 next-generation sequencing the launch of pyrosequencing by 454 life sciences in 2005 marked the beginning of the second generation of sequencing. massively parallel or next-generation sequencing (ngs) technologies eliminated the need for multiple personnel working on a genome by automating dna cleavage, amplification and parallel short-read sequencing on a single instrument, thereby lowering costs and increasing throughput.1,4 pyrosequencing relies on the release of pyrophosphates when nucleotides are washed over fixed dna clones in a dna polymerase-mediated reaction (figure 1). pyrophosphate takes part in a cascade of reactions that eventually give off light which is detected and interpreted by a computer program. ion torrent employs a similar technique except that it uses ph instead of luminescence to detect nucleotide addition, whereas the applied biosystems inc. solid™ system is a sequencing--by-ligation technique which detects fluorescence that results from the action of dna ligase on oligonucleotides (figure 1).5,6 figure 1: overview of shortand long-read sequencing technologies. short-read sequencing methods are displayed on the left, long-read sequencing methods are shown on the right. short-read sequencing methods are classified into sequencing by synthesis and sequencing by ligation. they all require clonal amplification; emulsion polymerase chain reaction for 454 pyrosequencing, solid™ and ion torrent, ssba is peculiar to illumina, solid-phase template walking is used for certain solid™ technologies. illumina, fluorescent-labelled dntp added to bridge amplified dna template; 454 pyrosequencing, empolymerase chain reaction-generated microbead-bound dna clone in picotitre well, dna polymerase is added to the well, nucleotides are washed over in turn, deoxynucleoside triphosphate incorporation monitored via pyrophosphate release; ion torrent, similar to 454 pyrosequencing, deoxynucleoside triphosphate incorporation monitored via h+ ions release detected by a ph sensor that uses complementary metal-oxide-semiconductor technology. solid™, microbead-bound dna template flanked by adapters is hybridized to a growing complementary strand, under the action of dna ligase. cpal, another sequencing by ligation technique not described in this paper, employed by complete genomics, anchor sequence and probes hybridize to dna template in a series of ligation reactions taking place on a nanoball. pacbio single-molecule real-time, sequencing takes place on zero-mode waveguide chip. dna polymerase at the bottom of the well, fluorescent nucleotides being added to the strand. oxford nanopore technology relies on changes in ion flow as nucleotides pass through the nanopore. solid™ and ion torrent have been in the ngs market since 2007 and 2010, whereas roche announced the phaseout of 454 pyrosequencing in 2013 after facing stiff competition in the market. the reversible terminator sequencing currently sold by illumina is the predominant ngs platform: a sequencing-by-synthesis approach that uses bridge amplification and fluorescent signals from the addition of deoxynucleoside triphosphate (dntp) to a growing complementary strand (figure 1). the signal is detected in real time by a coupled charge device camera and interpreted by computer software.1 next-generation sequencing technologies have made sequencing much easier, faster and cheaper than sanger sequencing. the august 2019 report from the national human genome research institute put the cost of sequencing a complete human genome at $942.00 united states dollars (usd). thanks to ngs, the reduction in cost of genome sequencing has beaten moore’s law prediction (figure 2), more evidently since 2008, about the same time that ngs became popular in sequencing centres.7 figure 2: the cost of sequencing a single human genome from 2001 to 2019. the y-axis represents the cost of sequencing a human genome, the x-axis is labelled from year 2001 to 2019. moore’s law predicts a biennial doubling of transistors on a microchip. the line drawn in the graph above illustrates moore’s law prediction of the cost of sequencing per human genome. it is shown clearly that the cost has been consistently lower than predicted by moore’s law since 2008. illumina, introduced in 2006, is the most robust of all ngs technologies. it quickly gained traction among scientists, because of its high throughput and affordability compared to 454 pyrosequencing, solid™ and ion torrent. in fact, illumina was the first company to fulfil the ‘$1000 genome’ promise with its hiseq x ten sequencer in 2014. sequencing a genome now costs below $1000.00 usd with ongoing efforts to reach the $100.00 usd target.3 where short reads come short all second-generation sequencing technologies have a common limitation – the inability to sequence long stretches of dna. to sequence a large genome like human dna using ngs, the dna has to be fragmented and amplified in clones of between 75 base pairs and 400 base pairs, hence the term ‘short-read sequencing’ (srs). computer programs are then used to assemble the random clones into a contiguous sequence.2 aside from the fact that polymerase chain reaction – a necessary step in srs – causes preferential amplification of repetitive dna, srs fails to generate sufficient overlap sequence from the dna fragments. this constitutes a major challenge for de novo sequencing of a highly complex and repetitive genome like the human genome.2 the detection of large sequence changes is another area of difficulty that is encountered using srs – a major barrier to studying structural variations.8 long-read sequencing third-generation sequencing technologies which are otherwise called long-read sequencing (lrs) technologies address the shortcomings of ngs. whereas the sanger and srs approaches cannot exceed read lengths of 1 kilobase pair, third-generation sequencing technologies read lengths are between 5 kilobase pairs and 30 kilobase pairs. the longest read length ever generated by a third-generation sequencing technology is 2 gigabase pairs.9 long-read sequencing methods sequence a single molecule – thus abolishing amplification bias – and generate a reasonable length of overlap sequence for better sequence assembly.1 there are two prominent lrs technologies namely, nanopore sequencing and the single-molecule real-time (smrt) sequencing.10 single-molecule real-time sequencing was commercially released in 2011 by pacific biosciences (pacbio). a smrt genome sequencer consists of millions of zero-mode waveguides – microscopic wells with dna polymerase fixed at the bottom. within each zero-mode waveguide, two hairpin adaptors are ligated to both ends of the molecule to form a circular single-stranded dna. with the aid of a primer complementary to the adaptors, dna polymerase is used to sequence a complementary strand. when a fluorescent nucleotide is added to the strand, a fluorescence of wavelength corresponding to the added nucleotide is given off. this signal is captured in real time by a coupled charge device camera and interpreted by a computer program.1 nanopore sequencing, released by oxford nanopore technologies in 2014, works by a different principle: threading the dna molecule through a 1.5 nm wide bioengineered channel embedded in a biological membrane. electrical current across the channel depends on which nucleotide is traversing the channel at the time. this variation is used to determine the base sequence of the nucleic acid.1 single-molecule real-time and nanopore sequencing check all of the boxes in terms of suitability for de novo sequencing, resolution of repeat sequences (e.g. human leukocyte antigen gene, centromere), detection of structural variations, epigenetics and transcriptome sequencing, among others. however, the accuracy per read and the throughput of lrs is poor compared to srs.1,10 moreover, there are challenges in the adaptability of lrs for sequencing different genome lengths, as lrs data processing takes longer for organisms with large genomes than for viral genomes.10 the high error rates of nanopore sequencing are attributed to the poor sensitivity of the nanopore and the inability to control the speed of dna translocation through the pore. whereas the errors in nanopore sequencing are systematic, smrt base-calling errors are random and can be reduced using circular consensus sequencing (ccs), a method that allows the dna to have multiple passes through the zero-mode waveguides. increasing the accuracy of smrt via ccs comes at a higher cost than using ngs.3 nanopore sequencing has improved over the years, although it is still not as accurate as ngs. ‘nano-promises, giga-prospects’ minion, a small-sized nanopore sequencing device similar to a universal serial bus flash drive, was used to sequence the ebola virus during the 2014-2016 outbreak in west africa in 60 min.3 the minion sequencer is much more affordable when compared with illumina and smrt considering the cost of instruments and consumables.9 considering its portability, affordability and its ability to generate ultra-long reads, the possibility of using minion to accurately determine larger genome sequences is of immense value to researchers and clinicians. it holds the answer to affordable genome sequencing in low-resource settings and at the point of care. with improving nanopore technology aimed at decreasing minion’s high error rates, these promises may not be far-fetched. the sequel ii 8m smrt cell, which was launched in 2019 by pacbio, has a ccs read accuracy of 99% and above. promethion, also released in 2019 by oxford nanopore technologies, generates ultra-long reads, although with less accuracy than smrt. in their efforts to beef up quality, both companies update their products regularly. the latest -double-sensored nanopore cell r10 and linear consensus sequencing (a similar method to smrt ccs) are expected to improve the accuracy of oxford nanopore technologies products. interestingly, lrs technologies are approaching srs in terms of cost and accuracy. using the sequel ii 8m smrt cell that was released, ccs reads can now be obtained for about $1500.00 – an eight-fold reduction in costs.3 less accurate long reads from the promethion flow cell can be obtained at about the same price.3,5 error rates for lrs used to be 12% – 15%5 but have now fallen to less than 1% for smrt and less than 5% for nanopore sequencers10 compared to an error rate of approximately 0.1% in ngs.3,5 situation report notwithstanding srs market dominance, lrs has broadened its range of applications in recent years as a result of significant improvements in accuracy and throughput. for instance, there has been an increase in the use of minion in metagenomics studies. lrs technologies are increasingly being used in epigenetics and transcriptomics as well.10 the technical difficulties associated with de novo assembly of short reads for large complex genomes will likely persist for the foreseeable future. the question then becomes whether lrs technologies will improve on their own shortcomings to take over the market. a paradigm shift in the prevailing technology is likely to occur if there is a major scientific breakthrough, such as the discovery of a dna polymerase with a longer lifespan for higher throughput and accuracy in smrt base calling, or a drastic improvement in the sensitivity and dna translocation speed in nanopore sequencing that matches the accuracy of srs technologies. however, such improvements are more likely to take place gradually rather than suddenly; i do not see that occurring in the next 10 years. lessons from the past history suggests a difficult transition from short-read to long-read technologies. when 454 pyrosequencing was introduced in 2005, it had a clear advantage over sanger sequencing in terms of throughput and cost. however, funding agencies and scientists were not immediately receptive to the new technology. many of the researchers had got used to sanger sequencing, whereas investors were mindful of their stakes. thus, it took 3 years before ngs gained wide acceptance in the scientific community.4,7 even a major scientific breakthrough in sequencing technologies could still take about 2–3 years before finding its footing in the market for wide acceptability. as of 2014, illumina controlled about 70% of the market for dna sequencers and accounted for 90% of global dna data.3 it is the biggest company on the genomic sequencing market with a worth of $43.6 billion usd. the company’s large and increasing customer base makes it difficult to change sides by motivating customers to buy their stocks.11 currently, no technology compares to illumina in terms of cost, throughput and accuracy. even if one were to arise today, it would still have a hard time replacing illumina, considering the niche that illumina has carved for itself in the market. the illumina-pacbio merger the potential of lrs, particularly smrt, is not unnoticed by market giant illumina, who entered talks in 2018 on a $1.2 billion usd merger with pacbio. both parties, however, terminated the deal owing to concerns from the united kingdom competition and markets authority and the united states federal trade commission about an emerging monopoly.12 one is left to imagine why illumina wanted a merger. sheer desire for monopoly or the prospect of a robust hybrid technology? anyway, the synergy of both generations of sequencing technology is worth considering. hybrid sequencing combines the throughput and accuracy of srs with the long read length of lrs. it is highly effective for genome polishing10 and can produce reference genomes for most organisms.3 outlook while it may be difficult to predict with certainty major breakthroughs capable of altering the narrative in genomic sequencing, it is apparent that lrs holds the key to many unanswered questions in genomics. long-read sequencing technologies, although constantly improving and gradually closing the gap on ngs in terms of accuracy and costs,3,5 have yet to attain their full potential. in this decade, i expect that lrs technologies will gain more recognition and acceptance as they are adapted for sequencing a variety of organisms. in the absence of an innovation in lrs technology that can significantly increase accuracy and throughput and reduce costs far beyond what srs technologies offer, i would argue that it is unlikely that lrs technologies completely replace srs technologies in the next 10 years. meanwhile, in the middle of these improvements in srs and lrs technologies, i expect to see increased efforts towards making hybrid sequencing more scalable. acknowledgements i am immensely grateful to my mentors and teachers dr j.o. olugbami and dr o.o. bello for their invaluable guidance and support. this article was adapted from the winning essay for the sanger institute prize 2020 organised by the wellcome sanger institute, hinxton, cambridge, united kingdom. competing interests the author declares that no competing interests exist. author’s contributions the author b.a.a. wrote and edited the manuscript. ethical considerations i confirm that ethical clearance was not needed for this paper. sources of support the author received no financial support for the research, authorship or publication of this article. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the author and do not necessarily reflect the official policy or position of any affiliated agency of the author. references heather jm, chain b. the sequence of sequencers: the history of sequencing dna. genomics. 2016;107(1):1–8. https://doi.org/10.1016/j.ygeno.2015.11.003 mardis er. dna sequencing technologies: 2006–2016. nat protoc. 2017;12(2):213–218. https://doi.org/10.1038/nprot.2016.182 giani am, gallo gr, gianfranceschi l, formenti g. long walk to genomics: history and current approaches to genome sequencing and assembly. comput struct biotechnol j. 2020;18:9–19. https://doi.org/10.1016/j.csbj.2019.11.002 schuster sc. next-generation sequencing transforms today’s biology. nat methods. 2008;5(1):16–18. https://doi.org/10.1038/nmeth1156 kchouk m, gibrat j, elloumi m. generations of sequencing technologies: from first to next generation. biol med. 2017;9(3):1–8. https://doi.org/10.4172/0974-8369.1000395 suresh ps, venkatesh t, tsutsumi r, shetty a. next-generation sequencing for endocrine cancers: recent advances and challenges. tumor biol. 2017;39(5):1–11. wetterstrand ka. dna sequencing costs: data from the nhgri genome sequencing program (gsp) [homepage on the internet]. 2019 [cited 2020 mar 06]. available from: https://www.genome.gov/about-genomics/fact-sheets/dna-sequencing-costs-data ho ss, urban ae, mills re. structural variation in the sequencing era. nature research. 2020;21:171–89. available from: https://www.nature.com/articles/s41576-019-0180-9 kraft f, kurth i. long-read sequencing in human genetics. medizinische genet. 2019;31:198–204. https://doi.org/10.1007/s11825-019-0249-z amarasinghe sl, su s, dong x, zappia l, ritchie me, gouil q. opportunities and challenges in long-read sequencing data analysis. genome biol. 2020;21(30):1–16. https://doi.org/10.1186/s13059-020-1935-5 berman k. genome sequencing stocks on the rise [homepage on the internet]. forbes; 2019 [cited 2020 mar 06]. available from: https://www.forbes.com/sites/kenberman/2019/02/21/genome-sequencing-stocks-on-the-rise/#76a851951f51 illumina, pacbio scrap $1.2b merger, citing ‘continued uncertainty’ [homepage on the internet]. genetic engineering and biotechnology news; 2020 [cited 2020 mar 06]. available from: https://www.genengnews.com/news/illumina-pacbio-scrap-1-2b-merger-citing-continued-uncertainty/ abstract introduction methods results discussion acknowledgements references about the author(s) lavendri govender department of biomedical and clinical technology, faculty of health sciences, durban university of technology, durban, south africa molecular research and development department, specialised laboratory services, south african national blood service, durban, south africa rosaley d. prakashchandra department of biomedical and clinical technology, faculty of health sciences, durban university of technology, durban, south africa pavitra pillay department of biomedical and clinical technology, faculty of health sciences, durban university of technology, durban, south africa ute jentsch medical department, specialised laboratory services, south african national blood service, durban, south africa citation govender l, prakashchandra rd, pillay p, jentsch u. molecular red cell genotyping of rare blood donors in south africa to enhance rare donor-patient blood matching. afr j lab med. 2021;10(1), a1400. https://doi.org/10.4102/ajlm.v10i1.1400 original research molecular red cell genotyping of rare blood donors in south africa to enhance rare donor-patient blood matching lavendri govender, rosaley d. prakashchandra, pavitra pillay, ute jentsch received: 21 sept. 2020; accepted: 14 may 2021; published: 27 sept. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: molecular red cell genotyping is devoid of serology limitations such as the scarcity of rare antisera and the possibility of inconclusive results due to biological interferences. blood incompatibility can result in immune transfusion reactions such as haemolytic transfusion reactions or haemolytic disease of the foetus and newborn. objective: the study aimed to use molecular red cell genotyping to identify rare blood group donors among south african blood donors. methods: red cell genotyping data were extracted retrospectively from the bids xt genotyping software in the immunohaematology reference laboratory from january 2015 to august 2016. the id core xt genotyping assay was used to identify the single nucleotide polymorphisms of 10 blood groups system alleles in 150 donors. associations between the resultant genotypes and predicted phenotypes, abo group, rhd type, race group and gender were studied. results: significant red cell genetic variability was noted among the numerous south african donor genotypes identified in this study. genotyping further confirmed the presence of at least one of the 16 rare genotypes in 50 donors. group o black donors were associated with two rare blood types, while several other rare blood types were found only in white donors, supporting an association between abo/rh subtype, race group and rare blood types. conclusion: targeted screening of donors for antigen-negative rare blood units for patients should be done to reduce the risk of haemolytic transfusion reactions and haemolytic disease of the foetus and newborn. keywords: red cell genotyping; genetic variations; rare blood types; donor-patient blood matching. introduction the antigens found on the surface of red blood cells determine the blood type of an individual. blood grouping is essential for donor-patient blood transfusion match.1 red cell antigen mismatch between donor and patient can result in alloimmunisation, which is the formation of red cell antibodies in the patient following transfusion with red cells that the patient lacks. clinically, alloimmunisation can result in mild to fatal haemolytic transfusion reactions.2 in neonates, alloimmunisation may cause mild to fatal haemolytic disease of the foetus or newborn.3,4 the current serological phenotyping method of red cell antibody detection used in south africa is simple and inexpensive. however, inconclusive serology results may result due to the interference from donor red blood cells in chronically transfused patients, a rare blood type causing false-positive results or autoantibodies in patients with autoimmune diseases.5 also, sourcing blood for patients with rare blood types is challenging due to the scarcity of commercial rare antisera for serological testing.6 the term rare blood type describes the absence of an antigen that is found normally in 99% of the country’s population and is described as: ‘negative for high-frequency-antigens (hfa)’ or the presence of antigens found in only 1% of the population termed ‘positive for low-frequency-antigens’ or the presence of an unusual or rare rh subtype in a very small percentage of the population (1:1000, 1:10 000).7 hence, rare commercial antisera are often not readily available and are expensive. therefore, they are not cost-effective for large-scale blood type screening.6 molecular genotyping overcomes the limitations of serology. the mapping of the human genome provided knowledge on the molecular backgrounds and polymorphisms of the blood groups that enabled the development of dna-based assays for red cell genotyping.8 genotyping utilises patients’ dna information to infer the red cell phenotype. genotyping assays have been widely used in transfusion medicine for over 20 years.8 this technology only became an affordable service in south africa in 2015 and has to date not translated into the genetic identification of rare and unique red cell genotypes in south africa or the wider african context. smaller studies in some african countries have been completed; however, these were limited to group-specific antigen coverage. moreover, the sample numbers were too low across the regions to be representative of the african diaspora and its multiple ethnic groups.9 therefore, this study aimed to provide the first comprehensive red cell genotyping of rare blood donors referred to the south african national blood service, immunohaematology reference laboratory (irl). methods ethical considerations full approval (ethical clearance number irec 011/17) was granted by the durban university of technology to complete the study as part of a master’s thesis. ethical approval was also granted by the south african national blood service human research ethics committee (certificate clearance number 2016/06). initial donor consent was provided by donors when completing the blood donor questionnaire at the time of blood donation. however, there were no human participants in this study as it was a retrospective review of red cell genotyping data only. all genotyping results were anonymised from donor details and blinded using designated study numbers only accessible to the researcher. study participants the south african national blood service provides a vein-to-vein blood service in eight of the nine south african provinces and the majority of the blood donations are successfully cross-matched for transfusion to patients. where routine cross-matching cannot be completed due to inconclusive serology results, the blood donation is referred to the national immunohaematology reference laboratory. this observational study retrospectively reviewed red cell genotyping data of 150 south african national blood service donors whose blood samples were referred to the immunohaematology reference laboratory between january 2015 and august 2016. the study population of 150 donors comprised 58 conveniently selected serology-determined rare blood type donors and 92 randomly selected group o, rhd+ donors (table 1). the selection bias for group o+ donors was because the south african national blood service donor recruitment strategy focuses on sourcing mainly group o+ blood since it is a common blood type in south africa and can be transfused to a+, b+, ab+ and o+ patients. table 1: percentage occurrence of the id core xt predicted phenotypes and genotypes for 150 south african blood donors genotyped at the south african national blood service immunohaematology reference laboratory from january 2015 to august 2016. the self-proclaimed race groups of the donors as stated on the blood donation donor questionnaire were collated to establish whether there is an association between red cell genotypes, rare blood types and race groups. donors indicated their self-proclaimed race groups as either black, mixed race, indian or white.10 these racial groups were introduced by apartheid and remain a description of the south african society with black south africans being the native black african population, mixed race south africans describing a person of mixed european (‘white’) and african (‘black’) or asian ancestry, as officially defined by the south african government from 1950 to 1991, indian south africans are south africans who descend from migrants who arrived from british-ruled india during the late 1800s and early 1900s and white south africans are south africans of european descent, as well as from certain parts of west asia. since the samples of donors were collected nationally within south africa, all race groups have representation in the study although not of equal distribution. dna extraction dna was extracted from blood collected in ethylene-diamine-tetraacetic-acid anticoagulated blood tubes using the maxwell as2000 (promega, madison, wisconsin, united states) instrument and the promega maxwell standard elution volume assay. dna was quantified using the nanodrop 2000 (thermofisher scientific, waltham, massachusetts, united states) then diluted to obtain a standard dna concentration of 20ng/µl with a purity falling between 1.7 and 1.9 as required for completion of the id core xt assay (progenika, derio, spain). id core xt assay to maximise resources, the immunohaematology reference laboratory utilised the id core xt assay kit (progenika/grifols, san antonio, texas, united states). the id core xt kit covers red cell antigens or alleles of 10 blood grouping systems: rhce, kell, kidd, duffy, mns, diego, dombrock, colton, cartwright and lutheran, totalling 37 red cell antigens in a single test. the id core xt kit was assayed in a luminex 200is analyser (luminex corporation, austin, texas, united states) to detect beads coated with specific single nucleotide polymorphisms. the bidsxt assay was used to exclude all invalid red cell genotypes that did not yield a bead count of greater than 30 or a median fluorescent intensity of 1000. data analysis the predicted phenotypes and genotypes were exported from the bids xt (progenika/grifols, san antonio, texas, united states) software to a powerbi software programme. simultaneously, demographic details of gender, race, abo group and rhd types were exported from the organisation’s information system to the business intelligence information technology database to study associations between these characteristics. database collation the occurrence of the genotypes and predicted phenotypes were analysed from the most to the least occurring among the white, black, indian and mixed south african race groups. rare blood types are either negative for hfa, positive for low-frequency-antigens or unusual, rare rh subtypes that occur in less than 1% in a population. however, due to the small sample size of the study and the 34 rare blood types that were conveniently added to the study, we could not use the less than 1% guideline. instead, the rare blood types currently listed on the south african rare donor file was used to separate the rare blood types – hrb–, hrs–, k–, jsb–, u–, kpb–, yta–, lub–, hy–, joa–, uvar+, cw+, r’r”, r”r, r’r’ and rzr1 – genetically tested in this study. results most of the study participants were men (63%, n = 95/150) with 80% of the race group distribution comprising 41% (n = 61) white donors and 39% (n = 59) black south african donors. the 2015–2016 red cell genotyping data was skewed by a selection bias for group o+ (71%, n = 107) donors which accounted for 33 rare blood types found in 32 donors. in comparison, group o– donors that made up 13% (n = 20) of the study population showed the presence of 15 rare blood types in 14 donors. the remaining 16% (n = 23) of the group a+/− and group b+/− had four rare blood types in four donors, cumulatively resulting in a total of 50 rare donors with 52 rare blood types at a molecular level. of the 58 serological known rare donors, 44 donors had the serologically defined rare blood types hrb–, hrs–, k–, u–, u-variant+, jsb, kpb–, r̎ r̍, rzr1, lub– and yta– that are covered by the id core xt. however, molecular genotype correlated with serotype in only 34 of the 44 donors (figure 1). ten of the 44 serology-defined rare blood types were absent upon molecular testing giving a 23% discrepancy rate between serotyping and genotyping. more so, rare blood types previously not detected by serology were found in two of these 10 donors (figure 1). fourteen of the 58 serologically known rare donors had one of the eight rare blood types: bombay oh, vel–, kna–, in(lu), lan–, dantu–, milton (mi ii) and henshaw that were not covered in the id core xt assay. therefore, these could not be confirmed genotypically (figure 1). figure 1: breakdown of the study population and the resultant total of 50 genetically identified donors based on retrospective red cell genotyping obtained from the south african national blood service, immunohaematology reference laboratory over the period january 2015 – august 2016. from among the 92 random donors, genetic and molecular screening identified an additional 14 donors with a total of 16 rare blood types. this brings the total number of rare donors confirmed using genotyping to 50, which comprises 33.3% of the study population (figure 1). rhd/rhce the most occurring rhd/rhce phenotype was the rh-positive ro (cde/cde) phenotype and was found in a majority (72%, n = 42/59) of the south african black donors. six black ro donors were associated with the rare blood genotype rhce*cear, rhce*cear and rhce*cear, rhce*ce[712g], representing the rare negative hfa, hrs–. in comparison, the white south african donors showed the most genetic variability, having all of the rh-negative and rh-positive subtypes. a rare rzr1 (cde/cde) phenotype occurred in one mixed race south african donor. the four black and three mixed race south african donors with the r’r (cde/cde) phenotype were associated with the rare blood genotype rhce*ce[733g,1006t], rhd*r’s-rhce*ce[733g,1006t] that is serologically referred to as the known negative for a hfa rh:-34 or hrb–. predominant among the indian south african donors was the rhce*ce, rhce*ce, r1r1 (cde/cde) genotype and predicted phenotype. kell the single nucleotide polymorphisms in id core xt cover the k/k, kpa/kpb and jsa/jsb antigens from the kell blood group system (table 1). the homozygous kel*k_kpb_jsb, kel*k_kpb_jsb was the most predominant kell blood group genotype found across the ethnicities in 84% (n = 126) of the study population. the remaining five kell genotypes were distributed among 16% (n = 24) of the race groups with the kel*k_kpa_jsb, kel*k_kpa_jsb (serologically referred to as the kpb-) phenotype being the least common kell genotype in this study. kidd the most occurring kidd genotype in the study population was the homozygous jk*a, jk*a kidd blood type. it was observed in 50% (n = 75) of the study population and the majority of the black and indian donors. the remaining 50% of the donors had the heterozygous jk*a, jk*b and the homozygous jk*b, jk*b. the three kidd genotypes were found in almost equal spread among the mixed race donors and the jk*a, jk*b was more common among the white donors. duffy the predominant occurrence of the gata mutation indicates the predicted fya–, fyb– phenotype as present in 31.3% (n = 47) of the study population (table 1). the fy*b_gata, fy*b_gata was predominant among the black donors while the fy*b, fy*b[265t]_fy*x was observed in one white donor. mns in the id core xt assay, the gypa and gypb genes identifying the mn and s-s-u alleles of the mns system are identified and reported individually. the relationship between the ss null alleles resulting in u-antigen variants (u-variant) and the gypb gene deletion resulting in a u-predicted phenotype is evident (table 1). the rare s-s-u– was found in only the black donors in this study. diego no di*a genotype was detected among study participants; however, the homozygous di*b, di*b was detected (table 1). dombrock, colton, cartwright, lutheran there was a slightly higher occurrence of the homozygous do*b, do*b than the do*a, do*b genotype, and there were four dombrock variants that occurred in less than 1% of the donors. the homozygous co*a, co*a, yt*a, yt*a and lu*b, lu*b genotypes occurred in more than 90% of the study population. a small percentage (2.7%, n = 4) of white donors had the co*a, co*b genotype while the yt*b, yt*b. lu*a, lu*a occurred in less than 1% of donors. the international society of blood transfusion’s categories of rare blood types include: negative for hfa, positive for low-frequency-antigens and rare unusual rh subtypes. there were 10 negative for hfas detected in 38 donors of which the hrb– and hrs– hfa were most common being found in 12 and 6 donors. the two positive for low-frequency-antigensme and four rare rh subtypes were found among seven donors. of the 52 rare blood types listed on table 2, 1% (n = 16) rare blood types were newly identified from 14 donors; two donors had two rare blood types each: hrs– with jsb– and u– with dob–. the 1% new rare blood types comprised of 3 hrb–, 3 jsb–, 2 u-variant (uvar+), 2 dob– (do*a, do*a_jo), 2 r’r”, 1 hrs–, 1 cecw, 1 r’r, and 1 u–. table 2: rare blood types by antigen type and frequency found among 150 donors genotyped between january 2012 and august 2016 at the south african national blood service, immunohaematology reference laboratory. discussion studying the red cell genetic variation among the four major south african race groups showed that the rh subtype ro is found in 42 out of 59 black donors, r1r1 in 10 out of 19 indian donors while six different rh subtypes are spread among the white donors. also, rare blood types such as the hrb– and hrs– were found more in black donors while other rare types such as k–, kpb– were found among white donors suggesting genetical association between rh subtype, race group and blood type. this knowledge allows for more effective rare donor screening strategies to be implemented to enhance rare donor-patient blood matching in south africa. in this study, screening of random group o+ donors identified 14 new rare donors that would have been missed by routine testing. hence, identifying markers suggesting a rare blood type in donors such as rh subtype, race and patterns of genetic variations will assist the development of algorithms for rare blood type testing and identification in donors. on comparing our results to those from historical serological prevalence studies,11 we observed that white and indian south africans share similar alleles for the rhce*ce/ce, *ce/ce, kel*k_kpb_jsb (k–), fy*a, fy*a and yt*a, yt*b genotypes.11 similarly, common red cell antigens among the black and mixed race south africans were the rhce*cde/cde, kel*k_kpb_jsb/jsa and fy*a/b_gata.11 according to the department of statistics south africa,10 the four major race groups are defined as black, white, indian and mixed race people while internationally and outside africa, the broadly defined ethnic groups are termed caucasian, african american, asian, other or mixed race. it has been proposed that global emigration patterns account for the red cell antigens of white south africans being related to caucasians, whereas black and mixed race south africans are similar to african americans and indian south africans similar to asians, and in some cases, caucasians.11,12,13,14 in this study, the presence of the rhce*cde/cde (r’r), *cde/cde (r’r”), *cde/cde(ro), kel*k (k–), kel*kpa (kpb–) and lu*a (lub–) in white south africans matched the findings in caucasian populations of italy, netherlands, america, spain, germany and austria.15,16,17,18,19 further, the rare e-hrband e-hrsfound among black south africans matched findings in the african american population.20,21,22 the js*a,js*a genotype (jsb–) was found in three black south africans and one indian donor which is not common in south africa. however, this phenotype is present in america and japan and is reportedly associated with asian populations.12,23 the detection of the rhce*cde/cde (ro) in all the race groups represented in the study contradicts the assumption of a race-specific blood type. interestingly, the rhce*cde/cde (ro) was most detected in the black south africans but only found in the netherlands.16 this finding suggests that this is a very rare blood type associated with african ancestry. the absence of the yta(yta–) cartwright antigen shown by the yt*b,yt*b genotype, was found in one indian south african but was not detected in a study by kahar and patel,24 which surveyed the prevalent red cell phenotypes in central india. the n of the yta(yta–) can be attributed to the small study size or area of india sampled. the ytaantigen was also identified in italy,15 america,17 spain,18 japan23 and canada25 prompting a decision to expand screening for this rare antigen in south africa to all race groups. similar to the european study by finning et al.,26 three of the negative for hfa js(b–), kp(b–) and jk(b–) were also found in the current study. although there have been suggested similarities between south african white donors and caucasians or europeans, it is evident that some uniqueness exists between the two geographic regions. the dib–, rhce*cern, dia–, mia+, cob+, lua+, dob+, which were unique to the european study by finning,26 were absent in the present study possibly due to the small sample size. more studies will be required to interrogate and investigate the underlying reasons for these potential differences at a genetic level or whether this pattern changes and becomes more similar when more donors are tested. the rhce*ce/ce genotype is a rare rh subtype (rzr1) that was found in one mixed race south african. a study completed by sharma et al.27 reported rzr1 to be found in 6% of native americans and 2.2% of indians in central india. the low prevalence antigen rhce*cw was found in two white south africans only. the id core xt assay does not differentiate the hrb– (rh:-34) from the hrb– (rh:-31). the formation of anti-hrb is due to the absence of the high-frequency antigen hrb (hrb–) and anti-hrb (rh:-31) is an ‘anti-e-like’ antibody. the absence of the ‘e’ antigen together with the hrb– (e-hrb)– indicates the true rare blood type. besides, the id core xt assay does not differentiate the hrs– (rh:-18) from hrs– (rh:-19) subtype. similar to the hrb– subtype, the absence of the ‘e’ antigen combined with the hrs– subtype indicates the true rare antigen type. this study had revealed a possible differentiation of the two hrb–/hrb– and hrs–/hrs– types due to the presence of specific single nucleotide polymorphisms in the genotype and in comparison to genotype patterns produced when historically known hrb– (rh:-34) samples are genotyped. this is reflected in table 2 where the r’s haplotype with the presence of the 733g and 1006t pointed to the rare hrb– type while the presence of homozygous cear or with the single nucleotide polymorphism 712g was an indication of the hrs– rare blood type. a total of 12 hrb– and six hrs– donors were reported in this study as these two rare types originated in south africa, hence found in more of our south african donor population.20,21 the rare negative high-frequency antigens k–, kp(b–), jk(b–), jo(a–), hy– and lu(b–) were found only in white donors and this is consistent with reports in europeans and caucasians from america and europe.28 the fy(a-b–) rare phenotype was not identified in the current study and may be attributed to the purposive sampling techniques used in the study. the gata mutation identified by the id core xt assay predicts fy(a–,b–); however, this is not the true rare phenotype, but rather an assay limitation. the genotype gypb*s, gypb*s or s– phenotype is currently not listed as a rare type in south africa as it is not difficult to locate; however, s– is listed on the rare donor files of spain18 and japan.23 although the s-s-u– phenotype predicted by the gypb*deletion is rare in caucasians and found mainly in african americans,17 it was found in one white and one black donor in the present study. this suggests the presence of this rare blood type among white south africans. interestingly, this study revealed many null alleles among the ss alleles of the mns blood group system, which was missed by serology. these are important as the null alleles were mainly associated with the presence of the u-variants. patients with u-variant phenotypes can only be given blood matched from u-variant donors or risk the formation of anti-u antibodies that will result in transfusion reactions. three red cell antigens, the jk(a-b–), co(a–) and ko, were not identified in the present study which could be explained by the small sample size. however, donors for these three antigens were previously active rare donors on the south african rare donor file but became lapsed donors having not donated blood in over 3 years.29 this highlights the need to increase stores of rare blood units in frozen storage so that patient-donor blood matching is not challenged by the lack of active blood donors. mass-scale cost-effective red cell genotyping can be used to screen donors routinely for this purpose in line with international practice as in the united states.30 the most important finding of this study was that among the mixed race group, 73% (8/11) of the donors showed the presence of rare antigens. thus, mass-scale rare donor screening among mixed race donors can increase the pool of donors with rare blood types. limitations a limiting factor of the id core xt assay is that several rare red cell antigens in south africa such as the rhnull, kn(a–), lan–, bombay oh, vel–, adult i–, ge–, inb–, rare abo subtypes and rhd/rhce hybrids are not covered by the assay. due to the small study sample size, genotypic findings must be confirmed in large-scale studies that include all south african ethnic groups that will be representative of the south african donor population. the study is the first comprehensive red cell genotyping study undertaken at the south african national blood service and for south africa. the extensive gene/antigen coverage of 10 blood groups systems in a single test translates to cost efficiencies, the possibility of high-throughput testing29 and easier detection of rare blood types present in more than one blood group. this study is also hypothesis-generating, and a few critical areas for future studies have been identified. conclusion this study has highlighted that random screening using molecular red cell genotyping increased the chances of finding rare donors by 15% (n = 14/92 random donors screened). it was further concluded that targeted screening using rh subtypes associated with race will increase the likelihood of identifying more rare blood donors. the competition between various commercial companies to provide faster, cost-effective genotyping kits with multiplex ability and high-throughput volumes has made it possible to introduce affordable mass-scale genotyping in south africa. this will also enable the creation of a substantial local south african red cell genotype database that can be expanded to include the rest of africa. acknowledgements we acknowledge the staff of the immunohaematology reference laboratory for the completion of red cell genotyping using the id core xt assay. we wish to express our gratitude to the supervisors of l.g.’s master’s thesis for their intellectual assistance. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions l.g. completed the study as part of her dissertation for her master’s in medical laboratory science completed in 2019 and is the main author of this manuscript. r.d.p., p.p. and u.j. supervised the findings of this work. all authors discussed the results and contributed to the final manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the authors confirm that the data supporting the findings of this study are available within the article. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references daniels g. human blood groups. john wiley & sons, hoboken, nj; 2008. suddock jt, crookston kp. transfusion reactions. instatpearls, treasure island, fl; 2019. daniels g, hadley a, soothill p. blood group antibodies in haemolytic disease of the fetus and newborn. alloimmune disorders of pregnancy. 2002;1:21–40. https://doi.org/10.1017/cbo9780511527043.004 urbaniak sj, greiss ma. rhd haemolytic disease of the fetus and the newborn. blood rev. 2000;14(1):44–61. https://doi.org/10.1054/blre.1999.0123 kutner j, mota m, conti f, et al. blood genotyping for improved outcomes in chronic transfusion patients: current and future perspectives. int j clin transfus med. 2014;2014(2):65–72. https://doi.org/10.2147/ijctm.s48394 wilkinson ds. clinical utility of genotyping human erythrocyte antigens. lab med. 2016;47(3):e28–e31. https://doi.org/10.1093/labmed/lmw014 nance st. global definitions of rare donors. isbt sci ser. 2013;8(1):23–27. https://doi.org/10.1111/voxs.12006 anstee dj. red cell genotyping and the future of pretransfusion testing. blood. 2009;114(2):248–256. https://doi.org/10.1182/blood-2008-11-146860 granier t, beley s, chiaroni j, et al. a comprehensive survey of both rhd and rhce allele frequencies in sub-saharan africa. transfusion. 2013:53(11pt2): 3009–3017. https://doi.org/10.1111/trf.12409 department of statistics south africa. community survey 2016, statistical release p0301, east london, eastern cape. (statssa.gov.za) [homepage on the internet]. statistical release report 2016. 2016 [cited 2020 feb 21]. available from: http://cs2016.statssa.gov.za/wp-content/uploads/2016/07/nt-30-06-2016-release-for-cs-2016-_statistical-releas_1-july-2016.pdf tishkoff sa, reed fa, friedlaender fr, et al. the genetic structure and history of africans and african americans. science [serial online]. 2009 [cited 2018 jul 19];324(5930):1035–1044. available from: http://science.sciencemag.org/ reid me, lomas-francis c, olsson ml. the blood group antigen factsbook. academic press, cambridge, ma; 2012. patin e, lopez m, grollemund r, et al. dispersals and genetic adaptation of bantu-speaking populations in africa and north america. science. 2017;356(6337):543–546. https://doi.org/10.1126/science.aal1988 ramerini m. dutch portuguese colonial history. marco ramerini, cape town; 1998. revelli n, villa ma, paccapelo c, et al. the lombardy rare donor programme. blood transfus. 2014;12(suppl 1):s249. luken js, danovic f, de haas m, et al. requests for red cells with rare blood types in the netherlands. immunohematology. 2016;32(2):51–52. https://doi.org/10.21307/immunohematology-2019-042 flickinger c. the american rare donor program. immunohematology. 2016;32(2) :71–73. https://doi.org/10.21307/immunohematology-2019-049 muñiz-diaz e, castro a, flores e, et al. the spanish program for rare blood donors. immunohematology. 2016;32(2):59–61. https://doi.org/10.21307/immunohematology-2019-046 hustinx h, lejon crottet s, scharberg ea. rare donor programs in switzerland, germany, and austria. immunohematology. 2016;32(2):63–66. https://doi.org/10.21307/immunohematology-2019-047 moores p. rh18 and hrs blood groups and antibodies. vox sanguinis. 1994;66(3):225–230. https://doi.org/10.1159/000462513 moores p, smart e. serology and genetics of the red blood cell factor rh34. vox sanguinis. 1991;61(2):122–129. https://doi.org/10.1159/000461336 poole j. the international rare donor panel. isbt sci ser. 2006;1(1):209. https://doi.org/10.1111/j.1751-2824.2006.00031.x tani y. rare donor program in japan. immunohematology. 2016;32(2):49–50. https://doi.org/10.21307/immunohematology-2019-041 kahar ma, patel rd. phenotype frequencies of blood group systems (rh, kell, kidd, duffy, mns, p, lewis, and lutheran) in blood donors of south gujarat, india. asian j transfus sci. 2014;8(1):51. https://doi.org/10.4103/0973-6247.126693 goldman m, st croix l. rare donor program: canadian blood services. immunohematology. 2016;32(1):15–16. https://doi.org/10.21307/immunohematology-2019-033 finning k, bhandari r, sellers f, et al. evaluation of red blood cell and platelet antigen genotyping platforms (id core xt/id hpa xt) in routine clinical practice. blood transfus. 2016;14(2):160. sharma dc, singhal s, rai s, et al. incidence of rh antigens, phenotype & probable genotype in the population of gwalior and chambal region, central india. int blood res rev. 2013;1(1):29–43. https://doi.org/10.9734/ibrr/2013/4616 avent nd, martinez a, flegel wa, et al. the bloodgen project of the european union, 2003–2009. transfus med hemother. 2009;36(3):162–167. https://doi.org/10.1159/000218192 smart e. south african rare donor panel. isbt sci ser. 2006;1(1):210–212. https://doi.org/10.1111/j.1751-2824.2006.00032.x noumsi gt, billingsley kl, mccaskill d, et al. effectiveness of high-throughput blood group genotyping on building a rare donor database. tranfusion. 2014;54:57a–57a. article information authors: coosje j. tuijn1 elizabeth msoka2 declare l. mushi3 marion sumari-de boer2 jaffu chilongola2,3 ankie van den broek4 affiliations: 1royal tropical institute (kit) biomedical research, amsterdam, the netherlands2kilimanjaro clinical research institute (kcri), moshi, tanzania 3kilimanjaro christian medical university college, tumaini university makumira, moshi, tanzania 4royal tropical institute (kit) health, amsterdam, the netherlands correspondence to: coosje tuijn postal address: médecins sans frontières plantage middenlaan 14, 1018 dd amsterdam, the netherlands dates: received: 10 july 2013 accepted: 25 feb. 2014 published: 23 july 2014 how to cite this article: tuijn cj, msoka e, mushi dl, sumari-de boer m, chilongola j, van den broek a. the interface between clinicians and laboratory staff: a field study in northern tanzania. afr j lab med. 2014;3(1), art. #126, 7 pages. http://dx.doi.org/10.4102/ ajlm.v3i1.126 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the interface between clinicians and laboratory staff: a field study in northern tanzania in this original research... open access • abstract • introduction • research method and design    • study design    • study population    • sample size, sampling procedures and data collection    • data analysis • ethical considerations • trustworthiness • results    • participants    • factors influencing the interface       • organisational factors    • personal factors • discussion • limitations of this study • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: strengthening the communication and professional relationships between clinicians and laboratory workers is essential in order to positively change clinicians’ attitudes about the reliability of diagnostic tests, enhancing the use of laboratory diagnostics and, ultimately, improving patient care. we developed an analytical framework to gain insight into the factors that influence communication amongst health professionals. objective: to explore whether the interaction between clinicians and laboratory workers influences the use of laboratory test results in clinical decision making. methods: four health facilities in northern tanzania were selected using convenience sampling, whereas study participants were selected using purposive sampling. the quantitative and qualitative data collection methods included self-administered questionnaires; semi-structured, individual interviews; in-depth, individual interviews; and/or focus group discussions with clinicians and laboratory workers. thematic content analyses were performed on qualitative data based on the framework. descriptive statistical analyses of quantitative data were conducted using microsoft excel. results: contact between clinicians and laboratory professionals is seldom institutionalised and collaboration is rare. the clinicians believe collaboration with laboratory staff is a challenge because of the gap in education levels. laboratory workers’ education levels are often lower than their positions require, leading to clinicians’ lack of respect for and confidence in laboratory professionals, which compromises the laboratory staff’s motivation. conclusions: hospital managers, clinicians and laboratory workers need to recognise the critical and complementary roles each professional plays and the importance of addressing the gap between them. field application of the framework proved successful, justifying the expansion of this study to a larger geographical area to include additional healthcare institutions. introduction top ↑ medical laboratories play a significant role in the diagnosis, monitoring and treatment of diseases; yet the efficacy of the information they provide may be questioned because of several factors, including the capacity of the laboratory workforce, the laboratory infrastructure and the availability of equipment and materials, especially in low-income countries. whilst improving the quality of laboratories is a solution, it does not always result in proper execution of tests.1,2,3 other obstacles that must also be considered are the cultural beliefs of the patients, attrition of healthcare workers, physicians’ attitudes and inadequate supplies of consumables.4medical laboratory services offer essential information for diagnoses and/or treatment plans. the communication and interactions between laboratory and clinical health workers can influence physicians’ request behaviour and treatment interventions. previous studies have shown that lack of communication is a barrier to effective healthcare.5,6,7,8,9improved communication between clinicians and laboratory workers is essential to changing clinicians’ attitudes about the reliability of diagnostic tests, possibly leading to increased use of laboratory diagnostics and, ultimately, improving patient care.5 this interface between clinicians and laboratory health workers is complex; the two groups may communicate face-to-face or by request and result forms, phone calls, text messages, e-mails or computerised forms. the factors that influence the mode of communication and shape the relationship between these two professional groups require further exploration. for this reason we constructed an analytical framework based on existing literature.10,11,12,13,14,15after further literature searches, analysis of guidelines for laboratories and discussion with experts, a conceptual model was developed.16 the model addresses the phases where clinicians and laboratory workers interact; the organisational and personal factors affecting their interface; and the socio-political, economic and cultural environment within which the health facility operates. the objective of this study was to demonstrate and test the analytical framework and to gain insight into the relationship between clinicians and laboratory workers and into the factors that influence their interface, with the intention of later scaling up the study using a calculated sample size. the analytical framework includes three phases of communication (pre-analytical, analytical, post-analytical) during which clinicians and laboratory workers interact (tables 1a and 1b).15 the testing process starts with a clinician ordering a test and sample collection, known as the pre-analytical phase. during the analytical phase, the sample is processed and analysed by laboratory staff. the post-analytical phase includes transfer of results from the laboratory back to the clinician. each phase consists of organisational factors, subdivided into ‘identity’ and ‘management’, as well as personal factors, subdivided into ‘individual’ and ‘professional’. the primary aim of the study was to explore whether the interaction between clinicians and laboratory workers influences the use of laboratory test results in clinical decision making. by means of the framework quantitative and qualitative tools were designed. the results of this study provide information on the importance of the interface between clinicians and laboratory workers and may form a basis for larger studies in the future. the implications of our findings are useful for health institutions in any country. table 1a: analytical framework. this framework was developed to test our conceptual model,16 and displays the organisational and personal factors playing a role during the three phases where clinicians and laboratory workers interact: pre-analytical, analytical and post-analytical. table 1b: analytical framework. this framework was developed to test our conceptual model,16 and displays the three phases where clinicians and laboratory workers interact: pre-analytical, analytical and post-analytical. each phase consists of organisational and personal factors (table 1a). research method and design top ↑ study design this was an exploratory study, employing both quantitative and qualitative methods. its purpose was to use tools to test the analytical framework and to better understand the factors that influence the interaction between clinical and laboratory workers. most participants took part in a focus group discussion (fgd) immediately following completion of an anonymous, self-administered questionnaire (saq). if there were fewer than three participants for a fgd, in-depth, individual interviews were conducted. semi-structured, individual interviews were used for hospital directors and heads of departments. fgds and in-depth, individual interviews followed the same format and covered the same topics. the assessment of the data collection tools was done at a private, not-for-profit, faith-based, district hospital, where a group of clinicians and laboratory staff were invited to assess the tools. study population the study population included hospital directors, heads of clinical and laboratory departments, clinicians and laboratory staff. staff came from three categories of health facilities: private, government; faith-based not-for-profit; and private for-profit. four hospitals participated in the study: a non-government referral hospital with 450 beds; a private not-for-profit hospital with 150 beds; a government regional hospital with 300 beds; and a private for-profit health centre with 50 beds. sample size, sampling procedures and data collection study sites were selected using convenience sampling, whereas study participants were selected using purposive sampling. as this was a pilot study, we did not determine a sample size. we contacted the sites and received verbal consent from the hospital directors for their participation. the interviewed staff members all signed written informed consent forms. initially, a total of 48 staff members were asked to participate, including six clinicians and six laboratory workers from each of the four sites. however, at the time the study was conducted, only 35 staff members were present: 18 clinicians and 17 laboratory personnel. amongst the latter were laboratory assistants and attendants who often perform the routine tests, namely, those who interacted most with clinicians (tables 2a and 2b). saqs were used to collect data from clinicians and laboratory staff at each hospital. after filling in the saqs, staff members participated in either an fgd or an in-depth, individual interview, allowing for the opportunity to elaborate on the saqs and further share their views. interviews were carried out with hospital directors and heads of departments. all interviews and fgds supplemented the saqs and were conducted in kiswahili for laboratory staff and in english for clinicians. interviews were recorded via tape recorder or note taking. as outlined in the study protocol, fgds involved six to 12 clinicians and three to nine laboratory staff members. these numbers were predetermined and agreed upon by the study team. data analysis data were collected within a period of five working days at the end of november 2011 and analysis was carried out throughout 2012. the individual interviews and fgds were transcribed, translated and analysed by a social scientist and a research nurse. qualitative data were analysed independently and manually, using a thematic framework approach involving data familiarisation, coding and development and categorisation of themes. coding of collected qualitative data was driven by the developed framework (tables 1a and 1b), whereby common words were sorted together, following an inductive method of code-creation. once a theme was identified and reviewed, categorisation and corresponding codes were developed to sort and organise the data. after reading through the data, the two independent researchers discussed the codes and themes until they agreed on each one. quotations were used to support and clarify the information provided using an editing analysis style. descriptive statistical analysis of quantitative data from the structured questionnaires were carried out using microsoft excel. ethical considerations top ↑ ethical clearance was obtained from the kilimanjaro christian medical university college research and the ethical review committee of tumaini university, makumira (research ethical clearance certificate number 448 from research proposal 467). verbal and written consent for the saqs, interviews and fgds was provided by study participants. confidentiality was assured at all stages of this study, through the use of coding for sites and participants. no names or data can be traced back to individual participants. trustworthiness top ↑ the results of this study are based on actual findings as described in the research method and design section. qualitative research (fgds and in-depth interviews) were complementary to results obtained from the saq. the experimental design of this exploratory study is reliable and valid and the procedures of qualitative and quantitative research used in this study are according to standardised methods, as laid out by varkevisser, pathmanathan and brownlee.17 results top ↑ participants pre-testing of the data collection tools, firstly, through discussions with clinical and laboratory staff at a referral hospital and secondly, by group discussions at a district hospital, allowed researchers to strengthen and modify the data collection methods. in total, 35 questionnaires were administered to 18 clinicians and 17 laboratory staff members, an estimated one-third of the official staff number, according to the heads of departments. factors influencing the interface organisational factors management factors: the analysis of the saqs showed that 11 of the 18 clinicians (61.1%) and eight of the 17 laboratory staff (47.1%) were aware of the availability of rules and guidelines for requesting tests and reporting results. of those remaining, one clinician (5.5%) and nine laboratory staff (52.9%) said there were no guidelines, whilst six clinicians (33.3%) did not know whether or not guidelines existed. these findings were also evident in the fgds. those who were aware that there are guidelines in place noted that there is little time to adhere to them because of staffing shortages and an insufficient supply of reagents. other factors that the study group cited as impacting on communication in relation to management of the organisation included the clinicians’ doubts about laboratory test results and uncertainty as to whether standard operational procedures are followed as well as the awareness of the persons to whom clinicians and laboratory staff report (figure 1 and figure 2). furthermore, the lack of competent and highly-educated laboratory staff was cited by clinicians as being a barrier to effective communication; in many health facilities, only laboratory attendants and assistants are present to perform tests and they sometimes lack the communication skills of a more highly-educated laboratory technician. in fgds with laboratory workers, it was noted that clinicians do not always use the test results with which they are provided. patients sometimes ask clinicians to prescribe treatment straight away as the waiting time to be tested in the laboratory can be very long. clinicians sometimes agree to this request in order to save time. laboratory staff also mentioned that nurses and sometimes patients play a role in the contact between the clinicians and laboratory staff. in some health facilities, nurses collect the sample request from the wards and are responsible for transfer of results from the laboratory back to the clinician. this supports the findings of the quantitative data analysis. figure 1: factors related to the management of the organisation, response of clinicians. figure 2: factors related to the management of the organisation, response of laboratory staff. identity: ten (55.5%) of the 18 clinicians and nine (52.9%) of the 17 laboratory workers involved in the survey worked in a referral hospital. others worked in a faith-based, public or private, for-profit facility or in a general laboratory or health facility. personal factors within the personal factors, individual and professional subfactors (qualitative data: quotes) were supportive of and in agreement with the quantitative data. individual factors: the personal factors of the clinicians (table 2a) and laboratory staff (table 2b) investigated in this study were ethnicity, religion, age and professional qualifications. data are displayed by gender, position in the organisation and the last time a course or workshop was attended. table 2a: the demographic distribution related to the personal identities of the clinicians participating in this field study. table 2b: the demographic distribution related to the personal identities of the laboratory workers participating in this study. professional factors: laboratory staff members indicated that clinicians regularly devalue their services. whilst seven of the 17 laboratory staff members (41.1%) believed that clinicians understand what laboratory workers do and six (35.3%) believed that clinicians know how to interpret the results when making clinical decisions, three (17.6%) noted that clinicians do not wait for laboratory test results before starting treatment and one (5.8%) noted that test ordering is not always specific. furthermore, in the fgds, nearly all of the laboratory staff members from public and faith-based organisations expressed that they lack recognition from clinicians, a sentiment also expressed by staff from the private health facility as being a contributing factor for their lack of motivation. further playing a part in the complicated relationship between clinicians and laboratory staff is the perceived frequency of use of test results for clinical decision making. only six clinicians (33.3%) and three laboratory workers (17.6%) claimed that test results are often used in clinical decision making. one laboratory staff member (5.8%) believed that clinicians never use the test results (figure 3 and figure 4). figure 3: professional factors. the perceptions of clinicians regarding the frequency with which test results are used for making clinical decisions. figure 4: professional factors. the perceptions of laboratory workers regarding the frequency with which test results are used for making clinical decisions. six of the 18 clinicians (33.3%) do not always trust the laboratory results; 10 (55.5%) mentioned that the waiting time for test results is too long; and eight (44.4%) were not satisfied with the type of tests that can be performed and also believed that the reporting of test results is not done properly. four clinicians (22.2%) believed that the quality of laboratory services was weak or substandard. several times during the fgds, laboratory workers noted that lack of equipment contributes to poor quality output. laboratory staff in the private hospitals pointed out the issue of lack of reagents and mentioned that expired reagents may be in use, further compromising clinicians’ confidence in the laboratory test results. in spite of the complicated relationship between clinicians and laboratory workers, a majority in both groups must interact on a daily basis (figure 5 and figure 6). yet, when grievances arise, the two groups use different avenues to address them. when laboratory staff members have problems with clinicians, they often discuss them within their own professional group; however, when a clinician has a complaint, he or she will often approach the individual laboratory worker or laboratory manager. whilst issues like these may be discussed broadly in staff meetings at most hospitals, the majority of those surveyed at the private not-for-profit hospital pointed out that the department was so small that organising meetings to discuss problems seemed unnecessary. figure 5: the frequency of professional contact between clinicians and laboratory workers. eight of 16 clinicians reported having daily interactions with laboratory workers. figure 6: the frequency of professional contact between clinicians and laboratory workers. eleven of 14 laboratory workers reported having daily professional contact with clinicians. poor reporting was identified as being a factor that contributed to inadequate communication between clinicians and laboratory staff. in some cases, either the handwriting was misinterpreted, or test requests or test results were incomplete. in addition, it was noted that communication only occurs between the groups when the need arises. whilst most staff members noted that communication and positive interactions between laboratory workers and clinicians are crucial, there is no managerial support, formalised system or motivation to maintain regular meetings or contact between clinicians and laboratory staff. discussion top ↑ the main objectives of this study were to test the analytical framework; to gain a better understanding of the factors that influence the interface between clinicians and laboratory health workers; and to investigate the impact of the use of laboratory test results on the way clinicians and laboratory workers interact to deliver effective and improved healthcare. our research results show that the roles of laboratory workers within the organisation are not determined by education levels, but by availability. according to clinicians, differences in levels of education lead to a lack of trust between clinicians and laboratory staff, impacting negatively on their collaboration and communication and creating a climate of distrust. poor communication between clinicians and laboratory staff further causes hostility when clinicians request a large number of tests, unaware of the high workload of the frequently understaffed laboratory. in all three phases of communication where clinicians and laboratory workers interact (pre-analytical phase: ordering of tests, sample collection; analytical phase: sample processing and analysis; post-analytical phase: results transfer), clinicians discuss their complaints and grievances with the laboratory staff more often than vice versa, suggesting that hierarchy plays a role in the dynamic between the two groups. despite their different perceptions of a variety of issues, the groups agreed that clinicians were sometimes reluctant to use test results for clinical decision making. all in all, the issue of ineffective communication between clinicians and laboratory staff on patient care and worker dissatisfaction remains largely unresolved, providing a major source of frustration for staff and resulting in inefficiency in expected outputs.567this study has increased the understanding of the interface between clinicians and laboratory workers and highlighted its importance in improving the quality of patient care. scaling up data collection in a larger group of health facilities is essential with regard to quantifying our findings. this will enable hospital managers to make suggestions for improvements, such as refresher training courses that cover communication skills, as well as involving clinical and laboratory staff, nurses and patients. the research may also motivate clinicians and laboratory managers to pay more attention to the international organization for standardization (iso)18 stipulations regarding communication. it is hoped that the findings from this study and similar future studies will, ultimately, improve the quality of patient care and communication between clinical and laboratory staff.19 limitations of this study top ↑ clinicians were sometimes rushed during the interview, as patients were waiting for assistance. only those staff who were on duty participated at the time of this study, which may have biased our results toward the perspectives of laboratory staff with lower education levels, since many more highly-educated staff were out in the field, in training, on holiday or had resigned. the perspectives of nurses and patients were not included in this study. laboratory staff often had difficulties in filling in the english saq. some questions were not clear and allowed multiple answers, which were adjusted for in the analysis. there were inconsistencies between the questionnaires for clinicians and those for laboratory workers. the outcome of our study was based mainly on qualitative data; the quantitative components were limited. in addition, the sample size of this study was too small to draw firm conclusions. as this was a pilot study, mainly descriptive statistical methods were used. this may limit the interpretation of the data presented, but it does provide valuable information on the interface between clinicians and laboratory workers and on the effectiveness of the framework to assess this interface. these insights may be used for future studies. conclusion top ↑ by combining quantitative and qualitative information, some insight emerged regarding the relationship between clinicians and laboratory workers and the perspectives that contribute to their sometimes problematic interactions. this explorative study has given us additional information on factors that influence the interface between clinicians and laboratory workers and shown the effectiveness of the analytical framework. the findings and discussions also provided information for the improvement of our analytical framework, indicating the need for inclusion of nurses and patients in future studies. the findings from this study underscore the relevance of the subject: the daily struggle of hospital managers, clinicians and laboratory workers to recognise the critical role each plays in providing efficient and reliable healthcare. performing this field study has provided information on the complexities of the interface between clinicians and laboratory staff and its impact on clinician decision making. the results justify expanding this study to a larger geographical area to include more health institutions. acknowledgements top ↑ we would like to thank all the staff of the hospital and clinic sites for their time and cooperation. a special thanks goes to the translators and data clerks at kilimanjaro clinical research institute (kcri). we would also like to acknowledge marjolein dieleman of the royal tropical institute (kit) health for her valuable input and critical assessment of this article. kind thanks to linda oskam (kit biomedical research) for her preliminary reading of the manuscript. this study would not have been possible without core funding from kcri and kit. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions a.vdb. (royal tropical institute [kit]), c.j.t. (royal tropical institute [kit]), biomedical research), d.l.m. (kilimanjaro christian medical university college), e.m. (kilimanjaro clinical research institute) and m.s-db. (kilimanjaro clinical research institute) set up the study design and developed the tools. a.vdb., c.j.t., d.l.m., e.m., j.c. (kilimanjaro christian medical university college; kilimanjaro clinical research institute) and m.s-db. performed the fieldwork. d.l.m., e.m. and m.s-db. carried out the qualitative data analysis, a.vdb. and c.j.t. analysed the quantitative data, c.j.t. wrote the report and all authors contributed to and approved the final manuscript. references top ↑ 1.cohen gm. access to diagnostics in support of hiv/aids and tuberculosis treatment in developing countries. aids. 2007;21(suppl 4):s81–s87. http://dx.doi.org/10.1097/01.aids.0000279710.47298.5c 2.ishengoma dr, rwegoshora rt, mdira ky, et al. health laboratories in the tanga region of tanzania: the quality of diagnostic services for malaria and other communicable diseases. ann trop med parasitol. 2009;103(5):441–453. http://dx.doi.org/10.1179/136485909x451726 3. nkengasong jn. a shifting paradigm in strengthening laboratory health systems for global health: acting now, acting collectively, but acting differently. am j clin pathol. 2010;134(3):359–360. http://dx.doi.org/10.1309/ajcpy5asuejyq5rk 4. polage cr, bedu-addo g, owusu-ofori a, et al. laboratory use in ghana: physician perception and practice. am j trop med hyg. 2006;75(3):526–531. 5.petti ca, polage cr, quinn tc, et al. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377–382. http://dx.doi.org/10.1086/499363 6. chilundo b, sundby j, aanestad m. analysing the quality of routine malaria data in mozambique. malaria journal. 2004;3:3. http://dx.doi.org/10.1186/1475-2875-3-3 7. leshabari mt, muhondwa ep, mwangu ma, et al. motivation of health care workers in tanzania: a case study of muhumbili national hospital. east afr j public health. 2008;5(1):32–37. http://dx.doi.org/10.4314/eajph.v5i1.38974 8. manongi rn, marchant tc, bygbjerg ic. improving motivation among primary health care workers in tanzania: a health worker perspective. hum resour health. 2006;4:6. http://dx.doi.org/10.1186/1478-4491-4-6 9. garcia p, hughes j, carcamo c, et al. training pharmacy workers in recognition, management, and prevention of stds: district-randomized controlled trial. bull world health organ. 2003;81(11):806–814. 10.carter j, müller-stöver i, östensen h, et al. good clinical diagnostic practice: a guide for clinicians in developing countries to the clinical diagnosis of disease and to making proper use of clinical diagnostic services. world health organization regional office for the eastern mediterranean: cairo; 2005. isbn: 978-92-9021-393-2. available from: applications.emro.who.int/dsaf/dsa236.pdf‎ 11.world health organization. strategic approach for the strengthening of laboratory services for tuberculosis control, 2006–2009 [document on the internet]. 2006 [cited 2014 mar 30]. available from: http://apps.who.int/iris/bitstream/10665/69303/1/who_htm_tb_2006.364_eng.pdf?ua=1 12.butao d, chafulumira f, felling b, et al. malawi: laboratory services and supply chain assessment. usaid/deliver project: arlington, va; 2009. 13.mepham so, squire sb, chisuwo l, et al. utilisation of laboratory services by health workers in a district hospital in malawi. j clin pathol. 2009;62(10):935–938. http://dx.doi.org/10.1136/jcp.2009.069062 14.may ta, clancy m, critchfield j, et al. reducing unnecessary inpatient laboratory testing in a teaching hospital. am j clin pathol. 2006;126(2):200–206. http://dx.doi.org/10.1309/wp59ym73l6cegx2f 15.plebani m. exploring the iceberg of errors in laboratory medicine. clin chim acta. 2009;404(1):16–23. http://dx.doi.org/10.1016/j.cca.2009.03.022 16.van den broek a, tuijn cj, van ’t klooster l, et al. understanding the interface between clinical and laboratory staff. afr j lab med. 2014;3(1), art. in press. 17.varkevisser cm, pathmanathan i, brownlee a. designing and conducting health systems research projects. volume 1: proposal development and fieldwork. kit publishers: amsterdam; international development research centre (idrc); who regional office for africa. isbn 90 6832 148 x. 18.international organization for standardization. iso15189:2007. medical laboratories – particular requirements for quality and competence [page on the internet]. 2007 [cited 2014 mar 30]. available from: http://www.iso.org/iso/catalogue_detail?csnumber=42641 19. carter jy, lema oe, wangai mw, et al. laboratory testing improves diagnosis and treatment outcomes in primary health care facilities. afr j lab med. 2012;1(1), art. #8, 6 pages. http://dx.doi.org/10.4102/ajlm.v1i1.8 abstract introduction methodology and data analysis review findings implications and recommendations acknowledgements references about the author(s) adesola olalekan department of medical laboratory science, university of lagos, idiaraba, lagos, nigeria centre for genomics of non-communicable diseases and personalized healthcare (cgnph), university of lagos, akoka, lagos, nigeria bamidele iwalokun centre for genomics of non-communicable diseases and personalized healthcare (cgnph), university of lagos, akoka, lagos, nigeria department of molecular biology and biotechnology, nigerian institute of medical research, yaba, lagos, nigeria oluwabukola m. akinloye department of medical laboratory science, oulton college, moncton, new brunswick, canada olayiwola popoola department of medical laboratory science, university of lagos, idiaraba, lagos, nigeria centre for genomics of non-communicable diseases and personalized healthcare (cgnph), university of lagos, akoka, lagos, nigeria titilola a. samuel centre for genomics of non-communicable diseases and personalized healthcare (cgnph), university of lagos, akoka, lagos, nigeria department of biochemistry, university of lagos, idiaraba, lagos, nigeria oluyemi akinloye department of medical laboratory science, university of lagos, idiaraba, lagos, nigeria centre for genomics of non-communicable diseases and personalized healthcare (cgnph), university of lagos, akoka, lagos, nigeria department of molecular biology and biotechnology, nigerian institute of medical research, yaba, lagos, nigeria citation olalekan a, iwalokun b, akinloye om, et al. covid-19 rapid diagnostic test could contain transmission in lowand middle-income countries. afr j lab med. 2020;9(1), a1255. https://doi.org/10.4102/ajlm.v9i1.1255 review article covid-19 rapid diagnostic test could contain transmission in lowand middle-income countries adesola olalekan, bamidele iwalokun, oluwabukola m. akinloye, olayiwola popoola, titilola a. samuel, oluyemi akinloye received: 26 apr. 2020; accepted: 07 aug. 2020; published: 30 sept. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: coronavirus disease 2019 (covid-19) caused by severe acute respiratory syndrome coronavirus-2 (sars-cov-2) has impacted heavily on global health. although real-time polymerase chain reaction (rt-pcr) is the current diagnostic method, challenges for lowand middle-income countries (lmics) necessitate cheaper, higher-throughput, reliable rapid diagnostic tests (rdts). objective: we reviewed the documented performance characteristics of available covid-19 rdts to understand their public health utility in the ongoing pandemic, especially in resource-scarce lmic settings. methods: using a scoping review methodology framework, common literature databases and documentary reports were searched up to 22 april 2020, irrespective of geographical location. the search terms included ‘sars-cov-2 and serological testing’ and ‘covid-19 and serological testing’. results: a total of 18 rdts produced in eight countries, namely china (6; 33.33%), the united states (4; 22.22%), germany (2; 11.11%), singapore (2; 11.11%), canada, kenya, korea and belgium (1 each; 5.56%), were evaluated. reported sensitivity ranged from 18.4% to 100% (average = 84.7%), whereas specificity ranged from 90.6% to 100% (average = 95.6%). the testing time ranged from 2 min to 30 min. of the 12 validated rdts, the igm/igg duo kit with non-colloidal gold labelling system was reported to elicit the highest sensitivity (98% – 100%) and specificity (98% – 99% for igg and 96% – 99% for igm). conclusion: we found reports of high sensitivity and specificity among the developed rdts that could complement rt-pcr for the detection of sars-cov-2 antibodies, especially for screening in lmics. however, it is necessary to validate these kits locally. keywords: coronavirus disease; covid-19; sars-cov-2; rapid diagnostic test; lowand middle-income countries. introduction coronavirus disease 2019 (covid-19) is an emerging respiratory disease that was first reported to the world health organization (who) as a cluster of pneumonia of unknown origin from wuhan, china, in december 2019.1 the unknown causative agent was found through deep sequencing to be severe acute respiratory syndrome coronavirus-2 (sars-cov-2) on 7 january 2020 and the disease covid-19 was named on 11 february 2020. in response, who declared covid-19 as a public health emergency of international concern on 30 january 2020 and a pandemic on 11 march 2020.1 as of 22 april 2020, an estimated 2 572 805 confirmed cases and 178 551 confirmed deaths from covid-19 had been reported.2 the first 10 cases in africa were reported in five countries (nigeria, algeria, morocco, egypt and senegal).3 although earlier cases of covid-19 in many lowand middle-income countries (lmics) were described as imported by travelers from china, italy, the united kingdom and germany, community transmission has now become the major cause of new covid-19 infections.3,4 early, rapid, large-scale diagnosis and accurate diagnosis of covid-19 is one of the key interventions for covid-19 containment in both high-income and lmic settings.3 the availability of the sars-cov-2 genome has led to the development and validation of various reverse transcriptase real-time polymerase chain reaction (rt-pcr) in vitro diagnostic test kits by different manufacturers for covid-19 diagnosis.5,6 this diagnostic test is based on the detection of genes encoding the envelope (e), spike (s), nucleoplasid (n), rna-dependent rna polymerase and open reading frame 1a/b (e.g. orf1ab, orf1a, orf1b) polyproteins within the genomic rna of sars-cov-2.5,6,7 due to lack of culture facilities, the rt-pcr method is currently the reference standard method of confirming covid-19 diagnosis in suspected cases globally. for epidemiological investigation, public health and clinical actions, rt-pcr has been shown to be very reliable at screening and confirming the diagnosis of covid-19 using upper respiratory (e.g. nasopharyngeal swab, oropharyngeal swab, throat swab and nasal swab) and lower respiratory (e.g. sputum and bronchioalveolar lavage) samples.7,8 real-time pcr has also been useful for monitoring viral rna shedding dynamics during the acute phase of the disease and viral rna decay and disappearance during the convalescence stage of the disease among survivors.9,10 however, in spite of its high analytical sensitivity its detection range is limited to 3.2 – < 10.0 rna copies per reaction.6,7,8 the rt-pcr method has been reported from studies done inside and outside china to also be prone to giving false negative results under certain conditions, thereby missing some covid-19 cases. these missed cases are therefore not isolated increasing community transmission.8,9,10 these conditions include insufficient or inappropriate sample for viral rna isolation, poor sample transportation to the laboratory, poor storage of the isolated rna samples, poor quality of the rt-pcr assay and poor timing for sample collection. the asymptomatic phase of sars-cov-2 infection – the first few days post infection onset and the convalescence phase ≥ 14 days post infection onset, especially in a missed infection, have been indicated as times when cases can be missed.7,8,9,10 poor quality rt-pcr assay is characterised by an inconsistent cycle threshold value and/or lack of amplification signal for one or two targeted genes. these missed cases are therefore not isolated increasing for sar-cov-2 detection.6,7,8 also, due to limited financial resources, the limited number of accredited molecular laboratories of biosafety level 2/3 and limited number of technical experts, the scaling up of rt-pcr for covid-19 diagnosis is limited in lmics.7,8,9,10 taken together, the above challenges of rt-pcr have necessitated the deployment of serological rapid diagnostic tests (rdts) for covid-19 diagnosis, which could identify asymptomatic and convalescent covid-19 cases undiagnosed by rt-pcr. covid-19 serological rdts are antigen-antibody based tests that detects sars-cov-2 igm and/or igg in human blood samples or sars-cov-2 viral antigen from respiratory samples within 15 min.8,11,12 unlike the rt-pcr protocols, serological tests require less expensive equipment, no technical expertise or electricity to run and very minimal biosafety requirements. also, unlike rdts that use small amounts of biological sample (10 ul – 20 ul) and have an average run time of 15 minutes, the rt-pcr protocols use large amounts of samples (150 ul – 200 ul) and have an average run time of about 90 minutes.6,7,8 these advantages of serological rdts have attracted serious attention for their use in large-scale covid-19 serological rdts are antigen-antibody based tests that detects sars-cov-2 igm and/or igg in human blood samples or sars-cov-2 viral antigen from respiratory samples within 15 min testing especially at the peripheral level of the health system and outside hospital settings in lmics. data from worldmeters show that african countries compared to other countries conducted fewer tests per population (figure 1). this lower testing power means relatively fewer cases can be detected. thus, the rollout of various rdt kits by different manufacturers could be a favourable development particularly for lmics as rdts can be easily scaled up for rapid covid-19 diagnosis.11,12 besides, rdts can provide additional sero-epidemiological data that will be used to determine the magnitude of covid-19 spread within a population. rdts achieve this by identifying active and previous symptomatic or asymptomatic cases; these data are then used to calculate case-fatality rate and determine the anti-sars-cov-2 immunity level of a community.11,12 however, to harness the various epidemiological and clinical usefulness of currently available covid-19 serological rdts, it is important to determine and/or validate their performance levels. in the present scoping review, the following research questions will be answered: (1) what are the currently available serological rdts for testing, (2) to what extent have these serological rdts been validated by their manufacturers and (3) what is the level of performance characteristics of these serological rdts? presently, the level of accuracy of many serological rdts available for use in lmics remains unclear, coupled with insufficient information about their strengths and limitations. this review will provide insight into the performance characteristics of these kits and enable evidenced-based decisions for their possible use in large-scale covid-19 testing and containment strategies in lmics. figure 1: distribution of severe acute respiratory syndrome coronavirus-2 burden and test per population in selected african countries. methodology and data analysis a scoping review was conducted using a methodology framework by arksey13 with modification as described by adhikari et al.14 this includes: (1) identifying a clear research objective and search strategies, (2) identifying relevant research articles, (3) selecting research articles, (4) extracting and charting of data, and (5) summarising, discussing, analysing and reporting the results. the online databases searched included google scholar, medrxiv, biorxiv and pubmed, as well as documentary reports and white paper publications from relevant online websites including who, the united states centers for disease control and prevention (cdc) and the nigeria centre for disease control (ncdc) for information on new rdts for covid-19 published up to 22 april 2020. the search terms used include ‘sars-cov-2 and testing’, ‘covid-19 and rapid test’ and ‘covid-19 and diagnostic kits’. diagnostic kits published for the confirmation of other coronaviruses, such as the coronavirus associated with the 2003 sars outbreak in asia and middle east respiratory syndrome-coronavirus, were excluded. all the members of the review teams were involved in paper search and selection and a consensus was reached through peer review. duplicated publications and those with insufficient information were removed. the extracted data included the name of the diagnostic kit, manufacturer, test performance based on sensitivity, specificity, predictive positive and negative values, test principles and special characteristics and testing time. the data were entered into excel (microsoft, redmond, washington, united states) and exported to statistical product and service solution version 23 (ibm spss inc., chicago, illinois, united states) for cleaning and analysis. review findings overall, 28 publications on coronavirus-based diagnostic kits that matched the goal of this publication were included in this study (figure 2). articles were excluded based on duplication and lack of information on detection principle, type of kit, performance characteristics and manufacturers’ details. all eligible publications on covid-19 diagnostic kits by country and performance as of 22 april 2020 were summarised in numbers and percentages using descriptive analysis. on the whole, a total of 18 serological rdt kits were included for analysis. of these, four were antigen rdts (22.2%), nine were total immunoglobulin rdts (50%) and five were igm/igg serological rdts (27.8%) (figure 3). these kits were produced in eight countries, namely china (6; 33.33%), the united states (4; 22.22%), germany (2; 11.11%), singapore (2; 11.11%) and kenya, canada, korea and belgium (1 each; 6.56%) (table 1). fourteen of the rdt kits are antibody-detection kits for use with blood, plasma or serum (77.8%), and four were antigen-detection kits for use with swab, sputum or blood (22.2%). the majority of these kits (13; 72.22%) use lateral flow membrane technology, whereas the remaining five (27.78%) use colloidal gold (figure 4). figure 2: prisma flow diagram showing the scoping review process. figure 3: distribution of the serological rapid diagnostic tests by testing principle. figure 4: distribution of the serological rapid diagnostic tests by testing platform. table 1: performance characteristics of newly developed severe acute respiratory syndrome coronavirus rapid diagnostic kits analysed in this review, 22 april 2020. in general, the sensitivity of the test kits irrespective of sample specification ranged from 18.4% to 100% and their specificity ranged from 90.6% to 100%. the pooled analysis revealed an average (range) sensitivity of 81.6% (72.9% – 88%) and specificity of 94.4% (88.2% – 97.5%). the sensitivity and specificity of lateral flow immunoassay membrane type rdt kits were in the range (average) of 84.4% – 100% (92.7%) and 90.6% – 100% (96%), respectively, and that of lateral flow immunoassay colloidal gold type were 18.4 – 99.1% (67.7%) and 91.7% – 100% (98.3%), respectively (figure 4). three of these kits, namely bodysphere rapid test (los angeles, california, united states), thermogenesis rapid covid-19 test kit (rancho cordova, california, united states) and nadal® covid-19 test kit (regensburg, germany), had a sensitivity of 99% – 100%. these three kits also had a specificity range of 91% – 100%. asides their better sensitivity and specificity compared to other rdts, these kits are for use with blood samples only, detect both igg and igm, and have shorter testing time of 2 – 10 min. the testing time for all the identified kits ranged from 2 to 30 min with an average testing time of 13.5 min (95% confidence interval = 10.8 min – 16.1 min). only two of the kits provided information on positive predicted value and negative predictive value (range = 87.5% – 100.0% to 26.2% – 96.2%). out of the 18 rdts identified, 6 (33%) were not subjected to performance validation by the manufacturers of the kits. two of four antigen detection kits, seven of nine total immunoglobulin and three of five igm + igg serological kits were validated for sensitivity and specificity using rt-pcr assay as the reference method (table 1).15 on the whole, eight of the 13 lateral flow immunoassay membrane type and four of the five lateral flow immunoassay colloidal gold type kits were validated. of the 12 serological rdts validated by rt-pcr, the igm/igg duo kit with non-colloidal gold labelling system was found to elicit the highest and acceptable sensitivity (98% – 100%) and specificity (98% – 99%) values for igg and specificity of 96% – 99% for igm compared to other rdt types and the counterpart colloidal gold system-based igm/igg duo kit (figure 5). figure 5: performance characteristics of the different serological rapid diagnostic tests by testing platform; lfia membrane (a) and colloidal gold devices (b). implications and recommendations the need to expand diagnostic testing in order to cope with the current spread of covid-19 infection in many settings in lmics where resources for rt-pcr are limited and difficult to sustain has made rdt kits for sars-cov-2 an important tool in the global fight against the covid-19 pandemic. for patients with suspected infection, rt-pcr is used to detect sars-cov-2 in sputum, throat and nasopharyngeal swab, and secretions of the lower respiratory tract samples such as bronchoalveolar lavage and bronchial washings.16,17,18 however, limited facilities and human resources for molecular testing using rt-pcr tends to slow down testing for covid-19 in resource-limited countries. it has been argued that rdts do not have sufficient evidence to support their use in the covid-19 pandemic and hence should be used only in a research setting.19 cassaniti et al. have earlier reported low sensitivity and specificity of serological assay which led to misdiagnosis of covid-19 in the vast majority of the patients in their study population.20 the who has emphasised that tests with inadequate quality may miss patients with active infection or falsely categorise patients as having the disease, further hampering disease control efforts, hence the need for questioning the performance of sars-cov-2 rdt kits.19,21 most manufacturers of the rdts have performance characteristics of the kits validated using the rt-pcr technique as the reference method. however, several publications have reported the possibility of false-negative results using rt-pcr.22 thus, the sensitivity and specificity data of reviewed kits should be understood in light of this bias. the declaration of covid-19 as a global pandemic and the huge concern of its transmission in lmics where hiv, tuberculosis and malaria are currently endemic have necessitated the need to scale up diagnostic testing to mitigate further spread and the rising number of covid-19 deaths outside china.23,24 in many settings in lmics, such as small communities, riverine areas, health posts and primary health centres, resources for rt-pcr are absent.23,24 this has made the development of serological rdts for the detection of specific sars-cov-2 antigens, anti-sars-cov-2 igm and anti-sars-cov-2 igg an attractive and very important tool in the global fight against the covid-19 pandemic in lmics. findings from the 18 serological rdt kits analysed in this review imply that three different types of serological rdts, antigen, total immunoglobulin, and combined igm and igg-based rdt with the ability to provide results between 2 min and 30 min are currently available for potential large-scale testing in lmics using five types of biological samples (nasopharyngeal swab, throat swab, whole blood, plasma and serum). due to challenges associated with more sensitive biological samples such as bronchoalveolar lavage and sputum, both nasopharyngeal and throat swabs are used for covid-19 testing by rt-pcr in many settings.7,8,9 also, whole blood, plasma or serum is often used as biological sample for rt-pcr for monitoring viremia to predict covid-19 severity during the acute stage of infection and viral clearance during the convalescent stage.7,8 the latter is currently used to inform hospital discharge decisions in many countries; use of different samples for diagnosis and viral clearance determination can negatively impact on discharge decision-making.8,9,10,12 a potential way of circumventing discharge decision errors is to employ a diagnostic tool that uses the same type of sample for both diagnosis and viraemia monitoring such as the sars-cov-2 antigen and specific anti-sars-cov-2 igm/igg duo detection kit identified in this review. this can be integrated into the local covid-19 management guidelines in lmics. this guideline is currently being used in malaysia and europe.25,26 the primary weakness of rt-pcr for covid-19 diagnosis lies in its inability to detect infection using nasopharyngeal samples collected outside the viral rna shedding period. the shedding period is characterised by presence of low viral rna, such as seen in asymptomatic, pre-symptom days (~2 days prior to symptom onset) and post-infection days (~14 post infection onset).26,27 also, the rt-pcr, may also miss infections due to poor sample collection and preparation as well as poor storage of isolated rna. these weaknesses can be addressed by serological rdts, which detect the more stable viral immunogenic proteins such as the s and n proteins, which persist more than rna or anti-sars-cov-2 igm and igg which have been reported to peak between 2 and 3 weeks and 17 days post infection onset.26,27 guo et al.28 reported an improvement of covid-19 identification by rt-pcr from 51.9% to 98.6% with the integration of an igm-based immunoassay. however, the results of sensitivity (18.4% – 100%) and specificity (90.6% – 100.0%) reported for 12 of the 18 reviewed serological rdt kits by their manufacturers imply that the currently available covid-19 rdts are not equally accurate and only a few of them pass the sensitivity and specificity benchmark of 95%. zainol et al.29 recently reported a sensitivity range of 72.7% – 100.0% and specificity range of 98.7% – 100.0% for igm/igg duo-based serological rdt kits for covid-19 in their review in which nine serological kits were analysed. the authors also reported a sensitivity range of 86.4% – 90.6% and a specificity of 99% for total immunoglobulin-based rdts. in brazil, castro et al.30 reported a mean (range) anti-sars-cov-2 igm sensitivity of 82% (76% – 87%) and specificity of 97% (96% – 98%) and anti-sars-cov-2 igg sensitivity of 97% (90% – 99%) and specificity of 98% (97% – 99%). although in this review, only 5 of the 18 serological rdt kits offered combined igm and igg detection, we also found a better performance characteristic for this type of rdt kit compared to the antigen and total immunoglobulin kits using non-colloidal gold labelling system with acceptable sensitivity (98% – 100%) and a specificity (98% – 99%) values for igg and specificity of 96% – 99% for igm, suggesting the ability of these kits to detect past infections, confirm true negative results and rule out false positive covid-19 testing results by rt-pcr. however, the performance of these kits to confirm recent infections seems to be below the benchmark of 95%, since they had a sensitivity range of 85% – 94%, which was even lower for colloidal gold labelling systems at 57.1%. meanwhile, the improvement offered by the antigen-based rdt kits in this review can be said to be none or marginal at 84.4% – 96%. another implication of these findings is that more than one serological rdt kit may be needed for a sars-cov-2 detection algorithm to improve confirmation and diagnosis of covid-19 by rt-pcr, if deployed in lmics. it is also important to note that 6 of the 18 reviewed serological rdt kits lacked reports on sensitivity and specificity, thus the accuracy in diagnosising covid-19 is unknown as at the time of this review. this finding further reiterates the difficulty associated with sars-cov-2 serological rdt kit validation by manufacturers, since rt-pcr the reference method targets viral rna instead of specific sars-cov-2 antibodies or antigens. a similar opinion has been shared by castrol et al.,30 given the well-documented differences in the kinetics of the viral rna (even between samples) and anti-sars-cov-2 antibodies in infected individuals. as of 01 april 2020, the death toll for covid-19 was over a million globally and the need for accurate intervention to stop transmission and re-infection of covid-19 is now extremely necessary. the who advises countries to improve the rate of testing to identify an infected individual for appropriate isolation and treatment. the availability of efficient and rapid diagnostics for covid-19 has been indicated as one of the mitigation strategies to control the pandemic. rapid diagnostic tests are cheaper and more readily available; thus, they might be more useful stopping transmission by rapidly identifying positive and previous cases particularly in lmics. these data will in turn be useful for both disease diagnosis and surveillance. the rdt will either detect the presence of viral proteins (antigens) expressed by the covid-19 virus or the presence of antibodies in the blood of covid-19-infected people.31,32 the performance of the kits has been shown to depend on several factors such as the onset of illness, the viral load in the specimen, the integrity of the specimen collected from suspected cases, processing, age, nutritional status, the severity of the disease, and certain medications or underlying disease condition, especially immune suppression diseases and the precise formulation of the reagents in the test kits.19 the lmics reported the lowest rate of testing per population with corresponding lower numbers of cases compared with developed countries. this may be an indication of limited testing resources and facilities due to the challenges associated with rt-pcr. therefore, there may be several cases in this population that are not detected with antecedent clinical implications. the use of rdts will not only help to detect currently infected or previously exposed individuals who have developed immunity as well as identify asymptomatic carriers. these will inform decisions for public health measures, for example, cases among a more igm-positive population may be an indication of a subclinical outbreak. the economic impact of movement restrictions and lockdowns in many of these countries is not well managed, adding unimaginable suffering in an already impoverished population. the use of rdts for the screening of covid-19 may help to determine individuals who are at lower risk and may be permitted to go back to work. when coupled with clinical symptoms and molecular testing, rdts may serve as a first-line tool for diagnosis and help to better understand the spread of diseases. although the covid-19 test kit market is in its infancy, the global covid-19 outbreak and up-surging cases are driving the demand for rdts, hence researchers throughout the world are striving to develop rdts to track infected people. to date, very few countries have succeeded in developing sars-cov-2 testing kits, while some are still working on improving the performance of their products. with the dedicated global efforts on preventing the spread of covid-19 and flattening the curve, significant improvement must have occurred in improving the performance of covid-19 test kits. increasing accessibility to testing among other interventions has improved the containment and transmission of the infection. while algorithms have been developed to limit testing to individuals that fulfil certain criteria, such as contact with the infected patients, clinical symptoms of covid-19, travelling history to epidemic countries, etc., testing the entire population has been recommended.33 resource limitations means most lmics can not cope with the up-surge of infection and transmission. while testing per population is high in developed countries with over 3 million tested in the united states, testing per population is still very low in developing countries with less than 10 000 tested in nigeria as of 22 april 2020. therefore, the addition of validated serological-based rdt even with lower performance characteristics compared to rt-pcr may serve as complementary tools to increase the rate of testing per population, especially in lmics where community transmission is now on the rise. therefore, the use of rdts operated as lateral flow immunochromatographic assays to detect both igm and igg on separate test lines using whole blood, plasma or serum samples is desirable for lmics. wang et al. reported that the combination of rt-pcr testing and clinical features for diagnosis of covid-19 facilitated the management of the sars-cov-2 outbreak in china,22 and covid-19 mass testing facilities have been strongly advocated to end the epidemic rapidly.34 the use of rdt will not only allow mass testing facilities in lmics but coupled with clinical features in symptomatic patients and molecular testing (rt-pcr) in asymptomatic populations may help to contain transmission in lmics. conclusion considering the peculiarity of lmics, especially their economic situation, the standard rt-pcr may not be able to cope with the testing needs of these countries because of limited infrastructure and human resources. generally, it is agreed that rapid testing techniques are useful for screening for early detection of symptomatic cases, which is crucial for averting community or hospital transmission and strengthening contact tracing and active surveillance. this review revealed considerable good performance of the rdt with manufacturer sensitivity and specificity using varieties of samples including blood samples. hence, the use of rdt kits in lmics may increase access to testing and better triaging of covid-19 patients. we, however, identified that most of the proposed rapid kits have not been optimised and validated. it is important that the kits undergo further validation with samples from countries of proposed use in reference to rt-pcr before use. acknowledgements competing interests the authors have declared that no competing interest exists. authors’ contributions o.a. conceived the idea for the study. o.a., b.i., and a.o. designed the study. a.o., t.a.s., o.m.a. and o.p. searched for published work. o.a. and b.i. reviewed and made the selection of eligible studies. a.o., t.a.s., o.m.a. and o.p. extracted and compiled the data. o.a., b.i. and a.o. analysed the data while b.i. and a.o. prepared the first draft of the article. o.a. did the final editing of the article. all authors contributed to the writing of the article and have seen and approved the final version. ethical considerations ethical clearance was not required for this study. funding information this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references zhu n, zhang d, wang w, et al. a novel coronavirus from patients with pneumonia in china, 2019. n engl j med. 2020;382(8):727–733. https://doi.org/10.1056/nejmoa2001017 dong e, du h, gardner l. an interactive web-based dashboard to track covid-19 in real time. lancet infect dis. 2020;20(5):533–534. https://doi.org/10.1016/s1473-3099(20)30120-1 nkengasong jn, mankoula w. looming threat of covid-19 infection in africa: act collectively, and fast. lancet (london, england). 2020;395(10227):841–842. https://doi.org/10.1016/s0140-6736(20)30464-5 khadka s, hashmi fk, usman m. preventing covid-19 in lowand middle-income countries. drugs ther perspect. 2020:1–3. https://doi.org/10.1007/s40267-020-00728-8 wang x, yao h, xu x, et al. limits of detection of six approved rt-pcr kits for the novel sars-coronavirus-2 (sars-cov-2). clin chem. 2020;66(7):977–979. https://doi.org/10.1093/clinchem/hvaa099 chan jf, yip cc, to kk, et al. improved molecular diagnosis of covid-19 by the novel, highly sensitive and specific covid-19-rdrp/hel real-time reverse transcription-pcr assay validated in vitro and with clinical specimens. j clin microbiol. 2020;58(5):e00310–e00320. https://doi.org/10.1128/jcm.00310-20 loeffelholz mj, tang y-w. laboratory diagnosis of emerging human coronavirus infections – the state of the art. emerg microb infect. 2020;9(1):747–756. https://doi.org/10.1080/22221751.2020.1745095 arevalo-rodriguez i, buitrago-garcia d, simancas-racines d, et al. false-negative results of initial rt-pcr assays for covid-19: a systematic review. medrxiv. 2020:2020.04.16.20066787. https://doi.org/10.1101/2020.04 younes n, al-sadeq dw, al-jighefee h, et al. challenges in laboratory diagnosis of the novel coronavirus sars-cov-2. viruses. 2020;12(6):582. https://doi.org/10.3390/v12060582 zhong l, chuan j, gong b, et al. detection of serum igm and igg for covid-19 diagnosis. sci china life sci. 2020;63(5):777–780. https://doi.org/10.1007/s11427-020-1688-9 haveri a, smura t, kuivanen s, et al. serological and molecular findings during sars-cov-2 infection: the first case study in finland, january to february 2020. euro surveill. 2020;25(11):2000266. https://doi.org/10.2807/1560-7917.es.2020.25.11.2000266 patel r, babady e, theel es, et al. report from the american society for microbiology covid-19 international summit, 23 march 2020: value of diagnostic testing for sars-cov-2/covid-19. mbio. 2020;11(2):e00722–20. https://doi.org/10.1128/mbio.00722-20 arksey h, o’malley l. scoping studies: towards a methodological framework. int j soc res methodol. 2005;8(1):19–32. https://doi.org/10.1080/1364557032000119616 adhikari sp, meng s, wu yj, et al. epidemiology, causes, clinical manifestation and diagnosis, prevention and control of coronavirus disease (covid-19) during the early outbreak period: a scoping review. infect dis poverty. 2020;9(1):020–00646. https://doi.org/10.1186/s40249-020-00646-x tahamtan a, ardebili a. real-time rt-pcr in covid-19 detection: issues affecting the results. expert rev mol diagn. 2020;20(5):453–454. https://doi.org/10.1080/14737159.2020.1757437 zhao j, yuan q, wang h, et al. antibody responses to sars-cov-2 in patients of novel coronavirus disease 2019. clin infect dis. 2020;ciaa344. https://doi.org/101093/cid/ciaa344 okba nma, muller ma, li w, et al. sars-cov-2 specific antibody responses in covid-19 patients. medrxiv. 2020:2020.03.18.20038059. gorse gj, donovan mm, patel gb. antibodies to coronaviruses are higher in older compared with younger adults and binding antibodies are more sensitive than neutralizing antibodies in identifying coronavirus-associated illnesses. j med virol. 2020;92(5):512–517. https://doi.org/10.1002/jmv.25715 who. advice on the use of point-of-care immunodiagnostic tests for covid-19 [homepage on the internet]. scientific brief 8 april 2020 [cited 2020 apr 08]. available from: who/2019-ncov/sci_brief/poc_immunodiagnostics/20201 cassaniti i, novazzi f, giardina f, et al. performance of vivadiag covid-19 igm/igg rapid test is inadequate for diagnosis of covid-19 in acute patients referring to emergency room department. j med virol. 2020 mar 30. https://doi.org/10.1002/jmv.25800 who. coronavirus disease (covid-19) technical guidance: laboratory testing for 2019-ncov in humans [homepage on the internet]. [cited 2020 apr 08]. available from: www.who.int; [cited 2020 apr 06]. wang y, kang h, liu x, tong z. combination of rt-qpcr testing and clinical features for diagnosis of covid-19 facilitates management of sars-cov-2 outbreak. j med virol. 2020;92(6):538–539. stackelberg o, esmaeilzadeh m, olsen b, lundkvist å. [rapid point-of-care serology testing for sars-cov-2]. lakartidningen, stockholm. 2020;117. malaysia moh. guidelines on covid-19 management in malaysia. 5th ed. the ministry of health malaysia: putrajaya; 2020. control. ecfdpa. novel coronavirus (sars-cov-2) discharge criteria for confirmed covid-19 cases – when is it safe to discharge covid-19 cases from the hospital or end home isolation? [homepage on the internet]. [cited 2020 apr 10]. available from: https://www.ecdceuropaeu/sites/default/files/documents/covid-19-discharge-criteria.pdf chan pk, ng kc, chan rc, et al. immunofluorescence assay for serologic diagnosis of sars. emerg infect dis. 2004;10(3):530–532. https://doi.org/10.3201/eid1003.030493 lee cy, lin rtp, renia l, ng lfp. serological approaches for covid-19: epidemiologic perspective on surveillance and control. front immunol. 2020;11:879. https://doi.org/10.3389/fimmu.2020.00879 guo l, ren l, yang s, et al. profiling early humoral response to diagnose novel coronavirus disease (covid-19). clin infect dis. 2020;71(15):778–785. https://doi.org/10.1093/cid/ciaa310 zainol rashid z, othman sn, abdul samat mn, ali uk, wong kk. diagnostic performance of covid-19 serology assays. malaysian j pathol. 2020;42(1):13–21. castro r, luz pm, wakimoto md, veloso vg, grinsztejn b, perazzo h. covid-19: a meta-analysis of diagnostic test accuracy of commercial assays registered in brazil. braz j infect dis. 2020;24(2):180–187. https://doi.org/10.1016/j.bjid.2020.04.003 zhang p, gao q, wang t, et al. evaluation of recombinant nucleocapsid and spike proteins for serological diagnosis of novel coronavirus disease 2019 (covid-19). medrxiv. 2020:2020.03.17.20036954. https://doi.org/10.1101/2020.03.17.20036954 liu y, liu y, diao b, et al. diagnostic indexes of a rapid igg/igm combined antibody test for sars-cov-2. medrxiv. 2020:2020.03.26.20044883. https://doi.org/10.1101/2020.03.26.20044883 julian p. covid-19 mass testing facilities could end the epidemic rapidly. bmj. 2020;368:m1163. https://doi.org/10.1136/bmj.m1163 bioconcept c. covid-19 ag resp-strip [homepage on the internet]. 2020 [cited 2020 apr 29]. available from: https://www.corisbiocom/products/human-field/covid-19.php (kemri) kmri. covid-19 results in 15 minutes: kemri starts manufacturing rapid test kits, kenya medical research institute (kemri) has started manufacturing covid-19 rapid test kits to ease the testing burden at the state’s facilities [homepage on the internet]. 2020 [cited 2020 apr 07]. available from: www.standardmediacoke/article/2001367222/kemri-starts-manufacturing-covid-19-rapid-test-kits cassette cc-ii. singapore’s camtech, jn medsys to increase production of covid-19 test kits. 2020 [homepage on the internet]. [cited 2020 apr 16]. available from: https://www.mobihealthnewscom/news/asia-pacific/singapore-s-camtech-jn-medsys-increase-production-covid-19-test-kits biosensor s. standard q covid-19 ag [homepage on the internet]. 2020 [cited 2020 apr 10]. available from: http://www.sdbiosensorcom/xe/product/7672 eua beua. bodysphere touts 2-minute covid-19 test [homepage on the internet]. 2020 [cited 2020 apr 02] available from: https://www.massdevicecom/fda-clears-bodysphere-2-minute-covid-19-test/ diagnostics oc. ortho clinical diagnostics pick up eua for covid-19 total antibody assay [homepage on the internet]. 2020 [cited 2020 apr 15]. available from: www.middionlinecom/ortho-clinical-diagnosics-picks-eva-covid-19-total-antibody-assay li z, yi y, luo x, et al. development and clinical application of a rapid igm-igg combined antibody test for sars-cov-2 infection diagnosis. j med virol. 2020. https://doi.org/10.1002/jmv.25727 fellmann f. jetzt beginnt die suche nach den genesenen. tages anzeiger. euroimmun medical laboratory diagnostics and epitope diagnostics; germany: lubeck. 2020. liu r, liu x, han h, et al. the comparative superiority of igm-igg antibody test to real-time reverse transcriptase pcr detection for sars-cov-2 infection diagnosis. preprints from medrxiv and biorxiv. 2020. https://doi.org/10.1101/2020.03.28.20045765 artronlab. one step novel coronavirus (covid-19) igm/igg antibody test kit [homepage on the internet]. 2020. available from: http://www.artronlabcom/products/covbrochure-ver2.pdf xiang j, yan m, li h, et al. evaluation of enzyme-linked immunoassay and colloidal goldimmunochromatographic assay kit for detection of novel coronavirus (sars-cov-2) causing an outbreak of pneumonia (covid-19). pre print medrxiv preprint https://doi.org/101101/202002272002878 thermogenesis. a rapid covid-19 igm/igg serological test for point-of-care [homepage on the internet]. [cited 2020 apr 09]. available from: https://www.thermogenesiscom/rapid-covid-19-point-of-care-diagnostic-test/ cassette at-niirt. 2019-ncov igg/igm rapid test cassette [homepge on the internet]. 2020 [cited 2020 feb 18]. available from: https://www.assaygeniecom/content/assay%20genie%20rapid%20covid%20poc%20test.pdf bd btw. biomedomics teams with bd to launch rapid covid-19 test [homepage on the internet]. [cited 2020 apr 06]. available from: https://www.ncbiotechorg/news/biomedomics-teams-bd-launch-rapid-covid-19-test kit tbtc-. nadalr covid-19 igg/igm test instruction manure [homepage on the internet]. 2020 [cited 2020 apr 20]. available from: https://www.dailymailcouk/news/article-8128327/test-test-covid-kits-10-minute-finger-prick-tests-mask-diagnose-instantly.html abstract introduction case presentation management and outcomes discussion acknowledgements references about the author(s) lebogang skosana department of medical microbiology, tshwane academic division, national health laboratory services, pretoria, south africa farzana ismail centre for tuberculosis, national institute of communicable diseases, johannesburg, south africa nontombi mbelle department of medical microbiology, tshwane academic division, national health laboratory services, pretoria, south africa mohamed said department of medical microbiology, tshwane academic division, national health laboratory services, pretoria, south africa citation skosana l, ismail f, mbelle n, said m. brucellosis – laboratory workers’ nightmare come true: a case study. afr j lab med. 2020;9(1), a1114. https://doi.org/10.4102/ajlm.v9i1.1114 case study brucellosis – laboratory workers’ nightmare come true: a case study lebogang skosana, farzana ismail, nontombi mbelle, mohamed said received: 22 oct. 2019; accepted: 25 june 2020; published: 29 sept. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract introduction: brucella spp. are rarely encountered organisms in the medical microbiology laboratory and, when encountered, can cause concern in laboratory workers. laboratory personnel may in fact develop serious disease as a result of this exposure. this case highlights shortcomings in recognition of brucella spp. from a patient presenting atypically as well as the follow-up and management of an infected patient. case presentation: the patient was an 8-year-old boy from a rural area of south africa who presented to an academic hospital with a bladder mass and history of enuresis in september 2016. brucella melitensis was isolated from a blood culture submitted to the laboratory. the child was subsequently treated for brucellosis in november 2016. management and outcome: the source of infection in the patient was traced to consumption of unpasteurised milk from a local farmer. the patient was treated with doxycycline 100 mg twice daily and rifampicin 600 mg daily for 6 weeks and completed treatment, however he was not followed up at our hospital. the laboratory personnel, however, did not handle the specimen as a biosafety level 3 pathogen as this organism is not commonly encountered; they were provided with prophylaxis for brucellosis (rifampicin and doxycycline). conclusion: brucella spp. is a dangerous pathogen, easily capable of causing significant exposure in an unsuspecting and unprepared laboratory. the case discusses the management of brucellosis in the infected patient as well as the management of laboratory exposure to brucella spp. our case also describes the public health response to a case of brucellosis. keywords: brucellosis; laboratory exposure; prophylaxis; public health; microbiology. introduction brucellosis is a zoonotic disease with a worldwide distribution that is caused by the brucella genus, which are gram-negative bacteria.1 there are approximately 500 000 brucellosis cases reported worldwide annually, however the figure may be much higher as the disease is under-reported, largely because of its non-specific signs and symptoms.2 transmission to humans is through direct contact with animal reservoirs and/or through consumption of infected milk and milk products.1,3 the most common clinical presentation is undulant fever, malaise and arthralgia, after an incubation period ranging from four weeks to several months.1 seven brucella species are potentially pathogenic to humans and each species has a preferred animal host.1,3,4 these include b. abortus (cattle), b. melitensis (sheep, goats), b. suis (swine), b. canis (dogs), b. ovis (sheep), b. ceti (cetaceans), b. pinnipedialis (pinnipeds) and b. inopinata (unknown host).1,3,4 brucellosis, particularly that caused by b. melitensis, is among the most frequently reported laboratory-acquired infections resulting from accidental or inadvertent exposure during aerosolisation procedures in the laboratory.3 this case aims to illustrate the holistic response required when a case of human brucellosis is encountered. ethical considerations this case study received ethical approval from the university of pretoria research ethics committee (ethics number: 412/2017). the patient’s mother was contacted and gave consent to publish by signing a consent form. case presentation the routine processing of blood culture specimens at the tshwane academic division microbiology laboratory (pretoria, south africa) includes removal of positive blood culture bottles from the bact/alert® (biomérieux, marcy-l’étoile, france) system and performing a gram stain, followed by direct sensitivity testing on those positive bottles. the bottle contents are then sub-cultured onto blood, chocolate and macconkey agar plates routinely. cultured plates are preliminarily examined after 10–12 hours of incubation and the chocolate plate is examined for any growth. if pure growth is noted, the plate is sent for identification using the vitek® 2 automated system (biomérieux, marcy-l’étoile france). in this case, the blood culture was taken from an 8-year-old boy from rural mpumalanga, south africa who was admitted to the steve biko academic hospital on 23 september 2016 and was being investigated for a bladder mass and a 2-year history of enuresis (figure 1). for this case isolate, the chocolate plate had pure growth of fine grey colonies after 14 h of incubation. this isolate was sent for vitek 2 for identification and was identified as b. melitensis. once this identification was noted, all further processing was done in a biological safety cabinet class 2 using biosafety level 3 precautions, as recommended by the united states centers for disease control and prevention.5 a gram stain from the culture plates revealed gram-negative coccobacilli which were catalase and oxidase positive and indole negative which was in keeping with a preliminary identification of brucella species. the positive b. melitensis culture was sent to the national institute of communicable diseases in sandringham, south africa on 24 september 2016, where the identification of the organism was confirmed using matrix-assisted laser desorption/ionisation time of flight (maldi-tof) mass spectrometry. the instrument used in this case was the vitek ms (biomérieux, marcy-l’étoile, france), instrument software version 1.5.0.4, myla version 4.5.1, knowledge base (database) version 3.2 (biomérieux, marcy-l’étoile, france). figure 1: significant timelines in the series of events related to the brucellosis case at steve biko hospital (pretoria, south africa) from september to november 2016. management and outcomes the blood culture was taken as part of ‘routine’ investigations as the child had no symptoms or clinical signs which may have led one to consider brucellosis as a possible differential diagnosis. the child was discharged once the blood samples were obtained. the outbreak response unit of the national institute of communicable diseases was alerted to the case on 25 september 2016 and began further investigations and follow-up of affected individuals and possible contacts. it was discovered that the child’s family had been consuming unpasteurised cow’s milk from a local farmer for many years. this farmer’s cattle were tested in late october/early november 2016 using standard south african veterinary testing guidelines.6 sixty-eight cows were tested and 13 were found to be positive for b. melitensis, using the rose bengal test, complement fixation test and serum agglutination test, all of which are serology-based tests. the affected cattle were then isolated, sent to an abattoir and slaughtered as stipulated in the control programme of brucellosis in animals of south africa – animal diseases act (act no. 35 of 1984 ss. 9.1, 9.2, 11).6 the farmer was instructed to send his milk to another local farm for pasteurisation until this farmer was able to pasteurise effectively on his own farm. the patient was treated with doxycycline 100 mg twice daily and rifampicin 600 mg daily for six weeks. he commenced treatment in the first week of november 2016. the treatment was initiated by the clinicians in his hometown and it was uncertain why he was started on the treatment six weeks following the initial blood culture result. soon after commencing with treatment, the patient reported side effects which included abdominal pain and vomiting. these were managed symptomatically, and the patient was able to complete treatment. the affected patient’s family was tested serologically for brucellosis and were found to be negative. prophylaxis was given to those family members who consented to receive it. the united states centers for disease control and prevention’s brucellosis laboratory exposure risk stratification tool was used to screen for exposed staff and staff exposures were stratified as minimal risk, low risk and high risk using these guidelines.5 twenty-one of seventy-two staff members were identified, who had either directly handled the specimen on an open bench or were within 1.5 metres of the person who handled the specimen on an open bench, thus rendering them at high risk for acquiring brucellosis.5 baseline serology was performed on all potentially exposed staff members and standard prophylaxis was provided by the employer. this comprised of rifampicin 600 mg oral daily for three weeks as well as doxycycline 100 mg twice daily for three weeks.5 one staff member was pregnant (in her second trimester) at the time, and subsequently received trimethroprim-sulfamethoxazole (160/800 mg) twice daily for three weeks.5 exposed staff were advised to undergo follow-up serology at 0, 6, 12, 18 and 24 weeks post-exposure.5 weekly symptom watch forms were completed and daily fever self-checks were done for six weeks as recommended by the centers for disease control and prevention and the world health organization.5,6 none of the staff members reported any symptoms for the duration of the symptom check. discussion the transmission of b. melitensis to humans could be through direct contact with infected cattle or the products of abortion of the infected cattle through breaks in the skin as well as through the consumption of unpasteurised milk and milk products.7 transmission can also occur as a result of laboratory exposure to the organism.7 brucella spp. have an infective dose of 10–100 organisms and are often aerosolised during routine laboratory processing of microbiology specimens on an open bench.5 this processing could include performing routine tests on the bench, such as the catalase, oxidase test and indole tests, as well as performing antimicrobial susceptibility testing. these procedures could lead to exposure and possible infection with the organism and can be prevented by ensuring that all processing of brucella spp. isolates is done in an appropriate biosafety cabinet employing biosafety level 3 precautions.5,6 once our laboratory had a preliminary identification of brucella spp., a decision was taken not to manipulate the culture further but to send the isolate to a reference lab with biosafety level 3 facilities for further processing. even though the gold standard for definitive diagnosis of brucella spp. is a positive culture, there is often a delay to final identification because of a number of factors: low index of suspicion, misidentification and unfamiliarity with the organism. the process of identifying exposed individuals and informing them of their risk of developing brucellosis caused much panic among laboratory personnel, and counselling may have been inadequate when addressing fears in the workplace. three staff members are known to have defaulted treatment because of a low perceived risk of acquiring brucellosis and also because of the intolerable side-effects of prophylactic drugs. results of serological testing did not show any significant rise in antibody titres which could have suggested acute infection. the laboratory had no standard operating procedure or policy for the management of possible exposure to a harmful organism before this case and were subsequently prompted to implement these. although b. melitensis mainly affects goats and sheep, cattle may also be infected as a result of indirect contact with goats and sheep.8 furthermore, the systems used in this case may have misidentified the species of brucella.8 the vitek® 2 automated system gram-negative card can only identify the species b. melitensis, while the vitek® maldi-tof ms (biomérieux, marcy-l’étoile, france) was found to correctly identify brucella spp. to the genus level with less correction for species-level identification.8 maldi-tof ms is a reliable method of species-level identification, provided that the brucella spp. reference library used in the database is regularly updated.9 the strengths of this case were the rapid follow-up of exposed laboratory workers and their thorough assessment and management, which managed to contain any potential laboratory outbreak of brucellosis. a limitation of this study was the lack of follow-up on the patient’s clinical progress by the microbiology laboratory. this could be attributed to the outbreak investigation unit having taken over the follow-up of the case, as directed by the public health response protocol. conclusion in conclusion, this case highlights the occupational exposure to b. melitensis as well as the shortcomings associated with the laboratory management thereof. brucella melitensis is a dangerous pathogen, easily capable of causing significant exposure in an unsuspecting and unprepared laboratory. laboratories must ensure that staff are frequently reminded of risks of exposure to such organisms. institutional policy documents should be updated and reviewed regularly, especially with regard to occupational hazards. collaboration between different sectors (eg. agriculture, health) is needed to ensure adequate surveillance and control efforts. acknowledgements competing interests the authors have declared that no competing interests exist. authors’ contributions l.s. collated data and wrote up the manuscript; f.i. collated data; n.m. collated data; and m.s. collated data and proofread the manuscript. all authors read and approved the final manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references bennett j, dolin r, blaser m. brucellosis (brucella species). in: bennett je, dolin r, blaser mj, editors. mandell, douglas, and bennett’s principles and practice of infectious diseases. philadelphia, pa: elsevier saunders; 2015. p. 2584–2585. godfroid j, al dahouk s, pappas g, et al. a ‘one health’ surveillance and control of brucellosis in developing countries: moving away from improvisation. comp immunol microbiol infect dis. 2013;36(3):241–248. https://doi.org/10.1016/j.cimid.2012.09.001 traxler rm, lehman mw, bosserman ea, guerra ma, smith tl. a literature review of laboratory-acquired brucellosis. j clin microbiol. 2013;51(9):3055–3062. https://doi.org/10.1128/jcm.00135-13 hull n, schumaker b. comparisons of brucellosis between human and veterinary medicine. infect ecol epidemiol. 2018;8(1):1500646. https://doi.org/10.1080/20008686.2018.1500846 us centers for disease control and prevention. brucellosis reference guide. assessing laboratory risk level and pep. atlanta, ga: us cdc; 2012. chisi ls, marageni y, naidoo p, zulu g, akol gw, van heerden h. an evaluation of serological tests in the diagnosis of bovine brucellosis in naturally infected cattle in kwazulu-natal province in south africa. j s afr vet assoc. 2017;88(1):e1–e7. https://doi.org/10.4102/jsava.v88i0.1381 corbel mj. brucellosis in humans and animals. geneva, switzerland: world health organization in collaboration with the food and agriculture organization of the united nations and world organisation for animal health; 2006. ferreira l, vega castaño s, sánchez-juanes f, gonzález-cabrero s, menegotto f, orduña-domingo a. identification of brucella by maldi-tof mass spectrometry. fast and reliable identification from agar plates and blood cultures. plos one. 2010;5(12):e14235. https://doi.org/10.1371/journal.pone.0014235 lista f, reubsaet f, santis r, et al. reliable identification at the species level of brucella isolates with maldi-tof-ms. bmc microbiol. 2011;11(1):267. https://doi.org/10.1186/1471-2180-11-267 response from attoh et al. acknowledgements references about the author(s) rujittika mungmungpuntipantip private practice, bangkok, thailand viroj wiwanitkit department of community medicine, dy patil university, pune, india citation mungmungpuntipantip r, wiwanitkit v. post-mortem diagnosis of covid-19. afr j lab med. 2021;10(1), a1471. https://doi.org/10.4102/ajlm.v10i1.1471 scientific letter post-mortem diagnosis of covid-19 rujittika mungmungpuntipantip, viroj wiwanitkit received: 26 nov. 2020; accepted: 23 dec. 2020; published: 24 mar. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. we would like to share our impression on the report ‘postmortem diagnosis of covid-19: antemortem challenges of three cases at the 37 military hospital, accra, ghana’.1 attoh et al. concluded that ‘the outcome of covid-19 testing is dependent on the sample type and accuracy of sampling amongst other factors’1 and suggested that ‘more autopsies are required to fully understand the pathogenesis of this disease in ghanaians’.1 indeed, post-mortem diagnosis of coronavirus disease 2019 (covid-19) is possible and there are many reports of the existence of pathogenic viruses in autopsy specimens.2,3 autopsy is also very useful for understanding the pathogenesis of this new disease. however, it must be performed with high caution. while there are no confirmed cases of the pathogen being spread from deceased patients, infection of forensic pathology workers has been reported.4 more autopsies might be recommended, but adequate biosafety and biosecurity, and other infection control precautions must be in place for these to occur. response from attoh et al. in our article we did not go into details on the methodology. although covid-19 is a category 3 infectious agent, negative pressure systems are unavailable in our country. therefore, a few modifications were made. post-mortems were performed using world health organization’s interim guidance for infection prevention and control for the safe management of a dead body in the context of covid-19.5 the structural design of the autopsy suites used lacked negative pressure systems with filters. as a result, extractors were fitted to provide unidirectional airflow away from the anatomical pathology team into the atmosphere as an appropriate technology. also, members of the anatomical pathology team were carefully selected to exclude those with chronic health conditions such as diabetes mellitus, and cardiovascular and respiratory diseases. it is interesting to note that none of the forensic pathologists nor the anatomical pathology technicians involved in the study have shown covid-19 symptoms up to today. acknowledgements competing interests the authors have declared that no competing interests exist. authors’ contributions r.m. and v.w. have equal contributions in giving ideas, drafting, analysing, writing and giving final approval for this submission. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references attoh sa, hobenu f, edusei l, et al. postmortem diagnosis of covid-19: antemortem challenges of three cases at the 37 military hospital, accra, ghana. afr j lab med. 2020 nov 3;9(1):a1290. https://doi.org/10.4102/ajlm.v9i1.1290 sawant ob, singh s, wright 3rd, re, et al. prevalence of sars-cov-2 in human post-mortem ocular tissues. ocul surf. 2020;nov 8;s1542-0124(20)30168–3. zijlstra jg, van meurs m, moser j. post-mortem diagnostics in covid-19 aki, more often but timely. j am soc nephrol. 2021;32(1):255. https://doi.org/10.1681/asn.2020091263 sriwijitalai w, wiwanitkit v. covid-19 in forensic medicine unit personnel: observation from thailand. j forensic leg med. 2020 may;72:101964. https://doi.org/10.1016/j.jflm.2020.101964 world health organization (who). infection prevention and control for the safe management of a dead body in the context of covid-19: interim guidance [homepage on the internet]. world health organization, 2020; p. 6. who reference number: who/2019-ncov/ipc_dbmgmt/2020.2. available from: https://www.who.int/publications/i/item/infection-prevention-and-control-for-the-safe-management-of-a-dead-body-in-the-context-of-covid-19-interim-guidance introduction antimicrobial resistance, a global menace coronavirus disease 2019, secondary bacterial infection and antimicrobial resistance coronavirus disease 2019, the environment and antimicrobial resistance antimicrobial resistance in africa in the era of coronavirus disease 2019 going forward lessons and conclusion acknowledgements references about the author(s) beverly egyir bacteriology department, noguchi memorial institute for medical research, university of ghana, accra, ghana noah obeng-nkrumah department of medical laboratory sciences, university of ghana, accra, ghana george b. kyei virology department, noguchi memorial institute for medical research, university of ghana, accra, ghana citation egyir b, obeng-nkrumah n, kyei gb. covid-19 pandemic and antimicrobial resistance: another call to strengthen laboratory diagnostic capacity in africa. afr j lab med. 2020;9(1), a1302. https://doi.org/10.4102/ajlm.v9i1.1302 opinion paper covid-19 pandemic and antimicrobial resistance: another call to strengthen laboratory diagnostic capacity in africa beverly egyir, noah obeng-nkrumah, george b. kyei received: 15 june 2020; accepted: 07 aug. 2020; published: 23 sept. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction the coronavirus disease 2019 (covid-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) resulted in 7 145 539 confirmed cases and 408 025 deaths by 03 june 2020.1 understandably, almost all attention has been on covid-19, which continues to take lives and stretch healthcare systems around the world. a critical issue receiving much less attention during this pandemic is the effect it could have on antimicrobial resistance (amr). with no proven therapy for covid-19, prescribers are more likely to use antibiotics indiscriminately for treatment and prevention of presumed bacterial co-infections. in high-income settings, where bacterial cultures can be done on a timely basis, such antibiotic regimens can be stopped within 48 hours. however, in africa, unneeded empiric antibiotic regimens are likely to be continued for a longer period of time due to lack of bacterial culture and antimicrobial susceptibility testing (ast) capabilities. in addition, the widespread use of hand sanitizers and antimicrobial soaps could exacerbate amr among healthcare workers and the general population. in this article, we discuss how the covid-19 pandemic could affect the already precarious amr situation in africa and discuss ways to minimise the effects on public health. antimicrobial resistance, a global menace antimicrobial resistance occurs when microbes change and render antimicrobial agents ineffective. this phenomenon is fuelled by overuse and misuse of antimicrobial agents in humans and animals. antimicrobial resistance leads to treatment failures, prolongs hospital stays, worsens clinical outcomes and makes surgical procedures and chemotherapy risky and unsafe. with increasing antibiotic resistance and few new antimicrobial agents in the production pipeline, it is imperative to monitor the epidemiology of bacteria species, especially respiratory pathogens to inform treatment decisions2,3 in the era of covid-19. it has been estimated that amr could lead to 10 million deaths by 2050 if nothing is done about the menace, and most of the deaths are likely to occur in asia and africa.4 actions such as infection prevention and control, antibiotic stewardships, capacity building (laboratory infrastructure and personnel) and surveillance are necessary to ameliorate the impact of amr. to support a global action plan on amr, the world health organization has developed the global antimicrobial resistance surveillance system to provide a standardised approach for collection, analysis and sharing of data related to amr to inform decision-making at national and international levels; surveillance of amr bacteria in humans and animals across the globe is key in tackling the problem of amr.5 unfortunately, this is hampered by the limited data from africa due to limited diagnostic microbiology infrastructure on the continent.6 coronavirus disease 2019, secondary bacterial infection and antimicrobial resistance viral respiratory infections can be complicated with secondary bacterial infections, commonly with streptococcus pneumoniae, haemophilus influenzae and staphylococcus aureus, resulting in increased severity and mortality.7 the majority of deaths from the 1918 influenza pandemic resulted from secondary bacterial infections.8 during the 2009 swine influenza pandemic, there was an increase in hospital pneumonia cases as a result of secondary bacterial pneumonia, which resulted in 29% – 55% of deaths.9 in a recent study on the outcome of covid-19 patients from the wuhan region of china, half of the non-survivors had secondary infections.10 of note, the majority (95%) of the patients in the study were given antibiotics.10 azithromycin, a broad-spectrum macrolide antibiotic (in combination with hydroxychloroquine) has become a common treatment for covid-19 patients in several parts of the world including africa11,12 without much evidence to support its use. it is worth noting that the world health organization has warned against indiscriminate use of antibiotics during covid-19 treatment.2 while antibiotics may not affect covid-19 directly, they are agents administered to prevent or treat secondary bacterial infections in covid-19 patients. therefore, the swell in the numbers of covid-19 patients could lead to the inappropriate use of antibiotics and subsequent selection of amr bacteria. in previous epidemics, there were indications of an increase in methicillin resistant s. aureus infections in hospitals, which was linked to heavy use of antimicrobial agents.13,14 it is therefore possible that covid-19 patients could also be battling with amr bacteria, in addition to the virus, resulting in poor patient outcomes. urgent studies are needed to define the bacteria aetiologies and amr bacteria that contribute most to mortality in covid-19 patients. coronavirus disease 2019, the environment and antimicrobial resistance from the onset of the pandemic, the general public has been constantly directed to regularly wash hands with soap and water, and use hand sanitizers. although such practices will help improve hygiene standards and reduce the spread of covid-19, they could have negative effects on amr. disinfectants and antimicrobial soaps contain biocides or antimicrobials. an increase in use of these agents may lead to an increase in their concentration in waste water and receiving water bodies; this may have a potentially negative impact, because the elevated concentrations may lead to selection of amr bacteria, posing a health risk to persons exposed to such environments.15 more research in africa is needed to determine how the widespread use of antimicrobial sanitizers and soaps affects the microbiome and contributes to amr. antimicrobial resistance in africa in the era of coronavirus disease 2019 the world health organization reports that there is limited data on amr prevalence from africa due to limited laboratory capacity and surveillance networks.6,16 a survey indicated that very few countries in africa have functional national surveillance systems for amr of common bacterial infections from community and hospitalised patients.17 an external quality assessment report indicated poor performance of ast in several african countries.18 quality assurance in ast is key for reporting and implementation of the global antimicrobial resistance surveillance system19; laboratories in africa have to be supported with the needed investments for better performance. with the paucity of data on amr, realistic evidence on how amr may compromise first-line empirical treatment in common bacterial infections is lacking on the continent.16 in the absence of microbiology and amr data, prescribers often manage clinical symptoms rather than specific bacteria,16 a situation that may fuel inappropriate use of antimicrobials and emergence of resistant microbes. in ghana, like most african countries, the majority of microbiology laboratories do not have the capacity to perform culture and ast tests.20 for the few laboratories that are able to do culture and ast, performing such tests in a standard way is a challenge often due to a lack of reference strains and up-to-date standard interpretation guidelines. in addition, culture of bacteria and ast are often not requested by clinicians, because of cost to patients and the long turn-around time that often render the results useless for patient care. altogether, data on amr bacteria to guide treatment decisions at local and national levels are scarce. newman et al.20 conducted the first nationwide amr surveillance in ghana between 2002 and 2003 and found bacteria species resistant to ceftriaxone (6.3%) and ciprofloxacin (11%).21 opintan et al. (2015), followed up with another nationwide survey between june 2014 and november 2014 and observed that > 50% of the commonly isolated bacteria species were resistant to third-generation cephalosporins and fluoroquinolones. in this study, the majority of the gram-negative bacteria species recovered were positive for extended spectrum beta lactamase; these organisms are resistant to a wide range of antimicrobial agents.22 in another study, a new delhi metallo-beta-lactamase-producing escherichia coli strain resistant to meropenem and belonging to st410 was detected in a urine sample from a hospitalised patient in the northern part of ghana. in this first report, the detected plasmid co-carried other resistance genes.23 this is disturbing, mainly because these organisms are resistant to the antimicrobials used as last-line treatment for severe bacterial infections.24 among female patients who presented with vaginal discharge, dysuria, intermenstrual bleeding or abdominal pain and men who presented with urethral discharge or dysuria, gonococcal isolates recovered from samples collected from five healthcare centres were resistant to tetracycline (100%), benzylpenicillin (91%) and ciprofloxacin (82%). one isolate resistant to cefixime (mic: 0.75 µg/ml) belonged to st1407, a pandemic resistant clone.25 a total of 520 methicillin susceptible s. aureus and 30 methicillin resistant s. aureus isolates were recovered from 1219 nasal swabs (community and hospital carriers) and 916 clinical isolates (blood, skin and soft tissues, wounds) in other studies; methicillin resistant s. aureus isolates detected belonged to global epidemic clones, including usa300.26,27,28,29 our review of more than 20 articles on amr from ghana for this write-up revealed that resistance of bacteria species (recovered from human, food and animals) to commonly used antimicrobial agents such as ampicillin, tetracycline, chloramphenicol and trimethoprim sulfamethoxazole was common. the situation is not different from other parts of the continent.16 the majority of amr data in ghana are from pockets of studies focusing on particular bacteria species recovered in select hospitals or research institutions. they therefore may not reflect the national amr situation and could be an underestimation of the actual magnitude of the amr problem in a country where self-medication is rampant and antimicrobial agents are often available without prescription.30 ghana is gradually gathering momentum to get to a stage of having a functional national system to monitor amr. the antimicrobial use and resistance policy and the national action plan on amr, which provide strategies and plans to guide amr data generation for evidence-based interventions at local, national and international levels, were launched by the president of ghana on 11 april 2018. the national action plan was fashioned along the lines of the objectives of the global action plan on amr.5 ghana also has a national amr working group; the group meets regularly to deliberate on amr issues in the country. to strengthen knowledge and evidence base through surveillance and research, ghana received the first fleming country grant, which is currently supporting a government-led system of collecting, analysing and reporting of amr and antimicrobial use data from humans and animals from 11 sentinel sites. these data will provide a national amr picture to inform treatment decisions at the national level. plans are underway to begin a pilot surveillance study. the prevalence and mortality rate of covid-19 in africa is lower compared to that of europe, america and asia. however, the rapid spread of the virus is another call to strengthen the laboratory diagnostic capacity in africa to perform standard antimicrobial susceptibility testing, especially of relevant respiratory pathogens to inform treatment decisions in healthcare facilities. importantly, ast data need to be collected yearly to support empiric treatment in hospitals. continuous surveillance is required on the african continent and across the globe to understand the epidemiology of amr pathogens, provide the needed data to guide and inform treatment decisions and policies and monitor resistance trends and emergence of new clones. in all of these actions, the role of networks and collaborations in enhancing the success of various interventions cannot be overemphasised. ownership and sustainability plans by governments and local authorities are key to maintaining the successes for continuous amr surveillance activities with local and international partners. capacity building (infrastructure and personnel) is a must-have and must be continuously improved to fight amr in ghana and africa. going forward one cannot blame the african prescribers for throwing the ‘kitchen sink’ of antibiotics at sick and dying covid-19 or other virally infected patients. rather, these practices could be reduced significantly, if physicians were empowered to order cultures and obtain results in a timely manner. doctors faced with negative culture results are more likely to stop antibiotics than if no results are available. with the necessary political will, african governments and academics can do a few things in line with the five strategic objectives of the global action plan on combating amr during the current pandemic and beyond. firstly, there is a need to raise awareness of amr among personnel in human and animal health, and agriculture, as well as among consumers, to ensure a proper understanding of amr pathogens and the impact of amr across sectors. there is an urgent need to build laboratory capacity to generate the required microbiology data through surveillance and research to inform treatment decisions, especially in urban centres where resistant organisms are often abundant. evidence-based prescribing and dispensing should be the way to go to optimise antimicrobial use. more automated systems like the genexpert platform used for tuberculosis could be repurposed for organisms like methicillin-resistant s. aureus. in addition to phenotypic methods used in the detection of resistance, genomic tools such as whole genome sequencing can be utilised to generate extensive data to expand our knowledge on the changing epidemiology of amr bacteria. during the covid-19 pandemic, urgent studies are needed to document the bacterial organisms responsible for co-infections at the local level to guide empiric therapy. due to the widespread use of hand sanitizers and antimicrobial soaps, research is needed to study healthcare workers and others for changes in the skin flora that they carry, which could be transmitted to vulnerable patients. healthcare institutions without antibiotic stewardship programmes must take steps to institute measures for rational antibiotic use. antibiotic stewardship optimises institutional antibiotic use and reduces the selection and spread of amr; similarly, infection prevention needs strengthening across the board to limit the spread of resistant bacteria. finally, the need for increased investment by african governments to drive development of new diagnostic tools, novel antimicrobial agents and vaccines cannot be over-emphasised. lessons and conclusion the amr menace has been described as a problem that knows no borders; resistant bacteria can be found in humans, animals, food and the environment. the current pandemic shows that we remain susceptible to infections for which we have no therapeutic options. the experience from covid-19 therefore should be another reminder of the life-threatening consequences of amr microbes, and the need for rapid capacity building (infrastructure and human resources) for surveillance of resistant bugs on the african continent. acknowledgements the authors of this manuscript are grateful to all other authors whose works were cited. competing interests the authors have declared that no competing interests exist. authors’ contributions b.e. conceived the idea and wrote the first draft. n.o.-n. and g.b.k. critically reviewed the content of the manuscript. ethical considerations no ethical clearance was needed for this study. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement data sharing is not applicable to this article as new data were not created or analysed. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references world health organisation. covid19 dashboard [homepage on the internet]. 2020. available from: https://covid19.who.int/?gclid=cjwkcajw2a32brbxeiwaucugieyvxpsw1ku6wc7kqlfeidlgzfchlmjynrxb0myg3kp3m7timtwjebocvl8qavd_bwe accessed 03 june, 2020. world health organisation. who [homepage on the internet]. 2020 [cited 2020 july 06]. available from: https://www.who.int/news-room/detail/01-06-2020-record-number-of-countries-contribute-data-revealing-disturbing-rates-of-antimicrobial-resistance moodley a, patel e. the straw that might break the camel’s back [homepage on the internet]. 2020 [cited 2020 july 06]. available from: https://www.ilri.org/news/straw-might-break-camel%e2%80%99s-back-exploring-link-between-covid-19-and-antibiotic-resistance-low?fbclid=iwar3xrwhcnesp4uyfz8jze1afoxfkc9g01uvwovqbhnryj-lhyx627buacr8 o’neil j. review on amr 2014: tackling drug resistant infections globally: final report [homepage on the internet]. 2014 [cited 2020 july 07]. available from: https://www.antimicrobialsworkinggroup.org/antimicrobial-resistance/ world health organisation. global action plan on antimicrobial resistance. geneva: who; 2015. world health organization. antimicrobial resistance: global report on surveillance [homepage on the internet]. 2014 [cited 2020 july 09]. available from: https://apps.who.int/iris/bitstream/10665/112642/1/9789241564748_eng.pdf?ua=1 morris de, cleary dw, clarke sc. secondary bacterial infections associated with influenza pandemics. front microbiol. 2017;8(1):1041. https://doi.org/10.3389/fmicb.2017.01041 morens dm, taubenberger jk, fauci as. predominant role of bacterial pneumonia as a cause of death in pandemic influenza: implications for pandemic influenza preparedness. j inf dis. 2008;198(7):962–970. https://doi.org/10.1086/591708 centers for disease control and prevention. bacterial co-infection in lung tissue specimens from fatal cases of 2009 pandemic influenza a (h1n1) – united states. morb mortal wkly rep. 2009;58(38):1071–1074. zhou f, yu t, du r, et al. clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study. lancet. 2020;395(10229):1054–1062. https://doi.org/10.1016/s0140-6736(20)30566-3 reardon s. antibiotic treatment for covid-19 complications could fuel resistant bacteria [homepage on the internet]. 2020 [cited 2020 may 27]. available from: https://www.sciencemag.org/news/2020/04/antibiotic-treatment-covid-19-complications-could-fuel-resistant-bacteria africanews [homepage on the internet]. 2020 [cited 2020 july 06]. available from: https://www.africanews.com/2020/05/27/covid-19-treatment-algeria-to-continue-using-hydroxychloroquine// yap fhy, gomersall cd, fung ksc, et al. increase in methicillin-resistant staphylococcus aureus acquisition rate and change in pathogen pattern associated with an outbreak of severe acute respiratory syndrome. clin inf dis. 2004;39(4):511–516. https://doi.org/10.1086/422641 european centre for disease prevention and control. ecdc country visit to italy to discuss antimicrobial resistance issues. stockholm: ecdc; 2017. murray ak. the novel coronavirus covid-19 outbreak: global implications for antimicrobial resistance. front microbiol. 2020;11(1):1020. https://doi.org/10.3389/fmicb.2020.01020 bernabé kj, langendorf c, ford n, ronat jb, murphy ra. antimicrobial resistance in west africa: a systematic review and meta-analysis. int j antimicrob agents. 2017;50(5);629–639. https://doi.org/10.1016/j.ijantimicag.2017.07.002 world health organisation. global database for antimicrobial resistance [homepage on the internet]. 2018/2019. available from: https://amrcountryprogress.org/ perovic o, yahaya aa, viljoen c, et al. external quality assessment of bacterial identification and antimicrobial susceptibility testing in african national public health laboratories, 2011–2016. trop med infect dis. 2019;13(4):144. https://doi.org/10.3390/tropicalmed4040144 world health organisation. global antimicrobial resistance surveillance system: manual for early implementation. geneva: who; 2015. newman mj, frimpong e, donkor es, opintan ja, asamoah-adu a. resistance to antimicrobial drugs in ghana. inf drug resist. 2011;4(1):215–220. https://doi.org/10.2147/idr.s21769 opintan ja, newman mj, arhin re, et al. laboratory-based nationwide surveillance of antimicrobial resistance in ghana. inf drug resist. 2015;(8):379–389. https://doi.org/10.2147/idr.s88725 opintan ja, newman mj. prevalence of antimicrobial resistant pathogens from blood cultures: results from a laboratory based nationwide surveillance in ghana. antimicrob resist infect control. 2017;6(1):64. https://doi.org/10.1186/s13756-017-0221-0 ayibieke a, sato w, mahazu s, et al. molecular characterisation of the ndm-1-encoding plasmid p2189-ndm in an escherichia coli st410 clinical isolate from ghana. plos one. 2018;13(12):e0209623. https://doi.org/10.1371/journal.pone.0209623 morrill hj, pogue jm, kaye ks, laplante kl. treatment options for carbapenem-resistant enterobacteriaceae infections. open forum infect dis. 2015;2(2):ofv050. https://doi.org/10.1093/ofid/ofv050 attram n, agbodzi b, dela h, et al. antimicrobial resistance (amr) and molecular characterization of neisseria gonorrhoeae in ghana, 2012–2015. plos one. 2019;14(10):e0223598. https://doi.org/10.1371/journal.pone.0223598 egyir b, guardabassi l, moneck s, newmann mj, addo kk, larsen ar. short communication: methicillin resistant staphylococcus aureus strains from ghana include usa300. j glob antimicrob resist. 2015;3(1):26–30. https://doi.org/10.1016/j.jgar.2014.11.006 egyir b, guardabassi l, sørum m, et al. molecular epidemiology and antimicrobial susceptibility of clinical staphylococcus aureus from healthcare institutions in ghana. plos one. 2014;9(2):e89716. https://doi.org/10.1371/journal.pone.0089716 egyir b, guardabassi l, esson j, et al. insights into nasal carriage of staphylococcus aureus in an urban and a rural community in ghana. plos one. 2014;9(4):e96119. https://doi.org/10.1371/journal.pone.0096119 egyir b, guardabassi l, nielsen ss, et al. prevalence of nasal carriage and diversity of staphylococcus aureus among inpatients and hospital staff at korle bu teaching hospital, ghana. j glob antimicrob resist. 2013;1(4):189–193. https://doi.org/10.1016/j.jgar.2013.05.006 donkor es, tetteh-quarcoo pb, nartey p, agyeman io. self-medication practices with antibiotics among tertiary level students in accra, ghana: a cross-sectional study. int j environ res public health. 2012;9(10):3519–3529. https://doi.org/10.3390/ijerph9103519 abstract introduction methods results discussion acknowledgements references about the author(s) candice l. hendricks department of paediatric haematology, faculty of health sciences, school of medicine, university of kwazulu-natal, durban, south africa department of paediatric haematology, inkosi albert luthuli central hospital, durban, south africa ashen naidoo department of haematology, faculty of health sciences, university of kwazulu-natal, durban, south africa national health laboratory service, inkosi albert luthuli central hospital, durban, south africa rajendra thejpal department of paediatric haematology, faculty of health sciences, school of medicine, university of kwazulu-natal, durban, south africa department of paediatric haematology, inkosi albert luthuli central hospital, durban, south africa nadine rapiti department of haematology, faculty of health sciences, university of kwazulu-natal, durban, south africa national health laboratory service, inkosi albert luthuli central hospital, durban, south africa beverley neethling department of paediatric haematology, faculty of health sciences, school of medicine, university of kwazulu-natal, durban, south africa department of paediatric haematology, inkosi albert luthuli central hospital, durban, south africa yasmin goga department of paediatric haematology, faculty of health sciences, school of medicine, university of kwazulu-natal, durban, south africa department of paediatric haematology, inkosi albert luthuli central hospital, durban, south africa suvarna buldeo department of haematology, faculty of health sciences, university of kwazulu-natal, durban, south africa national health laboratory service, inkosi albert luthuli central hospital, durban, south africa citation hendricks cl, naidoo a, thejpal r, et al. childhood aplastic anaemia with paroxysmal nocturnal haemoglobinuria clones: a retrospective single-centre study in south africa. afr j lab med. 2022;11(1), a1537. https://doi.org/10.4102/ajlm.v11i1.1537 original research childhood aplastic anaemia with paroxysmal nocturnal haemoglobinuria clones: a retrospective single-centre study in south africa candice l. hendricks, ashen naidoo, rajendra thejpal, nadine rapiti, beverley neethling, yasmin goga, suvarna buldeo received: 08 feb. 2021; accepted: 10 mar. 2022; published: 06 june 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: paroxysmal nocturnal haemoglobinuria (pnh) clones in children are rare but commonly associated with aplastic anaemia (aa) and myelodysplasia. objective: this study aimed to determine the prevalence of pnh clones in paediatric patients with idiopathic aa, identify differences in clinical and laboratory features and outcomes, and determine the impact of clone size on clinical presentation. methods: patients with confirmed idiopathic aa who were tested for pnh between september 2013 and january 2018 at the inkosi albert luthuli central hospital, durban, kwazulu-natal, south africa, were included. pnh clones were detected in neutrophils and monocytes by flow cytometry using fluorescent aerolysin, cd24, cd66b and cd14. results: twenty-nine children with aa were identified and 11 were excluded. ten patients (10/18, 55.6%) had pnh clones ranging from 0.11% to 24%. compared to the pnh-negative group, these children were older (median: 10 years vs 4 years, p = 0.02) and had significantly lower total white cell counts (median 1.7 × 109/l vs 3.2 × 109/l; p = 0.04). there was no difference in median absolute neutrophil count or haemoglobin concentration. four patients in each group received immunosuppressive therapy (ist). at six months, all four patients with pnh clones had responded, compared to one in the pnh-negative group. conclusion: more than half of children with aa had a pnh clone. the size of the clone did not impact clinical severity; however, ist use may positively impact prognosis. we recommend early initiation of ist in patients with aa to avoid delays associated with human leukocyte antigen typing. keywords: paroxysmal nocturnal haemoglobinuria clones; aplastic anaemia; paediatrics; flow cytometry; hla typing. introduction paroxysmal nocturnal haemoglobinuria (pnh) is an acquired haematopoietic stem cell disorder.1 it is rare in children and results from a somatic mutation in the glycophosphatidylinositol (gpi) glycan a gene on the short arm of the x chromosome.1,2,3 the gpi glycan a gene encodes gpi-anchored markers, including cd55 and cd59, which protect cells from complement-mediated attack.4,5 although the condition is classically described as presenting with nocturnal haemoglobinuria, this finding is not generally common and is particularly rare in children.4,6 patients may also present with bone marrow failure, and the presence of pnh clones has been shown in a large percentage of children with aplastic anaemia (aa).2,7 this association was described by ware et al. in 1991,8 where the largest initial paediatric pnh cohort was found to have features very different from their adult counterparts. since pnh clones are often found in aa, and patients with clinical pnh often progress to aa,9 what is the theory of association? the prevailing theory is that haematopoietic stem cells harbouring the pnh clone are resistant to the immune-mediated damage that occurs during aa and this promotes the expansion of these ‘unaffected’ cells,5,10 also termed the “escape mechanism”. according to luzatto,10 the progression of aa to pnh is thus “the rule rather than the exception”. very little knowledge exists on the difference in clinical presentation and response to treatment in children with and without pnh clones, particularly on the african continent. though aa is the most common presentation, there have been case series where thromboses and haemoglobinuria are documented.4 flow cytometry-based tests are the most accurate tests for the detection of pnh clones.7,11 classically, granulocytes and monocytes are analysed to determine the absence of gpi-linked proteins.3 red blood cells are not thought to represent the true size of the clone, as ongoing haemolysis and the presence of transfused cells usually confound results. paroxysmal nocturnal haemoglobinuria clone sizes < 1% require validation within each laboratory to determine accuracy. these smaller clone sizes are, however, becoming more important to identify, as their presence allows for future monitoring of clone size and the potential development of classic pnh.2 a previous study described the 10-year probability of developing pnh disease from a pnh clone to be 10.2%.2 historically, patients with aa were managed either supportively with transfusions or cured with a haematopoietic stem cell transplant (hsct).12,13 before immunosuppressive therapy (ist) became available, early reports from africa showed very high mortality rates in symptomatically treated patients.14,15 according to the north american pediatric aplastic anemia consortium, most centres would only opt for hsct as first-line therapy in patients presenting with acquired severe aa and who have a matched sibling donor.16 in the absence of a sibling donor, ist with anti-thymocyte globulin (atg) and cyclosporin becomes the best option.17,18 haematopoietic stem cell transplant from a matched unrelated-donor is only recommended for patients who do not respond to first-line ist. in our setting, the majority of patients are of recent african ancestry, where significant human leukocyte antigen (hla) diversity exists.19,20 all patients diagnosed with aa in our unit routinely have hla typing performed. if siblings from the same parents are present, they too are tested, and if they are a match, patients are referred for hsct. a donor search for local (south african) donors is also conducted via the south african bone marrow registry. uninsured patients treated within the state/public health system can be transplanted within the state/public sector if there is a compatible unrelated local donor identified from the registry. insured patients can in some instances access compatible international donors. human leukocyte antigen typing and the subsequent registry searches to find matches take time, and there is a low likelihood of finding an hla match for transplantation. this prolongs symptomatic treatment with the risk of multiple transfusion exposures. our study was conducted at a quaternary hospital in durban, south africa. the haematology laboratory provides in-house flow cytometry services and has been using the fluorescent aerolysin method to detect the presence of pnh clones in patients since september 2013. this study aimed to determine the prevalence of pnh clones in paediatric patients with aa, identify differences in clinical and laboratory features and treatment responses between patients with and without pnh clones, and determine the impact of clone size on clinical presentation. methods ethical considerations ethics approval was obtained from the biomedical research ethics committee of the university of kwazulu-natal (bca325/18). individual patient consent was not required due to the study being retrospective. data were password-protected, and patient confidentiality was maintained by only including patient hospital numbers on the final datasheets. study population the electronic database of the paediatric haematology-oncology unit of the inkosi albert luthuli central hospital, durban, south africa, was screened to identify patients with a diagnosis of aa between 01 september 2013 and 31 january 2018. the diagnosis was based on pancytopenia secondary to bone marrow hypoplasia in the absence of a clonal malignant disorder. patients who had an inherited bone marrow failure syndrome such as fanconi anaemia, or who were not tested for pnh, were excluded. the electronic medical files of these children were searched on the hospital information system (meditech, westwood, massachusetts, united states) as well as the laboratory information system (trak care, intersystems, cambridge, massachusetts, united states) to retrieve demographic and clinical information, as well as laboratory test results. patients with and without a pnh clone were compared with respect to demographics, laboratory parameters, presenting complaints, management and outcomes. the severity of aa was determined based on camitta’s criteria (cited by marsh et al.).21 haematuria was measured using a urine dipstick and therefore was not distinguished from haemoglobinuria. immunosuppressive therapy was administered in the form of rabbit or horse atg, where appropriate. all patients received premedication with antihistamines and antipyretics prior to the daily infusion, as well as intravenous methylprednisone daily during the administration of the drug course, followed by a course of oral prednisone and cyclosporin. response to ist was monitored at 6 months and 1-year post-administration. a response was deemed complete if full blood count parameters normalised (absolute neutrophil count [anc] ≥ 1.5 × 109/l, haemoglobin ≥ 11 g/dl, and platelet count ≥ 100 × 109/l), partial if there was transfusion independence in the presence of cytopenias, and none if there was continued transfusion dependence.22 laboratory analysis flow cytometry (facscanto ii, becton dickinson, san jose, california, united states) was performed to detect pnh clones using non-gpi-linked markers (cd15 and cd33) to identify the monocytes and neutrophils, and gpi-linked markers (fluorescent aerolysin, cd24, cd66b and cd14) to detect the loss of gpi anchors in a sequential gating strategy (figure 1). after performing doublet discrimination using forward scatter-area vs forward scatter-height and removing debris, all white blood cells were gated using cd45. neutrophils were then discriminated by gating on cd15-positive, cd33-dim and high-side scatter white blood cells. monocytes were discriminated by gating on cd33-bright, cd15-negative and low-side scatter white blood cells. neutrophil pnh clones were defined as clones negative for cd24, cd66b and fluorescent aerolysin, and monocyte pnh clones were clones negative for cd14 and fluorescent aerolysin. figure 1: gating strategy for paroxysmal nocturnal haemoglobinuria clones, south africa, september 2013 – january 2018. (a) all white blood cells gated using cd45; (b) gated neutrophils; (c) gated monocytes; (d, e, f) the dual-negative granulocytes; (g, h) dual-negative monocytes. a loss of two gpi-linked markers on both the monocyte and neutrophil lineage was required to detect a clone. all clone sizes were included as per the latest guidelines of the international clinical cytometry society/european society for clinical cell analysis,23 which classify clones as “pnh clone” for pnh populations > 1.0%, “minor population of pnh cells” or “minor pnh clone” for pnh populations between 0.1% and 1.0%, and “rare cells with gpi deficiency” or “rare cells with pnh phenotype” for pnh populations < 0.1%. for uniformity, we refer to all pnh populations, even if < 1.0%, as clones in this publication. data analysis statistical analysis was performed using graphpad prism version 8.4.3 (2020, san diego, california, united states). a multiple t-test which was used to determine the statistical difference between numerical variables using means (holm-sidak method) and a chi-square test or fisher’s exact test which was used for categorical variables. a p < 0.05 was considered statistically significant. results twenty-nine patients were diagnosed with aa during the study period, ten of whom had fanconi anaemia and were subsequently excluded (figure 2). of the remaining 19 patients, only one patient did not have pnh testing performed. eighteen patients were thus included. ten patients had a pnh clone (10/18; 55.6%); six of which were classified as having a “pnh clone” and four as having a “minor population of pnh cells”. figure 2: study inclusion and exclusion criteria for patients with aplastic anaemia at the inkosi albert luthuli central hospital, durban, south africa, september 2013 – january 2018. patients with a pnh clone were older compared to those without the clone (median age: 10 years vs 4 years; p = 0.02) (table 1). female patients made up the majority of patients in both the pnh-negative group (5/8; 62.5%) and the pnh-positive group (6/10; 60.0%). the full blood count results showed no difference in haemoglobin concentration and platelet count between the two groups. paroxysmal nocturnal haemoglobinuria-positive patients had a significantly lower median total white cell count of 1.7 × 109/l compared to 3.2 × 109/l in pnh-negative patients (p = 0.04). however, the median anc was similar in both groups: 0.26 × 109/l in the pnh-positive group and 0.32 × 109/l in the pnh-negative group. lactate dehydrogenase results were known in 80% (8/10) of pnh-positive and 50% (5/10) of pnh-negative patients and all results were within the normal range. all patients in the pnh-positive and pnh-negative groups had complete information relating to the reticulocyte production index, which was low in all patients. hypocellularity was also confirmed in all patients in both groups by bone marrow aspiration and trephine biopsy. table 1: clinical characteristics of paroxysmal nocturnal haemoglobinuria-positive and -negative patients at the inkosi albert luthuli central hospital, durban, south africa, september 2013 – january 2018. the majority of patients in both groups presented with severe or very severe aa. two patients without the pnh clone presented with haematuria, but this was not a presenting feature in any patients with the clone. bleeding from a peripheral site was noted in 70.0% of pnh-positive and 87.5% of pnh-negative patients, while no patients presented with evidence of thromboses. four pnh-positive (4/10; 40.0%) and four pnh-negative patients (4/8; 50.0%) were given ist. fifty percent of patients with the pnh clone died, compared to 37.5% among patients without the clone. seven of the ten (7/10; 70.00%) pnh-positive patients presented with bleeding (table 2). clone sizes in the granulocyte population and monocyte population in each patient ranged between 0.11% and 24.00%. five of the total pnh-positive patient cohort survived (5/10; 50.00%), four (40.00%) of whom received atg. the other survivor went on to receive an hsct and is still alive. the remaining five pnh-positive patients (5/10; 50.00%) died, two from sepsis and three from bleeding. one of these patients died while awaiting hla results. among those who died, the pnh clone sizes ranged from 0.30% to 15.00%. table 2: paroxysmal nocturnal haemoglobinuria clone sizes and clinical presentation and outcome of patients with aplastic anaemia at the inkosi albert luthuli central hospital, durban, south africa, september 2013 – january 2018. eight patients (four pnh-positive and four pnh-negative patients) in our cohort received atg. all patients received equine atg, and one patient subsequently received an additional dose of rabbit atg. one patient in the pnh-negative group died from sepsis a few days after infusion and was excluded from further analysis (figure 3). at both six and 12 months, one patient in the pnh-positive group had a complete response and the other three patients had a partial response. in the pnh-negative group, one patient had a partial response at six months while two did not respond. at 12 months, the patient with a partial response still had a partial response, one of the patients with no response had developed a partial response, while the other patient remained non-responsive despite receiving a second dose of atg. figure 3: response to immunosuppressive therapy among paroxysmal nocturnal haemoglobinuria population-positive and paroxysmal nocturnal haemoglobinuria population-negative patients at the inkosi albert luthuli central hospital, durban, south africa, september 2013 – january 2018. discussion paroxysmal nocturnal haemoglobinuria is rare in children and does not present classically. the presentation is normally bone marrow failure,12 which exists as a continuum with pnh.10,24 in this study we found a 55.6% prevalence of pnh clones in those children presenting with aa. the clone size had no impact on the severity of the clinical presentation, but patients appeared to respond well to ist. those with a pnh clone were also older than their pnh-negative counterparts and had lower total white cell counts. the differences in the presentation of pnh between adults and children have been described.8 a study reported in 1991 from the united states described that haemoglobinuria was detected in 50.0% of adult pnh patients compared to 15.0% among children, and only 25.0% of adult pnh patients had bone marrow failure compared to 58.0% among children.8 paroxysmal nocturnal haemoglobinuria clone sizes have been reported to be much lower in aa than in pnh disease,25 requiring high-sensitivity fluorescent aerolysin analysis to detect clones < 0.1%.26 no patients in our study presented with clinical pnh, in keeping with a report from japan also describing pnh clones in aa patients.2 the pnh clone prevalence in this study, at 55.6%, is much higher than the 12.9% prevalence observed in a paediatric cohort in india in 201527 that used a pnh clone cut-off of > 1.0%. when a pnh clone size of < 1.0% is included, as in our study, some studies show higher prevalence rates such as 41.0%28 and 46.0%.29 a study conducted in the united states30 found that 40.0% of patients were affected, even at a cut-off of 1.0%. in an adult cohort from tanzania, a 42.0% prevalence of pnh clones in aa patients was found.31 interestingly, this study also showed an overall higher aa prevalence of six cases per million per year, compared to two per million per year in europe and north america. a study among a paediatric cohort from egypt showed the presence of a pnh clone in 36.0% of patients using the cd59 immunohistochemical staining on bone marrow trephine biopsies.32 it must be noted that although we included very small clones in our study, the evidence for the impact of these minor clones on clinical presentation is not yet well established.27 it has also been shown that small populations of granulocytes bearing the pnh phenotype can even be found in healthy individuals33; any specific cut-off value may thus be arbitrary. the median age of children presenting with a pnh clone was significantly higher (p = 0.02). there is also evidence in the literature suggesting that pnh clones increase with age in children.27,34 in the current study, children without the pnh clone presented with a higher incidence of haematuria. as all patients presented with thrombocytopenia, the true reason for the haematuria could not be elucidated, particularly because a urine dipstick is unable to distinguish this from haemoglobinuria, the true mark of intravascular haemolysis. similarly, the median haemoglobin concentration of > 7 g/dl observed in all patients is likely due to blood transfusions received prior to referral. according to camitta’s criteria for the classification of aa severity (cited by marsh et al.),21 anc (< 0.5 × 109/l), reticulocyte count (< 20 × 109/l) and platelet count (< 20 × 109/l) are the predictors of severity. the presence of at least two of these three criteria indicates severe aa. a study in korea highlighted that the anc should be more heavily weighted, as it has the biggest impact on patient complications, is not influenced by transfusions, and can be readily used to classify severity in patients.35 in our cohort, 15 of the 18 patients had a neutrophil count of < 0.5 × 109/l, while the haemoglobin and platelet counts remained above the defined cut-off likely due to transfusions received, suggesting that anc may be a more important factor to consider in the diagnosis and severity grading of patients with aa. this study also found no association between pnh clone size and severity of clinical presentation. a study from india published in 2018 also showed no correlation between clone size and pnh symptoms in a large paediatric cohort.1 only three of the 100 patients in the study presented with haemolysis, all of whom had clone sizes > 10%, and one patient with a very small clone presented with thrombosis. this highlights the need for screening all patients for signs of clinical pnh, regardless of clone size. importantly, only one child in our cohort received an hsct. two patients had already developed platelet refractoriness and this may be due to the continuation of platelet transfusions while awaiting the hla results. our findings show an improved atg response in the pnh-positive cohort; however, the small size of the study population makes it impossible to reach a definitive conclusion. some studies have either shown, suggested or referenced an improved response to ist in patients with aa presenting with a pnh clone,18,27,36,37 or have found no difference in response.1,28,30 however, a recent meta-analysis that included 1236 participants from 11 studies showed a statistically significant improved response to ist in patients with pnh clones.38 in determining ist response in aa as a whole, the north american pediatric aplastic anemia consortium published results from 314 paediatric aa patients wherein complete response was observed in 59.8% of patients, and the 5-year event-free survival was 64.0%.39 relapsed or refractory disease is thus still an important factor to consider in the outcomes of these patients, with many either needing a second course of ist or an hsct. this group found that hsct in relapsed/refractory disease was the preferred treatment modality, rather than a second course of ist. while hsct has historically only been the preferred front-line therapy in patients having a matched sibling donor, a recent report by the united kingdom paediatric bmt working party, paediatric diseases working party and severe aplastic anaemia working party of the european society for blood and marrow transplantation showed that using a matched unrelated-donor upfront had similar results to matched sibling donors.40 importantly, patients with an upfront-matched unrelated-donor hsct had superior outcomes than patients who initially failed ist and then went on to receive a matched unrelated-donor hsct. regarding the timing of ist administration, it is important to note that in south africa, the genetic diversity of the population is such that less than 20.0% of patients (non-european) will have an hla match for hsct.41,42 although the paucity of african donors is certainly a problem, the diversity within the hla region is significant, even among typed donors.20,43 once abnormal cytogenetics, inherited bone marrow failure syndromes and malignancies have been excluded, early initiation of ist is recommended as a delay may lead to increased morbidity and risk of mortality. some studies suggest that the presence of a pnh clone in and of itself rules out an inherited bone marrow failure syndrome.1,29 this may further aid the timeous initiation of therapy. limitations the lower limit of quantitation or detection for pnh clones has not been defined by our laboratory. the quantitative values below 1.0% must thus be interpreted cautiously as the external quality assurance samples analysed in the laboratory have clone sizes greater than 1.0%. however, the external quality assurance programme used for minimal residual disease monitoring verifies the accuracy of the laboratory in defining clone sizes below 0.1% and the identification of very small clones in patients with bone marrow failure has been consistently reported in the literature. for this reason, we have opted to include all clones. the sample size is small; however, this is attributable to the rarity of pnh in the paediatric population and the lack of testing in all paediatric patients with aa. it is thus not possible to make any definitive conclusions regarding any observed differences between the two populations. additionally, the pnh-negative group was not monitored for the subsequent development of a pnh clone and the clone sizes in pnh-positive patients were not monitored and correlated to clinical outcomes. with clear evidence of the risk of developing clinical pnh in future,2 these are important factors to consider in the future management of these patients. conclusion our study found a very high prevalence of pnh clones in patients with aa. patients with pnh clones were older at presentation and had significantly lower total white cell counts. a higher clone size was not associated with a worse clinical presentation. patients with pnh clones who received ist survived. in similar settings where significant hla diversity exists and in the absence of a potential sibling donor, once malignancies and inherited bone marrow failure syndromes have been excluded, patients presenting with aa should be initiated on ist even while awaiting hla typing results. larger studies are needed to further determine the impact of pnh clones on the disease course in paediatric patients with aa. acknowledgements the authors acknowledge dr hamida van staaden who assisted with data collection for the project. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions c.l.h., a.n. and s.b. were responsible for conceptualisation of the study, protocol preparation, data analysis and write up of the manuscript; r.t., n.r., b.n. and y.g. were responsible for data analysis, and write up of the manuscript; c.l.h. collected the data. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability data supporting the findings of this study are available from the corresponding author, c.l.h., on request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references rahman k, mittal n, gupta r, et al. clinicopathological profile of paroxysmal nocturnal haemoglobinuria clone-positive aplastic anaemia paediatric patients – a single centre study from north india. int j lab hematol. 2018;40(5):604–610. https://doi.org/10.1111/ijlh.12875 narita a, muramatsu h, okuno y, et al. development of clinical paroxysmal nocturnal haemoglobinuria in children with aplastic anaemia. br j haematol. 2017;178(6):954–958. https://doi.org/10.1111/bjh.14790 fletcher m, whitby l, whitby a, barnett d. current international flow cytometric practices for the detection and monitoring of paroxysmal nocturnal haemoglobinuria clones: a uk neqas survey. cytom part b clin cytom. 2017;92(4):266–274. https://doi.org/10.1002/cyto.b.21368 van den heuvel-eibrink mm, bredius rgm, te winkel ml, et al. childhood paroxysmal nocturnal haemoglobinuria (pnh), a report of 11 cases in the netherlands. br j haematol. 2005;128(4):571–577. https://doi.org/10.1111/j.1365-2141.2004.05337.x luzzatto l, risitano am. advances in understanding the pathogenesis of acquired aplastic anaemia. br j haematol. 2018;182(6):758–776. https://doi.org/10.1111/bjh.15443 schubert j, röth a. update on paroxysmal nocturnal haemoglobinuria: on the long way to understand the principles of the disease. eur j haematol. 2015;94(6):464–473. https://doi.org/10.1111/ejh.12520 dezern ae, borowitz mj. iccs/escca consensus guidelines to detect gpi-deficient cells in paroxysmal nocturnal hemoglobinuria (pnh) and related disorders part 1 – clinical utility. cytom part b clin cytom. 2018;94(1):16–22. https://doi.org/10.1002/cyto.b.21608 ware re, hall se, rosse wf. paroxysmal nocturnal hemoglobinuria with onset in childhood and adolescence. n engl j med. 1991;325(14):991–996. https://doi.org/10.1056/nejm199110033251403 luzzatto l, bessler m, rotoli b. somatic mutations in paroxysmal nocturnal hemoglobinuria: a blessing in disguise? cell. 1997;88(1):1–4. https://doi.org/10.1016/s0092-8674(00)81850-4 luzzatto l. pnh phenotypes and their genesis. br j haematol. 2020;189(5):802–805. https://doi.org/10.1111/bjh.16473 hill a, richards sj, hillmen p. recent developments in the understanding and management of paroxysmal nocturnal haemoglobinuria. br j haematol. 2007;137(3):181–192. https://doi.org/10.1111/j.1365-2141.2007.06554.x andolina jr, reinish al, akhtar r, et al. successful reduced-intensity conditioning hematopoietic stem cell transplantation for paroxysmal nocturnal hemoglobinuria with aplastic anemia in two children. pediatr blood cancer. 2018;65(8):e27218. https://doi.org/10.1002/pbc.27218 luzzatto l, gianfaldoni g, notaro r. management of paroxysmal nocturnal haemoglobinuria: a personal view. br j haematol. 2011;153(6):709–720. https://doi.org/10.1111/j.1365-2141.2011.08690.x galiba atipo tsiba fo, kocko i, okouango ova guelongo jd, ondzotto ibatta ci, malanda f, elira dockekias a. severe aplastic anemia: management challenges at the university teaching hospital of brazzaville. rwanda med j. 2016;73(2):22–25. arewa op, akinola no. survival in primary a plastic anaemia; experience with 20 cases from a tertiary hospital in nigeria. afr health sci. 2009;9(4):290–293. williams da, bennett c, bertuch a, et al. diagnosis and treatment of pediatric acquired aplastic anemia (aaa): an initial survey of the north american pediatric aplastic anemia consortium (napaac). pediatr blood cancer. 2014;61(5):869–874. https://doi.org/10.1002/pbc.24875 cheung wc, lam cck, kwong yl. anti-thymocyte globulin treatment of marrow aplasia associated with paroxysmal nocturnal haemoglobinuria (pnh) resulted in haematological improvement due to an expansion of the pnh clone. br j haematol. 2003;120(2):325–328. https://doi.org/10.1046/j.1365-2141.2003.04046.x narita a, muramatsu h, sekiya y, et al. paroxysmal nocturnal hemoglobinuria and telomere length predicts response to immunosuppressive therapy in pediatric aplastic anemia. haematologica. 2015 dec 1;100(12):1546–1552. https://doi.org/10.3324/haematol.2015.132530 tshabalala m, mellet j, pepper ms. human leukocyte antigen diversity: a southern african perspective. j immunol res. 2015;2015(class i):1–11. https://doi.org/10.1155/2015/746151 viljoen im, hendricks cl, mellet j, pepper ms. perspectives on establishing a public cord blood inventory in south africa. cytotherapy. 2021;23(6):548–557. https://doi.org/10.1016/j.jcyt.2021.02.116 marsh jcw, ball se, cavenagh j, et al. guidelines for the diagnosis and management of aplastic anaemia. br j haematol. 2009;147(1):43–70. https://doi.org/10.1111/j.1365-2141.2009.07842.x mercuri a, farruggia p, timeus f, et al. a retrospective study of paroxysmal nocturnal hemoglobinuria in pediatric and adolescent patients. blood cells, mol dis. 2017;64:45–50. https://doi.org/10.1016/j.bcmd.2017.03.006 illingworth aj, marinov i, sutherland dr. sensitive and accurate identification of pnh clones based on iccs/escca pnh consensus guidelines – a summary. int j lab hematol. 2019;41(s1):73–81. https://doi.org/10.1111/ijlh.13011 olutogun t, cutini i, notaro r, luzzatto l. complement-mediated haemolysis and the role of blood transfusion in paroxysmal nocturnal haemoglobinuria. blood transfus. 2015;13(3):363–369. agarwal r, chapple p, brown m, szer j, juneja s. analysis of abnormal clones by the fluorescent aerolysin method in paroxysmal nocturnal haemoglobinuria and other marrow disorders. int j lab hematol. 2015;37(1):14–21. https://doi.org/10.1111/ijlh.12207 raza a, ravandi f, rastogi a, et al. a prospective multicenter study of paroxysmal nocturnal hemoglobinuria cells in patients with bone marrow failure. cytom part b clin cytom. 2014;86(3):175–182. https://doi.org/10.1002/cyto.b.21139 sreedharanunni s, sachdeva mus, bose p, varma n, bansal d, trehan a. frequency of paroxysmal nocturnal hemoglobinuria clones by multiparametric flow cytometry in pediatric aplastic anemia patients of indian ethnic origin. pediatr blood cancer. 2016;63(1):93–97. https://doi.org/10.1002/pbc.25691 timeus f, crescenzio n, longoni d, et al. paroxysmal nocturnal hemoglobinuria clones in children with acquired aplastic anemia: a multicentre study. plos one. 2014;9(7):e101948. https://doi.org/10.1371/journal.pone.0101948 dezern ae, symons hj, resar ls, borowitz mj, armanios my, brodsky ra. detection of paroxysmal nocturnal hemoglobinuria clones to exclude inherited bone marrow failure syndromes. eur j haematol. 2014;92(6):467–470. https://doi.org/10.1111/ejh.12299 scheinberg p, marte m, nunez o, young ns. paroxysmal nocturnal hemoglobinuria clones in severe aplastic anemia patients treated with horse anti-thymocyte globulin plus cyclosporine. haematologica. 2010;95(7):1075–1080. https://doi.org/10.3324/haematol.2009.017889 ally m, magesa p, luzzatto l. high frequency of acquired aplastic anemia in tanzania. am j hematol. 2019;94(4):e86–e88. https://doi.org/10.1002/ajh.25388 rizk s. screening for paroxysmal nocturnal hemoglobinuria (pnh) clone in egyptian children with aplastic anemia. j trop pediatr. 2002;48(3):132–137. https://doi.org/10.1093/tropej/48.3.132 araten dj, nafa k, pakdeesuwan k, luzzatto l. clonal populations of hematopoietic cells with paroxysmal nocturnal hemoglobinuria genotype and phenotype are present in normal individuals. proc natl acad sci. 1999;96(9):5209–5214. https://doi.org/10.1073/pnas.96.9.5209 reiss um, schwartz j, sakamoto km, et al. efficacy and safety of eculizumab in children and adolescents with paroxysmal nocturnal hemoglobinuria. pediatr blood cancer. 2014;61(9):1544–1550. https://doi.org/10.1002/pbc.25068 yoon hh, huh sj, lee jh, et al. should we still use camitta’s criteria for severe aplastic anemia? korean j hematol. 2012;47(2):126. https://doi.org/10.5045/kjh.2012.47.2.126 borowitz mj, craig fe, digiuseppe ja, et al. guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry. cytom part b clin cytom. 2010;78(4):211–230. https://doi.org/10.1002/cyto.b.20525 tutelman pr, aubert g, milner ra, dalal bi, schultz kr, deyell rj. paroxysmal nocturnal haemoglobinuria phenotype cells and leucocyte subset telomere length in childhood acquired aplastic anaemia. br j haematol. 2014;164(5):717–721. https://doi.org/10.1111/bjh.12656 tu j, pan h, li r, et al. pnh clones for aplastic anemia with immunosuppressive therapy: a systematic review and meta-analysis. acta haematol. 2021;144(1):34–43. https://doi.org/10.1159/000506387 rogers zr, nakano ta, olson ts, et al. immunosuppressive therapy for pediatric aplastic anemia: a north american pediatric aplastic anemia consortium study. haematologica. 2019;104(10):1974–1983. https://doi.org/10.3324/haematol.2018.206540 dufour c, veys p, carraro e, et al. similar outcome of upfront-unrelated and matched sibling stem cell transplantation in idiopathic paediatric aplastic anaemia. a study on behalf of the uk paediatric bmt working party, paediatric diseases working party and severe aplastic anaemia working party of ebmt br j haematol. 2015;171(4):585–594. https://doi.org/10.1111/bjh.13614 dessels c, alessandrini m, pepper ms. factors influencing the umbilical cord blood stem cell industry: an evolving treatment landscape. stem cells transl med. 2018;7(9):643–650. https://doi.org/10.1002/sctm.17-0244 tshabalala m, ingram c, schlaphoff t, borrill v, christoffels a, pepper ms. human leukocyte antigen-a, b, c, drb1, and dqb1 allele and haplotype frequencies in a subset of 237 donors in the south african bone marrow registry. j immunol res. 2018;2018:1–8. https://doi.org/10.1155/2018/2031571 cao k, moormann am, lyke ke, et al. differentiation between african populations is evidenced by the diversity of alleles and haplotypes of hla class i loci. tissue antigens. 2004;63(4):293–325. https://doi.org/10.1111/j.0001-2815.2004.00192.x acknowledgements references about the author(s) iruka n. okeke department of pharmaceutical microbiology, faculty of pharmacy, university of ibadan, ibadan, nigeria citation okeke in. african laboratory medicine in the time of covid-19. afr j lab med. 2020;9(1), a1447. https://doi.org/10.4102/ajlm.v9i1.1447 editorial african laboratory medicine in the time of covid-19 iruka n. okeke copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 2020 has been a turbulent year but one that fixed the importance of laboratory medicine in the eye of the global public. public health experts’ worst fears surrounding a hypothetical ‘agent x’ that spurred thousands of calls for ‘pandemic preparedness’ in the last half-decade have become eerily true and the importance of ‘testing’ is now broadly acknowledged. by the end of the first quarter of 2020, following its emergence in wuhan, china, agent x was named severe acute respiratory syndrome coronavirus 2 (sars-cov-2); it had caused infections in almost every country, and one of the worst pandemics of all time was underway (figure 1). instituting sars-cov-2 testing rapidly and reliably was one of the pivotal distinguishers between countries that got a handle on their coronavirus disease 2019 (covid-19) epidemics and those that did not. figure 1: in the time of covid-19. the importance of laboratory medicine has been made more visible by covid-19 than at any time this century. no country in the world was ready by the time covid-19 overwhelmed each national surveillance and health system. across africa, pandemic response planning began early and, as sars-cov-2 entered our continent relatively late, most african countries were able to deploy testing before the virus arrived.1,2 to their tremendous credit, african countries leveraged this testing advantage to implement pandemic infection control policies that likely combined with as yet unknown biological features to yield what are still now relatively temperate versions of covid-19 devastation. african countries have also continued to grow testing capacity so that, while we are not the necessary step ahead of the virus, most countries in africa are, to some degree, keeping pace with it. national epidemic responses depend on very local activities. published this year in the african journal of laboratory medicine (ajlm) is an exemplary local response to a covid-19 crisis in an african sars-cov-2 testing laboratory. the crisis was a laboratory outbreak that not only endangered the medical laboratory scientists, but also disabled the testing service when the need was greatest.3 among other things, the affected laboratory implemented heightened infection prevention and control measures and increased physical distancing. it also increased automation of sample processing, which reduced contact between staff and infected specimens, and will improve operations in the years to come. as another article from skosana et al.4 demonstrates, workplace infections are an ever-present risk in laboratories and so lessons learned from this pandemic will have broad application. at the patient level, a significant proportion of covid-19 survivors live for months with disabling sequelae in ‘long-covid’; the ultimate prognosis for this new disease remains unclear.5 covid-19 also has unclear long-haul implications on communities, on countries and on healthcare systems and their laboratories. the pandemic is a frontal challenge to weak systems, but it also offers opportunities to develop them and build resilience, if the response is implemented with this in mind.6 for example, future procurement could be templated on more generally applicable network approaches to procurement and supply chain management outlined by williams et al. for hiv, forestalling the procurement crises experienced in africa with covid-19 testing and personal protective equipment supplies.7,8 sars-cov-2 is not the only epidemic pathogen that circulated in africa in 2020. the democratic republic of congo declared a 21-month ebola outbreak that ended on 14 may 2020, but on 01 june 2020, a new outbreak began. nigeria has worked to contain lassa fever virus epidemics for most of the duration of the covid-19 pandemic. while these three feared viruses ravage, measles, cholera and other epidemics are also in play.8 one of the things that has become increasingly visible in the course of these epidemics is the central role laboratory medicine must play to contain them. volume 9, issue 1 of ajlm goes beyond chronicling a broad range of infectious disease catastrophes to outlining lessons learned that will strengthen health systems.3,9,10,11,12,13,14,15,16,17,18 while most pandemic activity has focused on reverse transcription polymerase chain reaction and other forms of testing to support the identification of cases and transmission chains,10 other domains of laboratory medicine have made important contributions that shed more light on the pathogenesis of the disease, and therefore how best to improve treatment.19,20 as clinical and laboratory health workers and the scientific community engaged in discoveries to combat the pandemic, another equally frenetic behind-the-scenes response took place in editorial offices as we, gratefully helped by volunteer reviewers, struggled under the mound of covid-19 submissions to bring to the fore discoveries that are most worthy of priority and long-term attention. in mid-may, relatively early in the pandemic, a science commentary reported that covid-19 researchers and policymakers were becoming overwhelmed by the literature in this brand new field with a 20-day doubling time on the number of articles indexed by major databases.21 by early november, that curve had flattened somewhat but, still, over 70 000 covid-19 articles were indexed in pubmed or posted on pre-print servers, awaiting assessment. the flood has spurred the creation of artificial intelligence approaches for curating the literature.21 but the bulk of the work required to review, improve and present these works to the drowning community is done manually by editors and volunteer reviewers. unsurprisingly, the pandemic has seen highly publicised discourse on research article quality, as well as some very high-profile retractions. the strain on peer review has also led to some misleading information driving covid-19 policy, prevention and therapeutics, some of which have been amplified by influential non-scientists. among the many lessons learned on the fly in this pandemic is that health crises bring an influx of emergency-care patients, specimens requiring immediate testing and a flood of literature. resilience is needed as much in scientific publishing as in health systems. pandemic preparedness must include plans for rapidly but effectively sifting through the literature flood to retrieve the knowledge most likely to reign in the emergency, which is the only way to stop the overwhelm on both fronts. as a regional journal addressing laboratory medicine on a continent that has seen multiple epidemics this year, we at ajlm quickly found that our standard workflows would not manage submissions fast and rigorously enough for a helpful pandemic response. at the same time, it was important for us to continue to process manuscripts across the journal’s scope. we did manage to ensure that the journal could contribute in valuable ways towards addressing the pandemic while at the same time ensuring that important articles not focused on the pressing problem of covid-19 were published. indeed, by responding to covid-19 but not succumbing to ’coviddisation’,22 we observed that articles submitted before the pandemic was declared were key to the covid-19 response. among these are the future of diagnostics special issue article by preiser and van zyl23 on pooled testing, with a focus on hiv, which included critical knowledge for aligning test-and-trace covid-19 needs with the extreme resource limitations that almost every country has seen associated with polymerase chain reaction testing. additional articles in that special issue also have direct relevance to strengthening testing capabilities in the pandemic.24,25 to meet the double demands of maintaining peer review and production of regular articles, the submission rate of which continues to rise, and processing pandemic submissions, the pandemic pushed us to alter our operations. ajlm’s new normal allows for a regular submission track, and a new fast track for articles containing knowledge that could be applied to the ongoing covid-19, lassa fever and ebola haemorrhagic fever outbreaks. this year, these ran in addition to the call to our special issue (volume 9, issue 2) on the future of diagnostics, which was guest edited by timothy amukele, noah fongwen and rosanna peeling.26 our fast track remains open and will include the many excellent articles we are processing now for volume 10. altogether, ajlm received 47 submissions through its viral epidemic fast track between april 2020 when we opened the track and october of the same year. only two of these epidemic articles addressed lassa fever, and none addressed ebola haemorrhagic fever, pointing to the disproportionately low scientific activity on local african epidemics. so far, just 10 of the covid-19 fast-tracked articles have been accepted. this acceptance rate compares with that for regular submissions but is unexpectedly low, because we expected predominantly pressing issues worthy of publication to come via the fast track. similar high submission, low acceptance rates have been reported by editors of other journals.27 do the low acceptance rates make all the extra hard work done on pandemic submissions by the editorial offices worth it? this question is important to us, because all three epidemics are still in play, our journal’s overall submission rate has continued to rise and our fast track is still open. the answer is undoubtedly yes. firstly, by overseeing the peer review of the manuscripts we received, we did help to shield frontline health workers and policymakers from the even larger deluge of covid-19 preprints that they would otherwise have had to navigate. this is an important function of peer review in general. secondly and more pointedly, we are proud to be publishing key articles that influence the course of the pandemic, particularly on the african continent, where the dynamics are different, the response, while variable, is largely commendable and resources are severely limited. given the sacrifices made in so many areas towards containing this pandemic, this is the least we could do. acknowledgements i thank the authors, reviewers, editorial office and publishers’ staff that made african journal of laboratory medicine’s necessary capacity surge in this pandemic possible. i am also grateful to frontline medical laboratory science and other health professionals for their selfless service through this pandemic. competing interests the author has declared that no competing interests exist. author’s contributions i conceived and wrote the editorial. disclaimer the views and opinions expressed in this article are those of the author and do not necessarily reflect the official policy or position of any affiliated agency of the author. references kapata n, ihekweazu c, ntoumi f, et al. is africa prepared for tackling the covid-19 (sars-cov-2) epidemic. lessons from past outbreaks, ongoing pan-african public health efforts, and implications for the future. int j infect dis. 2020;93:233–236. https://doi.org/10.1016/j.ijid.2020.02.049 senghore m, savi mk, gnangnon b, hanage wp, okeke in. leveraging africa’s preparedness towards the next phase of the covid-19 pandemic. lancet glob health. 2020;8(7):e884–e885. https://doi.org/10.1016/s2214-109x(20)30234-5 opperman cj, marais gjk, naidoo m, hsiao m, samodien n. response to a cluster of severe acute respiratory syndrome coronavirus 2 cases at a diagnostic laboratory. afr j lab med. 2020;9(1):a1307. https://doi.org/10.4102/ajlm.v9i1.1307 skosana l, ismail f, mbelle n, said m. brucellosis – laboratory workers’ nightmare come true: a case study. afr j lab med. 2020;9(1):a1114. https://doi.org/10.4102/ajlm.v9i1.1114 honigsbaum m, krishnan l. taking pandemic sequelae seriously: from the russian influenza to covid-19 long-haulers. lancet. 2020;396(10260):1389–1391. https://doi.org/10.1016/s0140-6736(20)32134-6 umaru fa. scaling up testing for covid-19 in africa: responding to the pandemic in ways that strengthen health systems. afr j lab med. 2020;9(1):a1244. https://doi.org/10.4102/ajlm.v9i1.1244 nkengasong jn, mankoula w. looming threat of covid-19 infection in africa: act collectively, and fast. lancet. 2020;395(10227):841–842. https://doi.org/10.1016/s0140-6736(20)30464-5 nkengasong jn, tessema sk. africa needs a new public health order to tackle infectious disease threats. cell. 2020;183(2):296–300. https://doi.org/10.1016/j.cell.2020.09.041 egyir b, obeng-nkrumah n, kyei gb. covid-19 pandemic and antimicrobial resistance: another call to strengthen laboratory diagnostic capacity in africa. afr j lab med. 2020;9(1):a1302. https://doi.org/10.4102/ajlm.v9i1.1302 olalekan a, iwalokun b, akinloye om, popoola o, samuel ta, akinloye o. covid-19 rapid diagnostic test could contain transmission in lowand middle-income countries. afr j lab med. 2020;9(1):a1255. https://doi.org/10.4102/ajlm.v9i1.1255 mitton b, rule r, mbelle n, van hougenhouck-tulleken w, said m. post-procedural bacillus cereus septic arthritis in a patient with systemic lupus erythematosus. afr j lab med. 2020;9(1):a1119. https://doi.org/10.4102/ajlm.v9i1.1119 govender s, mbambo l, nyirenda m, sebitloane m, abbai n. herpes simplex virus-2 infections in pregnant women from south africa: evaluation of the immunoflow rapid test. afr j lab med. 2020;9(1):a854. https://doi.org/10.4102/ajlm.v9i1.854 haumba sm, toda m, jeffries r, et al. prevalence of cryptococcal antigen (crag) among hiv-positive patients in eswatini, 2014–2015. afr j lab med. 2020;9(1):a933. https://doi.org/10.4102/ajlm.v9i1.933 madeira cm, azam ki, sato dn, khosa c, bhatt n, viegas so. evaluation of the ogawa-kudoh method for tuberculosis isolation in two health units in mozambique. afr j lab med. 2020;9(1):a929. https://doi.org/10.4102/ajlm.v9i1.929 pasipamire m, broughton e, mkhontfo m, maphalala g, simelane-vilane b, haumba s. detecting tuberculosis in pregnant and postpartum women in eswatini. afr j lab med. 2020;9(1):a837. https://doi.org/10.4102/ajlm.v9i1.837 sayed s, mutasa r, kaaya e, et al. establishing the college of pathologists of east, central and southern africa – the regional east central and southern africa college of pathology. afr j lab med. 2020;9(1):a979. https://doi.org/10.4102/ajlm.v9i1.979 mudenda v, malyangu e, sayed s, fleming k. addressing the shortage of pathologists in africa: creation of a mmed programme in pathology in zambia. afr j lab med. 2020;9(1):a974. https://doi.org/10.4102/ajlm.v9i1.974 demba rn, aradi sm, mwau m, mwanda wo. kaposi’s sarcoma-associated herpesvirus protein orf75 among hiv-1 patients in kenya. afr j lab med. 2020;9(1), a939. https://doi.org/10.4102/ajlm.v9i1.939 attoh sa, hobenu f, edusei l, et al. postmortem diagnosis of covid-19: antemortem challenges of three cases at the 37 military hospital, accra, ghana. afr j lab med. 2020;9(1):a1290. https://doi.org/10.4102/ajlm.v9i1.1290 admou b, hachimi a, samkaoui ma. how can clinical immunology laboratories contribute to the management of severe covid-19 cases in limited resource contexts? afr j lab med. 2020;9(1), a1282. https://doi.org/10.4102/ajlm.v9i1.1282 brainard j. scientists are drowning in covid-19 papers. can new tools keep them afloat. science. 2020 (13 may). https://doi.org/10.1126/science.abc7839 pai m. covidization of research: what are the risks? nat med. 2020;26(8):1159. https://doi.org/10.1038/s41591-020-1015-0 preiser w, van zyl gu. pooled testing: a tool to increase efficiency of infant hiv diagnosis and virological monitoring. afr j lab med. 2020;9(2):a1035. https://doi.org/10.4102/ajlm.v9i2.1035 emperador dm, mazzola lt, kelly-cirino c. an open-source molecular diagnostic platform approach for outbreak and epidemic preparedness. afr j lab med. 2020;9(2):a1017. https://doi.org/10.4102/ajlm.v9i2.1017 naidoo d, ihekweazu c. nigeria’s efforts to strengthen laboratory diagnostics – why access to reliable and affordable diagnostics is key to building resilient laboratory systems. afr j lab med. 2020;9(2):a1019. https://doi.org/10.4102/ajlm.v9i2.1019 fongwen n, boeras d, peeling rw, amukele t. connected diagnostics systems: the future of disease control in africa. afr j lab med. 2020;9(2):a1365. in press. the lancet global health. publishing in the time of covid-19. lancet glob health. 2020;8(7):e860. https://doi.org/10.1016/s2214-109x(20)30260-6 abstract background description of the intervention lessons learnt recommendations acknowledgements references about the author(s) lance d. presser global engagement program, mriglobal, gaithersburg, maryland, united states jeanette coffin global engagement program, mriglobal, gaithersburg, maryland, united states lamine koivogui centre de recherche et de formation en infectiologie de guinée, université gamal abdel nasser de conakry, conakry, guinea allan campbell central public health reference laboratory, freetown, sierra leone julian campbell central public health reference laboratory, freetown, sierra leone fatmata barrie central public health reference laboratory, freetown, sierra leone jone ngobeh central public health reference laboratory, freetown, sierra leone zein souma central public health reference laboratory, freetown, sierra leone samuel sorie central public health reference laboratory, freetown, sierra leone doris harding central public health reference laboratory, freetown, sierra leone alimou camara institut national de santé publique, conakry, guinea pepe tohonamou institut national de santé publique, conakry, guinea basala traore institut national de santé publique, conakry, guinea frank a. hamill global engagement program, mriglobal, gaithersburg, maryland, united states joe bogan global engagement program, mriglobal, gaithersburg, maryland, united states sharon altmann global engagement program, mriglobal, gaithersburg, maryland, united states casey ross global engagement program, mriglobal, gaithersburg, maryland, united states jay mansheim global engagement program, mriglobal, gaithersburg, maryland, united states robert hegerty global engagement program, mriglobal, gaithersburg, maryland, united states scott poynter global engagement program, mriglobal, gaithersburg, maryland, united states scott shearrer global engagement program, mriglobal, gaithersburg, maryland, united states carmen asbun global engagement program, mriglobal, gaithersburg, maryland, united states brendan karlstrand global engagement program, mriglobal, gaithersburg, maryland, united states phil davis global engagement program, mriglobal, gaithersburg, maryland, united states jane alam global engagement program, mriglobal, gaithersburg, maryland, united states david roberts global engagement program, mriglobal, gaithersburg, maryland, united states paul d. stamper global engagement program, mriglobal, gaithersburg, maryland, united states jean ndjomou global engagement program, mriglobal, gaithersburg, maryland, united states nadia wauquier global engagement program, mriglobal, gaithersburg, maryland, united states mohamed koroma global engagement program, mriglobal, gaithersburg, maryland, united states alhaji munu global engagement program, mriglobal, gaithersburg, maryland, united states jason mcclintock global engagement program, mriglobal, gaithersburg, maryland, united states mar mar global engagement program, mriglobal, gaithersburg, maryland, united states true burns global engagement program, mriglobal, gaithersburg, maryland, united states stephen krcha global engagement program, mriglobal, gaithersburg, maryland, united states citation presser ld, coffin j, koivogui l, et al. the deployment of mobile diagnostic laboratories for ebola virus disease diagnostics in sierra leone and guinea. afr j lab med. 2021;10(1), a1414. https://doi.org/10.4102/ajlm.v10i1.1414 lessons from the field the deployment of mobile diagnostic laboratories for ebola virus disease diagnostics in sierra leone and guinea lance d. presser, jeanette coffin, lamine koivogui, allan campbell, julian campbell, fatmata barrie, jone ngobeh, zein souma, samuel sorie, doris harding, alimou camara, pepe tohonamou, basala traore, frank a. hamill, joe bogan, sharon altmann, casey ross, jay mansheim, robert hegerty, scott poynter, scott shearrer, carmen asbun, brendan karlstrand, phil davis, jane alam, david roberts, paul d. stamper, jean ndjomou, nadia wauquier, mohamed koroma, alhaji munu, jason mcclintock, mar mar, true burns, stephen krcha received: 29 sept. 2020; accepted: 18 mar. 2021; published: 22 oct. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: ebola virus emerged in west africa in december 2013. the ease of mobility, porous borders, and lack of public health infrastructure led to the largest ebola virus disease (evd) outbreak to date. intervention: the 2013 evd outbreak signalled the need for laboratory diagnostic capabilities in areas without strong public health systems. as part of the united states’ department of defense response, mriglobal was contracted to design, fabricate, equip, deploy, and operate two mobile diagnostic laboratories (mdls). the first laboratory analysed blood samples from patients in an adjacent ebola treatment centre (etc) and buccal swabs from the deceased in the community in moyamba, sierra leone. the second laboratory was deployed to support an etc in conakry, guinea. the department of defense provided real-time quantitative reverse transcription polymerase chain reaction assays that were deployed and validated on-site. lessons learnt: prompt and accurate molecular diagnostics reduced sample turn-around times from over 24 h to under 4 h. experienced laboratory staff tested up to 110 samples per day and on-site engineering proved necessary for mdl setup and operation. as the ebola response slowed, the sustainment of the mdls’ operations was prioritised, including staff training and the transition of the mdls to local governments. training programmes for local staff were prepared in sierra leone and guinea. recommendations: the mriglobal mdl team significantly contributed to establishing increased laboratory capacity during the evd outbreak in west africa. using the mdls for molecular diagnosis is highly recommended until more sustainable solutions can be provided. keywords: ebola; ebola virus; sierra leone; guinea; diagnostics; laboratory capacity; service expansion; epidemic; outbreak; outbreak response; west africa; mobile diagnostic laboratories. background in march 2014, the world health organization was notified regarding a cluster of disease in guinea characterised by fever, severe diarrhoea, vomiting, and high fatality rate. eventually, the disease was identified as a novel strain of the ebola virus.1 further investigation suggested the first fatality of the outbreak had occurred in december 2013 in guinea.1 when the outbreak was declared over, 28 616 ebola virus disease (evd) cases including 11 310 total deaths had been reported.2 the unprecedented scale of this evd outbreak resulted in sustained human-to-human transmission, the consequences of which are still being elucidated. ebola virus is a biosafety level 4 (bsl-4) agent. specimen inactivation should be performed in at least a bsl-3 laboratory, after which routine diagnostic specimen testing can be performed in a bsl-2 laboratory. when the outbreak began, the closest bsl-3 laboratory was in nigeria and was being used for tuberculosis diagnostics. it was not capable of evd testing, in that it lacked polymerase chain reaction (pcr) machines and had no validated assay and reagents. similarly, the closest bsl-4 laboratory was situated in gabon, 3000 km away from freetown, and unable to assist with evd diagnostics timeously. none of the west african countries hit hardest by the evd outbreak (guinea, sierra leone, or liberia) had adequate evd diagnostic facilities; this necessitated the development of mobile diagnostic laboratory (mdl) units, and improvement of other laboratory capacities in the region to help control the outbreak. the united states department of defense initiated the cooperative biological engagement program in west africa through the defense threat reduction agency to contain the biological agent (ebola virus), enhance biosafety and biosecurity, and strengthen the region’s ability to detect, diagnose, and report public health emergencies of international concern to the world health organization.3 mriglobal was awarded the contract to design, assemble, equip, and deploy rapid response mdls for molecular detection of ebola virus in patient samples. at the invitation of the sierra leonean and guinean governments, as well as the department of defense, the non-profit organisation mriglobal designed, built, delivered, and operated mdls during the evd outbreak starting in december 2014. by 2016, mriglobal had shifted its focus from emergency response to a smooth transition of management, which included staff training and support to the central public health reference laboratory in sierra leone and the national institute of public health in guinea. we describe here the deployment of the mdls for the evd outbreak response and discuss the successes and challenges experienced. description of the intervention ethical considerations the evd outbreak response was declared a public health emergency of international concern by the world health organization on 08 august 2014. the standard operating procedures used for diagnostic testing were approved by the world health organization, the department of defense, and the ministry of health in guinea and sierra leone. diagnostic results were released as quickly as possible following specimen analyses. neither mriglobal nor the department of defense retained any samples as they were either destroyed or turned over to the host country. mobile diagnostic laboratory design from an engineering standpoint, the project’s goal was to build a mobile, self-sustained, self-contained (safe) laboratory ready for delivery in less than six weeks. mriglobal engineers selected 20-foot (~6.1 m) intermodal containers because they provide a rugged, watertight, customisable shell that can be easily transported. mriglobal has over 15 years of experience in designing, building, maintaining, and deploying similar containerised laboratories around the world. mriglobal engineers had to consider customisation such as including surfaces that were easy to decontaminate and providing attachment and stabilisation points for all pieces of equipment within the labs, including lighting, heating, ventilation, and air conditioning systems. mriglobal’s engineers and scientists worked together to design the laboratory to ensure safe handling and testing of samples and the accommodation of equipment required for operations in both countries. the laboratories were to include three separate areas – sample inactivation and extraction, reagent preparation, and quantitative reverse-transcription polymerase chain reaction (qrt-pcr) areas inside the two laboratory containers. the first container housed the sample inactivation and extraction area where infectious samples would be processed and inactivated inside of a class ii type b2 biological safety cabinet (bsc). four bscs were placed in the inactivation laboratory to handle the anticipated sample volume. after delivery, modifications to the container were necessary to allow for sample pass-through between the two containers and to properly exhaust the bscs (figure 1c). figure 1: mriglobal mobile diagnostic laboratory that was deployed in response to the west africa ebola virus outbreak. (a) interior of polymerase chain reaction laboratory unit. (b) interior of master mix laboratory. (c) interior of inactivation and extraction laboratory unit. (d) interior of locker room. a partition was built inside the second laboratory container to make two separate work areas for the reagent preparation area and the qrt-pcr area (figure 1a and 1b). due to concerns about possible contamination in the reagent preparation area, additional air handling equipment was used to provide positive pressure to the reagent preparation area, thus ensuring it would remain clean. office space, supplies store, personal protective equipment (ppe) locker rooms, restroom, shower room, and a tool warehouse were also deemed necessary. a standard container was used to provide both office space and supply storage. mriglobal worked with a mobile restroom manufacturer to design a mobile trailer that would provide restroom and shower facilities as well as a locker room (figure 1d). an additional small container was used as the tool warehouse and office space for the on-site engineer. mobile diagnostic laboratory transportation and installation mriglobal was responsible for arranging transportation of these laboratories to guinea and sierra leone. to best satisfy the schedule requirements, air cargo was used. the aviastar-sp antonov an-124 ruslan (figure 2) was the only aeroplane option due to some issues including the mdl size and cargo weight and the runway length in guinea and sierra leone. figure 2: transport of mriglobal mdl from the united states to guinea and sierra leone. (a) antonov an-124 aircraft used to transport the mriglobal mdl from kansas city, missouri, united states, to guinea and sierra leone sites. (b) interior of antonov an-124 aircraft, loaded with mdl units. (c) truck unloading mdl units in moyamba, sierra leone. (d) hydraulic system used to unload mdl units in moyamba, sierra leone. once the equipment arrived at the sites, laboratory equipment was unpacked. the bscs were certified by the on-site engineers and the electrical lines were run. however, the engineering team was faced with technical challenges relating to the electrical power supply, safe water and sewer connections, laundry facilities, biohazard waste disposal, and internet connectivity. the mdls were built with the united states electrical standards of 120 volts and 60 hertz. these electrical standards are not shared in guinea or sierra leone and this resulted in difficulties replacing or servicing equipment. also, there was the issue of an unstable power supply. to address the unstable power supply, two diesel generators were purchased, and fuel contracts were established. each generator could provide light for the entire laboratory system on its own. however, the generators were significantly larger than necessary, resulting in reduced efficiency of the generators and increased fuel expenses. an automatic transfer switch was used to continuously monitor the power produced by the generator. to address water availability and to provide a sewer connection and a laundry facility, a small container was used to house the water equipment for the laboratory, including the two safety showers which were located directly outside of the laboratory exit. the container housed a water pump, pressure bladder tanks, and the laundry facility. the system was designed so it could accept water from a supplied water line or a tank stored on top of the water container, depending on what was available when the laboratory reached its final destination. the restroom and shower trailers were built with black water storage tanks but were also capable of being connected to a septic or sewer system. to ensure proper disposal of biohazard waste, a medical incinerator (elastec mediburn, carmi, illinois, united states) capable of temperatures above 1000 °c was installed. this ensured all infectious and pathological waste generated by the laboratory was safely disposed of. the environment in guinea and sierra leone also presented challenges. the mdls were consistently exposed to high temperatures, high humidity, and heavy rains, which resulted in the rapid decomposition of many elements of the mdls. these were addressed using guidelines and assessment tools provided in the ‘report on the status of emerging and dangerous pathogen laboratory network bsl-3 in select countries in the african region’.4 when possible, repairs and parts were sourced locally. when local repairs or parts were not available, they were included in quarterly laboratory supply shipments that originated in the united states. staff composition and worksite layout in sierra leone, the mdl arrived on 18 december 2014 and sample testing started on 12 january 2015 (25 days). in guinea, the time between the arrival of the mdl arrival and the start of sample testing was shortened to 13 days (21 april 2015 to 04 may 2015). the composition of the mdl team was a rotation of four scientists, two engineers, and multiple drivers at each site. the initial site layout for both sierra leone and guinea is shown in figure 3. figure 3: worksite layout for the mriglobal mobile diagnostic laboratory, moyamba, sierra leone. each laboratory unit is labelled appropriately. specimen collection blood and swab specimens were delivered to the mdl sites in conakry, guinea and moyamba, sierra leone, primarily by motorbike couriers or from the adjacent ebola treatment centre. when receiving the specimens, the staff wore coveralls, sleeves, and double gloves. ppe was stored and donned in the locker room unit (figure 1d). the surface of the specimen bucket and the sample packaging bag were disinfected by spraying with 0.5% hypochlorite solution. specimen inactivation and nucleic acid extraction the mriglobal ebola response team inactivated samples in the inactivation and extraction laboratory unit (figure 1c) wearing appropriate ppe. the ppe included coveralls, sleeves, triple gloves, and a powered air-purifying respirator system. the sample transport container was disinfected with 0.5% hypochlorite solution and opened within the bsc. for samples requiring malaria testing, the malaria ag p.f. test (sd bioline, chicago, illinois, united states) was performed. whole blood or plasma sample inactivation was performed using trizol liquid sample (thermofisher, waltham, massachusetts, united states). following inactivation, sample rna was extracted using the ez1 virus mini kit version 2.0 in the ez1 advanced xl hardware platform (qiagen, hilden, germany). extracted rna from samples was immediately sent for qrt-pcr amplification or stored at –20 °c. quantitative reverse transcription polymerase chain reaction assays the qrt-pcr assays were performed using the ebola zaire taqman and ebola zaire taqman-mgb assays provided by the department of defense and joint program manager-medical countermeasure systems critical reagents program (figure 1a). the assays were authorised for use on the 7500 fast dx real-time pcr instrument (applied biosystems, foster city, california, united states).5 the multiplex pcr steps were programmed as follows: stage 1 – 15 min at 50 °c, stage 2 – 5 min at 95 °c, stage 3 – 1 s at 95 °c, 26 s at 60 °c, stage 4 – 30 s at 40 °c.5 specimen storage the mdl was equipped with –20 °c freezers. as a result, only short-term (< 14 days) storage of patient samples was maintained. the specimens were well packed and the surface disinfected by 0.5% hypochlorite solution before storage. the mdl sites were guarded round the clock and all freezers and containers were locked. test result release samples from patients were assigned an individual, unique identification number by the emergency operations, established by each country’s ministry of health. when a sample was collected, the clinician was asked to complete a ‘viral haemorrhagic fever case investigation form’. the sample and the investigation form were marked with the case identification number and patient name and promptly transported to the laboratory. the case identification number provided a unique number for tracking the specimen from the patient, and the results of the specimen. the information in the test report included the case identification number, the ct value determined by qrt-pcr, and the confirmed result (yes, no, or suspect). according to the agreement with the health authorities, the mriglobal ebola response team did not contact hospitals directly. instead, mriglobal submitted the test report to the world health organization and a local emergency operations centre, which delivered consistent and timely results to each hospital and treatment centre. transfer of ownership the united states government supported the transfer of ownership of the supplies, materials, and equipment to the ministry of health of guinea on 23 september 2016 and to the ministry of health of sierra leone on 16 march 2017. personnel from each recipient country were trained before the transfer of ownership to allow for long-term independent maintenance of the improved diagnostic capabilities. in preparation for the transfer of ownership to the ministry of health of guinea, mriglobal developed and provided a series of trainings, each followed by a test and evaluation. training included tabletop exercise and a field operational demonstration. the tabletop exercise was designed to engage ministries of health and local and international partners in a collaborative effort to define how these new facilities and capabilities can best be merged into the existing national laboratory response systems and to capture stakeholder recommendations for improving long-term sustainability. the field training exercise was observed by evaluators and referees to gauge the capability of the newly trained staff in performing essential laboratory functions safely and effectively. the transfer of laboratory capacity to sierra leone and guinea is aimed at helping both countries fulfil the ‘core capacity requirements for surveillance and response’ as outlined in the international health regulations 2005 annex 1.6 other partner capabilities many other international partners played a role in the evd response in west africa, and there were numerous types of laboratories and laboratory diagnostics deployed. the dutch government deployed three mdl units, the chinese government deployed one mdl unit and built a bsl-3 laboratory and hospital outside of freetown, sierra leone, and the united states centers for disease control and prevention established a field laboratory in bo, sierra leone, alongside a host of other partner activities.7,8,9,10 the two mdl laboratories that were deployed by mriglobal tested 18 624 total samples without a safety incident. having adequate laboratory capacity, provided almost entirely by international partners, was key to meeting sample turn-around time criteria, proper diagnosis, contact tracing, and ultimately containment of the evd outbreak. lessons learnt the challenge assigned to mriglobal by the department of defense was to quickly deploy safe and effective laboratory diagnostic capabilities to sierra leone and guinea to address the evd outbreak. numerous international partners were or became involved in guinea and sierra leone, including but not limited to the united states centers for disease control and prevention, the chinese centre for disease control and prevention, médecins sans frontières international, public health england, world health organization, partners in health, the dutch government,8 and a consortium of nigerian scientists with support from the european union and african union. mriglobal operated the mdls commissioned by department of defense in guinea and sierra leone and tested thousands of samples safely. there were numerous challenges and lessons learned while establishing the mdls in guinea and sierra leone. many of the challenges were resolved by collaborating with the host government and other international partners. the primary goal of the mdls was to provide a biologically safe laboratory to perform timely and quality diagnostics. however, without the dedicated support of engineering and logistics staff, the project would not have achieved a high level of success. recommendations the mriglobal mdls in both conakry and freetown are still in use and will continue to be utilised by both countries, as well as international partners in the future. the diagnostic testing that is being performed in both laboratories has expanded over the past few years to include assays for influenza, severe acute respiratory syndrome coronavirus 2, dengue, chikungunya, zika, and more. using the mdls for their molecular diagnostics is highly recommended until more sustainable solutions can be provided. since their initial deployment, the mdls in sierra leone and guinea have increased both countries’ integrated disease surveillance and response systems, and adherence to international health regulations.6 in both sierra leone and guinea, molecular testing for severe acute respiratory syndrome coronavirus 2 was performed using the capacity provided by the mdl. the mriglobal mdl provides a reproducible, strategic solution for the rapid deployment of molecular diagnostics in resource-limited settings. the strength of the mriglobal mdl is the ability to rapidly build and deploy it to almost anywhere in the world. however, the mriglobal mdl is expensive (other organisations deployed significantly cheaper laboratory operations that were of greater or equal sample testing efficiency and safety) and in resource-limited settings the mdls are extremely challenging to maintain. therefore, the deployment of mdls should be carefully considered, given the cost and context. acknowledgements the authors express their deep gratitude to many partners, including the sierra leone and guinea health authorities, various donors, and local and international organisations whose contributions have helped support efforts to build quality clinical and public health laboratory systems. the authors would like to thank the support staff at mriglobal, and the staff at the us embassy in guinea and sierra leone. the authors deeply appreciate the translation services provided by david tolno and michel haba in conakry. the views expressed in the submitted article are the authors’ own, not an official position of mriglobal or the funding agency responsible, and no official endorsement should be inferred. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions l.d.p., j.c., l.k., a.c., j.c., f.b., j.n., z.s., s. sorie, d.h., a.c., p.t., b.t., f.a.h., j.b., s.a., c.r., j.m., r.h., s.p., s. shearrer, c.a., b.k., p.d., j.a., d.r., p.s., j.n., n.w., m.k., a.m., j.m., m.m. and t.b. contributed to the design and implementation of the research, to the analysis of the results, and to the writing of the manuscript. sources of support funding for the study was received from the united states defense threat reduction agency cooperative biological engagement program contracts hdtra1-08-d-0008 and hdtra1-15-c-0007. data availability data sharing is not applicable to this article, as no new data were created or analysed in this study. disclaimer the views expressed in the submitted article are the authors’ own, not an official position of mriglobal or the funding agency responsible, and no official endorsement should be inferred. references baize s, pannetier d, oestereich l, et al. emergence of zaire ebola virus in guinea. n engl j med. 2014;371:1418–1425. https://doi.org/10.1056/nejmoa1404505 world health organization. who ebola situation report [homepage on the internet]. 2016 [cited 2019 mar 06]. available from: http://apps.who.int/ebola/ebola-situation-reports federal select agent program. select agents and toxins list [homepage on the internet]. 2017 [cited 2019 mar 06]. available from: https://www.selectagents.gov/selectagentsandtoxinslist.html world health organization. report on the status of edpln bsl-3 in select countries in the african region, december 2016 [homepage on the internet]. 2017 [cited 2019 jun 12]. available from: https://reliefweb.int/report/world/report-status-edpln-bsl-3-select-countries-african-region-december-2016 2014 ebola virus emergency use authorizations. ez1 real-time rt-pcr assay (dod) – october 10, 2014 [homepage on the internet]. 2014 [cited 2019 jun 12]. available from: https://www.fda.gov/media/89984/download world health organization. international health regulations 2005 third edition [homepage on the internet]. 2016 [cited 2020 dec 15]. available from: https://www.who.int/publications/i/item/9789241580496 zhang y, yan g, wang c, et al. rapid deployment of a mobile biosafety level-3 laboratory in sierra leone during the 2014 ebola virus epidemic. plos negl trop dis. 2017;11(5):e0005622. https://doi.org/10.1371/journal.pntd.0005622 reusken c, smit p, pas s, et al. ebola virus laboratory response: the three dutch mobile laboratories in liberia and sierra leone. clin microbiol infect dis. 2016;1(4):1–7. https://doi.org/10.15761/cmid.1000s1003 nigeria mobile lab in sierra leone: bringing skills learned in one outbreak to another [homepage on the internet]. 2015 [cited 2020 aug 11]. available from: https://www.afro.who.int/news/nigerian-mobile-lab-sierra-leone-bringing-skills-learned-one-outbreak-another sealy tk, erickson br, taboy ch, et al. laboratory response to ebola – west africa and united states. mmwr suppl. 2016;65(suppl 3):44–49. https://doi.org/10.15585/mmwr.su6503a7 abstract background description of the intervention lessons learnt recommendations acknowledgements references about the author(s) mary kirk i-tech, department of global health, schools of public health and medicine, university of washington, seattle, washington, united states paul h. assoa international training and education center for health (i-tech), abidjan, côte d’ivoire casey iiams-hauser i-tech, department of global health, schools of public health and medicine, university of washington, seattle, washington, united states yves-rolland kouabenan international training and education center for health (i-tech), abidjan, côte d’ivoire jennifer antilla department of global health, university of washington, seattle, washington, united states caleb steele-lane i-tech, department of global health, schools of public health and medicine, university of washington, seattle, washington, united states greg rossum i-tech, department of global health, schools of public health and medicine, university of washington, seattle, washington, united states pascal komena international training and education center for health (i-tech), abidjan, côte d’ivoire patricia sadate ngatchou i-tech, department of global health, schools of public health and medicine, university of washington, seattle, washington, united states nadine abiola international training and education center for health (i-tech), abidjan, côte d’ivoire alain kouakou direction de l’informatique et de l’information sanitaire (diis), ministry of health, abidjan, côte d’ivoire adama pongathie direction de l’informatique et de l’information sanitaire (diis), ministry of health, abidjan, côte d’ivoire jean b. koffi division of global hiv and tuberculosis, centers for disease control and prevention, abidjan, côte d’ivoire christiane adje division of global hiv and tuberculosis, centers for disease control and prevention, abidjan, côte d’ivoire lucy a. perrone i-tech, department of global health, schools of public health and medicine, university of washington, seattle, washington, united states citation kirk m, assoa ph, iiams-hauser c, et al. adaptation of an electronic dashboard to monitor hiv viral load testing in côte d’ivoire. afr j lab med. 2021;10(1), a1284. https://doi.org/10.4102/ajlm.v10i1.1284 lessons from the field adaptation of an electronic dashboard to monitor hiv viral load testing in côte d’ivoire mary kirk, paul h. assoa, casey iiams-hauser, yves-rolland kouabenan, jennifer antilla, caleb steele-lane, greg rossum, pascal komena, patricia sadate ngatchou, nadine abiola, alain kouakou, adama pongathie, jean b. koffi, christiane adje, lucy a. perrone received: 03 june 2020; accepted: 06 jan. 2021; published: 17 may 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the ministère de le santé et de l’hygiène publique in côte d’ivoire and the international community have invested in health information systems in côte d’ivoire since 2009, including electronic laboratory information systems. these systems have been implemented in more than 80 laboratories to date and capture all test results produced from these laboratories, including hiv viral load (vl) testing. in 2018 the national hiv programme in côte d’ivoire requested international support to develop real-time tools such as dashboards to aggregate and display test-specific data such as hiv vl testing to support the country’s programmatic response to hiv. intervention: the vl dashboard was adapted in 2018 using source software code obtained from the kenyan ministry of health and modified for the ivorian context. the dashboard enables users to assess relevant clinical data from all ivoirians living with hiv who undergo vl testing through dashboard data visualisations, including the number of vl tests, kinds of samples tested, and vl levels stratified by demographics and geographic location. lessons learnt: the vl dashboard enables rapid analysis of vl testing data from across the country and enables the national hiv programme, donors and partners to respond rapidly to issues pertaining to access, turn-around times and others. recommendations: adapting existing open-source software is an effective and efficient way to implement transformative tools such as dashboards. the vl dashboard will likely be an essential tool for côte d’ivoire to meet the united nations programme on hiv/aids 90-90-90 targets. keywords: hiv viral load; electronic dashboard; patient monitoring; laboratory information system; côte d’ivoire. background as of 2018, côte d’ivoire had approximately 460 000 people living with hiv1 and an estimated hiv prevalence rate of 2.6%,1 the highest in west africa.2 to address unmet goals for the joint united nations programme on aids/hiv 90-90-90 targets, côte d’ivoire adopted the test and start approach in 2015, a proven cost-effective and lifesaving intervention.3 côte d’ivoire relies on viral load (vl) testing as the gold standard for clinical monitoring of hiv, yet vl testing remains limited in côte d’ivoire despite 17 laboratories having molecular testing capability. côte d’ivoire is working to scale up access to vl testing and optimise how test results are utilised for effective anti-retroviral therapy monitoring in pateints.4 the national hiv programme and the healthcare workforce in côte d’ivoire have been advocating with the international donor community for effective, user-friendly health information systems and monitoring tools.5 in response to this request, the united states centers for disease control and prevention engaged the international training and education center for health (i-tech) at the university of washington in 2009 to support the ministère de le santé et de l’hygiene publique and the national hiv programme (programme nationale de lutte contre le sida [pnls]) to improve the quality of care of people living with hiv by strengthening electronic laboratory information systems as part of the national health information system architecture. openelis (electronic laboratory information systems) (oe) is an open-source electronic laboratory information system that tracks laboratory processes and outputs and was created in the united states for public health laboratories as a software and business process framework.6 starting in 2009, oe was adapted for côte d’ivoire with i-tech’s support and 82 laboratories to date are implementing the system across the nation. although implementation of oe continues to expand and efforts are underway to interlink individual operating sites and their data through a centrally located consolidated server, obtaining aggregated hiv-related testing data from all sites remains challenging. in 2018 the ministère de le santé et de l’hygiene publique requested support to develop and deploy a dashboard solution for hiv vl testing. dashboards are increasingly used in clinic-based interventions7 as well as in population-based disease assessments.8 these dashboards can empower stakeholders to make decisions with greater efficiency over time.9 one primary function of dashboards is displaying data so that stakeholders can analyse key metrics to monitor progress to targets, identify challenges and mobilise to improve service quality.10 dashboards have been used for reporting purposes in hiv programme monitoring efforts. in uganda, option b+ usage has been documented since 2013 via a nationwide weekly reporting system using short message service technology.11 kenya also uses a dashboard to support the national review of vl and infant virologic test programme data through the integration of open-source laboratory information systems with cloud-based servers.12 namibia developed a pharmaceutical management information dashboard that interlinks four pharmaceutical information tools to serve as a platform for analysis and dissemination to improve anti-retroviral therapy delivery.13 here we describe the development of the vl dashboard for côte d’ivoire which is connected to the oe system and aggregates site-level oe data that can be viewed on a publicly available website. these visualisations permit the rapid examination of hiv-related testing data in côte d’ivoire, including spatial and temporal attributes of vl testing coverage, and support timely programme policymaking. description of the intervention ethical considerations ethical clearance for this project was given by the university of washington, united states centers for disease control and prevention and ivorian institutional review board. all de-identified data are publicly available on the dashboard website. dashboard development in october 2017, i-tech coordinated with the united states agency for international development, clinton health access initiative, and the kenyan ministry of health to access the software source code for the kenyan vl dashboard (https://viralload.nascop.org/).14 using this software source code as a starting point, the ivorian vl dashboard was developed to interface with the oe system in côte d’ivoire and designed to extract and aggregate vl test-specific data. populating the dashboard with data initially occurred through the manual extraction of a comma-separated value file from each oe system at each laboratory. trained staff then exported this file to data quality analysts through remote connection access or email. a data validation check was then conducted to ensure that data were not missing or inaccurate, otherwise it was sent back to the laboratories for correction in the oe system. once individual laboratory data were validated and complete, these cleaned data were then manually uploaded to the vl dashboard (note that a direct link from the oe systems to the vl dashboard through a national consolidated server is in its pilot phase as of september 2020, and this automation will remove the need for manual data management processes). the visualisations that populate the vl dashboard were adapted from the kenyan dashboard to the ivorian context, including geography, language, and data sources. dashboard features the vl dashboard was designed to display a range of data visualisations that can be sorted by month, year and region (figure 1). the primary source data for ‘trends by testing sample’ (test sample type by month) can be exported to microsoft excel (microsoft corp. redmond, washington, united states); the remaining data visualisations are available as graphic downloads. test sample type (dried blood spot or ethylenediaminetetraacetic acid plasma), demographic data (age, sex, and region), test results (≥/< 1000 copies, invalid, or undetectable), and clinical justification for the vl test can be visualised on the vl dashboard. the turn-around times of the clinical vl samples can also be visualised, with the graphic displaying times from collection of the sample to reception at a laboratory, reception to processing, and processing to sample validation. data submitted by each laboratory are available by regionally filtering data, with test numbers by results shown on the ‘region sites outcomes’ graph on the dashboard. a ‘visitor counter’ tracking feature was added to the dashboard in january 2020, showing the total number of visits, number of visits to a specific page of the dashboard, and the number of people online. figure 1: number of viral load tests in côte d’ivoire by region, october – december 2019. the 20 regions of the country are covered by 17 public laboratories that are capable of molecular testing for hiv viral load. the capital abidjan is divided into two jurisdictions based on geospatial parameters with the most people living with hiv located in abidjan 2. this graph was produced directly from the viral load dashboard. lessons learnt the vl dashboard was completed in june 2018 (figure 2). in july 2018, initial pilot data from the dashboard were presented to ministère de le santé et de l’hygiene publique and donor stakeholders at a technical working group meeting for final dashboard approval. the website became publicly available in july 2018 (https://chargevirale.openelisci.org/vl_dashboard/), with an initial 10 vl testing laboratories successfully transmitting data to the dashboard via oe. the remaining seven laboratories started data sharing from january 2019, and retrospective data from october 2016–2018 were obtained, cleaned, and uploaded into the dashboard. the vl dashboard successfully reported data for all people living with hiv who received vl testing services in côte d’ivoire, with each unique test event recorded in the dashboard. data from october 2016 to december 2019 indicated that a total of 622 500 samples were tested for vl, of which 452 896 (72.8%) samples were from women and 169 604 (27.2%) from men. regional disaggregation of the data accounts for 616 812 vl tests, with the remaining 5688 tests having lost location traceability and are currently under investigation. of the 616 812 traceable vl tests, the two abidjan catchment areas accounted for 40.6% (n = 252 510) of the tests in the dashboard. of the vl tests reported in the dashboard, 89.6% were from patients aged 25 years or older. since the dashboard became active, the sample processing rate has increased, reflecting increased vl testing access and scale-up and increased test demand after national prioritisation in 2016 (figure 3). data from october 2016 to december 2019 indicate that 56.9% (354 184 tests) of people living with hiv receiving anti-retroviral therapy have viral suppression. people living with hiv aged 25 years or older had the highest suppression rate (79%), and paediatric and adolescent patients (aged 0–19 years) had the lowest rates. paediatric (age < 10 years) viral suppression rates ranged from 47.6% to 56.9%. adolescent (age 10–19 years) suppression rates ranged from 53.9% to 55.2%. although the dashboard indicated that the national average turn-around time for ethylenediaminetetraacetic acid plasma samples decreased from more than 50 days to 16 days (figure 4), this does not yet meet the national target of under 14 days. viral load results obtained from tests performed on dried blood spots were introduced in 2018. figure 2: timeline of viral load dashboard development. the development of the viral load dashboard began in october 2017 and a pilot version was completed in july 2018. following adjustments to the software code the dashboard was finalised in january 2019 and is now populating data from all viral load testing laboratories. figure 3: number of viral load tests reported in côte d’ivoire, october 2016 – december 2019. the viral load dashboard captured all viral load testing data since its beginning in côte d’ivoire, october 2016. as the country committed to scaling up viral load testing access to people living with hiv, the number of total tests increased. the data represented above show this trend, which is expected to continue. figure 4: viral load testing turn-around time by sample type and by year that each type of viral load test was used. (a) ethylenediaminetetraacetic acid plasma 2016–2019; (b) dried blood spots 2018–2019, in côte d’ivoire. turn-around time is further disaggregated by collection to reception (c-r), reception to processing (r-p), and processing to validation (p-v). the adaptation, development and launch of the vl dashboard in côte d’ivoire is a collaborative success story and a milestone achievement for the national hiv programme and for international donors such as the united states president’s emergency plan for aids relief. the information available through the dashboard will help accelerate decision-making and programmatic response time,11 especially the ability to monitor key subpopulations. for example, paediatric and adolescent viral suppression rates are observably lower (53.4%) than adults aged 25 years or older (79.8%), and the dashboard is being used to monitor this population subset and track their clinical outcomes. it is noted that these vl suppression rates by age groups mirror trends among paediatric, adolescent, and adult patients in other resource-limited countries.15 this kind of information is critical to inform programmes and initiatives like the determined, resilient, empowered, aids-free, mentored, and safe, which serves adolescent girls and young women. in another example of the utility of the dashboard, although only 23.9% of all people living with hiv in côte d’ivoire1 live in the capital region of abidjan, dashboard data from october 2016 through december 2019 show that a large proportion of vl samples come from this region (n = 252 510 tests, 40.6%; figure 2). this finding suggests that more people from abidjan are receiving vl tests compared with other provinces and these data are now informing programmatic prioritisation. organisations in côte d’ivoire are now rapidly utilising vl dashboard information in programmatic and technical working group meetings. the pnls uses the data and visualisations from the dashboard in monthly reviews with implementing partners and other ministerial departments. the data from the vl dashboard is trusted in the pnls’s decision-making processes. one of the key success factors in the development of the dashboard was the collaborative nature of the work in each step of the process: from planning to design and implementation. the willingness of the developers of the kenyan dashboard to share the source code immensely benefited côte d’ivoire, as it shortened product launch time, reduced potential costs and prevented duplication of efforts to implement a dashboard for ivorian vl testing services. in the adaptation phase, i-tech worked closely with the direction de l’informatique et l’information sanitaire, the pnls, and the côte d’ivoire national hiv reference laboratory in monthly technical working group meetings. the technical working group first reviewed the data available in oe and mapped the data to the indicator calculations. after the vl dashboard was launched, these high-functioning technical working groups provided a forum to address challenges and improve the dashboard and included representatives from the direction de l’informatique et l’information sanitaire, pnls, laboratoire national de la santé public, and implementing partners. this ensured that all stakeholders were involved in the process. the vl dashboard has been a helpful tool in monitoring hiv trends towards meeting the ‘third 90’ united nations programme on hiv/aids goal and additional developments are underway to enhance features and improve clinical utilisation. the vl dashboard is also a model for the new early infant diagnosis dashboard launched in may 2020.16 completion of the consolidated national server, which is in its pilot phase since september 2020, will allow for rapid data importation from oe to the vl dashboard. plans are also underway to transfer ownership of the dashboard’s internet protocol address, currently owned by the university of washington, to the ministère de le santé et de l’hygiene publique-direction de l’informatique et l’information sanitaire. recommendations dashboards are helpful tools for public health programmes. the vl dashboard in côte d’ivoire is poised to be a transformational tool that improves the national response to hiv by displaying key data including the number of vl tests, kinds of samples tested, and vl levels stratified by demographics and geographic location. this dashboard enables rapid analysis of vl testing data from testing points across the country and will enable the pnls and other implementing partners to respond rapidly to vl testing access, delayed turn-around time, and data quality issues. the vl dashboard will help côte d’ivoire scale up vl monitoring to help meet the united nations programme on hiv/aids 90-90-90 targets. acknowledgements we thank colleagues at the united states centers for disease control and prevention for their support in developing the dashboard, colleagues in the ministère de le santé et de l’hygiene publique, clinical partner implementers, and all users of oe and the vl dashboard for providing valuable feedback for improving these tools. we thank clinton health access initiative/united states agency for international development kenya for sharing the vl dashboard software code. we thank eric zakpa, armande kouame, marisa van osdale, rama ouattara, and cat koehn for their programmatic and operational support. this project was determined to be non-research by the university of washington institutional review board, and the project was approved by the national institutional review board committee in côte d’ivoire, le comite’ national d’ethique des sciences de la vie et de la sante’. this project was also reviewed in accordance with the centers for disease control and prevention human research protection procedures and was determined to be a non-research programme evaluation. competing interests the authors have declared that no competing interests exist. authors’ contributions p.h.a., c.i.-h., c.s.-l., g.r., j.a., p.k., p.s.n., a.k., and a.p. conceived and designed the work. y.-r.k., m.k., n.a., and l.a.p. acquired, analysed, and interpreted data. p.h.a., c.i.-h., m.k., and l.a.p. wrote the original draft. j.b.k. and c.a. provided project funding and technical direction. y.r.k., m.k., n.a., g.r., and l.a.p. reviewed, revised, and critically edited the draft. all authors provided final approval of the version to be published and agree to be accountable for all aspects of the work. sources of support this work has been supported by the president’s emergency plan for aids relief through the united states centers for disease control and prevention under the terms of the cooperative agreement awarded to the university of washington-i-tech (6 nu2ggh001968-03-02). data availability the data that support the findings of this study are publicly available at: https://chargevirale.openelisci.org/vl_dashboard/. disclaimer the findings and conclusion in this report are those of the authors and do not necessarily represent the official position of the funding agencies. references unaids. aidsinfo: côte d’ivoire data sheet [homepage on the internet]. 2018 [cited 2020 jan 20]. available from: https://aidsinfo.unaids.org/ gbd 2015 collaborators. estimates of global, regional and national incidence, prevalence, and mortality of hiv, 1980–2015: the global burden of disease study 2015. lancet. 2016;3:e361–e387. estill j, egger m, blaser n, et al. cost-effectiveness of point-of-care viral load monitoring of antiretroviral therapy in resource-limited settings: mathematical modelling study. aids. 2013;27(9):1483–1492. https://doi.org/10.1097/qad.0b013e328360a4e5 maheu-giroux m, vesga j, diabaté s, et al. population-level impact of an accelerated hiv response plan to reach the unaids 90-90-90 target in côte d’ivoire: insights from mathematical modeling. plos med. 2017;14(6):e1002321. https://doi.org/10.1371/journal.pmed.1002321 rowan bh, robinson j, granato a, et al. workforce patterns in the prevention of mother to child transmission of hiv in côte d’ivoire: a qualitative model. hum resour health. 2018:16(4). https://doi.org/10.1186/s12960-018-0268-x university of washington. openelis global: a global lis. viewed 29 january 2021, from https://sites.google.com/site/openelisglobal/ enane la, vreeman rc, foster c. retention and adherence: global challenges for the long-term care of adolescents and young adults living with hiv. curr opin hiv aids. 2018;13(3):212–219. https://doi.org/10.1097/coh.0000000000000459 crofts j, moyo j, ndebele w, et al. adaption and implementation of local maternity dashboards in a zimbabwean hospital to drive clinical improvement. bull world health organ. 2014;92(2):146–152. https://doi.org/10.2471/blt.13.124347 cheng ck, ip dk, cowling bj, ho lm, leung gm, lau eh. digital dashboard design using multiple data streams for disease surveillance with influenza surveillance as an example. j med internet res. 2011;13(4):e85. https://doi.org/10.2196/jmir.1658 world health organization. considerations for developing a monitoring and evaluation framework for viral load testing. 2019; who/cds/hiv/19.5. radin ak, abutu aa, okwero ma, et al. confronting challenges in monitoring and evaluation: innovation in the context of the global plan towards the elimination of new hiv infections among children by 2015 and keeping their mothers alive. j acquir immune defic syndr. 2017;75(suppl 1):s66–s75. https://doi.org/10.1097/qai.0000000000001313 vrazo ac, sullivan d, ryan phelps b. eliminating mother-to-child transmission of hiv by 2030: 5 strategies to ensure continued progress. glob health sci pract. 2018;6(2):249–256. https://doi.org/10.9745/ghsp-d-17-00097 mabirizi d, phulu b, churfo w. implementing an integrated pharmaceutical management information system for antiretrovirals and other medicines: lessons from namibia. glob health sci pract. 2018;6(4):723–735. https://doi.org/10.9745/ghsp-d-18-00157 national aids/std control program (nascop), ministry of health government of kenya. viral load dashboard. viewed 29 january 2021, from https://viralload.nascop.org/ gous nm, onyebujoh pc, abimiku a, et al. the role of connected diagnostics in strengthening regional, national and continental african disease surveillance. afr j lab med. 2018;7(2):a775. https://doi.org/10.4102/ajlm.v7i2 ministère de le santé et de l’hygiène publique (mshp), government of côte d’ivoire. eid dashboard. 2020. viewed 29 january 2021, from https://chargevirale.openelisci.org/eid_dashboard/en/ article information authors: linda m. parsons1 akos somoskovi2 evan lee2 chinnambedu n. paramasivan2 miriam schneidman3 deborah birx1 giorgio roscigno2 john nkengasong1 affiliations: 1global aids program, us centers for disease control and prevention, atlanta, usa2foundation for innovative new diagnostics, geneva, switzerland 3the world bank, washington, dc, usa correspondence to: john nkengasong postal address: 1600 clifton road, atlanta ga 30333, usa dates: received: 15 apr. 2011 accepted: 19 jan. 2012 published: 11 june 2012 how to cite this article: parsons lm, somoskovi a, lee e, et al. global health: integrating national laboratory health systems and services in resource-limited settings. afr j lab med. 2012;1(1), art. #11, 5 pages. http://dx.doi.org/10.4102/ ajlm.v1i1.11 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. global health: integrating national laboratory health systems and services in resource-limited settings in this original research... open access • abstract • introduction • what does integration of laboratory systems and services mean? • advantages of integrated laboratory systems • disease-specific programmes impede laboratory services integration • strategies for integrating laboratory systems and services    • policy and strategic planning    • standardising testing and equipment and coordinating with partners    • joint coordination with clinicians    • implementation • practical examples of integrated laboratory capacity in the field    • integration of tb laboratory capacity in hiv laboratories    • integration of hiv testing in a tb laboratory    • avian influenza laboratory support in hiv laboratories • human resources training • recommendations and conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ laboratory systems worldwide are challenged not only by the need to compete for scarce resources with other sections of national health care programmes, but also with the lack of understanding of the critical role that laboratories play in the accurate diagnosis and monitoring of patients suffering from high-burdens of disease. an effective approach to establishing cost-effective laboratory systems that provide rapid and accurate test results for optimal impact on patient care is to move away from disease-specific programmes and establish integrated laboratory services. an integrated laboratory network provides all primary diagnostic services needed for care and treatment without requiring patients to go to different laboratory facilities for specific tests. such a network focuses on providing quality-assured basic laboratory testing through the use of common specimen collection, reporting and diagnostic platforms that can be used across diseases. an integrated laboratory system also provides specimen transport to specialised laboratories and an environment conducive to the introduction and use of new and more complex technologies that would benefit the patient population and public health systems as a whole. as such, this article described various strategies for, and practical examples of, the successful integration of laboratory services. introduction top ↑ human immunodeficiency virus (hiv) and/or aids, tuberculosis (tb) and malaria remain major threats that undermine the health of global populations and account for approximately five million deaths every year.1 a lack of adequate laboratory capacity in resource-poor, high-burden countries presents a significant barrier in providing appropriate diagnosis, care and treatment to patients infected with these and other emerging pathogens.1, 2, 3 modernisation and quality improvement of laboratory services would greatly improve the control of these diseases and support other initiatives.4 decades of underinvestment in national laboratory programmes have resulted in deficiencies in the infrastructure, equipment and human resources that are necessary for effective, quality-assured laboratory services.5 increases in international funding have been unprecedented in recent years,6,7 but programmes for major diseases are still organised vertically as silos, including the establishment of similar testing technologies for different diseases. this has led to wasted resources because of duplications of equipment and training and inefficient use of human capacity. during this time of increased awareness and programmatic scale-up, it is imperative to establish integrated national laboratory systems to optimise quality, efficient and cost-effective testing. what does integration of laboratory systems and services mean? top ↑ the demand for laboratory services to meet the diagnostic and treatment needs for hiv has helped drive investment in new and renovated laboratories to provide comprehensive laboratory diagnostic services for monitoring patients on anti-retroviral therapy with cd4, chemistry, haematology, molecular tests such as measuring plasma ribonucleic acid (rna) levels, testing of hiv-1 drug resistance mutations and testing for opportunistic infections. however, this expansion and investment in laboratory capacity should be optimised to serve the needs of other diseases of public health importance, provide general diagnostic services to support clinical care and support other public health priorities such as re-emerging infectious diseases and chronic diseases. an integrated laboratory network can be defined as one that has the ability to provide all primary diagnostic services needed for the care and treatment of patients without requiring them to go to different laboratories for specific tests. to meet these requirements the network should, (1) be focused on providing quality-assured basic laboratory testing, (2) use common specimen collection, timely reporting and diagnostic platforms that can be used across diseases within the same facility and (3) increase capacity for introducing and using new and more complex technologies. advantages of integrated laboratory systems top ↑ integrated systems have several advantages. firstly, and most importantly, they can support timely and comprehensive care and treatment without the costs and delays associated with referral to specialised facilities for particular diagnostic exams. an integrated system can benefit patients and clinical staff at hospitals located in urban centres, as well as patients served at the community level in remote areas. secondly, new diagnostic platforms, such as microscopes with dual light and fluorescent capacity and molecular diagnostic equipment, have the potential to perform assays for more than one disease. an integrated laboratory approach will ensure that these tools can be used optimally by cross-trained technical staff. thirdly, integrating diagnostic services for different diseases within the same facility helps avoid duplication of investments in infrastructure and laboratory supporting systems, such as specimen transport, supply chain management and information systems. lastly, an integrated approach to training can help ensure standardisation of core laboratory issues, such as quality assurance and standard operating procedures, as well as ensure the more efficient delivery of training. the examples below show how these approaches are both practical and successful. disease-specific programmes impede laboratory services integration top ↑ laboratory systems in resource-poor countries are challenged by, (1) dilapidated infrastructures, (2) lack of funding for developing and implementing national policies, strategic planning, and quality management systems, (3) unlinked referral and reporting services and (4) inadequate human resources, including lack of organised in-service training and long-term career pathways. the difficulty of dealing with all these challenges separately by each programme should provide sufficient reason to encourage integration of services. however, overcoming the traditional vertical approach in which higher level public health laboratories have been established to provide support for disease-specific ministerial directorates and programmes has proven difficult. furthermore, an unintentional negative effect of the disease-specific approach to laboratory strengthening has been the neglect of the core public health laboratory functions. most often, integrated laboratory services are usually found only at the district, sub-district and primary health centre levels. however, establishment of parallel laboratories within a national health care system is neither cost-effective nor conducive to efficient and coordinated patient care. strategies for integrating laboratory systems and services top ↑ integration of laboratory systems and services at different levels of the health system will ensure optimisation of the investments in laboratory-system strengthening by ministries of health, as well as those of international donors, such as the global fund to fight aids, tb, and malaria, the world bank, the us president’s emergency plan for aids relief (pepfar), and others. in the past, donor funding mechanisms have contributed to the fragmentation of laboratory capacities because they usually have been earmarked for disease specific efforts; although, recently, there is a trend towards greater flexibility. various strategies for integration are outlined in the subsections below. policy and strategic planning national laboratory systems (ideally composed of a tiered network of diagnostic laboratories and a national public health reference laboratory) must be capable of providing accurate, timely and cost-effective testing that is in line with each country’s programmatic goals and available clinical interventions. it is necessary to define at which level of a national laboratory network certain services or diagnostic platforms should be performed. these decisions should be based on testing complexity, throughput, specimen referral requirements and the needs of the public health programme and patient population being served. laboratories are complex systems that include components such as personnel, equipment, supplies and infrastructure, as well as support systems for information management, purchasing and inventory, and systems for evaluation and continuous improvement, such as internal and external quality assessment and occurrence management. developing and implementing a national laboratory policy and strategic plan that ensures integrated capacity at each level of the network can enable countries to work with partners and donors in defining specific objectives, setting standards and allocating appropriate funding and personnel for sustainable laboratory services.8 standardising testing and equipment and coordinating with partners laboratory infrastructure, test menus, technology, platforms and commodities should be standardised in each country to avoid duplication, as agreed upon by programme and laboratory representatives from 13 resource-poor countries in the maputo declaration of 2008.9 this approach requires strong leadership and coordination by the local ministries, along with partner and donor compliance, and includes many benefits, such as reduced procurement costs for commodities, easier implementation of quality assurance programmes and integration of multi-focused testing that uses shared equipment. for example, partners could work together in developing specialised facilities that would accommodate instruments for molecular diagnostics of tb, hiv and other diseases, rather than building separate labs dedicated to only one disease. training, equipment maintenance and quality management systems could be standardised to allow technicians to work using similar techniques across diseases. joint coordination amongst partners at the onset of a laboratory-related project could enable each partner to play a preferred role in areas where they have a relative advantage or expertise. joint coordination with clinicians strengthening of national laboratory systems depends on close partnerships – beyond the laboratory facility itself – with technical and clinical professionals, healthcare managers at the community, regional and national levels, and public health programmes. clinicians should support, facilitate and demand high-quality and responsive laboratory support for appropriate patient care. clinicians should also be involved in ensuring that testing algorithms are cost-effective, are based on sound evidence and have a relevant impact on clinical decision-making and patient-important outcomes.10 implementation there are two basic aspects to implementing integrated services within laboratories. the first is focused on provision of adequate services for patients presenting with particular clinical symptoms indicative of major infectious diseases. for example, an integrated laboratory should have the capacity to adequately monitor hiv-positive individuals for tb, malaria, or other opportunistic infections, or to provide rapid molecular testing for multi-drugresistant tb (mdr tb) in hiv and tb co-infected patients in order to improve infection control and treatment outcomes (figure 1). conversely, this laboratory should also be able to screen specimens from tb and malaria patients for hiv and provide other routine monitoring (clinical chemistry and haematology before and during treatment for hiv, tb, and malaria) when required. the second aspect focuses on the establishment of integrated diagnostic platforms or instruments that can be used for a variety of tests. the simplest example can be an integrated system for collecting and transporting specimens for tb and hiv testing or monitoring. a further step can be the integration of microscopy testing for tb, malaria and other parasitic pathogens. the most promising approach for cross-cutting disease testing is offered by the molecular platform. polymerase chain reaction (pcr) molecular testing can be used to detect rapidly and identify a wide range of viral and bacterial pathogens, including those that cause endemic diseases such as hiv, tb and malaria, and emerging infectious diseases such as new strains of influenza. practical examples of integrated laboratory capacity in the field top ↑ integration of tb laboratory capacity in hiv laboratories in 2006, a state of the art bio-safety level (bsl)-2 central laboratory was established at the ethiopian health and nutrition research institute (ehnri) in addis ababa through pepfar funding to support the laboratory diagnosis of hiv patients. since then, technical support has been provided by international partners to help establish testing and training capacity for hiv serology and external quality assessment (eqa), hiv incidence, cd4 counts, biochemistry, haematology and molecular testing for viral load and early infant diagnosis (eid). beginning in 2008, support was provided through the foundation for innovative new diagnostics (find) to create two bsl-3 tb testing laboratories in addis ababa: the first at the hiv laboratory at ehnri, to host the national reference laboratory for tuberculosis (nrlt), and the second at st. peter’s hospital, a central hospital responsible for the care of tb and hiv co-infected patients experiencing tb treatment failure or relapse. the testing capacity of the two laboratories now includes liquid and solid growth detection and drug susceptibility testing for tb, lateral-flow immuno-assay for identification of tb and rapid detection of mdr tb by the molecular line probe assay (lpa). the newly renovated molecular unit at st. peter’s will also be used to restart hiv viral load testing at the hospital, a function that had been stopped in the past because of problems related to inadequate infrastructure for molecular testing. in addition, molecular lpa testing for mdr tb will soon be introduced in four ethiopian regional laboratories that are currently performing hiv dna pcr for eid. in nigeria, a multifaceted, integrated, tiered laboratory programme has been established by the institute of human virology at the university of maryland to support a pepfar-funded scale-up of aids care and treatment in 26 states.11 services provided by the laboratory network include hiv rapid tests, adult cd4 counts, paediatric cd4 percentages, haematology, blood chemistries, syphilis serology, cryptococcal antigen test, tb smear microscopy, culture, drug susceptibility testing (dst), and molecular lpa. use of appropriate technology at all service levels and a robust quality assurance programme provide high quality integrated laboratory services. integration of hiv testing in a tb laboratory the national tb reference laboratory (ntrl) located at the queen elizabeth ii hospital in maseru, lesotho, was renovated by find and other partners to provide capacity for tb culture and molecular diagnostics. following strengthening of tb smear microscopy testing through training and the establishment of a quality assurance programme, the ntrl was first renovated to create a bsl-3 facility to implement tb culture, dst and rapid immunoassay-based identification of tb, with eqa provided from south africa.12 then tb lpa and hiv dna pcr were implemented in an adjacent, newly constructed clean-room facility, resulting in the establishment of integrated tb and hiv testing in a single facility. by using an integrated approach, implementation of novel molecular tb diagnostics paved the way to introduce hiv molecular testing capacity in the country. avian influenza laboratory support in hiv laboratories molecular hiv laboratories in africa have been called on to respond rapidly to laboratory surveillance for outbreaks of avian influenza. during the 2005 outbreak of avian influenza, the pepfar-supported global aids program laboratory in entebbe, uganda, provided training for about 60 laboratory experts on the use of pcr diagnosis for avian influenza. also in 2006, the pepfar-supported laboratory at asokoro hospital in abuja, nigeria, was instrumental in providing diagnostic support for an investigation of an avian influenza outbreak in nigeria.13 planning is ongoing for the provision of support for localised outbreaks of h1n1 (swine) influenza. figure 1: the framework of integrated laboratory services that addresses levels of a tiered laboratory network in developing countries. human resources training top ↑ the african centre for integrated laboratory training (acilt) was established in 2008 to develop and offer hands-on training courses for front-line laboratory staff. acilt’s vision is to provide for a healthier africa through quality laboratory practices to combat major infectious diseases. hosted by the south african national institute for communicable diseases and the national health laboratory service, acilt has a governance board consisting of experts from collaborating international institutions.before establishing acilt, needs assessments were performed in 10 resource-poor african countries to determine which topics would be most important for building capacity amongst laboratory personnel. courses were then developed collaboratively by acilt and international partners including the us centers for disease control and prevention (cdc), find and the world health organization to ensure standardisation. as of early august 2011, 720 participants from 29 countries had participated in more than 20 hands-on training courses covering: hiv dna pcr for eid, hiv incidence assay, tb culture and identification, eqa for hiv rapid testing, national laboratory strategic planning, biosafety, laboratory management, and laboratory accreditation. acilt satellite courses are offered at sites outside of south africa. a two-week course on tb culture and drug susceptibility testing was recently offered to south-east asian participants in bangkok and one-week courses on the molecular lpa testing for mdr tb have been hosted by the new laboratory facility at ehnri, at the national tb reference laboratory of lesotho, and at the institut pasteur in abidjan, côte d’ivoire. acilt tb culture and molecular lpa course materials have also been translated into french and portuguese through a partnership with the american society for microbiology. recommendations and conclusion top ↑ an integrated network of laboratories is indispensable for providing national support for global health initiatives for hiv, tb, malaria and other public health priorities. however, an integrated national laboratory network should be able to provide all needed primary diagnostic services for patients at each level of service without requiring patients to go to different facilities for specific tests. such laboratory services might be delivered either directly at defined levels of the network or indirectly by using an integrated specimen collection, referral and reporting system. the extent of integration at different levels of the laboratory system should always be based on the complexity, throughput and specimen referral requirements of the particular diagnostic platforms. last but not least, an integrated system allows increased capacity and preparedness for implementation of new technologies to improve the quality of current diagnostics and to address newly emerging public health concerns. this article has presented examples to demonstrate how some resource-limited countries have benefited from an integrated approach for clinical laboratory strengthening. these strategies could be replicated more broadly. acknowledgements top ↑ use of trade names is for identification only and does not constitute endorsement by the us department of health and human services, the public health service, or the cdc. financial support has been received from the global aids program of the national center for hiv, sexually transmitted diseases (std), and tb prevention at the cdc. the findings and conclusions in this article are those of the authors and do not necessarily represent the views of the funding agency. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions j.n. (us centers for disease control and prevention) was the project leader. j.n. l.m.p. (us centers for disease control and prevention) a.s., (foundation for innovative new diagnostics) e.l. (foundation for innovative new diagnostics) c.n.p., (foundation for innovative new diagnostics) m.s., (the world bank) d.b.(us centers for disease control and prevention) and g.r. foundation for innovative new diagnostics) were responsible for experimental design and wrote the manuscript. references top ↑ 1. vittoria m. granich r. gilks cf, et al. the global fight against hiv/aids, tuberculosis and malaria. am j clin pathol. 2009;131:844–848. http://dx.doi.org/10.1309/ajcp5xhdb1pnaeyt, pmid:19461091 2. birx d, desouza m, nkengasong jn. laboratory challenges in the scaling up of hiv, tb and malaria programs. am j clin pathol. 2009;131:849–851. http://dx.doi.org/10.1309/ajcpgh89qdswfons, pmid:19461092 3. petti ca, polage cr, quinn tc, ronald ar, sande ma. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42:377–382. http://dx.doi.org/10.1086/499363, pmid:16392084 4. frieden tm, teklehaimanot a, chideya s, farmer p, kim jy, raviglione mc. a roadmap to control malaria, tuberculosis and human immunodeficiency virus/aids. arch intern med. 2009;169:1650–1652. http://dx.doi.org/10.1001/archinternmed.2009.309, pmid:19822819 5. marchal b, cavalli a, kegels g. global health actors claim to support health system strengthening – is this reality or rhetoric? plos med. 2009;6:1–5. 6. ravishankar n, gubbins p, cooley rj, et al. financing of global health: tracking development assistance for health from 1990 to 2007. lancet. 2009;373:2113–2124. http://dx.doi.org/10.1016/s0140-6736(09)60881-3 7. yu d, souteyrand y, banda ma, kaufman j, perriëns jh. investment in hiv/aids programs: does it help strengthen health systems in developing countries? global health. 2008;4:8. http://dx.doi.org/10.1186/1744-8603-4-8, pmid:18796148, pmcid:2556650 8. nkengasong jn, mesele t, orloff s, et al. critical role of developing national strategic plans as a guide to strengthen laboratory health systems in resource-poor settings. am j clin pathol. 2009;131:852–857. http://dx.doi.org/10.1309/ajcpc51blobbpakc, pmid:19461093 9. world health organization. the maputo declaration on strengthening of laboratory systems [document on the internet]. c.2008 [cited 2009 nov 16]. maputo: world health organization; 2008. available from: http://www.who.int/diagnostics_laboratory/maputo-declaration_2008.pdf 10. pai m, ramsay a, o’brian r. comprehensive new resource for evidence-based tb diagnosis. expert rev mol diagn. 2009;9:637–639. http://dx.doi.org/10.1586/erm.09.48, pmid:19817547 11. abimiku ag. building laboratory infrastructure to support scale-up of hiv/aids treatment, care and prevention. am j clin pathol. 2009;131:875–886. http://dx.doi.org/10.1309/ajcpelmg6gx6rqsm, pmid:19461097 12. paramasivan cn, lee e, kao k, et al. experience establishing tuberculosis laboratory capacity in a developing country setting. int j tuberc lung dis. 2009;14:59–54. 13. ortiz jr, katz ma, mahmoud mn, et al. lack of evidence of avian-to-human transmission of avian influenza a (h5n1) virus among poultry workers, kano, nigeria, 2006. j infect dis. 2007;196:1685–1691. http://dx.doi.org/10.1086/522158, pmid:18008254 abstract background description of the intervention lessons learnt recommendations acknowledgements references about the author(s) abdourahamane yacouba faculté des sciences de la santé, université abdou moumouni, niamey, nigerlaboratory team covid-19, niamey, niger adamou lagaré laboratory team covid-19, niamey, nigercentre de recherche médicale et sanitaire, niamey, niger daouada alhousseini maiga faculté des sciences de la santé, université abdou moumouni, niamey, nigerlaboratory team covid-19, niamey, niger halimatou moumouni sambo laboratory team covid-19, niamey, niger direction des laboratoires de santé, ministère de la santé publique, niamey, niger sani ousmane laboratory team covid-19, niamey, niger centre de recherche médicale et sanitaire, niamey, niger zelika hamidou harouna laboratory team covid-19, niamey, niger hôpital national amirou boubacar diallo, niamey, niger boubacar marou faculté des sciences de la santé, université abdou moumouni, niamey, niger maman k. sanoussi laboratory team covid-19, niamey, niger balki aoula laboratory team covid-19, niamey, niger ali amadou laboratory team covid-19, niamey, niger hôpital de l’amitié niger-turquie, niamey, niger hassane boureima laboratory team covid-19, niamey, niger hôpital général de référence de maradi, maradi, niger saidou amatagas laboratory team covid-19, niamey, niger hôpital national de zinder, zinder, niger abdoulaye ousmane faculté des sciences de la santé, université dan dicko dankoulodo de maradi, maradi, niger eric adehossi faculté des sciences de la santé, université abdou moumouni, niamey, niger covid-19 experts group, niamey, niger saidou mamadou faculté des sciences de la santé, université abdou moumouni, niamey, niger covid-19 experts group, niamey, niger citation yacouba a, lagaré a, alhousseini maiga d, et al. laboratory organisation and management of sars-cov-2 infection in niger, west africa. afr j lab med. 2020;9(1), a1308. https://doi.org/10.4102/ajlm.v9i1.1308 lessons from the field laboratory organisation and management of sars-cov-2 infection in niger, west africa abdourahamane yacouba, adamou lagaré, daouada alhousseini maiga, halimatou moumouni sambo, sani ousmane, zelika hamidou harouna, boubacar marou, maman k. sanoussi, balki aoula, ali amadou, hassane boureima, saidou amatagas, abdoulaye ousmane, eric adehossi, saidou mamadou received: 20 june 2020; accepted: 01 oct. 2020; published: 21 dec. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: as the coronavirus disease 2019 (covid-19) pandemic unfolds, laboratory services have been identified as key to its containment. this article outlines the laboratory organisation and management and control interventions in niger. intervention: the capitol city of niger, niamey, adopted a ‘national covid-19 emergency preparedness and response plan’ to strengthen the preparedness of the country for the detection of severe acute respiratory syndrome coronavirus-2. laboratory training and diagnostic capacity building were supported by existing active clinical and research laboratories for more rapid and practicable responses. the national reference laboratory for respiratory viruses located at the centre de recherche médicale et sanitaire was designated as the reference centre for covid-19 testing. the national plan for covid-19 testing is being gradually adopted in other regions of the country in response to the rapidly evolving covid-19 emergency and to ensure a more rapid turn-around time. lessons learnt: after the decentralisation of covid-19 testing to other regions of the country, turn-around times were reduced from 48–72 h to 12–24 h. reducing turn-around times allowed niger to reduce the length of patients’ stays in hospitals and isolation facilities. shortages in testing capacity must be anticipated and addressed. in an effort to reduce risk of shortages and increase availability of reagents and consumables, niamey diversified real-time reverse transcriptase–polymerase chain reaction kits for severe acute respiratory syndrome coronavirus-2 detection. recommendations: continued investment in training programmes and laboratory strategy is needed in order to strengthen niger’s laboratory capacity against the outbreak. keywords: severe acute respiratory syndrome coronavirus-2; sars-cov-2; coronavirus disease 2019; covid-19; laboratory diagnosis; west africa; niger. background in december 2019, a new viral respiratory infection emerged. the infection was first detected and reported in the chinese province of hubei and has since spread globally, affecting people and socio-economic development in both developing and developed countries. the disease, later named the coronavirus disease 2019 (covid-19) and declared a global pandemic by the world health organization, is caused by a species of coronavirus known as severe acute respiratory syndrome coronavirus-2 (sars-cov-2), which belongs to the family coronaviridae and the genus betacoronavirus.1,2,3 worldwide, real-time reverse transcriptase–polymerase chain reaction (rrt-pcr) is the gold standard method used to detect the presence of sars-cov-2 rna in clinical specimens. as of 15 june 2020, 175 503 laboratory-confirmed covid-19 cases were reported in africa, with 4111 deaths.4 these figures make africa the continent least affected by this pandemic, with the most significantly affected countries in the region being south africa (70 038 cases), nigeria (16 085 cases), ghana (11 422 cases), and algeria (10 919 cases).4 the index case in the republic of niger was detected on 19 march 2020. this was an imported case involving a 36-year old man who arrived by road from burkina faso. since the official declaration of the index case, a total of 980 cases have been confirmed as of 15 june 2020.5 as this pandemic unfolds, laboratory services have been identified as key to containment efforts. this article outlines the laboratory organisation and management and control interventions in niger. description of the intervention ethical consideration this article followed all ethical standards for research without direct contact with human or animal subjects. organisational response severe acute respiratory syndrome coronavirus-2 is an emerging respiratory pathogen spreading rapidly through the general population. niamey, the capitol city of niger, adopted the ‘national covid-19 emergency preparedness and response plan’ (available from https://tinyurl.com/y2x3vpmt) to strengthen niger’s preparedness for the detection of sars-cov-2. this plan details the procedures for containment, contact tracing and screening based on the risk of spread. the plan is structured around five major strategic axes including reinforcement of coordination, strengthening of epidemiological surveillance, strengthening of health services capacities, reinforcement of risk communication and community engagement, and, lastly, the creation of isolation facilities. eight committees have been set up to implement the national covid-19 emergency preparedness and response plan, one of which is the laboratory and research team. the national covid-19 emergency preparedness and response plan provides $16 484 884.48 (united states dollars) to enable expedited building and implementation of sars-cov-2 testing capacity in the eight regions of the country in tune with the expansion of the pandemic. the laboratory and research team, which is charged with the responsibility of improving laboratory capacity and capability, is structured into three groups comprising a pre-analytical group (sample collection, inactivation, identification numbers), an analytical group (rrt-pcr testing) and a post-analytical group (validation and reporting test results). for more rapid and practicable responses, laboratory diagnostic capacity building is being supported by existing active clinical and research laboratories. the national reference laboratory for respiratory viruses located at the centre de recherche médicale et sanitaire (cermes) was designated as the reference centre for covid-19 testing. the veterinary research laboratory ‘laboratoire central de l’elevage’, the national reference laboratory for hiv and tuberculosis, the national hospital of niamey and the research institute for sustainable development supported the laboratory response by providing equipment for pcr techniques. moreover, seven laboratory technicians from these centres, who have had extensive training on pcr techniques, were mobilised by the ministry of health, in addition to the cermes technicians, to support the response by performing the rrt-pcr testing at cermes, which received and analysed samples collected from across the country. as part of covid-19 response, niamey and its partners provided logistical air support for sample transport from remote regions such as diffa, zinder, maradi, tahoua and agadez. gradually, the national plan on covid-19 testing is being adopted in other regions of the country in response to the rapidly evolving covid-19 emergency and to ensure a more rapid turn-around time (tat). this adoption involves gradual decentralisation of the sars-cov-2 rna rrt-pcr assays to three regions, tahoua, maradi and zinder, located 550, 662 and 891 kilometres from niamey (figure 1). the choice of these regions was based on several factors, including the distance from the capital city, the number of close contacts in these regions, the number of confirmed cases at the time of the decentralisation and the capacity of the laboratory facilities on site. figure 1: laboratory organisation and adaptation for covid-19 pandemic in niger. the 68 cases (in red) recorded on 11 april 2020 denote the peak between 19 march 2020 (index case) and 15 june 2020. the tahoua laboratory analysed samples collected from the tahoua and agadez regions. samples collected from the zinder and diffa regions were analysed in zinder, whereas the maradi laboratory analysed samples collected in the maradi region. samples collected from the niamey, dosso and tillabery regions were analysed at cermes. logistics and testing capacity are handled by cermes, which reported the availability of the reagents and materials needed to sample patients and to perform the rrt-pcr on a weekly basis to the ministry of health. laboratory diagnosis severe acute respiratory syndrome coronavirus-2 is classified as a risk group 3 human pathogen, similar to middle east respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus.6 the interim guidance from the world health organization suggests that non-propagative processing and handling of specimens containing sars-cov-2 must be performed in standard biosafety level-2 laboratories.7,8 consequently, niamey leveraged the safe practices and techniques, safety equipment and appropriate facility design of cermes to reduce or eliminate exposure of laboratory workers to sars-cov-2-containing materials according to the world health organization requirements.7 before the decentralisation of sars-cov-2 rna rrt-pcr assays to other regions, samples collected from across the country were sent to cermes. samples from remote regions such as diffa, zinder and agadez were transported using flights in accordance with category b transportation regulations.9 in an effort to help close contact management, sample collection was performed during home visits. ribonucleic acid extraction from samples was performed manually using qiaamp® viral rna mini kit (250) (qiagen gmbh, hilden, germany) or nucleic acid isolation or purification reagent (daan gene co., ltd, guangzhou, china) according to the manufacturer’s instructions. two teams of four laboratory technicians were dedicated to rna extraction at cermes. these teams were often overworked due to the large volume of samples handled. qualitative rrt-pcr assays were performed using the nucleic acid testing kit (daan gene co., ltd, guangzhou, china).10 two target genes, the open reading frame1ab (orf1ab) and nucleocapsid protein (n), were simultaneously amplified. a cycle threshold value lower than 40 was defined as a positive test result, and a cycle threshold value of 40 or more was defined as a negative outcome according to the manufacturer’s protocol. alternatively, rrt-pcr targeting the rdrp gene of the sars-cov-2 or sars-like coronavirus was performed using the lightmix® modular sars-cov-2 rdrp (tib molbiol, berlin, germany). positive samples for rdrp gene were confirmed by performance of rrt-pcr for the detection of the e gene using lightmix® sarbecov e gene plus equine arteritis virus control (tib molbiol, berlin, germany). given the extraordinary demand for reagents and consumables, risk of supply shortages became the main issue. niamey experienced a shortage of rna extraction kits and this led to the prioritisation of the testing of vulnerable people, health professionals and patients requiring hospitalisation. as of 15 june 2020, a total of 5386 samples had been tested for sars-cov-2 in niger. of these, 980 (18.2%) were confirmed positive (figure 2). all test results, positive or negative, were immediately reported to the national committee, which is responsible for communication on the pandemic. the highest number of new daily confirmed cases (68 cases) was detected on 11 april 2020. figure 2: covid-19 situation in niger as of 15 june 2020. (a) numbers of sars-cov-2 rna-positive patients. (b) map of niger showing the distribution of sars-cov-2-positive patients according to region. in march and april 2020, before the decentralisation of sars-cov-2 rna rrt-pcr assays to other regions, the number of samples received exceeded the capacity of the single centralised laboratory earmarked for the testing. consequently, tats were between 48 h and 72 h. this tat length often created a disconnect between clinicians and laboratory staff. indeed, according to the clinicians, the tat of 48–72 h delayed treatment and increased patients’ length of stay, particularly in isolation facilities where patients are often asymptomatic. after decentralisation, on 15 june, 2020, the tat had decreased significantly to 12 h at maradi and between 12 h and 24 h at tahoua, zinder and cermes, niamey. lessons learnt experience gained in the management of previous outbreaks (rift valley fever and meningitis) helped niamey to build a quick response to the covid-19 pandemic. however, additional control efforts are needed to improve the laboratory strategy and response against covid-19 in the country. firstly, a well-coordinated laboratory strategy and operational plan is needed in order to address the shortcomings of the existing plan. moreover, considering the importance of improved sars-cov-2 laboratory capacity, the country should provide extensive training to laboratory technicians in preparation for rapid expansion of laboratory diagnostic capacity. secondly, laboratory tat is critical in determining the success of both the laboratory response programme and the management of patients. reducing the tat allowed reductions in patients’ length of stay in hospitals and isolation facilities. thirdly, communication between clinical and laboratory staff needs to be improved in order to ensure that laboratory results are understandable to clinicians. fourthly, the shortages in testing capacity need to be anticipated and addressed. if capacity is exceeded, priority should be given to the testing of vulnerable patients, health professionals and patients requiring hospitalisation. niamey diversified rrt-pcr kits for sars-cov-2 detection to reduce the risk of shortages and increase the availability of reagents. it has been demonstrated that rna extraction kits from different manufacturers are interchangeable.11 however, diversifying the type of rrt-pcr kits for sars-cov-2 detection can lead to some variation in the detection rate between kits.12 it has been well documented that the limit of detection of different rrt-pcr kits can differ substantially.13,14 therefore, care must be taken in the interpretation of the results when using different kits for the monitoring of patients. fifthly, the overwhelming number of specimens to test highlighted the need for fast methods to extract viral rna. ribonucleic acid extraction from samples was performed manually in niger. in addition to the high risk of contamination, manual extraction of viral rna is time consuming and requires a large number of laboratory professionals. lastly, amplification of multiple target genes (e.g. n, e, rprd genes) could be used to avoid invalid results and increase sensitivity for detection of new genomic variants.15,16 recommendations despite tat improvement in the laboratory management of covid-19 testing, several additional control efforts are needed to improve the laboratory response against covid-19 in niger. considering the importance of improved sars-cov-2 laboratory capacity, continued investment in training programmes and laboratory strategy is needed to systematically guide the laboratory response against the outbreak. acknowledgements authors would like to acknowledge the contributions of the national covid-19 emergency preparedness and response committee for the technical, resource and logistical contributions, the government of niger, the ministry of health and all its partners for the good support that formed the basis of a favourable working environment for the response against covid-19 pandemic in niger. we would also like to thank dr ahmed olowo-okere of usmanu danfodiyo university, sokoto-nigeria, for language editing. competing interests the authors have declared that no competing interests exist. authors’ contributions a.y. and s.m. conceived and designed the study. data acquisition was done by a.l. the draft manuscript was written by a.y., while b.m., d.a.m., h.m.s., h.b., s.o., e.a., s.m., m.k.s, z.h.h., h.b., s.a., a.o., a.a. and b.a. critically reviewed the manuscript. all authors have read and agreed to the final version of this manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement the data are available from the corresponding author upon reasonable request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references bbc news. coronavirus disease named covid-19 [homepage on the internet]. [cited 2020 may 31]. available from: https://www.bbc.com/news/world-asia-china-51466362 who director-general’s opening remarks at the media briefing on covid-19 – 25 may 2020 [homepage on the internet]. [cited 2020 may 31]. available from: https://www.who.int/dg/speeches/detail/who-director-general-s-opening-remarks-at-the-media-briefing-on-covid-19---25-may-2020 gorbalenya ae, baker sc, baric rs, et al. the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2. nat microbiol. 2020;5(5):536–544. https://doi.org/10.1038/s41564-020-0695-z world health organisation. coronavirus disease (covid-19) situation reports [homepage on the internet]. [cited 2020 jun 16]. available from: https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports coronavirus: le niger enregistre son premier cas. sikafinance.com [homepage on the internet]. [cited 2020 aug 09]. available from: https://www.sikafinance.com/marches/coronavirus-le-niger-enregistre-son-premier-cas_21336 iwen pc, stiles kl, pentella ma. safety considerations in the laboratory testing of specimens suspected or known to contain the severe acute respiratory syndrome coronavirus 2 (sars-cov-2). am j clin pathol. 2020 apr 15;153(5):567–570. https://doi.org/10.1093/ajcp/aqaa047 world health organisation. laboratory testing for coronavirus disease 2019 (covid-19) in suspected human cases: interim guidance, 2 march 2020 [homepage on the internet]. world health organization; 2020. [cited 2020 sep 05]. available from: https://www.who/covid-19/laboratory/2020.4 world health organisation. laboratory biosafety guidance related to the novel coronavirus (2019-ncov) [homepage on the internet]. world health organization; 2020 [cited 2020 sep 18]. available from: https://www.who.int/docs/default-source/coronaviruse/laboratory-biosafety-novel-coronavirus-version-1-1.pdf?sfvrsn=912a9847_2.%20accessed%20march%207,%202020 world health organisation. guidance on regulations for the transport of infectious substances [homepage on the internet]. [cited 2020 sep 05]. available from: https://www.who.int/csr/resources/publications/biosafety/who_cds_csr_lyo_2005_22r%20.pdf?ua=1 world health organisation. who emergency use listing for in vitro diagnostics (ivds) detecting sars-cov-2 nucleic acid [homepage on the internet]. [cited 2020 sep 18]. available from: https://www.who.int/diagnostics_laboratory/200710_eul_sars_cov2_product_list.pdf?ua=1 lim kl, johari na, wong st, et al. a novel strategy for community screening of sars-cov-2 (covid-19): sample pooling method. plos one. 2020;15(8):e0238417. https://doi.org/10.1371/journal.pone.0238417 van kasteren pb, van der veer b, van den brink s, et al. comparison of seven commercial rt-pcr diagnostic kits for covid-19. j clin virol. 2020 may 8;128:104412. https://doi.org/10.1016/j.jcv.2020.104412 hogan ca, sahoo mk, huang c, et al. comparison of the panther fusion and a laboratory-developed test targeting the envelope gene for detection of sars-cov-2. j clin virol. 2020 jun 1;127:104383. https://doi.org/10.1016/j.jcv.2020.104383 wang x, yao h, xu x, et al. limits of detection of 6 approved rt–pcr kits for the novel sars-coronavirus-2 (sars-cov-2). clin chem. 2020 jul 1;66(7):977–979. https://doi.org/10.1093/clinchem/hvaa099 tahamtan a, ardebili a. real-time rt-pcr in covid-19 detection: issues affecting the results. expert rev mol diagn. 2020 may 3;20(5):453–454. https://doi.org/10.1080/14737159.2020.1757437 penarrubia al, ruiz m, porco r, et al. multiple assays in a real-time rt-pcr sars-cov-2 panel can mitigate the risk of loss of sensitivity by new genomic variants during the covid-19 outbreak. int j infect dis. 2020;97:225–229. https://doi.org/10.1016/j.ijid.2020.06.027 abstract introduction methods results discussion acknowledgements references about the author(s) kyle degruy centers for disease control and prevention, division of global hiv and tb, international laboratory branch, atlanta, georgia, united states katherine klein centers for disease control and prevention, division of tb elimination, laboratory branch, atlanta, georgia, united states zilma rey centers for disease control and prevention, division of global hiv and tb, international laboratory branch, atlanta, georgia, united states patricia hall centers for disease control and prevention, division of global hiv and tb, international laboratory branch, atlanta, georgia, united states andrea kim centers for disease control and prevention, division of global hiv and tb, international laboratory branch, atlanta, georgia, united states heather alexander centers for disease control and prevention, division of global hiv and tb, international laboratory branch, atlanta, georgia, united states citation degruy k, klein k, rey z, hall p, kim a, alexander h. development of dried tube specimens for xpert mtb/rif proficiency testing. afr j lab med. 2020;9(1), a1166. https://doi.org/10.4102/ajlm.v9i1.1166 original research development of dried tube specimens for xpert mtb/rif proficiency testing kyle degruy, katherine klein, zilma rey, patricia hall, andrea kim, heather alexander received: 14 jan. 2020; accepted: 24 june 2020; published: 29 sept. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: proficiency testing (pt) is part of a comprehensive quality assurance programme, which is critical to ensuring patients receive accurate and reliable diagnostic testing. implementation of the cepheid xpert® mtb/rif assay to aid in the diagnosis of tuberculosis has expanded rapidly in recent years; however, pt material for xpert mtb/rif is not readily available in many resource-limited settings. objective: to develop an accurate and precise pt material based on the dried tube specimen (dts) method, using supplies and reagents available in most tuberculosis culture laboratories. methods: dried tube specimens were produced at the united states centers for disease control and prevention from 2013 to 2015 by inactivating liquid cultures of well-characterised mycobacterial strains. ten percent of dts produced were tested with xpert mtb/rif and evaluated for accuracy and precision. results: validation testing across eight rounds of pt demonstrated that dts are highly accurate, achieving an average of 96.8% concordance with the xpert mtb/rif results from the original mycobacterial strains. dried tube specimen testing was also precise, with cycle threshold standard deviations below two cycles when inherent test cartridge variability was low. conclusion: dried tube specimens can be produced using equipment already present in tuberculosis culture laboratories, making xpert mtb/rif pt scale-up more feasible in resource-limited settings. use of dts may fill the gap in tuberculosis laboratory access to external quality assessment, which is an essential component of a comprehensive continuous quality improvement programme. keywords: external quality assessment; eqa; xpert mtb/rif; proficiency testing; dried tube specimen; dts. introduction the world health organization (who) estimated that 10.0 million people developed tuberculosis and 1.5 million died from tuberculosis disease worldwide in 2018.1 human immunodeficiency virus contributes to this epidemic, with 860 000 (8.6%) of the 10.0 million tuberculosis patients also being co-infected with hiv. approximately 251 000 people living with human immunodeficiency virus died from tuberculosis in 2018.1 rapid tuberculosis diagnosis and treatment initiation are therefore critical to reducing tuberculosis-related deaths and ongoing transmission in communities. access to rapid, quality diagnostic testing is a significant barrier to global tuberculosis elimination.2 the who estimated that approximately 3 million cases of tuberculosis disease in 2018 went undiagnosed or unreported, and only 186 772 of the estimated 500 000 people who developed either multidrug-resistant or rifampicin-resistant tuberculosis were diagnosed.1 the implementation of liquid culture and molecular methods have shortened the time-to-detection of mycobacterium tuberculosis and time-to-determination of the m. tuberculosis drug susceptibility profile.3,4 the cepheid xpert® mtb/rif assay (cepheid, sunnyvale, california, united states) is a cartridge-based, hemi-nested, real-time polymerase chain reaction assay detecting m. tuberculosis and genetic mutations associated with resistance to rifampicin.5 this assay provides test results within two hours of beginning the test when compared to conventional culture and phenotypic drug susceptibility testing, which may take eight weeks or longer.5,6 the who recommends xpert mtb/rif as one of the initial diagnostic tests for all individuals evaluated for tuberculosis.7 quality assurance (qa) is vital in the clinical laboratory for accuracy and reliability of diagnostic testing. clinicians depend on accurate and rapid results for appropriate patient diagnosis and treatment.8 rapid implementation of the xpert mtb/rif assay occurred in many resource-limited settings to quickly increase capacity for tuberculosis diagnosis and treatment initiation; unfortunately, qa activities have not kept pace with the expansion of xpert mtb/rif testing. when we initiated development of the dried tube specimen (dts) for the xpert mtb/rif external quality assessment (eqa) procedure in 2012, only 386 of 907 (43%) global xpert mtb/rif testing sites participated in eqa, and only 77 of 318 (24%) african testing sites, participated in eqa.9 quality assessment programmes for xpert mtb/rif are needed to ensure patients receive accurate tuberculosis diagnostics, rifampicin resistance testing, and follow-up test results. a comprehensive qa plan for xpert mtb/rif includes documented training and competency assessment for all operators, verification of the genexpert instrument or modules, new lot testing, temperature monitoring for testing site and kit storage, routine monitoring of quality indicators, and eqa, including onsite supervisory visits and participation in proficiency testing (pt).10,11 proficiency testing provides opportunities for the laboratory to identify errors in the testing process and implement systems to detect and prevent those errors.12 although a limited number of xpert mtb/rif pt panels are currently available to testing sites in resource-limited settings, many must be procured from commercial international providers.13 this may be cost prohibitive, and transport of infectious material can be complicated.14 because of these constraints, as well as the rapidly expanding number of testing sites, there is a critical need for countries to implement a simple and sustainable national pt programme in order to ensure the accuracy of xpert mtb/rif results. we modified the dts method originally developed for hiv rapid test pt to produce pt panels for the xpert mtb/rif assay.15 the novel dts-based xpert mtb/rif pt panel methodology described here was developed to address the need for countries to sustainably produce their own pt panels for the xpert mtb/rif assay. methods ethical considerations no patient specimen collection was required, and no patient identifiers were recorded on quality assurance tools. participation in the xpert mtb/rif quality assurance and pt programme was voluntary and free of charge. no incentives were provided. this study was approved by the cdc center for global health, division of human research protection (cgh hsr tracking # 2014-082). dried tube specimen panel preparation dried tube specimens for this study were prepared at the united states centers for disease control and prevention (cdc; atlanta, georgia, united states), international laboratory branch between 2013 and 2015. the xpert mtb/rif cartridge captures and lyses intact m. tuberculosis bacilli.5 dried tube specimens were developed using whole cell inactivation in order to preserve the cell structure of the selected mycobacterial strains. this more closely simulates dna extraction from a patient specimen. all dts preparation steps were performed in a biosafety level 3 containment laboratory with personal protective equipment, including respiratory protection. dried tube specimens were prepared using well-characterised rifampicin-resistant and rifampicin-susceptible m. tuberculosis strains and non-tuberculous mycobacteria strains obtained from the american type culture collection, who proficiency testing challenges, and the cdc’s collection of laboratory-derived m. tuberculosis strains. m. tuberculosis strains were characterised using phenotypic and genotypic methods, including the middlebrook 7h10 method of proportion drug susceptibility testing and sanger sequencing of the rpob gene.6,16 strains were inoculated into panta-supplemented bactec® mgit (mycobacteria growth indicator tube) 7 ml tubes (modified middlebrook 7h9 broth base) (becton, dickinson and company; sparks, maryland, united states) and incubated in the bactec® mgit 960® instrument (becton, dickinson and company; sparks, maryland, united states) until flagged positive for culture growth. cultures were then incubated an additional 4–6 days at 37 °c in an auxiliary incubator and inoculated onto middlebrook 7h10 agar and blood agar (remel, lenexa, kansas, united states) for enumeration of mycobacteria and contamination checks. mycobacteria were inactivated using a 2:1 ratio of xpert mtb/rif sample reagent (sr) (cepheid; sunnyvale, california, united states) to mgit culture in sterile 50 ml conical tubes (figure 1). the suspensions were incubated at 15 °c – 30 °c, and vortexed for 30 seconds every 15 minutes for a total of two hours. mgit culture/sr suspensions were neutralised with 20 ml – 25 ml of in-house prepared, sterile phosphate buffer ph 6.8 and centrifuged at 3000 × g for 15 min. the pellets were washed with an additional 45 ml phosphate buffer, centrifuged, and the supernatants were discarded. pellets were then resuspended in 5 ml of phosphate buffer, transferred to sterile glass tubes with 5–10 3 mm glass beads (thermo fisher scientific, waltham, massachusetts, united states), vortexed for 5 min, and allowed to settle for 15 min. supernatants above the beads were transferred to new sterile glass tubes, labelled as stock suspensions, and stored at 2 °c – 8 °c. inactivation verification was performed by inoculating 0.5 ml of stock suspension into mgit tubes supplemented with panta and incubating for two cycles for a total of 84 days. only stocks testing negative for growth were considered for panel preparation and distribution. figure 1: overview of dried tube specimens for xpert mtb/rif preparation procedure, 2013–2015, united states centers for disease control and prevention (atlanta, georgia, united states). pre-testing xpert mtb/rif pre-testing of five dts from each stock was conducted to evaluate dts accuracy and precision on a limited scale prior to preparing large numbers of pt samples. dried tube specimens were prepared by diluting an aliquot of each stock suspension 1:10 with in-house prepared, sterile saline, adding food grade dye at a concentration of 0.1% (volume/volume) to each dilution, and transferring 100 µl of each stock dilution into five 4 ml cryovials. cryovials were left uncapped in a class ii biosafety cabinet (bsc) until all liquid evaporated and aliquots were visibly dry. dried tube specimen samples were then capped and stored in the dark at room temperature until tested. dried tube specimens were rehydrated with 2.5 ml of sr, shaken vigorously 20 times and incubated at room temperature for 15 min with additional shaking repeated after 10 min (figure 2). the xpert mtb/rif cartridges were inoculated with approximately 2.0 ml of rehydrated samples using the transfer pipette provided with the kit, and tested immediately on a genexpert iv or genexpert viii using genexpert dx software version 4.0 (cepheid, sunnyvale, california, united states), according to manufacturer’s instructions.17 dried tube specimens prepared in 2013 were tested with xpert mtb/rif assay g4 research use only cartridges. dried tube specimens prepared in 2014 and 2015 were tested with food and drug administration-cleared xpert mtb/rif us in vitro diagnostic (ivd) cartridges. figure 2: testing dried tube specimens using xpert mtb/rif, 2013–2015, united states centers for disease control and prevention (atlanta, georgia, united states). to ensure that dts yielded accurate xpert mtb/rif results, m. tuberculosis detection and rifampicin resistance results from dts pre-testing were verified against the expected xpert mtb/rif results for the parent strain (as determined by prior phenotypic and genotypic testing). results were considered acceptable if the qualitative m. tuberculosis detection and rifampicin resistance results matched the expected results for the parent strain for all five pre-tested dts from the stock. precision was evaluated to verify uniform suspension of the organism in the stock using the cycle threshold (ct) standard deviation (sd). in line with previous studies, probe a was selected for most analysis as it was the first probe to reach the detection threshold.18 the sds of probe a ct values were calculated for all dts and compared between different samples within each panel. probe c ct values were used when probe a did not bind. the sd for the cartridge internal specimen processing control (spc) ct was also calculated for all samples and panels to investigate whether inherent xpert mtb/rif cartridge variability contributed to variation in dts ct values. to ensure that adequate amounts of inactivated organism were present in the dts and that the integrity of dna was not compromised during inactivation, the average xpert mtb/rif semi-quantitative result (i.e. the category assigned to the amount of m. tuberculosis dna based on the ct of the first probe detected) was required to fall in the medium (ct = 16–22) to low (ct = 22–28) range. stock selection and dried tube specimen panel validation the five most precise and accurate stocks (i.e. the stocks with the lowest ct sd and lowest mean ct with dts all yielding expected test results during pre-testing) were selected to aliquot dts for use in the pt programme. each xpert mtb/rif pt panel included one dts per stock, for a total of five samples. eight unique panels were prepared for use in the pt programme from 2013 (2013-a, 2013-b, 2013-c, and 2013-d) to 2015 (2014-a, 2015-a, 2015-b, and 2015-c) as described above. a strict bsc cleaning protocol (1% sodium hypochlorite followed by 70% ethanol rinse of all inner walls, sash, work surface and under work surface) was employed before aliquoting each panel. in most cases, only one sample was dried at a time. in situations where more than one sample was dried in the same bsc at the same time, all dts had the same expected result. an aliquoting order was routinely followed: (1) ‘tuberculosis not detected’ (non-tuberculous mycobacteria) samples were first aliquoted, then dried, capped, and removed; (2) ‘tuberculosis detected, rifampicin resistance not detected’ samples followed; and finally (3) ‘tuberculosis detected, rifampicin resistance detected’ samples were aliquoted last. no culture manipulation was performed in the bsc while dts samples were drying. once prepared, panels were validated by confirming accurate and precise test results for 10% of the dts produced using the same methodologies outlined above. ten dts from each stock underwent validation for panel 2013-a (50 total), 18 for panel 2013-b (90 total), 25 for panels 2013-c, 2013-d, and 2014-a (125 total per panel), 50 for panels 2015-a and 2015-b (250 total per panel), and 55 for panel 2015-c (275 total). results across all eight panels, the mean ct values for probe a (or probe c when probe a did not bind) ranged from 19.3 to 26.2, which were within the acceptable xpert mtb/rif semi-quantitative range of medium to low (data not shown). the spc ct ranged from 0.0 to 40.8 across all eight panels with a mean ct range of 23.8–27.8. when panel validation results were compared with the expected xpert mtb/rif results for the parent strain, dts tested from panels 2013-a through 2015-c ranged from 96.0% to 98.5% concordant (table 1). only three (0.2%) of the 1290 dts tested were discordant, and of those, two were false negative and one was false positive. a total of eight (0.6%) dts samples, including four from 2013-b, three from 2013-c, and one from 2013-d, yielded indeterminate rifampicin results. when examining cts for all discordant and rifampicin-indeterminate results, we found that the cts for the test cartridge spc were consistently high (35.3–38.2) with the exception of one discordant result from 2015-b (25.7). table 1: accuracy of dried tube specimen xpert mtb/rif test results collected during panel validation, 2013–2015, united states centers for disease control and prevention (atlanta, georgia, united states). a total of 30 errors were encountered while testing the 1290 dts, which resulted in an overall error rate of 2.3% (table 2). this rate ranged from 0.0% to 4.4% across panels (table 1). errors encompassed six different error codes (table 2). no ‘invalid’ or ‘no result’ results were observed. table 2: errors received during validation of dried tube specimens for 2013–2015 proficiency testing panels, 2013–2015, united states centers for disease control and prevention (atlanta, georgia, united states). while dts samples were found to be accurate (96.8% concordance overall), sample precision varied, with ct sd values ranging from 0.9 to 6.2 (figure 3). the sd of the cartridge spc also varied among dts pt samples, ranging from 0.7 to 10.5. we observed higher variability in probe a ct (sd: 1.0–6.2) and spc ct values (sd: 1.4–10.5) for panels produced in 2013 and tested with xpert mtb/rif assay g4 cartridges than for those produced in 2014 (1.0–1.9; 0.9–3.3) and 2015 (0.9–1.7; 0.7–2.9), which were tested with food and drug administration-cleared xpert mtb/rif us ivd cartridges. figure 3: standard deviations of probe a and specimen processing control cycle threshold values for dried tube specimens containing mycobacterium tuberculosis complex tested during panel validation, 2013–2015, united states centers for disease control and prevention (atlanta, georgia, united states). discussion the dts method was originally developed for hiv rapid test pt and subsequently used to produce pt panels for other assays such as syphilis, hiv viral load and malaria rapid test, with good results.15,19,20,21 this study demonstrates that an accurate and precise m. tuberculosis pt panel for the xpert mtb/rif assay can also be produced using the dts technique. the panel validation process verifies the accuracy and precision of each dts panel produced and adheres to clinical and laboratory standards institute recommendations for characterising molecular pt materials.12 the dts panels produced according to this methodology were accurate, achieving 96.8% test result concordance with parental stocks for the detection of m. tuberculosis and rifampicin resistance. interestingly, the discordant and indeterminate samples, with the exception of the 2015-b false negative discordant sample, had spc ct values above 35. a previous quantitative assessment by blakemore et al. found that xpert mtb/rif assay results with spc ct values above 34 could be quantified inaccurately.22 thus, it is possible that the majority (2 of 3) of the discordant results and all 8 of the rif indeterminate results in the dts panel evaluations could be due to factors inherent to the xpert mtb/rif assay cartridges. the overall error rate was relatively low (2.3%), and no trend in error rates was observed over the study period. eighty percent of the errors observed were due to error codes 5006 and 5007 (probe check failure). these errors often are associated with the incorrect sample volume added to the cartridge, incorrect filling or bubbles in the cartridge reaction tube, or probe integrity issues.10 the remaining errors (codes 2005, 2014, 2037 and 5011) are associated with instrument or cartridge performance. additionally, the dts method delivers a consistent amount of m. tuberculosis to each sample, such that testing of dts prepared from the same stock yielded probe a ct values with an sd similar to the sd of the spc internal control ct values in most cases. there is no current standard for molecular pt variability. however, the standard deviations observed for dts produced in 2014 and 2015 and tested with food and drug administration-cleared xpert mtb/rif us ivd cartridges are in line with those seen by scott et al. using a similar inactivation technique.18 xpert mtb/rif sr has been shown to effectively inactivate mycobacteria while leaving the cells intact.18 although this evaluation did not assess the cell structure post-inactivation, other investigators confirmed the presence of whole bacilli following sr-mediated inactivation of m. tuberculosis using flow cytometry.18 inoculation of dts stock suspensions into mgit culture provides confirmation of inactivation prior to distribution of dts. furthermore, the incubation of mgit culture for 4–6 days post-positivity did not adversely affect inactivation with sr and was found to consistently yield dts samples with the desired m. tuberculosis dna concentration for dts preparation and testing. while the accuracy and precision of dts are similar to those reported for other pt panels, the simplicity of matrix preparation and feasibility for transfer to low-resource settings set the dts xpert mtb/rif pt panel apart.13 additionally, since the procedure is similar to specimen processing for tb culture, much of the laboratory expertise, technical skill, equipment (e.g., class ii bsc, safety centrifuge, bactec mgit 960 or 320 instrument, vortex, and an auxiliary incubator), and consumables necessary for dts preparation already exist in tuberculosis culture laboratories in resource-limited countries. many national tuberculosis reference laboratories in these settings have implemented bactec mgit 960 culture and drug susceptibility testing in recent years, making mgit 7 ml tube media the most common liquid media for m. tuberculosis culture and detection.23 lastly, since dts are prepared within the tubes utilised for sample rehydration, testing of dts does not require additional laboratory supplies. not only are expertise, equipment, and supplies readily available, but dts are also estimated to be low cost. regarding transport, parekh et al. reported that the use of dts for hiv pt eliminates the need for cold chain and results in a significant reduction in shipping costs.15 reagent costs are also low, as approximately 500 dts can be made from a single inactivated mgit culture. it is therefore possible for one culture laboratory, such as a national tuberculosis reference laboratory, to produce and validate enough dts to provide pt material for all testing sites in a country. the dts method can thus provide resource-limited countries with a suitable pt material to create and manage their own sustainable xpert mtb/rif pt programme. external quality assessment using dts can serve as an important component of a laboratory qa programme, which is necessary to ensure that patients receive accurate and reliable diagnostic testing services.11 challenges and limitations the primary challenge associated with producing dts was variability between ct values from separate aliquots of the same dilution. however, the inherent cartridge-to-cartridge variability in ct values that was observed when comparing the sd of probe a to the sd of the spc likely plays a role in the variability observed in dts ct values. after the xpert mtb/rif assay gained food and drug administration clearance in 2013, we transitioned from using xpert mtb/rif assay g4 research use only cartridges in 2013 to xpert mtb/rif us ivd cartridges in 2014 and 2015. the range of sds of probe a ct values decreased from 1.0–6.2 in 2013 to 0.9–1.9 in 2014–2015. this decrease further suggests that the ct value variability and high sd values observed were likely due to inconsistencies between cartridges. however, the variability of dts could also be due to mycobacteria (particularly m. tuberculosis) clumping in mgit cultures, leading to an uneven dts stock suspension and difficulty in achieving equal numbers of bacilli in each aliquot. additional evaluations are underway to improve both the accuracy and precision of dts. another limitation of using the dts technique for pt is that the dts sample type (inactivated, dried mycobacteria) does not resemble the most commonly tested clinical sample type, sputum. while the use of dts for pt does not allow for the complete evaluation of all the procedural steps or potential pitfalls involved in xpert mtb/rif testing of sputum, it could be an adequate substitute. for example, creating lyophilised, m. tuberculosis-spiked sputum samples would involve the purchase and maintenance of expensive equipment, such as lyophilisers, and increase biosafety risk for laboratorians. alternately, the use of liquid samples increases biohazard risk to both shipping and laboratory personnel and increases transportation costs. therefore, the safety and cost-saving benefits of dts may outweigh potential disadvantages associated with assessing proficiency of xpert mtb/rif testing using dried, inactivated m. tuberculosis in resource-limited settings. recommendations and next steps we are continuing to study and refine the technique for producing dts, including the investigation of heat inactivation of m. tuberculosis for improved biosafety, stability and sample accuracy and precision. since development of this novel pt technology, a voluntary xpert mtb/rif dts-based pt programme has been rolled out to 26 countries and united states-affiliated territories, as described in ‘a global proficiency testing program for xpert mtb/rif using dried tube specimens’ by klein et al.24 in addition, in 2016 we began the transfer of dts technology to countries as part of a pt package that includes feedback and corrective actions to sustainably improve the quality of international diagnostic testing services. in 2019, the scope of the pt programme was increased to include the xpert mrb/rif ultra assay (cepheid, sunnyvale, california, united states) where the same panel prepared for xpert mtb/rif pt was also successfully validated for use as pt for the xpert mtb/rif ultra assay. conclusion proficiency testing for the xpert mtb/rif assay, as part of a comprehensive qa programme, should be a priority for every national tuberculosis programme. the dts technique creates accurate, precise, and safe pt panels. designing this preparation procedure around the availability of equipment and reagents in national tuberculosis reference laboratories in resource-limited countries promotes the transfer of dts technology to both national and regional programmes and assists countries in implementing consistent, sustainable eqa programmes. acknowledgements we thank bharat parekh for his inspiration and guidance, and adeboye adelekan, elizabeth prentince and wendy stevens for assistance accessing laboratories willing to pre-test dts panels. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions k.d. and h.a. conceived and designed the experiments, k.d. and k.k. performed the experiments and analysed the data, and k.d., k.k., z.r. a.k. and p.h. wrote the paper. k.d., k.k., z.r., p.h., a.k. and h.a. reviewed and approved the manuscript. sources of support this research has been supported by the president’s emergency plan for aids relief (pepfar) through the centers for disease control and prevention, atlanta georgia, united states. data availability statement data access level is public without identifying information of proficiency testers, testing sites and country. disclaimer the findings and conclusions in this paper are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention or other funding agencies. references world health organization. global tuberculosis report 2019 [homepage on the internet]. c2019 [cited 2020 may 20]. geneva: who press. available from: https://www.who.int/tb/publications/global_report/en/ world health organization. the end tb strategy [homepage on the internet]. c2014 [cited 2020 may 20]. geneva: who press. available from: https://www.who.int/tb/strategy/end_tb_strategy.pdf?ua=1 scarparo c, piccoli p, rigon a, ruggiero g, ricordi p, piersimoni c. evaluation of the bactec mgit 960 in comparison with bactec460 tb for detection and recovery of mycobacteria from clinical specimens evaluation of the bactec mgit 960 in comparison with bactec460 tb for detection and recovery of mycobacteria from clinical specimens. diagn microbiol infect dis. 2002;44(2):157–161. https://doi.org/10.1016/s0732-8893(02)00437-6 barnard m, albert h, coetzee g, o’brien r, bosman me. rapid molecular screening for multidrug-resistant tuberculosis in a high-volume public health laboratory in south africa. am j respir crit care med. 2008;177(7):787–792. https://doi.org/10.1164/rccm.200709-1436oc helb d, jones m, story e, et al. rapid detection of mycobacterium tuberculosis and rifampin resistance by use of on-demand, near-patient technology. j clin microbiol. 2010;48(1):229–237. https://doi.org/10.1128/jcm.01463-09 kent pt, kubica gp. public health mycobacteriology: a guide for the level iii laboratory. atlanta, ga: centers for disease control; 1985. world health organization. rapid communication: molecular assays as initial tests for the diagnosis of tuberculosis and rifampicin [homepage on the internet]. c2020 [cited 2020 may 20]. geneva: who press. available from: https://www.who.int/publications-detail/rapid-communication-molecular-assays-as-initial-tests-for-the-diagnosis-of-tuberculosis-and-rifampicin-resistance ondoa p, datema t, keita-sow ms, et al. a new matrix for scoring the functionality of national laboratory networks in africa: introducing the labnet scorecard. afr j lab med. 2016;5(3):498. https://doi.org/10.4102/ajlm.v5i3.498 world health organization. download data as csv files [homepage on the internet]. n.d. [cited 2020 may 20]. geneva: laboratory diagnostic services. available from: https://www.who.int/tb/country/data/download/en/ global laboratory initiative. training package on xpert mtb/rif [homepage on the internet]. geneva: world health organization; 2016 [cited 2020 may 21]. available from: http://www.stoptb.org/wg/gli/trainingpackage_xpert_mtb_rif.asp global laboratory initiative. practical guide to implementing a quality assurance system for xpert mtb/rif testing [homepage on the internet]. geneva: world health organization; 2019 [cited 2020 may 25]. available from: http://www.stoptb.org/wg/gli/pgiqas.asp clinical and laboratory standards institute. design of molecular proficiency testing/external quality assessment; approved guideline. 2nd ed. clsi document mm14-a2. wayne, pa: clinical and laboratory standards institute, 2013; p. 18–19. scott l, albert h, gilpin c, alexander h, degruy k, stevens w. multicenter feasibility study to assess external quality assessment panels for xpert mtb/rif assay in south africa. j clin microbiol. 2014;52(7):2493–2499. https://doi.org/10.1128/jcm.03533-13 international air transport association. dangerous goods regulations. 61st ed. montreal: international air transport association, 2020; p. 151–155. parekh bs, anyanwu j, patel h, et al. dried tube specimens: a simple and cost-effective method for preparation of hiv proficiency testing panels and quality control materials for use in resource-limited settings. j virol methods. 2010;163(2):295–300. https://doi.org/10.1016/j.jviromet.2009.10.013 morlock gp, plikaytis bb, crawford jt. characterization of spontaneous, in vitro-selected, rifampin-resistant mutants of mycobacterium tuberculosis strain h37rv. antimicrob agents chemother. 2000;44(12):3298–3301. https://doi.org/10.1128/aac.44.12.3298-3301.2000 xpert mtb/rif assay product insert [homepage on the internet]. sunnyvale, ca: cepheid; c2019 [cited 2020 may 20]. available from: https://www.cepheid.com/package%20insert%20files/xpert-mtb-rif-english-package-insert-301-1404-rev-f.pdf scott le, gous n, cunningham be, et al. dried culture spots for xpert mtb/rif external quality assessment: results of a phase 1 pilot study in south africa. j clin microbiol. 2011;49(12):4356–4360. https://doi.org/10.1128/jcm.05167-11 benzaken as, bazzo ml, galban e, et al. external quality assurance with dried tube specimens (dts) for point-of-care syphilis and hiv tests: experience in an indigenous population screening programme in the brazilian amazon. sex transm infect. 2014;90(1):14–18. https://doi.org/10.1136/sextrans-2013-051181 ramos a, nguyen s, garcia a, subbarao s, nkengasong jn, ellenberger d. generation of dried tube specimen for hiv-1 viral load proficiency test panels: a cost-effective alternative for external quality assessment programs. j virol methods. 2012;188(1–2):1–5. https://doi.org/10.1016/j.jviromet.2012.11.036 tamiru a, boulanger l, chang ma, malone jl, aidoo m. field assessment of dried plasmodium falciparum samples for malaria rapid diagnostic test quality control and proficiency testing in ethiopia. malar j. 2015;14:11. https://doi.org/10.1186/s12936-014-0524-z blakemore r, nabeta p, davidow al, et al. a multisite assessment of the quantitative capabilities of the xpert mtb/rif assay. am j respir crit care med. 2011;184(9):1076–1084. https://doi.org/10.1164/rccm.201103-0536oc foundation for innovative new diagnostics. liquid culture and drug susceptibility testing [homepage on the internet]. switzerland: foundation for innovative new diagnostics; c2016 [cited 2020 may 20]. available from: https://www.finddx.org/wp-content/uploads/2016/02/mgit_manual_nov2006.pdf klein k, degruy k, rey z, et al. a global proficiency testing program for xpert mtb/rif using dried tube specimens, 2013–2015. afr j lab med. 2020. forthcoming. article information author: corena de beer1 monika esser2 wolfgang preiser1 affiliation: 1department of pathology (division of medical virology), university of stellenbosch, south africa2department of pathology (immunology unit), university of stellenbosch, south africa correspondence to: corena de beer postal address: department of pathology, university of stellenbosch, po box 19063, tygerberg 7505, south africa dates: received: 09 dec. 2012 accepted: 15 aug. 2012 published: 15 oct. 2012 how to cite this article: de beer c, esser m, preiser w. optimising automation of a manual enzyme-linked immunosorbent assay. afr j lab med. 2011;1(1), art. #15, 3 pages. http://dx.doi.org/10.4102/ ajlm.v1i1.15 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. optimising automation of a manual enzyme-linked immunosorbent assay in this original research... open access • abstract • introduction • materials and methods    • automation of the mk016 enzyme-linked immunosorbent assay • results • discussion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ objective: enzyme-linked immunosorbent assays (elisas) are widely used to quantify immunoglobulin levels induced by infection or vaccination. compared to conventional manual assays, automated elisa systems offer more accurate and reproducible results, faster turnaround times and cost effectiveness due to the use of multianalyte reagents.design: the vacczyme™ human anti-haemophilus influenzae type b (hib) kit (mk016) from the binding site company was optimised to be used on an automated biorad phd™ system in the immunology laboratory (national health laboratory service) in tygerberg, south africa. methods: an automated elisa system that uses individual well incubation was compared to a manual method that uses whole-plate incubation. results: results were calculated from calibration curves constructed with each assay. marked differences in calibration curves were observed for the two methods. the automated method produced lower-than-recommended optical density values and resulted in invalid calibration curves and diagnostic results. a comparison of the individual steps of the two methods showed a difference of 10 minutes per incubation cycle. all incubation steps of the automated method were subsequently increased from 30 minutes to 40 minutes. several comparative assays were performed according to the amended protocol and all calibration curves obtained were valid. calibrators and controls were also included as samples in different positions and orders on the plate and all results were valid. conclusion: proper validation is vital before converting manual elisa assays to automated or semi-automated methods. introduction top ↑ quantitative analytical methods have advanced significantly since the development of the enzyme immunoassay (eia) and enzyme-linked immunosorbent assay (elisa) in 1971 by the groups of perlmann and engvall, and schuurs and van weemen, respectively.1,2,3 before this, the only method for performing immunoassays was the radioimmunoassay (ria), which was first described by yalow and berson in 1960.4however, the ria had several shortcomings, for example the potential health threat of radioactivity, short half-lives of radioisotopes, cumbersome radioactive waste disposal, expensive counting equipment, etc.3,5,6 an important shift from radioisotope-labelled liquid-phase assays to solid-phase assays occurred in 1968. miles and hales7 developed an immuno-radiometric technique, which used radioactively labelled antibodies instead of labelled antigens for measuring insulin in human plasma. plastic tubes were subsequently coated with the antigen or antibody to create a solid-phase or immunosorbent platform. 8 modern commercial elisa/eia kits use 96-well microtitre plates, where either an antigen or an antibody is noncovalently bound to a solid-phase support. these methods are widely employed by laboratories and manufacturing companies for microbiological, virological and other serological diagnostic tests, validation of assays and general quality control. although automated pipetting devices have been used for more than two decades, the high cost associated with the technique remains a major limiting factor in developing countries and smaller laboratories.3 materials and methods top ↑ the immunology unit of the national health laboratory service (nhls) in tygerberg, south africa, uses elisas for serological quantification of antibody levels. before installation of the biorad phd™ system, all assays had been performed manually. the system performs sample dilution, dispenses patient samples and reagents into the microplate, and performs temperature-specific incubation and washing according to pre-defined protocols. automation of the mk016 enzyme-linked immunosorbent assay the 96-well microtitre plate included in the vacczyme™ human anti-haemophilus influenzae type b (hib) kit (mk016) from the binding site company (birmingham, united kingdom) is precoated with the hib capsular polysaccharide antigen conjugated to human serum albumin. in addition to controls and other reagents, the kit also contains five calibrators (0.1 mg/l – 9.0 mg/l) to construct a five-point calibration curve. concentration (logarithmic scale) is plotted against optical density (linear scale) to produce the calibration curve. the quantification range for anti-hib antibody concentration is 0.11 mg/l – 9.0 mg/l.9prediluted samples and controls were pipetted into the plate and incubated for 30 minutes. unbound proteins were removed by a wash step before addition of conjugate (purified peroxidase-labelled rabbit anti-human g-chain-specific immunoglobulin g). after a further 30 minutes of incubation, another wash step was applied to remove all excess conjugate. substrate (3.3’,5.5’ tetramethylbenzidine) was then added, which induced a colour change (from the characteristic serum colour to blue) over 30 minutes. phosphoric acid was then added to stop colour development. the optical density (od) was measured spectrophotometrically at 450 nm and the intensity of the final colour is proportional to the concentration of antibody present in the sample. a list of assays that are validated on the phd™ system is available from biorad (www.bio-rad.com; phd™ validated assay list). results top ↑ analyses with elisa kits from the biorad list produced valid results in our laboratory and these methods were automated without any problems. however, when performing the non-validated mk016 assay on the phd™ system, the calibrators did not produce the required od values recommended by the quality control sheet included in the kit (figure 1). figure 1: vacczyme™ anti-hib enzyme-linked immunosorbent assays calibration curve as provided on the quality control sheet. the highest calibrator reached an od of only 1.417 ± 0.245 (range 1.252–1.813) instead of 2.500 as specified on the quality control certificate. although the lower calibrators were associated with smaller margins of error, they showed a similar trend. all results calculated from this calibration curve were therefore too low and hence invalid.comparison of the individual steps of the automated and manual methods identified a difference of 10 minutes in all incubation periods. the phd™ system times incubation for each well individually and proceeds to the next step only once the exact incubation time has been reached for that specific well. however, timing of manual assays usually starts only after reagents have been added to the last well of the plate; i.e. well incubation is timed rather than plate incubation. in an effort to address this difference, all incubation steps were increased from 30 minutes to 40 minutes on the phd™ system. the duration of the washing steps of the two methods was very similar and therefore not regarded as contributing to the discrepant results. the duration of the washing steps was therefore left unchanged. a total of 16 calibration curves were subsequently generated from the phd™ system to validate the adjusted protocol. all the od values obtained produced acceptable calibration curves (figure 2) and the mean od value for the highest calibrator reached 2.295 ± 0.171 (median = 2.405; range = 1.934–2.553). the recommended values for the high and low controls are 2.4 mg/l – 3.6 mg/l and < 0.35 mg/l, respectively. the high and low controls as used on the phd™ system produced results of 2.538 ± 0.094 mg/l and < 0.35 mg/l, respectively. figure 2: vacczyme™ anti-hib enzyme-linked immunosorbent assay calibration curves before and after optimisation. to confirm our findings, calibrators and controls were included in different positions and orders on the plate. values obtained for these tests were within 5% of the required ranges, which suggested that the amended protocol had a uniform effect on all individual wells of the plate (data not shown). discussion top ↑ automated systems, such as the biorad phd™ instrument, are extremely useful and accurate in assessing immune responses to specific antigens following disease or vaccination. the major advantages of automation or semi-automation include the use of volumes as low as 1 ml, increased accuracy and reproducibility of results, better use of expensive skilled labour, faster overall laboratory turnaround time, ability to perform multiple assays simultaneously and cost effectiveness due to use of multianalyte reagents. use of an automated system also eliminates pipette volume variation and handling errors.accurate, reproducible and reliable laboratory results are crucial for patient management and care, such as initiating treatment in patients with possible immunodeficiency and revaccination of children with insufficient protection following routine childhood vaccination. it is furthermore important for evaluation of study cohort results or establishing reference ranges for specific populations or age groups. this study emphasises the importance of optimisation and validation whenever changing protocols or reagents in order to produce valid and accurate results. in the case of the mk016 assay it was not possible to transfer the protocol established for the manual method to the automated system without modification. after troubleshooting all the individual steps of both methods, a methodological difference was identified and addressed. the modified protocol was scrutinised by repeated measurements of samples of known concentration in different assays and plate positions before amending the existing protocol. acknowledgements top ↑ we would like to thank allere healthcare (pty) ltd (bedfordview, south africa) for donating test kits for optimisation. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. authors’ contributions c.d.b. was the project leader and responsible for experimental and project design, as well as performing the experiments and writing the manuscript. m.e. was the head of the laboratory where the experiments were conducted and w.p. provided valuable input into the final manuscript. references top ↑ 1. engvall e, perlman p. enzyme-linked immunosorbent assay (elisa). quantitative assay of immunoglobulin g. immunochemistry. 1971;8(9):871–874. http://dx.doi.org/10.1016/0019-2791(71)90454-x2. van weemen bk, schuurs ah. immunoassay using antigen-enzyme conjugates. febs lett. 1971;15(3):232–236. http://dx.doi.org/10.1016/0014-5793(71)80319-8 3. lequin r. enzyme immunoassay (eia)/enzyme-linked immunosorbent assay (elisa). clin chem. 2005;51(12):2415–2418. http://dx.doi.org/10.1373/clinchem.2005.051532 4. yalow r, berson s. immunoassay of endogenous plasma insulin in man. j clin invest 1960;39(7):1157–1175. http://dx.doi.org/10.1172/jci104130 5. gosling jp. a decade of development in immunoassay methodology. clin chem. 1990;36(8):1408–1427. 6. engvall e. the elisa, enzyme-linked immunosorbent assay. clin chem. 2010;56(2):319–320. http://dx.doi.org/10.1373/clinchem.2009.127803 7. miles lem, hales cn. labelled antibodies and immunological assay systems. nature 1968;219:186–189. http://dx.doi.org/10.1038/219186a0 8. engvall e, jonsson k, perlmann p. enzyme-linked immunosorbent assay, elisa. ii. quantitative assay of protein antigen, immunoglobulin g, by means of enzyme-labeled antigen and antibody-coated tubes. biochem biophys acta 1971;251:427–434. http://dx.doi.org/10.1016/0005-2795(71)90132-2 9. package insert, 24 may 2006. vacczyme™ human anti haemophilus influenzae type b (hib) enzyme immunoassay kit mk016. the binding site, birmingham, uk. abstract introduction methods results discussion acknowledgements references about the author(s) lindi-marie coetzee national health laboratory service (nhls), johannesburg, south africa department of molecular medicine and haematology, university of the witwatersrand, johannesburg, south africa naseem cassim national health laboratory service (nhls), johannesburg, south africa department of molecular medicine and haematology, university of the witwatersrand, johannesburg, south africa deborah k. glencross national health laboratory service (nhls), johannesburg, south africa department of molecular medicine and haematology, university of the witwatersrand, johannesburg, south africa citation coetzee l-m, cassim n, glencross dk. weekly laboratory turn-around time identifies poor performance masked by aggregated reporting. afr j lab med. 2020;9(1), a1102. https://doi.org/10.4102/ajlm.v9i1.1102 note: additional supporting information may be found in the online version of this article as supplementary document 1. original research weekly laboratory turn-around time identifies poor performance masked by aggregated reporting lindi-marie coetzee, naseem cassim, deborah k. glencross received: 18 sept. 2019; accepted: 14 sept. 2020; published: 21 dec. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: high-level monthly, quarterly and annual turn-around time (tat) reports are used to assess laboratory performance across the national health laboratory service in south africa. individual laboratory performances are masked by aggregate tat reporting across network of testing facilities. objective: this study investigated weekly tat reporting to identify laboratory inefficiencies for intervention. methods: cd4 tat data were extracted for 46 laboratories from the corporate data warehouse for the 2016/2017 financial period. the total tat median, 75th percentile and percentage of samples meeting organisational tat cut-off (90% within 40 hours) were calculated. total tat was reported at national, provincial and laboratory levels. provincial tat performance was classified as markedly or moderately poor, satisfactory and good based on the percentage of samples that met the cut-off. the pre-analytical, testing and result review tat component times were calculated. results: median annual tat was 18.8 h, 75th percentile was 25 h and percentage within cut-off was 92% (n = 3 332 599). corresponding 75th percentiles of component tat were 10 h (pre-analytical), 22 h testing and 1.6 h review. provincial 75th percentile tat varied from 17.6 h to 34.1 h, with three good (n = 13 laboratories), four satisfactory (n = 24 laboratories) and two poor performers (n = 9 laboratories) provinces. weekly tat analysis showed 12/46 laboratories (28.6%) without outlier weeks, 31/46 (73.8%) with 1–10 outlier weeks and 3/46 (6.5%) with more than 10 (highest of 20/52 weeks) outlier weeks. conclusion: masked tat under-performances were revealed by weekly tat analyses, identifying poorly performing laboratories needing immediate intervention; tat component analyses identified specific areas for improvement. keywords: cd4; turnaround time; laboratory performance; outliers; weekly reporting. introduction in south africa, public health facilities across 52 districts provide patient care through primary healthcare (phc) services, district, regional and tertiary hospitals. a wide spectrum of tests can be requested and submitted to the nearest pathology laboratory of the national health laboratory service (nhls). the nhls is the choice laboratory service provider of the south africa national department of health (ndoh). a network of more than 266 laboratories are strategically placed around the country to optimally accommodate the needs of local communities (urban and rural).1,2 routine laboratory tests have a predetermined total turn-around time (tat) cut-off that ensures that tests are processed within the required timeframe to effect the appropriate clinical intervention. total tat is defined as the time from first registration of a sample on the laboratory information system (lis) to the time a result is reviewed and released to the requesting physician. tat is in part determined by (1) the window of testing from venepuncture as prescribed by the test manufacturers, (2) time validity of sample integrity (e.g. how many hours or days before erythrocytes in blood samples die and cannot bind to antibodies effectively) and (3) the clinician timeline (emergency or quick-resulted laboratory testing vs routine laboratory testing).3,4,5 laboratory tat reflects the time taken for processing a sample and is a direct indicator of laboratory performance and an integral measure of efficiency where delays can impact patient management.3,4,5,6,7 hiv-associated tests like hiv viral load (vl) and cd4 counts, like all nhls laboratory tests, have strict predetermined organisational tat cut-offs, set to reflect treatment guidelines requirements and standards of care for hiv management by local authorities and the world health organization (who)8,9. these guidelines call for the availability of a cd4 result within 7–14 days.9 to meet this standard, the nhls has set a within-organisation standard of 40 h for 90% of all cd4 testing to be completed and results released. the accurate reporting of tat depends on the quality of data collected through the lis, that is, the inclusion of automated system date and timestamps at various time points in the journey from patient venesection to result review.10 for samples sent to nhls laboratories, four major date and timestamps are electronically collected and used to calculate tat components2,10: (1) pre-analytical time (lab-to-lab), that is, the time from first registration at any nhls source laboratory to referral receipt at the designated testing laboratory, (2) analytical time (reg-to-test), time from registration at the testing laboratory to result transmission to the lis, and (3) post-analytical time (test-to-review), time from test transmission onto the lis to result review and verification by a senior laboratory staff member (i.e. results become available for the requesting physician or nurse to access). total tat is the summation of all three tat components. the limitation of current tat reporting is that it commences when a sample is registered at a source laboratory (nearest to testing site), that is, the time lapse from patient venesection to sample arrival at the laboratory is not included in the pre-analytical tat.10 the majority of cd4 samples tested in nhls laboratories originate from public health clinics of the ndoh, with no electronic system linked to lis. for true clinical tat assessment and impact on patient care, the time from sample collection to result receipt should be tracked, but this remains a challenge.11 laboratory test tat in the nhls is monitored at national, provincial and laboratory level, with annual,12 quarterly and monthly reports generated routinely.10 traditionally, the mean tat is reported, but retrospective data analysis confirmed a non-gaussian tat distribution.4 taking this into account, cd4 tat reporting was upgraded to include the median, 75th percentile and percentage within cut-off to better reflect performance. the concept of classifying laboratory performance was also developed: laboratories are reported as good, satisfactory or poor, based on the 75th percentile and percentage within tat cut-off value quadrants as described in a recent publication.10 the current monthly reports are effective at giving management a snapshot of the cd4 programme and overall (global) laboratory performance10 but cannot be used for timely interventions. underlying problems with tat are not detected in real time, thus corrective actions are taken retrospectively, days or weeks after they occurred.10 more frequent reporting was thus recommended in addition to traditional tat reporting to enable more meaningful and timely interventions to improve laboratory performance. using these described tat parameters and classification, a weekly tat dashboard was developed and rolled out nationally in 2018 for monitoring the tat of some tests (the most requested hiv, tuberculosis and non-communicable diseases tests). turn-around time data will inform corrective action such as additional test operator training.13,14 although the cut-off values for cd4 testing changed from 85% within 48 h to 90% within 40 h in the 2016/2017 financial year, the concept and wording of laboratory performance classification were retained as managers were well acquainted with these terms. the aim of this article is to describe how weekly review of cd4 tat analysis can enable the identification of non-compliant laboratories to facilitate effective and timely corrective action and ensure continuous quality management for improved service delivery. data analysed represents performance prior to the national implementation of the weekly tat dashboard. methods ethical considerations ethics clearance was obtained from the university of the witwatersrand (m1706108). no patient identifiers were used for this study and laboratories and provinces were anonymised. cd4 turn-around time data cd4 tat data were extracted from the corporate data warehouse for the financial period april 2016 to march 2017 (2016/2017 financial year) for 46 cd4 testing laboratories. total tat was calculated for each sample tested and reported for 52 weeks, together with the tat component data. data analysis included the calculation of the median, 75th percentile and the percentage of samples with a tat within the stipulated organisation cut-off per week. this was reported per laboratory and per province (aggregated data of laboratories within each of the nine provinces). performance classification was introduced at provincial level and based on the percentage samples within tat cut-off as follows: (a) ≥ 95%: good performance, (b) 90.0% – 94.9%: satisfactory performance; (c) 85.0% – 89.9%: moderate to poor performance and (d) 80.0% – 84.99%: poor performance. performance thus refers to the degree of compliance with nhls tat cut-off. the number of weeks that provinces and laboratories did not achieve the 40 h cut-off was reported. outlier weeks were defined as weeks where the total tat of all samples tested did not achieve 90% with a tat under 40 h. additional data analysis was done on the weekly laboratory data to describe the tat component contribution to total tat per laboratory per week and included: (1) lab-to-lab tat, (2) reg-to-test tat and (3) test-to-review tat. the target times set for each tat component are (1) 14 h, (2) 24 h and (3) 2 h. although tat component analysis by laboratory is distributed weekly, for this study, only specific laboratories were selected to represent different levels of compliance and performance categories to demonstrate how individual tat components affects total tat. outlying laboratory tat components (> 24 h and < 2 h) were correlated with beckman coulter engineer logs to verify the impact of instrument downtime on prolonged tat (data not shown). laboratory site visits were conducted to assess root cause analysis for below standard tat (< 90% processed for > 40 h) performance identified. statistical analysis data were prepared and analysed using sas version 9.4 (cary, north carolina, united states) and graphpad software (san diego, california, united states). the nine provinces were numbered 1–9, with individual laboratories within a province assigned a number and labelled accordingly (i.e. 1.5 represents province 1 and laboratory 5). box and whisker plots were created for individual laboratory data over 52 weeks. national total test volumes and tat was plotted against the 50th and 75th percentiles in a bar graph. provincial total tat was plotted as 75th percentile per performance category per week. individual laboratory distribution of 75th percentiles per performance category was plotted, indicating high (> 350 samples per day), medium (150-350 samples per day) and low volume facilities (< 150 samples per day). component tat was plotted as stacked bar graphs, showing the 75th percentile for pre-analytical, testing, and review tat for selected laboratories representing the four performance categories. results global annual cd4 total turn-around time overview in this study 3 332 599 cd4 test tat were analysed. for the 2016/2017 financial year, the national median tat for all cd4 tests was 18 h with a 75th percentile of 23 h (table 1). overall, 91% of all samples met the stipulated cut-off of 40 h, indicating good overall laboratory performance for meeting organisational criteria for cd4 testing. the matched organisational component median (and tat 75th percentiles) for lab-to-lab tat was 6.3 (10 h), reg-to-test tat was 17.3 (22 h) and test-to-review tat was 1.3 (2.1 h). table 1: national annual national health laboratory service cd4 total turn-around time for all samples tested during the 2016/2017 financial year. national weekly total turn-around time the weekly distribution of the 50th percentile (median) and 75th percentile showed good consistency despite fluctuations in test volumes across the network of testing laboratories (n = 52) (figure 1). the median ranged from 14 h to 18 h, while the 75th percentile ranged from 21 h to 26 h (figure 1). test volumes fluctuated between 23 681 to 80 821 per week (mean of 64 088 tests weekly). figure 1: national total turn-around time of national health laboratory service cd4 tests per week for the 2016/2017 financial year. the 50th percentile (median, green circles), 75th percentile (red circles) and volume of samples (grey bars) are depicted. provincial total turn-around time distribution (75th percentile) per testing week annual global tat distribution did not identify any poor performance over 52 weeks. to identify poor performances, national tat were analysed per province. the number of cd4 tests ranged from 65 395 (lowest) to 1 066 137 (highest) (table 2). the percentage of samples tested within the 40-h tat cut-off ranged from 82% to 98%. provincial performance classification was made based on the latter percentage per province, as a to d (as described above). a minimum of three laboratories represented each province. table 2: annual provincial national health laboratory service cd4 data, indicating test volumes, the 75th percentile total turn-around time, the percentage of samples within turn-around time cut-off and the number of representative laboratories for 2016/2017 financial year. three provinces (3, 7 and 9) were classified as category a (good performance). these laboratories were able to maintain all cd4 reporting within organisation-stipulated tat at greater than 95% and 75th percentile tat of 21.7 h (province 3), 17.6 h (province 7) and 2.08 h (province 9). four provinces (1, 2, 4 and 8) were categorised b (satisfactory performance) having 90% – 94.9% of samples meeting the tat cut-off; 75th percentile values for these provinces were 23 h, 25.1 h, 23.4 h and 18.9 h, respectively. two provinces failed to meet the tat cut-off and were classified as categories c (moderate poor performance; province 6) and d (markedly poor performance province 5), indicating that less than 90% of samples met the cd4 tat. within the latter provincial performance clusters (c and d), the 75th percentile reported was 28.8 h and 34.2 h. no weekly outliers (weeks where total tat did not meet 90% < 40 h) were noted in the three good performance provinces (3, 7, and 9; comprising n = 13 individual laboratories; figure 2a). the 75th percentile total tat for these provinces never exceeded 30 h during the test period. figure 2b describes the four provinces with satisfactory performance (1, 2, 4 and 8, representing 24 individual laboratories; table 2). among this group, province 2 and 4 showed better consistency, easily meeting the tat cut-off throughout the test period. province 1 had a week with 75th percentile value of 37 h while province 8 had two weeks with 75th percentile values of 38 h and 40h. figure 2c represents province 6 comprising five laboratories, categorised as a moderate or poor performer, due to inconsistency, especially during weeks 24–34 of 2016, with two weeks having a 75th percentile tat of over 40 h. after week 35 of 2016, performance stabilised and 75th percentile values corrected to within cut-off values. one province (four individual laboratories; figure 2d) was classified as a markedly poor performer and characterised by inconsistency and repeated failure to meet the cut-off with less than 85% of reported tests meeting stipulated organisational tat cut-off (10 weeks exceeding 40 h). figure 2: weekly national 75th percentile cd4 turn-around time of nine provinces per performance category for the 2016/2017 financial year. (a) good performance (n = 3 provinces); (b) satisfactory performance (n = 4 provinces); (c) moderate to poor performance (n = 1 province) and (d) markedly poor performance (n = 1 province). individual laboratory total turn-around time by week and performance category the different performance levels identified at provincial level still masked the performance and contribution of individual laboratories to provincial performance. scatter plots were constructed to visualise the performance of individual laboratories over 52 weeks per provincial performance category. results showed that irrespective of the provincial performance classification (figures 3a–d), individual laboratory performance included good, satisfactory and poor performance laboratories. figure 3: scatter plots of the 75th percentile total turnaround time of individual laboratories in the national health laboratory service within the provincial performance classification groups a to d for the 2016/2017 financial year. the overall 75th percentile total turn-around time per laboratory is indicated above each plot. (a) good performance (n = 13 laboratories); (b) satisfactory performance (n = 24 laboratories), (c) moderate to poor performance (n = 5 laboratories), and (d) markedly poor performance (n = 4 laboratories). high-volume (blue circles), medium volume (light blue squares) and low-volume (pink triangles) laboratories are indicated. the red line in each graphs represents the target total tat. individual median total tat is indicated above each representative laboratory. the 75th percentile across 52 weeks for the good performance provinces (3 provinces and 13 laboratories) showed good overall compliance (tight clumping of weekly 75th percentile values) where the overall 75th percentile for the whole period ranged from 5.9 h (laboratory 7.1) to 24.8 h (laboratory 9.2) (figure 3a). similarly, the satisfactory performance provinces (n = 24 laboratories) (figure 3b) had a 75th percentile ranging from 10 h (laboratory 4.3) to 34.2 h (laboratory 4.4). the provinces having moderately poor performance had variable tat between 9 h (laboratory 6.4) to 35.7 h (laboratory 6.2) (figure 3c) while markedly poor performance provinces had tat 75th percentile values ranging from 17.2 h (laboratory 5.4) to 39.7 h (laboratory 5.5) (figure 3d). overall, four laboratories recorded a 52-week median tat of more than 30 h. the number of weeks that laboratories did not meet the cut-off criteria of 90% with tat under 40 h varied among categories and laboratories (0–22 weeks), where 12 of 46 laboratories (irrespective of performance category) had zero outlying weeks (26%), 25/46 (54%) more than 5 outlying weeks and 6 (13%) between 6 and 10 outlying weeks. only three laboratories showed outliers for more than 10 weeks where cut-off was not met (6.5%). case examples of individual laboratory component turn-around time analysis figure 4a shows a good performer high-volume laboratory, doing more than 350 samples per day with no outlying weeks (exceeding 40 h cut-off). over the 52 weeks, 86 559 tests were performed by this laboratory. a lab-to-lab 75th percentile of 9.5 h was reported (ranging from 3 h to 16 h), with a reg-to-test of 8.8 h (range from 6 h to 17 h) and a test-to-review of 1.3 h (range from 0 h to 4 h). more than 98% of all samples tested had a total tat of under 40 h. figure 4: stacked bar graphs showing examples of individual laboratory weekly performance for 2016/2017 financial year. 75th percentile turn-around time components color-coded: lab-to-lab (orange), reg-to-test (blue) and test-to-review (green), with cut-off of 40 h (red dotted lines). (a) good performance (laboratory a); (b) satisfactory performance (laboratory b); (c) moderate to poor performance (laboratory c) and (d) markedly poor performance (laboratory d). figure 4b represents a satisfactory performance laboratory, doing 63 998 samples for the period. it reported nine non-consecutive weeks of outliers (exceeding 40 h). of these outlier weeks, eight were due to prolonged reg-to-test (testing delay), where this component contributed between 27 h and 87 h to the total tat reported for these weeks. one week (week 51 of 2016) had an extended lab-to-lab value of 65 h, due to confirmed challenges with logistics. test-to-review ranged from 0 h to 6 h and, as such, had no contribution to the outlying weeks. extended reg-to-test (within laboratory tat) seen in weeks 6–11 of 2017 correlated with instrument downtime based on data provided by beckman coulter call centre log on engineers dispatched. the laboratory represented in figure 4c (moderate to poor performance) tested 77 640 samples during the 52 weeks and had an overall lab-to-lab 75th percentile of 17.7 h (ranging from 10 h to 78 h per week), with 11 weeks exceeding the target total tat (highest recorded total tat of 129 h). the lab-to-lab component (orange bars) contributed to total tat outlying weeks during weeks 21, 30–32, and 48 of 2016 and weeks 11 and 18 of 2017. reg-to-test (within laboratory tat) was the leading cause of outliers noticed during weeks 30–32, 47–48 of 2016 and weeks 11, 15 and 18 of 2016. the combined extended tat in two components (i.e. pre-analytical or lab-to-lab and analytical or reg-to-test) for weeks 30–32 of 2016 and weeks 15 and 18 of 2017 contributed to the total tat for this laboratory to fall into a category of 85% – 90% of samples within the tat target of 40 h. the laboratory contributing the most outlying weeks to group d (figure 3d) was analysed for component tat. this laboratory had 18 weeks of exceeding the target total tat. this was for the most part due to prolonged within laboratory tat (blue bars, figure 4d). from week 31 to 45 of 2016 the reg-to-test component contributed as much as 81 h (week 40 of 2016) to the weekly tat. during this period, some weeks also experienced prolonged test-to-review times of up to 25 h (week 1 of 2017). discussion tat remains a key performance indicator of laboratory service efficiency.4,14 the parameters reported (mean vs median) and time intervals of reporting impacts the utilisation of tat as a means to identify and address non-compliance to organisational cut-offs. definitions of tat may vary and depend on the test (routine or emergency), priority of reporting (immediate or delayed clinical intervention), the population served and activities or components measured.4 the clinical outcome, needs and responsibilities of management determine how tat information is used to ensure that there are no unnecessary delays in result reporting. across the nhls, tat information typically remains the jurisdiction of the testing laboratory where the laboratory manager uses this data to identify problems and initiate corrective action; the individual laboratory has sole and direct access to its own daily or weekly tat data.3,4,5 tat monitoring is however critical for priority programmes, such as hiv and tuberculosis,2,12 where individual laboratories monitor their respective test tat, while the organisation is responsible for reporting performance of the network of laboratories. relevant updated information on the efficiency of service delivery is vital in this context for risk assessment and timely intervention to ensure the continued excellence of service delivery10 and meeting dire local hiv and tuberculosis programme needs.9 ideally, sample-by-sample real-time reporting of tat would be the preferred way to monitor and assist laboratories in the identification of specific service delivery and related tat challenges. hierarchical global overview (usually annual) tat reporting is however the simplest and most widely used, but masks poor performance, as confirmed by data from this study. interrogating the weekly data by drilling down to laboratory level at weekly intervals, enables the identification of outliers and poor performers. this study showed that lower hierarchical levels, as well as shorter time periods, can unveil problematic and inefficient testing laboratories (figure 2 and figure 3). adding weekly tat component analysis at laboratory level further identifies problematic testing weeks and possible causes of prolonged tat (figure 4 and box 1). box 1: summary of study findings. the most common issues that impacted on prolonged lab-to-lab (pre-analytical) tat were delays in transport or sample collection from clinics to testing laboratories and changes in courier routes and pick up times. reg-to-test times (laboratory tat) were prolonged due to instrument downtime, lack of trained staff to operate testing instruments, delayed sample registration in the testing facility, challenges with reagent availability and timely delivery, and staff strikes.15 delays in the test-to-review phase, were mostly due to lack of result auto-authorisation in high-volume laboratories, nonavailability of result authorising staff when samples are analysed during night shifts or where problems with connectivity of the trackcare lis were reported. several references focus on the main causes of tat delays.5,16,17,18,19,20,21,22 the main objective of this study, however, was not to describe causes of delayed tat, but to emphasise the importance of tat monitoring with shorter time intervals as a means of more proactive interventions for sustained good performance across network of laboratories. data reported here demonstrate the need for more frequent tat reporting in effective performance management.10,13,15,23,24 an interactive weekly tat dashboard was introduced nationally in 2018 for the most requested tests across the nhls. frequent performance reporting can be effective in identifying challenges with meeting target tat cut-offs allowing for timely interventions.13 weekly assessment of tat and tat components not only identifies problematic testing laboratories or days with tat challenges, but also enables the identification of individual outlier samples that can be investigated (root cause analysis) to assess causes of tat delays. based on the data presented in this study, further refinement of the current reporting platforms is recommended to include daily reporting for rapid proactive intervention. a further recommendation is to integrate daily quality control tests, external quality assessment testing and equipment downtime supplier call-out data into the reporting to assist with focused troubleshooting and interventions. turn-around time monitoring and reporting are however guided by the requirements of the end user and will continue to be available at various time intervals for laboratory network management to assess overall trends, with weekly or daily reports to laboratory and programme managers enabling timely proactive intervention to ensure optimal laboratory performance and timely patient result reporting. limitations the monitoring of tat in the nhls is currently limited as end-to-end sample tracking system is absent. the tat reported in this article thus only represents the time from first registration on the lis to review of the result. conclusion national and provincial analysis of tat mask individual laboratory performance; therefore, tat analysis by week and by laboratory is recommended to highlight laboratory tat inefficiencies. root-cause analyses were able to identify pre-analytical, analytical or post-analytical factors contributing to performance. tat data was used to categorise performance at the provincial and laboratory level. this study used the concept of zooming in to lower levels and shorter times of tat reporting to identify possible non-compliant laboratories. in conclusion: (1) outlying weeks are not prescribed by provincial or laboratory classification of performance, (2) performance did not correlate to the size of the laboratory (i.e. test volumes of high, medium and low), (3) there were laboratories that had no outlier weeks during the analysed period that can serve as model laboratories for setting performance standards and good reproducibility of week-on-week performance across a network of testing laboratories. acknowledgements this article was in part presented as a poster at the african society for laboratory medicine meeting in 2018, abuja, nigeria (id:ps-2-3b-070), 10–13 december 2018. the authors thank area, business and laboratory managers in the nhls for their feedback on the use of the weekly tat dashboard. thank you to the central data warehouse for the availability of data. competing interests the authors have declared that no competing interests exist. authors’ contributions d.k.g. supervised the study by providing leadership and oversight as the project leader. l.-m.c. and n.c. designed the study, developed the methodology and conducted the research, data analysis, initial write-up and review. d.k.g. reviewed the data, provided editorial comments and technical input. all authors contributed to the manuscript development. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement data is available as online supplementary document 1. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references national health laboratory service (nhls). nhls annual report 2014/15 sandringham [homepage on the internet]. johannesburg: national health laboratory service; 2015 [cited 2017 jul 26]. available from: http://intranet.nhls.ac.za/assets/files/policy/nhls_annual_report_2015.pdf glencross dk, coetzee l, cassim n. an integrated tiered service delivery model (itsdm) based on local cd4 testing demands can improve turn-around times and save costs whilst ensuring accessible and scalable cd4 services across a national programme. plos one. 2014;9(12):e114727. https://doi.org/10.1371/journal.pone.0114727 goswami b, singh b, chawla r, gupta vk, mallika v. turn around time (tat) as a benchmark of laboratory performance. indian j clin biochem. 2010;25(4):376–379. https://doi.org/10.1007/s12291-010-0056-4 hawkins rc. laboratory turnaround time. clin biochem rev. 2007;28(4):179–194. pati hp, singh g. turnaround time (tat): difference in concept for laboratory and clinician. indian j hematol blood transfus. 2014;30(2):81–84. https://doi.org/10.1007/s12288-012-0214-3 valenstein p. laboratory turnaround time. am j clin pathol. 1996;105(6):676–688. https://doi.org/10.1093/ajcp/105.6.676 valenstein pn, emancipator k. sensitivity, specificity, and reproducibility of four measures of laboratory turnaround time. am j clin pathol. 1989;91(4):452–457. https://doi.org/10.1093/ajcp/91.4.452 world health organisation. guidelines for managing advanced hiv disease and rapid initiation of antiretroviral therapy [homepage on the internet]. policy brief. geneva: who; 2017 [cited 2019 jul 16]. available from: https://www.who.int/hiv/pub/toolkits/advanced-hiv-disease-policy/en/ national department of health (ndoh). national consolidated guidelines for the prevention of mother-to-child transmission of hiv (pmtct) and the management of hiv in children, adolescents and adults [homepage on the internet]. pretoria; 2015 [cited 2019 jul 16]. available from: https://sahivsoc.org/files/art%20guidelines%2015052015.pdf coetzee l, cassim n, glencross dk. using laboratory data to categorise cd4 laboratory turn-around-time performance across a national programme. afr j lab med. 2018;7(1):a665. https://doi.org/10.4102/ajlm.v7i1.665 stotler ba, kratz a. determination of turnaround time in the clinical laboratory: “accessioning-to-result” time does not always accurately reflect laboratory performance. am j clin pathol. 2012;138(5):724–729. https://doi.org/10.1309/ajcpyhbt9oqrm8dx national health laboratory service (nhls). annual report 2017/18 [homepage on the internet]. johannesburg: national health laboratory service (nhls); 2018 [cited 2018 dec 10]. available from: http://www.nhls.ac.za/assets/files/an_report/nhls_ar_2018.pdf cassim n, coetzee lm, tepper mee, perelson l, glencross dk. timely delivery of laboratory efficiency information, part ii: assessing the impact of a turn-around time dashboard at a high-volume laboratory. afr j lab med. 2020;9(2):a948. https://doi.org/10.4102/ajlm.v9i2.948 cassim n, coetzee l-m, tepper m, motlonye b, glencross dk, editors. tat as a risk model for operational services. johannesburg: pathred. 2017. coetzee l, cassim n, tepper m, glencross dk. the importance of reporting individual weekly laboratory turn-around-time (tat) to identify outliers and underperformance masked during global annual tat review. aslm conference; 2018 dec 10–13; abuja; p. poster: id ps-2.3b-070. cakirca g. the evaluation of error types and turnaround time of preanalytical phase in biochemistry and hematology laboratories. iran j pathol. 2018;13(2):173–178. https://doi.org/10.30699/ijp.13.2.173 chauhan kp, trivedi ap, patel d, gami b, haridas n. monitoring and root cause analysis of clinical biochemistry turn around time at an academic hospital. indian j clin biochem. 2014;29(4):505–509. https://doi.org/10.1007/s12291-013-0397-x jalili m, shalileh k, mojtahed a, mojtahed m, moradi-lakeh m. identifying causes of laboratory turnaround time delay in the emergency department. arch iran med. 2012;15(12):759–763. https://doi.org/0121512/aim.008 khalifa m, khalid p. improving laboratory results turnaround time by reducing pre analytical phase. stud health technol inform. 2014;202:71–74. https://doi.org/10.3233/978-1-61499-423-7-71 lou ah, elnenaei mo, sadek i, thompson s, crocker bd, nassar ba. multiple preand post-analytical lean approaches to the improvement of the laboratory turnaround time in a large core laboratory. clin biochem. 2017;50(15):864–869. https://doi.org/10.1016/j.clinbiochem.2017.04.019 minchella pa, chipungu g, kim aa, et al. specimen origin, type and testing laboratory are linked to longer turnaround times for hiv viral load testing in malawi. plos one. 2017;12(2):e0173009. https://doi.org/10.1371/journal.pone.0173009 saathoff am, macdonald r, krenzischek e. effectiveness of specimen collection technology in the reduction of collection turnaround time and mislabeled specimens in emergency, medical-surgical, critical care, and maternal child health departments. comput inform nurs. 2018;36(3):133–139. https://doi.org/10.1097/cin.0000000000000402 cassim n, tepper me, coetzee lm, glencross dk. timely delivery of laboratory efficiency information, part i: developing an interactive turnaround time dashboard at a high-volume laboratory. afr j lab med. 2020;9(2):a947. https://doi.org/10.4102/ajlm.v9i2.947 coetzee l, cassim n, tepper m, glencross dk. standardizing individual laboratory turnaround time (tat) performance amongst laboratories in a cd4 testing network. pathcape 2018: 56th international fsasp congress; 2018 aug 16–18; stellenbosch. ajlm 9(1)_2020_contents.indd http://www.ajlmonline.org open access table of contents i original research kaposi’s sarcoma-associated herpesvirus protein orf75 among hiv-1 patients in kenya rodgers n. demba, sylviah m. aradi, matilu mwau, walter o. mwanda african journal of laboratory medicine | vol 9, no 1 | a939 | 25 august 2020 original research prevalence and risk factors for red blood cell alloimmunisation among sickle cell patients in mwanza city, tanzania erius tebuka, mwesige charles, jeffer o. bhuko african journal of laboratory medicine | vol 9, no 1 | a823 | 10 september 2020 original research fluorescence microscopy for the diagnosis of smear-negative pulmonary tuberculosis in ethiopia gemeda abebe, dossegnaw aragaw, mulualem tadesse african journal of laboratory medicine | vol 9, no 1 | a810 | 28 september 2020 original research using systematized nomenclature of medicine clinical term codes to assign histological findings for prostate biopsies in the gauteng province, south africa: lessons learnt naseem cassim, ahsan ahmad, reubina wadee, jaya a. george, deborah k. glencross african journal of laboratory medicine | vol 9, no 1 | a909 | 28 september 2020 original research development of dried tube specimens for xpert mtb/rif proficiency testing kyle degruy, katherine klein, zilma rey, patricia hall, andrea kim, heather alexander african journal of laboratory medicine | vol 9, no 1 | a1166 | 29 september 2020 original research endometrial sampling at an academic hospital in south africa: histological findings, lessons learnt and interesting surprises reena d. mohanlal african journal of laboratory medicine | vol 9, no 1 | a1038 | 29 september 2020 original research serological detection of brucellosis among febrile, malaria-negative children and domesticated dogs in an urban african setting john b. kalule, joseph tomusange, teddy namatovu african journal of laboratory medicine | vol 9, no 1 | a864 | 30 september 2020 original research review of the stepwise laboratory quality improvement process towards accreditation (slipta) version 2:2015 tjeerd a.m. datema, linda oskam, jacqueline e.w. broerse, paul r. klatser african journal of laboratory medicine | vol 9, no 1 | a1068 | 28 october 2020 original research postmortem diagnosis of covid-19: antemortem challenges of three cases at the 37 military hospital, accra, ghana seth a. attoh, frederick hobenu, lawrence edusei, kwasi agyemanbediako, clement t. laryea, edward o. nyarko, michael k. amedi, richard h. asmah, edward asumanu, mary mcaddy, anthony maison, godwin nyarko, raymond d. fatchu, kafui akakpo african journal of laboratory medicine | vol 9, no 1 | a1290 | 03 november 2020 56 62 67 73 82 90 97 103 110 page i of iii table of contents i editorial african laboratory medicine in the time of covid-19 iruka n. okeke african journal of laboratory medicine | vol 9, no 1 | a1447 | 21 december 2020 original research diagnostic outcomes of bone marrow aspirate and trephine biopsies performed at a hospital in kwazulu-natal, south africa wanda s. tshabalala, somasundram pillay, douglas p.k. wilson african journal of laboratory medicine | vol 9, no 1 | a1028 | 25 february 2020 original research evaluation of corrective actions of feedback from clinicians on clinical laboratory services at bamenda regional hospital laboratory, cameroon victor n. fondoh, charles n. awasom, rebecca enow-tanjong, richard m. fondoh, patrick njukeng, judith shang, julianna ndasi, moses samje, claris n. muluh, thompson n. kinge african journal of laboratory medicine | vol 9, no 1 | a843 | 23 march 2020 original research clinical staff knowledge and awareness of point-of-care-testing best practices at tygerberg hospital, south africa thumeka p. jalavu, megan rensburg, rajiv erasmus african journal of laboratory medicine | vol 9, no 1 | a853 | 16 july 2020 original research evaluation of the ogawa-kudoh method for tuberculosis isolation in two health units in mozambique carla m. madeira, khalide i. azam, daisy n. sato, celso khosa, nilesh bhatt, sofia o. viegas african journal of laboratory medicine | vol 9, no 1 | a929 | 20 july 2020 original research prevalence of cryptococcal antigen (crag) among hiv-positive patients in eswatini, 2014–2015 samson m. haumba, mitsuru toda, rossana jeffries, peter ehrenkranz, munyaradzi pasipamire, trong ao, nomthandazo lukhele, sikhathele mazibuko, mandzisi mkhontfo, rachel m. smith, tom chiller african journal of laboratory medicine | vol 9, no 1 | a933 | 29 july 2020 original research detecting tuberculosis in pregnant and postpartum women in eswatini munyaradzi pasipamire, edward broughton, mandzisi mkhontfo, gugu maphalala, batsabile simelane-vilane, samson haumba african journal of laboratory medicine | vol 9, no 1 | a837 | 30 july 2020 original research prevalence of urinary schistosomiasis amongst primary school children in ikwo and ohaukwu communities of ebonyi state, nigeria nse o. umoh, chimezie f. nwamini, nyoho j. inyang, anthony n. umo, victor u. usanga, amos nworie, michael o. elom, boniface n. ukwah african journal of laboratory medicine | vol 9, no 1 | a812 | 24 august 2020 original research prevalence and aetiology of moderate and severe thrombocytopenia in a tertiary and quaternary centre in kwazulu-natal ayanda g.p. jali, bongani b. nkambule african journal of laboratory medicine | vol 9, no 1 | a799 | 24 august 2020 1 4 10 17 25 30 37 46 51 vol 9, no 1 (2020) issn: 2225-2002 (print) | issn: 2225-2010 (online)african journal of laboratory medicine http://www.ajlmonline.org open access table of contents ii original research adult and paediatric haematology and clinical chemistry laboratory reference limits for liberia mark w. kieh, sarah m. browne, greg a. grandits, julie blie, jestina w. doe-anderson, marie l. hoover, bionca davis, cavan s. reilly, james d. neaton, h. clifford lane, stephen b. kennedy african journal of laboratory medicine | vol 9, no 1 | a1080 | 25 november 2020 original research infant hiv diagnosis and turn-around time for testing in malawi, 2015 hammad ali, peter minchella, geoffrey chipungu, evelyn kim, james kandulu, dalitso midiani, andrea kim, mahesh swaminathan, steve gutreuter, john nkengasong, daniel singer african journal of laboratory medicine | vol 9, no 1 | a904 | 26 november 2020 original research a global proficiency testing programme for xpert® mtb/rif using dried tube specimens, 2013–2015 katherine klein, kyle degruy, zilma rey, patricia hall, andrea kim, steve gutreuter, heather alexander african journal of laboratory medicine | vol 9, no 1 | a1167 | 27 november 2020 original research prevalence and antifungal susceptibility of gastrointestinal candidiasis among diabetic patients: a cross-sectional study anthony p. oyom, emmanuel okello, victoria acam, christine aramo, bashir mwambi, john c. okiria, caesar oyet african journal of laboratory medicine | vol 9, no 1 | a997 | 10 december 2020 original research evaluation of ceftriaxone-sulbactam-disodium edetate adjuvant combination against multi-drug resistant gram-negative organisms shilpi gupta, mahadevan kumar, shelinder p.s. shergill, kundan tandel african journal of laboratory medicine | vol 9, no 1 | a991 | 10 december 2020 original research validation of phase for deriving n-acetyltransferase 2 haplotypes in the western cape mixed ancestry population celeste swart, surita meldau, chad m. centner, adrian d. marais, fierdoz omar african journal of laboratory medicine | vol 9, no 1 | a988 | 17 december 2020 original research sensitivity and specificity of mean platelet volume as a laboratory marker for irritable bowel syndrome: can it be added to rome criteria? mostafa vaghari-tabari, soheila moein, durdi qujeq, mehrdad kashifard, haydeh alaoddolehei, karimollah hajian-tilaki african journal of laboratory medicine | vol 9, no 1 | a1001 | 21 december 2020 original research serologic evidence of seasonal influenza a and b in hiv patients on combined antiretroviral therapy in lagos, nigeria abdulazeez a. anjorin, barakat a. adepoju african journal of laboratory medicine | vol 9, no 1 | a1048 | 21 december 2020 original research haematological indices of sickle cell patients with chronic leg ulcers on compression therapy oluwatoyin a. babalola, ayodele ogunkeyede, abayomi b. odetunde, foluke fasola, anthony a. oni, chinedum p. babalola, adeyinka g. falusi african journal of laboratory medicine | vol 9, no 1 | a1037 | 21 december 2020 118 126 133 141 148 154 161 166 172 original research categorising specimen referral delays for cd4 testing: how interlaboratory distances and travel times impact turn-around time across a national laboratory service in south africa naseem cassim, lindi m. coetzee, deborah k. glencross african journal of laboratory medicine | vol 9, no 1 | a1120 | 21 december 2020 original research weekly laboratory turn-around time identifies poor performance masked by aggregated reporting lindi-marie coetzee, naseem cassim, deborah k. glencross african journal of laboratory medicine | vol 9, no 1 | a1102 | 21 december 2020 original research haematological reference intervals for healthy adults in bamenda, cameroon victor n. fondoh, richard m. fondoh, charles n. awasom, pefoule l. edith, winlove a. ntungwen, bong roland, rebeca enow-tanjong, patrick njukeng, judith shang, egbe p. egbengu, talkmore maruta, akazong etheline, robert leke, ayuk leo, denis nsame african journal of laboratory medicine | vol 9, no 1 | a1193 | 21 december 2020 original research biochemical changes in whole blood stored for transfusion at bungoma county referral hospital, kenya phidelis m. marabi, stanslaus musyoki, angela amayo african journal of laboratory medicine | vol 9, no 1 | a1182 | 21 december 2020 lessons from the field h3africa partnerships to empower clinical research sites to generate highquality biological samples talishiea croxton, ndidi agala, emmanuel jonathan, olasinbo balogun, petronilla j. ozumba, enzenwa onyemata, shefiya lawal, manmak mamven, samuel ajayi, sylvia e. melikam, mayowa owolabi, bruce ovbiagele, dwomoa adu, akinlolu ojo, christine m. beiswanger, alash’le abimiku african journal of laboratory medicine | vol 9, no 1 | a935 | 18 march 2020 lessons from the field addressing the shortage of pathologists in africa: creation of a mmed programme in pathology in zambia victor mudenda, evans malyangu, shahin sayed, kenneth fleming african journal of laboratory medicine | vol 9, no 1 | a974 | 03 june 2020 lessons from the field establishing the college of pathologists of east, central and southern africa – the regional east central and southern africa college of pathology shahin sayed, rudo mutasa, ephata kaaya, victor mudenda, erasmus rajiv, edda vuhahula, jamilla rajab, robert lukande, edwin walong, angela mutuku, kenneth fleming african journal of laboratory medicine | vol 9, no 1 | a979 | 03 june 2020 lessons from the field laboratory organisation and management of sars-cov-2 infection in niger, west africa abdourahamane yacouba, adamou lagaré, daouada alhousseini maiga, halimatou moumouni sambo, sani ousmane, zelika hamidou harouna, boubacar marou, maman k. sanoussi, balki aoula, ali amadou, hassane boureima, saidou amatagas, abdoulaye ousmane, eric adehossi, saidou mamadou african journal of laboratory medicine | vol 9, no 1 | a1308 | 21 december 2020 180 187 195 203 208 217 224 232 page ii of iii http://www.ajlmonline.org open access table of contents iii brief report herpes simplex virus-2 infections in pregnant women from south africa: evaluation of the immunoflow rapid test shanthie govender, lungile mbambo, makandwe nyirenda, motshedisi sebitloane, nathlee abbai african journal of laboratory medicine | vol 9, no 1 | a854 | 31 august 2020 brief report rapid testing for respiratory syncytial virus in a resource-limited paediatric intensive care setting howard newman, donald tshabalala, sikhumbuzo mabunda, nokwazi nkosi, candice carelson african journal of laboratory medicine | vol 9, no 1 | a1084 | 08 december 2020 brief report critical success factors for vietnamese laboratories striving to implement quality management systems cathy robinson, james johnson, katy yao, hien bui african journal of laboratory medicine | vol 9, no 1 | a937 | 18 december 2020 case study post-procedural bacillus cereus septic arthritis in a patient with systemic lupus erythematosus barend mitton, roxanne rule, nontombi mbelle, wesley van hougenhouck-tulleken, mohamed said african journal of laboratory medicine | vol 9, no 1 | a1119 | 20 august 2020 case study response to a cluster of severe acute respiratory syndrome coronavirus 2 cases at a diagnostic laboratory christoffel j. opperman, gert j.k. marais, michelle naidoo, marvin hsiao, nazlee samodien african journal of laboratory medicine | vol 9, no 1 | a1307 | 25 august 2020 case study brucellosis – laboratory workers’ nightmare come true: a case study lebogang skosana, farzana ismail, nontombi mbelle, mohamed said african journal of laboratory medicine | vol 9, no 1 | a1114 | 29 september 2020 review article the network approach to laboratory procurement and supply chain management: addressing the system issues to enhance hiv viral load scale-up jason williams, dianna edgil, matthew wattleworth, clement ndongmo, joel kuritsky african journal of laboratory medicine | vol 9, no 1 | a1022 | 13 august 2020 review article covid-19 rapid diagnostic test could contain transmission in lowand middle-income countries adesola olalekan, bamidele iwalokun, oluwabukola m. akinloye, olayiwola popoola, titilola a. samuel, oluyemi akinloye african journal of laboratory medicine | vol 9, no 1 | a1255 | 30 september 2020 237 242 247 251 255 259 263 272 opinion paper scaling up testing for covid-19 in africa: responding to the pandemic in ways that strengthen health systems farouk a. umaru african journal of laboratory medicine | vol 9, no 1 | a1244 | 14 may 2020 opinion paper establishing diagnostic training programs in resource-poor settings: the case of sierra leone lance d. presser african journal of laboratory medicine | vol 9, no 1 | a889 | 15 june 2020 opinion paper covid-19 pandemic and antimicrobial resistance: another call to strengthen laboratory diagnostic capacity in africa beverly egyir, noah obeng-nkrumah, george b. kyei african journal of laboratory medicine | vol 9, no 1 | a1302 | 23 september 2020 opinion paper how can clinical immunology laboratories contribute to the management of severe covid-19 cases in limited resource contexts? brahim admou, abdelhamid hachimi, mohammed abdenasser samkaoui african journal of laboratory medicine | vol 9, no 1 | a1282 | 02 november 2020 opinion paper covid-19 during malaria transmission season in africa and why we should be prepared: an example from senegal khadim diongue, mamadou a. diallo african journal of laboratory medicine | vol 9, no 1 | a1332 | 18 november 2020 opinion paper will long-read sequencing technologies replace short-read sequencing technologies in the next 10 years? boluwatife a. adewale african journal of laboratory medicine | vol 9, no 1 | a1340 | 26 november 2020 scientific letter inflammatory markers as predictors of mortality in covid-19 infection awadia a. ahmeidi, ashraf musa, hind s. ahmed, adel a. elahmar, ryhana b. goota, ibtihal a. ahmed, abdelhakam h. ali, mushal allam, mozan o. hassan african journal of laboratory medicine | vol 9, no 1 | a1298 | 21 december 2020 reviewer acknowledgement african journal of laboratory medicine | vol 9, no 1 | a1445 | 21 december 2020 280 282 287 291 294 297 302 304 page iii of iii abstract introduction molecular diagnostics for epidemic preparedness new diagnostic development approach acknowledgements references about the author(s) devy m. emperador foundation for innovative and new diagnostics, geneva, switzerland laura t. mazzola foundation for innovative and new diagnostics, san francisco, california, united states cassandra kelly-cirino foundation for innovative and new diagnostics, geneva, switzerland citation emperado dm, mazzola lt, kelly-cirino c. an open-source molecular diagnostic platform approach for outbreak and epidemic preparedness. afr j lab med. 2020;9(2), a1017. https://doi.org/10.4102/ajlm.v9i2.1017 lessons from the field an open-source molecular diagnostic platform approach for outbreak and epidemic preparedness devy m. emperador, laura t. mazzola, cassandra kelly-cirino received: 29 mar. 2019; accepted: 08 july 2020; published: 28 sept. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: diagnostic development for outbreak pathogens has typically followed a disease-specific reactive rather than proactive response. given the diversity of outbreak pathogens, particularly those prioritised by the world health organization research and development blueprint, a more flexible and proactive approach to epidemic preparedness is needed to expand access to critical molecular diagnostic tests in peripheral and resource-constrained deployment settings. objective: new and more sustainable directives are needed to spur the development of high-quality products, particularly for epidemics more often found in lowand middle-income countries. to leverage and de-risk the development process, we present the benefits and challenges of an open-source business model for co-development of molecular diagnostic tests for decentralised settings. methods: we identify key outbreak pathogens that are available only for testing in high infrastructure laboratories and compare in-country installed base platforms that could be leveraged for menu expansion. key strengths and challenges for development are highlighted for both platform and assay developers, with discussion of how to leverage and de-risk the process through an open-source development model. results: depending on the specific partner strengths, options for partnership roles are presented. the proposed open-source business model addresses the particular challenges in the detection of outbreakand epidemic-prone pathogens in lowand middle-income countries, reduces development and deployment risks to support outbreak response, strengthens diagnostic capacity and creates a viable market for product developers. conclusion: we hope this model for a collaborative and open-source approach for molecular diagnostics serves to encourage stakeholders to consider co-development partnerships to improve outbreak preparedness and epidemic/pandemic response. keywords: diagnostics; development; outbreak; preparedness; infectious disease; test development. introduction diagnostics are a fundamental component of a successful outbreak response by enabling surveillance and early detection, rapid and appropriate patient management, and objective evaluation of outbreak containment, in addition to facilitating therapeutic and vaccine development.1,2,3,4 for outbreak and epidemic-prone pathogens, diagnostic development has typically followed a disease-specific reactive approach. faced with significant mortality and morbidity during the 2013–2016 ebola virus disease and 2015–2017 zika virus epidemics, substantial global funding was made available, resulting in the rapid development of new diagnostics, therapeutics and vaccines.5,6,7,8 however, only a handful of the diagnostic tests survived to become commercial products, and few found a sustainable market once the epidemics were declared contained and public funding was diverted elsewhere.5,9,10 a shift toward a more proactive and nuanced development strategy for outbreaks and epidemics is needed, especially to support diagnostic capacity in lowand middle-income countries (lmics). some epidemic-prone diseases are endemic in certain regions, requiring diagnostics that can differentiate active disease from background exposure. for example, lassa fever affects many countries in west africa, with resurging high rates of cases during the dry season.11,12,13 likewise, some emergent diseases share overlapping geographies and similar clinical symptoms (e.g. lassa fever and ebola virus disease, middle east respiratory syndrome coronavirus and influenza), an additional challenge for rapid and definitive diagnosis.2,14 rapid pathogen identification can be critical when early detection can direct lifesaving treatment and intervention, such as antibiotics for confirmed bacterial infections15 or vector control vaccination campaigns during a yellow fever outbreak.2 there are common challenges to the deployment of diagnostic tests during outbreaks.16 early detection and contact tracing at the first stages of an outbreak are critical to rapid containment. similarly, a rapid test turn-around time enables patients to be triaged both quickly and appropriately. in settings of endemic diseases with similar clinical indications, it will be invaluable to rapidly differentiate and identify the pathogen responsible for the outbreak. this kind of dynamic outbreak response demands access to diagnostics across a wide range of infrastructure, from reference laboratories to near-patient (npt) hospital laboratories to point-of-care (poc) clinics and community testing. in particular, the priority pathogens identified by the world health organization research and development blueprint17 present a challenge for diagnostic development for epidemic preparedness by any single developer, as the pathogens cover a wide range in clinical presentation, transmission mode, specimen type, lineage diversity, geography and outbreak frequency. a more flexible approach is needed to rapidly develop or adapt diagnostic tests for a wide variety of pathogens, which can be implemented in outbreak-appropriate deployment settings.18,19,20 molecular diagnostics for epidemic preparedness molecular diagnostics (mdx) plays an important role in disease diagnosis (detection and confirmation of active infection), case management and surveillance, as well as a supporting role in therapeutic and vaccine trials. due to the processing complexity and risks of contamination, mdx testing capacity is traditionally available only at reference-level or high-resourced laboratories, which can significantly delay patient diagnosis and intervention during an outbreak when specimen transport is a challenge. molecular diagnostics testing is particularly useful at peripheral health facilities, where sick patients first present, to allow for pathogen detection. however, peripheral settings typically have limited resources for laboratory testing: npt settings describe hospital and clinic-associated laboratories or mobile units, which support a minimal level of laboratory infrastructure and personnel; poc settings typically lack any laboratory infrastructure, including bedside, primary care and community-based testing.21 near-patient/point-of-care mdx platforms have been designed specifically to meet the requirements for rapid pathogen detection in low-infrastructure settings,22,23,24,25,26 with a compact and automated design for a simplified ‘sample in, result out’ processing.18,19,27,28 near-patient/point-of-care mdx target product profiles have been described in detail.29,30,31,32 successful implementation has been demonstrated in decentralised laboratories in lmics for diseases including hiv, tuberculosis, zika virus, ebola virus disease and influenza,22,23,24,33,34,35,36 suggesting that a broader menu of assays could be developed for these npt/poc platforms for outbreak detection. outbreak pathogen molecular diagnostics opportunities table 1 presents a list of outbreak pathogens identified as high priority pathogens for medical countermeasures due to their high epidemic and pandemic potential.37,38,39 most of these diseases have mdx tests (reagent kits) that are commercially available, but appropriate only for use in high infrastructure research laboratories. in some cases, the mdx test has not been validated clinically or approved for diagnostic use (i.e. research-use-only kit), while in other cases, particularly for newer pathogens, mdx tests are available only as published protocols from trusted reference laboratories.17,38,40 there is potential for available laboratory kits to be re-engineered for npt/poc testing, including kits for viral haemorrhagic pathogens41,42,43,44, arboviral diseases,45,46,47,48 respiratory pathogens49,50,51 and biothreat pathogens (including disease x).39,52,53 table 1: availability of molecular diagnostic tests for select outbreak pathogens. new diagnostic development approach traditional assay development model tests for novel diseases are typically developed in academic or national reference laboratories which have the expertise, infrastructure and resources for rapid development and validation of mdx tests. these ‘in-house’ or laboratory-developed mdx tests are published and distributed as protocols from an internationally trusted source.44,54,55,56 however, the test methods require a high level of expertise and are generally limited in deployment to other reference-level laboratories. commercial products can enable broader deployment, where the mdx test is manufactured as quality-assured kits that contain all of the necessary pre-measured test reagents. in the traditional development model, a commercial platform developer builds a portfolio of proprietary assays, using a cartridge specific to the developer’s instrument platform. the instrument, cartridge, reagents and consumables are manufactured by the platform developer and supported as a ‘sole source’ provision. commercial development requires significant financial resources from the developer, with the cost of test development, manufacturing, clinical validation and regulatory approval generally considered to be an investment toward sustainable sales. companies require some profit margin in order to sustain their business and further develop test menus. when the anticipated markets are small, such as for disease with limited or intermittent outbreaks, or where prices are constrained to the cost of goods (as is typical with global health/lmic markets), then there is little incentive for commercial development. in many resource-constrained settings, alternative test options are not affordable or simply not available. to support rapid menu expansion during new or repeated outbreaks, a more flexible concept for outbreak pathogen test development is needed. open-source diagnostic model in lmic settings, leveraging the existing diagnostic infrastructure is key to expanding the capacity for disease management and outbreak preparedness. rather than requiring repeated de novo investment, existing platforms could be utilised for test menu expansion, building on the existing human resources, laboratory testing capacity and supply chains. a similar approach has seen success in the integration of tuberculosisand hiv-testing at peripheral health centres.57 for outbreak response, more flexible initiatives are needed to support rapid test development and deployment scenarios. here we describe a new open-source business model for mdx test development using existing platforms for npt/poc diagnostic testing. as a key innovation, the open-source development model introduces an open-architecture cartridge designed to facilitate rapid development of tests by outside assay developers, where the platform developer provides an open, sterile cartridge that can be used by assay developers (academic or commercial) to rapidly prototype mdx tests for the existing platform. this development model leverages the strength of each developer, namely the available install base of a platform within relevant lmics and the availability of reagent kits for the detection of outbreak pathogens, albeit for high infrastructure laboratory use. as noted in table 2, this open-source development model can serve to decouple some of the risks for new test deployment. table 2: co-development leverage and risk reduction. this open-source model is intended to catalyse a more rapid approach to the development of diagnostic tests for decentralised or low-infrastructure settings, and to provide a more flexible approach to manufacturing tests for novel and endemic pathogens for lmics through co-development (table 3). variations on the co-development partnership include collaborative research, test kit sub-contract manufacturing, and licensing or acquisition. these variations each require specific research and development and supply agreements, as well as marketing and product support agreements between the partners. in some cases, a highly successful development partnership could lead to downstream acquisition. even in cases with limited sales, the ability to rapidly develop and deploy npt/poc tests for novel pathogens is valuable in improving local outbreak response. table 3: co-development roles for open-source model. model benefits and challenges an open-source model for diagnostics can provide a more flexible and cost-effective approach to mdx deployment in peripheral settings, provided that commercial developers can find mutual benefit in a strategic partnership rather than traditional competition. the co-development model outlined above is intended to leverage the differential strengths between assay and platform developers by decoupling the investment risk for test menu expansion to include pathogens that may have limited but critically important deployment. by leveraging existing assays and npt/poc infrastructure, new tests can be rapidly re-engineered with a more cost-effective and ‘on demand’ approach for manufacturing. for the platform developer, a comprehensive assay menu expands the user base, which may provide more resilient and sustainable market volumes for diagnostic manufacturing. for the assay developer, their existing assay portfolio is expanded to include testing across a broad range of settings.58,59,60,61 designed to utilise existing lmic resources, an acknowledged potential disadvantage of this business model is reduced competition in favour of entrenched player market stability and product availability. novel molecular platforms must be evaluated, both for their potential implementation in lmics, as well as their openness to work with partners in developing assays, particularly for outbreak-prone diseases. to support this new development model, innovative partnerships and finance solutions are needed, especially for proactive development and commercial viability. here, the role of product development partners can serve as both matchmaker and co-investor to incentivise co-development partnerships.62 the open-source model is currently being piloted by the foundation for innovative and new diagnostics to partner diagnostic assay and platform developers in support of outbreak preparedness in the near term (2–5 years).58 similarly, this development model would also benefit from support from the global health community toward sustainable market commitments, pooled procurement mechanisms, and funding for test stockpiling to establish more sustainable supply chains and support long term commitments to diagnostic manufacturers.16 some of these mechanisms are in place to address similar challenges in the vaccines sector and should be explored and adapted for diagnostics.63 conclusion despite the influx of interest at the onset of a new outbreak, it is more often the case that research and development costs for new diagnostics may be too high relative to market size to provide a sufficient incentive for product development, and sustained support can be particularly challenging, for pathogens that present seasonal or sporadic markets. alternative business models for diagnostic development could help reduce the high costs associated with bringing a new pathogen test to market. an open-source development partnership model can combine the strengths of commercial developers to reduce development the cost and time to market, while supporting existing supply chain infrastructure, training and proficiency.29,32 certainly, there are additional upstream and downstream challenges for pathogen test development. for new or poorly understood outbreaks, there are uncertainties in pathogen identification, epidemiology and disease kinetics. newly developed tests require additional resources for clinical validation, for which well-characterised specimens and calibration standards may be difficult to obtain.16 commercial products also require international and/or in-country regulatory approval, though this may be a lower hurdle for assays that were previously validated as laboratory tests. here, product development partnerships can also serve as matchmakers to allow for sample sharing between research sites and developers with promising technologies, as well as to leverage in-country experience to support evaluation studies in lmics that meet the requirements for regulatory approval. the availability of appropriate diagnostics is a key feature for outbreak and epidemic preparedness. early diagnosis of patients at initial point-of-care will enable timely patient and population interventions and support national surveillance systems for emerging and re-emerging diseases. collaborative efforts are needed to develop npt/poc diagnostics for all world health organization priority pathogens, to ensure access to high-quality diagnostics where testing is needed and to enable innovative funding mechanisms to support proactive development and market sustainability. we hope this article serves to encourage all diagnostic stakeholders (industry, academia, non-for-profit, non-governmental-organisations, governments) to foster partnerships in development and consider the open-source model early in the development of new diagnostics. acknowledgements competing interests d.m.e. and c.k.-c. are employees of find, while l.t.m. is a consultant at find; find is a non-profit organisation and product development partnership that works with industry partners to support diagnostic development. authors’ contributions d.m.e. and c.k.-c. were responsible for concept design. d.m.e. and l.t.m. performed literature reviews and co-wrote the manuscript. c.k.-c. reviewed and approved the final version of the manuscript. ethical consideration the authors confirm that ethical clearance was not required for this study. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors; c.k.-c., d.m.e. and l.t.m. are employed by find. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency to the authors. references perkins md, dye c, balasegaram m, et al. diagnostic preparedness for infectious disease outbreaks. lancet. 2017;390:2211–2214. https://doi.org/10.1016/s0140-6736(17)31224-2 wilder-smith a, gubler dj, weaver sc, monath tp, heymann dl, scott tw. epidemic arboviral diseases: priorities for research and public health. lancet infect dis. 2017;17:e101–e106. https://doi.org/10.1016/s1473-3099(16)30518-7 kost gj, ferguson wj, hoe j, truong a-t, banpavichit a, kongpila s. the ebola spatial care path(tm): accelerating point-of-care diagnosis, decision making, and community resilience in outbreaks. am j disaster med. 2015;10:121–143. https://doi.org/10.5055/ajdm.2015.0196 find. diagnostics for epidemic preparedness [homepage on the internet]. c2018 [cited 2018 sep 28]. available from: https://www.finddx.org/wp-content/uploads/2018/05/find_outbreak-strategy_web.pdf emperador dm, mazzola lt, wonderly trainor b, chua a, kelly-cirino cd. diagnostics for filovirus detection: impact of recent outbreaks on the diagnostic landscape. bmj glob health. 2019;4:e001112. https://doi.org/10.1136/bmjgh-2018-001112 perkins md, kessel m. what ebola tells us about outbreak diagnostic readiness. nat biotechnol. 2015;33:464–469. https://doi.org/10.1038/nbt.3215 who. ebola | additional documents [homepage on the internet]. rd bluepr. c2018 [cited 2018 nov 26]. available from: http://www.who.int/blueprint/priority-diseases/key-action/ebola-additional-documents/en/ fda. 2014 ebola virus emergency use authorizations [homepage on the internet]. c2018 [cited 2018 nov 26]. available from: https://www.fda.gov/medicaldevices/safety/emergencysituations/ucm161496.htm#ebola theel es, hata dj. diagnostic testing for zika virus: a postoutbreak update. j clin microbiol. 2018;56. https://doi.org/10.1128/jcm.01972-17 cnops l, de smet b, mbala-kingebeni p, van griensven j, ahuka-mundeke s, arien kk. where are the ebola diagnostics from last time? nature. 2019;565:419–421. https://doi.org/10.1038/d41586-019-00212-y ehichioya du, hass m, olschläger s, et al. lassa fever, nigeria, 2005–2008. emerg infect dis. 2010;16:1040–1041. https://doi.org/10.3201/eid1606.100080 cdc. lassa fever [homepage on the internet]. cdc; c2017 [cited 2017 dec 08]. available from: https://www.cdc.gov/vhf/lassa/index.html. who. lassa fever [homepage on the internet]. who; c2017 [cited 2017 dec 08]. available from: http://www.who.int/csr/disease/lassafever/en/. racsa ld, kraft cs, olinger gg, hensley le. viral hemorrhagic fever diagnostics. clin infect dis. 2016;62:214–219. https://doi.org/10.1093/cid/civ792 dittrich s, tadesse bt, moussy f, et al. target product profile for a diagnostic assay to differentiate between bacterial and non-bacterial infections and reduce antimicrobial overuse in resource-limited settings: an expert consensus. plos one. 2016;11:e0161721. kelly-cirino cd, nkengasong j, kettler h, et al. importance of diagnostics in epidemic and pandemic preparedness. bmj glob health. 2019;4:e001179. https://doi.org/10.1136/bmjgh-2018-001179 who. who | r&d blueprint for action to prevent epidemics [homepage on the internet]. who r&d blueprint. c2017 [cited 2017 sep 12]. available from: http://www.who.int/blueprint/en/. roberts t, bygrave h, fajardo e, ford n. challenges and opportunities for the implementation of virological testing in resource-limited settings. j int aids soc. 2012;15:17324. wang s, lifson ma, inci f, liang l-g, sheng y-f, demirci u. advances in addressing technical challenges of point-of-care diagnostics in resource-limited settings. expert rev mol diagn. 2016;16:449–459. https://doi.org/10.1586/14737159.2016.1142877 peeling rw, boeras di, nkengasong j. re-imagining the future of diagnosis of neglected tropical diseases. comput struct biotechnol j. 2017;15:271–274. who. guidance for procurement of in vitro diagnostics and related laboratory items and equipment. who: geneva; 2017. jani iv, meggi b, vubil a, et al. evaluation of the whole-blood alere q nat point-of-care rna assay for hiv-1 viral load monitoring in a primary health care setting in mozambique. j clin microbiol. 2016;54:2104–2108. moyo s, mohammed t, wirth ke, et al. point-of-care cepheid xpert hiv-1 viral load test in rural african communities is feasible and reliable. j clin microbiol. 2016;54:3050–3055. https://doi.org/10.1128/jcm.01594-16 goel n, ritchie av, mtapuri-zinyowera s, et al. performance of the samba i and ii hiv-1 semi-q tests for viral load monitoring at the point-of-care. j virol methods. 2017;244:39–45. drain pk, hyle ep, noubary f, et al. diagnostic point-of-care tests in resource-limited settings. lancet infect dis. 2014;14:239–249. https://doi.org/10.1016/s1473-3099(13)70250-0 gupta e, agarwala p, kumar g, maiwall r, sarin sk. point-of-care testing (poct) in molecular diagnostics: performance evaluation of genexpert hcv rna test in diagnosing and monitoring of hcv infection. j clin virol. 2017;88:46–51. https://doi.org/10.1016/j.jcv.2017.01.006 pai np, vadnais c, denkinger c, engel n, pai m. point-of-care testing for infectious diseases: diversity, complexity, and barriers in lowand middle-income countries. plos med. 2012;9:e1001306. palamountain km, baker j, cowan ep, et al. perspectives on introduction and implementation of new point-of-care diagnostic tests. j infect dis. 2012;205 suppl 2:s181–s190. https://doi.org/10.1093/infdis/jis203 who, msf, find. a multiplex multi-analyte diagnostic platform [homepage on the internet]. c2018 [cited 2018 sep 01]. available from: http://www.who.int/medical_devices/tpp_20180327_final.pdf path. developing a point-of-care multiplexed diagnostic system for low-resource settings in developing countries [homepage on the internet]. path; c2008 [cited 2018 nov 26]. available from: https://path.org/resources/developing-a-point-of-care-multiplexed-diagnostic-system-for-low-resource-settings-in-developing-countries/ bmgf. develop technologies that allow assessment of multiple conditions and pathogens at point-of-care [homepage on the internet]. global grand challenges; c2011 [cited 2018 nov 26]. available from: https://gcgh.grandchallenges.org/challenge/develop-technologies-allow-assessment-multiple-conditions-and-pathogens-point-care. unitaid. multi-disease diagnostic landscape for integrated management of hiv, hcv, tb and other coinfections [homepage on the internet]. unitaid; c2018 [cited 2018 sep 28]. available from: https://unitaid.eu/assets/multi-disease-diagnostics-landscape-for-integrated-management-of-hiv-hcv-tb-and-other-coinfections-january-2018.pdf clark dj, tyson j, sails ad, krishna s, staines hm. the current landscape of nucleic acid tests for filovirus detection. j clin virol. 2018;103:27–36. https://doi.org/10.1016/j.jcv.2018.03.005 fallah mp, skrip la, raftery p, et al. bolstering community cooperation in ebola resurgence protocols: combining field blood draw and point-of-care diagnosis. plos med. 2017;14:e1002227. raftery p, condell o, wasunna c, et al. establishing ebola virus disease (evd) diagnostics using genexpert technology at a mobile laboratory in liberia: impact on outbreak response, case management and laboratory systems strengthening. plos negl trop dis. 2018;12:e0006135. https://doi.org/10.1371/journal.pntd.0006135 ndegwa lk, emukule g, uyeki tm, et al. evaluation of the point-of-care becton dickinson veritortm rapid influenza diagnostic test in kenya, 2013–2014. bmc infect dis. 2017;17:60. who. disease commodity packages [homepage on the internet]. who; c2020 [cited 15 january 2020]. available from: https://www.who.int/emergencies/what-we-do/prevention-readiness/disease-commodity-packages/en/. who. disease outbreaks [homepage on the internet]. who health emergencies programme. c2018 [cited 2018 sep 25]. available from: http://www.who.int/emergencies/diseases/en/ who. prioritizing diseases for research and development in emergency contexts [homepage on the internet]. c2020 [cited 2020 feb 03]. available from: https://www.who.int/activities/prioritizing-diseases-for-research-and-development-in-emergency-contexts morens dm, fauci as. emerging infectious diseases: threats to human health and global stability. plos pathog. 2013;9:e1003467. https://doi.org/10.1371/journal.ppat.1003467 drosten c, göttig s, schilling s, et al. rapid detection and quantification of rna of ebola and marburg viruses, lassa virus, crimean-congo hemorrhagic fever virus, rift valley fever virus, dengue virus, and yellow fever virus by real-time reverse transcription-pcr. j clin microbiol. 2002;40:2323–2330. rieger t, kerber r, el halas h, et al. evaluation of realstar reverse transcription-polymerase chain reaction kits for filovirus detection in the laboratory and field. j infect dis. 2016;214:s243–s249. https://doi.org/10.1093/infdis/jiw246 pang z, li a, li j, et al. comprehensive multiplex one-step real-time taqman qrt-pcr assays for detection and quantification of hemorrhagic fever viruses. plos one. 2014;9:e95635. he j, kraft aj, fan j, et al. simultaneous detection of cdc category ‘a’ dna and rna bioterrorism agents by use of multiplex pcr & rt-pcr enzyme hybridization assays. viruses. 2009;1:441–459. priye a, bird sw, light yk, ball cs, negrete oa, meagher rj. a smartphone-based diagnostic platform for rapid detection of zika, chikungunya, and dengue viruses. sci rep. 2017;7:44778. https://doi.org/10.1038/srep44778 ganguli a, ornob a, yu h, et al. hands-free smartphone-based diagnostics for simultaneous detection of zika, chikungunya, and dengue at point-of-care. biomed microdevices. 2017;19:73. https://doi.org/10.1007/s10544-017-0209-9 yaren o, alto bw, gangodkar pv, et al. point of sampling detection of zika virus within a multiplexed kit capable of detecting dengue and chikungunya. bmc infect dis. 2017;17:293. https://doi.org/10.1186/s12879-017-2382-0 interim guidance for zika virus testing of urine – united states, 2016. mmwr morb mortal wkly rep. 2016;65:474. lee jm, lee jh, kim yk. laboratory impact of rapid molecular tests used for the detection of respiratory pathogens. clin lab. 2018;64:1545–1551. https://doi.org/10.7754/clin.lab.2018.180411 chen h, weng h, lin m, et al. the clinical significance of filmarray respiratory panel in diagnosing community-acquired pneumonia. biomed res int. 2017;2017:7320859. https://doi.org/10.1155/2017/7320859 malhotra b, swamy ma, reddy pvj, kumar n, tiwari jk. evaluation of custom multiplex real – time rt – pcr in comparison to fast – track diagnostics respiratory 21 pathogens kit for detection of multiple respiratory viruses. virol j. 2016;13:91. gay-andrieu f, magassouba n, picot v, et al. clinical evaluation of the biofire filmarray(®) biothreat-e test for the diagnosis of ebola virus disease in guinea. j clin virol. 2017;92:20–24. gunnell mk, lovelace cd, satterfield ba, moore ea, o’neill kl, robison ra. a multiplex real-time pcr assay for the detection and differentiation of francisella tularensis subspecies. j med microbiol. 2012;61:1525–1531. https://doi.org/10.1099/jmm.0.046631-0 williams ha, dunville rl, gerber si, et al. cdc’s early response to a novel viral disease, middle east respiratory syndrome coronavirus (mers-cov), september 2012–may 2014. public health rep. 2015;130:307–317. cdc. cdc provides test protocol for novel coronavirus, preps kits for local use [homepage on the internet]. genomeweb; c2020 [cited 2020 jan 29]. available from: https://www.genomeweb.com/pcr/cdc-provides-test-protocol-novel-coronavirus-preps-kits-local-use who. laboratory testing for middle east respiratory syndrome coronavirus [homepage on the internet]. who; c2018 [cited 2018 mar 21]. available from: http://www.who.int/csr/disease/coronavirus_infections/mers-laboratory-testing/en/ davies m-a, pinto j, bras m. getting to 90-90-90 in paediatric hiv: what is needed? j int aids soc. 2015;18:20770. https://doi.org/10.7448/ias.18.7.20770 find. find pilots partnership-based business model for outbreak response as first investment in new epidemic preparedness strategy [homepage on the internet]. c2018 [cited 2018 aug 23]. available from: https://www.finddx.org/news/find-pilots-partnership-based-business-model-outbreak-response-first-investment-new-epidemic-preparedness-strategy/ blink dx. c2020 [cited 2020 jan 29]. available from: https://www.blink-dx.com/our-model genomeweb. speedx, labcorp will codevelop mdx tests [homepage on the internet]. c2020 [cited 2020 jan 29]. available from: https://www.genomeweb.com/business-news/speedx-labcorp-will-codevelop-mdx-tests genomeweb | 360dx. speedx mycoplasma genitalium resistance test on cepheid system receives ce-ivd [homepage on the internet]. 360dx; c2020 [cited 2020 jan 29]. available from: https://www.360dx.com/pcr/speedx-mycoplasma-genitalium-resistance-test-cepheid-system-receives-ce-ivd mahoney rt. product development partnerships: case studies of a new mechanism for health technology innovation. health res policy syst. 2011;9:33. https://doi.org/10.1186/1478-4505-9-33 gavi. gavi ‘effective and fit for purpose’ [homepage on the internet]. c2017 [cited 2019 mar 29]. available from: https://www.gavi.org/library/news/statements/2017/gavi--effective-and-fit-for-purpose-/ acknowledgements references about the author(s) farouk a. umaru department of global public health, united states pharmacopeia, rockville, maryland, united states citation umaru fa. scaling up testing for covid-19 in africa: responding to the pandemic in ways that strengthen health systems. afr j lab med. 2020;9(1), a1244. https://doi.org/10.4102/ajlm.v9i1.1244 opinion paper scaling up testing for covid-19 in africa: responding to the pandemic in ways that strengthen health systems farouk a. umaru received: 10 apr. 2020; accepted: 25 apr. 2020; published: 14 may 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. in the midst of responding to the coronavirus disease 2019 (covid-19) pandemic, public health practitioners, agencies and the private sector are partnering to provide urgent emergency solutions to the ongoing crisis. in the words of world health organization director general, dr tedros ghebreyesus, a critical component of this response is to ‘test, test and test’. this need for testing continues to spur multiple innovations in testing techniques, strategies and applications. as of 08 april 2020, more than 48 different in vitro diagnostic devices for covid-19 diagnosis were listed on the world health organization website under the international medical devices regulatory forum jurisdiction as having received emergency use authorization (eua) from nine countries, with china authorising 19 devices or technologies (including antibody test kits).1 although no country in africa has issued an eua on any of these devices, it is very likely that most of these devices may be marketed or distributed on the continent. while developed countries like the united states, italy and spain have struggled to cope with large-scale testing on multiple devices, many countries in africa are disproportionately hit by the need for testing because of severe limitations in testing technologies. the lack of africa-issued euas on emerging technologies specific to severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the virus responsible for covid-19, may continue to handicap africa’s response to the pandemic. but, should african regulatory agencies or the africa centres for disease control and prevention (cdc) begin to issue euas for emerging technologies, with limited validation information in response to the covid-19 pandemic? african union member states, through the efforts of africa cdc and partners, have received technical support to use existing real-time polymerase chain reaction (rt-pcr) instruments to conduct testing, mostly at national reference or equivalent laboratories. although this technology may be inadequate to entirely meet the scale of testing required for covid-19 (because of limited numbers of instruments), these instruments are within the existing tiered laboratory network. leveraging existing rt-pcr instruments for covid-19 diagnosis is an important step in strengthening health systems on the continent for future emergency pandemics. responding to the current pandemic in ways that strengthen health systems and that go beyond emergency solutions to consider long-term solutions will benefit the continent as a whole. the ebola outbreak in west africa provides useful lessons on how emergency responses can impact health systems.2 during the ebola outbreak, novel technologies were provided to countries without consideration to the existing tiered laboratory network. as a consequence, some countries have been unable to incorporate those novel technologies into their laboratory networks, which impacts the overall sustainability of their health systems. it is time to remind both national and regional communities on the continent to think beyond the current covid-19 pandemic so that when africa emerges on the other side, its health systems will be stronger and more prepared to respond to the next one. central questions to keep in mind during the covid-19 response include: how will countries absorb multiple novel technologies within their health systems post-covid-19? how will emergency-use-authorised in vitro diagnostics be part of national tiered laboratory systems post-pandemic? what role will manufacturers play in initiating long-term evaluation procedures for covid-19 technologies? will these technologies be left to countries to manage without adequate support, guidance or capacity? answers to these questions are critical now. it is therefore imperative that national regulatory agencies, diagnostics manufacturers and national diagnostics technical working groups not ‘rush’ into issuing or adopting euas for new and untested devices outside their networks, but to consider the long-term impact of those technologies on their health systems. some of these approaches may include: update the current rt-pcr instruments to incorporate covid-19 testing. as the gold standard for viral testing, countries must work with their existing rt-pcr technology manufacturers to upgrade reagents, kits and software to accommodate covid-19.3 the latest eua from the united states food and drug administration for the cepheid xpress cartridge on genexpert instruments (cepheid, sunnyvale, california, united states) and the abbott r-sars-cov-2 reagents on abbott m2000 instrument (abbott laboratories, chicago, illinois, united states) are typical examples.4 national regulatory agencies should develop guidelines that outline clear and unambiguous procedures for issuing eua for new technologies. these guidelines should incorporate manufacturers’ plans to work with national agencies to incorporate new devices into existing tiered networks as euas expire. national regulatory agencies should limit eua approvals to devices that employ the gold standard of rt-pcr in their technologies over antigen-antibody-based, lateral-flow rapid diagnostic test kits, which may not demonstrate comparable sensitivity and specificity to sars-cov-2 as with rt-pcr instruments. in cases where rapid diagnostic tests are considered (because of urgency to scale up testing), scientifically prudent testing algorithms must be developed by national stakeholders and enforced. in this algorithm, any positive covid-19 sample from a rapid diagnostic test should be accompanied an rt-pcr-based confirmatory test. in addition, a percentage of negative test samples should also be confirmed with rt-pcr, in order to continuously monitor and confirm the specificity and sensitivity of rapid diagnostic tests. national regulatory agencies should seek the support of international technical partners, including the world health association, africa cdc, the african society for laboratory medicine and other non-governmental organisations such as the united states pharmacopeia and foundation for innovative new diagnostics, to help support and build capacity to rapidly scale up testing for enhanced case management and long-term emergency preparedness. these strategies and others, supported by national stakeholders, will support african countries in strengthening systems and improve preparedness for emerging pandemics, while building sustainable laboratory systems to help support better healthcare across the continent. acknowledgements the manuscript went through internal united states pharmacopeia technical and editorial process workflow. no need to mention individuals. competing interests the author has declared that no competing interest exists. authors’ contributions f.a.u. was the sole author of this article. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sector. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the author and do not necessarily reflect the official policy or position of any affiliated agency of the author. references world health organization. in vitro diagnostics and laboratory technology: prequalification of ivds and medical devices. [homepage on the internet]. geneva: world health organization; c2020 [cited 2020 apr 8]. available from: https://www.who.int/diagnostics_laboratory/200408_imdrf_collated_table_8_april_2020.pdf?ua=1 kieny m-p, evans db, schumets g, et al. health-system resilience: reflection on the ebola crisis in western africa. geneva: world health organization 2014;92:850. https://doi.org/10.2471/blt.14.149278 world health organization. emergency use listing (eul), weekly update. [homepage on the internet]. date [cited 2020 apr 4]. available from: https://www.who.int/diagnostics_laboratory/200403_eul_covid19_ivd_update.pdf?ua=1 united states food & drug administration (fda). xpert xpress sars-cov-2 test. [email discussion on the internet] 2020 march 20 [cited 2020 apr 8]. available from: https://www.fda.gov/media/136316/download abstract introduction methods results discussion acknowledgements references about the author(s) modupe a. kuti department of chemical pathology, faculty of basic medical sciences, college of medicine, university of ibadan, ibadan, nigeriadepartment of chemical pathology, university college hospital, ibadan, nigeria olabisi t. bamidele department of chemical pathology, university college hospital, ibadan, nigeria chioma t. udeh department of chemical pathology, university college hospital, ibadan, nigeria bola j. eseile department of chemical pathology, university college hospital, ibadan, nigeria olajumoke a. ogundeji department of chemical pathology, university college hospital, ibadan, nigeria citation kuti ma, bamidele ot, udeh ct, eseile bj, ogundeji oa. appropriate use of plasma glucose tests for diagnosis of diabetes mellitus in ibadan, nigeria. afr j lab med. 2022;11(1), a1433. https://doi.org/10.4102/ajlm.v11i1.1433 original research appropriate use of plasma glucose tests for diagnosis of diabetes mellitus in ibadan, nigeria modupe a. kuti, olabisi t. bamidele, chioma t. udeh, bola j. eseile, olajumoke a. ogundeji received: 23 oct. 2020; accepted: 07 feb. 2022; published: 29 apr. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: diabetes mellitus is a growing epidemic in africa. its diagnosis relies exclusively on laboratory evidence, which differs based on clinical circumstances. objective: the study described the appropriateness of plasma glucose test requests per the american diabetes association criteria. methods: we reviewed the plasma glucose test requests received by the chemical pathology laboratory of the university college hospital, ibadan, nigeria between june 2018 and november 2018. the american diabetes association diabetes diagnostic criteria were used to define the appropriateness of test requests and determine the potential for ill-informed clinical decisions. results: four hundred and twenty-three requisition forms were included, with the majority from the medical wards/clinics (72.3%); the most frequent reason for a plasma glucose test was systemic hypertension (28.6%). fasting plasma glucose was most requested (254; 60.0%). one hundred and sixteen (27.4%) requests were potentially inappropriate, with the 2-h postprandial plasma glucose (2hppg) test requests (83; 71.6%) being the most inappropriate. the difference in the proportion of inappropriate requests was not statistically significantly between medical or surgical wards/clinics (odds ratio 1.131, 95% confidence interval 0.709–1.803, p = 0.605). inappropriate requests in six cases may have triggered inappropriate action. conclusion: a third of the glucose tests requested for querying diabetes mellitus may have been inappropriate. results of such testing may trigger inappropriate clinical action. to improve the quality of care and for economic reasons, laboratories should have programmes to improve the appropriate use of their services. keywords: diabetes mellitus; glucose; utilisation management; laboratory; guidelines; fasting; postprandial; random. introduction the socio-economic burden of a diabetes mellitus (dm) diagnosis is substantial for persons in low and middle-income countries relative to persons in higher-income countries.1 the absence of robust national health insurance schemes in low and middle-income countries means that this burden is borne mainly by patients and their families. the total healthcare cost of persons with dm has been estimated to be about four times higher than for persons with normal glucose tolerance.2 this cost includes those incurred from admissions, outpatient visits, laboratory tests, medication, and treatment trips, as well as productivity losses by patients and caregivers. the rapidly rising prevalence of dm imposes an increasing significant strain on fragile national health systems,which are already buckling under a huge infectious disease burden. estimates of dm prevalence from urban populations in kenya, cameroon, and south africa range between 10% and 12%.3,4,5 a recent lancet commission on the burden of dm in sub-saharan africa reported that dm and its complications are costly to patients and that national health systems are largely unable to cope with the current burden of the disease.6 one of the fundamental requirements for appropriate public health response to this epidemic is accurate diagnosis of dm. the diagnosis of dm relies exclusively on biochemical evidence of a specific degree of glucose intolerance. the current accepted diagnostic criteria for dm in the non-pregnant adult were initially published by the american diabetes association in 1998 and were adopted by the world health organization in 1999.7,8 the laboratory tests referenced in the criteria are fasting plasma glucose (fpg), 2-h post-load glucose (2hplg) during an oral glucose tolerance test (ogtt), and random plasma glucose (rpg). in 2010, glycated haemoglobin was listed as a further diagnostic criterion.9 other than glycated haemoglobin, all the glucose criteria require that specific patient preparation steps or clinical symptoms be present. the fpg sample is collected after an overnight fast of at least 8 h, while the 2hplg sample is obtained 2 h after the patient consumes a standard glucose load of 75 g of anhydrous glucose. for rpg to be used as a dm diagnostic criterion, the presence of either classic symptoms of dm or hyperglycaemic crisis is required. this suggests that rpg is not recommended for screening of the asymptomatic person and 2hplg should not be used interchangeably with 2-h postprandial plasma glucose (2hppg). the latter test is performed on a sample taken after the consumption of the individual’s regular diet, which will vary significantly from person to person. two-hour postprandial plasma glucose has utility in assessing glycaemic control in persons with known dm.10 the study aimed to determine the appropriateness of the glucose test requests per the american diabetes association dm diagnosis criteria.11 furthermore, for tests that were ordered contrary to recommended guidelines (inappropriate tests), we examined the potential for consequent inappropriate clinical action based on the result of such tests. methods ethical considerations ethical approval was obtained from the university of ibadan/university college hospital ethics committee with a national health research ethics committee nhrec/05/01/2008a. the ethical committee assigned number for the study was ui/ec/19/0630. this was an analysis of secondary data and did not retrieve any patient-identifiable information or involve contact with any human participants. patient consent was therefore not required. all glucose results were anonymised from patient details and study numbers accessible only to the researchers were used during analysis. study setting the university college hospital, ibadan is a public tertiary hospital located in an urban setting in the south western region of nigeria. the hospital is served by a chemical pathology laboratory that receives about 33 000 samples annually, including blood, urine, and cerebrospinal, ascitic and pleural fluids. tests provided include those assessing for renal, liver, metabolic, endocrine, and neoplastic diseases. for a non-pregnant adult, the laboratory offers two stand-alone glucose tests – fpg and rpg – as well as two glucose profiles – fpg/2hppg and fpg/2hplg. study design this cross-sectional study reviewed all request forms from the wards/clinics of the hospital from june and november 2018 for plasma glucose tests. to be included in this study, the section for clinical information was required to contain information about the patient such as presenting symptoms, signs, working diagnosis, or treatment. any form with no clinical information or with clear information that the patient was previously diagnosed as having dm was excluded. definition of test request inappropriateness the appropriateness of the tests was defined using the american diabetes association dm diagnostic criteria (box 1).12 the following test requests were deemed as probably inappropriate requests for dm diagnosis: all 2hppgtest requests. all rpg test requests in which the clinical details on the requisition form included none of the classic symptoms of dm (polyuria, polydipsia, and weight loss). box 1: criteria for the diagnosis of diabetes mellitus by the american diabetes association (2018). the potential for inappropriate clinical action was deemed to be present if: the result of an inappropriately requested rpg exceeded 11.1 mmol/l, and/or the fpg was < 7.1 mmol/l and the 2hppg was > 11.1 mmol/l. data analysis statistical analysis was performed using ibm® statistical package for social sciences (spss®) version 25 (2017, ibm corp, armonk, new york, united states). proportions are presented as numbers (percent) and were compared using the chi-square test. results were considered significant if p was 0.05 or less, with a 95% confidence interval. results four hundred and twenty-three request forms satisfied the inclusion criteria. medical outpatient clinics and wards accounted for 306 (72.3%) of these requests. overall, the most common reason for a glucose test request was to evaluate systemic hypertension (table 1). the surgical clinics and wards most commonly requested a glucose test for evaluating neoplastic disease (benign or malignant). a one-off fpg was the most requested glucose test in both specialities, accounting for 254 (60.0%) of all requests (table 2). the usage pattern for the different tests/profiles was significantly different between both specialities (x2 = 138.911 [p < 0.001]); the surgical specialities requested more ogtt, mostly for postnatal assessment of persons with previous gestational dm. table 1: clinical information on requisition forms received in the chemical pathology laboratory, university college hospital, ibadan nigeria (june 2018 – november 2018). table 2: distribution of glucose requests by speciality of source ward/clinics of the university college hospital, ibadan, nigeria (june 2018 – november 2018). the fpg level was normal (< 5.5 mmol/l) in 77.7% (303) of cases, impaired (5.5 mmol/l – 6.9 mmol/l) in 15.1% (n = 59), and diabetic (≥ 7.0 mmol/l) in 7.2% (n = 28). the glucose level was < 11.1 mmol/l for 88% (n = 73) 2hppg, 88.7% (n = 47) 2-h ogtt and 100% (n = 33) rpg requests. one hundred and sixteen (27.4%) of the test requests were potentially inappropriate, comprising 83 (71.6%) requests for fpg/2hppg and 33 (28.4%) requests for rpg (table 3). all of the ogtt requests were appropriate. inappropriate test requests from the medical wards and clinics (n = 82; 70.7%) were higher than those from the surgical wards and clinics (48 tests; p = –0.629) (table 2). of the 83 inappropriately requested fpg/2hppg, the fpg was < 7 mmol/l while the 2hppg was > 11.1 mmol/l in 6 (7.2%) cases. the fpg and accompanying 2hppg were < 7 mmol/l and < 11.1 mmol/l in 71 (85.5%) cases. fasting plasma glucose > 7 mmol/l/2hppg < 11.1 mmol/l was seen in one case and fpg > 7 mmol/l/2hppg > 11.1 mmol/l was seen in another five cases. table 3: clinical information against inappropriately requested glucose tests in the chemical pathology laboratory, university college hospital, ibadan nigeria (june 2018 – november 2018). discussion despite the annual publication of the american diabetes association standards for the laboratory diagnosis of dm for over 20 years, this study provides evidence that there exists a need to increase the uptake of its recommendations in routine practice in our setting. nearly one out of every three requests for glucose tests, in our setting, was found to be potentially inappropriate. inappropriate requests were mostly due to requests for 2hppg as a dm screening test. reports from both nigeria and cameroon indicate that primary care physicians, who represent the bulk of physicians providing dm screening and care, have poor knowledge of the diabetes clinical guideline criteria.13,14 over 70% of the participants in both studies were not reliably diagnosed with dm using the requested glucose tests.13,14 the primary consequence of an inappropriate glucose test is the waste of the patient’s money for an unnecessary test. the wastage is particularly costly in a region where most patients are uninsured and thus pay out of pocket.15 money spent on unnecessary testing may reduce money for necessary treatment. there is also a cumulative cost to the health systems. glucose tests are relatively inexpensive, however, increased requests over time will drain laboratory resources.16, 17 a further consequence of inappropriate testing is a false negative test result. as observed in this study, the majority of the requests for rpg returned values below a ‘diagnostic’ threshold. there is no guarantee that the screened patients were all glucose tolerant. reports suggest that the sensitivity of an rpg value greater than or equal to 7 mmol/l for identifying asymptomatic persons with dm may be less than 70%.18,19 these reports concluded that it would be inappropriate to use the rpg test alone as a dm screening test. thus, an additional test is required for definitive diagnosis of dm, thereby increasing costs incurred by patients. we examined whether the requests from surgical wards and clinics were more likely to be inappropriate compared to those from medical wards and clinics. reports suggest that certain practices by surgeons may increase the likelihood of inappropriate test requests. charani et al.20 reported that surgeons frequently left care decisions perceived as non-surgical to junior team members. this practice is reportedly associated with an increased likelihood of inappropriate laboratory test requests.21,22 our study, however, did not observe a significant difference in rates of inappropriate test requests from the surgical wards and clinics compared to the medical ones. a possible explanation for this observed lack of significant difference in inappropriate test requests between both wards and clinics in this study might be due to, the much higher number of test requests from the medical areas consequently increasing the number of inappropriate requests. this may thus cancel out the effect of the delegation of duties within the surgical areas. we also note that, although a 2hppg > 11.1 mmol/l may be suggestive of the presence of dm, it may not be used as conclusive evidence of the disease, as current criteria for dm diagnosis do not include it. glycaemia > 11.1 mmol/l may be observed as part of a physiologic stress response.23 the appropriate response, therefore, to such a result should be an evaluation for dm using tests listed in the currently accepted criteria. any other clinical course, such as commencing treatment or re-ordering the same test, would be inappropriate. the present study has demonstrated that a significant percentage of requests for plasma glucose, a routine and frequently test, may be inappropriate. similarly, estimates from a teaching hospital in austria suggest that as many as 60% – 70% of high throughput tests such as potassium and lactate dehydrogenase may have been ordered inappropriately or in clinical situations where they had only minor relevance.24 depending on the criteria used, estimates of request inappropriateness from the united states range between 5% and 95% of tests.25 we have also shown here the potential for inappropriate clinical action following an inappropriate laboraory test requests. a 20-year review of malpractice claims in insurance companies that provide cover for patients in over 40 academic and non-academic hospitals in the united states found 181 claims that involved diagnostic errors and resulted in harm to the patient. over 50% of these claims were attributable to a failure to order an appropriate test.26 inappropriate test usage wastes resources and may result in actual harm to the patient. predictably, a system to manage physician test utilisation has value for patients, the physicians, and the health care system, with significant positive yields in quality of care and economic savings.27,28 such a system should be characterised by an understanding of the multitude of factors that influence test requisition. these range from diagnostic, therapeutic/prognostic, patient-related, physician-related and policy/organisation factors.29 depending on the specific intention of such a management programme, a wide range of approaches is available.30,31 these approaches include the use of utilisation audits, as in the current study, analytical algorithms, test guidelines, formularies and, where applicable, clinical decision support systems and changes to computerised provider order entry. implementing utilisation management initiatives requires the collaboration of key stakeholders, including pathologists, laboratorians and other professionals who contribute to the health care process.28 pathologists, whose background training enables them to observe testing behaviour patterns, suggest alternatives, manage testing algorithms and provide interpretative services, could show leadership in this regard.28,32,33 limitations the study was limited by its reliance on the information included in the submitted requisition forms. this information may not have correctly reflected the entire clinical status of the patient being evaluated. conclusion in conclusion, we have demonstrated the suboptimal usage of glucose tests in dm screening, with the speciality of the requesting physician as a potential factor. this provides evidence that laboratories should engage in programmes, such as standard diagnosis awareness campaigns, directed at improving the appropriate use of their services. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions m.a.k. conceived the article and analysed the data; o.t.b. and c.t.u. conceived the article and collected the data; all authors were involved in writing and approval of the manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability due to the nature of this research, participants of this study did not agree for their data to be shared publicly, so supporting data is not available. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references seuring t, archangelidi o, suhrcke m. the economic costs of type 2 diabetes: a global systematic review. pharmacoeconomics. 2015;33(8):811–831. https://doi.org/10.1007/s40273-015-0268-9 bermudez-tamayo c, besançon s, johri m, assa s, brown jb, ramaiya k. direct and indirect costs of diabetes mellitus in mali: a case-control study. plos one. 2017;12(5):e0176128. https://doi.org/10.1371/journal.pone.0176128 peer n, steyn k, lombard c, lambert ev, vythilingum b, levitt ns. rising diabetes prevalence among urban-dwelling black south africans. plos one. 2012;7(9):e43336. https://doi.org/10.1371/journal.pone.0043336 ploubidis gb, mathenge w, de stavola b, grundy e, foster a, kuper h. socioeconomic position and later life prevalence of hypertension, diabetes and visual impairment in nakuru, kenya. int j public health. 2013;58(1):133–141. https://doi.org/10.1007/s00038-012-0389-2 echouffo-tcheugui jb, dzudie a, epacka me, et al. prevalence and determinants of undiagnosed diabetes in an urban sub-saharan african population. prim care diabetes. 2012;6(3):229–234. https://doi.org/10.1016/j.pcd.2012.05.002 atun r, davies ji, gale eam, et al. diabetes in sub-saharan africa: from clinical care to health policy. lancet diabetes endocrinol. 2017;5(8):622–667. https://doi.org/10.1016/s2213-8587(17)30181-x gavin jr, alberti kgmm, davidson mb, et al. report of the expert committee on the diagnosis and classification of diabetes mellitus. diabetes care. 1998;21(suppl 1):s5–s19. https://doi.org/10.2337/diacare.21.1.s5 alberti kgmm, zimmet pz, consultation who. definition, diagnosis and classification of diabetes mellitus and its complications. part 1: diagnosis and classification of diabetes mellitus. provisional report of a who consultation. diabet med. 1998;15(7):539–553. https://doi.org/10.1002/(sici)1096-9136(199807)15:7%3c539::aid-dia668%3e3.0.co;2-s american diabetes association. diagnosis and classification of diabetes mellitus. diabetes care. 2010;33(suppl 1):s62–s69. https://doi.org/10.2337/dc10-s062 abougalambou ssi, ahmed no, abougalambou as. a study evaluating the postprandial plasma glucose control among type 2 diabetes patients attending a teaching hospital in malaysia. diabetes metab syndr clin res rev. 2017;11:s445–s449. https://doi.org/10.1016/j.dsx.2017.03.033 american diabetes association. classification and diagnosis of diabetes: standards of medical care in diabetes 2018. diabetes care. 2018;41(suppl 1):s13–s27. https://doi.org/10.2337/dc18-s002 american diabetes association. standards of medical care in diabetes – 2013. diabetes care. 2013;36(suppl 1):s11–s66. https://doi.org/10.2337/dc13-s011 jingi am, nansseu jrn, noubiap jjn. primary care physicians’ practice regarding diabetes mellitus diagnosis, evaluation and management in the west region of cameroon. bmc endocr disord. 2015;15(1):18. https://doi.org/10.1186/s12902-015-0016-3 ugwu e, young e, nkpozi m. diabetes care knowledge and practice among primary care physicians in southeast nigeria: a cross-sectional study. bmc fam pract. 2020;21(1):128. https://doi.org/10.1186/s12875-020-01202-0 okebukola po, brieger wr. providing universal health insurance coverage in nigeria. int q commun health educ. 2016;36(4):241–246. https://doi.org/10.1177/0272684x16657451 horton s, fleming ka, kuti m, et al. the top 25 laboratory tests by volume and revenue in five different countries. am j clin pathol. 2019;151(5):446–451. https://doi.org/10.1093/ajcp/aqy165 moloney tw, rogers de. medical technology – a different view of the contentious debate over costs. n engl j med. 1979;301(26):1413–1419. https://doi.org/10.1056/nejm197912273012603 qiao q, keinänen-kiukaanniemi s, rajala u, uusimäki a, kivelä sl. random capillary whole blood glucose test as a screening test for diabetes mellitus in a middle-aged population. scand j clin lab invest. 1995;55(1):3–8. https://doi.org/10.3109/00365519509075372 simmons d, williams dr. random blood glucose as a screening test for diabetes in a bi-ethnic population. diabet med. 1994;11(9):830–835. https://doi.org/10.1111/j.1464-5491.1994.tb00364.x charani e, ahmad r, rawson tm, castro-sanchèz e, tarrant c, holmes ah. the differences in antibiotic decision-making between acute surgical and acute medical teams: an ethnographic study of culture and team dynamics. clin infect dis. 2019;69(1):12–20. https://doi.org/10.1093/cid/ciy844 mughal z, narayanan a, gupta v, reay-jones n. clinical need-directed blood tests: a step in saving the nhs? ann clin biochem. 2016;53(5):568–574. https://doi.org/10.1177/0004563215617782 chu kh, wagholikar as, greenslade jh, o’dwyer ja, brown af. sustained reductions in emergency department laboratory test orders: impact of a simple intervention. postgrad med j. 2013;89(1056):566–571. https://doi.org/10.1136/postgradmedj-2012-130833 dungan km, braithwaite ss, preiser jc. stress hyperglycaemia. lancet. 2009;373(9677):1798–1807. https://doi.org/10.1016/s0140-6736(09)60553-5 cadamuro j, gaksch m, wiedemann h, et al. are laboratory tests always needed? frequency and causes of laboratory overuse in a hospital setting. clin biochem. 2018;54:85–91. https://doi.org/10.1016/j.clinbiochem.2018.01.024 van walraven c, david naylor c. do we know what inappropriate laboratory utilization is? a systematic review of laboratory clinical audits. j am med assoc. 1998;280(6):550–558. https://doi.org/10.1001/jama.280.6.550 gandhi tk, kachalia a, thomas ej, et al. missed and delayed diagnoses in the ambulatory setting: a study of closed malpractice claims. ann intern med. 2006;145(7):488–496. https://doi.org/10.7326/0003-4819-145-7-200610030-00006 plebani m, zaninotto m, faggian d. utilization management: a european perspective. clin chim acta. 2014;427:137–141. https://doi.org/10.1016/j.cca.2013.03.002 reichard kk, wood aj. laboratory test utilization management. general principles and applications in hematopathology. surg pathol clin. 2016;9(1):1–10. https://doi.org/10.1016/j.path.2015.10.002 whiting p, toerien m, de salis i, et al. a review identifies and classifies reasons for ordering diagnostic tests. j clin epidemiol. 2007;60(10):981–989. https://doi.org/10.1016/j.jclinepi.2007.01.012 wilson ml. decreasing inappropriate laboratory test utilization controlling costs and improving quality of care. am j clin pathol. 2015;143(5):614–616. https://doi.org/10.1309/ajcphqodm9xywlz9 baird g. the laboratory test utilization management toolbox. biochem medica. 2014;24(2):223–234. https://doi.org/10.11613/bm.2014.025 zhao x, bo l, zhao h, li l, zhou y, wang h. descriptive study of the relationship between the subclinical carotid disease and biomarkers, carotid femoral pulse wave velocity in patients with hypertension. clin exp hypertens. 2018;40(3):274–280. https://doi.org/10.1080/10641963.2017.1368537 ducatman bs, ducatman am, crawford jm, laposata m, sanfilippo f. the value proposition for pathologists: a population health approach. acad pathol. 2020;7. https://doi.org/10.1177/2374289519898857 global targets are not enough cooperation hinges on wealthy nations doing more acknowledgements references about the author(s) lukoye atwoli east african medical journal, nairobi, kenya abdullah h. baqui journal of health population and nutrition, baltimore, maryland, united states of america thomas benfield danish medical journal, copenhagen, denmark raffaella bosurgi plos medicine, cambridge, united kingdom fiona godlee the bmj, london, united kingdom stephen hancocks british dental journal, london, united kingdom richard horton the lancet, london, united kingdom laurie laybourn-langton uk health alliance on climate change, london, united kingdom carlos augusto monteiro revista de saúde pública, são paulo, brazil ian norman international journal of nursing studies, london, united kingdom kirsten patrick canadian medical association journal, ottawa, ontario, canada nigel praities pharmaceutical journal, london, united kingdom marcel g.m. olde rikkert the dutch journal of medicine, amsterdam, netherlands eric j. rubin the new england journal of medicine, boston, massachusetts, united states of america peush sahni national medical journal of india, new delhi, delhi, india richard smith uk health alliance on climate change, london, united kingdom nicholas j. talley medical journal of australia, sydney, new south wales, australia sue turale international nursing review, geneva, switzerland damián vázquez pan american journal of public health, washington, dc, united states of america citation atwoli l, baqui ah, benfield t, et al. call for emergency action to limit global temperature increases, restore biodiversity, and protect health. afr j lab med. 2021;10(1), a1707. https://doi.org/10.4102/ajlm.v10i1.1707 editorial call for emergency action to limit global temperature increases, restore biodiversity, and protect health lukoye atwoli, abdullah h. baqui, thomas benfield, raffaella bosurgi, fiona godlee, stephen hancocks, richard horton, laurie laybourn-langton, carlos augusto monteiro, ian norman, kirsten patrick, nigel praities, marcel g.m. olde rikkert, eric j. rubin, peush sahni, richard smith, nicholas j. talley, sue turale, damián vázquez copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. wealthy nations must do much more, much faster the un general assembly in september 2021 will bring countries together at a critical time for marshalling collective action to tackle the global environmental crisis. they will meet again at the biodiversity summit in kunming, china, and the climate conference (cop26) in glasgow, uk. ahead of these pivotal meetings, we—the editors of health journals worldwide—call for urgent action to keep average global temperature increases below 1.5°c, halt the destruction of nature, and protect health. health is already being harmed by global temperature increases and the destruction of the natural world, a state of affairs health professionals have been bringing attention to for decades.1 the science is unequivocal; a global increase of 1.5°c above the pre-industrial average and the continued loss of biodiversity risk catastrophic harm to health that will be impossible to reverse.2,3 despite the world’s necessary preoccupation with covid-19, we cannot wait for the pandemic to pass to rapidly reduce emissions. reflecting the severity of the moment, this editorial appears in health journals across the world. we are united in recognising that only fundamental and equitable changes to societies will reverse our current trajectory. the risks to health of increases above 1.5°c are now well established.2 indeed, no temperature rise is “safe.” in the past 20 years, heat related mortality among people aged over 65 has increased by more than 50%.4 higher temperatures have brought increased dehydration and renal function loss, dermatological malignancies, tropical infections, adverse mental health outcomes, pregnancy complications, allergies, and cardiovascular and pulmonary morbidity and mortality.5,6 harms disproportionately affect the most vulnerable, including among children, older populations, ethnic minorities, poorer communities, and those with underlying health problems.2,4 global heating is also contributing to the decline in global yield potential for major crops, falling by 1.8–5.6% since 1981; this, together with the effects of extreme weather and soil depletion, is hampering efforts to reduce undernutrition.4 thriving ecosystems are essential to human health, and the widespread destruction of nature, including habitats and species, is eroding water and food security and increasing the chance of pandemics.3,7,8 the consequences of the environmental crisis fall disproportionately on those countries and communities that have contributed least to the problem and are least able to mitigate the harms. yet no country, no matter how wealthy, can shield itself from these impacts. allowing the consequences to fall disproportionately on the most vulnerable will breed more conflict, food insecurity, forced displacement, and zoonotic disease—with severe implications for all countries and communities. as with the covid-19 pandemic, we are globally as strong as our weakest member. rises above 1.5°c increase the chance of reaching tipping points in natural systems that could lock the world into an acutely unstable state. this would critically impair our ability to mitigate harms and to prevent catastrophic, runaway environmental change.9,10 global targets are not enough encouragingly, many governments, financial institutions, and businesses are setting targets to reach net-zero emissions, including targets for 2030. the cost of renewable energy is dropping rapidly. many countries are aiming to protect at least 30% of the world’s land and oceans by 2030.11 these promises are not enough. targets are easy to set and hard to achieve. they are yet to be matched with credible short and longer term plans to accelerate cleaner technologies and transform societies. emissions reduction plans do not adequately incorporate health considerations.12 concern is growing that temperature rises above 1.5°c are beginning to be seen as inevitable, or even acceptable, to powerful members of the global community.13 relatedly, current strategies for reducing emissions to net zero by the middle of the century implausibly assume that the world will acquire great capabilities to remove greenhouse gases from the atmosphere.14,15 this insufficient action means that temperature increases are likely to be well in excess of 2°c,16 a catastrophic outcome for health and environmental stability. critically, the destruction of nature does not have parity of esteem with the climate element of the crisis, and every single global target to restore biodiversity loss by 2020 was missed.17 this is an overall environmental crisis.18 health professionals are united with environmental scientists, businesses, and many others in rejecting that this outcome is inevitable. more can and must be done now—in glasgow and kunming—and in the immediate years that follow. we join health professionals worldwide who have already supported calls for rapid action.1,19 equity must be at the centre of the global response. contributing a fair share to the global effort means that reduction commitments must account for the cumulative, historical contribution each country has made to emissions, as well as its current emissions and capacity to respond. wealthier countries will have to cut emissions more quickly, making reductions by 2030 beyond those currently proposed20,21 and reaching net-zero emissions before 2050. similar targets and emergency action are needed for biodiversity loss and the wider destruction of the natural world. to achieve these targets, governments must make fundamental changes to how our societies and economies are organised and how we live. the current strategy of encouraging markets to swap dirty for cleaner technologies is not enough. governments must intervene to support the redesign of transport systems, cities, production and distribution of food, markets for financial investments, health systems, and much more. global coordination is needed to ensure that the rush for cleaner technologies does not come at the cost of more environmental destruction and human exploitation. many governments met the threat of the covid-19 pandemic with unprecedented funding. the environmental crisis demands a similar emergency response. huge investment will be needed, beyond what is being considered or delivered anywhere in the world. but such investments will produce huge positive health and economic outcomes. these include high quality jobs, reduced air pollution, increased physical activity, and improved housing and diet. better air quality alone would realise health benefits that easily offset the global costs of emissions reductions.22 these measures will also improve the social and economic determinants of health, the poor state of which may have made populations more vulnerable to the covid-19 pandemic.23 but the changes cannot be achieved through a return to damaging austerity policies or the continuation of the large inequalities of wealth and power within and between countries. cooperation hinges on wealthy nations doing more in particular, countries that have disproportionately created the environmental crisis must do more to support low and middle income countries to build cleaner, healthier, and more resilient societies. high income countries must meet and go beyond their outstanding commitment to provide $100bn a year, making up for any shortfall in 2020 and increasing contributions to and beyond 2025. funding must be equally split mitigation and adaptation, including improving the resilience of health systems. financing should be through grants rather than loans, building local capabilities and truly empowering communities, and should come alongside forgiving large debts, which constrain the agency of so many low income countries. additional funding must be marshalled to compensate for inevitable loss and damage caused by the consequences of the environmental crisis. as health professionals, we must do all we can to aid the transition to a sustainable, fairer, resilient, and healthier world. alongside acting to reduce the harm from the environmental crisis, we should proactively contribute to global prevention of further damage and action on the root causes of the crisis. we must hold global leaders to account and continue to educate others about the health risks of the crisis. we must join in the work to achieve environmentally sustainable health systems before 2040, recognising that this will mean changing clinical practice. health institutions have already divested more than $42bn of assets from fossil fuels; others should join them.4 the greatest threat to global public health is the continued failure of world leaders to keep the global temperature rise below 1.5°c and to restore nature. urgent, society-wide changes must be made and will lead to a fairer and healthier world. we, as editors of health journals, call for governments and other leaders to act, marking 2021 as the year that the world finally changes course. competing interests: we have read and understood bmj policy on declaration of interests and fg serves on the executive committee for the uk health alliance on climate change and is a trustee of the eden project. rs is the chair of patients know best, has stock in unitedhealth group, has done consultancy work for oxford pharmagenesis, and is chair of the lancet commission of the value of death. none further declared. provenance and peer review: commissioned; not externally peer reviewed. this editorial is being published simultaneously in many international journals. please see the full list here: https://www.bmj.com/content/full-list-authors-and-signatories-climate-emergency-editorial-september-2021 this is an open access article distributed in accordance with the terms of the creative commons attribution (cc by 4.0) license, which permits others to distribute, remix, adapt and build upon this work, for commercial use, provided the original work is properly cited. see: http://creativecommons.org/licenses/by/4.0/ acknowledgements competing interests we have read and understood bmj policy on declaration of interests and fg serves on the executive committee for the uk health alliance on climate change and is a trustee of the eden project. rs is the chair of patients know best, has stock in unitedhealth group, has done consultancy work for oxford pharmagenesis, and is chair of the lancet commission of the value of death. none further declared. authors’ contributions laurie laybourn-langton conceived of the idea, coordinated its development and delivery, and led drafting along with richard smith. all authors contributed significantly to the editorial content. funding information laurie laybourn-langton’s time was funded by the climate and health council. respective authors were paid by their employers. data availability the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. disclaimer this editorial is being published simultaneously in many international journals. please see the full list here: https://www.bmj.com/content/full-list-authors-and-signatories-climate-emergency-editorial-september-2021. references in support of a health recovery [homepage on the internet]. available from: https://healthyrecovery.net intergovernmental panel on climate change. summary for policymakers. in: global warming of 1.5°c. an ipcc special report on the impacts of global warming of 1.5°c above pre-industrial levels and related global greenhouse gas emission pathways, in the context of strengthening the global response to the threat of climate change, sustainable development, and efforts to eradicate poverty [homepage on the internet]. 2018. available from: https://www.ipcc.ch/sr15/ intergovernmental science-policy platform on biodiversity and ecosystem services. summary for policymakers: the global assessment report on biodiversity and ecosystem services [homepage on the internet]. 2019. available from: https://ipbes.net/sites/default/files/2020-02/ipbes_global_assessment_report_summary_for_policymakers_en.pdf watts n, amann m, arnell n, et al. the 2020 report of the lancet countdown on health and climate change: responding to converging crises. lancet. 2021;397:129–170. rocque rj, beaudoin c, ndjaboue r, et al. health effects of climate change: an overview of systematic reviews. bmj open. 2021;11(6):e046333. https://doi.org/10.1136/bmjopen-2020-046333 haines a, ebi k. the imperative for climate action to protect health. n engl j med. 2019;380:263–273. https://doi.org/10.1056/nejmra1807873 united nations environment programme and international livestock research institute. preventing the next pandemic: zoonotic diseases and how to break the chain of transmission [homepage on the internet]. 2020. available from: https://72d37324-5089-459c-8f70-271d19427cf2.filesusr.com/ugd/056cf4_b5b2fc067f094dd3b2250cda15c47acd.pdf ipcc. 2019: summary for policymakers. in: climate change and land: an ipcc special report on climate change, desertification, land degradation, sustainable land management, food security, and greenhouse gas fluxes in terrestrial ecosystems. forthcoming. lenton tm, rockström j, gaffney o, et al. climate tipping points—too risky to bet against. nature. 2019;575:592–595. https://doi.org/10.1038/d41586-019-03595-0 wunderling n, donges jf, kurths j, winkelmann r. interacting tipping elements increase risk of climate domino effects under global warming. earth system dynamics discussions; 2020, p. 1–21. high ambition coalition [homepage on the internet]. available from: https://www.hacfornatureandpeople.org global climate and health alliance. are national climate commitments enough to protect our health? [homepage on the internet]. available from: https://climateandhealthalliance.org/initiatives/healthy-ndcs/ndc-scorecards/ climate strikers: open letter to eu leaders on why their new climate law is ‘surrender’ [homepage on the internet]. carbon brief 2020. available from: https://www.carbonbrief.org/climate-strikers-open-letter-to-eu-leaders-on-why-their-new-climate-law-is-surrender fajardy m, köberle a, macdowell n, fantuzzi a. beccs deployment: a reality check [homepage on the internet]. grantham institute briefing paper 28, 2019. available from: https://www.imperial.ac.uk/media/imperial-college/grantham-institute/public/publications/briefing-papers/beccs-deployment---a-reality-check.pdf anderson k, peters g. the trouble with negative emissions. science. 2016;354 (6309):182–183. https://doi.org/10.1126/science.aah4567 climate action tracker [homepage on the internet]. available from: https://climateactiontracker.org secretariat of the convention on biological diversity. global biodiversity outlook 5 [homepage on the internet]. 2020. available from: https://www.cbd.int/gbo5 steffen w, richardson k, rockström j, et al. sustainability. planetary boundaries: guiding human development on a changing planet. science. 2015; 347(6223):1259855. https://doi.org10.1126/science.1259855 uk health alliance. our calls for action [homepage on the internet]. available from: http://www.ukhealthalliance.org/cop26/ climate action tracker. warming projections global update: may 2021 [homepage on the internet]. available from: https://climateactiontracker.org/documents/853/cat_2021-05-04_briefing_global-update_climate-summit-momentum.pdf united nations environment programme. emissions gap report 2020. unep; 2020. markandya a, sampedro j, smith sj, et al. health co-benefits from air pollution and mitigation costs of the paris agreement: a modelling study. lancet planet health. 2018;2:e126–e133. https:doi.org/10.1016/s2542-5196(18)30029-9 paremoer l, nandi s, serag h, baum f. covid-19 pandemic and the social determinants of health. bmj. 2021;372:n129. https://doi.org/10.1136/bmj.n129 references about the author(s) iruka n. okeke department of pharmaceutical microbiology, faculty of pharmacy, university of ibadan, ibadan, nigeria citation okeke in. towards a fiercely urgent expansion of laboratory medicine in africa. afr j lab med. 2021;10(1), a1785. https://doi.org/10.4102/ajlm.v10i1.1785 editorial towards a fiercely urgent expansion of laboratory medicine in africa iruka n. okeke copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the african journal of laboratory medicine (ajlm) has published its 10th annual volume! our journal was established in 2011 on the tails of rapid and unprecedented growth of laboratory medicine in africa that began in the current century.1 the journal’s first nine volumes published articles covering virtually all scientific, social science and practice specialties in, or studying, laboratory medicine. the ajlm has also proudly curated six forward-looking special issues focused on the strengthening laboratory management towards accreditation programme (2014),2 quality assurance in point-of-care testing for hiv (2016),3 the role of the laboratory in global health security (2016),4 strengthening tuberculosis diagnostic networks in africa (2017),5 african laboratories in antimicrobial resistance surveillance (2018)6 and the future of diagnostics (2020).7 it has been especially rewarding to steer our journal in the last four years of its first decade and to simultaneously watch the reputational quality my predecessors established maintained, while growing what is now the pre-eminent laboratory medicine journal on the african continent. working during a pandemic has made the last two extraordinarily productive years doubly strenuous, for us and all other biomedical science editorial offices. at the end of 2020, our valiant, but inaccurate, assessment was that our essential pandemic contributions might be temporary.8 one year later, it is clear that the battle against coronavirus disease 2019 (covid-19) will continue for some time to come and, therefore, how we operate now must become part of our future. it is perhaps fitting that i reflect on the fact that, while the scope of our journal is very broad, 8 of 32 articles published in volume 10, excluding this editorial, relate to the pandemic. covid-19 articles make roughly a quarter of our pages; this reflects the enormous effort that laboratory medicine researchers and practitioners have put into the response. it serves as one of many records of the heavy burden the pandemic has placed on the african continent, which has made giant strides in developing laboratory medicine in the last decade. there are 24 non-covid-19 articles in volume 10 (2021), while the number of such articles published pre-pandemic in 2018 and 2019 was 17 and 29. thus, we have added pandemic scientific publishing onto our usual, and growing, ajlm output. nonetheless, we have not diverted all our focus towards the response to a single disease. for centuries, africa has been fighting infections without necessary laboratory medicine support;9 a new journal was born to document the african laboratory’s role a decade ago,1 but this substantial upsurge in published papers – just one reading of diagnostic progress – happened now. therefore, in a sense, at least some of our growth was pandemic-driven. the pandemic also forces us to accept that there is some predictability that new diseases of unpredictable nature could emerge in the future, and that scientific knowledge and laboratory capacities must be extended to meet them. a more recent, but equally pressing, rise in non-communicable disease prevalence adds to the essentiality of, and demand for, effective laboratory medicine and improved health systems, which can offer needed laboratory support for high-quality care. for all of these reasons, we should expect our journal to grow geometrically in the next decade. our editors, readers and authors must keep pace with the rising magnitude and increased diversity of laboratory operations that support clinical care. no doubt, growth is essential, but how quickly does it need to take place? synthetic evidence has shown that most patients in africa still do not have access to needed diagnostics for managing their illnesses,10 despite advances in laboratory medicine on the continent in the past two decades. even for severe acute respiratory syndrome coronavirus 2 (sars-cov-2), where we have seen phenomenal growth and a truly impressive scale of testing and laboratory-led surveillance,11,12 access to laboratory medicine for covid-19 on the african continent is still suboptimal for public health and patient management. the experts that author and read our articles are working frenetically to close the gap. the answer to the question of ‘how quickly?’ must be ‘very’. the response needed to close africa’s diagnostic gaps is not only critically important, it is fiercely urgent. the value of health systems, and the will to maintain them, will become recognisable only when the best evidence, often from laboratories, forms the basis for patient treatment and management.13 moreso, every missed or mistreated illness due to diagnostic and laboratory insufficiency is a prolonged or, often, fatal illness. once the need for any change becomes apparent, harder pushes are needed to make change happen and sometimes events coincide to produce a landscape that will help ensure that the effort invested will field results. the pandemic, although a time of great difficulty, has created such a time for laboratory medicine. we must leverage current impetus for better laboratory diagnostics to avert future disaster. one of the oft-quoted speeches of the united states’ martin luther king jr enjoins us to push when opportunity stands in front of emergency: we are now faced with the fact that tomorrow is today. we are confronted with the fierce urgency of now. in this unfolding conundrum of life and history, there ‘is’ such a thing as being too late. this is no time for apathy or complacency. this is a time for vigorous and positive action.14 as our journal, and the african society for laboratory medicine that owns it, move into their second decades, we must consider the need to grow not only expansively, but also quickly and purposefully as part of a multipronged, continent-wide and locally proclaimed effort to enhance health for africans.15 we must just as fiercely move from the motivating rhetoric of another place and era to now, and we must hold our selves, as well as others, to account.16 this is particularly true because the current pandemic has shown us that rapid but rigorous growth is possible in laboratory medicine and powerful tools exist to help get us there.17,18 we have now seen that the price of ‘rapid’ growth is insignificant compared to the cost of not growing responsively is far responsively. we must, therefore, plan to scale up laboratory medicine accordingly. references luman e. the african journal of laboratory medicine – advancing laboratory medicine and science in africa. afr j lab med. 2012;1(1):1. https://doi.org/10.4102/ajlm.v1i1.86 noble ma, martin r, ndihokubwayo j-b. making great strides in medical laboratory quality. afr j lab med. 2016;5(2):1–2. murtagh mm. quality assurance for point-of-care diagnostic testing: it is not negotiable. afr j lab med. 2016;5(2):1–2. https://doi.org/10.4102/ajlm.v5i2.554 peter t, keita m-s, nkengasong j. building laboratory capacity to combat disease outbreaks in africa. afr j lab med. 2016;5(3):1–2. https://doi.org/10.4102/ajlm.v5i3.579 piatek a. tuberculosis diagnostic networks: moving beyond the laboratory to end tuberculosis in africa. afr j lab med. 2017;6(2):1–2. https://doi.org/10.4102/ajlm.v6i2.608 kariuki s, keddy kh, antonio m, okeke in. antimicrobial resistance surveillance in africa: successes, gaps and a roadmap for the future. afr j lab med. 2018;7(2):1–2. https://doi.org/10.4102/ajlm.v7i2.924 fongwen n, boeras d, peeling rw, amukele t. connected diagnostics systems: the future of disease control in africa. afr j lab med. 2020;9(2):a1365. https://doi.org/10.4102/ajlm.v9i2.1365 okeke in. african laboratory medicine in the time of covid-19. afr j lab med. 2020;9(1):1–3. https://doi.org/10.4102/ajlm.v9i1.1447 okeke in. divining without seeds: the case for strengthening laboratory medicine in africa. ithaca, ny: ilr/cornell university press, 2011; p. 222. fleming ka, horton s, wilson ml, et al. the lancet commission on diagnostics: transforming access to diagnostics. lancet. 2021, p. 1–54. https://doi.org/10.1016/s0140-6736(21)00673-5 kapata n, ihekweazu c, ntoumi f, et al. is africa prepared for tackling the covid-19 (sars-cov-2) epidemic. lessons from past outbreaks, ongoing pan-african public health efforts, and implications for the future. int j infect dis. 2020;93:233–236. https://doi.org/10.1016/j.ijid.2020.02.049 wilkinson e, giovanetti m, tegally h, et al. a year of genomic surveillance reveals how the sars-cov-2 pandemic unfolded in africa. science. 2021;374(6566):423–431. https://doi.org/10.1126/science.abj4336 ondoa p, oskam l, loembe mm, okeke in. transforming access to diagnostics: how to turn good intentions into action? lancet. 2021. https://doi.org/10.1016/s0140-6736(21)02182-6 king ml, washington jm. a testament of hope: the essential writings of martin luther king, jr. new york: harper & row; 1986, p. 231–244. nkengasong jn, tessema sk. africa needs a new public health order to tackle infectious disease threats. cell. 2020;183(2):296–300. https://doi.org/10.1016/j.cell.2020.09.041 sirleaf ej, clark h. achieving vaccination justice: a call for global cooperation. plos global public health. 2021;1(10):e0000036. https://doi.org/10.1371/journal.pgph.0000036 okeke in, ihekweazu c. the importance of molecular diagnostics for infectious diseases in low-resource settings. nat rev microbiol. 2021;19:547–548. https://doi.org/10.1038/s41579-021-00598-5 gous nm, onyebujoh pc, abimiku a, macek c, takle j. the role of connected diagnostics in strengthening regional, national and continental african disease surveillance. afr j lab med. 2018;7(2):775. https://doi.org/10.4102/ajlm.v7i2.775 abstract background description of the intervention lessons learnt recommendations acknowledgements references about the author(s) iriagbonse i. osaigbovo department of medical microbiology, school of medicine, college of medical sciences, university of benin, benin city, nigeriadepartment of medical microbiology, university of benin teaching hospital, benin city, nigeria isaac o. igbarumah molecular virology laboratory, university of benin teaching hospital, benin city, nigeria ekene b. muoebonam institute of lassa fever research and control, irrua specialist teaching hospital, irrua, nigeria darlington e. obaseki department of anatomic pathology, school of medicine, college of medical sciences, university of benin, benin city, nigeriadepartment of anatomic pathology, university of benin teaching hospital, benin city, nigeria citation osaigbovo ii, igbarumah io, muoebonam eb, obaseki de. setting up a molecular diagnostic laboratory for sars-cov-2 testing: experience of a single centre in a resource-constrained setting. afr j lab med. 2021;10(1), a1326. https://doi.org/10.4102/ajlm.v10i1.1326 lessons from the field setting up a molecular diagnostic laboratory for sars-cov-2 testing: experience of a single centre in a resource-constrained setting iriagbonse i. osaigbovo, isaac o. igbarumah, ekene b. muoebonam, darlington e. obaseki received: 08 july 2020; accepted: 04 feb. 2021; published: 30 mar. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: molecular detection of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is at the forefront of the global response to the coronavirus disease 2019 (covid-19) pandemic. however, molecular diagnostic capabilities are poorly developed in many african countries. efforts by the nigeria centre for disease control and other public health agencies to scale up facilities for molecular testing across the continent are well documented, but there are few accounts from the laboratories at the frontline. intervention: as part of an institutional response to the covid-19 pandemic, the university of benin teaching hospital, benin city, nigeria, signed a memorandum of understanding with a world bank-supported institution to obtain a non-proprietary testing platform, renovated an existing molecular virology laboratory and validated the test process to make sars-cov-2 testing readily available for decision-making by frontline health workers. these efforts resulted in the university of benin teaching hospital’s inclusion in the nigeria centre for disease control covid-19 molecular laboratory network. the laboratory achieved a turnover of 12 123 tests within 7 months of operation. challenges faced and dealt with include incompatible equipment, limited skilled manpower, unstable (unreliable) electric power supply, disrupted procurement and supply chain, and significant overhead costs. lessons learnt: molecular diagnostic capability is essential in laboratory preparedness for pandemic response and can be achieved by establishing collaborative networks in low-resource settings. recommendations: molecular diagnostic capabilities attained during the covid-19 pandemic should be maintained by governmental support of the local biotechnology sector, collaboration with partners and stakeholders and the expansion of diagnostics to include other diseases of public health importance. keywords: covid-19; coronavirus disease 2019; laboratory; nigeria; molecular diagnosis; sars-cov-2; severe acute respiratory syndrome coronavirus 2; ubth; university of benin teaching hospital; resource-constrained. background coronavirus disease 2019 (covid-19) caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) was first described following an outbreak of atypical pneumonia in china in late 2019.1 it has since escalated into a global pandemic with over 36 million cases and more than a million deaths worldwide as of 10 october 2020.2 laboratory testing to detect, isolate and treat cases is key to the containment of the virus; establishing widespread diagnostic capacity has been a strategy of successful response in countries like south korea.3 to date, molecular methods, particularly real-time reverse transcriptase polymerase chain reaction (pcr), remain the mainstay of diagnostic testing.4 molecular diagnostic capability is in short supply in many parts of sub-saharan africa. in nigeria, available molecular diagnostic capabilities arose from vertical disease programme models for hiv and lassa fever in the 2000s.5,6,7 since the 2014 west african ebola outbreak, which emphasised the need for strong public health systems, the nigeria centre for disease control (ncdc) has gradually developed a network of laboratories capable of molecular diagnosis of infections of public health importance.8,9 this enabled nigeria to establish diagnostic capacity for sars-cov-2 testing in three laboratories within one month of the report of covid-19 cases from china and before the detection of the first case in sub-saharan africa on 27 february 2020.10,11 by leveraging already existing infrastructure for hiv, lassa fever and drug-resistant tuberculosis, the number of public and private laboratories able to test for sars-cov-2 has been gradually and consistently scaled up to 90 laboratories distributed across all 36 states and the federal capital territory of nigeria as of 08 october 2020 (figure 1a). the tremendous efforts by ncdc and other public health agencies across africa to scale up testing are well documented in the scientific literature.11 it is desirable that laboratory testing for sars-cov-2 is made available close to the ‘frontline’ of the pandemic, that is, within hospitals whenever feasible and safe, as this enables clinical teams to make critical decisions about inpatient management.12 these decisions include whether to isolate patients, thereby preventing the nosocomial spread of covid-19, and how to allocate scarce personal protective equipment (ppe). however, there is a paucity of accounts from laboratories at the frontline of testing and diagnosis. figure 1: covid-19 laboratory network in nigeria and edo state, 2020. (a) map of nigeria showing nigeria centre for disease control covid-19 molecular laboratory network (public laboratories only). (b) map of edo state showing local government areas and the locations of university of benin teaching hospital molecular virology laboratory and the institute of lassa fever research and control, irrua specialist teaching hospital. two public laboratories in the ncdc network for covid-19 testing are domiciled in edo state, nigeria (figure 1b). one of these, the institute of lassa fever research and control (ilfrc), irrua specialist teaching hospital, is a pioneer laboratory conscripted early in the fight against covid-19. the other, located at the university of benin teaching hospital (ubth) and included in the network on 10 may 2020, was set up because the management envisioned frontline diagnostic testing as a critical aspect of institutional response to the pandemic. this article describes the steps taken to set up testing for sars-cov-2 in the ubth. site description the ubth is an 850-bed tertiary hospital located in the south-south geopolitical zone of nigeria. it serves edo state which has a landmass of 17 802 square kilometres and neighbouring states delta, kogi, anambra and ondo, providing both primary and referral heathcare services. located in the state capital benin city, the institution is at the forefront of the state’s response to combating the pandemic.13 as of 31 december 2020, 2870 cases of laboratory-confirmed covid-19 have been reported in edo state.14 the molecular virology laboratory in ubth is a biosafety level 2 containment facility. it was set up in 2005 and moved to its current location in 2011 with the support of the institute of human virology of nigeria to offer early infant diagnosis and viral load detection services for the united states president’s emergency plan for aids relief aids care treatment in nigeria programme in the state.5 this partnership provided training in pcr diagnostic testing for laboratory scientists as well as laboratory equipment including a conventional pcr machine (later replaced with the roche cobas® taqman 96, a proprietary real-time pcr machine not compatible with currently accessible sars-cov-2 assays). other important equipment and items that were already in place in the laboratory include biosafety cabinets, fixed angle micro-centrifuge, bench centrifuge, heating blocks, freezers, an uninterrupted power supply and back-up generator. besides hiv testing, the laboratory also recently joined the ncdc network for yellow fever and measles testing following a favourable performance in a laboratory audit exercise conducted in december 2019. the staff of six consists of three laboratory scientists, one laboratory technician and two data clerks. description of the intervention ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. planning and preparation memorandum of understanding and polymerase chain reaction machine setup to expedite the commencement of testing while sourcing funds to purchase a new pcr machine, the hospital collaborated with the centre for excellence in reproductive health innovation, university of benin, nigeria. the centre for excellence in reproductive health innovation is one of the several world bank-funded centres for excellence in african higher education institutions initiated to promote homegrown and regional research networks. the collaboration with centre for excellence in reproductive health innovation was made official by signing a memorandum of understanding on 15 april 2020. this memorandum of understanding clearly states the purpose of the agreement, which is to jointly operate a covid-19 testing unit in ubth. the areas and scope of cooperation (including equipment, personnel and biosafety, provision of consumables and data collection for research purposes), implementation and the mode of handling intellectual property that may arise from the collaboration were also spelt out. following the signing of the memorandum of understanding, a new magnetic induction cycler real-time dx48 pcr instrument (biomolecular systems, upper coomera, queensland, australia) belonging to the centre for excellence in reproductive health innovation was transferred to the ubth molecular laboratory to conduct sars-cov-2 testing. the machine is an open (non-proprietary) platform compatible for use with many assays (figure 2). figure 2: magnetic induction cycler real-time dx48 pcr instrument obtained from centre for excellence in reproductive health innovation, edo state, nigeria, 2020. renovation of existing laboratory physical infrastructure the existing laboratory was ill-designed for molecular testing of highly infectious diseases like sars-cov-2. clean and dirty areas were not demarcated, that is, staff had to take the specimens through the master mix preparation area to get to the extraction room. the laboratory was thus physically reconfigured and renovated to demarcate the dirty (red) zone from clean areas to comply with a strictly unidirectional flow of movement. the cleanroom or section for mastermix preparation room, extraction room with a biosafety cabinet and centrifuge, and amplification and detection room for real-time detection of nucleic acid (figure 3) were each equipped with a supply of ppe, multi-channel pipette and other equipment and consumables to prevent contamination. for increased biosafety, an exit room for doffing ppe and showering was created to ensure a strictly unidirectional movement and prevent contamination of administrative and office areas. a well-demarcated sample reception area was also created. figure 3: amplification and detection room for real-time detection of nucleic acid, edo state, nigeria, 2020. online mentorship and training to gain proficiency in the use of the machine, the chief scientist, other laboratory scientists and staff in the laboratory underwent hands-on training which, due to the lock-down situation in many states and ban on interstate travel in nigeria, was done via online video conferencing. an experienced molecular biologist, conversant with the magnetic induction cycler testing platform and sars-cov-2 molecular testing provided the training. sessions included the development of standard operating protocols and job aids specific for sars-cov-2 testing, biosafety protocols applicable to the virus and appropriate waste disposal. manpower reinforcement staff strength was reinforced by redeploying two scientists from the general medical microbiology laboratory and two data clerks. this scaled up the number of staff to 10: five laboratory scientists, one laboratory technician and four data clerks. the new staff received orientation and training for their specific tasks. specifically, the laboratory technician is responsible for specimen reception and registration, the laboratory scientists carry out the various stages of reverse transcription-pcr and run quality control checks, while the data clerks are responsible for uploading data from the case investigation forms and test results onto online surveillance platforms. stockpiling of personal protective equipment personal protective equipment items and stock were increased to ensure adequate protection of laboratory staff. personal protective equipment items included gloves, gowns, goggles, footwear, face shields, face masks and respirators. evaluation and validation by nigeria centre for disease control site visit by officials of institute of lassa fever research and control as a regional reference laboratory for covid-19 testing, ilfrc officials represented ncdc for the laboratory assessment visit using a checklist designed by the ncdc. areas of assessment included biosafety checks, availability of ppe, a waste management plan, staff strength, equipment and stock management. on-site mentorship, training and evaluation an experienced molecular scientist from the ilfrc was deployed to provide technical support to the laboratory staff in workflow optimisation, infection prevention and control and handling of laboratory data. the ubth laboratory scientists were also evaluated for competence in conducting the test using the national algorithm, laboratory safety, environmental cleaning and decontamination protocols. validation equipment validation was done using commercial standards and controls (liferiver, shanghai zj biotech, shanghai, china), while assay runs were validated by inter-laboratory comparison with ilfrc. duplicate samples were collected from all suspected cases in the facility: one sample was sent to ilfrc while the other was processed and analysed by the on-site laboratory. inter-laboratory concordance was measured and independent testing was commenced when 100% concordance was achieved in 40 samples. operations following a successful validation process and on the recommendation of the site evaluators from ilfrc, the ubth molecular laboratory was included in the ncdc sars-cov-2 testing network on 10 may 2020. the testing protocol begins with inactivation of each sample, typically a nasopharyngeal swab and oropharyngeal swab in the same tube of viral transport medium, in a biosafety cabinet (figure 4) using an external lysis buffer reagent, which accompanies the commercial rna extraction kit. the sample is then moved to the rna extraction room where rna is extracted manually using an rna isolation kit (shanghai zj biotech, shanghai, china). five microlitres (μl) of the extracted rna is then added to 20 μl of prepared master mix (composed of 19 μl supermix and 1 μl enzyme mix) in the reaction tube. polymerase chain reaction amplification was achieved using a novel coronavirus real-time multiplex reverse transcription-pcr kit (liferiver, shanghai zj biotech, shanghai, china) on a magnetic induction cycler real-time dx48 pcr instrument (biomolecular systems, upper coomera, queensland, australia). results were interpreted based on the detection of the envelope (e), nucleocapsid (n) and open reading frame 1ab (orf1ab) genes. for a test to be considered positive, at least two of these genes, which must include the orf1ab, must have been detected at the recommended cycle threshold (ct) of less than 41. figure 4: sample inactivation room with a biosafety cabinet, edo state, nigeria, 2020. due to the open nature of the test platform, we were able to use other commercial rna extraction and detection kits, for instance daan gene (da an gene co. limited of sun yat-sen university, guangzhou, china) was used when the liferiver kit (liferiver, shanghai zj biotech, shanghai, china) was not available. the real-time outbreak and epidemic surveillance software surveillance outbreak response management and analysis system, employed by ncdc, was installed in the laboratory information management system and data clerks were trained by ncdc officials on test data imputation. quality assurance was emphasised along the entire testing pathway. pre-analytical conditions were addressed by a clinical pathologist closely liaising with clinical staff on proper specimen collection, handling and transport. quality control was ensured in each run by the use of appropriate positive, negative and internal controls. equally, data were collected and analysed to monitor key performance indicators such as turnaround time, positivity and specimen rejection rates. results in seven months of operation, the laboratory has performed 12 123 assays on ubth patient samples, referral samples from other health facilities and community samples from the edo state government-driven community screening and testing efforts. before the inclusion of the laboratory into the covid-19 testing network, all samples from ubth had to be sent in a once-daily trip to ilfrc which is over 100 km away and the result turnaround time was 3–4 days. the availability of on-site sars-cov-2 testing has shortened the turnaround time to 1–2 days as sample transportation-related delays in test turnaround time have been eliminated. the shortened sample collection-to-test result turnaround time expedites crucial patient care decision-making such as treatment initiation, triaging of patients in emergency departments, allocation of scarce ppe and other actions that prevent nosocomial spread. the renovations carried out in the laboratory improved the workflow and minimised the risks of contamination by ensuring that laboratory staff only move from clean areas to the dirty area (amplification area containing nucleic acids). challenges establishing sars-cov-2 testing amid the covid-19 pandemic has been problematic even in the most developed countries.15,16 general challenges include a slowdown in global manufacturing of sampling materials like swabs and viral transport medium, difficulties in shipping and procurement of commercial testing kits, contamination of molecular diagnostic reagents, lack of performance data for covid-19 testing kits that have been approved for emergency use, lack of positive control materials for covid-19 testing and shortage of ppe, among others. these obstacles are, expectedly, more pronounced in resource-poor settings, which also have their unique challenges.17 currently, sars-cov-2 testing in nigerian public health facilities is offered at no cost to individual patients and clients. although reagents and consumables are provided by ncdc, the supply is often irregular and testing institutions bear the brunt of significant overhead running costs. to mitigate the impact of delays in supply of testing reagents, ample lead time is given in making requests for reagents to prevent stock-outs. in addition, the dedicated 30 kilovolt-amperes (kva) back-up generator had to be replaced with a 60 kva to cope with the unstable electric power supply to prevent disruptions in testing and ensure proper storage of samples (table 1). table 1: challenges of setting up of sars-cov-2 testing in university of benin teaching hospital and mitigating actions taken, edo state, nigeria, 2020. lessons learnt the experience of setting up the molecular laboratory during the covid-19 pandemic emphasised the need for laboratory preparedness as a major aspect of institutional response to infectious disease outbreaks and other emergencies. molecular diagnostic capability is key to this laboratory preparedness and can be achieved by establishing collaborative networks in low-resource settings. firstly, ubth was able to obtain a platform for testing by signing a collaborative memorandum of understanding with a funded research centre in the university. secondly, despite travel restrictions, the laboratory staff were able to get the laboratory running via remote mentoring using web conferencing technology. the mentorship and guidance from the reference laboratory in the state was also invaluable particularly on the laboratory design, on-site evaluation and testing capacity validation. another lesson learnt is the value of non-proprietary molecular testing platforms that allow for flexibility and capacity to scale up in emergencies. this became especially evident when there were difficulties in accessing a particular commercial assay as the flexibility of the platform allowed us to easily switch to other available assay kits. recommendations the covid-19 pandemic has demonstrated the essential role of diagnostics in the control of infectious diseases and sparked renewed interest in molecular technologies in low-resource settings, including nigeria. we recommend that the capabilities attained are sustained by governmental support of the local biotechnology sector, continued surveillance for sars-cov-2 beyond the pandemic period, fostering collaboration and broadening the scope to include other diseases of public health importance. we expand on these recommendations below. it is hoped that the rekindled interest in molecular diagnostics will bring about a burgeoning biotechnology sector including a network of biomedical engineering services and local sources for laboratory reagents and consumables. these are necessary for sustaining efforts at the institutional healthcare level. indeed, this is already occurring in nigeria; for instance, the viral transport medium required for the shipment of samples that was being imported at the start of the pandemic is now locally produced and supplied by the national veterinary research institute in vom, jos. laboratories with the capacity to develop and validate quality in-house assays should be supported to reduce the over-reliance on external commercial sources. if sars-cov-2 becomes endemic in the population, surveillance for covid-19 will need to be maintained. low complexity, rapid point of care molecular testing platforms already in use in many hospitals can be leveraged to carry out smaller-scale testing and surveillance in the post-pandemic period when prevalence rates are likely to be lower. for instance, the xpress sars-cov-2 test cartridge can be used in the genexpert® (cepheid, sunnyvale, california, united states) machine that is already in use for diagnosis of tuberculosis in many hospitals. costs can be further lowered and throughput increased by deploying a pooled sample testing strategy after validating.18 there are no guarantees that government support for supplies of reagents, consumables and other logistics will last beyond the pandemic period. sustaining operations in a molecular laboratory is capital intensive and it is pertinent to consider how this will be achieved in the future. as observed in several other contexts, capital investments in laboratory equipment are difficult to recoup especially where test volumes are low and overhead maintenance costs are high.5,19 collaboration with an institution that had external funding was pivotal to the sars-cov-2 testing initiative in ubth and collaboration will be what sustains capabilities attained going forward. coordinated and goal-directed partnerships with national and regional stakeholders, research institutes, global donor agencies, international governing bodies and biomedical industries are required.19 also, the trained workforce will need to be retained utilising incentives and new hands will need to be recruited. while maintaining surveillance for sars-cov-2, laboratories, including ubth, will need to broaden their focus to include other diseases of public health importance that can attract collaboration from both ncdc and external donor agencies invested in global health. lassa fever, a disease endemic to many parts of nigeria, is an attractive candidate. the world health organization also prescribes molecular diagnosis for some other relevant infectious diseases like sexually transmitted diseases and dengue fever in the essential diagnostics list currently in its second edition.20 additionally, the covid-19 outbreak has impressed the need to better define the epidemiology of severe acute respiratory illnesses of viral aetiology. these viruses are best detected by molecular means and it will be worthwhile attaining the tools necessary to differentiate them from the novel coronavirus.21 in conclusion, the successful setup of sars-cov-2 testing in ubth was predicated on collaborative efforts, established quality management systems culture and innovativeness. whatever the future focus of the molecular laboratory, it is pertinent that molecular diagnostic infrastructure is kept patent as part of both the laboratory and institutional preparedness plans. this will ensure swift mobilisation to deal with infectious disease crises that will almost inevitably arise in the future. acknowledgements the authors acknowledge the centre for excellence in reproductive health innovation and the centre leader, professor friday okonofua, for providing the magnetic induction cycler real-time dx48 polymerase chain reaction instrument used in this project, the nigeria centre for disease control and its director-general, dr chikwe ihekweazu, for expediting activation of the laboratory, the chief medical director of irrua specialist teaching hospital, professor sylvanus okogbenin, and the director of the institute of lassa fever research and control, irrua specialist teaching hospital, dr ephraim ogbaini-emovon, for assessing the laboratory and contributing to its design. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions i.i.o., i.o.i., e.b.m. and d.e.o. contributed equally to the design and implementation of the research, to the analysis of the results and to the writing of the manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability data sharing does not apply to this article as no new data were created or analysed. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references zhu n, zhang d, wang w, et al. a novel coronavirus from patients with pneumonia in china, 2019. n engl j med. 2020;382(8):727–733. https://doi.org/10.1056/nejmoa2001017 world health organization. coronavirus disease (covid-19) weekly update on covid-19, 9th october, 2020 [homepage on the internet]. [cited 2020 oct 10]. available from: https://www.who.int/publications/m/item/weekly-update-on-covid-19-9-october-2020 oh j, lee j, schwarz d, ratcliffe hl, markuns j, hirschhorn lr. national response to covid-19 in the republic of korea and lessons learned for other countries. health syst reform. 2020;6(1):e1753464. https://doi.org/10.1080/23288604.2020.1753464 loeffelholz mj, tang yw. laboratory diagnosis of emerging human coronavirus infections-the state of the art. emerg microbes infect. 2020;9(1):747–756. https://doi.org/10.1080/22221751.2020.1745095 abimiku ag. building laboratory infrastructure to support scale-up of hiv/aids treatment, care and prevention: in-country experience. am j clin pathol. 2009;131(6):875–886. https://doi.org/10.1309/ajcpelmg6gx6rqsm omilabu sa, badaru so, okokhere p, et al. lassa fever, nigeria, 2003 and 2004. emerg infect dis. 2005;11(10):1642–1644. https://doi.org/10.3201/eid1110.041343 asogun d, adomeh d, ehimuan j, et al. molecular diagnostics for lassa fever at irrua specialist teaching hospital, nigeria: lessons learnt from two years of laboratory operation. plos negl trop dis. 2012;6(9):e1839. https://doi.org/10.1371/journal.pntd.0001839 njidda am, oyebanji o, obasanya j, et al. the nigeria centre for disease control. bmj glob health. 2018;3(2):e000712. https://doi.org/10.1136/bmjgh-2018-000712 kapata n, ihekweazu c, ntoumi f, et al. is africa prepared for tackling the covid-19 (sars-cov-2) epidemic? lessons from past outbreaks, ongoing pan-african public health efforts, and implications for the future. int j infect dis. 2020;93(2020):233–236. https://doi.org/10.1016/j.ijid.2020.02.049 nigeria centre for disease control. national strategy to scale up access to coronavirus disease testing in nigeria [homepage on the internet]. 2020 [cited 2020 jun 10]. available from: https://covid19.ncdc.gov.ng/media/files/covid19testingstrategy_2zwbqwh.pdf ihekweazu c, agogo e. africa’s response to covid-19. bmc med. 2020;18:151. https://doi.org/10.1186/s12916-020-01622-w binnicker mj. emergence of a novel coronavirus disease (covid-19) and the importance of diagnostic testing: why partnership between clinical laboratories, public health agencies, and industry is essential to control the outbreak. clin chem. 2020;66(5):664–666. https://doi.org/10.1093/clinchem/hvaa071 university of benin teaching hospital. about ubth [homepage on the internet]. [cited 2021 jan 11]. available from: https://ubth.org/general-information/#about nigeria centre for disease control. progression of covid-19 cases in nigeria. [cited 2021 jan 12]. available from: https://covid19.ncdc.gov.ng/progression/ babiker a, myers cw, hill ce, guarner j. sars-cov-2 testing: trials and tribulations. am j clin pathol. 2020;153(6):706–708. https://doi.org/10.1093/ajcp/aqaa052 mögling r, meijer a, berginc n, et al. delayed laboratory response to covid-19 caused by molecular diagnostic contamination. emerg infect dis. 2020;26(8):1944–1946. https://doi.org/10.3201/eid2608.201843 kobia f, gitaka j. covid-19: are africa’s diagnostic challenges blunting response effectiveness. aas open res. 2020;3:4. https://doi.org/10.12688/aasopenres.13061.1 becker mg, taylor t, kiazyk s, cabiles dr, meyers af, sandstorm pa. recommendations for sample pooling on the cepheid genexpert system using the cepheid xpert xpress sars-cov-2 assay. biorxiv preprint. in press 2020. https://doi.org/10.1101/2020.05.14.097287 zhang hl, omondi mw, musyoka am, et al. challenges of maintaining good clinical laboratory practices in low-resource settings: a health program evaluation framework case study from east africa. am j clin pathol. 2016;146(2):199–206. https://doi.org/10.1093/ajcp/aqw083 world health organization 2018. world health organization model list of essential in vitro diagnostics [homepage on the internet]. 2nd ed. geneva: world health organization [cited 2020 jun 14]. available from: https://www.who.int/medical_devices/publications/second_who_model_list_of_essential_in_vitro_diagnostics/en/ somerville lk, ratnamohan vm, dwyer de, kok j. molecular diagnosis of respiratory viruses. pathology. 2015;47(3):243–249. https://doi.org/10.1097/pat.0000000000000240 abstract introduction methods results discussion acknowledgements references about the author(s) mark w. kieh partnership for research on ebola virus in liberia (prevail), new kru town, monrovia, liberia sarah m. browne partnership for research on ebola virus in liberia (prevail), new kru town, monrovia, liberia greg a. grandits division of biostatistics, university of minnesota, minneapolis, minnesota, united states julie blie partnership for research on ebola virus in liberia (prevail), new kru town, monrovia, liberia jestina w. doe-anderson leidos biochemical research, fredrick, maryland, united states marie l. hoover advanced biomedical laboratories, cinnaminson, new jersey, united states bionca davis division of biostatistics, university of minnesota, minneapolis, minnesota, united states cavan s. reilly division of biostatistics, university of minnesota, minneapolis, minnesota, united states james d. neaton division of biostatistics, university of minnesota, minneapolis, minnesota, united states h. clifford lane division of clinical research, national institute of allergy and infectious diseases, national institute of health, bethesda, maryland, united states stephen b. kennedy partnership for research on ebola virus in liberia (prevail), new kru town, monrovia, liberialiberian college of physicians and surgeons, monrovia, liberia citation kieh mw, browne sm, grandits ga, et al. adult and paediatric haematology and clinical chemistry laboratory reference limits for liberia. afr j lab med. 2020;9(1), a1080. https://doi.org/10.4102/ajlm.v9i1.1080 original research adult and paediatric haematology and clinical chemistry laboratory reference limits for liberia mark w. kieh, sarah m. browne, greg a. grandits, julie blie, jestina w. doe-anderson, marie l. hoover, bionca davis, cavan s. reilly, james d. neaton, h. clifford lane, stephen b. kennedy received: 20 aug. 2019; accepted: 12 aug. 2020; published: 25 nov. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: as more research is conducted in liberia, there is a need for laboratory reference limits for common chemistry and haematology values based on a healthy population. reference limits from the united states may not be applicable. objective: the aim of this study was to present laboratory reference ranges from a liberian population and compare them to united states ranges. methods: serum chemistry and haematology values from 2529 adults and 694 children and adolescents obtained from two studies conducted in liberia between 2015 to 2017 were used to determine reference limits. after removing outliers, the reference limits defined by the 2.5th and 97.5th percentiles were determined by sex in three age groups (6–11, 12–17, and 18+ years). results: the median (interquartile range) of adults was 29 (23, 37) years; 44% were female. the median (interquartile range) for children and adolescents was 12 (9, 15) years; 53% were female. several reference ranges determined using liberian participants differed from those in the us. for chemistries, a high percentage of both adults and children/adolescents had high serum chloride levels based on united states ranges. for haematology, a high percentage of liberian participants had haemoglobin and related assays below the lower limit of united states ranges. conclusion: chemistry and haematology reference intervals determined for a liberian population of healthy individuals should be considered for establishing eligibility criteria and monitoring of laboratory adverse events for clinical trials as well as for use in clinical settings in liberia and perhaps for other countries in western africa. keywords: reference ranges; liberia; chemistries; haematology; paediatric. introduction reference limits for clinical laboratory tests for healthy adults and children in liberia are not available based on a liberian population. in a clinical setting, reference limits determined from a healthy population can provide useful information for making decisions based on laboratory reports. in addition, in a research setting, reference limits are often used for inclusion and exclusion criteria, for assessing possible adverse effects of treatment, and for the diagnosis of outcomes. differences in reference limits for haematology and chemistry measurements have been reported between united states (us) reference intervals and reference intervals determined for healthy individuals in other african countries.1,2,3,4,5,6,7,8,9,10,11,12,13,14 these differences have been attributed to differences in socioeconomic status, diet, physical exercise, environmental pathogens and altitude. the partnership for research on ebola virus in liberia (prevail) initiated several studies beginning in 2015. our experience carrying out this research motivated the determination of reference limits based on healthy people living in liberia who enrolled in two of the studies. for example, in one of these studies, a vaccine trial for the prevention of ebola virus disease (evd), a large percentage of apparently healthy participants had initial laboratory test result values outside the limits considered ‘normal’. the purpose of this paper is three-fold: (1) to describe chemistry and haematology test results for a large number of apparently healthy adults and children in liberia; (2) to use that data to define reference limits for use in future research projects in liberia; and (3) to compare the reference limits determined based on liberian participants with those based on us participants. methods ethical considerations we used data from two studies. prevail i and prevail iii, to determine the reference limits. all participants aged 18 years and older provided written informed consent. a parent or guardian signed a written informed consent for all participants under age 18 years, and children aged 9 years and older also signed a written assent. both study protocols were approved by the national research ethics board of liberia and the institutional review board of the united states national institutes of health; protocol identification number 15-i-n071 (prevail i) and 15-i-0122 (prevail iii). study design and sample selection the prevail i study was a phase 2 placebo-controlled randomised trial that evaluated the efficacy and safety of two vaccines to prevent evd. the prevail iii study was a cohort study of evd survivors and their close contacts. close contacts were identified by survivors and either lived with the survivor at the time of diagnosis or after discharge from the ebola treatment unit, or were sexual partners after discharge. the study design, methods and results of prevail i and prevail iii have been described elsewhere.15,16,17 study participants used to determine reference limits in prevail i, volunteers aged 18 years or older were enrolled over a three-month period beginning in february 2015 at redemption hospital in monrovia, liberia. the trial excluded participants with a history of evd, those with a temperature of more than 38 ºc, and women who were pregnant or breast-feeding. the following additional exclusions were made for these analyses: (1) participants with a history of high blood pressure, diabetes or cancer; (2) participants with hiv or syphilis infection based on blood testing; and (3) participants with antibody levels against the ebola virus surface glycoprotein greater than or equal to 548 enzyme-linked immunosorbent assay units (eu)/ml which was considered indicative of past ebola infection. these additional exclusions were made to remove potentially unhealthy participants that could have abnormal laboratory values as a result of medical conditions. in prevail iii, close contacts of evd survivors of any age, identified by evd survivors, were enrolled between 2015 and 2017. close contacts were enrolled at three sites: john f. kennedy (jfk) medical centre and duport road clinic (both in monrovia), and c.h. rennie hospital (a more rural site about 70 km north of monrovia). for participants in prevail iii, the following additional exclusions were made for these analyses: (1) participants with a history of high blood pressure, diabetes, cancer, stroke or ischemic heart disease; (2) participants with hiv or syphilis based on blood testing; and (3) participants with antibody levels against the ebola virus surface glycoprotein greater than or equal to 548 eu/ml. a map showing the locations of the sites where participants were enrolled in prevail i and iii is provided in figure 1. monrovia is a coastal city located on the atlantic coast, with elevation just above sea level. figure 1: map of liberia and location of study sites. laboratory measurements laboratories were established at redemption hospital, jfk medical centre, and c.h. rennie hospital. for participants enrolled in prevail i, all laboratory testing was done at redemption hospital. for participants enrolled in prevail iii, the testing was done at jfk hospital (jfk participants) or c.h. rennie hospital (c.h. rennie and duport road participants), with occasional backup testing at redemption hospital. the same laboratory equipment and methods for blood drawing, specimen labelling and testing were used by all three laboratories, with written documentation of all procedures provided in standard operating procedures. all samples were typically analysed within 4 h. calibrators and controls (vender and third-party) were run daily at each site to maintain quality assurance. the vendors provided precision data for each analyte. calibrators and control samples were run before the study samples, and results needed to be within the accepted range in order for the study samples to be analysed. in both studies, the participants were seen in the morning for their baseline clinic visits, during which non-fasting venous blood specimens were obtained. blood was collected in serum separator tubes for chemistry analyses and ethylenediaminetetraacetic acid tubes for haematology. specimens were then transferred to the respective laboratory. all samples were accessioned, centrifuged (if required) and then analysed on benchtop instrumentation: haematology using cell dyn ruby (abbott diagnostics, abbott park, illinois, united states), and chemistries using trademark name for alfa wassermann’s first chemistry analyzer (ace) alera or ace axcel (alfa wassermann, west caldwell, new jersey, united states), with alera or axcel used interchangeably. in addition to assessing hiv and syphilis serostatus, a complete blood count was obtained with differential and platelet count, aspirate aminotransferase, alanine aminotransferase, creatinine, potassium, chloride and sodium. alcohol intake was not ascertained in either study. each day reports were generated giving chemistry and haematology results for each participant and indicating values that were outside ‘normal’ limits based on us values. data analysis laboratory data for prevail i and iii were combined for these analyses. as a first step, outliers for each test were removed after box-cox transformation (restricting transformation to the identity, square root or log) using a method proposed by tukey:18 values 1.5 × interquartile range above the 75th percentile or 1.5 × interquartile range below the 25th percentile were removed. this was done because the information collected to exclude participants with chronic conditions was minimal and the goal was to identify a ‘healthy’ population. following this step, for each laboratory test, the reference limits defined by the 2.5th and 97.5th percentiles were determined based on a commonly used non-parametric approach.19,20 percentile values were back-transformed to the original scale. because it is important that reference ranges consider age and sex, this process was performed separately for each sex and age group (6–11, 12–17, and 18 years and over). the median and reference ranges are cited for male and female individuals in each of the three age groups. the percentage of participants both below and above published us reference ranges21 are cited for each laboratory value. the results found in this study and for other countries in west africa are given for reader reference. no formal statistical tests were made for these comparisons. non-parametric tests were conducted for differences in medians between male and female participants and among the three age groups by sex. all analyses were performed using sas version 9.4 (sas institute, cary, north carolina, united states). p-values < 0.001 were regarded as statistically significant. results of the 3986 participants enrolled in prevail i and iii, 3223 met the eligibility criteria for these analyses (figure 2). the adults enrolled in prevail i that are included in these analyses had a median age of 29 years, 33% were female, and the median body mass index was 21.6 kg/m2 (table 1). the adults in prevail iii that are included in these analyses had similar age distributions as those of the prevail i adults, but included a higher percentage of women and had a slightly higher median body mass index. the median age for the children/adolescents was 12 years, 53% were female, and the median body mass index was 17.4 kg/m2. figure 2: flow diagram for inclusion of participants for determination of reference levels: monrovia, liberia 2015–2017. table 1: baseline characteristics of study participants in laboratory analysis: monrovia, liberia 2015–2017. laboratory median levels and reference limits by age and gender many laboratory median levels and corresponding reference ranges differed significantly by sex and age (table 2). for example, the median and reference ranges for white blood cell (wbc) count in male participants aged 6–11 years was 7.18 (4.61–11.92) x 103/μl compared to 5.77 (3.63–9.72) x 103/μl in adult female participants. table 2: median and reference ranges for chemistries and haematology by gender and age group: monrovia, liberia 2015–2017. for chemistry, adult men had higher liver enzymes, creatinine, and potassium levels, with lower serum chloride and sodium levels than adult women (p < 0.001). in general, the differences between sexes were less pronounced in children/adolescents. for example, the median serum creatinine levels were nearly identical for male and female individuals aged 6–11 years and only slightly higher for male than female individuals aged 12–17 years. for haematology values, adult men had lower wbc counts but higher red blood cell counts and haemoglobin levels than adult women (p < 0.001). platelet counts were higher in adult women than men. the differences in haematology values between sexes were much smaller in the youngest age group (ages 6–11 years) and were not statistically significant. for serum chemistry, the median aminotransferase was inversely related to age for both male and female participants, whereas serum creatinine was strongly positively related to age for both sexes (p < 0.001). the median serum sodium increased with age for both sexes. for haematology, the wbc counts were highest for ages 6–11 years for both sexes and the red blood cell counts increased with age for male but not female participants. the median haemoglobin levels increased with age in both sexes but the relationship was stronger in male participants. platelets decreased with age for both male and female participants. comparison with united states reference ranges for chemistries, a high percentage of both adults and children/adolescents would be classified as having high serum chloride levels based on the us reference standard (table 3). similarly, several haematology factors would be classified as out of range for both adults and children/adolescents using the us standard. a high percentage of both children/adolescent and adult participants had haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, and haematocrit levels below the lower limit. in addition, 20% – 25% of liberian adults had wbc counts and neutrophils below the us reference standard. table 3: percentage of study participants outside of us1 reference intervals: monrovia, liberia 2015-2017 reference ranges for other west african countries medians and reference limits for our liberian study showed some differences compared to reference limits reported for ghana, mali, and gambia (table 4);4,10,13,14 chemistry values were only available from ghana. liver enzymes were lower in liberia than in ghana. potassium, sodium, and chloride levels were also lower in the liberian population than in the ghanaian population. the median and reference limits for wbc counts were similar for men in liberia and mali. the median wbc counts for women in liberia were higher than in the other west african countries. the red blood cell counts for men in liberia were similar to both ghana and mali, while the red blood cell counts for women in liberia were higher than those in ghana. the haemoglobin levels were similar in the liberian population as compared to the other west african countries. platelet counts differed among countries but were always higher in women than men in each country. comparison of medians among countries were not always consistent with the reference ranges, suggesting differences in variability between populations. table 4: comparisons of adult liberian reference ranges with other adult african countries reference ranges, monrovia, liberia 2015–2017. discussion to determine reference limits for common clinical laboratory test results for people in liberia by age and sex, we used baseline data collected at the time of enrolment in two research studies conducted during the west african ebola epidemic. one study enrolled healthy adults in a vaccine trial16 and the other enrolled adults and children who were close contacts of individuals who survived ebola.17 ‘unhealthy’ participants were excluded based on medical history and for positive hiv or syphilis tests. our goal was to select individuals for determining reference intervals and to use recommended statistical methods for determining these intervals as outlined by the clinical and laboratory standards institute22 that could be used to interpret an individual’s laboratory test results and could be used in the design of future clinical research studies. we found that for several laboratory tests, the reference limits based on the data from this liberian population differed greatly from the us-based reference limits. for example, the haemoglobin reference ranges were much lower in this study than the us-based reference values. based on the us reference limits, one could imply that a high proportion of liberians have ‘abnormal’ low haemoglobin levels. other reference limits that differed considerably were chloride, for which the liberian population had higher reference limits, and mean corpuscular volume, mean corpuscular haemoglobin, haematocrit and mean platelet volume, for which the liberian population had lower reference limits. differences between values found in this study compared to us levels could be due to several reasons, including genetics, environmental factors, subclinical disease, and laboratory equipment and/or methods. the observed differences are unlikely to be because of laboratory differences, as the laboratories set up in liberia used standard equipment that is also used in the us for chemistries and haematology. the reference limits from our study also confirm that separate values for many laboratory parameters should be considered for men and women and for adults and children/adolescents. the adult women in our study had significantly higher wbc counts than men. the children/adolescents also had higher wbc counts, lower creatinine levels, and lower haemoglobin levels than adults. many clinical trials use reference limits and tables for grading laboratory toxicities, such as the division of aids table for grading the severity of adult and paediatric adverse events,23 to define eligibility criteria and to grade adverse events during follow-up. the difference in reference limits between those estimated for liberia and us limits could impact the number of participants found to be eligible for a trial, as well as the percentage developing adverse events based on laboratory test results. for trials conducted in other parts of africa this has been the case. eller et al.9 studied the impact of using us reference limits in uganda to screen participants for an hiv vaccine trial. they found that us reference limits led to more exclusions during screening for a phase 1 vaccine trial than the use of their reference limits derived from people living in uganda. they also noted that the division of aids toxicity table did not reflect locally established reference limits, and the lower limit for neutrophils they had estimated would qualify as a grade 2 adverse event. segolodi et al.7 reported that many healthy volunteers for an hiv pre-exposure prophylaxis trial conducted in botswana had abnormal amylase results according to us-derived reference values. zeh et al.24 reported that over 58% of participants would have been excluded from a trial in kenya using us reference limits as compared to reference limits determined for the local population. in addition, 40% of otherwise healthy study participants would have been considered to have a grade 1–4 laboratory-based adverse event based on the division of aids toxicity table. there may be pros and cons to using liberian versus us reference limits for reporting adverse events. the burden associated with reporting lower-severity grade events,25 particularly those based solely on laboratory results and not associated with symptoms, would suggest using reference limits from local populations. on the other hand, in early phase research of novel treatments such as the ebola studies on which this research is based, it may be more prudent to use more conservative limits such as the us reference limits until safety is established. a strength of this study is the large number of participants (> 2500 adults and nearly 700 children/adolescents) studied with common laboratory methods in two research studies, allowing for more precise estimates of laboratory percentiles on which the reference limits are based. to our knowledge, this is the first report of laboratory reference limits from a liberian population. we recommend that additional laboratory-based studies be conducted to establish suitable laboratory reference limits for liberia. limitations a limitation to this study is that we based our definition of ‘healthy’ participants largely on a self-reported medical history. while we used statistical methods that attempted to remove outliers to establish a ‘healthy’ population, and that typically removed about 1% – 3% of participants from the group where normal ranges were calculated using 2.5% and 97.5% percentiles, our cohort is likely to include some participants with undiagnosed illnesses. conclusion in conclusion, we present the chemistry and haematology reference intervals from a liberian population of healthy individuals for men and women and for children/adolescents and adults. these levels should be considered for screening and monitoring participants in clinical trials in liberia and perhaps other countries in west africa. acknowledgements the authors would like to thank the many participants in the prevail i and prevail iii studies whose data were used for this manuscript. competing interests the authors have declared that no competing interests exist. authors’ contributions h.c.l. and s.b.k. conceived and designed the studies; m.w.k., j.d.n. and g.a.g. wrote the article; g.a.g. and c.s.r. analysed the data; m.l.h. set up and conducted the laboratory testing in liberia. clinical support and data collection were provided by m.w.k., s.m.b. and j.b; j.w.d.-a. and b.d. contributed to the editing of the manuscript. j.w.d.-a. and b.d. were also involved in the collection of data and monitoring of the study. source of support united states national institute of allergy and infectious diseases. p1 principal investigators: stephen kennedy, fotorma bolay, and h. clifford lane, p3 principal investigators: mosoka fallah and michael sneller. data availability statement the data used for this manuscript is available upon request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references mekonnen z, amuamuta a, mulu w, et al. clinical chemistry reference intervals of healthy adult populations in gojjam zones of amhara national regional state, northwest ethiopia. plos one. 2017;12(9):e0184665. https://doi.org/10.1371/journal.pone.0184665 karita e, ketter n, price ma, et al. clsi-derived hematology and biochemistry reference intervals for healthy adults in eastern and southern africa. plos one. 2009;4(2):e4401. https://doi.org/10.1371/journal.pone.0004401 odhiambo c, oyaro b, odipo r, et al. evaluation of locally established reference intervals for hematology and biochemistry parameters in western kenya. plos one. 2015;10(4):e0123140. https://doi.org/10.1371/journal.pone.0123140 kone b, maiga m, baya b, et al. establishing reference ranges of hematological parameters from malian healthy adults. j blood lymph. 2017;7(1):pii 154. samaneka wp, mandozana g, tinago w, et al. adult hematology and clinical chemistry laboratory reference ranges in a zimbabwean population. plos one. 2016;11(11):e0165821. https://doi.org/10.1371/journal.pone.0165821 tembe n, joaquim o, alfai e, et al. reference values for clinical laboratory parameters in young adults in maputo, mozambique. plos one. 2014;9(5):e97391. https://doi.org/10.1371/journal.pone.0097391 segolodi tm, henderson fl, rose ce, et al. normal laboratory reference intervals among healthy adults screened for a hiv pre-exposure prophylaxis clinical trial in botswana. plos one. 2014;9(4):e93034. https://doi.org/10.1371/journal.pone.0093034 miri-dashe t, osawe s, tokdung m, et al. comprehensive reference ranges for hematology and clinical chemistry laboratory parameters derived from normal nigerian adults. plos one. 2014;9(5):e93919. https://doi.org/10.1371/journal.pone.0093919 eller la, eller ma, ouma b, et al. reference intervals in healthy adult ugandan blood donors and their impact on conducting international vaccine trials. plos one. 2008;3(12):e3919. https://doi.org/10.1371/journal.pone.0003919 adetifa imo, hill pc, jeffries dj, jackson dj, ibanga hb, bah g, et al. haematological values from a gambian cohort – possible reference range for a west african population. int j lab hematol. 2009;31(6):615–622. https://doi.org/10.1111/j.1751-553x.2008.01087.x mine m, moyo s, stevens p, et al. immunohaetological reference values for hiv-negative healthy adults in botswana. afr j lab med. 2011;1(1): art. #5, 7 pages. https://doi.org/10.4102/ajlm.v1i1.5 saathof e, schneider p, kleinfeldt v, et al. laboratory reference values for healthy adults from southern tanzania. trop med int health. 2008;13(5):612–625. https://doi.org/10.1111/j.1365-3156.2008.02047.x dosoo dk, kayan k, adu-gyasi d, et al. haematologial and biochemical reference values for healthy adults in the middle belt of ghana. plos one. 2012;7(4):e36308. https://doi.org/10.1371/journal.pone.0036308 addai-mensah o, gyamfi d, duneeh rv, et al. determination of haematological reference ranges in heathy adults in three regions in ghana. biomed res int. 2019; feb 5 2019:id7467512. https://doi.org/10.1155/2019/7467512 kennedy sb, neaton jd, lane hc, et al. implementation of an ebola virus disease vaccine clinical trial during the ebola epidemic in liberia: design, procedures and challenges. clin trials. 2016;13(1):49–56. https://doi.org/10.1177/1740774515621037 kennedy sb, bolay f, kieh m, et al. phase 2 placebo-controlled trial of two vaccines to prevent ebola in liberia. n engl j med. 2017;377(15):1438–1447. https://doi.org/10.1056/nejmoa1614067 the prevail iii study group. a longitudinal study of ebola sequelae in liberia. n engl j med. 2019;380(10):924–934. https://doi.org/10.1056/nejmoa1805435 tukey jw. exploratory data analysis. reading, ma: addison-wesley; 1977. national committee on clinical laboratory standards (nccls). how to define and determine reference intervals in the clinical laboratory; approved guideline. vol. 20(13). 2nd ed. wayne, pa: nccls c28-a2; 2000. reed ah, henry rj, mason wb. influence of statistical method on the resulting estimate of normal range. clin chem. 1971;17:275–284. https://doi.org/10.1093/clinchem/17.4.275 kratz a, ferraro m, sluss pm, lewandrowski kb. laboratory reference values. n engl j med. 2004;351;1548–1563. https://doi.org/10.1056/nejmcpc049016 clinical and laboratory institute (clsi). defining, establishing, and verifying reference intervals in the clinical laboratory; approved guideline – third edition. clsi document ep28-a3c (isbn 1-56238-683-4). wayne, pa: clinical and laboratory standards institute; 2008. u.s. department of health and human services, national institutes of health, national institute of allergy and infectious diseases, division of aids (daids). table for grading the severity of adult and pediatric adverse events, version 2.0 [homepage on the internet]. c2014 [cited 2019 aug 15]. available from: https://rsc.niaid.nih.gov/sites/default/files/daids-aegrading-table-v2-nov2014.pdf zeh ce, odhiambo co, mills la. laboratory reference intervals in africa. london, uk: intech open; 2012. https://doi.org/10.5772/48250 chou vb, omer sb, hussian h, et al. the costs associated with adverse event procedures for an international hiv clinical trial determined by activity-based costing. j acquir immune defic syndr. 2007;46(4):426–432. https://doi.org/10.1097/qai.0b013e318156ee37 abstract background excess laboratory capacity challenges with the scale-up of viral load testing in sub-saharan africa solutions: procurement and supply chain management acknowledgements references about the author(s) jason williams supply chain division, united states agency for international development (usaid), crystal city, virginia, united states dianna edgil supply chain division, united states agency for international development (usaid), crystal city, virginia, united states matthew wattleworth global health supply chain program, procurement and supply management (ghsc-psm), arlington, virginia, united states clement ndongmo global health supply chain program, procurement and supply management (ghsc-psm), arlington, virginia, united states joel kuritsky supply chain division, united states agency for international development (usaid), crystal city, virginia, united states citation williams j, edgil d, wattleworth m, ndongmo c, kuritsky j. the network approach to laboratory procurement and supply chain management: addressing the system issues to enhance hiv viral load scale-up. afr j lab med. 2020;9(1), a1022. https://doi.org/10.4102/ajlm.v9i1.1022 review article the network approach to laboratory procurement and supply chain management: addressing the system issues to enhance hiv viral load scale-up jason williams, dianna edgil, matthew wattleworth, clement ndongmo, joel kuritsky received: 01 apr. 2019; accepted: 15 may 2020; published: 13 aug. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract investment in viral load scale-up in order to control the hiv epidemic and meet the joint united nations programme on hiv and aids (unaids) ‘90-90-90’ goals has prompted the president’s emergency plan for aids relief and countries to increase their investment in viral load and infant virological testing. this has resulted in the increased procurement of molecular-based instruments, with many countries having challenges to effectively procure and place these products. in response to these challenges, the global laboratory stakeholder community has developed an informed ‘network approach’ to guide placement strategies. this article defines and describes the ‘network approach’ for laboratory procurement and supply chain management to assist countries in developing a strategic instrument procurement and placement strategy. the four key pillars of the approach should be performed in a stepwise fashion, with regular reviews. the approach is comprised of (1) laboratory network optimisation, (2) forecasting and supply planning, (3) the development of effective procurement and strategic sourcing to develop ‘all-inclusive’ contracts that provide transparent pricing, and the establishment of clear service and maintenance expectations and key performance indicators and (4) performance management to increase communication and planning, and promote issue resolution. investments in the network approach will enable countries to strengthen laboratory systems and ready them for future laboratory needs. these disease-agnostic networks will be poised to improve overall national disease surveillance and assist countries in responding to disease outbreaks and other chronic diseases. keywords: laboratory networks; molecular scale-up; optimisation; supply chain; laboratory. background of the 36.9 million people living with hiv, approximately half (21.7 million) are on antiretroviral therapy, and of those, four out of five are virally suppressed.1 ensuring patients are on the most effective treatments relies on the availability and use of viral load (vl) testing. for this to be successful, clinicians must order the test, samples must be transported to the laboratory and results must be returned. the achievement of the third ‘90’ of the ‘90-90-90’ strategy of the joint united nations programme on hiv and aids (unaids), to ensure that 90% of patients that are on hiv treatment are virally suppressed, depends on the scale-up of laboratory capacity with an effective sample transport network and an efficient laboratory–clinic interface that facilitates responses to patient management issues related to adherence and treatment failure. in 2013, the world health organization included vl monitoring in its treatment guidelines, with subsequent guidance and a recommendation for its use in 2014 and 2015.2,3,4 the addition of vl testing as the cornerstone of the unaids, ‘90-90-90’ strategy has resulted in an investment in vl testing globally.5 investments have been made to assist ministries of health in revising treatment policies, building laboratory capacity, and training and sensitising clinicians and patients on testing. to facilitate scale-up, there has been an effort to increase efficiencies by promoting procurement coordination between donors, to improve transparent pricing for reagents, and to implement procurement principles to address service and maintenance. the goal for coordination is to create a network of diagnostic capability that is nested within a broader public health response towards laboratory development. as countries have attempted to take vl testing to scale, reoccurring challenges continue to surface, which include difficulty with procurement and sample transport, delays in the return of results and the need to increase clinical demand.6,7,8 these challenges have an impact on the ability to increase testing and ensure quality services. in order to address this, we promote a ‘network approach’. by this we mean the use of a systematic strategy that aligns capacity with utilisation, promotes efficiency in the procurement and placement of machines, enables collaboration between donors and countries, and focuses on the development of efficient sample transport and result return. the purpose of this article is to identify the key aspects of this approach and provide critical considerations for countries to improve performance and create network efficiencies in order to reach their diagnostic goals. excess laboratory capacity in most countries, instrument capacity is higher than needed, requiring significant growth in testing in order for these products to be optimally used. even with a phased approach to the scale-up of vl testing, as recommended by the world health organization,3 only a portion of coverage goals have been achieved. in zimbabwe, for example, the national vl testing coverage target was established at 21% in 2015, but only 5.6% coverage was achieved by year’s end, largely due to challenges with resource mobilisation and coordination, equipment procurement and specimen transport.9 by june 2016, of seven countries surveyed, four (tanzania, côte d’ivoire, malawi and uganda) were performing less than 25% of the necessary vl tests needed for patients on antiretroviral therapy.10 in 2016, médecins sans frontières (msf) estimated the coverage of vl monitoring across seven sub-saharan african countries to be variable, ranging between 32% and 91%.6 data on infant virological testing (ivt) show less than 50% coverage within the first six weeks of life in many sub-saharan african countries.7 the world health organization’s survey of data on diagnostic instruments from 2013, which assessed the scale-up of vl testing in 127 countries, showed that vl testing capacity was available to conduct 1.2 tests per person on antiretroviral therapy, but only 0.5 tests per person were conducted. this results in a capacity utilisation rate of only 36.5%.11 more recently, reports from major molecular instrument manufacturers demonstrate that countries continue to increase the number of instruments. between may 2016 and may 2018, testing capacity in 21 african countries increased from over 15 million to 20.5 million tests, with an increase of 164 large molecular instruments (manufacturer reported, see figure 1 and figure 2). in most countries, existing instrument numbers and capacity are not limiting factors associated with the scale-up of vl testing. figure 1: reported manufacturer instrument counts in sub-saharan africa (may 2016 to may 2018, an increase from 405 to 569 instruments). figure 2: diagnostic capacity estimates in sub-saharan africa (may 2018 – 20 580 136 tests). past scale-up efforts for cd4 testing resulted in uncoordinated procurement and underutilisation of instruments, suboptimal network expansion and a lack of service maintenance coverage across laboratory networks.11,12 as vl testing replaces cd4 in most sub-saharan countries to monitor antiretroviral therapy, many of these issues are again resurfacing, including uncoordinated instrument management strategies.6,13 lessons learned from the implementation of cd4 testing indicate the need for a more efficient model of procurement and service provision for vl and ivt programmes. challenges with the scale-up of viral load testing in sub-saharan africa we have identified four challenges that programmes must address in order to take vl testing to scale: (1) donor and stakeholder coordination and transparent pricing, (2) inconsistency of reagent availability (forecasting and supply planning), (3) ensuring functional instrumentation and (4) suboptimal laboratory network planning and sample transport strategies. challenge 1 – donor and stakeholder coordination and transparent pricing coordination between partners and governments to ensure the distribution of resources according to programme needs has been challenging, frequently resulting in the over-procurement or under-procurement of instruments and reagents that do not meet the testing needs of programmes.14,15 one core aspect of the alignment of effective global procurement is to create transparency in pricing, leveraging volumes and donor investments as part of negotiating influence. pricing variability across countries has been described as a limiting factor to scale-up,16 creating challenges with budgeting. many countries with budget limitations have historically paid more per test due to lower testing volumes, with more difficult infrastructural challenges to overcome as part of service delivery. without coordination, donors can inadvertently undermine the ability to negotiate cost-effective testing strategies, with an end result of diminishing testing pools across instrument types, limiting negotiating influence and undermining volume pricing for tests performed nationally. to clearly understand pricing, it is important to unpack costs for fair comparisons. for example, per test costs could be calculated based on the primary reagent only, or may include reagents, consumables, shipping and distribution. pricing depends on volumes, instrument type, sample type, local versus international procurement, mode of import, inclusive service, maintenance costs, logistics costs, vendor management of reagent inventories and reagent rental or leasing arrangements. all of these components influence pricing for comparative purposes. the global fund (gf) has negotiated global access pricing for low and middle-income countries. the two most commonly used molecular brands for vl testing and ivt are roche molecular diagnostics and abbott molecular inc. commodity-related prices are set at a rate of $9.40 per test for roche, which includes reagents and consumables, with ex-works terms (goods are available at the seller’s or manufacturer’s site and must be transported by the buyer), whereas abbott’s pricing is based on volumes and duration commitments.17 the abbott’s approach has resulted in pricing variability across countries of between $10.50 and $22.50 per test for core reagents, with an additional $2.50 for the necessary calibrators, controls and added consumables. this brings abbott’s pricing to between $13.50 and $23.60 per test. yet, based on volumes and multi-year commitments as well as national negotiating influence, some countries have further reduced these prices. it should be noted that pricing schemes offered by roche can also have country-specific variability due to ‘free carrier’ pricing (where the seller arranges and pays for shipping to the country of export) included in the reagent pricing, with shipping details not separately itemised. this creates challenges during budgetary planning sessions, since it becomes difficult to predict shipping costs and ensure that the global ex-works $9.40 reagent and consumable pricing is adhered to. transparency in total cost breakdown is needed, as there is a perception that the pricing offered is different from the gf-published $9.40 per test pricing. challenge 2 – inconsistent reagent availability (forecasting and supply planning) reagent availability has been highlighted as one core obstacle to the scale-up of vl testing.6,7,16 although reagent availability is a critical aspect of ensuring vl testing, stock-outs are a symptom of broader supply chain system issues and data flow challenges that have a negative impact on scale-up. challenge 3 – instrument functionality due to inadequate or absent service and maintenance ensuring adequate service and maintenance, warranty and preventive maintenance coverage for equipment is a significant challenge. to date, instrument and vendor oversight has been managed on an instrument-by-instrument basis, with limited coordination of management strategies, sometimes independently by stakeholders in the same country. this has resulted in multiple, separate contracts for individual instruments, often negotiated on different timelines, using non-standardised terms and with limited consistency in contract oversight and management. key performance indicators and reporting requirements that can be used to monitor vendor performance have not historically been included in contracts. this makes adhering to existing service contracts and the monitoring of vendor performance difficult, limiting both vendor accountability and the development of appropriate maintenance strategies. challenge 4 – weak laboratory and sample referral networks given the ever-changing laboratory network environment, sample transport and referral networks have grown organically, and do not necessarily reflect an efficient network approach. these networks quickly become outdated and require adjustments to not only reflect national needs (e.g. the addition of other diagnostics demands, point-of-care, the integration of new specimens, backup sample transport in the event of equipment breakdown), but also to look for efficiencies, and possible integration. ultimately, the effects of a fragmented sample referral network can be far-reaching, ranging from increased operational costs across the entire network to improperly placed instruments and limited instrument utilisation. solutions: procurement and supply chain management to effectively address these existing challenges, a holistic approach or network approach is needed, which spans four major building blocks or elements that are described below and summarised in figure 3. figure 3: the network approach to laboratory procurement and supply management. diagnostic network optimisation the concept behind a network approach is to shift to an all-inclusive reagent rental scheme (rrs) or reagent service scheme (rss) that serves all existing and new instruments. this approach would be national, and not specific to a stakeholder, donor or disease. a vendor-specific instrument contract would contain terms and conditions that are informed collectively by all stakeholders. this approach would require all stakeholders to take stock of existing instruments, service contracts and procurement pricing schemes, and establish jointly renegotiated terms that take all stakeholder investments and contributions to the overall network into consideration. revised pricing schemes could potentially include: a national cost and contract structure that allows for volume growth and instrument expansion within a complete network, irrespective of the disease type or programme area, and that can be accessed by all stakeholders. a cost structure translated into an ‘all-inclusive per test cost’ spread across all instruments of the same brands within the network to include: cost options as part of network expansion that would account for existing legacy instruments and the development of new contract models (e.g. leasing and rentals) that facilitate the introduction of new instruments under standardised pricing schemes inclusive service and maintenance data solutions that would include patient result transmission, as well as instrument and user performance network staff training and consistency additional technology support that could assist in site-level efficiencies (barcode use, sample processing and workflow evaluations) enhanced commodity management strategies to ensure reagent availability (to include vendor-managed inventory) the goal of a network approach is to improve instrument utilisation by aligning capacity with demand: introducing standardised national pricing schemes, irrespective of the procurement mechanism, thereby enabling continuous service contract coverage providing opportunities to amortise instrument costs into reagent costs, in order to lower startup costs associated with scale-up sharing and assigning the longer-term management and mitigation of risks associated with instrumentation onto manufacturers and local vendors providing a no-cost option for instrument replacements due to high failure rates, capacity issues (upgrading) or even outdated technology. a network approach focuses on developing a baseline understanding of the current national vl testing network, including capacity and equipment utilisation, then exploring more efficient network options. once a refined network is adopted, planning and procurement must be coordinated among all stakeholders to avert the addition of more instrumentation that may not be included in the planned diagnostic network, and ensuring the constant supply of reagents and consumables. in support of coordinated planning, it is important to develop criteria for the placement of additional machines, including point-of-care or near to point-of-care platforms and higher throughput platforms which all stakeholders would be required to adhere to. negotiated agreements should look to the bundling of services (including connectivity). contractual requirements for data sharing (downtime, testing protocols, specimen types, etc.) will facilitate management of the network in real time and improve vendor accountability. to advance a network approach, it is important to look beyond a lowest price per test and focus on the total cost of ownership; initial per test costs will likely be higher, but the longer-term strategy will benefit the network. evidence-based optimisation of laboratory network factors determining the success of vl and ivt testing programmes include laboratory infrastructure and instrumentation, logistics, specimen transport, clinical implementation, and monitoring and evaluation. while it is helpful to coordinate procurement and service maintenance under a network approach, a limited understanding of reagent consumption, testing demand, laboratory performance and human resource capacity can undermine the functionality of a network. in cases of network expansion or revision, it may be necessary to carry out an analysis toward the goal of optimising the network. an approach to laboratory network optimisation would focus on the use of geographical information systems mapping tools (e.g. laboratory efficiency and quality improvement planning [labeqip] software and supply chain guru™ – llamasoft18,19), to map laboratory network parameters, including instrument locations and utilisation, testing demands, quality assurance, human resources, sample transport lanes, specimen types, demographic needs, costing components and partner performance data. labeqip is a software tool developed by united states agency for international development (usaid) and llamasoft, which is managed by the global health supply chain – procurement and supply management (ghsc-psm). it is a geographical information, systems-based solution that can improve laboratory network efficiency and advance quality service delivery through data-driven optimisation and modelling. virtual modelling, prior to instrument placement, or as part of formalising an overall shift in testing strategies, is a critical component in informing the approaches to laboratory network optimisation. labeqip has most often been used to strategically plan the design of laboratory networks, the placement of equipment, the planning of sample referrals and the improvement of instrument utilisation. labeqip and supply chain guru™ have been used in nigeria, cameroon, rwanda, eswatini, zimbabwe and zambia with support from the president’s emergency plan for aids relief (pepfar), gf, the clinton health access initiative (chai) and ghsc-psm, to develop virtual strategies to integrate hiv-tuberculosis sample transport, reduce instrument footprints to improve operational costs, and virtually place instruments to determine the impact on laboratory testing demands and instrument capacity requirements. labeqip can also be used to inform the integration of point-of-care technologies, and to assist in prioritisation and instrument rebalancing due to overburdened or underburdened testing demands. demand forecasting and supply planning in the initial phase of scale-up, programmes often use demographic or target-based forecasts. a demographic forecast takes the number of patients who are on antiretroviral treatment and multiplies that number by the number of vl tests per patient; a target-based forecast does the same, but uses the national or programme annual treatment numbers. both types of forecast invariably overestimate commodity demand, as they do not account for unreliable laboratory or logistics information systems and poor reporting, poor site-level stock management practices, uncoordinated instruments and instrument failure.6 further, during a period of rapid scale-up, historical consumption and procurement are not reliable indicators of future consumption. usaid and pepfar, through its supply chain implementing partner, ghsc-psm, has increased procurement of vl testing reagents from just over $7 million in 2014 to nearly $90 million in 2018. a linear projection of vl testing demand based on historical procurement in 2016 would have forecast approximately $37 million of vl testing-related procurement by 2017, increasing to $47 million in 2018. actual 2017 vl testing procurement data reflected almost $6 million in ghsc-psm expenditures, with over $90 million in procurement moving into 2018, an underestimation of about 44% and 48% if linear projections were used (figure 4). figure 4: linear projection of all the president’s emergency plan for aids relief viral load testing demand based on 2013 to 2016 historical procurement (projected 2016), compared to actual 2017/2018 procurement data. to address forecasting challenges, usaid promotes forlab (forlabplus.com), which was developed by usaid and chai and is managed by ghsc-psm, for national laboratory forecasting. forlab has been used in more than 21 countries since its launch in 2013. forlab includes forecasting commodity needs using a mixed methodology approach to improve accuracy and to provide consistent and greater transparency in national forecasting exercises. forlab includes demographic and morbidity data, service statistics and logistics data on commodity consumption in an effort to triangulate multiple forecasting methods to derive a best-fit procurement plan that can be used by stakeholders to establish realistic budgets and supply planning activities.19,20,21,22,23,24 forlab is a data-driven tool and works well when data are available and, when data are of a high quality, it can precisely predict need. however, poor site-level reporting can reduce its forecasting accuracy. when highlighting stock-outs as a limiting factor associated with the scale-up of vl testing, it is important to acknowledge that there are many factors outside the supply chain that can impede improvements in reagent availability, which must be addressed concurrently. as programmes scale up, site-level storage space can become a challenge, causing the dispersal of reagents across various locations within a particular laboratory. product dispersion can make routine stock management tasks more laborious and reduce reporting frequency and accuracy. as programmes scale up, it may be necessary to increase reagent distribution frequency to sites, if commodity storage is limited, for example from quarterly to monthly. for this to be successful, there is a need to ensure consistent and reliable stock status visibility. an additional factor not related to supply chains that has an impact on reagent availability includes early visibility into new instrument introductions, as without coordination additional reagents may not be available to support extra instrumentation. in order to prevent stock-outs, programmes need effective data flow from testing sites to the central warehouse to guide inventory management practices and product distribution mechanisms. effective supply chains are data-driven and require constant input on service delivery performance. accurate and consistent commodity stock levels and consumption reporting improves supply chain systems, allowing for accurate forecasting, timely procurements and improved visibility for manufacturers to assist with manufacturing lead times for large order quantities. without these consistent and reliable inputs through logistic management information systems or laboratory information management systems, it becomes increasingly difficult to prescribe effective procurement and supply chain interventions to reduce stock-out situations. strategic procurement and sourcing to address pricing variability within countries, it is critical to negotiate national pricing schemes. national testing volumes should be aggregated to negotiate a consistent price that all stakeholders can achieve. donors, host-country counterparts and manufacturers can work collaboratively with aggregated volumes to derive transparent pricing schemes and mutually agreed upon prices that include additional service offerings outside of just reagents and consumables. recent coordination with the gf has resulted in price transparency in haiti, the democratic republic of congo and cameroon, with initial price reductions of approximately $21.00 to $16.50, and then further to $13.50 for reagent costs. efforts are still in process to promote further reductions as scale-up continues in these countries, as well as to include more comprehensive service packages. this includes service, maintenance and data management, as well as standardised reporting requirements informed by agreed upon key performance indicators as part of a price-per-test scheme. the pepfar has currently renegotiated all of its existing vl/eid procurement contracts to significantly lower all-inclusive pricing schemes. a formal press-release will be announced shortly after the publication of this paper. the pepfar has engaged gf to push further transparent pricing reductions, with additional itemised system costing options, including all-inclusive reagent rental, service and maintenance, data management systems, as well as possible vendor-managed inventory. all future instrument investments and reagent procurement strategies should use rrs for new instruments, as well as inclusive rss for existing instrumentation. the pepfar’s current country operational planning technical guidance emphasises the use of rrs for instrument expansion, driving countries towards a more systems-focused approach. currently, south africa, kenya, uganda21,23 and, more recently, mozambique, haiti and nigeria are taking advantage of rrs or a combination of rrs and rss. currently, usaid is working with ghsc-psm to introduce more dynamic rrs agreements in nigeria, mozambique, haiti and zambia. by moving to a rrs or a rss approach, countries can amortise their initial capital investment for the scale-up and servicing of vl-testing instruments within their reagent pricing scheme, offsetting initial scale-up costs and expanding instrument coverage, as well as ensuring complete service contract availability. in order to assist countries in this approach, usaid developed a ‘12 question’ approach designed to help countries think through the use, placement and servicing of laboratory instruments prior to initiating procurement or rrs or rss arrangements (box 1, figure 5).18 figure 5: approach for negotiating reagent rental agreements (linked to 12-question approach). box 1: the ‘12 question’ approach to instrument procurement and placement. monitoring instrument and vendor performance when considering rrs or rss contracts, it is critical to establish defined expectations. contracts should be negotiated collaboratively with all stakeholders and donors. a harmonised set of key performance indicators (table 1) should be developed and should include: minimum response times for instrument repairs, training, logistics, and instrument and end-user performance. clear thresholds should be established for instrument failure frequencies, and service providers should be held accountable for responding to site-level failures that go beyond these established thresholds. contracts should dictate a standardised monthly and quarterly reporting format to assist in addressing site or instrument-specific challenges, as well as vendor service delivery issues. the contract should also define at least quarterly meetings with the supplier to review performance and work collaboratively to solve problems and address any performance issues. contracts should also clearly delineate lists of parts to be made available in-country for high-failure parts, minimum service technician requirements, possible data solutions for patient result transmission, and monitoring instrument and end-user performance. table 1: illustrative key performance indicators used to monitor vendor service and instrument performance through service contracts. conclusion the current effort to scale up vl testing and ivt has been significant. gains have been achieved within national laboratory networks to scale up vl testing and ivt, but there is still a need to ensure sound investments in laboratory infrastructure and instrumentation, without overlooking the supportive structures of logistics, clinical components, and monitoring and evaluation protocols. there are lessons learned from past scale-up efforts for cd4 testing, with the current global strategy to ensure procurement coordination across donors, standardising and ensuring transparent pricing for reagents, and implementing general procurement principles that aim to address some of the main supply chain and service challenges. however, these global strategies must be translated into operational plans at a country level. to be successful, all stakeholders will need to embrace the full cycle of the network approach for laboratory procurement and supply chain management; take stock of existing instruments, service contracts and procurement pricing schemes; and establish jointly renegotiated terms that leverage all stakeholder investments. countries that have successfully scaled up vl testing and ivt have focused on making these commitments and have thereby reduced the risk of equipment failure and commodity stock-outs two critical challenges to the success of vl testing and ivt programmes. while each of the four pillars of the network approach for procurement and supply management can support elements of the supply chain, true transformation of the laboratory network is only possible through embracing all four of the strategic pillars in a stepwise approach, with each phase in the cycle continuing to inform the next step. in the longer term, these investments and the broader network approach will not only address some of the more immediate challenges, but will also enable countries to strengthen laboratory systems and ready themselves for implementing future laboratory needs. these disease-agnostic molecular networks will be poised to improve overall national disease surveillance and assist countries in responding to disease outbreak responses and other chronic diseases. in addition, such networks will position countries to address sustainable strategies for laboratories in future health agendas. acknowledgements competing interests we declare that we have no financial or personal relationships that may have inappropriately influenced us in writing this article. authors’ contributions j.w. was the nigeria and zimbabwe laboratory network optimisation lead, d.e. was the eswatini laboratory network optimisation lead, m.w. was the zimbabwe procurement and supply management technical lead in network optimisation, and c.n. was the eswatini procurement and supply management technical lead in network optimisation. all leads contributed to the development and implementation of the laboratory network approach strategy. all authors, including j.k., were involved in technical content review and narrative development. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the content in this manuscript is that of the authors and does not necessarily reflect the view of the united states president’s emergency plan for aids relief, the united states agency for international development or the united states government. references joint united nations programme on hiv and aids (unaids). global hiv & aids statistics – 2020 fact sheet [homepage on the internet]. [cited 2018 aug 03]. available from: https://www.unaids.org/en/resources/fact-sheet world health organization (who). 2013 treatment guidelines [homepage on the internet]. [cited 2019 feb 11]. available from: https://apps.who.int/iris/bitstream/handle/10665/85326/9789241505734_eng.pdf;jsessionid=371608d14cd62d7d49780e6e9fa85ad8?sequence=1 world health organization (who). 2014 considerations for viral load implementation [homepage on the internet]. [cited 2019 jan 17]. available from: https://www.who.int/hiv/pub/arv/viral-load-testing-technical-update/en/ world health organization (who). 2015 guideline on when to start antiretroviral therapy and on pre-exposure prophylaxis for hiv [homepage on the internet]. [cited 2019 jan 17]. available from: https://www.who.int/hiv/pub/guidelines/earlyrelease-arv/en/ joint united nations programme on hiv and aids (unaids). 90/90/90 strategy [homepage on the internet]. [cited 2019 mar 21]. available from: https://www.unaids.org/sites/default/files/media_asset/90-90-90_en.pdf médecins sans frontières (msf). report: making viral load routine, successes and challenges in the implementation of routine hiv viral load monitoring, part i [homepage on the internet]. the viral load laboratory. 2016 [cited 2019 jan 17]. available from: https://www.msf.org/sites/msf.org/files/making_viral_load_routine_part_1_programmatic_strategies.pdf médecins sans frontières (msf). report: making viral load routine, successes and challenges in the implementation of routine hiv viral load monitoring, part ii [homepage on the internet]. the viral load laboratory. [cited 2019 jan 17]. available from: https://www.msf.org/sites/msf.org/files/making_viral_load_routine_part_2_the_viral_load_laboratory.pdf stevens w, marshall tm. challenges in implementing hiv load testing in south africa. j infect dis. 2010;201(suppl 1):s78–s84. https://doi.org/10.1086/650383 kilmarx ph, simbi r. progress and challenges in scaling up laboratory monitoring of hiv treatment. plos med. 2016;13(8):e1002089. https://doi.org/10.1371/journal.pmed.1002089 lecher s, williams j, fonjungo pn, et al. progress with scale-up of hiv viral load monitoring – seven sub-saharan african countries, january 2015–june 2016. mmwr morb mortal wkly rep. 2016;65(47):1332–1335. https://doi.org/10.15585/mmwr.mm6547a2 habiyambere v, ford n, low-beer d, et al. availability and use of hiv monitoring and early infant diagnosis technologies in who member states in 2011–2013: analysis of annual surveys at the facility level. plos med. 2016;13(8):e1002088. https://doi.org/10.1371/journal.pmed.1002088 edgil d, williams j, smith p, kuritsky j. optimising the laboratory supply chain: the key to effective laboratory services. afr j lab med. 2014;3(1):art. #101:1–7. https://doi.org/10.4102/ajlm.v3i1.101 williams j, umaru f, edgil d, kuritsky j. progress in harmonizing tiered hiv laboratory systems: challenges and opportunities in 8 african countries. glob health sci pract. 2016;4(3):467–480. https://doi.org/10.9745/ghsp-d-16-00004 mwisongo a, nabyonga-orem j. global health initiatives in africa – governance, priorities, harmonisation and alignment. bmc health serv res. 2016;16(suppl 4):212. https://doi.org/10.1186/s12913-016-1448-9 alemnji g, onyebujoh p, nkengasong jn. improving laboratory efficiencies to scale-up hiv viral load testing. curr opin hiv & aids. 2017;12(2):165–170. https://doi.org/10.1097/coh.0000000000000346 world health organization. the availability and use of diagnostics for hiv: a 2012/2013 who survey of lowand middle-income countries [homepage on the internet]. 2014 [cited 2019 feb 11]. available from: https://apps.who.int/iris/bitstream/handle/10665/147213/9789241507905_eng.pdf?sequence=1 hiv viral load and early infant diagnosis selection and procurement, information tool [homepage on the internet]. version 2. 2017 [cited 2019 mar 21]. available from: https://www.theglobalfund.org/media/5765/psm_viralloadearlyinfantdiagnosis_content_en.pdf?u=636679306940000000 world health organization. guidance for procurement of in vitro diagnostics and related laboratory items and equipment [homepage on the internet]. 2nd ed. 2017 [cited 2019 mar 21]. available from: https://apps.who.int/iris/bitstream/10665/255577/1/9789241512558-eng.pdf joint united nations programme on hiv and aids (usaids). forlab laboratory quantification tool and labeqip software tool [homepage on the internet]. [cited 2018 jul 30]. available from: https://www.ghsupplychain.org/resources/other-resources joint united nations programme on hiv and aids (usaids). deliver project, task order 4. quantification of health commodities: a guide to forecasting and supply planning for procurement. arlington, va: usaid; 2014 peter t, zeh c, katz z, et al. scaling up hiv viral load – lessons from the large-scale implementation of hiv early infant diagnosis and cd4 testing. j int aids soc. 2017;20(suppl 7):e25008. https://doi.org/10.1002/jia2.25008 global laboratory initiative. gli guide to tb specimen referral systems and integrated networks [homepage on the internet]. [cited 2019 jul 30]. available from: http://www.stoptb.org/wg/gli/assets/documents/gli_guide_specimens_web_ready.pdf roberts t, cohn j, bonner k, hargreaves s. scale-up of routine viral load testing in resource-poor settings: current and future implementation challenges. clin infect dis. 2016;62(8):1043–1048. https://doi.org/10.1093/cid/ciw001 marinucci f, medina-moreno s, paterniti ad, wattleworth m, redfield r. decentralization of cd4 testing in resource-limited settings: 7 years of experience in six african countries. cytometry. part a: j int soc anal cytol. 2011;79(5):368–374. https://doi.org/10.1002/cyto.a.21064 abstract introduction methods results discussion acknowledgements references about the author(s) erica-mari nell division of haematological pathology, department of pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa national health laboratory service, tygerberg hospital, cape town, south africa zivanai c. chapanduka division of haematological pathology, department of pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa national health laboratory service, tygerberg hospital, cape town, south africa citation nell e, chapanduka zc. aetiology of pancytopenia: experience of a south african tertiary academic centre. afr j lab med. 2022;11(1), a1645. https://doi.org/10.4102/ajlm.v11i1.1645 original research aetiology of pancytopenia: experience of a south african tertiary academic centre erica-mari nell, zivanai c. chapanduka received: 10 june 2021; accepted: 18 feb. 2022; published: 31 may 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: pancytopenia is a manifestation of numerous disease entities. the causes of pancytopenia differ with geographic region, socio-economic factors and hiv prevalence. awareness of the common causes of pancytopenia may aid timely diagnosis. objective: this study aimed to determine the aetiology of pancytopenia in a south african population. methods: a retrospective observational study of adult patients presenting with pancytopenia at tygerberg academic hospital, south africa, from january 2016 to december 2017 was performed. data on pancytopenia cases were obtained from the laboratory information system and utilised to determine the causes of pancytopenia. results: a total of 673 cases of pancytopenia were identified. the most common causes of pancytopenia were chemoradiation therapy (25%), sepsis (18%), haematological malignancy (9%), advanced hiv (7%), and megaloblastic anaemia (6%). the diagnostic yield of bone marrow examinations (bme) was 57% (n = 52/91). the aetiology of pancytopenia differed according to age, with malignancy being a more common cause of pancytopenia among the elderly. conclusion: several easily recognisable and treatable conditions can manifest as pancytopenia. prompt management of such conditions, notably sepsis and megaloblastic anaemia, can result in the resolution of the cytopenias and negate the need for a bme. however, haematological malignancy and unexplained pancytopenia strongly rely on a bme to establish a diagnosis. pancytopenia investigations, when guided by appropriate clinic-laboratory findings, can promptly identify the underlying aetiology, while also identifying cases where an expedited bme is required. this is valuable in resource-conscious medicine. keywords: pancytopenia; sepsis; hiv; haematological malignancy; nutritional deficiency; megaloblastic anaemia; aging. introduction the term pancytopenia is used to describe a reduction in all three haematopoietic cell lines in the peripheral blood, namely erythrocytes, leukocytes and platelets. pancytopenia is not a disease entity, but rather the manifestation of several diseases and signifies the need for investigation. chemotherapy and radiation therapy are predictable causes of pancytopenia.1 the occurrence of pancytopenia in the absence of chemotherapy or radiation therapy is a common diagnostic dilemma and a bone marrow examination (bme) is recommended if the cause of pancytopenia cannot be otherwise ascertained. pancytopenia can also be caused by central inadequate production or peripheral destruction/sequestration,1 and the incidence of the various causes of pancytopenia differs based on geographic, socio-economic, dietary and other factors. in india, numerous studies have highlighted megaloblastic anaemia and aplastic anaemia as the commonly encountered causes of pancytopenia.2,3,4,5,6,7,8,9,10,11,12,13,14,15 studies from neighbouring countries, including pakistan,16,17,18,19,20 bangladesh21,22 and nepal,23,24,25 show similar findings. climate also affects the aetiology of pancytopenia by affecting disease transmission.26 as such, malaria, which is endemic in bangladesh, was found to be the most common cause of pancytopenia in a bangladeshi study.22 in high-income countries, however, the disease profile is quite different. studies done in france, sweden, south korea, oman and the united states demonstrate that haematological malignancies (hms) are the most common cause of pancytopenia.27,28,29,30,31,32 a study in mexico revealed a mix of hm and megaloblastic anaemia as the most common causes,33 demonstrating the role that socio-economic status and access to healthcare has on the aetiology of pancytopenia. in africa, the causes of pancytopenia follow the pattern of poor nutrition and poor access to healthcare. studies performed in zimbabwe, djibouti, morocco and tunisia showed that megaloblastic anaemia was the most common cause of pancytopenia.34,35,36,37 furthermore, hiv was found to be the third most common cause of pancytopenia in both the zimbabwean and djiboutian studies.34,35 to our knowledge, a study by retief and heyns performed in bloemfontein in 1976 is the only previous study investigating the aetiology of pancytopenia in south africa.38 the most common cause of pancytopenia as identified in that study was bone marrow aplasia (49.0%), and over two-thirds of those cases were attributed to radiation or chemotherapy. infectious agents (9.7%) were the second most common cause, followed by megaloblastic anaemia (9.6%). the hiv pandemic started in 1981, thus the effect of hiv on pancytopenia was not seen in the retief and heyns study.39 there is currently a high burden of hiv infection in south africa, and by the middle of 2019, 13.5% of south africans, that is 7.97 million people, were living with hiv.40 cytopenias are commonly observed in patients with hiv infection and are often multifactorial in aetiology.41 while cytopenias in hiv are common, the prevalence of pancytopenia in hiv clinics in african countries is low: 0.7% in an ethiopian study and 0.5% in a ugandan study.42,43 the pancytopenia prevalence is much higher (8.7%) in hiv clinics in puerto rico.44 this study aimed to contribute to the body of knowledge regarding the aetiology of pancytopenia in a developing country with a high burden of hiv and to assess the most common causes of pancytopenia across different age groups. methods ethical considerations this study was approved by the human research ethics committee review board of stellenbosch university (study approval number: s18/08/171) and all research was performed in accordance with relevant regulations with anonymised data. the requirement for informed consent was waived by the ethics committee due to the retrospective nature of the study. setting, specimens and defining criteria a retrospective cross-sectional descriptive study was conducted over a two-year period. all adult patients with new-onset pancytopenia who were treated at tygerberg academic hospital, cape town, south africa, from 01 january 2016 to 31 december 2017 were identified and included in our study. pancytopenia was defined as leucocyte count < 4 × 109/l, haemoglobin < 10 g/dl, and a platelet count < 100 × 109/l. these parameters are comparable with other studies investigating the aetiology of pancytopenia.6,7,14,15,16,21,45,46 patients were excluded from the study if they received clinical care at another facility even though the bme was reported at tygerberg academic hospital. data collection and interpretation the data were obtained from the laboratory information system (lis) of the national health laboratory service. an lis search was conducted to identify cases meeting the study criteria for pancytopenia. the identified pancytopenia cases were reviewed on the national health laboratory service lis to identify the cause of the pancytopenia. information retrieved from the lis included the reports of peripheral blood smears, bmes, vitamin b12 and serum folate levels, iron studies, viral studies, mycobacterium tuberculosis culture and genexpert® tests (cepheid, sunnyvale, california, united states), malaria rapid tests (binaxnow malaria, abbott laboratories, chicago, illinois, united states) and thick and thin smears, autoimmune screens, sepsis markers, and blood cultures. in addition, information on patient age, gender, hiv status, cd4 count, and hiv viral load was obtained. advanced hiv was defined as a cd4 count < 200 cells/µl.47,48 since the aetiology of cytopenias in hiv is multifactorial, evidence of contributing factors such as folate deficiency, opportunistic infection or malignancy were sought in hiv-positive patients. in the absence of any other cause of cytopenias, and if the cd4 count was < 200 cells/µl, the pancytopenia was attributed to the advanced hiv. data analysis statistical analysis was done in conjunction with the biostatistics department of stellenbosch university. percentages were calculated for categorical variables. age was presented as mean and standard deviation. non-parametric data such as hiv viral load, cd4 count, time to bme and time to resolution of pancytopenia were presented as medians and interquartile ranges. pearson’s chi-square tests were used to assess the statistical differences in the frequencies of causes of pancytopenia between the hiv-positive and negative groups, as well as between different age groups. the data were analysed using statistical package for the social sciences version 25 (ibm corp., armonk, new york, united states). results a total of 695 cases of new-onset pancytopenia were identified within the two-year period. further investigation showed that seven cases had platelet clumping with a subsequent platelet count above 100 × 109/l, and 15 cases were due to sample dilution, having been collected from a cannulated infusion vein. thus the total number of true pancytopenia cases was 673 over the specified period (figure 1). figure 1: flow diagram of case selection and stratification to identify the causes of pancytopenia at tygerberg academic hospital, cape town, south africa, from january 2016 to december 2017. true pancytopenia was confirmed in 673 cases. chemoradiation therapy was found to be the cause in 166 cases, leaving 507 non-iatrogenic cases with unidentified underlying aetiologies. of those, 91 patients had a bme and the cause of pancytopenia was identified in 52 of these patients. in 16 patients whose bme showed non-specific findings, a cause for pancytopenia was found using information available on the lis. using information available on the lis, the cause of pancytopenia was identified in 340 of the remaining 416 patients who did not have a bme. of the 673 patients, 273 (41%) were male and 400 (59%) were female. the mean age at which pancytopenia was diagnosed was 44 ± 15 years (range: 18–87 years). most common causes of pancytopenia chemotherapy and/or radiation therapy was the most common (25%; 166/673) cause of pancytopenia (figure 2). the chemoradiation therapy was for the treatment of non-haematological malignancies (51%; 85/166) and hm (49%; 81/166) (figure 3). figure 2: frequency of different causes of pancytopenia among adult patients at tygerberg academic hospital, cape town, south africa, from january 2016 to december 2017. figure 3: pancytopenia cases at tygerberg academic hospital, cape town, south africa, from january 2016 to december 2017 for which chemoradiation therapy was the cause. (a) site of nhm, (b) haematological malignancy (hm) subtypes. the most common causes of pancytopenia among the remaining 507 (75%) cases were sepsis (18%; 122/673), hm (9%; 62/673), advanced hiv with no other identifiable cause (7%; 49/673) and megaloblastic anaemia (6%; 42/673). a cause for pancytopenia could not be established in 15% (98/673) of cases. haematological conditions were found to be common causes of pancytopenia. these included hm (9%; 62/673) and aplastic anaemia (3%; 20/673). six of the 20 patients with aplastic anaemia had a paroxysmal nocturnal haematuria clone. serum vitamin b12, folate and ferritin levels were measured in 280 of the 507 patients with non-iatrogenic pancytopenia. of these, 12% (33/280) had isolated folate deficiency and 3% (9/280) had vitamin b12 deficiency. infection contributed to a considerable proportion of pancytopenia cases. sepsis was the most common non-iatrogenic cause of pancytopenia. in 80% of sepsis cases (97/122), a blood culture was positive for an organism. gram-negative bacteria were more commonly cultured (61%; 74/122) compared to gram-positive bacteria (16%; 19/122). the remaining four cultures were positive for candida albicans. in 62% (76/122) of sepsis-associated pancytopenia cases, the pancytopenia resolved. the median time to resolution of pancytopenia was 2 days (interquartile range: 1–6). twenty-three (3%; 23/673) patients had positive mycobacterium tuberculosis cultures/genexpert®; the majority (19/23) of these were hiv-positive patients. nine (1%; 9/673) patients had malaria. description of pancytopenia in patients with hiv of the 507 non-iatrogenic pancytopenia cases, 41% (207/507) were hiv-positive, 47% (236/507) were hiv-negative, and the hiv status was unknown in 13% (64/507). the median cd4 count was 94 cells/µl (interquartile range: 33–202 cells/µl, n = 175 patients tested) and the median hiv viral load was 1096 copies/ml (interquartile range lower than detectable limit – 91 560 copies/ml, n = 113 patients tested). the viral load was undetectable in 14% (29/207) of the hiv-positive patients with pancytopenia, and advanced hiv was seen in 68% (141/207). the cd4 count was not performed in 27 hiv-positive patients with pancytopenia, thus the proportion of patients with advanced hiv may be underestimated. further investigation in patients with advanced hiv revealed additional contributing causes of pancytopenia, including sepsis (22%; 31/141), folate deficiency (13%; 18/141) and tuberculosis (10%; 14/141). moreover, advanced hiv was the only identifiable cause of pancytopenia in 24% (49/207) of the hiv-positive patients, accounting for 7% of all cases (49/673). the aetiology of pancytopenia differed between hiv-positive and hiv-negative patients (figure 4). haematological malignancies and aplastic anaemia were significantly more common in hiv-negative patients (p < 0.0001), while folate deficiency (p = 0.004), tuberculosis (p < 0.0001) and thrombotic microangiopathy (p = 0.011) were more common among hiv-positive patients. there was no significant difference in the proportion of sepsis cases between the hiv-positive and hiv-negative groups (p = 0.66). figure 4: proportions of various causes of pancytopenia among hiv-positive and hiv-negative patients at tygerberg academic hospital, cape town, south africa, from january 2016 to december 2017. bone marrow examination to determine pancytopenia aetiology bone marrow examination was performed to determine the cause of pancytopenia in 91 of the non-iatrogenic cases and this revealed the cause of pancytopenia in 52 cases (57%; 52/91). thirty-six of these had hm (40%; 36/91), nine had aplastic anaemia (10%; 9/91), and three had non-haematological malignancies (3%; 3/91). the most common hms were myelodysplastic syndrome (11%) and acute leukaemia (10%). notably, in four of the nine acute leukaemia cases, there were scanty or no blasts (< 1%) on the peripheral blood smear. in the remaining 39 bmes, no specific bone marrow pathology could be identified. causes of pancytopenia in different age groups the aetiology of pancytopenia also varied according to age (figure 5). while sepsis was the most common cause of pancytopenia among young (18–39 years old) and middle-aged (40–59 years old) patients, hm was the most common cause of pancytopenia in the oldest age group (60–89 years old). there was a significantly higher proportion of patients diagnosed with hm (p < 0.0001) and non-haematological malignancies (p = 0.012) among patients aged 60–89 years old. the increase in hm seen in the 60–89-year-old category was mainly due to an increase in the number of myelodysplastic syndrome cases. the number of cases of sepsis, megaloblastic anaemia and aplastic anaemia did not significantly differ between the age categories. figure 5: most common causes of non-treatment-related pancytopenia among adult patients at tygerberg academic hospital, cape town, south africa, from january 2016 to december 2017. (a) all patients (n = 507), (b) 18–39 years old (n = 250), (c) 40–59 years old (n = 172), (d) 60–89 years old (n = 85). discussion this study showed that chemoradiation therapy was the most common cause of pancytopenia in adult patients at a south african tertiary academic centre. the most common non-treatment-related causes of pancytopenia were sepsis, hm, advanced hiv, and megaloblastic anaemia. the aetiology of pancytopenia as found in our study is comparable to the findings of a previous south african tertiary institution study.38 their study showed chemoradiation therapy to be the most common cause of pancytopenia and revealed infection as another prominent cause.38 haematological malignancy, which was not a prominent cause of pancytopenia in their study, was the third most common cause of pancytopenia in this study. infection, megaloblastic anaemia and aplastic anaemia are common causes of pancytopenia in developing countries while hm is the most common cause in developed countries (table 1). in comparison to the aetiologies of pancytopenia described by studies across the world, our study shows features common to both developing and developed countries. this is similar to the findings of a study performed in mexico where the most common causes of pancytopenia were hm and megaloblastic anaemia.32 in south africa, there are considerable differences in access to health services between socio-economic groups, and the poor face many predisposing factors that are recognised as social determinants of ill health.49 hiv and other communicable diseases are concentrated among the lower socio-economic groups of south africa.50 this may explain why infections contributed considerably to the aetiology of pancytopenia in our study. table 1: most common causes of pancytopenia across different studies globally, 1976–2019. advanced hiv was found to be one of the common causes of pancytopenia in our study, similar to studies performed in zimbabwe, djibouti, india and the united states.12,32,34,35 in our study, 41% of patients were hiv-positive. the majority of these patients had advanced hiv, and in a quarter of these, advanced hiv was found to be the only identifiable cause of pancytopenia. additionally, our study showed that hiv status alters the aetiology of pancytopenia; folate deficiency, tuberculosis and thrombotic microangiopathy were significantly more common among hiv-positive patients, while aplastic anaemia and hm were significantly more common among hiv-negative patients. interestingly, despite the known risk of hiv-associated lymphomas in advanced hiv,51 we did not find an increase in hm-associated pancytopenia in hiv-positive patients. this may be because lymphomas do not typically cause pancytopenia even when there is bone marrow infiltration.52 haematological malignancies were a more common cause of pancytopenia in our study than in other studies performed in developing nations (table 1). the burden of cancer is highest in affluent regions and is thought to be associated with diet, lifestyle and environmental exposures. with increasing urbanisation and globalisation, developing nations are increasingly exposed to diet and lifestyle changes, including smoking, a sedentary lifestyle and obesity, which were previously mainly seen in developed societies.53 in addition, developing nations have an increased risk of viral infection, including infection with oncoviruses, which can lead to malignancy.53 expensive diagnostic approaches are also increasingly available in tertiary centres, allowing for easier diagnosis of malignancy, which may be missed in rural regions or countries with fewer resources. these factors are likely contributing to the changing landscape of pancytopenia aetiology in south africa. megaloblastic anaemia as a cause of pancytopenia was not as prevalent in our study as in studies performed in india3,4,5,7,8,9,10,11,13,15 and the rest of africa.34,35,36,37 this is likely because the south african government introduced legislation in 2003 for the mandatory folate fortification of the staple maize meal and wheat flour.57,58 nevertheless, folate deficiency was more common in hiv-positive patients than in hiv-negative patients in this study, likely reflecting poor folate absorption due to hiv-associated gastrointestinal disease. importantly, six of the 48 patients with megaloblastic anaemia in our study had an underlying pathology leading to depletion of folate reserve. this highlights the importance of follow-up in patients with haematinic deficiencies and the importance of further investigation if there is a poor response to the haematinic replacement or clinical suspicion of an underlying condition. the aetiology of pancytopenia also varied with age. there were significantly more malignancies, both haematological and non-haematological, in the age group 60–89 years. this is comparable to the findings of studies in turkey and iran.45,46 globally, malignancies are not a more common cause of pancytopenia in older patients. in studies conducted in india, megaloblastic anaemia was found to be the most common cause of pancytopenia, with no noticeably higher frequency of malignancy among older people.6,12,15 the substantial variation in the frequency of malignancy as a cause of pancytopenia in the elderly is most likely due to regional risk factors for megaloblastic anaemia, as well as variable availability of expensive diagnostic approaches such as flow cytometry, cytogenetics and next-generation sequencing for the diagnosis of malignancy. the findings of this study highlight that good clinical and laboratory correlation is required for prompt and cost-effective identification of the underlying cause of pancytopenia to guide management. chemoradiation is a predictable cause of pancytopenia; however, cell counts should also be monitored, and if the cell counts do not improve, investigation for vitamin b12/folate deficiency or marrow infiltration should be performed. sepsis was another easily identifiable cause of pancytopenia in our study. the median time to resolution of sepsis-induced pancytopenia was two days. therefore, when sepsis is treated empirically, cell counts should be monitored for recovery. should cell counts fail to recover, further investigation is required. vitamin b12 and folate deficiency are important to exclude in cases of pancytopenia. in cases where haematinic deficiency is confirmed based on serum levels, poor response to replacement therapy may indicate underlying marrow pathology requiring bme. knowledge of the vitamin b12 and folate serum levels are also important for the interpretation of bmes, especially if myelodysplastic syndrome is suspected. additionally, if there are features of myelodysplastic syndrome, acute leukaemia or bone marrow infiltration (such as unexplained leucoerythroblastic reaction) in peripheral blood or, importantly, if the pancytopenia is unexplained, a bme should be fast-tracked in the investigation of the pancytopenia. furthermore, in patients with hiv, vitamin b12 and folate deficiency should be excluded early, as folate deficiency was found to be common in the hiv patients in our study. moreover, in hiv-positive patients, there should be a thorough investigation for tuberculosis, and a bme is recommended to investigate marrow infiltration by opportunistic infections and malignancy. in older patients, there should be a high clinical suspicion of malignancy. ultimately, bme remains a valuable procedure for the investigation of unexplained pancytopenia. limitations one of the limitations of our study was that only retrospective information available on the lis was used to identify the cause of pancytopenia. as a result, the contribution of drugs and co-morbidities was unknown and a cause for pancytopenia could not be identified in 14% of patients. the world health organization defines advanced hiv as either cd4 count < 200/µl or clinical stage iii or stage iv disease. however, the clinical stage of hiv disease was not known in this study. thus the definition of advanced hiv was based only on cd4 count, and the patients with advanced hiv may be underestimated.48 clinicopathological correlation would also have improved the study. furthermore, it is noteworthy that our study was performed in a tertiary hospital, thus selection bias may have resulted in chemoradiation therapy being the most common cause of pancytopenia. socio-economic, geographical and ethnic factors are known to influence the aetiology of pancytopenia. it is noteworthy, however, that differences in study design also influence the described aetiology of pancytopenia reported by different studies. study designs differ in their definitions of pancytopenia and whether clinical or laboratory data or both were used. additionally, some studies only evaluate bme patients, while some include all patients with pancytopenia. some studies may also include children, and others may include patients that had chemotherapy or radiation therapy. these differences influence the top causes of pancytopenia between different studies. conclusion this study shows that the most common causes of new-onset pancytopenia in adults at a south african tertiary academic centre are chemoradiation therapy, sepsis, hm, advanced hiv and megaloblastic anaemia. these results demonstrate the need for the prompt recognition and treatment of sepsis and megaloblastic anaemia, early recognition of hiv and initiation of antiretroviral therapy, and a thorough investigation for malignancy. integration of clinical, laboratory and radiological findings to guide investigation of pancytopenia allows for prompt diagnosis, while also elucidating where bme should be expedited in the investigation of pancytopenia. acknowledgements we would like to acknowledge mr wessel kleinhans for his assistance with data extraction from the laboratory information system. competing interests the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. authors’ contributions e.n. was the primary investigator and partook in data collection, analysis and writing of the manuscript. z.c.c. supervised the research, assisted in critical appraisal of the work and contributed to the writing of the manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial or non-profit sectors. data availability all data generated or analysed during this study are included in this article. disclaimer the views expressed in the submitted article are our own and not an official position of the institution. references weinzierl ep, arber da. the differential diagnosis and bone marrow evaluation of new-onset pancytopenia. am j clin pathol. 2013;139(1):9–29. https://doi.org/10.1309/ajcp50aeeygrewuz varma n, dash s. a reappraisal of underlying pathology in adult patients presenting with pancytopenia. trop geogr med. 1992;44(4):322–327. tilak v, jain r. pancytopenia – a clinico-hematologic analysis of 77 cases. indian j pathol microbiol. 1999;42(4):399–404. kumar r, kalra sp, kumar h, anand ac, madan h. pancytopenia – a six year study. j assoc physicians india. 2001;49:1078–1081. khunger jm, arulselvi s, sharma u, ranga s, talib vh. pancytopenia – a clinico haematological study of 200 cases. indian j pathol microbiol. 2002;45(3):375–379. devi pm, laishram rs, sharma ps, singh am, singh mk, singh ym. clinico-hematological profile of pancytopenia in manipur, india. kuwait med j. 2008;40(3):221–224. gayathri bn, rao ks. pancytopenia: a clinico hematological study. j lab physicians. 2011;3(1):15–20. https://doi.org/10.4103/0974-2727.78555 rangaswamy m, prabhu, nandini nm, manjunath gv. bone marrow examination in pancytopenia. j indian med assoc. 2012;110(8):560–562, 566. raphael v, khonglah y, dey b, gogoi p, bhuyan a. pancytopenia – an etiological profile. turk j hematol. 2012;29(1):85–86. https://doi.org/10.5505/tjh.2012.98360 premkumar m, gupta n, singh t, velpandian t. cobalamin and folic acid status in relation to the etiopathogenesis of pancytopenia in adults at a tertiary care centre in north india. anemia. 2012;2012:article id 707402. https://doi.org/10.1155/2012/707402 mir ta, bhat mh, raina aa. etiological profile of pancytopenia in a tertiary care hospital of kashmir valley. int j sci res. 2015;4(10):1186–1189. jain a, naniwadekar m. an etiological reappraisal of pancytopenia – largest series reported to date from a single tertiary care teaching hospital. bmc hematol. 2013;13(10):1–10. https://doi.org/10.1186/2052-1839-13-10 dasgupta s, mandal p, chakrabarti s. etiology of pancytopenia: an observation from a referral medical institution of eastern region of india. j lab physicians. 2015;7(2):90–95. https://doi.org/10.4103/0974-2727.163136 santra g, das bk. a cross-sectional study of the clinical profile and aetiological spectrum of pancytopenia in a tertiary care centre. singapore med j. 2010;51(10):806–812. thakkar b, bhavsar u, trivedi n, agnihotri a. a study of pancytopenia in adult patients more than 12 years of age in north west region of saurashtra. natl j med res. 2013;3(1):48–52. niazi m, raziq f. the incidence of underlying pathology in pancytopenia – an experience of 89 cases. j postgrad med inst (peshawar – pakistan). 2004;18(1):76–79. tariq m, basri r, khan nu, amin s. aetiology of pancytopenia. prof med j. 2010;17(2):252–256. https://doi.org/10.29309/tpmj/2010.17.02.2371 aziz t, ali l, ansari t, liaquat h, shah s, ara j. pancytopenia: megaloblastic anemia is still the commonest cause. pak j med sci. 2010;26(1):132–136. makheja kd, maheshwari bk, arain s, kumar s, kumari s, vikash. the common causes leading to pancytopenia in patients presenting to tertiary care hospital. pak j med sci. 2013;29(5):1108–1111. https://doi.org/10.12669/pjms.295.3458 rehmani th, arif m, heraid s, arif s, ahmad r, saeed m. spectrum of pancytopenia: a tertiary care experience. prof med j. 2016;23(5):620–626. https://doi.org/10.29309/tpmj/2016.23.05.1594 biswas pk, sardar mh, saha gc, et al. etiological and clinical spectrum of pancytopenia based on bone marrow examination. j med. 2019;20(2):68–71. https://doi.org/10.3329/jom.v20i2.42005 basavaiah sh, rai s, suresh pk, shivaprasad sm, khandelia b. clinicopathological diversity of pancytopenia: a series of 400 cases. j clin diagnostic res. 2018;12(3):ec01–ec05. https://doi.org/10.7860/jcdr/2018/32116.11227 jha a, sayami g, adhikari rc, panta ad, jha r. bone marrow examination in cases of pancytopenia. j nepal med assoc. 2008;47(169):12–17. https://doi.org/10.31729/jnma.209 lakhey a, talwar o, singh v, shiva raj k. clinico-hematological study of pancytopenia. j pathol nepal. 2012;2(3):207–210. https://doi.org/10.3126/jpn.v2i3.6023 pathak r, jha a, sayami g. evaluation of bone marrow in patients with pancytopenia. j pathol nepal. 2012;2(4):265–271. https://doi.org/10.3126/jpn.v2i4.6875 national research council (us) committee on climate, ecosystems, infectious diseases, and human health. analytical approaches to studying climate/disease linkages. in: national research council, editor. under the weather: climate, ecosystems, and infectious disease. washington, d.c.: national academy press. 2001; p. 59–79. https://doi.org/10.17226/10025 imbert m, scoazec jy, mary jy, jouzult h, rochant h, sultan c. adult patients presenting with pancytopenia: a reappraisal of underlying pathology and diagnostic procedures in 213 cases. hematol pathol. 1989;3(4):159–167. keisu m, ost a. diagnoses in patients with severe pancytopenia suspected of having aplastic anemia. eur j haematol. 1990;45(1):11–14. https://doi.org/10.1111/j.1600-0609.1990.tb00407.x bae m-h, cho y-u, kim b, et al. pancytopenia or bicytopenia in a korean tertiary care center; etiological profile based on bone marrow examination and suggestion for diagnostic approach. blood. 2015;126(23):5610. https://doi.org/10.1182/blood.v126.23.5610.5610 al-khalisi ka, al-zubaidy as, rhaima m. pancytopenia adult patients at baghdad teaching hospital. iraqi acad sci j. 2011;10(4):441–448. weinzierl ep, arber da. bone marrow evaluation in new-onset pancytopenia. hum pathol. 2013;44(6):1154–1164. https://doi.org/10.1016/j.humpath.2012.10.006 devitt ka, lunde jh, lewis mr. new onset pancytopenia in adults: a review of underlying pathologies and their associated clinical and laboratory findings. leuk lymphoma. 2014;55(5):1099–1105. https://doi.org/10.3109/10428194.2013.821703 vargas-carretero cj, fernandez-vargas oe, ron-magaña al, padilla-ortega ja, ron-guerrero cs, barrera-chairez e. etiology and clinico-hematological profile of pancytopenia: experience of a mexican tertiary care center and review of the literature. hematology. 2019;24(1):399–404. savage dg, allen rh, gangaidzo it, et al. pancytopenia in zimbabwe. am j med sci. 1999;317(1):22–32. https://doi.org/10.1097/00000441-199901000-00004 lavigne c, lavigne e, massenet d, binet c, brémond jl, prigent d. role of vitamin deficiency in pancytopenia in djibouti. findings in a series of 81 consecutive patients. med trop (mars). 2005;65(1):59–63. nafil h, tazi i, sifsalam m, bouchtia m, mahmal l. profil étiologique des pancytopénies chez l’adulte à marrakech (maroc). east mediterr health j. 2012;18(5):532–536. klii r, chaaben i, kechida m, et al. profil étiologique des cytopénies dans un service de médecine interne: à propos de 103 cas. la rev médecine interne. 2016;37:a145. https://doi.org/10.1016/j.revmed.2016.10.144 retief fp, heyns a.d.p. pansitopenie en aplastiese anemie: ’n retrospektiewe beoordeling. s afr med j. 1976;50(34):1318–1322. a timeline of hiv and aids [homepage on the internet]. hiv.gov. n.d. [cited 2019 dec 19]. available from: https://www.hiv.gov/hiv-basics/overview/history/hiv-and-aids-timeline statistical release: mid-year population estimates 2019 [homepage on the internet]. 2019. available from: http://www.statssa.gov.za/publications/p0302/p03022019.pdf opie j. haematological complications of hiv infection. s afr med j. 2012;102(6):465–468. https://doi.org/10.7196/samj.5595 enawgaw b, alem m, addis z, melku m. determination of hematological and immunological parameters among hiv positive patients taking highly active antiretroviral treatment and treatment naïve in the antiretroviral therapy clinic of gondar university hospital, gondar, northwest ethiopia. bmc hematol. 2014;14:article number 8. https://doi.org/10.1186/2052-1839-14-8 kyeyune r, saathoff e, ezeamama ae, loscher t, fawzi w, guwatudde d. prevalence and correlates of cytopenias in hiv-infected adults initiating highly active antiretroviral therapy in uganda. bmc infect dis. 2014;14(1):1–10. https://doi.org/10.1186/1471-2334-14-496 santiago-rodríguez ej, mayor am, fernández-santos dm, hunter-mellado rf. profile of hiv-infected hispanics with pancytopenia. int j environ res public health. 2015;13(38):1–7. https://doi.org/10.3390/ijerph13010038 yokuş o, gedik h. etiological causes of pancytopenia: a report of 137 cases. avicenna j med. 2016;6(4):109–112. https://doi.org/10.4103/2231-0770.191447 jalaeikhoo h, mohammad s, kashfi h, azimzadeh p, narimani a. acute myeloid leukemia as the main cause of pancytopenia in iranian population. iran j pathol. 2017;12(3):265–271. https://doi.org/10.30699/ijp.2017.25647 waldrop g, doherty m, vitoria m, ford n. stable patients and patients with advanced disease: consensus definitions to support sustained scale up of antiretroviral therapy. trop med int health. 2016;21(9):1124–1130. https://doi.org/10.1111/tmi.12746 who. advanced hiv disease. n.d. [cited 2019 dec 17]. available from: https://www.who.int/teams/global-hiv-hepatitis-and-stis-programmes/hiv/treatment/advanced-hiv-disease marmot m, friel s, bell r, houweling ta, taylor s. closing the gap in a generation: health equity through action on the social determinants of health. lancet. 2008;372(9650):1661–1669. https://doi.org/10.1016/s0140-6736(08)61690-6 ataguba je, akazili j, mcintyre d. socioeconomic-related health inequality in south africa: evidence from general household surveys. int j equity health. 2011;10(48): https://doi.org/10.1186/1475-9276-10–48 rios a. hiv-related hematological malignancies: a concise review. clin lymphoma myeloma leuk. 2014;14:s96–s103. https://doi.org/10.1016/j.clml.2014.06.020 conlan mg, armitage jo, bast m, weisenburger dd. clinical significance of hematologic parameters in non-hodgkin’s lymphoma at diagnosis. cancer. 1991;67(5):1389–1395. https://doi.org/10.1002/1097-0142(19910301)67:5<1389::aid-cncr2820670519>3.0.co;2-q kanavos p. the rising burden of cancer in the developing world. ann oncol. 2006;17(supplement 8):viii15–viii23. https://doi.org/10.1093/annonc/mdl983 tareen sm, bajwa ma, tariq mm, babar s, tareen am. pancytopenia in two national ethnic groups of baluchistan. j ayub med coll abbottabad. 23(2):82–86. hamid ga, shukry sar. patterns of pancytopenia in yemen. turk j haematol. 2008;25(2):71–74. azaad ma, li y, zhang q, wang h. detection of pancytopenia associated with clinical manifestation and their final diagnosis. open j blood dis. 2015;5(3):17–30. https://doi.org/10.4236/ojbd.2015.53004 modjadji s, alberts m, mamabolo r. folate and iron status of south african non-pregnanat rural women of childbearing age, before and after fortification of foods. s afr j clin nutr. 2008;20(3):89–95. https://doi.org/10.1080/16070658.2007.11734132 steyn np, wolmarans p, nel jh, bourne lt. national fortification of staple foods can make a significant contribution to micronutrient intake of south african adults. public health nutr. 2008;11(3):307–313. https://doi.org/10.1017/s136898000700033x abstract introduction methods results discussion acknowledgements references about the author(s) benard m. mutua department of medical laboratory sciences, school of public health biomedical sciences and technology, masinde muliro university of science and technology, kakamega, kenya george sowayi department of medical laboratory sciences, school of public health biomedical sciences and technology, masinde muliro university of science and technology, kakamega, kenya patrick okoth department of biological sciences, school of natural and applied sciences, masinde muliro university of science and technology, kakamega, kenya citation mutua bm, sowayi g, okoth p. red cell distribution width as a surrogate marker of haemoglobinopathies in western kenya. afr j lab med. 2022;11(1), a1644. https://doi.org/10.4102/ajlm.v11i1.1644 project research registration: project number: 407653 original research red cell distribution width as a surrogate marker of haemoglobinopathies in western kenya benard m. mutua, george sowayi, patrick okoth received: 09 june 2021; accepted: 11 feb. 2022; published: 29 apr. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: haemoglobinopathies are inherited haemoglobin disorders that result in anaemia characterised by erythrocyte anisopoikilocytosis. red cell distribution width (rdw) measures anisopoikiloytosis and is readily reported by haematology analysers as a complete blood count parameter. the utility of rdw as a diagnostic marker of haemoglobinopathies in kenya remains undetermined and undocumented. objective: this study aimed to determine the diagnostic efficacy of rdw in discriminating haemoglobinopathy and haemoglobinopathy-free cases in kenya. methods: the case-control study used randomly selected haematology analyser outputs for haemoglobinopathy-free (241, 49.4%) and haemoglobinopathy cases (247, 50.1%) aged 1 month to 66 years old tested in the aga khan hospital, kisumu, and its satellite centres in western kenya from 01 january 2015 to 31 december 2020. results were verified using high performance liquid chromatography. the receiver operating characteristic (roc) curve was used to evaluate the diagnostic power of rdw as a biomarker for sickle cell disease (scd) and sickle cell trait phenotypes and β-thalassaemia. results: the rdw showed diagnostic significance in scd phenotypes at 21.1 roc curve coordinate with 67.7% sensitivity, 90.0% specificity, 0.789 accuracy, 70.5% positive predictive validity, 88.8% negative predictive validity, 6.77 positive likelihood ratio, 0.36 negative likelihood ratio and 18.94 (11.4–31.4) odds ratio. conclusion: an rdw of 21.1% is potentially a predictor of scd haemoglobin phenotypes and should be included in the haematology screening algorithm as a critical value, above which suspected cases qualify to be investigated for scd. keywords: red cell distribution width; surrogate marker; biomarker; haemoglobinopathies; patients; western kenya. introduction haemoglobinopathies, thalassaemia syndromes, and structural haemoglobinopathy variants – haemoglobin s, haemoglobin e, haemoglobin c – are hereditary haemoglobin disorders resulting from mutations in genes encoding the haemoglobin polypeptide chain. these structural haemoglobin disorders result in functionally impaired molecules; impairment includes inefficient oxygen supply and susceptibility to destruction by the victim’s reticuloendothelial system, with consequential fatal or life-threatening severe anaemia and hypoxia.1,2 the world health organization reports that approximately 7% of the global population carry an inherited haemoglobin disorder gene and that about 300 000 infants are born with severe haemoglobin disorders annually, with over 200 000 being born in the sub-saharan african countries.1,3 if proper measures are not put in place, it is estimated that between 2010 and 2050, 14 282 000 babies will be born with sickle cell disease (scd), of which 82% will be born in sub-saharan african countries.4 haemoglobinopathies are neglected but increasing global health problems; children with scd who live in sub-saharan africa have an estimated high mortality rate of 50% – 80% by age 5.1,5,6,7,8,9,10,11 parents who are carriers of haemoglobinopathies have a 25% risk of genetically transferring potentially severe disorders to offspring, thus making prevention and control of these disorders difficult. therefore, due to the recessive character of haemoglobinopathy inheritance, researchers have recommended screening for the carrier state in potentially susceptible populations.12,13 studies have shown that the kenyan population has a significant burden of haemoglobin disorders that remains undocumented as with many other sub-saharan african countries. non-surveillance is due to the high financial cost of haemoglobinopathy laboratory tests. these tests include the world health organization recommended haemoglobin electrophoresis and genotyping tests for population and newborn screening.11,14 consequently, a simple model to unmask cases needs to be established. laboratory testing is the surest way to diagnose blood disorders; however, most affected children in african countries continue to die in early childhood, usually undiagnosed, due to the lack of effective programmes for early detection and treatment. a prospective cohort study in the kilifi area of kenya documented a 50% – 90% mortality rate in children under five years with scd, which was consistent with the 50% – 80% mortality rate recorded among undiagnosed and untreated children across africa. the authors recommended prioritising scd diagnosis and management health research in africa. the current study sought to determine the potency of red cell distribution width (rdw) as a surrogate marker for haemoglobinopathies circulating in a vulnerable population in resource-poor settings of western kenya.4,5,7,6 the kenyan regions served by the aga khan hospital kisumu, being within the malaria holoendemic region of western kenya, lie in the lake victoria economic block region which is known to have a high burden of haemoglobinopathies, particularly sickle cell haemoglobinopathy.11,15,16 the need for a less costly haemoglobinopathies laboratory testing method in western kenya is imperative. previous studies have demonstrated the utility of rdw as a discriminatory marker of iron deficiency anaemia from other microcytic anaemias while other studies have demonstrated the ability of this haematological parameter to discriminate iron deficiency anaemia and thalassaemias. the haematological index or parameter, rdw, seems able to discriminate haemoglobinopathies generally from other erythrocyte disorders associated with anaemia.17,18,19,20,21,22 this makes rdw a potentially simpler, cheaper, faster, and potentially dependable laboratory assay method for haemoglobinopathy detection suitable in low-income settings of western kenya. a sickling test was recommended for screening scd in children since it proved to have high sensitivity and specificity compared to solubility test and peripheral blood film in a study done in uganda. however, unknown adult haemoglobinopathy carriers were left out by the same study; thus, the present study sought to unmask these carriers from the general population.23 the rdw measures variation in red blood cell sizes (anisocytosis) and shapes (poikilocytosis) in cell volume within the red cell population. these are erythrocyte phenotypic features that are commonly abnormal in the presence of haemoglobinopathy.24 the rdw is one of the haematological indices routinely generated by automated haematology analysers in clinical laboratory assays; therefore, it is a simple, faster, cheaper, and widely used test in routine practice as part of a full haemogram report. the rdw has been studied as a significant entity in various disease pathogeneses involving erythrocyte size and shape variations24,25,26 it is derived and presented as a coefficient of variation. studies have shown the potential utility of the rdw as a marker for laboratory detection of haemoglobinopathies, but there is a paucity of data on its use in kenya. genetic variation and environmentally and socioculturally imposed epigenetic changes have been shown to influence gene-phenotype.27 it is uncertain if the rdw values and their relationship with haemoglobin phenotypes on populations in other geographical settings can apply to the kenyan scenario, especially the malaria holoendemic lake victoria basin. the overall goal of the study was to contribute to improving the chances of survival of infants and children with haemoglobinopathies by enabling financial access to timely laboratory testing through a dependable but affordable assay method. its main objective was to establish the overall accuracy of the rdw as a surrogate marker of haemoglobinopathies among age-mixed patients. methods ethical considerations ethical approval was granted by the masinde muliro university ethical review committee (reference mmu/cor:403012 vol 3(03) and national commission of science and technology (nacosti) (ref. 407653). permission to collect data was approved by aga khan hospital, kisumu ethics committee (reference adm/007/089) thus patients’ consent was not needed. the raw data was stored by the principal investigator in restricted rooms and electronic data was coded to maintain anonymity in password proof computers. sample size determination sample size calculation was performed using cochrans’s formula for sample size determination in case-control and other comparative studies.28 assuming 19% prevalence of α-thalassaemia and sickle cell among children enrolled in a malaria vaccine clinical trial study done at kombewa in lake victoria basin, western kenya, a confidence level of 95% and a precision level of 5%, sample size of 237 was obtained; since this was a two-arm study (case-control), an equal control of 237 was needed, giving a minimum required sample size of 474.16 study design this was a hospital-based cross-sectional retrospective comparative study of 488 randomly selected high performance liquid chromatography confirmed haemoglobinopathy, but non-iron deficiency, subjects (cases) (n = 247, 50.1%) and haemoglobinopathy-free and non-anaemic results (control group) (n = 241, 49.4%) with corresponding complete blood counts from hospital databases for the aga khan hospital, kisumu, and its western kenya satellites. data collection data were obtained from the laboratory database on patients examined at the hospital’s haematology laboratory for the past five years from 01 january 2015 to 31 december 2020. complete blood count reports were performed using various sysmex analysers (kx-21n, xp 300, sysmex xnl 330, symex xs 500i and sysmex xs1000i; sysmex corporations, kobe, japan). the cases were individuals who were confirmed for various haemoglobinopathies using a high performance liquid chromatography (bio-rad d10) machine (bio-rad laboratories, hercules, california, united states). excluded cases included those without their respective complete blood counts reports, those with confirmed leukaemia, and those cases that had received transfusion in the past three months. the control group consisted of individuals presumed free from disorders normally associated with abnormality of red erythrocyte shape and size (including haemolytic, macrocytic or iron deficiency anaemia) and haemoglobinopathy-free. these were age-mixed people electrophoretically confirmed to have normal haemoglobin (haemoglobin aa genotype) and had haemoglobin concentrations of ≥ 9.5 g/dl for ≤ 5-year-olds, ≥ 10.5 g/dl for ≤ 12-year-olds and ≥ 11 g/dl for ≥ 13-year-olds.29 all participants had rdw results as part of the complete blood counts from automated haematology analysers, but cases had additional haemoglobin profiles. data analysis statistical package for social sciences version 20 (spss inc., chicago, illinois, united states) was used to analyse data with kolmogorov-smirnov and shapiro-wilks tests. these tests revealed that the control group was a skewed (non-normal) distribution (p < 0.05); thus, a non-parametric statistics test, the kruskal-wallis h-test, was used to assess the rdw variation within haemoglobinopathy variants while the mann whitney u-test was used to compare the rdw variations between groups as recommended by nahm, 2016.30 accordingly, the normal rdw reference values were derived from the control group as the upper limit of 95% confidence interval (ci) of the median. data were summarised as a median and interquartile range for the rdw and percentage for haemoglobinopathy presence. the clinical utility of the rdw was studied through receiver operating characteristic (roc) curves analysis to assess its diagnostic efficacy in differentiating diseased (haemoglobinopathy) from non-diseased (haemoglobinopathy-free) population. the sensitivity and specificity at optimal points by use of youden index, plus predictive values, likelihood ratio (lr), and odds ratio (or) at the 5% significance level (p = 0.05) were determined.31 results demographic characteristics of study participants the proportions of the control group (49.4%, n = 241) and the case group (50.6%, n = 247) did not differ significantly (p = 0.740) in a total of 488 individuals (table 1). there was no significant difference (p = 0.502) between the number of male (43.9%, n = 214) and female (56.1%, n = 274) participants. the majority of the participants were from the kisumu station (49.0%, n = 239), followed by busia (15.4%, n = 75) and homabay (12.3%, n = 60; p < 0.001). the rest of the participants were distributed among the rest of the locations. there was a statistically significant variation in scd frequencies across the three age groups: ≤ 5-year-olds (42.4%, n = 207), ≤ 12-year-olds (19.9%, n = 97), and ≥ 13-year-olds (37.7%, n = 184; p < 0.001). table 1: demographic characteristics of study participants and rdw in control and case (haemoglobinopathies) groups in western kenya, 01 january 2015 – 31 december 2020. rdw in haemoglobinopathy phenotyping the median rdw difference was 6.2 between the case (20.7, interquartile range [iqr] = 8.3) and the control groups (14.5, iqr = 2.7; 95%, ci = 9.1–19.9; p < 0.001). the rdw median for scd phenotypes were: haemoglobin ss genotype, 25.4 (iqr = 5.5); haemoglobin ss genotype + β-thalassemia, 23.3 (iqr = 7.9); and haemoglobin ss genotype + haemoglobin f, 20.9 (iqr = 5.5) which were significantly higher compared to the control group (14.5, iqr = 2.7; 95% ci = 9.1–19.9; p < 0.001). individuals with pure haemoglobin as genotype had a significantly higher (p < 0.001) rdw median of 16.4 (iqr = 6.5) compared to the control group (14.5, iqr = 2.7; 95% ci = 9.1–19.9). the rdw was high in haemoglobin as genotype + haemoglobin f (24.2, p = 0.449), haemoglobin as genotype + β-thalassemia (20.9, iqr = 10.5; p = 0.791) and β-thalassaemia (19.9, iqr = 8.6; p = 1.00) patients but the difference was not statistically significant when compared to the control group (14.5, iqr = 2.7; 95% ci = 9.1–19.9). diagnostic efficacy of the rdw in haemoglobinopathies at given optimal points, the rdw demonstrated its diagnostic efficacy by marking some haemoglobinopathies with a high sensitivity, specifity, youden index and asymptotic significance (p < 0.001) with their roc curves flowing upwards on the left side of the curve (table 2; figure 1). on the other hand, the diagnostic utility of rdw for some haemoglobinopathies was marked by low sensitivity, specifity and youden index and did not have asymptotic significance with their roc curve flowing along the diagonal line (figure 2 and figure 3). table 2: summary of rdw predictive ability in haemoglobin disorders in western kenya, 01 january 2015 – 31 december 2020. figure 1: red cell distribution width, roc curve in scd phenotypes (haemoglobin ss genotype, haemoglobin ss genotype + haemoglobin f, haemoglobin ss genotype + β-thalassaemia) in western kenya, 01 january 2015 – 31 december 2020. (a) red cell distribution width roc curve in diagnosis of homozygous scd. (b) red cell distribution width roc curve in diagnosis of sickle cell disease with haemoglobin f. (c) red cell distribution width roc curve in diagnosis of scd with β-thalassaemia. figure 2: red cell distribution width roc curve in sickle cell triat phenotypes (haemoglobin as genotype and β-thalassaemia) in western kenya, 01 january 2015 – 31 december 2020. (a) red cell distribution width, receiver operating characteristic curve for sickle cell trait flowing along diagonal line of the roc curve. (b) red cell distribution width, receiver operating characteristic curve for sickle cell trait+β-thalassaemia also flowing along the diagonal line of the roc curve. figure 3: red cell distribution width roc curve in β-thalassaemia in western kenya from 01 january 2015 – 31 december 2020. red cell distribution roc curve in sct phenotyping the rdw proportion for pure haemoglobin as genotype was 21.1% (n = 103; roc curve flowed along the diagonal line with a youden index = 0.501; p = 0.976) for diagnosis of pure sickle cell trait (sct) phenotype (table 2, figure 2). similarly, the rdw roc curve coordinates at the optimal point of 19.8 did not have diagnostic significance (p = 0.399, sensitivity, 50%, specificity 70%; curve flowed along the diagonal line with a low youden index = 0.600) for the diagnosis of haemoglobin as genotype + β-thalassaemia. red cell distribution roc curve in scd phenotyping the rdw proportion for haemoglobin ss genotype was 9.2% (n = 45; optimal point = 21.1; p < 0.001; accuracy = 0.892; sensitivity = 86.7%; specificity = 80.0%) (table 2, figure 1) pushing the curve to the left upper side of the roc curve. the proportion for haemoglobin ss genotype and haemoglobin f was 4.1% (n = 20; optimal point = 21.8; p < 0.001; sensitivity = 70.0%; specificity = 67.6%; youden index = 0.766), with the roc curve flowing above the diagonal line. a haemoglobin ss genotype + β-thalassaemia prevalence of 12.7% (n = 62) was obtained and at 17.7 the rdw optimal point (p < 0.001; sensitivity = 78.0%; specifity = 64.5%; youden index = 0.805). roc curve in β-thalassaemia the rdw optimal point of 16.8 for β-thalassaemia was not significant (p = 0.706). the roc curve flowed along the diagonal line area under curve = 0.539 with sensitivity = 63% and specificity = 60%) (table 2; figure 3). sensitivity and specificity of rdw in scd phenotype diagnosis the roc curves grouped haemoglobinopathies into two groups serving as an excellent significant (p < 0.001) biomarker in scd phenotypes diagnosis; but it was poor in the diagnosis of sct phenotypes and β-thalassemia (low youden index, sensitivity, and specificity) (table 2). therefore, the efficacy of rdw as a biomarker for use in scd phenotype diagnosis was evaluated in a single roc curve (figure 4) giving a sensitivity of 67.7%, specificity of 90.0% and an accuracy of 0.789 at an optimal point of 21.1 (table 2). figure 4: red cell distribution width roc curve in haemoglobin ss phenotypes in western kenya, 01 janauary 2015 – 31 december 2020. this roc curve demonstrates the efficacy of rdw at 21.1% optimal point in scd (haemoglobin ss) phenotypes diagnosis. predictive validity, likelihood and or of rdw in scd phenotyping the rdw at 21.1 optimal value, recorded 70.5% positive predictive validity and 88.8% negative predictive validity in scd phenotypes diagnosis (table 2). similarly, the same optimal value had a 6.77 positive likelihood ratio (lr+), 0.36 negative likelihood ratio (lr-) and 18.94 or in the diagnosis of haemoglobin ss phenotypes. discussion the rdw was able to diagnose scd phenotypes significantly (p < 0.001), but could not diagnose sct phenotypes and β-thalassaemia (p > 0.05; low youden index, sensitivity, and specificity). the overall accuracy of the rdw proved to be an excellent biomarker for scd haemoglobinoathies; thus, unknown (seemingly haemoglobinopathy-free) cases with rdw above 21.1 need to be confirmed using advanced technology. therefore, countries with limited financial resources who have not implemented newborn and population screening can use this potential biomarker as a cost-effective approach. a worthless test has a youden index of 0.5, poor sensitivity of 50%, a specificity of about 50% and a roc curve that flows along the diagonal line and thus is unable to distinguish diseased from non-diseased individuals.31 the rdw could not serve as a biomarker for pure sct (p = 0.976) and sct+β-thalassaemia (p = 0.399) diagnoses; both had low youden index, sensitivity and specifity. in homozygous scd, the rdw had a sensitivity of 86.7%, specificity of 80% and an accuracy of 0.892 which are features of a significant (p < 0.001) biomarker at 21.1 roc curve coordinate.31 similar findings of elevated rdw were documented by webster and castro32 with homozygous scd having the highest rdw, followed by heterozygous scd+β-thalassaemia and then scd and haemoglobin f. the rdw proved to be an excellent diagnostic marker for scd and haemoglobin f at 20.8 roc curve coordinate where a sensitivity of 70.0% and a specificity of 67.6% were obtained with a high accuracy of 0.766. the roc curve demonstrated that the rdw was an excellent diagnostic marker (p < 0.001) in detecting scd+β-thalassaemia with an accuracy of 0.805. the β-thalassaemia roc curve flowed along the diagonal line with poor diagnostic significance (p = 0.706) similar to qurtom et al’s report33 of β-thalassaemia having a normal or mildly elevated rdw at a mean of 15.4 ± 1.21, meaning it is not possible to use the rdw to differentiate β-thalassemia from the healthy population. the rdw roc curves were able to discriminate (p < 0.001) scd phenotypes from the healthy population but could not diagnose sct phenotypes and β-thalassaemia. the optimal rdw value for the overall efficacy in diagnosing scd phenotypes was 21.1 (sensitivity = 67.7%; specificity = 90.0%; accuracy = 0.789). this means that in a population, an rdw > 21.1 can identify 67.7% of the individuals as having scd while an rdw of < 21.1 will identify 90% of individuals who would test negative for scd. this implies that at 21.1, the rdw can be used as an optimal diagnostic biomarker for scd phenotypes in western kenya. a positive predictive value tells how likely an individual will test positive for a given disease. in regard to the present study, patients with rdw > 21.1 were 70.5% likely to test positive for scd indicating 29.5% would still test negative for scd even when the rdw was > 21.1. negative predictive value tells how likely an individual will be to test negative for a particular disease meaning 88.1% of patients with rdw < 21.1 would be free from scd while 11.9% would still test positive. therefore, the rdw is proving to be a good diagnostic biomarker for scd phenotypes among haemoglobinopathies listed in the present study. to this end, this is the first-ever attempt to determine the likelihood of using the rdw value to diagnose scd phenotype in western kenya. likelihood ratios are clinically more useful than sensitivity and specificity in determining the usefulness of diagnostic tests. the positive likelihood ratio (lr+) expresses how likely a test is going to correctly diagnose the presence of the condition where the greater the lr+, the more likely the test is going to give a true positive diagnosis. the rdw > 21.1 had a lr+ of 6.77, meaning any individual having the rdw > 21.1 is 6.77 times more likely to test positive for scd. negative likelihood ratio (lr–) is defined by akobeng34 as the ratio of how likely a test will correctly diagnose the absence of a condition whose value is usually < 1; the closer the value gets to zero, the better the test is correctly indicating the absence of the condition. regarding the present study, lr– was 0.36, meaning the probability of a person with the rdw < 21.1, is 0.36 times less likely to be free from scd. it is important to note a test having both lr+ and lr– close to 1 has little influence to predict the presence or absence of a disease and is, therefore, worthless in clinical practice. on the same optimal value, an or of 18.9 was obtained meaning that individuals with the rdw > 21.1 were 18.9 times at greater risk of having scd haemoglobinopathy compared to those with the rdw < 21.1. limitations sickle cell traits +haemoglobin f and +β-thalassaemia recorded abnormally elevated rdw that did not have statistical significance due to the small sample size. conclusion these findings indicate that the rdw is a promising diagnostic marker for scd phenotypes; thus, rdw of 21.1 should be included in the haematology policy screening algorithm as a critical value above which the unknown cases qualify to be investigated for sickle cell haemoglobinopathy. however, the data used were retrospective and hence the diagnostic utility of this haematological index for haemoglobinopathy should be explored further using prospective data. a confirmatory test would still be needed and therefore provision and use of powered mini-electrophoretic equipment will be appropriate in low-resource settings. acknowledgements we are grateful to mr. raphael kitonga for allowing us to use his home-based library and its accessories in addition to steadfast moral support. we appreciate the aga khan hospital, kisumu laboratory manager lucy mathenge and dr simon onsongo head of the pathology department for supporting us during data collection. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions b.m.m. conceptualised the study, analysed data, wrote the original draft of the manuscript and critical reviews. g.s. conceptualised the study, and contributed to the validation and critical reviews. p.o. conceptualised the study, gave critical technical expert advice on the study, validation and gave critical reviews on the manuscript. sources of support the authors received no financial support for the research, authorship, or publication of this article. data availability data supporting the findings of this study is available upon request from the corresponding author, b.m.m. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references weatherall d. the inherited disorders of haemoglobin: an increasingly neglected global health burden. indian j med res. 2011;134(4):493. ilesanmi oo. pathological basis of symptoms and crises in sickle cell disorder: implications for counseling and psychotherapy. hematol rep. 2010;2(1):e2. https://doi.org/10.4081/hr.2010.e2 piety nz, yang x, kanter j, vignes sm, george a, shevkoplyas ss. validation of a low-cost paper-based screening test for sickle cell anemia. plos one. 2016;11(1):e0144901. https://doi.org/10.1371/journal.pone.0144901 debaun mr, galadanci na, vichinsky ep. sickle cell disease in sub-saharan africa. alphen aan den rijn: wolters kluwer uptodate;2019. makani j, cox se, soka d, et al. mortality in sickle cell anemia in africa: a prospective cohort study in tanzania. plos one. 2011;6(2):e14699. https://doi.org/10.1371/journal.pone.0014699 arishi wa, al-hadrami ha, zourob m. techniques for the detection of sickle cell disease: a review. micromachines. 2021;12(5):519. https://doi.org/10.3390/mi12050519 uyoga s, macharia aw, mochamah g, et al. the epidemiology of sickle cell disease in children recruited in infancy in kilifi, kenya: a prospective cohort study. the lancet global health. 2019;7(10):e1458–e1466. https://doi.org/10.1016/s2214-109x(19)30328-6 chakravorty s, williams tn. sickle cell disease: a neglected chronic disease of increasing global health importance. arch dis child. 2015;100(1):48–53. https://doi.org/10.1136/archdischild-2013-303773 grosse sd, odame i, atrash hk, amendah dd, piel fb, williams tn. sickle cell disease in africa: a neglected cause of early childhood mortality. am j prev med. 2011;41(6):s398–s405. https://doi.org/10.1016/j.amepre.2011.09.013 modell b, darlison m. global epidemiology of haemoglobin disorders and derived service indicators. bulletin of the world health organization. 2008;86:480–487. https://doi.org/10.2471/blt.06.036673 suchdev ps, ruth lj, earley m, macharia a, williams tn. the burden and consequences of inherited blood disorders among young children in western kenya. matern child nutr. 2014;10(1):135–144. https://doi.org/10.1111/j.1740-8709.2012.00454.x sawaimul kd, iqbal mb, sawaimul vd, kambale t, hanmante r. study to identify the role of high-performance liquid chromatography in detecting haemoglobinopathies in antenatal patients. indian j pathol oncol. 2018;5(1):6–11. https://doi.org/10.18231/2394-6792.2018.0002 trent rj. diagnosis of the haemoglobinopathies. clin biochemist rev. 2006;27(1):27. tluway f, makani j. sickle cell disease in africa: an overview of the integrated approach to health, research, education, and advocacy in tanzania, 2004–2016. br j haematol. 2017;177(6):919–929. https://doi.org/10.1111/bjh.14594 kosiyo p, otieno w, gitaka j, munde eo, ouma c. association between haematological parameters and sickle cell genotypes in children with plasmodium falciparum malaria resident in kisumu county in western kenya. bmc infect dis. 2020;20(1):1–1. https://doi.org/10.1186/s12879-020-05625-z kifude cm. prevalence of sickle cell and α-thalassemia traits in children enrolled in a malaria vaccine clinical trial, in kombewa, western kenya. doctoral dissertation. nairobi: jesuit historical institute in africa, kenyatta university;2007. available from: http://thesisbank.jhia.ac.ke/id/eprint/2505 sarah b, sheikh k, shah t. red cell distribution width is early marker for detection of iron deficiency anemia during pregnancy. j liaquat university med health sci. 2018;17(3):165–169. https://doi.org/10.22442/jlumhs.181730571 aulakh r, sohi i, singh t, kakkar n. red cell distribution width (rdw) in the diagnosis of iron deficiency with microcytic hypochromic anemia. indian j pediatr. 2009;76(3):265–268. https://doi.org/10.1007/s12098-009-0014-4 eldibany mm, totonchi kf, joseph nj, rhone d. usefulness of certain red blood cell indices in diagnosing and differentiating thalassemia trait from iron-deficiency anemia. am j clin pathol. 1999;111(5):676–682. https://doi.org/10.1093/ajcp/111.5.676 matos jf, dusse l, borges kb, de castro rl, coura-vital w, carvalho md. a new index to discriminate between iron deficiency anemia and thalassemia trait. revista brasileira de hematologia e hemoterapia. 2016;38(3):214–219. https://doi.org/10.1016/j.bjhh.2016.05.011 miri-moghaddam e, sargolzaie n. cut off determination of discrimination indices in differential diagnosis between iron deficiency anemia and β-thalassemia minor. int j hematol oncol stem cell res. 2014;8(2):27. sharma a, sharma m, sharma v. evaluation of red cell distribution width in the diagnosis of iron deficiency anemia. int j res med sci. 2016;4(9):3733–3736. https://doi.org/10.18203/2320-6012.ijrms20162603 okwi al, byarugaba w, parkes a, ocaido m. the reliability of sickling and solubility tests and peripheral blood film method for sickle cell disease screening at district health centers in uganda. clin mother child health. 2010;7(1)1–5. https://doi.org/10.4303/cmch/c101947 ramby al, goodman dm, wald el, weiss sl. red blood cell distribution width as a pragmatic marker for outcome in pediatric critical illness. plos one. 2015;10(6):e0129258. https://doi.org/10.1371/journal.pone.0129258 nishad an, de silva is, perera hl, pathmeswaran a, kastutiratne ka, premawardhena ap. role of red cell distribution width in screening for hb e trait in population screening for hemoglobin disorders. j pediatr hematol oncol. 2014;36(8):e490–e492. https://doi.org/10.1097/mph.0000000000000052 thame m, grandison y, mason k, et al. the red cell distribution width in sickle cell disease – is it of clinical value? clin lab haematol. 1991;13(3):229–237. https://doi.org/10.1111/j.1365-2257.1991.tb00277.x saulnier km, dupras c. race in the postgenomic era: social epigenetics calling for interdisciplinary ethical safeguards. am j bioeth. 2017;17(9):58–60. https://doi.org/10.1080/15265161.2017.1353182 nam jm. sample size determination for case-control studies and the comparison of stratified and unstratified analyses. biometrics. 1992;48(2):389–395. https://doi.org/10.2307/2532298 wacholder s, mclaughlin jk, silverman dt, mandel js. selection of controls in case-control studies: i. principles. am j epidemiol. 1992;135(9):1019–1028. https://doi.org/10.1093/oxfordjournals.aje.a116396 nahm fs. nonparametric statistical tests for the continuous data: the basic concept and the practical use. korean j anesthesiol. 2016;69(1):8. https://doi.org/10.4097/kjae.2016.69.1.8 ruopp md, perkins nj, whitcomb bw, schisterman ef. youden index and optimal cut-point estimated from observations affected by a lower limit of detection. biom j. 2008;50(3):419–430. https://doi.org/10.1002/bimj.200710415 webster pa, castro os. red cell distribution width in sickle cell disease. ann clin lab sci. 1986;16(4):274–277. qurtom ha, al-saleh qa, lubani mm, et al. the value of red cell distribution width in the diagnosis of anaemia in children. eur j pediatrics. 1989;148(8):745–748. https://doi.org/10.1007/bf00443100 akobeng ak. understanding diagnostic tests 2: likelihood ratios, pre-and post-test probabilities, and their use in clinical practice. acta paediatr. 2007;96(4):487–491. https://doi.org/10.1111/j.1651-2227.2006.00179.x abstract introduction methods results discussion acknowledgements references about the author(s) faithful makita-chingombe international pharmacology specialty laboratory, school of pharmacy, university of zimbabwe college of health sciences, harare, zimbabwe anthony t. podany antiviral pharmacology laboratory, university of nebraska medical center, omaha, nebraska, united states timothy mykris antiviral pharmacology laboratory, university of nebraska medical center, omaha, nebraska, united states farai muzambi international pharmacology specialty laboratory, school of pharmacy, university of zimbabwe college of health sciences, harare, zimbabwe richard w. browne translational pharmacology research core, center for integrated global biomedical sciences, school of pharmacy and pharmaceutical sciences, university at buffalo, buffalo, new york, united states andrew j. ocque translational pharmacology research core, center for integrated global biomedical sciences, school of pharmacy and pharmaceutical sciences, university at buffalo, buffalo, new york, united states robin difrancesco translational pharmacology research core, center for integrated global biomedical sciences, school of pharmacy and pharmaceutical sciences, university at buffalo, buffalo, new york, united states lee c. winchester antiviral pharmacology laboratory, university of nebraska medical center, omaha, nebraska, united states courtney v. fletcher antiviral pharmacology laboratory, university of nebraska medical center, omaha, nebraska, united states tinashe mudzviti international pharmacology specialty laboratory, school of pharmacy, university of zimbabwe college of health sciences, harare, zimbabwe charles c. maponga international pharmacology specialty laboratory, school of pharmacy, university of zimbabwe college of health sciences, harare, zimbabwe translational pharmacology research core, center for integrated global biomedical sciences, school of pharmacy and pharmaceutical sciences, university at buffalo, buffalo, new york, united states gene d. morse translational pharmacology research core, center for integrated global biomedical sciences, school of pharmacy and pharmaceutical sciences, university at buffalo, buffalo, new york, united states citation makita-chingombe f, podany at, mykris t, et al. cross-validation of a high-performance liquid chromatography nevirapine plasma assay in a resource-limited setting in zimbabwe. afr j lab med. 2021;10(1), a1264. https://doi.org/10.4102/ajlm.v10i1.1264 brief report cross-validation of a high-performance liquid chromatography nevirapine plasma assay in a resource-limited setting in zimbabwe faithful makita-chingombe, anthony t. podany, timothy mykris, farai muzambi, richard w. browne, andrew j. ocque, robin difrancesco, lee c. winchester, courtney v. fletcher, tinashe mudzviti, charles c. maponga, gene d. morse received: 08 may 2020; accepted: 24 feb. 2021; published: 08 july 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract an international hiv pharmacology specialty laboratory (psl) was established at the university of zimbabwe to increase bioanalytical and investigator capacities. quantitation of plasma nevirapine in samples from the aids clinical trials group protocol 5279 was compared between the university of nebraska medical center psl and the university of zimbabwe psl. both psls employed internally developed methods utilising reverse-phase high-performance liquid chromatography with ultraviolet detection. eighty-seven percent of the cross-validation results exhibited ± 20% difference. keywords: cross-validation; hiv; root-cause analysis; high-performance liquid chromatography. introduction the university of zimbabwe (uz) international hiv pharmacology speciality laboratory (ipsl) was established to increase hiv pharmacology-related research and support research investigator capacity in africa.1 the uz-ipsl performs drug development assay and clinical research specimens analyses, specifically drug-drug interactions, pharmacokinetic and pharmacodynamic data analyses. the clinical pharmacology quality assurance programme, which monitors all psls within the aids clinical trials group, provided technical guidance to and developed a comparative nevirapine validation study for the uz-ipsl to analyse replicate hiv clinical samples. validation of the nevirapine bioanalytics method2 was essential at the time because of nevirapine use in the sub-saharan region and results were generated by a reverse-phase high-performance liquid chromatography (hplc) system using ultraviolet detection with gradient-elution separation.3,4,5 this report compares nevirapine bioanalysis at two laboratories: the united states-based, university of nebraska medical center pharmacology specialty laboratory (unmc-psl) and the uz-ipsl. the unmc-psl served as the aids clinical trials group 5279 (a5279) protocol designated laboratory. the unmc-psl, during the time of the a5279 study, participated in the clinical pharmacology quality assurance proficiency testing (pt) programme to assure nevirapine assay performance and accuracy. although each psl validated the nevirapine assay separately, the lack of data comparing the bioanalytical capacity of pharmacology laboratories in low-middle income countries using clinical samples (rather than spiked plasma samples) was the justification for this project. therefore, implementation of the project was the last stage in determining uz-ipsl’s readiness to begin assaying human samples for clinical trials. methods ethical considerations data files used in this comparison study were de-identified and clinical pharmacology quality assurance was blinded to any data associated with samples. this article followed all ethical standards for research without direct contact with human or animal subjects. project design the uz-ipsl utilised the a5279 replicate samples (n = 95) that were available at the uz college of health sciences clinical trial research center (uzchs-ctrc) for cross-validation of nevirapine assays between the unmc-psl and the uz-ipsl. a5279 was a multi-centre, phase iii clinical trial investigating the use of short-course rifapentine or isoniazid for the prevention of active tuberculosis in hiv-positive adults.6 in a5279, nevirapine pharmacokinetics when co-administered with rifapentine and isoniazid was one of the exploratory objectives. all a5279 primary aliquots collected from the uz aids clinical trials group ctrc were analysed for nevirapine by the protocol designated psl at the unmc-psl. a5279 sample collection and processing plasma samples for the quantitation of nevirapine concentrations were obtained from a5279 study participants. blood was collected into potassium ethylenediaminetetraacetic acid collection tubes and transported on ice to the processing laboratory within 1 h where the plasma was separated from cells by centrifugation (1200 × g, 10 min, 4 °c). the plasma aliquots were frozen (–70 °c) and shipped to the unmc-psl. the uzchs-ctrc retained secondary aliquots for the uz-ipsl cross-validation assay. university of nebraska medical center psl nevirapine assay a5279 primary plasma aliquots were shipped from uzchs-ctrc on dry ice to the unmc-psl for determination of the nevirapine. the unmc-psl nevirapine determination assay had a quantitation range of 25 ng/ml to 10 000 ng/ml. samples measuring above the quantitation range were diluted and reanalysed using a validated dilution protocol. solid-phase extraction was utilised to prepare samples for analysis. the unmc-psl utilised a waters e2695 hplc coupled to a waters 2489 ultraviolet (waters, milford, massachusetts, united states) detector, which was controlled with empower 2 (waters, milford, massachusetts, united states) software as the analytical platform. the performance of the assay was compliant with the united states food and drug administration bioanalytical guidelines for method validation.7 university of zimbabwe international hiv psl nevirapine assay replicate plasma aliquots (n = 95) from a5279 available at the uzchs-ctrc were shipped to the uz-ipsl for determination of the nevirapine concentration for cross-validation. the uz-ipsl measured nevirapine using a validated hplc-ultraviolet assay detailed previously.2 the uz-ipsl chromatographic system consisted of a shimadzu lc20a hplc using ultraviolet photodiode array detection (model spd-m20a) and labsolutions software (version 5.8; kyoto, japan). the assay quantitation range was 500 ng/ml to 15 000 ng/ml. samples that were above the quantitation range were diluted and reanalysed. the performance of the assay was compliant with united states food and drug administration bioanalytical guidelines. peak purity for nevirapine was assessed to confirm the absence of interferences from potential impurities also extracted from the patient’s sample. a5279 samples were assayed over five runs; the two last runs included samples that were above the upper limit of quantification and were diluted (1:8) according to the validated dilution protocol.2 data management and analysis the uz-ipsl nevirapine results were submitted to the aids clinical trials group data management center portal using the data submission utility (frontier science foundation inc., brookline, massachusetts, united states). the a5279 study team approved the release of the unmc a5279 nevirapine concentration results for comparison with the uz-ipsl results. the original nevirapine concentration data from the unmc-psl were used in the specified a5279 protocol pharmacokinetics analysis; no uz-ipsl derived pharmacokinetics data were used in the per-protocol study analysis. the data management center compiled the data and provided the de-identified data files to the clinical pharmacology quality assurance pt unit to perform blinded, comparative statistical analyses of the nevirapine results. after examination (omnibus 2) and finalisation of the data, the analyses of the paired t-test, deming regression and bland-altman analyses were completed using prism graphpad (version 8.3; san diego, california, united states) software. results ninety-five replicate plasma aliquots were bioanalysed. the nevirapine concentration range in the analysed samples was ~2000 ng/ml – 34 000 ng/ml (~17-fold). of this, 97% (n = 92) ranged between 2000 ng/ml and 20 000 ng/ml and the remaining 3% (n = 3) were above 20 000 ng/ml and not contiguous (table 1, figure 1a)9. the nevirapine concentration values from the laboratories were tested for normality and only data ≤ 15 000 ng/ml or less were normally distributed. data 15 000 ng/ml reflected the upper limit of the uz-ipsl assay and was appropriate because it more closely represented the previously reported nevirapine steady state concentrations (5086 ng/ml – 13 368 ng/ml or 19.1 um to 50.2 um).3,4,5 of the data, 93% (n = 88) were 15 000 ng/ml or less (table 1)9 while 7% (n = 7) were above this value. therefore, the seven sample pairs were excluded from the method comparison. after exclusion, an additional sample pair was identified as a significant outlier using the grubbs test and was also excluded. figure 1: differences in nevirapine concentrations assayed at unmc-psl (omaha, nebraska, united states, october 2017) and uz-ipsl (harare, zimbabwe, may 2018); (a) scatter plot (n = 95 data points). (b) deming regression plot of 87 data points (outliers excluded). boxes in (a) indicate unmc upper limit of quantitation (dashed) and uz-ipls upper limit of quantitation (solid lines). table 1: a comparative study of nevirapine assay results conducted at the university of zimbabwe international hiv pharmacology specialty laboratory (harare, zimbabwe, may 2018) and the university of nebraska medical center pharmacology specialty laboratory (omaha, nebraska, united states, october, 2017). the two-tailed paired t-test of the final data set (n = 87) (table 1)9 showed uz-ipsl nevirapine values were significantly higher with a constant error of 3% – 4% more than unmc-psl values. the deming regression analysis (figure 1b)9 and the bland-altman plots (figure 2)9 further confirmed the higher values from the uz-ipsl. as shown in figure 2a9, regression analysis using the difference plot indicated an increasing proportional difference between the clinical pharmacology laboratories as the concentration of nevirapine increased. however, when the data were normalised by concentration, using the % difference analysis (figure 2b)9, the correlation was no longer significant. figure 2: differences in nevirapine samples (n = 87) assayed at unmc-psl (omaha, nebraska, united states, october 2017) and uz-ipsl (harare, zimbabwe, may 2018). (a) bland-altman difference plot indicating proportionally higher difference at high concentrations (mean difference = −430). (b) bland-altman percentage difference on normalisation by concentration, correlation not significant (mean % deviation = −4.49). shaded areas present confidence interval limits for mean (green shade) and agreement limits (red shade). discussion drug concentrations for nevirapine reported in the literature for hiv-positive individuals indicate a steady state maximum of 5740 ng/ml (5000–7440) and minimum of 3730 ng/ml (3200–5080)8. both nevirapine assays from the two laboratories can detect concentrations that might be below the target concentration for virologic suppression or above the upper limit of the therapeutic range. although the uz-ipsl nevirapine concentrations were higher than those of unmc-psl and this difference was statistically significant, it is unlikely to have clinical significance. the united states food and drug administration does not provide a window of acceptance for cross-validation studies but allows laboratories to determine a priori the acceptance criteria. using the established acceptance window for clinical pharmacology quality assurance pt samples, about 20% of the final target value, and expected concentrations of nevirapine, this difference was acceptable. the outcomes of the cross-validation also presented an opportunity to perform a root-cause analysis. the uz-ipsl’s trend of pt outcomes before and during the a5279 sample analysis period was explored. while all pt results were satisfactory, agreement of initial pt sample analyses showed unbiased accuracy, while during subsequent analyses which coincided with a5279 sample analysis, the uz-ipsl values exhibited a biased, high trend (4% – 19%) as compared to the final target values of that pt round. this shift in bias and accuracy was provided within the pt report and the uz-ipsl was able to complete a root-cause analysis and remediate as needed. limitations one limitation to consider is that the calibration ranges for the two equipment sets used for the nevirapine analysis were not identical. future comparisons of this nature may benefit from evaluations where the calibration range is the same for both laboratories. in addition, limited data points above 20 000 ng/ml entailed difficulties in quantitation of proportional error at that level. conclusion the cross-validation study provided evidence that the uz-ipsl performance using a nevirapine assay that was validated in their laboratory was acceptable and correlated with the results of an experienced psl. this study afforded the uz-ipsl a valuable opportunity to implement operations using its validated nevirapine assay for the analysis of samples obtained from a clinical research protocol and adopt procedures for handling of protocol specimens based on experience. furthermore, the outcomes of the cross-validation emphasised the value of pt and provided an occasion to perform root-cause analysis. acknowledgements the uz-ipsl acknowledges the contribution of dr marshall munjoma for his support during the laboratory work that led to the development of this manuscript. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions f.m.-c. was the project leader and f.m. was the laboratory technologist. t.mudzviti and c.c.m. provided manuscript review and supervised the uz-ipsl activities for the project. a.t.p. was clinical pharmacologist and c.v.f. was the psl director and a5279 pharmacology investigator. t.mykris and l.c.w. were laboratory technologists. a.j.o., r.w.b., r.d. and g.d.m. provided clinical pharmacology quality assurance project design and data analysis. all authors critically revised intellectual content and approved the final version to be published. sources of support university of zimbabwe ipsl: project was funded by the united states national institute of allergy and infectious diseases, national institutes of health (aids clinical trials group grant 5um1ai106701-04). university of nebraska psl: project was funded by the united states national institute of allergy and infectious diseases, national institutes of health, department of health and human services awards atp (k23 ai134307), and cvf (um ai106701). clinical pharmacology quality assurance program: this project has been funded in whole or in part with federal funds from the united states national institute of allergy and infectious diseases, national institutes of health and the department of health and human services, under contract numbers hhsn272201500006c and hhsn272200800019c. center for aids research: this publication was made possible through core services and support from the university of rochester center for aids research, a united states national institutes of health-funded programme (p30 ai078498). data availability data are available from (1) clinical pharmacolog quality assurance program translational pharmacology research core, center for integrated global biomedical sciences, school of pharmacy and pharmaceutical sciences, university at buffalo, state university of new york, buffalo, new york, united states, (2) international psl, school of pharmacy, university of zimbabwe college of health sciences, po box a178, avondale, harare, zimbabwe and (3) antiviral pharmacology laboratory, university of nebraska medical center, omaha, nebraska, united states. disclaimer the content is solely the responsibility of the authors and does not necessarily represent the official views of the united states national institutes of health. references mtisi tj, maponga c, monera-penduka tg, et al. strategic establishment of an international pharmacology specialty laboratory in a resource-limited setting. afr j lab med. 2018;7(1):a659. https://doi.org/10.4102/ajlm.v7i1.659 makita-chingombe f, ocque aj, difrancesco r, et al. development and validation of a high performance liquid chromatography method to determine nevirapine in plasma in a resource-limited setting. afr j lab med. 2019;8(1):a880. https://doi.org/10.4102/ajlm.v8i1.880 bolaris ma, keller ma, robbins bl, podany at, fletcher cv. nevirapine plasma concentrations in human immunodeficiency virus-exposed neonates receiving high-dose nevirapine prophylaxis as part of 3-drug regimen. j pediatric infect dis soc. 2017 mar 1;6(1):102–104. https://doi.org/10.1093/jpids/piv084 pav jw, rowland ls, korpalski dj. hplc-uv method for the quantitation of nevirapine in biological matrices following solid phase extraction. j pharm biomed anal. 1999;20(1–2):91–98. https://doi.org/10.1016/s0731-7085(98)00312-4 fan-havard p, liu z, chou m, et al. pharmacokinetics of phase i nevirapine metabolites following a single dose and at steady state. antimicrob agents chemother. 2013 may;57(5):2154–2160. https://doi.org/10.1128/aac.02294-12 swindells s, ramchandani r, gupta a, et al. one month of rifapentine plus isoniazid to prevent hiv-related tuberculosis. n engl j med. 2019;380:1001–1011. httrps://doi.org/10.1056/nejmoa1806808 united states food and drug administration. guidance for industry: bioanalytical method validation. rockville, md: fda; 2001. nevirapine pk fact sheet [homepage on the internet]. 2016 [cited 2020 mar 19]. https://liverpool-hiv-hep.s3.amazonaws.com/prescribing_resources/pdfs/000/000/059/original/hiv_factsheet_nvp_2016_mar.pdf?1520612265 clinical pharmacology quality assurance, proficiency testing unit. 2019. blind nevirapine comparison report [not published]. article information authors: mohammed abdel-maksoud1 rania abdel-khalek2 atef el-gendy2 rawia f. gamal3 hemmat m. abdelhady3 brent l. house1 affiliations: 1global disease detection and response program, us naval medical research unit, egypt 2bacterial and parasitic disease research program, us naval medical research unit, egypt 3faculty of agriculture, ain shams university, department of microbiology, egypt correspondence to: mohammed abdel-maksoud email: mohamed.abdelmaksoud.eg@med.navy.mil postal address: us naval medical research unit number 3, commanding officer, psc 452, box 5000, code 304 fpo ae 09835-0007 dates: received: 09 dec. 2013 accepted: 02 oct. 2014 published: 14 may 2015 how to cite this article: abdel-maksoud m, abdel-khalek r, el-gendy a, gamal rf, abdelhady hm, house bl. genetic characterisation of multidrug-resistant salmonella enterica serotypes isolated from poultry in cairo, egypt. afr j lab med. 2015;4(1), art. #158, 7 pages. http://dx.doi.org/10.4102/ajlm.v4i1.158 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. genetic characterisation of multidrug-resistant salmonella enterica serotypes isolated from poultry in cairo, egypt in this original research... open access • abstract • introduction • research methods and design    • isolation and identification of salmonella from poultry meat, egg and faecal samples    • serogrouping and serotyping of salmonella isolates    • antibiotic susceptibility testing and detection of extended-spectrum β-lactamase production    • detection of antimicrobial resistance genes    • detection and characterisation of integrons    • statistical analysis • results • discussion • limitations • conclusion • acknowledgements    • competing interests    • disclaimer    • copyright assignment statement    • authors’ contributions • references abstract top ↑ background: food-borne diseases pose serious health problems, affecting public health and economic development worldwide. methods: salmonella was isolated from samples of chicken parts, skin samples of whole chicken carcasses, raw egg yolks, eggshells and chicken faeces. resulting isolates were characterised by serogrouping, serotyping, antimicrobial susceptibility testing and detection of extended-spectrum β-lactamase (esbl) production. antibiotic resistance genes and integrons were identified by polymerase chain reaction (pcr). results: the detection rates of salmonella were 60%, 64% and 62% in chicken parts, skin, and faeces, respectively, whereas the egg yolks and eggshells were uniformly negative. salmonella kentucky and s. enteritidis serotypes comprised 43.6% and 2.6% of the isolates, respectively, whilst s. typhimurium was absent. variable resistance rates were observed against 16 antibiotics; 97% were resistant to sulfamethoxazole, 96% to nalidixic acid and tetracycline and 76% to ampicillin. multidrug resistance was detected in 82% (64/78) of the isolates and esbl production was detected in 8% (6/78). the β-lactamase blatem-1 gene was detected in 57.6% and blashv-1 in 6.8% of the isolates, whilst the blaoxa gene was absent. the sul1 gene was detected in 97.3% and the sul2 gene in 5.3% of the isolates. sixty-four of the 78 isolates (82%) were positive for the integrase gene (int i) from class 1 integrons, whilst int ii was absent. conclusion: this study reveals the presence of an alarming number of multidrug-resistant salmonella isolates in the local poultry markets in cairo. the high levels of drug resistance suggest an emerging problem that could impact negatively on efforts to prevent and treat poultry and poultry-transmitted human diseases in egypt. introduction top ↑ food-borne diseases caused by non-typhoid salmonella present an important public health problem that impacts significantly on the economy in many parts of the world. the main source of infection is food of animal origin, such as poultry, eggs, milk, beef and pork. in addition, fruits and vegetables have been implicated as vehicles for salmonella transmission.1 antibiotics are used extensively to prevent or treat microbial infections in veterinary medicine. microbial infections may be detected at various levels in animal products and disseminated into the environment when manure is applied to fields.2 in the last two decades, antimicrobial resistance has emerged quickly amongst salmonella isolates, creating a serious health hazard worldwide.1 although fluoroquinolones have recently been used as the drug of choice to treat gastrointestinal infections in humans, resistant strains have since emerged and have been associated with increased illness and death.3 whereas fluoroquinolones are contraindicated because of toxicity, cephalosporins have also been used to treat salmonellosis, particularly in children. however, resistance to this class of drugs appeared in 1992, mainly because of the emergence of extended-spectrum β-lactamases (esbls).4 esbls comprise rapidly evolving groups of β-lactamases, capable of hydrolysing (and thus inactivating) third-generation cephalosporins and aztreonam, which are inhibited by the β-lactamase inhibitor, clavulanic acid.5 the esbls are encoded frequently by genes located on r-plasmids, which often carry additional genes encoding resistance to other drug classes (e.g. fluoroquinolones and aminoglycosides).6 esbl-producing strains of bacteria are emerging worldwide, particularly amongst the enterobacteriaceae7 where the exchange of multidrug-resistant (mdr) plasmids between members of the family is common. this mdr can lead to severe limitations in treatment options for infection with these microorganisms, which are responsible for nearly half of all human infections.8 the presence of integron gene sequences has been identified as a primary method by which bacteria can acquire existing antimicrobial resistance genes. each integron sequence is unique in that it acts as a site-specific recombination system capable of capturing or excising novel genetic elements called ‘gene cassettes’. these gene cassettes are promotorless genes with a recombination site known as a 59-base element or attc located at the 3′ end of the gene. the gene cassettes code for a wide range of antimicrobial resistant determinants.9 only class 1 and 2 integrons have been detected in salmonella, with class 1 being the most predominant.10 in egypt, antibiotic resistance has been reported amongst human salmonella isolates, including salmonella enterica serovar typhi (s. typhi) and other diarrhoeagenic strains.11,12 however, with no national salmonella surveillance centre to provide reliable statistical data, little is known about food-borne salmonellosis in egypt. the present study was undertaken to determine the contamination rates of different salmonella serotypes in chicken eggs, raw chicken meat and related environmental samples in poultry markets in cairo, egypt; and to characterise the identified isolates by serotype, antimicrobial susceptibility testing (ast) profiles and esbl production. we also examined selected isolates to identify the presence of common antibiotic-resistance genes and integrons. research methods and design top ↑ isolation and identification of salmonella from poultry meat, egg and faecal samples a total of 165 samples were collected between december 2011 and may 2012 from 18 poultry markets (mostly street markets and retail shops that sell meat and live birds) distributed throughout 5 geographical locations in cairo governorate. the food samples were collected from 62 chicken meat parts (20 boneless breasts, 19 cloacae, 10 livers, 8 gizzards and 5 wings), 22 skin pieces from slaughtered birds, 30 raw egg yolks and shells from another 30 eggs. the faecal samples were collected from 21 separate chicken faeces specimens. the chicken parts and carcass samples were taken from different birds; all samples were cultured within 2 hours. the faecal samples were obtained from the same 18 poultry markets. salmonella strains were isolated and identified according to standard methods,13 and colonies that exhibited typical biochemical reactions were further confirmed as salmonella using the api 20e identification kit (biomerieux, craponne, france). serogrouping and serotyping of salmonella isolates biochemically identified salmonella isolates were serogrouped initially by slide agglutination using commercially-available salmonella o antiserum (difco laboratories, detroit, mi, united states). because of funding limitations, only serogroup b, c2 and d isolates were then serotyped for s. typhimurium, s. enteritidis and s. kentucky according to the kauffman white scheme,14 using salmonella h antisera (sifin, germany; statens, denmark). antibiotic susceptibility testing and detection of extended-spectrum β-lactamase production antimicrobial susceptibilities to tetracyclines (tetracycline), sulphonamides (sulfamethoxazole trimethoprim/sulphamethoxazole), quinolones (nalidixic acid), penicillins (ampicillin), penicillin/β -lactamase inhibitor combinations (ticarcillin/clavulanate, ampicillin/sulbactam), phenicols (chloramphenicol), fluoroqinolones (ciprofloxacin), aminoglycosides (streptomycin, gentamicin, amikacin), monobactams (aztreonam), cephalosporins (cefotaxime, ceftriaxone, ceftazidime, cefepime) and carbapenems (imipenem) were determined using kirby-bauer disc diffusion according to clinical and laboratory standards institute (clsi)15 guidelines. in addition, minimum inhibitory concentration (mic) was determined using e-test methods (ab biodisk, solana, sweden), also according to clsi guidelines. mdr salmonella was defined as any isolate that showed resistance to at least three different classes of antibiotics.11 screening for esbl production in salmonella isolates was done using the standard procedure of measuring the zones of inhibition surrounding cefotaxime and ceftazidime discs versus cefotaxime-clavulanic acid and ceftazidime-clavulanic acid discs, respectively. any isolate with a ≥ 5 mm increase in zone diameter for either antibiotic tested in combination with clavulanic acid, versus without clavulanic acid, was considered to be an esbl producer.15 the reference strains escherichia coli atcc 25922 and staphylococcus aureus atcc 25923 were used to verify the quality and accuracy of testing procedures. detection of antimicrobial resistance genes salmonella-isolate dna was purified using the dna-boiling method suggested by sambrook, fritsch and maniatis.16. the following genes implicated in antimicrobial resistance were detected by pcr amplification: for β-lactam resistance – blatem-1, blashv-1 and blaoxa-1; for sulphonamide resistance – sul1 and sul2. the primer sets and assay conditions used for amplification were as described previously.17,18 detection and characterisation of integrons the presence of class 1 and 2 integrase-coding genes (int i and int ii) were detected by pcr with specific primers.17,19 primers 5’-conserved segment (cs) and 3’-cs described by levesque,20 targeting the inserted gene cassette regions of class 1 integrons, were used to determine these regions. statistical analysis for statistical analyses to detect significant differences between antibiotic resistance rates, p values were determined by using student's t-test in microsoft® office excel 2010 (microsoft corp., redmond, wa, united states). results top ↑ salmonella isolates were recovered from 64 of the 165 samples collected, including 60% of chicken meat samples, 64% of chicken carcasses (skin) samples and 62% of chicken faeces samples. no salmonella was isolated from raw egg yolk or eggshell samples. serogroups identified amongst the salmonella isolates were b, c1, c2, and d. fifty-one samples yielded isolates from one serogroup and 12 samples yielded isolates from 2 serogroups, whilst one sample from skin yielded 3 serogroups. of the 78 salmonella isolates obtained, group c2 was the predominant group found in chicken meat (table 1). serotypes identified included s. kentucky (43.6%) and s. enteritidis (2.6%) (table 2). s. typhimurium was not identified. table 1: distribution of salmonella serogroups isolated from 144 poultry samples and 21 faecal samples collected between december 2011 and may 2012. table 2: distribution of serotypes for the 78 salmonella isolates recovered from poultry and faecal samples collected between december 2011 and may 2012. overall, there was a high level of antibiotic resistance found amongst the salmonella isolates (table 3). resistance was detected to 16 out of 18 antibiotics tested, whilst all salmonella isolates were susceptible to imipenem and cefepime. isolates demonstrated high levels of resistance to sulfamethoxazole (97%), nalidixic acid and tetracycline (96%), ampicillin (76%), ticarcillin/clavulanate (67%), chloramphenicol (56%) and ciprofloxacin (46%). multidrug resistance was observed amongst 82% (64/78) of the isolates, with 59 isolates (76%) resistant to more than 5 antibiotics. esbl production was detected in 8% (6/78), with all 6 being highly resistant to multiple antibiotics when compared to non-esbl producing strains. when compared with other salmonella serotypes, the s. kentucky isolates showed higher resistance rates to the majority of antibiotics tested, reaching statistical significance against ciprofloxacin and ticarcillin/clavulanate (p < 0.01). sixteen (46%) s. kentucky strains were resistant to at least 8 antibiotics. table 3: percent antibiotic resistance and mic range of s. kentucky, other salmonella serotypes and esbl-producing salmonella isolates from poultry meat and faecal samples. amongst the 75 strains resistant to nalidixic acid, 36 were resistant to the related ciprofloxacin (mic range of 4–12 μg/ml). imipenem showed the lowest mic values, followed by cefepime and amikacin, all of which were found to be effective against salmonella strains isolated from poultry in cairo. on the other hand, the highest mic values were obtained against streptomycin, followed by nalidixic acid and ampicillin (table 3). table 4 lists the resistance genes detected in the salmonella isolates. the most frequent β-lactam gene identified amongst ampicillin resistant isolates was blatem-1, detected in 57.6% of the isolates, followed by blashv-1 which was identified in 6.8%. the blaoxa-1 gene was not detected in any isolate in this study. regarding sulphonamide resistance genes, the presence of the sul1 gene was detected in 97.3% of the isolates and the sul2 gene in 5.3%. four isolates possessed both the sul1 and sul2 genes (table 4). table 4: antimicrobial resistance genes and the resistance phenotype of s. enterica strains isolated from poultry meat and faecal samples (n = 78). sixty-four of the 78 isolates (82%) were positive for the int i gene, whilst int ii was absent. the 5′and 3′-cs regions were identified in 46.8% of the int i positive isolates. eight types of class 1 integrons were detected for the salmonella spp. isolates, including the 1950 bp, 1550 bp, 1200 bp, 1100 bp, 1000 bp, 700 bp, a combination of 950 bp and 1200 bp and a combination of 1100 bp and 1550 bp integrons. five s. kentucky isolates resistant to ciprofloxacin carried the 1950 bp class 1 integron (table 5). table 5: characteristics of class 1 integron-carrying multidrug-resistant s. enterica strains isolated from poultry meat and faecal samples (n = 64). discussion top ↑ the objectives of this study were to determine the frequency of salmonella contamination of chicken eggs, meat and faeces in poultry markets in cairo, egypt and to identify prevalent serotypes and antibiotic susceptibility profiles, including esbl production. the results from this study revealed levels of salmonella-contamination in fresh chicken meat of 60% – 64%, which are higher than those previously reported in assiut, egypt from frozen chicken legs and fillet samples (36% – 52%);21 in senegal from chicken carcasses (32%);22 and in ethiopia from raw chicken meat and giblets (18%).23 in contrast to a study conducted by del cerro et al.24 which reported that faeces from chickens were positive for salmonella by culture in 39% of the samples tested, our study demonstrated a 62% positivity rate for salmonella isolation from faecal specimens. the high contamination rates seen in this study may, at least in part, be explained by the lack of hygienic slaughtering processes that occur commonly at small shops, away from the modern abattoirs available for mass slaughtering of poultry. at these shops, slaughtering is manual, rudimentary and may take place either indoors or outdoors. usually, one person provides all the labour, including live bird care, cleaning, slaughtering, de-feathering and evisceration, increasing the likelihood of cross-contamination amongst birds. in this study, serogroups b, c1 and c2 accounted for 97% of the isolates from chicken meat. a similar study performed in the pacific northwest, in the united states25 also found that serogroups b and c comprised the majority (95%) of all salmonella isolated from poultry and the poultry environment. a study in saudi arabia26 reported that 64% of the isolates from poultry and the poultry environment were from groups b and c. in the current study, there were only two isolates assigned to serogroup d, subsequently serotyped as s. enteritidis. in humans, s. enteritidis and s. typhimurium have been reported to be the two most prevalent salmonella serotypes in many regions of the world.26 in addition, a study in turkey demonstrated that s. enteritidis was the most prevalent salmonella serotype isolated from chicken meat.27 however, a study in senegal identified only 6 s. enteritidis serotypes out of 90 salmonella strains isolated.28 another study in sudan identified 2 chicken and 2 human origin s. kentucky strains resistant to both ciprofloxacin and norfloxacin out of 64 salmonella isolates studied.29 interestingly, studies in france,30 switzerland31 and the slovak republic32 have reported that infection with s. kentucky strains resistant to ciprofloxacin was associated with travel to egypt, morocco and other countries in the north african region. corroborating these studies, our results indicate that the most prevalent serotype in cairo, egypt is s. kentucky, with high rates of resistance to ciprofloxacin. s. enteritidis was isolated at a very low rate, and s. typhimurium was not detected at all. this is comparable to the results from senegal,27,29 where s. kentucky was also found to be the most prevalent serotype (30% of the total isolates). the observed low isolation rates for s. enteritidis and s. typhimurium in this study may result from replacement by other serotypes (54% were not serotyped in this study). the remarkably high rates of antibiotic resistance exhibited by salmonella strains from this study, particularly against sulfamethoxazole (97.4%), nalidixic acid (96.2%), tetracycline (96.2%), ampicillin (75.6%) and streptomycin (35.9%), are probably because of the early introduction and consequent widespread use of these antibiotics in veterinary and human medicine in egypt. the high resistance rates to nalidixic acid and ciprofloxacin are of particular note, since quinolones have been considered one of the last options for the treatment of mdr salmonella. all s. kentucky strains tested in this study were mdr and demonstrated significantly higher rates of resistance than other salmonella serotypes to ciprofloxacin (97% vs. 2.5%; p < 0.01) and ticarcillin/clavulanate (94% vs. 42.5%; p < 0.01). the correlation between antibiogram and serotype suggests poor infection control practices in the poultry production industry, which may facilitate the spread of these mdr s. kentucky strains. the most common mode of bacterial-acquired resistance to β-lactam antibiotics is the β -lactamase enzyme.34 in this study, 30 of 48 poultry meat isolates and 4 of 11 faeces isolates that were ampicillin resistant possessed the blatem gene. this result is in agreement with other findings.1,17 recently, esbl acquisition rates by salmonella, in particular, have arisen worldwide. in an earlier study conducted on salmonella isolates from poultry in egypt, 5% of the isolates belonging to serovar poona produced esbls.12 in this study 8% (n = 6) of salmonella isolates demonstrated esbl production, two of them belonging to serovar kentucky. the detection of an esbl phenotype in poultry meat and faecal samples in this study may indicate a lack of infectious disease barriers amongst clinics, humans and animals. in addition, approximately half of all antibiotics produced worldwide (many of which are used routinely in humans) are used in animals to prevent infection and consequently improve production.35 unfortunately, this leads to the development of resistant bacteria in animals that can infect humans directly or transfer antibiotic resistance genes to other human pathogens.36 sulphonamides are amongst the most commonly-used antibiotics for food animal production worldwide.37 these compounds are bacteriostatic antimicrobial drugs that act by means of competitive inhibition of the enzymes involved in the synthesis of tetrahydrofolic acid. sulphonamides compete with the structural analogue p-aminobenzoic acid binding to dihydropteroate synthetase (dhps), a catalytic enzyme in the folic acid biosynthesis pathway, thus inhibiting the formation of dihydrofolic acid.38 sulphonamide resistance in salmonella isolates has been attributed to the presence of an extra sul gene, which expresses an insensitive form of dhps. in this study, the presence of the sul1 gene was detected in 74 of 76 sulphonamide-resistant isolates. the pcr results were consistent with the antimicrobial susceptibility phenotypes; the sul1 and/or sul2 genes were detected in 97.4% of the sulphonamide-resistant salmonella isolates. other studies have found this gene to be present at moderate to high rates in retail meats and other foods.1,17,39 integrons are genetic elements that are able to recognise and capture mobile gene cassettes carrying the antibiotic resistance genes, which leads to mdr distribution and the subsequent limitation of treatment options for infectious diseases.40 in this study, pcr screening results of 78 salmonella isolates detected class 1 integrons in 62 (79.5%) isolates. very few studies have investigated class 1 integrons in salmonella isolates from human, poultry and faeces isolates in egypt. the presence of integrons was examined in 21 salmonella isolates from diseased broiler chickens in egypt where the researchers identified class 1 and class 2 integrons in 42.9% and 14.3% of the isolates, respectively.41 lower detection rates were obtained in a study of food isolates in germany (65%).1 a study by antunes, machad and peixe42 showed, in a large survey of 1183 salmonella isolates from various animal, human and food sources, that 75% carried class 1 integrons. class 1 integrons have been detected in s. kentucky.43 in our study, 5 isolates of s. kentucky carried the 1950 bp class 1 integron. multidrug resistance was observed amongst 82% (64/78) of the isolates, with 59 isolates (76%) being resistant to more than 5 antibiotics. our study indicates that 88.1% of the 59 mdr isolates harboured class 1 integrons, whilst none of the mdr isolates carried class 2 integrons. several groups have reported that integron-containing isolates are more antibiotic resistant than those isolates obtained from comparable patients which were lacking an integron.44 limitations top ↑ samples included in this study were collected from the cairo governorate during the six-month period from december 2011 to may 2012. thus, the results of this study may not be generalisable to other regions or seasons. more studies are needed on samples collected from the nile delta and upper egypt governorates and during different seasons. conclusion top ↑ in conclusion, this study demonstrated a relatively high prevalence of salmonella-contaminated poultry products, with s. kentucky the most prevalent serotype, in poultry markets in cairo, egypt. in addition, this study revealed significant mdr rates, particularly carried by s. kentucky serovar strains, against the β-lactam and fluoroquinolone (e.g., ciprofloxacin) classes of antibiotics. ultimately, these trends may limit treatment options and contribute to treatment failure and increased death rates. more comprehensive studies are needed to better determine the prevalence and antibiotic resistance patterns of salmonella-contaminated poultry meat and its products. more serotypes should be utilised in identification and be included in a national surveillance database to allow comparisons with findings within egypt and from other countries in the region. this surveillance should include antimicrobial susceptibility profiles to track the emergence and exacerbation of existing drug resistance amongst salmonella and other food-borne disease pathogens. acknowledgements top ↑ competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. disclaimer the views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the us department of the navy, the us department of defense, the us government or the egyptian ministry of health and population. copyright assignment statement the authors are employees of the us government. this work was prepared as part of their official duties. title 17 u.s.c. §105 provides that ‘copyright protection under this title is not available for any work of the united states government.’ title 17 u.s.c. §101 defines a us government work as a work prepared by a military service member or employee of the us government as part of that person's official duties. authors’ contributions m.a-m. (global disease detection and response program, us naval medical research unit) developed the concept, processed the samples and participated in writing the manuscript. b.l.h. (global disease detection and response program, us naval medical research unit), r.f.g. (ain shams university) and h.m.a. (ain shams university) developed the concept, analysed the results, wrote and reviewed the manuscript. r.a-k. and a.e.-g. (both bacterial and parasitic disease research program, us naval medical research unit) participated and supervised the molecular biology work and helped in analysing the results and writing the manuscript. references top ↑ miko a, pries k, schroeter a, et al. molecular mechanisms of resistance in multidrug-resistant serovars of salmonella enterica isolated from foods in germany. j antimicrob chemother. 2005;56(6):1025–1033. http://dx.doi.org/10.1093/jac/dki365 kümmerer k. resistance in the environment. j antimicrob chemother. 2004;54(2):311–320. http://dx.doi.org/10.1093/jac/dkh325 helms m, simonsen j, mølbak k. quinolone resistance is associated with increased risk of invasive illness or death during infection with salmonella serotype typhimurium. j infect dis. 2004;190(9):1652–1654. http://dx.doi.org/10.1086/424570 aarestrup fm, hasman h, olsen i, et al. international spread of blacmy-2mediated cephalosporin resistance in a multiresistant salmonella enterica serovar heidelberg isolate stemming from the importation of a boar by denmark from canada. antimicrob agents chemother. 2004;48(5):1916–1917. http://dx.doi.org/10.1128/aac.48.5.1916-1917.2004 paterson dl, bonomo ra. extended-spectrum β-lactamases: a clinical update. clin microbiol rev. 2005;18(4):657–686. http://dx.doi.org/10.1128/cmr.18.4.657-686.2005 siu lk, lo jyc, yuen ky, et al. β-lactamases in shigella flexneri isolates from hong kong and shanghai and a novel oxa-1-like β-lactamase, oxa-30. antimicrob agents chemother. 2000;44(8):2034–2038. http://dx.doi.org/10.1128/aac.44.8.2034-2038.2000 bouchillon sk, johnson bm, hoban dj, et al. determining incidence of extended spectrum beta-lacamase producing enterobacteriaceae, vancomycin-resistant enterococcus faecium and methicillin-resistant staphylococcus aureus in 38 centres from 17 countries: the pearls study 2001–2002. int j antimicrob agents. 2004;24(2):119–124. http://dx.doi.org/10.1016/j.ijantimicag.2004.01.010 fluit ac. towards more virulent and antibiotic-resistant salmonella? fems immunol med microbiol. 2005;43(1):1–11. http://dx.doi.org/10.1016/j.femsim.2004.10.007 recchia gd, hall rm. gene cassettes: a new class of mobile element. microbiology. 1995;141(pt 12):3015–3027. http://dx.doi.org/10.1099/13500872-141-12-3015 rodriguez i, rodicio mr, herrera-león s, et al. class 1 integrons in multidrug-resistant non-typhoidal salmonella enterica isolated in spain between 2002 and 2004. int j antimicrob agents. 2008;32(2):158–164. http://dx.doi.org/10.1016/j.ijantimicag.2008.03.005 wasfy mo, frenck r, ismail tf, et al. trends of multiple-drug resistance among salmonella serotype typhi isolates during a 14-year period in egypt. clin infect dis. 2002;35(10):1265–1268. http://dx.doi.org/10.1086/343052 aouf a, messai y, salama ms, et al. resistance to β-lactams of human and veterinary salmonella isolates in egypt and algeria. afr j microbiol res. 2011;5(7):802–808. international organization for standardization (iso). iso 6579:2002 microbiology of food and animal feeding stuffs – shorizontal method for the detection of salmonella spp. geneva, switzerland: iso; 2002. popoff my. antigenic formulas of the salmonella serovars, 8th ed. paris, france: who collaborating center for reference and research on salmonella, institute pasteur; 2001. clinical and laboratory standards institute. performance standards for antimicrobial susceptibility testing: twentieth informational supplement (m 100-s20). wayne, pa: clsi; 2010. sambrook j, fritsch ef, maniatis t. molecular cloning: a laboratory manual. 2nd ed. cold spring harbor, ny: cold spring harbor laboratory press; 1989. chen s, zhao s, white dg, et al. characterization of multiple-antimicrobial-resistant salmonella serovars isolated from retail meats. appl environ microbiol. 2004;70(1):1–7. http://dx.doi.org/10.1128/aem.70.1.1-7.2004 kerrn mb, klemmensen t, frimodt-møller n, et al. susceptibility of danish escherichia coli strains isolated from urinary tract infections and bacteraemia, and distribution of sul genes conferring sulphonamide resistance. j antimicrob chemother. 2002;50(4):513–516. http://dx.doi.org/10.1093/jac/dkf164 goldstein c, lee md, sanchez s, et al. incidence of class 1 and 2 integrases in clinical and commensal bacteria from livestock, companion animals, and exotics. antimicrob agents chemother. 2001;45(3):723–726. http://dx.doi.org/10.1128/aac.45.3.723-726.2001 lévesque c, piché l, larose c, et al. pcr mapping of integrons reveals several novel combinations of resistance genes. antimicrob agents chemother. 1995; 39(1):185–191. http://dx.doi.org/10.1128/aac.39.1.185 hassanein r, hassan ali sf, abd el-malek am, et al. detection and identification of salmonella species in minced beef and chicken meats by using multiplex pcr in assiut city. vet world. 2011;4(1):5–11. http://dx.doi.org/10.5455/vetworld.2011.5-11 cardinale e, perrier gros-claude jd, tall f, et al. prevalence of salmonella and campylobacter in retail chicken carcasses in senegal. revue élev méd vét pays trop. 2003;56(1–2):13–16. tibaijuka b, molla b, hildebrandt g, et al. antimicrobial susceptibilities of salmonella isolated from retail raw chicken meat and giblets in ethiopia. bull anim hlth prod afr. 2002;50(2):86–95. del cerro, a, soto sm, landeras e, et al. pcr-based procedures in detection and dna-fingerprinting of salmonella from samples of animal origin. food microbiology. 2002;19(6):567–575. http://dx.doi.org/10.1006/fmic.2002.0512 roy p, dhillon, as, lauerman lh, et al. results of salmonella isolation from poultry products, poultry, poultry environment, and other characteristics. avian dis. 2002;46(1):17–24. http://dx.doi.org/10.1637/0005-2086(2002)046[0017:rosifp]2.0.co;2 al-nakhli hm, al-ogaily zh, nassar tj. representative salmonella serovars isolated from poultry and poultry environments in saudi arabia. rev sci tech. 1999;18(3):700–709. hendriksen rss, vieira ar, karlsmose s, et al. global monitoring of salmonella serovar distribution from the world health organization global foodborne infections network country data bank: results of quality assured laboratories from 2001 to 2007. foodborne pathog dis. 2011;8(8):887–900. http://dx.doi.org/10.1089/fpd.2010.0787 goncagül g, günaydın e, carlı kt. prevalence of salmonella serogroups in chicken meat. turk j vet anim sci. 2005;29:103–106. bada-alambedji r, fofana a, seydi m, et al. antimicrobial resistance of salmonella isolated from poultry carcasses in dakar (senegal). braz j microbiol. 2006;37(4):510–515. http://dx.doi.org/10.1590/s1517-83822006000400020 elmadiena mm, el hussein aa, muckle ca, et al. antimicrobial susceptibility and multi-drug resistance of salmonella enterica subspecies enterica serovars in sudan. trop anim health prod. 2013;45(5):1113–1118. http://dx.doi.org/10.1007/s11250-012-0334-7 le hello s, hendriksen rs, doublet b, et al. international spread of an epidemic population of salmonella enterica serotype kentucky st198 resistant to ciprofloxacin. j infect dis. 2011;204(5):675–684. http://dx.doi.org/10.1093/infdis/jir409 bonalli m, stephan r, käppeli u, et al. salmonella enterica serotype kentucky associated with human infections in switzerland: genotype and resistance trends 2004–2009. food res int. 2011;45(2):953–957. http://dx.doi.org/10.1016/j.foodres.2011.04.051 majtán v, matján t, matján j, et al. salmonella enterica serovar kentucky: antimicrobial resistance and molecular analysis of clinical isolates from the slovak republic. jpn j infect dis. 2006;59(6):358–362. livermore dm, canton r, gniadkowski m, et al. ctx-m: changing the face of esbls in europe. j antimicrob chemother. 2007;59(2):165–174. http://dx.doi.org/10.1093/jac/dkl483 greenwood d, finch r, davey p, et al. antimicrobial chemotherapy. new york: oxford university press; 2007. stürenburg e, mack d. extended spectrum β-lactamases: implications for the clinical microbiology laboratory, therapy, and infection control. j infect dis. 2003;47(4):273–295. pezzella c, ricci a, digiannatale, e. et al. tetracycline andstreptomycin resistance genes, transposons, and plasmids in salmonella enterica isolates from animals in italy. antimicrob agents chemother. 2004;48(3):903–908. http://dx.doi.org/10.1128/aac.48.3.903-908.2004 sköld o. sulfonamide resistance: mechanisms and trends. drug resist updat. 2000;3(3):155–160. http://dx.doi.org/10.1054/drup.2000.0146 peirano g, agersø y, aarestrup fm, et al. occurrence of integrons and antimicrobial resistance genes among salmonella enterica from brazil. j antimicrob chemother. 2006;58(2):305–309. http://dx.doi.org/10.1093/jac/dkl248 stokes hw, hall rm. a novel family of potentially mobile dna elements encoding site-specific gene-integration functions: integrons. mol microbiol. 1989;3(12):1669–1683. http://dx.doi.org/10.1111/j.1365-2958.1989.tb00153.x ahmed am, shimamoto t. genetic analysis of multiple antimicrobial resistance in salmonella isolated from diseased broilers in egypt. microbiol immunol. 2012;56(4):254–261. http://dx.doi.org/10.1111/j.1348-0421.2012.00429.x antunes p, machado j, and peixe l. characterization of antimicrobial resistance and class 1 and 2 integrons in salmonella enterica isolates from different sources in portugal. j antimicrob chemother. 2006;58:297–304. http://dx.doi.org/10.1093/jac/dkl242 levings rs, partridge sr, lightfoot d, et al. new integron-associated gene cassette encoding a 3-n-aminoglycoside acetyltransferase. antimicrob agents chemother. 2005;49(3):1238–1241. http://dx.doi.org/10.1128/aac.49.3.1238-1241.2005 fluit ac, schmitz fj. resistance integrons and super integrons. clin microbiol infect. 2004;10(4):272–288. http://dx.doi.org/10.1111/j.1198-743x.2004.00858.x abstract introduction methods results discussion acknowledgements references about the author(s) adon chawe laboratory department, st. francis mission hospital, katete, zambia ruth l. mfune department of clinical sciences, faculty of medicine, michael chilufya school of medicine, copperbelt university, ndola, zambia paul m. syapiila department of clinical sciences, faculty of medicine, michael chilufya school of medicine, copperbelt university, ndola, zambia sharon d. zimba department of clinical sciences, faculty of medicine, chikankata college of biomedical sciences, chikankata, zambia pipina a. vlahakis department of basic science, faculty of medicine, michael chilufya school of medicine, copperbelt university, ndola, zambia samson mwale department of biomedical sciences, tropical diseases research centre, ndola, zambia kapambwe mwape department of clinical sciences, faculty of medicine, michael chilufya school of medicine, copperbelt university, ndola, zambia memory chirambo-kalolekesha department of clinical sciences, faculty of medicine, michael chilufya school of medicine, copperbelt university, ndola, zambia misheck chileshe laboratory department, mary begg health services, ndola, zambia joseph mutale laboratory department, kabompo district hospital, kabompo, zambia tobela mudenda department of pathology, ndola teaching hospital, ndola, zambia grace manda laboratory department, kalomo district hospital, kalomo, zambia victor daka department of clinical sciences, faculty of medicine, michael chilufya school of medicine, copperbelt university, ndola, zambia citation chawe a, mfune rl, syapiila pm, et al. knowledge, attitude and practices of covid-19 among medical laboratory professionals in zambia. afr j lab med. 2021;10(1), a1403. https://doi.org/10.4102/ajlm.v10i1.1403 original research knowledge, attitude and practices of covid-19 among medical laboratory professionals in zambia adon chawe, ruth l. mfune, paul m. syapiila, sharon d. zimba, pipina a. vlahakis, samson mwale, kapambwe mwape, memory chirambo-kalolekesha, misheck chileshe, joseph mutale, tobela mudenda, grace manda, victor daka received: 21 sept. 2020; accepted: 06 jan. 2021; published: 04 mar. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: coronavirus disease 2019 (covid-19) is a novel disease that has spread to nearly every country worldwide. medical laboratory professionals are key in the fight against covid-19 as they provide confirmatory diagnosis for subsequent management and mitigation of the disease. objective: this study investigated the knowledge, attitude and practices of covid-19 and their predictors among medical laboratory personnel in zambia. methods: we conducted a cross-sectional study among medical laboratory professionals in zambia from 10 to 29 june 2020. data were collected using google forms and exported to statistical package for social sciences version 23 for statistical analysis. independent predictors of covid-19 knowledge and practices were determined. adjusted odds ratios (aor) and their 95% confidence intervals (ci) are reported. results: a total of 208 medical laboratory professionals, 58.2% male, participated in the study. the majority of respondents had good knowledge (84.1%) and practice (75.0%) regarding covid-19. predictors of good knowledge included having a bachelor’s degree (aor: 5.0, ci: 1.13–22.19) and having prior covid-19 related training (aor: 8.83, ci: 2.03–38.44). predictors of good practice included having a master’s or doctor of philosophy (phd) qualification (aor: 5.23, ci: 1.15–23.87) and having prior covid-19 related training (aor: 14.01, ci: 6.47–30.36). conclusion: our findings revealed that medical laboratory professionals in zambia have good knowledge regarding covid-19. there is need for continuous professional development to ensure that medical laboratory professionals are well informed and aware of best practices to aid in curbing the pandemic. keywords: covid-19; medical laboratory professional; knowledge; attitude; practices. introduction coronavirus disease 2019 (covid-19) is a respiratory disease caused by severe acute respiratory syndrome coronavirus 2.1 coronaviruses have been known to affect humans, infecting the respiratory tract and causing infections ranging from mild to severe.2 in the past, different strains of coronaviruses have caused severe acute respiratory syndrome and middle east respiratory syndrome. research has shown that covid-19 is more contagious compared to the previous outbreaks, but less lethal.3 transmission occurs through the inhalation of droplets or contact with surfaces that have been contaminated with the severe acute respiratory syndrome coronavirus 2. as of this writing, there is currently no known vaccine or documented specific treatment for covid-19 disease.4 drugs that show potential to treat critically ill patients are still being investigated for safety and efficacy.4,5,6 prevention and control of the spread of covid-19 is done by social distancing, wearing face masks to prevent both the inhalation and transmission of infectious droplets, as well as effective hand hygiene by regularly washing hands or using alcohol-based hand sanitisers.7,8 the first cases of covid-19 were reported in december 2019 in wuhan, hubei province, central china. since then, the disease has spread to nearly every country worldwide leading to the world health organization declaring covid-19 a pandemic on 11 march 2020.9 as of 27 october 2020 there were 44 055 642 confirmed cases and 1 168 306 deaths reported globally, with 1 740 720 confirmed cases and 38 830 deaths reported in africa.10 in zambia, 16 243 confirmed cases and 348 deaths were reported as of 27 october 2020, including a high proportion of community deaths.11,12 few publications and national situation reports exist that detail the number of healthcare workers (hcws) including medical laboratory personnel around the world infected with covid-19.13 in china where the disease first started, 2055 hcws were infected as of 24 march 2020.14 in the united states, 9282 hcws were infected as of 14 april 2020.15 the world health organization estimates that over 1000 hcws in africa had been infected with covid-19 by 23 july 2020.16 information on the source of infection among hcws in countries that have recorded the disease remains limited. medical laboratory professionals are key personnel in the diagnosis of covid-19 in zambia. although they are not in the front line, which precludes them from prominence, their role in providing confirmatory diagnosis is the main basis upon which cases are identified and clinical management is instituted. the work areas of biomedical laboratory professionals are very hazardous due to both suspected and unsuspected infectious agents. lack of knowledge and good attitude, as well as poor laboratory practices, can have a twofold effect. firstly, a wrong diagnosis leading to wrong patient management could portend severe consequences for the patient as well as undermine transmission prevention efforts; this is particularly true with covid-19. secondly, poor attitude and practices could result in safety incidents (such as infection transmission) which could be deleterious to both the concerned staff and their immediate environment, including co-workers, families and patients or laboratory clients.17 therefore, this study aimed at evaluating the knowledge, attitudes and practices of medical laboratory professionals in zambia who are integral in the diagnosis of covid-19. methods ethical considerations ethical clearance for this study was obtained from the tropical diseases research centre institution review board (registration number: 00002911). the questionnaire contained an information sheet regarding the study and an informed consent statement for participants to agree to participate or not. all participants who declined to take part in the study were immediately withdrawn and could not proceed to respond to the questionnaire. access to the online portal for the questionnaire was limited to investigators who were assigned user rights by the principal investigator to ensure confidentiality of collected data. sample size determination we used the methods for calculating the sample size for prevalence studies as described by pourhoseingholi and others.18 assuming an 82% prevalence of good knowledge and practice regarding covid-19 obtained from a similar study among hcws,19 a confidence level of 95% and a precision level of 5%, we obtained a minimum required sample size of 227 participants. by extrapolating to a finite population of 1900 medical laboratory personnel officially recognised under the medical laboratory register of the biomedical society of zambia, we obtained a final sample size of 204 participants. study design this cross-sectional survey was conducted among 208 medical laboratory professionals in zambia. due to covid-19 related restrictions imposed during this period, it was not feasible to conduct face-to-face interviews and therefore we administered an online questionnaire using google forms (alphabet, inc., mountain view, california, united states). data collection content validation of the questionnaire was done by administering the questionnaire to faculty in the biomedical sciences unit at the copperbelt university, ndola, zambia. the results from the pre-test were not included in the final analysis but were used to modify the questionnaire based on feedback. the questionnaire had two main components: demographics and knowledge, attitudes and practices. the demographic section had 8 questions while the knowledge, attitudes and practices section was divided into the following subsections: knowledge (8 questions), attitude (5 questions) and practices (13 questions). to ensure that only target respondents participated in the survey we distributed the link to the survey through the email database and whatsapp (facebook inc, menlo park, california, united states) group facilitated by the biomedical society of zambia during data collection from 10 to 29 june 2020. a total of 750 participants were availed the link for the questionnaire. there were 210 medical laboratory personnel who participated in the study, giving a response rate of 28%. participants who consented to participate in the study by checking the consent box in the preliminary page of the questionnaire were allowed to proceed with the rest of the questionnaire. only participants who completed the questionnaire by clicking on the ‘submit’ button had their responses recorded. data management and statistical analysis collected data were downloaded and cleaned in microsoft excel (microsoft corp., redmond, washington, united states) and exported to statistical package for social sciences version 23 (ibm corp., armonk, new york, united states) for statistical analysis. to ensure the internal consistency and reliability of the data, we used cronbach’s alpha coefficient according to methods described in a previous study.20 we obtained an alpha value of 0.645, indicating adequate reliability.21 the outcome variables in this study were covid-19 knowledge levels and practice towards covid-19. attitude was investigated but limited to descriptive analysis due to the limited number of questions. eight questions were used to assess covid-19 knowledge, each scoring 1 point for a correct answer. thirteen questions were asked to determine whether a participant had good practice towards covid-19 or not, each scoring 1 point for positive practice and 0 for negative practice. bloom’s cut-off of 80%22 was used to determine good knowledge and good practice. questions were adapted from a previous study.23 to identify factors that predict good knowledge and practice towards covid-19, bivariate analysis was performed. all factors that were statistically significant as reported by 95% confidence interval (ci) in bivariate logistic regression were included in a forward stepwise multivariate logistic regression model to identify factors that were independently associated with good knowledge and practice. results demographic characteristics a total of 208 medical laboratory professionals from seven provinces of zambia took part in this study. there were more men (n = 121, 58.2%) than women. half of the participants were aged 20–29 years while more than half had a diploma. most of the respondents were from a hospital laboratory (n = 134, 64.4%) (table 1). table 1: demographic characteristics of medical laboratory professionals, zambia, june 2020. participants showed good knowledge regarding awareness of practical measures to stop the spread of covid-19 (n = 208, 100.0%), the techniques used to test for covid-19 (n = 208, 100.0%) and symptomatic management of covid-19 (n = 202, 97.1%) (table 2 and table 3). one-quarter (n = 52, 25.0%) of the participants reported being involved in sampling for covid-19 while slightly over a third (n = 73, 35.1%) reported having been trained in sample handing and transportation. table 2: characteristics of responses for knowledge and practice towards covid-19, zambia, june 2020.† table 3: characteristics of responses for knowledge and practice towards covid-19, zambia, june 2020.† factors associated with covid-19 knowledge a high proportion of respondents were knowledgeable about covid-19 (n = 175, 84.1%). bivariate logistic regression analysis showed that having a bachelor’s degree compared to having a certificate or diploma (crude odds ratio (or): 4.68, ci: 1.07–20.44) and covid-19 training (crude or: 8.72, ci: 2.02–37.65) among participants were associated with covid-19 knowledge. therefore, participants with higher academic qualifications and covid-19 training were 4.68 and 8.72 times more likely to have good covid-19 knowledge (table 4). table 4: covid-19 knowledge of participants and associated factors, zambia, june 2020. practice towards covid-19 by medical laboratory personnel poor practices towards covid-19 were recorded among three-quarters of the participants. having a master’s degree or a doctor of philosophy compared to having a certificate or diploma (crude or: 4.51, 95% ci: 1.30–15.70), private or research laboratories compared to clinic or health centre laboratories (crude or: 3.09, 95% ci: 1.01–9.45) and covid-19 training (crude or: 12.97, 95% ci: 6.19–27.18) were associated with good covid-19 practices (table 5). table 5: covid-19 practices of participants and associated factors, zambia, june 2020. attitude towards covid-19 among medical laboratory personnel about 93.8% of participants reported that they would accept isolation from the community if diagnosed with covid-19. a few (46.6%) would accept to be vaccinated against covid-19 if a vaccine was available. on the other hand, many participants (97.6%) were ready to take part in anti-epidemic community activities. our study revealed that 77.9% were confident that zambia could win the battle against the covid-19 virus (table 6). table 6: attitude of biomedical professionals towards coronavirus disease 2019, zambia, june 2020. factors independently associated with good knowledge and practice about covid-19 after controlling for possible confounding factors, having a higher current qualification (bachelor’s degree) and covid-19 training were independently associated with good covid-19 knowledge among biomedical professionals in zambia. similarly, having a higher current qualification (master’s degree or a doctor of philosophy) and covid-19 training were independently associated with good covid-19 practice (table 7). table 7: factors independently associated with good covid-19 knowledge and practice, zambia, june 2020. discussion very few studies worldwide have documented knowledge, attitudes and practices among hcws towards covid-19 due to the novel nature of the disease.22 our study findings showed that the majority of medical laboratory professionals in zambia had good knowledge of covid-19. the level of knowledge on covid-19 among participants was similar irrespective of their gender, age and laboratory facility. these findings are encouraging as they indicate that there are no inherent differences in knowledge of covid-19 among groups based on unique demographic characteristics in the population. the majority of participants exhibited poor practices towards covid-19 contrary to findings from uganda and nepal.19,22 differences in knowledge and practice regarding covid-19 have been reported before in a study by asemahagn.24 most participants with poor practices were those who had certificate qualifications, those without prior covid-19 training and those from clinic and health centre laboratories. this could be attributed to limited resources, health information and laboratory materials in most clinic and health centre laboratories found in rural areas. poor practices can lead to delayed or wrong laboratory diagnosis, leading to poor patient management or safety incidents that could harm the personnel and their immediate co-workers, families and patients or laboratory clients.25 current qualification and covid-19 training among participants were significantly associated with good covid-19 knowledge, a finding similar to that obtained by a study in vietnam,26 but contrary to the findings of bhagavathula and others.27 our findings show that participants with higher academic qualifications and covid-19 training were 4.68 and 8.72 times more likely to have good covid-19 knowledge. the current qualification was also significantly associated with good covid-19 practices which agrees with study findings from uganda.22 on the other hand, the type of laboratory facility and covid-19 training were significantly associated with good covid-19 practices. this shows that participants who received covid-19 training were 12.97 times more likely to have good practices towards covid-19 and general infection prevention; this is in agreement with similar studies in nepal and ethiopia.19,28 limitations the study may be susceptible to self-presentation bias as it was based on an online questionnaire. the study was limited by the level of responses available and could have been strengthened by increasing the range of answers using, for example, a five-point likert scale. the questions on attitude were limited and therefore not powerful enough to generate meaningful conclusions on the attitude of respondents. the study could not capture dropouts and respondents who refused consent as only individuals who consented and submitted the questionnaire had their responses recorded. conclusion our study found that medical laboratory professionals in zambia have good knowledge regarding covid-19. the current qualification and covid-19 training were independently associated with covid-19 knowledge and practice. as cases of covid-19 continue to be recorded in the country, there is need for continuous professional development among medical laboratory personnel as a key intervention in improving their contribution to covid-19 control efforts. acknowledgements the authors are grateful to the biomedical society of zambia and its members for facilitating the distribution of questionnaires to the medical laboratory personnel. competing interests the authors declare that they have no financial or personal relationships that may have influenced the writing of this article. authors’ contributions a.c., r.l.m., g.m. and v.d. conceptualised the study. a.c., s.d.z., p.a.v., s.m., k.m., m.c.-k., j.m. and t.m. developed the data collection tools. a.c., p.m.s., m.c. and v.d. performed the formal analysis and interpretation. a.c., r.l.m., p.a.v., k.m. and v.d. wrote the first draft manuscript. all authors read and approved the final manuscript. sources of support this research was not supported by any external funding. data availability statement the data analysed in this study can be made available on request from the corresponding author. disclaimer the views and opinions expressed in this article are solely of the authors and do not reflect the official policy or position of any affiliated organisation of the authors. references modi pd, nair g, uppe a, et al. covid-19 awareness among healthcare students and professionals in mumbai metropolitan region: a questionnaire-based survey. cureus. 2020;124. https://doi.org/10.7759/cureus.7514 madjid m, safavi-naeini p, solomon sd, vardeny o. potential effects of coronaviruses on the cardiovascular system: a review jama cardiol. 2020;5:831–840. https://doi/org10.1001/jamacardio.2020.1286 zhong bl, luo w, li hm, et al. knowledge, attitudes, and practices towards covid-19 among chinese residents during the rapid rise period of the covid-19 outbreak: a quick online cross-sectional survey. int j biol sci. 2020;16(10):1745–1752. https://doi.org/10.7150/ijbs.45221 jean ss, lee pi, hsueh pr. treatment options for covid-19: the reality and challenges. j microbiol immunol infect [serial online]. 2020 [cited 2020 sep 13];53(3):436–443. https://doi.org/10.1016/j.jmii.2020.03.034 li l, li r, wu z, et al. therapeutic strategies for critically ill patients with covid-19. ann intensive care [serial online]. 2020;10:45. https://doi.org/10.1186/s13613-020-00661-z wu r, wang l, kuo hcd, et al. an update on current therapeutic drugs treating covid-19. curr pharmacol rep. 2020;6:56–70. https://doi.org/10.1007/s40495-020-00216-7 peng y, pei c, zheng y, et al. knowledge, attitude and practice associated with covid-19 among university students: a cross-sectional survey in china. bmc public health. 2020, 1292. https://doi.org/10.21203/rs.3.rs-21185/v1 güner r, hasanoğlu i̇, aktaş f. covid-19: prevention and control measures in community. turkish j med sci. 2020;50:571–577. https://doi.org/10.3906/sag-2004-146 cucinotta d, vanelli m. who declares covid-19 a pandemic. acta biomedica. 2020;91:157–160. https://doi.org/10.23750/abm.v91i1.9397 who. coronavirus disease (covid-19) situation reports [homepage on the internet]. 2020 [cited 2020 sep 17]. available from: https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports/ moh. zambia covid-19 general dasboard [homepage on the internet]. 2020 [cited 2020 sep 17]. available from: https://rtc-planning.maps.arcgis.com/apps/opsdashboard/index.html#/3b3a01c1d8444932ba075fb44b119b63 chileshe m, mulenga d, mfune rl, et al. increased number of brought-in-dead cases with covid-19: is it due to poor health-seeking behaviour among the zambian population? pan afr med j [serial online]. 2020 [cited 2020 oct 14];37. available from: https://www.panafrican-med-journal.com/content/article/37/136/fulls who. situation report-82 highlights [homepage on the internet]. geneva; 2020 [cited 2020 sep 13]. available from: http://weekly.chinacdc.cn/en/article/id/e53946e2-c6c4-41e9-9a9b-fea8db1a8f51 wang h, liu y, hu k, et al. healthcare workers’ stress when caring for covid-19 patients: an altruistic perspective. nurs ethics [serial online]. 2020;27(7):1490–1500. https://doi.org/10.1177/0969733020934146 burrer sl, de perio ma, hughes mm, et al. characteristics of health care personnel with covid-19 – united states, february 12–april 9, 2020. mmwr morb mortal wkly rep [serial online]. 2020 [cited 2020 sep 13];69(15):477–481. available from: http://www.cdc.gov/mmwr/volumes/69/wr/mm6915e6.htm?s_cid=mm6915e6_w who. over 10 000 health workers in africa infected with covid-19 [homepage on the internet]. who, regional office for africa. 2020 [cited 2020 sep 17]. available from: https://www.afro.who.int/news/over-10-000-health-workers-africa-infected-covid-19 tait fn, mburu c, gikunju j. occupational safety and health status of medical laboratories in kajiado county, kenya. pan afr med j [serial online]. 2018;29:65. https://doi.org/10.11604/pamj.2018.29.65.12578 pourhoseingholi ma, vahedi m, rahimzadeh m. sample size calculation in medical studies. gastroenterol hepatol bed bench [serial online]. 2013 [cited 2020 oct 23];6(1):14–17. available from: https://pubmed.ncbi.nlm.nih.gov/24834239/ nepal r, sapkota k, adhikari k, et al. knowledge, attitude and practice regarding covid-19 among healthcare workers in chitwan, nepal. 2020 [cited 2020 sep 14];in press. https://doi.org/10.21203/rs.3.rs-26774/v1 azlan aa, hamzah mr, sern tj, ayub sh, mohamad e. public knowledge, attitudes and practices towards covid-19: a cross-sectional study in malaysia. plos one. 2020;15(5):e0233668. https://doi.org/10.1371/journal.pone.0233668 taber ks. the use of cronbach’s alpha when developing and reporting research instruments in science education. res sci educ. 2018;48(6):1273–1296. https://doi.org/10.1007/s11165-016-9602-2 olum r, chekwech g, wekha g, nassozi dr, bongomin f. coronavirus disease-2019: knowledge, attitude, and practices of health care workers at makerere university teaching hospitals, uganda. front public heal. 2020;8:181. https://doi.org/10.3389/fpubh.2020.00181 akalu y, ayelign b, molla md. knowledge, attitude and practice towards covid-19 among chronic disease patients at addis zemen hospital, northwest ethiopia. infect drug resist [serial online]. 2020 [cited 2020 oct 26];13:1949–1960. available from: https://www.dovepress.com/knowledge-attitude-and-practice-towards-covid-19-among-chronic-disease-peer-reviewed-article-idr asemahagn ma. factors determining the knowledge and prevention practice of healthcare workers towards covid-19 in amhara region, ethiopia: a cross-sectional survey. trop med health. 2020;48:72. https://doi.org/10.1186/s41182-020-00254-3 nakhleh re, nosé v, colasacco c, et al. interpretive diagnostic error reduction in surgical pathology and cytology: guideline from the college of american pathologists pathology and laboratory quality center and the association of directors of anatomic and surgical pathology. arch pathol lab med. 2016;140(1):29–40. giao h, le an p, thi ngoc han n, van khanh t, kim ngan v, van tam v. knowledge and attitude toward covid-19 among healthcare workers at district 2 hospital, ho chi minh city. asian pac j trop med. 2020;13(6):6–11. https://coi.org/10.4103/1995-7645.280396 bhagavathula as, aldhaleei wa, rahmani j, mahabadi ma, bandari dk. knowledge and perceptions of covid-19 among health care workers: cross-sectional study. jmir public heal surveill. 2020;6(2):e19160. geberemariyam bs, donka gm, wordofa b. assessment of knowledge and practices of healthcare workers towards infection prevention and associated factors in healthcare facilities of west arsi district, southeast ethiopia: a facility-based cross-sectional study. arch public heal. 2018;76(69). https://doi.org/10.1186/s13690-018-0314-0 abstract introduction methods results discussion acknowledgements references about the author(s) cailin nieuwenhuizen department of haematology, faculty of health sciences, university of pretoria, pretoria, south africa department of haematology, tshwane academic division, national health laboratory service, pretoria, south africa tshiphiri netshidzivhani department of haematology, faculty of health sciences, university of pretoria, pretoria, south africa department of haematology, tshwane academic division, national health laboratory service, pretoria, south africa johan potgieter department of haematology, faculty of health sciences, university of pretoria, pretoria, south africa department of haematology, tshwane academic division, national health laboratory service, pretoria, south africa citation nieuwenhuizen c, netshidzivhani t, potgieter j. establishment of haemoglobin a2 reference intervals in pretoria, south africa: a retrospective secondary data analysis. afr j lab med. 2022;11(1), a1841. https://doi.org/10.4102/ajlm.v11i1.1841 original research establishment of haemoglobin a2 reference intervals in pretoria, south africa: a retrospective secondary data analysis cailin nieuwenhuizen, tshiphiri netshidzivhani, johan potgieter received: 28 jan. 2022; accepted: 20 apr. 2022; published: 12 aug. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: haemoglobinopathies are one of the most common inherited diseases worldwide. quantification of haemoglobin a2 is necessary for the diagnosis of the beta thalassaemia trait. in this context, it is important to have a reliable reference interval for haemoglobin a2 and a local reference range for south africa has not been established. objective: this study aimed to establish reference intervals for haemoglobin a2 using stored patient laboratory data. methods: this descriptive study used retrospective data to evaluate haemoglobin a2 levels determined using high-performance liquid chromatography at the national health laboratory service haematology laboratory in pretoria, south africa. all tests performed from 01 october 2012 to 31 december 2020 were screened for inclusion; of these, 144 patients’ data met the selection criteria. the reference interval was calculated using descriptive statistics (mean and standard deviation) with a 95% confidence interval. results: analysed data from enrolled patients showed a normal distribution. the mean age of the patients was 40 years (range: 3–84 years). the reference interval for haemoglobin a2 calculated from this data was 2.3% – 3.6%. the minimum haemoglobin a2 was 2.3% and the maximum was 3.9% with a mean of 2.95% and a standard deviation of 0.357%. conclusion: a normal reference interval has been established for the population served by the laboratory that will assist with accurate diagnosis of the beta thalassaemia trait. this reference interval may also be useful to other laboratories that employ the same technology, especially smaller laboratories where obtaining a sufficiently large number of normal controls may be challenging. keywords: haemoglobin a2; reference range; reference interval; beta thalassemia; high-performance liquid chromatography. introduction reference intervals are an integral part of any functioning clinical laboratory. reference intervals are often the endpoint used by clinicians in the management of patients and in clinical decision-making. it has been estimated that 80% of medical decisions are made from laboratory results.1 the value of the results produced by a laboratory is largely influenced by the quality of the reference interval used in the interpretation of that result.2 an example of where reference intervals are of diagnostic utility is in the diagnosis of the beta thalassaemia trait. the increase in haemoglobin a2 (hba2) level is the most important laboratory parameter for the identification of carriers of beta thalassaemia, and is considered diagnostic in the appropriate clinical context.3 accurate quantification of hba2 in the haematology laboratory is therefore essential to allow for routine diagnosis of the beta thalassaemia trait.4 this highlights the value of reliable reference intervals. beta thalassaemia is inherited in an autosomal recessive pattern and the beta thalassaemia trait is estimated to have a prevalence of 1.5% worldwide, affecting approximately 80–90 million people.5 africa has a considerable disease burden in terms of haemoglobinopathies and in particular beta thalassaemia with 1520 conceptions affected annually, western africa accounting for the majority of cases.6 reference intervals are established through a validation process with a statistically adequate number, ideally 120, of healthy reference individuals.7 this needs to be done for all reagents and instrument combinations. however, even the clinical and laboratory standards institute guidelines recognise that this is not feasible for many laboratories and finding a cohort of 120 healthy individuals is not feasible for every test.8 alternatively, reference intervals may be verified, with only 20 samples needed, or transferred provided that the analytic system and the test population are comparable.8 however, verification and transfer of reference intervals is not ideal and these methods have their own disadvantages.8 every laboratory that performs hba2 testing is responsible for establishing its own reference interval. this is done by quantifying the hba2 percentage in a cohort of healthy adults who do not have iron deficiency or the thalassaemia trait.3 the reference interval is a range that should be calculated including individuals with characteristics that are comparable to the reference group so that the reference interval can be correctly applied to the population serviced by the laboratory.7 given the importance of hba2 reference ranges, considerable work has been done internationally on normal reference intervals for hba2. however, there is a paucity of literature from africa with no published reference intervals for hba2. in keeping with good laboratory practice, a need was identified to determine hba2 reference intervals in a local african population for the department of haematology of the national health laboratory service (nhls), tshwane academic division (tad), pretoria, south africa. using a sufficient number of results from medical records would provide a healthy cohort for establishing a reference interval without the need to recruit healthy individuals. this is particularly useful for a test that is not routinely requested and only offered by specialised laboratories. in order to establish a reference interval for hba2 without the limitations inherent to traditional methods of establishing reference intervals, we made use of previously reported normal high-performance liquid chromatography (hplc) results from the nhls department of haematology. methods ethical considerations approval was obtained from the academic affairs, research and quality assurance department of the nhls. the study protocol was approved by the faculty of health sciences research ethics committee of the university of pretoria (protocol number 518/2019). patient consent was waived by the ethics committee as the study was conducted using historical data. participants’ information was treated with confidentiality. each participant was allocated a unique study number to ensure anonymity. study design and setting this was a descriptive study using retrospective data from blood test results stored in the laboratory information system of the nhls at the department of haematology, tad, and vermaak and partners path care pathology group. study population and sampling strategy the study population comprised patients investigated by tad, nhls, and included patients from both high-income and low-income settings and represented a variety of ethnicities. all hplc results of tests performed at the nhls tad from 01 october 2012 to 31 december 2020 were screened for inclusion (figure 1). the following selection criteria were applied: hplc performed within the study period and normal hplc results of patients who also had a corresponding complete blood counts (cbc) were included in this study. results of individual patients were excluded if they were aged two years or younger, were anaemic (haemoglobin < 12.5 g/dl), had a mean cell volume < 75 fl or > 100 fl, had a mean corpuscular haemoglobin below 27 pg, had a red cell distribution width of > 15% or had variant haemoglobin, inclusive of haemoglobin s, detected in their haemoglobin electrophoretic result. participants that did not meet the selection criteria or met the exclusion criteria were excluded from the study and subsequent analysis. a minimum sample size of 120 patient is required in order to establish a reference interval.8 figure 1: flow diagram of the study design, pretoria, south africa, 01 october 2012 – 31 december 2020. data collection apart from hplc reports, corresponding results of cbc, thyroid stimulating hormone, serum folate, serum vitamin b12 and ferritin level, performed within a week of the taking of the hplc specimen, were evaluated. only normal hplc results were included in the analysis. the hba2 levels were determined in the haematology laboratory using the hplc d10 instrument (bio-rad® laboratories, hercules, california, united states). the d10 instrument uses ion-exchange hplc technology to analyse haemoglobin. all analyses were performed according to good laboratory practice and the manufacturer’s recommendations. the haematology laboratory at the nhls tad has been accredited by the south african national accreditation system. appropriate controls and calibrators were used throughout the study. the lyphochek® hemoglobin a2 control level 1 and 2 (bio-rad laboratories, irvine, california, united states) were used as controls and the d10tm dual program hba2/f/a1c calibrator/diluent set (bio-rad® laboratories, hercules, california, united states) was used for calibration. the blood test results listed above were captured in a microsoft excel (microsoft corporation, redmond, washington, united states) spreadsheet. exclusion criteria were applied after which 144 samples with normal hba2 levels were identified, and these were used for calculation of reference intervals. data analysis the descriptive statistics mean, median, standard deviation and inter-quartile range, with 95% confidence interval, and the 2.5th and 97.5th percentiles were used to describe the continuous variables such as hba2 levels. the two-sample t-test, or non-parametric alternative were used to compare group means. pearson’s correlation was used to measure correlations between hba2 levels and age, as well as other continuous variables such as cbc parameters. tests were evaluated at 5% level of significance. all analyses were done using stata 15 (statacorp, college station, texas, united states) software. results the mean age of the 144 patients included in this data analysis was 40 years (range 3–84 years). the study population comprised 67 female and 77 male patients. after a single outlier of 4.3% was excluded from the analysis, the mean hba2 value was 2.95% with the range between 2.2% and 3.9%. the standard deviation was 0.357% (figure 2). figure 2: box-plot of haemoglobin a2 distribution, pretoria, south africa, 01 october 2012 – 31 december 2020. data were normally distributed as indicated in the kernel density estimation (figure 3). the hba2 reference interval established from this data set was 2.3% – 3.6%. figure 3: kernel density estimate demonstrating the normal distribution of data, pretoria, south africa, 01 october 2012 – 31 december 2020. a sex comparison was performed by t-test to compare the mean hba2 of male patients to that of female patients in an attempt to identify possible bias. no significant difference was found (p = 0.1328). the correlation between age and hba2 was also assessed. a trend towards lower hba2 values with increased age was appreciated (figure 4), with a pearson correlation coefficient of –0.2826. figure 4: scatterplot of haemoglobin a2 and age distribution, pretoria, south africa, 01 october 2012 – 31 december 2020. discussion the hba2 reference interval established from this data set was 2.3% – 3.6%. normal reference intervals for hba2 have been published for other patient populations. a study performed at the leiden university in the netherlands, using the variant classic hplc (bio-rad®) platform, reported a hba2 reference interval of 2.3% – 3.5%.3 despite the difference in study population, the reference interval determined in this current study is comparable to that of the leiden group which used a similar method, that is, hplc (bio-rad®) technology. han et al. reported a reference interval of 2.3% – 3.1% for hba2 in a chinese population of reproductive age.9 however, these investigators used a capillarys2 instrument (sebia, france) to generate their data.9 most evidence suggests that hba2 of > 4% is indicative of beta thalassaemia trait with almost 100% sensitivity and 90% specificity.10 a grey zone between 3.1 and 3.9 is generally accepted as reported in a comprehensive review.11 this often poses a diagnostic challenge. studies have been conducted to identify the presence of mutations in these individuals.10,11,12,13,14 giambona et al. found that 80% of patients in this group in an italian population were negative for molecular defects, and the most significant finding was the presence of beta thalassaemia gene mutations found mostly in patients with hba2 in the region of 3.5–3.9 and mean cell volume < 80 fl.11 the upper limit of the normal reference interval established in this current study does fall within the previously described ‘grey zone’. however, in the presence of a normal cbc, the possibility of an underlying carrier state in these patients remains small. this highlights the importance of interpreting hba2 within the clinical context, taking into consideration the cbc parameters and iron studies. there appears to be a weak association between a decreasing hba2 value and increasing age; this was true even when looking at subsets of age and when excluding patients aged 70 years or older. although this trend was seen, when calculating the reference interval by age, the reference interval remained 2.3–3.6 when rounded to one decimal place. therefore, this was not a significant finding. in this current study we used data available on the laboratory information system in order to establish a reference interval. this represents a novel approach in our setting. data mining is emerging as an alternative to the traditional direct a priori method. data mining makes use of electronic data records and statistical techniques to determine the healthy population within a data set in order to establish a reference interval.15 the electronic data records may be obtained from insurance claims, electronic health records as well as a variety of other sources.16 all sources require data capturing platforms which allow for database management in order to deal with the enormous volume of information currently being generated as well as the complexity of analysing and interpreting the data. the analytical methods that have been employed to establish reference intervals include the hoffmann method, bhatacharya method and more recently the truncated maximum likelihood method.15 the truncated maximum likelihood method employs complex statistical algorithms that make use of maximum likelihood estimation and require 4000 data points in order to establish a robust reference interval.15 currently there is still hesitancy regarding the use of indirect methods but it is likely to be used to establish many reference intervals in the future. indirect methods may be particularly useful for tests that are not routinely performed as screening tests in the healthy population.17 data mining has many advantages: it is less costly as the blood results of large cohorts of individuals are readily available, it is faster, and it can even be considered more ethical.15 a study conducted by katayev et al. showed that reference intervals could be reliably and reproducibly established using data mining. reference intervals were calculated for eight analytes and were found to be comparable to already accepted published reference intervals.17 although our study was small and the data were captured manually, it does highlight the potential of using laboratory information records to glean valuable data with relatively low cost and fewer limitations than are inherent to establishing reference intervals in the conventional manner by direct population sampling. limitations one of the limitations of this study was the inability to exclude all confounding factors. although our study could be improved on by only including patients who have been tested for all confounding factors for hba2, this would only be feasible if a large number of data sets were included. another option would be to use a larger cohort and an algorithm that does not require the exclusion of all confounders. a normal cbc was used as a surrogate marker of a nutritional deficiency, and this remains a limitation. it should be noted that all published confounders are not routinely considered when establishing reference intervals for hba2 or when interpreting hplc results. conclusion a normal hba2 reference interval of 2.3% – 3.6% has been established for the population served by the laboratory. this will assist with the interpretation of results. the reference interval could also be useful to other laboratories, especially smaller laboratories where obtaining a sufficiently large number of normal controls may be challenging. the inability to exclude all of the confounding factors that influence hba2 levels needs further research. acknowledgements we thank prof. r. pool for his insight and guidance and dr d. toi for his assistance in proofreading the article. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions j.p. and t.n. conceived of the presented idea. c.n. developed the theory and performed the computations. j.p., c.n. and t.n. verified the analytical methods. j.p. supervised the findings of this work. all authors discussed the results and contributed to the final manuscript. sources of support funding has been provided by the university of pretoria development fund. data availability the data that support the findings of this study are available on request from the corresponding author, c.n. disclaimer the views expressed in the submitted article are the authors’ own and not an official position of the institution. references castellone dd. establishing reference intervals in the coagulation laboratory. int j lab hematol. 2017;39(s1):121–127. https://doi.org/10.1111/ijlh.12661 jones g, barker a. reference intervals. clin biochem rev. 2008;29(suppl 1):s93–s97. mosca a, paleari r, ivaldi g, galanello r, giordano p. the role of haemoglobin a2 testing in the diagnosis of thalassaemias and related haemoglobinopathies. j clin pathol. 2009;62(1):13–17. https://doi.org/10.1136/jcp.2008.056945 stephens ad, angastiniotis m, baysal e, et al. icsh recommendations for the measurement of haemoglobin a₂. int j lab hematol. 2012;34(1):1–13. https://doi.org/10.1111/j.1751-553x.2011.01368.x galanello r, origa r. beta-thalassemia. orphanet j rare dis. 2010;5(1):11. https://doi.org/10.1186/1750-1172-5-11 modell b, darlison m. global epidemiology of haemoglobin disorders and derived service indicators. bull world health organ. 2008;86(6):480–487. https://doi.org/10.2471/blt.06.036673 katayev a, balciza c, seccombe dw. establishing reference intervals for clinical laboratory test results: is there a better way? am j clin pathol. 2010;133(2):180–186. https://doi.org/10.1309/ajcpn5bmtsf1cdyp boyd j. defining, establishing, and verifying reference intervals in the clinical laboratory: approved guidelines. document c28-a3. wayne, pa: clinical and laboratory standards institute; 2010. han wp, huang l, li yy, et al. reference intervals for hba2 and hbf and cut-off value of hba2 for β-thalassemia carrier screening in a guizhou population of reproductive age. clin biochem. 2019;65:24–28. https://doi.org/10.1016/j.clinbiochem.2018.11.007 greene dn, vaughn cp, crews bo, agarwal am. advances in detection of hemoglobinopathies. clin chim acta. 2015;439:50–57. https://doi.org/10.1016/j.cca.2014.10.006 giambona a, passarello c, renda d, maggio a. the significance of the hemoglobin a2 value in screening for hemoglobinopathies. clin biochem. 2009;42(18):1786–1796. https://doi.org/10.1016/j.clinbiochem.2009.06.026 giambona a, passarello c, vinciguerra m, et al. significance of borderline hemoglobin a2 values in an italian population with a high prevalence of beta-thalassemia. haematologica. 2008;93(9):1380–1384. tamagnini gp, gonçalves p, ribeiro ml, et al. beta-thalassemia mutations in the portuguese; high frequencies of two alleles in restricted populations. hemoglobin. 1993;17(1):31–40. efremov dg, dimovski aj, baysal e, et al. possible factors influencing the haemoglobin and fetal haemoglobin levels in patients with beta-thalassaemia due to a homozygosity for the ivs-i-6 (t-->c) mutation. british journal of haematology. 1994;86(4):824–830. bohn mk, adeli k. application of the tml method to big data analytics and reference interval harmonization. j lab med. 2021;45(2):79–85. https://doi.org/10.1515/labmed-2020-0133 yang j, li y, liu q, et al. brief introduction of medical database and data mining technology in big data era. j evid based med. 2020;13(1):57–69. https://doi.org/10.1111/jebm.12373 katayev a, fleming jk, luo d, fisher ah, sharp tm. reference intervals data mining: no longer a probability paper method. am j clin pathol. 2015;143(1):134–142. https://doi.org/10.1309/ajcpqprnib54wfkj ajlm 10(1)_2021_contents.indd http://www.ajlmonline.org open access table of contents original research an oral history of medical laboratory development in francophone west african countries winny koster, albert g. ndione, mourfou adama, ibrehima guindo, iyane sow, souleymane diallo, jean sakandé, pascale ondoa african journal of laboratory medicine | vol 10, no 1 | a1157 | 16 march 2021 original research multicentre study of the burden of multidrug-resistant bacteria in the aetiology of infected diabetic foot ulcers adeyemi t. adeyemo, babatope kolawole, vincent o. rotimi, aaron o. aboderin african journal of laboratory medicine | vol 10, no 1 | a1261 | 23 march 2021 original research the growth patterns of the medical technology profession in south africa malcolm t. ellapen, terry j. ellapen, yvonne paul african journal of laboratory medicine | vol 10, no 1 | a1164 | 23 april 2021 original research effects of tnf-α and il-10-819 t>c single nucleotide polymorphisms on urogenital schistosomiasis in preschool children in zimbabwe amos marume, theresa chimponda, arthur vengesai, caroline mushayi, jaclyn mann, takafira mduluza african journal of laboratory medicine | vol 10, no 1 | a1138 | 29 april 2021 original research in-depth investigation of turn-around time of full blood count tests requested from a clinical haematology outpatient department in cape town, south africa leonard mutema, zivanai chapanduka, fungai musaigwa, nomusa mashigo african journal of laboratory medicine | vol 10, no 1 | a1318 | 29 april 2021 original research improving laboratory quality and capacity through leadership and management training: lessons from zambia 2016–2018 felicity gopolang, fales zulu-mwamba, davy nsama, annika kruuner, dailes nsofwa, ishmael kasvosve, royce gomo, tiny motlhabane, bhavna chohan, olusegun soge, daniel osterhage, nancy campbell, michael noble, ann downer, jean-frederic flandin, anya nartker, catherine koehn, linda k. nonde, aaron shibemba, clement b. ndongmo, martin steinau, lucy a. perrone african journal of laboratory medicine | vol 10, no 1 | a1225 | 30 april 2021 original research molecular characterisation of npm1 and flt3-itd mutations in a central south african adult de novo acute myeloid leukaemia cohort jean f. kloppers, andré de kock, johané cronjé, anne-cecilia van marle african journal of laboratory medicine | vol 10, no 1 | a1363 | 30 june 2021 original research could ante-mortem computed tomography be useful in forensic pathology of traumatic intracranial haemorrhage? mmachuene i. hlahla, moshibudi j. selatole african journal of laboratory medicine | vol 10, no 1 | a1040 | 29 july 2021 39 49 59 64 71 77 86 92 page i of iii table of contents editorial call for emergency action to limit global temperature increases, restore biodiversity, and protect health lukoye atwoli, abdullah h. baqui, thomas benfield, raffaella bosurgi, fiona godlee, stephen hancocks, richard horton, laurie laybournlangton, carlos augusto monteiro, ian norman, kirsten patrick, nigel praities, marcel g.m. olde rikkert, eric j. rubin, peush sahni, richard smith, nicholas j. talley, sue turale, damián vázquez african journal of laboratory medicine | vol 10, no 1 | a1707 | 20 september 2021 editorial towards a fiercely urgent expansion of laboratory medicine in africa iruka n. okeke african journal of laboratory medicine | vol 10, no 1 | a1785 | 17 december 2021 opinion paper maintaining routine hiv and tuberculosis testing services in sub-saharan african countries in the context of covid-19: lessons learnt and opportunities for improvement collins o. odhiambo, anafi mataka, marguerite massinga loembe, pascale ondoa african journal of laboratory medicine | vol 10, no 1 | a1413 | 17 june 2021 opinion paper impact of covid-19 on blood donation and supply in africa kenneth b. david, knovicks simfukwe, mohamed b. musa, steven munharo, don e. lucero-prisno iii african journal of laboratory medicine | vol 10, no 1 | a1408 | 25 october 2021 original research performance of international aids vaccine initiative african clinical research laboratories in standardised elispot and peripheral blood mononuclear cell processing in support of hiv vaccine clinical trials robert k. langat, bashir farah, jackton indangasi, simon ogola, gloria omosa-manyonyi, omu anzala, jean bizimana, emmanuel tekirya, caroline ngetsa, moses silwamba, enoch muyanja, paramesh chetty, maureen jangano, nancy hills, jill gilmour, len dally, josephine h. cox, peter hayes african journal of laboratory medicine | vol 10, no 1 | a1056 | 17 february 2021 original research knowledge, attitude and practices of covid-19 among medical laboratory professionals in zambia adon chawe, ruth l. mfune, paul m. syapiila, sharon d. zimba, pipina a. vlahakis, samson mwale, kapambwe mwape, memory chirambo-kalolekesha, misheck chileshe, joseph mutale, tobela mudenda, grace manda, victor daka african journal of laboratory medicine | vol 10, no 1 | a1403 | 04 march 2021 original research involvement of cd95 and ligand in cd4+ t-cell and cd8+ t-cell depletion and hepatic cytolysis in patients with chronic viral hepatitis b franklin s. azebaze agueguia, paul talla, marie c. okomo assoumou, graeme b. jacobs, cedric h. mbakam, elise guiedem, martha tongo mesembe, emilia lyonga, george mondinde ikomey african journal of laboratory medicine | vol 10, no 1 | a1224 | 15 march 2021 1 4 6 10 13 26 33 vol 10, no 1 (2021) issn: 2225-2002 (print) | issn: 2225-2010 (online)african journal of laboratory medicine http://www.ajlmonline.org open access table of contents original research comparison of 24-hour versus random urine samples for determination and quantification of bence jones protein in a south african population ashandree reddy, nadine rapiti, verena gounden african journal of laboratory medicine | vol 10, no 1 | a1228 | 04 august 2021 original research operational analysis of the national sickle cell screening programme in the republic of uganda arielle g. hernandez, charles kiyaga, thad a. howard, isaac ssewanyana, grace ndeezi, jane r. aceng, russell e. ware african journal of laboratory medicine | vol 10, no 1 | a1303 | 12 august 2021 original research higher proportion of non-classical and intermediate monocytes in newly diagnosed multiple myeloma patients in egypt: a possible prognostic marker asmaa m. zahran, hanaa nafady-hego, sawsan m. moeen, hanan a. eltyb, mohammed m. wahman, asmaa nafady african journal of laboratory medicine | vol 10, no 1 | a1296 | 25 august 2021 original research pre-evaluation assessment of serological-based covid-19 point-of-care lateral flow assays in kenya james h. kimotho, abdiaziz a. gosar, ronald inyangala, paulyne wairimu, fred siyoi, damaris matoke-muhia, cecilia wanjala, jeremiah zablon, moses orina, lucy muita, jacqueline thiga, lameck nyabuti, eunice wainaina, joseph mwangi, alice mumbi, samuel omari, ann wanjiru, samson m. nzou, missiani ochwoto african journal of laboratory medicine | vol 10, no 1 | a1317 | 17 september 2021 original research molecular red cell genotyping of rare blood donors in south africa to enhance rare donor-patient blood matching lavendri govender, rosaley d. prakashchandra, pavitra pillay, ute jentsch african journal of laboratory medicine | vol 10, no 1 | a1400 | 27 september 2021 original research establishing the cost of xpert mtb/rif mobile testing in high-burden peri-mining communities in south africa naseem cassim, lindi m. coetzee, abel l. makuraj, wendy s. stevens, deborah k. glencross african journal of laboratory medicine | vol 10, no 1 | a1229 | 30 november 2021 original research relationship between amino acid ratios and decline in estimated glomerular filtration rate in diabetic and non-diabetic patients in south africa thapelo mbhele, donald m. tanyanyiwa, refilwe j. moepya, sindeep bhana, maya m. makatini african journal of laboratory medicine | vol 10, no 1 | a1398 | 10 december 2021 original research effect of polyethylene glycol 20 000 on protein extraction efficiency of formalin-fixed paraffin-embedded tissues in south africa sophia rossouw, hocine bendou, liam bell, jonathan rigby, alan christoffels african journal of laboratory medicine | vol 10, no 1 | a1122 | 17 december 2021 scientific letter post-mortem diagnosis of covid-19 rujittika mungmungpuntipantip, viroj wiwanitkit african journal of laboratory medicine | vol 10, no 1 | a1471 | 24 march 2021 97 104 112 120 126 134 141 148 158 scientific letter biobanking of covid-19 specimens during the pandemic: the need for enhanced biosafety olayinka s. ilesanmi, aanuoluwapo a. afolabi african journal of laboratory medicine | vol 10, no 1 | a1379 | 21 october 2021 lessons from the field from benchmarking to best practices: lessons from the laboratory quality improvement programme at the military teaching hospital in cotonou, benin alban zohoun, tatiana b. agbodandé, angélique kpadé, raliatou o. goga, rené gainsi, paul balè, bibata m. sambo, remi charlebois, rachel crane, michele merkel, ludovic anani, ekaterina milgotina african journal of laboratory medicine | vol 10, no 1 | a1057 | 11 february 2021 lessons from the field the deployment of mobile diagnostic laboratories for ebola virus disease diagnostics in sierra leone and guinea lance d. presser, jeanette coffin, lamine koivogui, allan campbell, julian campbell, fatmata barrie, jone ngobeh, zein souma, samuel sorie, doris harding, alimou camara, pepe tohonamou, basala traore, frank a. hamill, joe bogan, sharon altmann, casey ross, jay mansheim, robert hegerty, scott poynter, scott shearrer, carmen asbun, brendan karlstrand, phil davis, jane alam, david roberts, paul d. stamper, jean ndjomou, nadia wauquier, mohamed koroma, alhaji munu, jason mcclintock, mar mar, true burns, stephen krcha african journal of laboratory medicine | vol 10, no 1 | a1414 | 22 october 2021 lessons from the field setting up a molecular diagnostic laboratory for sars-cov-2 testing: experience of a single centre in a resource-constrained setting iriagbonse i. osaigbovo, isaac o. igbarumah, ekene b. muoebonam, darlington e. obaseki african journal of laboratory medicine | vol 10, no 1 | a1326 | 30 march 2021 lessons from the field adaptation of an electronic dashboard to monitor hiv viral load testing in côte d’ivoire mary kirk, paul h. assoa, casey iiams-hauser, yves-rolland kouabenan, jennifer antilla, caleb steele-lane, greg rossum, pascal komena, patricia sadate ngatchou, nadine abiola, alain kouakou, adama pongathie, jean b. koffi, christiane adje, lucy a. perrone african journal of laboratory medicine | vol 10, no 1 | a1284 | 17 may 2021 lessons from the field strategic site selection for placement of hiv early infant diagnosis pointof-care technology within a national diagnostic network in lesotho anafi mataka, esther a.j. tumbare, tsietso motsoane, david holtzman, monkoe leqheka, kolisang phatsoane, emma sacks, anthony isavwa, appolinaire tiam african journal of laboratory medicine | vol 10, no 1 | a1156 | 24 august 2021 lessons from the field african countries established covid-19 testing in one month: here’s how they did it timothy amukele, ryland n. spence african journal of laboratory medicine | vol 10, no 1 | a1457 | 15 december 2021 case study emphysematous pyelonephritis in an infant from sokoto, north-western nigeria fatima b. jiya, paul k. ibitoye, nma m. jiya, maryam amodu-sanni, yahaya mohammed, dada m. aquib, lukman k. coker african journal of laboratory medicine | vol 10, no 1 | a1181 | 26 april 2021 160 162 169 175 182 188 194 200 page ii of iii http://www.ajlmonline.org open access table of contents brief report epstein-barr virus, human papillomavirus and herpes simplex virus 2 co-presence severely dysregulates mirna expression jude o. okoye, anthony a. ngokere, charles c. onyenekwe, olaposi omotuyi, deborah i. dada african journal of laboratory medicine | vol 10, no 1 | a975 | 16 march 2021 brief report performance of taqman probes for the detection of sexually transmitted infections in south african women nireshni mitchev, ravesh singh, nigel garrett, veron ramsuran, abraham j. niehaus, koleka p. mlisana african journal of laboratory medicine | vol 10, no 1 | a1124 | 31 march 2021 brief report cross-validation of a high-performance liquid chromatography nevirapine plasma assay in a resource-limited setting in zimbabwe faithful makita-chingombe, anthony t. podany, timothy mykris, farai muzambi, richard w. browne, andrew j. ocque, robin difrancesco, lee c. winchester, courtney v. fletcher, tinashe mudzviti, charles c. maponga, gene d. morse african journal of laboratory medicine | vol 10, no 1 | a1264 | 08 july 2021 204 214 218 brief report re-decontamination of liquid mycobacterial cultures: additional mycobacterium tuberculosis yield in the era of xpert mtb/rif ultra in cape town, south africa dawood da costa, pieter nel african journal of laboratory medicine | vol 10, no 1 | a1529 | 10 december 2021 correction corrigendum: brucellosis – laboratory workers’ nightmare come true: a case study lebogang skosana, farzana ismail, nontombi mbelle, mohamed said african journal of laboratory medicine | vol 10, no 1 | a1690 | 22 november 2021 reviewer acknowledgement african journal of laboratory medicine | vol 10, no 1 | a1812 | 22 december 2021 223 226 227 page iii of iii abstract introduction methods results discussion acknowledgements references about the author(s) ivy j. mutai phage biology laboratory, institute of primate research, nairobi, kenya department of biochemistry, biotechnology and microbiology, faculty of pure and applied sciences, kenyatta university, nairobi, kenya angela a. juma phage biology laboratory, institute of primate research, nairobi, kenya martin i. inyimili department of human anatomy, university of nairobi, nairobi, kenya atunga nyachieo phage biology laboratory, institute of primate research, nairobi, kenya anthony k. nyamache department of biochemistry, biotechnology and microbiology, faculty of pure and applied sciences, kenyatta university, nairobi, kenya citation mutai ij, juma aa, inyimili mi, nyachieo a, nyamache ak. efficacy of diversely isolated lytic phages against multi-drug resistant enterobacter cloacae isolates in kenya. afr j lab med. 2022;11(1), a1673. https://doi.org/10.4102/ajlm.v11i1.1673 original research efficacy of diversely isolated lytic phages against multi-drug resistant enterobacter cloacae isolates in kenya ivy j. mutai, angela a. juma, martin i. inyimili, atunga nyachieo, anthony k. nyamache received: 13 july 2021; accepted: 04 may 2022; published: 11 aug. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: enterobacter cloacae causes nosocomial infections in 15% of patients in lowand middle-income countries with emergence of carbapenem resistance. the utilisation of bacteriophages for therapeutic purposes is crucial for eradicating these resistant bacterial strains. objective: this study evaluated the efficacy of lytic phages on bacterial isolates of e. cloacae and determined their stability in various physicochemical conditions. methods: twenty-nine lytic phages were isolated from the waste water of six informal settlements in nairobi county, kenya, from july 2019 to december 2020 and cross-reacted with 30 anonymised clinical isolates of e. cloacae. six phages were then selected for physicochemical property studies. phages were described as potent upon lysing any bacterial strain in the panel. results: selected phages were stable at 4 °c – 50 °c with a 5.1% decrease in titre in four of six phages and a 1.8% increase in titre in two of six phages at 50 °c. the phages were efficient following two weeks incubation at 4 °c with optimal activity at human body temperature (37 °c) and an optimal ph of 7.5. phages were active at 0.002 m and 0.015 m concentrations of ca2+ ions. the efficiency of all phages decreased with increased exposure to ultraviolet light. all phages (n = 29) showed cross-reactivity against anonymised clinical isolates of e. cloacae strains (n = 30). the most potent phage lysed 67.0% of bacterial strains; the least potent phage lysed 27.0%. conclusion: this study reveals the existence of therapeutic phages in kenya that are potent enough for treatment of multi-drug resistant e. cloacae. keywords: lytic phages; enterobacter cloacae; multi-drug resistance; nosocomial infections; nairobi county; kenya. introduction enterobacter cloacae is a gram-negative rod-shaped bacteria that is associated with nosocomial infections.1 this organism is naturally resistant to ampicillin, amoxicillin/clavulanic, cephamycin and the firstand second-generation cephalosporins due to chromosomally encoded ampc β-lactamase.1 enterobacter cloacae is a normal flora of the human gastrointestinal tract, which can cause opportunistic infections among immunocompromised individuals, the elderly and newborns.1,2 this organism causes septicemia, endocarditis, urinary tract infections, wound infections, and meningitis in newborns.1,2 these infections can lead to prolonged hospitalisation and higher cost of treatments, increased antibiotic use and pressure leading to the development of antimicrobial resistance in the hospital. in addition, the rates of morbidity and mortality among critical patients can also increase.1,2 enterobacter cloacae causes nosocomial infections that affect at least 7% of patients in high-income countries; such infections are twice as high (15%) in the lowand middle-income countries.3 enterobacter cloacae is ranked as the third most predominant organism causing nosocomial infections and the second most predominant carbapenem-resistant organism, according to studies conducted in the united states.1,4,5,6 according to nyangacha et al., e. cloacae resistance was observed among patients suffering from tungiasis, or jigger disease, a secondary infection in a study done in the western part of kenya with the following resistance profiles: ampicillin (75.0%), amoxicillin-clavulanic acid (25.0%), tetracycline (50.0%), ceftazidime (25.0%) and cefuroxime (25.0%).7 globally, the emergence and spread of carbapenemase producers e. cloacae has been reported with the prevalence rate of 59.5% being reported in japan and china in three tertiary hospitals.8,9,10 this record of a high rate of drug resistance is a worrying trend for this organism.10 there is a growing need to conduct active surveillance of e. cloacae, in order to control and prevent further spread of drug resistance to other low-prevalence countries.8,10,11 in order to control the current rise in antimicrobial resistance, it is clear that an alternative to the use of antibiotics is urgently needed. bacteriophages (phages) have been proposed as an alternative to antibiotics based on reported evidence.12,13 bacteriophages are obligate parasites; hence, they infect bacteria and form potential antimicrobial agents capable of killing bacteria, including the drug resistant strains.11,13,14 phages naturally multiply through feeding on the bacteria leading to their lysis which in the process regulates bacterial populations within the ecosystems.13,14,15 since phages are very specific to bacteria, their lysis process could be exploited for the development and production of new therapeutic agents.11 given this revelation, phage hunting is now pursued to aid in disease management, as an alternative to elimination and prevention of multi-drug resistant (mdr) strains.10,11 however, e. cloacae is a bacteria of medical importance, but there is limited information on the application of bacteriophages as alternative antimicrobial agents against this bacteria.1,2,5 additionally, there is scarce information on studies of lytic phages against e. cloacae in kenya. the efficacy of phages in treatment of disease-causing bacteria has been exhibited by numerous reports. on the other hand, there is scarce information about their physicochemical properties. furthermore, the stability of kenyan phages under various extreme conditions of alkalinity, acidity, high and low temperatures, ultraviolet exposure, salinity and storage has not been fully studied. we argue that determining the effect of external factors that could influence the yield and potency of phage preparations is important as one prepares phages as an alternative to antibiotics. this study, therefore, evaluated the efficacy of lytic phages in vitro on a panel of bacterial isolates of e. cloacae and their stability under different physicochemical environments.. methods ethical considerations ethical clearance was not required for this study, since there were no human subjects or animals models used in this research; however, this study was registered with the institutional research and ethics committee (iserc) of the institute of primate research (ipr/irc/2014). the bacterial isolates used in this study were anonymised clinical isolates obtained from the kenya medical research institute (kemri) center for microbiology (seru#2767), in nairobi, kenya. for environmental waste water collection, an approval was issued by nairobi water and sewerage company (nawasco#ncwsc/trg14/109). sample collection environmental waste water collection was done by the institute of primate research phage biology group from july to august 2019. samples were collected from six informal settlements: kibera, dandora, kariobangi, huruma, mathare and korogocho, all in nairobi county, kenya. three samples per settlement were collected making a total of 18 samples. dark screw cap containers (to prevent direct light) were used to collect the samples which were transported to the institute of primate research phage laboratory using cooler boxes as the secondary containment followed by storage at 4 °c and processed within three weeks. the three samples per settlement were named using number designations in the following order: 1, 2 and 3. bacterial isolation and identification an mdr isolate of e. cloacae isolated from environmental waste water was used for phage isolation. this bacterium was identified using culture media, microscopic and biochemical examination using vitek ii machine (biomérieux, marcy-l’etoile, france) for identification and antimicrobial susceptibility of bacteria.16 a panel of anonymised clinical bacterial isolates (not evaluated for antimicrobial susceptility test) of e. cloacae (n = 30) were subjected to the phages to assess cross infectivity. phage isolation phages were isolated through an enrichment method according to the methods described by akhtar et al. and clokie et al. with slight modifications.17,18 briefly, 30 ml of each environmental waste water was centrifuged at 10 000 × gravitation for 10 min (centrific™, centrifuge fisher scientific, waltham, massachusetts, united states). the supernatant was mixed with an equivalent volume of tryptose soy broth (tsb) (himedia, mumbai, india), and inoculated with 1 ml of 18 h-old mdr e. cloacae culture. the mixture was incubated overnight at 37 °c in a shaker incubator at 120 rotations per minute (lab-line® incubator-shaker, waltham, massachusetts, united states). the cultures were then centrifuged at 10 000 × gravitation for 10 min, and the supernatant sterilised using a 0.22 µm syringe filter (millipore, merck, darmstadt, germany) and stored at 4 °c for use in the spot test. in vitro screening for phages (spot testing) phages were screened through spot test procedures according to clokie and kropinski (2010) with slight modifications.18 briefly, a lawn of 24 h-old mdr e. cloacae isolate (100 µl) was prepared in soft agar (0.7%) with tsb on a tryptose soy agar (tsa) plate (himedia, mumbai, india). ten-fold serial dilutions of the phage filtrate were prepared and 5 µl of each dilution was spotted on a well-labelled plate with dilutions ranging from 10−1 to 10−8 followed by overnight incubation at 37 °c. observation of plaques (clear-patched regions) on the bacterial lawn was recorded as positive for the phage. for plates with distinct plaques, the well-isolated plaques were harvested using a sterile pasteur pipette and suspended in 200 µl sterile saline magnesium (sm) buffer (100 mm sodium chloride, 10 mm magnesium sulphate, 50 mm tris-hcl, ph 7.5 and 0.01% weight by volume gelatin), vortexed and incubated at room temperature for 1 h before centrifuging at 4000 gravitation for 5 min to remove any remaining debris, labelled and stored at 4 °c. preparation and titration of phage lysate (plaque assay) for plates without well-isolated plaques, the double agar overlay method was employed with slight modifications.19 briefly, 100 µl of the 10-fold serial dilution of the lysate (from the spot with the least number of plaques) was mixed with 100 µl of 18 h-old e. cloacae in 6 ml molten agar (0.7% agar with tsb) and dispensed on tsa plate (1.5% agar with tsb) medium and allowed to solidify before incubation at 37 °c for 18 h. well-isolated plaques were identified and marked before harvesting each plaque using a sterile pasteur pipette, vortexed and incubated at room temperature for 1 h before centrifuging at 4000 gravitation for 5 min to remove any remaining debris, labelled well and stored at 4 °c. calculation of phage titre in plates with distinct plaques for the spot test and plaque assay, the plaques were counted respective to their dilution factors employing the following formula: effect of temperature on phage titre phage adsorption rates on the host bacterium were recorded at the temperatures 4 °c, 10 °c, 20 °c, 37 °c and 50 °c. then 100 µl of actively growing host strain cultures in tsb to an optical density of 600 nm (od600) of 0.6 was used to make an overlay on tsa plates. the selected phages were then incubated at these different temperatures for 1 h and then placed at room temperature (20 °c – 27 °c) for 30 min prior to performing spot tests. the outcomes were given as a log10 of phage titre.20,21,22 effect of ph on phage titre the effect of ph 2, 5, 7, 9, 11 and 13 on phage titre and viability of phages was studied in tsa plates by the spot test method.18 the sm buffer was adjusted to the desired ph by the use of naoh and hcl. the actual ph of the sm was determined with a ph meter (hanna instruments inc. woonsocket, rhode island, united states) and this was used as a control or standard. the adjusted sm buffers were used for serial dilutions of the phages under study. dilutions of the phage stocks were done to get the working dilution factors. roughly, 106 pfu/ml of 20 µl phages (individual phages) was added to 180 µl of sm buffer, after an earlier adjustment of ph (2–11), in eppendorf tubes, followed by 30 min of incubation at 37 °c. the remaining phages were determined by spotting the phages in different ph and the outcomes indicated as pfu/ml.23 influence of ca2+ ions on phage titre and phage stability the effect of the divalent cations on bacterial lysis and phage adsorption was investigated by varying the concentration of cacl2: 0.000 m, 0.005 m, 0.010 m and 0.015 m in soft agar during preparation. plaque assays were conducted in duplicate, followed by overnight incubation at 37 °c. phage titre was determined at the different salt concentrations as log10 and comparisons made with the increase in salt concentrations.21 storage stability of enterobacter cloacae phages stability of the phages during storage was investigated using a previously described method with slight modifications.24 briefly, 3 ml of the selected phages with known phage titre were aliquoted into 15 ml centrifuge tubes and wrapped with aluminium foil to prevent direct light and kept at –20 °c, 4 °c, and 37 °c for two weeks. the spot test method was used to determine the effectiveness and the efficiency of the phages after storage. effect of ultraviolet light on phage titre the spot test method was used to determine the effect of ultraviolet light on phage irradiation with various modifications.25 briefly, 10 µl of each phage of known titre from each site was aliquoted into five sterile pcr tubes and labelled as 0, 5, 10, 15 and 20 min of ultraviolet light exposure. the phages for each time period were placed in a biosafety cabinet level 2 (290 nm – 320 nm, bsc-2, haier, tokyo, japan) and the ultraviolet light turned on for the required time; the phages were then removed simultaneously. polymerase chain reaction (pcr) tubes have been found to permeate ultraviolet light rays and affect the integrity of dna; hence, the tubes were capped during this study. host range determination of phages to ensure the specificity of bacteriophages, their effect on other bacterial genera and species was investigated. the anti-bactericidal efficacy of individual phages (n = 29) was evaluated through the spot test method against each individual e. cloacae isolate (n = 30) as described by kutter (2009).23 staphylococcus aureus, a bacteria from another genera, was used as negative control. a volume of 5 µl of individual phage stock was spotted on a tsa plate with a lawn of 100 µl overnight cultured host bacteria in soft agar, which was examined for bacterial lysis after 18 h – 24 h. the spot tests were performed in duplicate. a clear zone was considered as a positive infection in the tests and negative with no cross-reactivity.26 a phage was termed ‘potent’ upon lysing any bacterial strain in the host range panel. a phage with the widest spectrum of lysis activity on the tested bacterial strains was termed the ‘most potent’, while a phage with the lowest spectrum of activity on the tested bacterial strains was termed the ‘least potent’. a bacteria that was sensitive to a phage infection was termed ‘susceptible’. data analysis all the experiments were performed in triplicate and the mean values obtained. statistical entry was carried out and given treatment in microsoft excel 20 for windows (microsoft, redmond, washington, united states) and epidemiological information (epi info7tm, centers for disease control and prevention, atlanta, georgia, united states). one-way analysis of variance using statistical package for social sciences version 20 (spss inc., chicago, illinois, united states) was used to determine significant differences at p < 0.05. the surviving phage population obtained in each study were converted to log10 pfu/ml. data presentation was performed using graphpad prism version 5.00 for windows (graphpad software, san diego, california, united states). results antimicrobial susceptibility profile of the host bacterium (enterobacter cloacae) this isolate was resistant to five classes of antibiotics namely: beta-lactams, penicillins (ticarcillin or clavulanic acid, and piperacillin), cephalosporins (cefuroxime, cefuroxime axetil, ceftriaxone, and cefepime), monobactams (aztreonam), chloramphenicols (amphenicol), tetracycline (tetracycline, minocycline), quinolones (levofloxacin) and sulphonamides (trimethoprim). it was susceptible only to: meropenem (carbapenem) and tigelcycline (glycylcycline). isolated phages a total of 29 phage strains were isolated, with four phages from dandora, five phages from huruma, five phages from kibera, five phages from kariobangi, seven phages from korogocho and three phages from mathare. these phages were named using number-letter designations according to the sample source. for example, from kibera 1 settlement: the phages were named kibera 1a, 1b, and 1c. all the phages were subjected to host range studies. from the 29 phages isolated from the six sources, one phage from each source that had complete lytic properties and a lytic zone diameter of ≥ 3 mm was selected for study of physicochemical properties. effect of temperature on phage titre the isolated phages were stable from 4 °c to 50 °c (figure 1). there was a slight decrease in phage titre in four out of six phages at 50 °c, and two out of six phages had a slight increase in phage titre at 50 °c. in addition, one out of five phages had a constant titre from 4 °c to 30 °c. figure 1: effect of temperature on phages isolated in nairobi county, kenya, july 2019 to december 2020. phages were named based on source and phage strain number for example huruma 1b (p12) means huruma 1b source; phage strain number 12. effect of ph on phage titre no phages had any lytic activity at ph 2. all the phages were stable from ph 5 to 11 ph (slightly acidic to strong base). no phages had activity at ph 13 (figure 2). figure 2: effect of ph on phages isolated in nairobi county, kenya, july 2019 to december 2020. phages were named based on source and phage strain number for example huruma 1b (p12) means huruma 1b source; phage strain number 12. influence of ca2+ ions on phage titre and phage stability the addition of calcium chloride (0.002 m – 0.015 m) salt increased the adsorption rate and phage titre of phages in three out of five of the phages and a decrease in two out of five phages (figure 3). figure 3: effect of salt on phages isolated in nairobi county, kenya, july 2019 to december 2020. phages were named based on source and phage strain number, for example huruma 1b (p12) means huruma 1b source; phage strain number 12. storage stability of enterobacter cloacae phages all the phages were stable and efficacious at 4 °c and 37 °c. there was minimal or no activity at −20 °c following two weeks of storage (figure 4). figure 4: effect of storage on phage effectiveness and stability isolated in nairobi county, kenya, july 2019 to december 2020. the plates show the stability and lysis activity of: (a) huruma 1b phage, (b) dandora 3a phage, (c) kibera 1b phage, (d) korogocho 3a phage, (e) kariobangi 3b3 phage, and (f) mathare 1a phage at 4 °c, 37 °c and −20 °c following 2 weeks of storage. these phages were isolated in nairobi county, kenya, between july 2019 and december 2020. the phages were stable and efficacious at 4 °c and 37 °c with minimal or no activity at −20 °c. effect of ultraviolet light on phage titre exposure to ultraviolet light (290 nm – 320 nm) resulted in decreased phage titre from the 5th min to the 15th min. at the 20th min, there was no activity at all, which indicated that all the phages had been sterilised (figure 5). figure 5: effect of ultraviolet light exposure on phage survivability isolated in nairobi county, kenya, july 2019 to december 2020. the plates show the effect of ultraviolet light exposure from 0 min, 5 min, 10 min, 15 min and 20 min on: (a) huruma 1b phage, (b) dandora 3a phage, (c) kibera 1b phage, (d) korogocho 3a phage, (e) kariobangi 3b3 phage, and (f) mathare 1a phage isolated in nairobi county, kenya, between july 2019 and december 2020. the selected phages had decreased activity with increase in time from the 5th min to the 15th min, and no activity at all at the 20th min because all the phages had been destroyed. host range spot test analysis on enterobacter cloacae bacterial isolates all the isolated e. cloacae phages (n = 29) showed cross-reactivity against e. cloacae strains (n = 30) (figure 6). in order of individual phage potency from the highest to the least, the reactivity levels were: 67% (1 phage), 63% (3 phages), 60% (4 phages), 57% (5 phages), 53% (12 phages), 50% (1 phage), 47% (1 phage) and 27% (2 phages). according to the most susceptible bacteria to phages from the highest to the least, the following data were obtained: 100% (8), 97% (1), 93% (6), 86% (1), 79% (1), 31% (1), 21% (1), 14% (1), 10% (2), 7% (2) and 0% (7) (figure 7). figure 6: percentage potency of isolated phages on enterobacter cloacae strains isolated in nairobi county, kenya, july 2019 to december 2020. figure 7: susceptibility of enterobacter cloacae bacteria to isolated phages in nairobi county, kenya, july 2019 to december 2020. discussion this study found that the e. cloacae isolates from environmental waste water were resistant to five classes of antibiotics and hence termed as a mdr organisms. this is an indication that our environment habours mdr isolates that could be pathogenic to human and animal health. the antimicrobial profile of this isolate made it a good candidate for phage isolation. all of the isolated e. cloacae phages showed cross-reactivity against e. cloacae strains with the most potent phage lysing 67% of the bacterial strains and the least potent phage lysing 27% of the bacterial strains. a bacteria that was sensitive to a phage infection was termed as susceptible, with the highest susceptibility rate at 100% and the least susceptible at 0%. these results have similarities with previous studies done in india in 2019, on the host range of e. cloacae phage, which was able to lyse four species of the bacteria.27 additionally, in a study done in portugal in 2015, the use of three phages of e. cloacae as a cocktail for inactivation of urinary tract infections increased the potency of the phages in killing mdr e. cloacae.28 this specificity of the phages to the host bacteria is being exploited for therapeutic purposes in the treatment of various mdr bacteria.29 the specificity of the lytic activity is a characteristic that has been utilised for the development and production of novel therapeutic agents.11 host-range specificity in phage therapy is one of the major advantages for its success while it spares the commensal microbes from destruction during remedy.30 the specificity of the phages to their host bacteria is attributed to the phage host receptors involved in recognition, interaction and adsorption during attachment.31 additionally, the receptors are recognised by the ends of the virion’s long tail fibres of the phage towards the host bacteria.32 the stability of the e. cloacae phages obtained from this study varied from 4 °c to 50 °c. this stability concurs with e. cloacae phages previously isolated in lahore, pakistan.33 additionally, the phage titre fluctuated with different temperature conditions: there was a slight decrease in phage titre in four of six phages at 50 °c while two of six phages had a slight increase in phage titre at 50 °c. in addition, one of five of the phages had a constant titre from 4 °c to 30°c. the observed variation was also observed in a study done in iowa, united states, with the yield of phages being highly dependent on temperature.34,35 the elucidation of phage stability at different temperatures is needed to establish phage effectiveness as alternative therapeutic agents. from our ph stability studies, it is evident that the isolated e. cloacae phages were not stable in very acidic environments such as ph 2. this could be associated with the denaturation of the phage protein coat at low ph and stability being attained at basic ph above 5, with optimal activity in ph-neutral conditions (ph 7.5).36 however, the inactivation and reduction of the lytic activity of the phages decreased in high alkaline conditions (ph 11–13). this could be attributed to dissociation of the capsid protein due to high concentrations of hydrogen and hydroxyl ion in the solution.36 these findings concur with findings from previous studies done in portugal and poland with optimal phage stability at neutral ph (7.5).24,37 the ability of these phages to survive in the neutral ph (7.5) could be exploited or utilised in various applications such as sterilisation of hospital equipment, industrial mass production and for therapeutic purposes in patients with mdr infections of e. cloacae. in our study, there was increased activity of the isolated phages in 0.002 m and 0.015 m concentrations of ca2+ ions. but there was a slight drop of phage activity (1.79% drop) at 0.05 m ca2+ concentration in four out of six6 phages. in a study done in ireland in 2015, calcium was found to accelerate the phage lytic cycle with an impact on dairy fermentations.38 some phages require the cation for nucleic acid injection, efficient adsorption to cell wall binding sites and enhanced stability.38,39 the addition of the salts in our study corresponded with the above findings. salt availability also aids in the penetration processes of the phage genome into the host cytoplasm.39 a slight drop in phage titre of some phages might have been caused by the increase in growth of phage aggregates that might have resulted from neutralisation of the negatively charged moieties on the phage surface by cation binding with an increase in calcium salt concentration.40 all the phages in the current study were stable and efficacious at refrigerated temperature (4 °c) with optimal activity at human body temperature (37 °c) and minimal or no activity after being frozen (−20 °c) for two weeks. the crystal structure of ice destroys phages at −20 °c; hence, this storage is highly discouraged.41,42 the viability of phages at 4 °c has also been reported in other studies.24,37,43 a 5% – 10% glycerol addition to the phage suspension possibly warrants safe viability and infectivity for 30 days or longer-term storage at −20 °c or −70 °c.44 ultraviolet light is known to kill viruses and bacteria cells by disrupting their dna by damaging the thymine bases through creating a reaction between molecules or creating dimers.45 in our study, phages had decreased activity after exposure to ultraviolet light, and efficiency and efficacy decreased upon continuous sterilisation up to the 15th min and no activity at the 20th min. this effect of irradiation has also been reported in previous studies done in the united states in 2002 and 1947.25,46 in addition, a study conducted in china in 2020 reported that ultraviolet light potentially reduced phage titres in pathogen reduction quality.47 limitations in host range determination, a panel of 30 anonymised clinical isolates of e. cloacae and one staphylococcus aureus (control) were used for cross-reactivity studies. however, no antimicrobial susceptibility studies for these bacterial isolates were carried out. conclusion this study reveals the existence of the most potent lytic phages that are effective on mdr e. cloacae isolates found in kenya. the existence of diverse phage strains from the sampled areas provided an effective cocktail of phages that could be used as antimicrobial agents. findings from this study demonstrate that physicochemical properties influence the efficacy of phages in their antimicrobial activities and are worth considering. acknowledgements this work was inspired and partly funded by dr elizabeth (betty) kutter (the evergreen state college, united states). we appreciate ipr for the laboratory space and partial funding support. we acknowledge dr lilian musila (kemri, center for microbiology) for laboratory technical support. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions all authors approved the study and submission. i.j.m., a.k.n. and a.n. conceived the idea. i.j.m. carried out the experiment supervised by a.k.n. and a.n., a.a.j. and m.i.i. offered technical support. i.j.m., a.k.n. and a.n. wrote the manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability the authors confirm that the data supporting the findings of this study are available within the article. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references mezzatesta ml, gona f, stefani s. enterobacter cloacae complex: clinical impact and emerging antibiotic resistance. future microbiol. 2012;7(7):887–902. https://doi.org/10.2217/fmb.12.61 salimiyan rizi k, ghazvini k, farsiani h. clinical and pathogenesis overview of enterobacter infections. rev clin med. 2020;6(4):146–154. allegranzi b, nejad sb, combescure c, et al. burden of endemic health-care-associated infection in developing countries: systematic review and meta-analysis. lancet. 2011;377(9761):228–141. https://doi.org/10.1016/s0140-6736(10)61458-4 davin-regli a, lavigne j-p, pagès j-m. enterobacter spp.: update on taxonomy, clinical aspects, and emerging antimicrobial resistance. clin microbiol rev. 2019;32(4):e00002–e00019. https://doi.org/10.1128/cmr.00002-19 annavajhala mk, gomez-simmonds a, uhlemann a-c. multidrug-resistant enterobacter cloacae complex emerging as a global, diversifying threat. front microbiol. 2019;10:44. https://doi.org/10.3389/fmicb.2019.00044 rauwers aw, voor in ’t holt af, buijs jg, et al. high prevalence rate of digestive tract bacteria in duodenoscopes: a nationwide study. gut. 2018;67(9):1637–1645. https://doi.org/10.1136/gutjnl-2017-315082 nyangacha rm, odongo d, oyieke f, et al. secondary bacterial infections and antibiotic resistance among tungiasis patients in western, kenya. plos negl trop dis. 2017;11(9):e0005901. https://doi.org/10.1371/journal.pntd.0005901 nishida s, matsunaga n, kamimura y, ishigaki s, furukawa t, ono y. emergence of enterobacter cloacae complex co-producing imp-10 and ctx-m, and klebsiella pneumoniae producing vim-1 in clinical isolates in japan. microorganisms. 2020;8(11):1816. https://doi.org/10.3390/microorganisms8111816 letarov av, kulikov ee. determination of the bacteriophage host range: culture-based approach. bacteriophage therapy. methods mol biol. 2018;1693:75–84. https://doi.org/10.1007/978-1-4939-7395-8_7 cai y, chen c, zhao m, et al. high prevalence of metallo-β-lactamase-producing enterobacter cloacae from three tertiary hospitals in china. front microbiol. 2019;10:1610. https://doi.org/10.3389/fmicb.2019.01610 wang i-n. lysis timing and bacteriophage fitness. genetics. 2006;172(1):17–26. https://doi.org/10.1534/genetics.105.045922 abatángelo v, bacci np, boncompain ca, et al. broad-range lytic bacteriophages that kill staphylococcus aureus local field strains. plos one. 2017;12(7):e0181671. https://doi.org/10.1371/journal.pone.0181671 oduor jmo, onkoba n, maloba f, nyachieo a. experimental phage therapy against haematogenous multi-drug resistant staphylococcus aureus pneumonia in mice. afr j lab med. 2016;5(1):435. https://doi.org/10.4102/ajlm.v5i1.435 nyachieo a. phage training reaches east africa. phage directory. 2019;54(1):1–15. chang y, shin h, lee j-h, park cj, paik s-y, ryu s. isolation and genome characterization of the virulent staphylococcus aureus bacteriophage sa97. viruses. 2015;7(10):5225–5242. https://doi.org/10.3390/v7102870 abbas ya, radhi gf. rapid identification of enterobacter spp. islated from hospitals in basrah province by automated system (vitek®2 compact). ea j. 2016;1(1): 9–20. akhtar m, viazis s, diez-gonzalez f. isolation, identification and characterization of lytic, wide host range bacteriophages from waste effluents against salmonella enterica serovars. food contr. 2014;38:67–74. https://doi.org/10.1016/j.foodcont.2013.09.064 clokie mrj, kropinski a, editors. bacteriophages: methods and protocols, volume 1: isolation, characterization, and interactions. softcover reprint of hardcover. 1st ed. new york, ny: humana; 2010, 330 p. wommack ke, williamson ke, helton rr, bench sr, winget dm. methods for the isolation of viruses from environmental samples. in: clokie mrj, kropinski am, editors. bacteriophages: methods and protocols, volume 1: isolation, characterization, and interactions. totowa, nj: humana press; 2009, p. 3–14. https://doi.org/10.1007/978-1-60327-164-6_1 lu z, breidt f. escherichia coli o157:h7 bacteriophage φ241 isolated from an industrial cucumber fermentation at high acidity and salinity. front microbiol. 2015;6:67. https://doi.org/10.3389/fmicb.2015.00067 capra ml, quiberoni a, reinheimer j. phages of lactobacillus casei/paracasei: response to environmental factors and interaction with collection and commercial strains. j appl microbiol. 2006;100(2):334–342. https://doi.org/10.1111/j.1365-2672.2005.02767.x adams mh. the stability of bacterial viruses in solutions of salts. j gen physiol. 1949;32(5):579–594. https://doi.org/10.1085/jgp.32.5.579 kutter e. phage host range and efficiency of plating. in: clokie mrj, kropinski am, editors. bacteriophages: methods and protocols, volume 1: isolation, characterization, and interactions. totowa, nj: humana press; 2009, p. 141–149. jończyk e, kłak m, międzybrodzki r, górski a. the influence of external factors on bacteriophages-review. folia microbiol. 2011;56(3):191–200. https://doi.org/10.1007/s12223-011-0039-8 greene gi, babel fj. effect of ultraviolet irradiation on bacteriophage active against streptococcus lactis1. j dairy sci. 1948;31(6):509–515. https://doi.org/10.3168/jds.s0022-0302(48)92234-6 havelaar ah, hogeboom wm. factors affecting the enumeration of coliphages in sewage and sewage-polluted waters. antonie van leeuwenhoek. 1983;49(4–5):387–397. manohar p, tamhankar aj, lundborg cs, nachimuthu r. therapeutic characterization and efficacy of bacteriophage cocktails infecting escherichia coli, klebsiella pneumoniae, and enterobacter species. front microbiol. 2019;10:574. https://doi.org/10.3389/fmicb.2019.00574 pereira s, pereira c, santos l, klumpp j, almeida a. potential of phage cocktails in the inactivation of enterobacter cloacae – an in vitro study in a buffer solution and in urine samples. virus res. 2016;211:199–208. https://doi.org/10.1016/j.virusres.2015.10.025 popova av, zhilenkov el, myakinina vp, krasilnikova vm, volozhantsev nv. isolation and characterization of wide host range lytic bacteriophage ap22 infecting acinetobacter baumannii. fems microbiol lett. 2012;332(1):40–46. https://doi.org/10.1111/j.1574-6968.2012.02573.x gill jj, hyman p. phage choice, isolation, and preparation for phage therapy. curr pharm biotechnol. 2010;11(1):2–14. https://doi.org/10.2174/138920110790725311 bertozzi silva j, storms z, sauvageau d. host receptors for bacteriophage adsorption. fems microbiol lett. 2016;363(4). https://doi.org/10.1093/femsle/fnw002 drexler k, riede i, montag d, eschbach ml, henning u. receptor specificity of the escherichia coli t-even type phage ox2. mutational alterations in host range mutants. j mol biol. 1989;207(4):797–803. https://doi.org/10.1016/0022-2836(89)90245-3 khawaja k, abbas z, rehman s. isolation and characterization of lytic phagestse1-3 against enterobacter cloacae. open life sci. 2016;11(1):287–292. https://doi.org/10.1515/biol-2016-0038 wilkowske hh, nelson fe, parmelee ce. heat inactivation of bacteriophage strains active against lactic streptococci. appl microbiol. 1954;2(5):250–253. https://doi.org/10.1128/am.2.5.250-253.1954 taj m, ling j, bing l, et al. effect of dilution, temperature and ph on the lysis activity of t4 phage against e. coli bl21. j anim plant sci. 2014;24(4):2014–1252. feng yy, ong sl, hu jy, tan xl, ng wj. effects of ph and temperature on the survival of coliphages ms2 and qbeta. j indian microbiol biotechnol. 2003;30(9):549–552. https://doi.org/10.1007/s10295-003-0080-y nobrega fl, costa ar, santos jf, et al. genetically manipulated phages with improved ph resistance for oral administration in veterinary medicine. sci rep. 2016;6(1):39235. https://doi.org/10.1038/srep39235 mahony j, tremblay dm, labrie sj, moineau s, van sinderen d. investigating the requirement for calcium during lactococcal phage infection. int j food microbiol. 2015;201:47–51. https://doi.org/10.1016/j.ijfoodmicro.2015.02.017 chhibber s, kaur t, kaur s. essential role of calcium in the infection process of broad-spectrum methicillin-resistant staphylococcus aureus bacteriophage. j basic microbiol. 2014;54(8):775–780. https://doi.org/10.1002/jobm.201300051 ul haq i, chaudhry wn, andleeb s, qadri i. isolation and partial characterization of a virulent bacteriophage ihq1 specific for aeromonas punctata from stream water. microb ecol. 2012;63(4):954–963. https://doi.org/10.1007/s00248-011-9944-2 gould ea. methods for long-term virus preservation. mol biotechnol. 1999;13(1):57–66. https://doi.org/10.1385/mb:13:1:57 warren jc, hatch mt. survival of t3 coliphage in varied extracellular environments. i. viability of the coliphage during storage and in aerosols1. appl microbiol. 1969;17(2):256–261. https://doi.org/10.1128/am.17.2.256-261.1969 litt pk, jaroni d. isolation and physiomorphological characterization of escherichia coli o157:h7-infecting bacteriophages recovered from beef cattle operations. int j microbiol. 2017;2017:7013236. https://doi.org/10.1155/2017/7013236 olson mr, axler rp, hicks re. effects of freezing and storage temperature on ms2 viability. j virol methods. 2004;122(2):147–152. https://doi.org/10.1016/j.jviromet.2004.08.010 cutler td, zimmerman jj. ultraviolet irradiation and the mechanisms underlying its inactivation of infectious agents. anim health res rev. 2011;12(1):15–23. https://doi.org/10.1017/s1466252311000016 hazem a. effects of temperatures, ph-values, ultra-violet light, ethanol and chloroform on the growth of isolated thermophilic bacillus phages. new microbiol. 2002;25(4):469–476. yin y, li l, gong l, xu h, liu z. effects of riboflavin and ultraviolet light treatment on pathogen reduction and platelets. transfusion. 2020;60(11):2647–2654. https://doi.org/10.1111/trf.16053 abstract introduction methods results discussion acknowledgements references about the author(s) kamela l. mahlakwane division of medical virology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa division of medical virology, tygerberg hospital, national health laboratory service, cape town, south africa wolfgang preiser division of medical virology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa division of medical virology, tygerberg hospital, national health laboratory service, cape town, south africa nokwazi nkosi division of medical virology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa division of medical virology, tygerberg hospital, national health laboratory service, cape town, south africa nasheen naidoo division of clinical pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africadivision of clinical pathology, tygerberg hospital, national health laboratory service, cape town, south africa gert van zyl division of medical virology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa division of medical virology, tygerberg hospital, national health laboratory service, cape town, south africa citation mahlakwane kl, preiser w, nkosi n, naidoo n, van zyl g. delays in hiv-1 infant polymerase chain reaction testing may leave children without confirmed diagnoses in the western cape province, south africa. afr j lab med. 2022;11(1), a1485. https://doi.org/10.4102/ajlm.v11i1.1485 original research delays in hiv-1 infant polymerase chain reaction testing may leave children without confirmed diagnoses in the western cape province, south africa kamela l. mahlakwane, wolfgang preiser, nokwazi nkosi, nasheen naidoo, gert van zyl received: 04 dec. 2020; accepted: 24 mar. 2022; published: 23 june 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: early diagnosis and confirmation of hiv infection in newborns is crucial for expedited initiation of antiretroviral therapy. confirmatory testing must be done for all children with a reactive hiv pcr result. there is no comprehensive data on confirmatory testing and hiv pcr test request rejections at national health laboratory service laboratories in south africa. objective: this study assessed the metrics of routine infant hiv pcr testing at the tygerberg hospital virology laboratory, cape town, western cape, south africa, including the proportion of rejected test requests, turn-around time (tat), and rate of confirmatory testing. methods: we retrospectively reviewed laboratory-based data on all hiv pcr tests performed on children ≤ 24 months old (n = 43 346) and data on rejected hiv pcr requests (n = 1479) at the tygerberg virology laboratory over two years (2017–2019). data from sample collection to release of results were analysed to assess the tat and follow-up patterns. results: the proportion of rejected hiv pcr requests was 3.3%; 83.9% of these were rejected for various pre-analytical reasons. most of the test results (89.2%) met the required 96-h tat. of the reactive initial test results, 53.5% had a follow-up sample tested, of which 93.1% were positive. of the initial indeterminate results, 74.7% were negative on follow-up testing. conclusion: a high proportion of hiv pcr requests were rejected for pre-analytical reasons. the high number of initial reactive tests without evidence of follow-up suggests that a shorter tat is required to allow confirmatory testing before children are discharged. keywords: infant hiv pcr; confirmatory testing; early infant diagnosis; eid; laboratory diagnosis; antiretroviral therapy; turn-around time; follow-up testing. introduction the early diagnosis of hiv infection in infants is very important.1,2 infants who are initiated on antiretroviral therapy (art) within seven days of life are four times more likely to achieve early viral suppression than those who commence art later.3,4 the south african national guidelines on the prevention of mother-to-child transmission of hiv recommend that hiv polymerase chain reaction (pcr) tests be conducted within 3–6 days of birth and that art should be immediately initiated in all children with detectable hiv nucleic acid while awaiting the follow-up hiv pcr results.5 early initiation of art is associated with improved virological, immunological, and clinical outcomes.6,7,8,9,10 early diagnosis of hiv in infants is done by testing for viral nucleic acid (dna and/or rna) in blood samples, usually by pcr.11 current guidelines, including those issued by the western cape department of health and south african national department of health, recommend the testing of all hiv-exposed infants by pcr within seven days of birth (i.e., birth pcr), at approximately 10 weeks of age for those who tested negative at birth, at 18 weeks for high-risk infants who received extended prophylactic art for 12 weeks, at six months, and after cessation of breastfeeding.5,12,13 the post-breastfeeding hiv test may either be by pcr if the child is younger than 18 months of age or by serology if the child is older.5,11,12 the world health organization recommends that hiv antibody testing be done after at least three months post-breastfeeding to allow for hiv antibody development and to prevent missing hiv infection in the last few days of breastfeeding.13 a negative antibody test in an infant older than 18 months is confirmation that the infant is not infected.13 although the world health organization recommends hiv pcr testing for infants ≤ 18 months old, the age cut-off for hiv pcr testing at national health laboratory service (nhls) laboratories is 24 months rather than 18 months. this is based on studies that showed that maternal hiv antibody clearance took longer than 18 months in some perinatally exposed infants, with seroreversion rates of 89.3%, 94.2%, and 100.0% at ages 12, 18, and 24 months, respectively.14,15 nevertheless, this policy has not been formalised in the laboratory diagnostic booklet, the western cape provincial testing guidelines, or the national department of health hiv testing guidelines. given the consequences of hiv infection (such as lifelong art), a follow-up sample must be collected as soon as possible from all children with a reactive infant pcr result to confirm the diagnosis or detect false-positive initial results.11,16 however, a negative test result following an initial reactive result does not necessarily constitute false positivity, particularly in infants who are on prophylaxis or art. clinical actions taken based on false-positive results may have dire biomedical (exposure to drug toxicity and side effects), psychosocial (possible stigma and negative impact on life outlook and future relationships), financial (art costs), and medico-legal implications.16 once initiated on art, it may become impossible to distinguish a virally suppressed child from one who was never infected.4 in south africa, the nhls provides laboratory testing for the public health sector, providing healthcare for approximately 85% of the population who do not have medical insurance.17 the nhls aims to issue results for at least 80% of infant pcr tests within a 96-h turn-around time (tat).18 this tat is defined by the nhls19 as the time interval between sample reception and the release of results by a virologist (i.e., laboratory tat). this tat does not consider the periods between sample collection and reception, or between the review of results and the receipt of results by the patient. for this study, we refer to the period between sample collection and authorisation of results as ‘clinical tat’. the nhls performs all hiv early infant diagnosis testing using the roche® cobas® ampliprep/cobas® taqman® system (roche® molecular systems, inc., branchburg, new jersey, united states) in a network of centralised laboratories.17 results of this assay could be positive, negative, or indeterminate. indeterminate qualitative hiv pcr results on the roche® cobas® ampliprep/cobas® taqman® system were first described in the tygerberg hospital virology laboratory in 2012.20,21 during the period covered by this study, indeterminate results were defined as a cycle threshold of ≥ 33, or a relative fluorescence intensity below five.22 these diagnostic criteria have since been revised,23,24,25,26 mostly based on collated data, which showed cycle threshold value cut-offs to be the best predictor of reproducible positive results. the cycle threshold value of 33 was shown to most precisely differentiate clear positive from irreproducible cases.23 in the tygerberg virology laboratory, hiv pcr testing is done only once on each sample unless the results are invalid, in which case a single retest is done on the remnant sample. samples that produce invalid results for the second time are rejected with this reason attached: ‘failed after repeated attempts’. all other results (i.e., negative, positive, or indeterminate) are released as such, with no repeat testing of the remnant sample. the world health organization reports that most women and their newborn infants are likely to be discharged within one to two days following uncomplicated vaginal delivery, and within two to four days following uncomplicated caesarean delivery.27 thus, infant birth hiv pcr results should, ideally, be available before the child gets discharged to allow immediate optimal management (prophylaxis, art, or confirmatory testing). hiv pcr tat strategies should thus seek to address this challenge. in this study, a laboratory-based retrospective review was conducted to determine the proportion of rejected infant hiv pcr requests, assess laboratory conformance with tat requirements, and assess whether there were confirmatory tests done following reactive infant hiv pcr results performed on the roche® cobas® ampliprep/cobas® taqman® system. methods ethical considerations this study was approved by the health research ethics committee (hrec) of stellenbosch university (reference number: s19/03/053). the hrec does not require patient consent when residual clinical samples are used for research if the research findings will not impact the patient or change their management in any way. access to the extracted laboratory data was limited to the researchers. study design and setting this is a retrospective review of data generated at the tygerberg hospital virology laboratory in cape town, western cape, south africa, between july 2017 and june 2019. the data was downloaded from the laboratory information system (lis) of the tygerberg laboratory (trakcare lab, intersystems corporation, cambridge, massachusetts, united states). the tygerberg virology laboratory renders diagnostic virological pathology services to tygerberg hospital and other health facilities from its drainage areas. all samples received in the laboratory go through the routine process of sample reception, registration and analysis, as well as review of results. all hiv-1 qualitative pcr results from ethylenediaminetetraacetic acid-anticoagulated whole blood and dried blood spot samples from children aged ≤ 24 months that were tested at the tygerberg virology laboratory between 01 july 2017 and 30 june 2019 were included in this study. samples from patients older than 24 months (n = 534) or with unknown age (n = 287), quality control samples (n = 29), and samples with coded names or surnames (n = 15) were excluded. a total of 43 346 tests were included in this study. data collection rejection of test requests a raw data set of rejected hiv pcr requests for children aged ≤ 24 months was extracted from the lis (n = 1479) and assessed to determine the pattern of hiv pcr request rejections by the laboratory. infant pcr test requests may be rejected for various reasons, as included in the lis and nhls standard operating procedure for sample rejection. these reasons may be pre-analytical (e.g. ‘unsuitable age’ for children aged > 24 months) or analytical (e.g. ‘laboratory error’). samples from other peripheral laboratories are referred both digitally and physically. digital referrals that are not accompanied by the sample are rejected as ‘lost in transit’ or ‘specimen not received’ after consultation with the referring laboratory or the requesting clinician. all ethylenediaminetetraacetic acid-anticoagulated samples are discarded after seven days from the date of collection. samples that are not tested within this period are rejected as ‘sample too old’. the term ‘insufficient specimen’ refers to remnant samples with initial invalid results and insufficient sample volume for repeat testing. the classification of these rejections as pre-analytical is, therefore, arbitrary. turn-around time laboratory tat was calculated based on the nhls definition,19 while clinical tat was analysed as earlier defined. the time points were extracted from the lis as recorded during each step of the testing process. the results were then stratified using a 96-h tat cut-off. follow-up testing all results were grouped using the unique laboratory code, that is, the medical record number, which is assigned to each patient and remains the same for all subsequent test requests. the time-to-follow-up was calculated as the number of days between the first and second sample collection dates as recorded on the lis. in line with the guidelines, all positive and indeterminate results were expected to have a follow-up sample sent for confirmatory testing as soon as possible, while all the negative samples were expected to be followed up as per schedule. negative follow-up test results were further checked to determine if a third follow-up sample was tested. data analysis data were analysed using microsoft excel 2010 version 14 (microsoft corporation, redmond, washington, united states). rejected test requests were stratified by reason for rejection and further summarised into pre-analytical and analytical reasons for rejection. results rejected test sets out of the 1479 rejected samples, 1241 (83.9%) were rejected for pre-analytical reasons, and 238 (16.1%) for analytical reasons (figure 1). ‘duplicate requests’ (21.3%), ‘insufficient specimen’ (21.1%), and ‘specimen not received’ (16.1%) were the most common pre-analytical reasons for sample rejections (table 1). other reasons that each constituted less than 1% of the reasons for test rejection were grouped as ‘other various reasons’. figure 1: pre-analytical and analytical reasons for hiv polymerase chain reaction test rejection at tygerberg hospital, cape town, south africa, between july 2017 and june 2019. table 1: reasons for rejection of hiv polymerase chain reaction test requests at tygerberg hospital, cape town, south africa, between july 2017 and june 2019. demographics of the 43 346 samples that were tested for infant hiv pcr, 27 978 (64.5%) were the patients’ first or initial hiv pcr samples, while 18 393 (42.4%) samples were collected at birth. among the 9585 patients who were tested for the first time after seven days of life (i.e. beyond birth pcr), 5715 (59.6%) were tested between day eight and week 12 of life (median = 10 weeks; interquartile range [iqr] = 8–11 weeks). the proportions of male (49.9%) and female patients (49.8%) were similar, with the sex of 149 (0.3%) patients being unknown. turn-around time in total 38 653 (89.2%) test results were within the 96-h laboratory tat cut-off (median = 44 h; iqr = 31–64), while the remaining 4693 (10.8%) were not (median = 116 h; iqr = 106–137) (figure 2). figure 2: numbers of hiv pcr requests, rejected samples, and follow-up tests conducted at tygerberg hospital, cape town, south africa, between july 2017 and june 2019. however, for the clinical tat, only 33 245 (76.7%) samples were signed out within 96 h (median = 57 h; iqr = 47–73), while 10 101 (23.3%) test results were released after 96 h (median = 119 h; iqr = 105–140). the median tat from sample collection to sample registration was 17 h (iqr = 8–24). follow-up hiv pcr test results of the 520 (1.9%) reactive initial results, approximately half were followed up by testing of a subsequent sample. similarly, 9409 (50.3%) of the initial negative results had subsequent samples tested, with only 1264 (13.4%) of these tested at 10 weeks of age (6–12 weeks of life) as per the national hiv testing guidelines. nine (0.7%) of these 10-week samples had positive or indeterminate results. it should be noted that some of the follow-up results may have not been linked to the initial samples and may have been missed in this analysis. of the 520 initial reactive tests, 251 (48.3%) were tested at birth, with 62.9% (n = 158) of those subjected to confirmatory testing. the remaining 269 (51.7%) were tested after seven days of life, and 44.6% (n = 120) of those had confirmatory testing conducted. the reactive results were further analysed to assess the agreement between initial and confirmatory results. one hundred and eighty-nine (93.1%) patients with initial positive results were also positive following confirmatory testing, while five (2.5%) were indeterminate, and nine (4.4%) had discordant results (negative on follow-up testing) (table 2). further scrutiny of these nine results revealed that all of them had a third follow-up hiv pcr test done, with four testing positive and five testing negative. of the 75 patients with initial indeterminate results who had a confirmatory sample tested, 56 (74.7%) had negative results. of these, 30 (53.6%) had a third hiv pcr follow-up test done, 24 (42.9%) had no third follow-up hiv pcr or hiv viral load test conducted, while two patients had hiv viral load follow-up tests conducted, rather than an hiv pcr test (table 3). among the 30 patients who had a third hiv pcr test conducted, 29 (96.7%) were negative. one of two patients who had hiv viral load follow-up tests done had detectable hiv viral load (862 328 copies per millilitre). the presence of detectable hiv nucleic acid in this quantitative molecular test is confirmatory of hiv infection. table 2: comparison of results of the initial reactive infant polymerase chain reaction tests and confirmatory tests conducted at tygerberg hospital, cape town, south africa, between july 2017 and june 2019. table 3: follow-up tests for samples that initially tested indeterminate at tygerberg hospital, cape town, south africa, between july 2017 and june 2019. urgency of follow-up samples of the 278 follow-up tests conducted, 147 (52.9%) were performed more than seven days after the initial test (mean: 82 days; range: 8–606 days). among the 131 (47.1%) follow-up samples tested within the first seven days of life, only 52 (39.7%) were tested within three days of the initial test. discussion in this study, we determined that 3.3% of infant hiv pcr requests were rejected, and that the majority of the test results met the tat requirements. infant hiv pcr requests were rejected for various reasons, mainly due to duplicated requests, insufficient sample volumes, and loss of specimen in transit. these rejections may occur when more than one clinician requests the same test for the same patient, when a sample is received in the laboratory with a volume lower than that required for the test, or when test requests are not accompanied by the patient’s sample. the proportion of rejected samples is concerningly high and negatively impacts the early diagnosis and management of hiv-positive infants. comparative data from other settings is limited, thus making it difficult to determine the rejection threshold across laboratories. an overall appraisal of the tat showed that the tygerberg virology laboratory meets the standards set by the nhls national strategic plan. however, the average laboratory tat was longer when only positive and indeterminate results, which were the focus of our study, were considered; 14.9% of these results were released after the required 96 h. the clinical tat was also longer, with only 76.7% of results released within 96 h. the clinical tat may thus require some revisions to enable the expedition of early infant diagnosis and art initiation. point-of-care hiv pcr testing has been shown to accomplish same-day diagnosis for infants,28 with a significantly higher number of infants accessing confirmatory testing,29 thereby assisting with rapid art initiation. this study also found that only 39.7% of the reactive confirmatory tests were done within three days after the initial test. on average, most newborns would have been discharged from the hospital within three days of birth. if hiv birth pcr test results are received within this period, newborns with positive results are likely to be initiated on art before being discharged, thus reducing the number of patients lost to follow-up.28 although new mothers are required to visit the clinic within 6 days after birth, the 6-day maternal postnatal clinic visit was reported to be at 58.0% in the western cape between 2017 and 2019.30 the delay in follow-up testing (over seven days after initial testing) in 59.3% of patients in this study may be as a result of this low uptake or because our 96-h tat only accounts for the processes within the laboratory. while 1.9% of initial test results were reactive, only 53.5% of these had the prescribed confirmatory test done. it is unknown whether the other reactive results (46.5%) had confirmatory tests done, and this may mean that many hiv-positive patients may not have been initiated on art, or that a substantial proportion would have been started on art without confirmatory test results, effectively exposing some uninfected children to lifelong art. the 53.5% follow-up test rate is similar to the 53.0% confirmatory test rate reported in a study in tshwane, south africa.31 this proportion may be different from what is seen in clinical practice31 as some of the patients, particularly those aged > 18 months, may have had either hiv viral load or serological follow-up tests. these patients would have been missed in our study, as we focused specifically on hiv pcr results. the discordant negative results in 4.4% of patients with an initial reactive result indicate that these initial reactive results may have been false reactive or may be due to a rapid decline in hiv nucleic acid concentration following exposure to highly active art in infants receiving antiretroviral drugs (as prevention of mother-to-child transmission or combination art), making it difficult to make a definitive diagnosis of hiv infection. most children with indeterminate results tested negative on subsequent follow-up tests. again, this could either indicate that a large proportion of these indeterminate results were initially false reactive, or may indicate rapid hiv nucleic acid decay in children who are receiving art for prophylaxis, which may be two or three drugs in high-risk cases.4,32 it should, however, be noted that the indeterminate cut-offs used in this study were based on the reproducibility of a particular assay, with a particular chemistry and software, and within a different prevention of mother-to-child transmission context. these were later improved. recently, hiv diagnostic criteria have been revised to address this diagnostic dilemma.23,24,25,26 only 13.4% of patients with negative birth pcr results received a follow-up test within 10 weeks as per schedule; about half (50.3%) were later followed up after 10 weeks. this is consistent with other similar local studies on 10-week hiv pcr follow-up tests.33 at ten weeks, 0.7% of the infants in this study tested positive or indeterminate on hiv pcr, which is lower than the national hiv prevalence of 0.9% and the national strategic plan target of 1.3% at 10 weeks.30 it is, however, higher than the reported western cape rate of 0.5%.30 it should be noted that while these patients could have been missed at birth following intrauterine infection, they also could have been infected either perinatally or postnatally. just over half of the infants who required confirmatory pcr testing received such tests. it remains unclear whether the patients who were not followed up were hiv positive or not. a small proportion of babies received the 10-week follow-up test. the patients who did not receive this test may only be seen in the hospital later in life when they are sick. clinician education on hiv testing guidelines may help to reduce this number, and strengthening systems to reduce the time between first and confirmatory hiv pcr tests could also be beneficial. most of the indeterminate results were negative on the second and third follow-up tests. it was not known if patients were receiving prevention of mother-to-child transmission regimens or combination art during the periods of observation, as that may have resulted in undetectable hiv nucleic acid. the new early infant diagnosis criteria, as well as a separate and independent hiv pcr testing platform, may help resolve the challenges presented by indeterminate hiv pcr results. limitations our interpretation of the tat data did not account for the current workflow practice, in which hiv pcr testing is processed only during weekdays (monday to friday). the fact that no tests are done on weekends may result in tat variability. this study is thus not indicative of laboratories that operate a shift system (i.e., a 24-h service). occasionally, hiv pcr results also fail to transmit to the lis, further delaying the tat. another limitation of the study was the classification of indeterminate results using outdated criteria that have since been revised. some of these results may now be re-classified as positive. follow-up tests were reconciled using a unique laboratory code (medical record number) for each patient. however, we noted that some patients may have erroneously had more than one medical record number. this could have occurred in instances where samples from babies were labelled with the mother’s name (i.e., baby of xyz) and then later labelled with the baby’s name, resulting in non-reconciled patient profiles. this might have contributed to the under-reporting of follow-up tests, as these would have been missed. we, however, compared data provided on actual request forms with those captured by the lis and found them to match. also, some patients could have moved to other health facilities outside of our testing site. it must be noted that this study was limited to the tygerberg hospital virology laboratory, and thus may not be representative of the whole western cape region (i.e. groote schuur hospital and green point complex). the magnitude of this data concern is unknown due to our inability to assess the number of follow-up results that were not linked with the initial patient results. conclusion a high proportion of infant hiv pcr requests were rejected for various reasons. hiv pcr testing tat at the tygerberg laboratory is at par with the national requirements. however, this 96-h laboratory tat may need to be revised as it may negatively impact the number of children who receive confirmatory testing after a positive result. a larger study is recommended for a clearer appreciation of the hiv pcr testing challenges. acknowledgements the authors of this manuscript would like to extend their gratitude to the national health laboratory service for allowing them to use its data. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions k.l.m. conceptualised the research project, carried out the investigation and analytical calculations, and wrote the original draft. w.p. supervised the project and edited and reviewed the final version of the manuscript. n. nkosi provided critical feedback and helped shape the research, analyse the data, and edit the manuscript. n. naidoo helped with the analytical calculations and the drafting and editing of the manuscript. g.v.z. supervised the project and helped with the editing and review of the final version of the manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the data that support the findings of this study are available on request from the corresponding author, k.l.m. the data are not publicly available due to their containing information that could compromise the privacy of patients whose samples were used in this research project. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the national health laboratory service or university of stellenbosch. references mofenson lm, cohn j, sacks e. challenges in the early infant hiv diagnosis and treatment cascade. j acquir immune defic syndr. 2020;(84 suppl 1):s1–s4. https://doi.org/10.1097/qai.0000000000002366 webb k, chitiyo v, mahachi n, et al. brief report: improving early infant diagnosis observations: estimates of timely hiv testing and mortality among hiv-exposed infants. j acquir immune defic syndr. 2020;83(3):235–239. https://doi.org/10.1097/qai.0000000000002263 dominguez-rodriguez s, tagarro a, palma p, et al. reduced time to suppression among neonates with hiv initiating antiretroviral therapy within 7 days after birth. j acquir immune defic syndr. 2019;82(5):483–490. https://doi.org/10.1097/qai.0000000000002188 veldsman ka, maritz j, isaacs s, et al. rapid decline of hiv-1 dna and rna in infants starting very early antiretroviral therapy may pose a diagnostic challenge. aids. 2018;32(5):629–634. https://doi.org/10.1097/qad.0000000000001739 department of health. guideline for the prevention of mother to child transmission of communicable infections. health, editor. pretoria: doh; 2019. lilian rr, kalk e, technau kg, sherman gg. birth diagnosis of hiv infection in infants to reduce infant mortality and monitor for elimination of mother-to-child transmission. pediatr infect dis j. 2013;32(10):10801085. https://doi.org/10.1097/inf.0b013e318290622e violari a, cotton mf, kuhn l, et al. a child with perinatal hiv infection and long-term sustained virological control following antiretroviral treatment cessation. nat commun. 2019;10(1):412. https://doi.org/10.1038/s41467-019-08311-0 marston m, becquet r, zaba b, et al. net survival of perinatally and postnatally hiv-infected children: a pooled analysis of individual data from sub-saharan africa. int j epidemiol. 2011;40(2):385–396. https://doi.org/10.1093/ije/dyq255 cotton mf, violari a, otwombe k, et al. early time-limited antiretroviral therapy versus deferred therapy in south african infants infected with hiv: results from the children with hiv early antiretroviral (cher) randomised trial. lancet. 2013;382(9904):1555–1563. https://doi.org/10.1016/s0140-6736(13)61409-9 frigati l, wynberg e, maritz j, holgate s, cotton mf, rabie h. antiretroviral treatment initiated in the first month of life. pediatr infect dis j. 2017;36(6):584–587. https://doi.org/10.1097/inf.0000000000001504 world health organization. early detection of hiv infection in infants and children – guidance note on the selection of technology for the early diagnosis of hiv in infants and children. geneva: world health organization; 2010. mazanderani ah, moyo f, kufa t, sherman gg. brief report: declining baseline viremia and escalating discordant hiv-1 confirmatory results within south africa’s early infant diagnosis program, 2010–2016. j acquir immune defic syndr. 2018;77(2):212–216. https://doi.org/10.1097/qai.0000000000001581 world health organization. updated recommendations on hiv prevention, infant diagnosis, antiretroviral initiation and monitoring. geneva: who; 2021. baroncelli s, galluzzo cm, liotta g, et al. dynamics of immunoglobulin g subclasses during the first two years of life in malawian infants born to hiv-positive mothers. bmc pediatr. 2020;20(1):181. https://doi.org/10.1186/s12887-020-02091-z liu a, zhang l, zhang x, et al. delayed seroreversion of specifical antibody against hiv in hiv-exposed infants: a retrospective cohort study. hiv med. 2020;21(11):718–721. https://doi.org/10.1111/hiv.13026 dunning l, francke ja, mallampati d, et al. the value of confirmatory testing in early infant hiv diagnosis programmes in south africa: a cost-effectiveness analysis. plos med. 2017;14(11):e1002446. sherman gg, lilian rr, bhardwaj s, candy s, barron p. laboratory information system data demonstrate successful implementation of the prevention of mother-to-child transmission programme in south africa. s afr med j. 2014;104(3 suppl 1):235–238. https://doi.org/10.7196/samj.7598 national health laboratory service. national laboratory health service strategicplan for the fiscal years 2015/16–2019/20. johannesburg: nhls; 2016. national health laboratory service. strategic plan for the fiscal years 2015/16–2019/20. johannesburg: nhls; 2016. maritz j, preiser w, van zyl gu. establishing diagnostic cut-off criteria for the cobas ampliprep/cobas taqman hiv-1 qualitative test through validation against the amplicor dna test v1.5 for infant diagnosis using dried blood spots. j clin virol. 2012;53(2):106–109. https://doi.org/10.1016/j.jcv.2011.12.002 maritz j, van zyl gu, preiser w. irreproducible positive results on the cobas ampliprep/cobas taqman hiv-1 qual test are different qualitatively from confirmed positive results. j med virol. 2014;86(1):82–87. https://doi.org/10.1002/jmv.23811 haeri mazanderani a, moyo f, sherman gg. missed diagnostic opportunities within south africa’s early infant diagnosis program, 2010–2015. plos one. 2017;12(5):e0177173. https://doi.org/10.1371/journal.pone.0177173 haeri mazanderani a, moyo f, kufa t, maritz j, sherman gg. differentiating clearly positive from indeterminate results: a review of irreproducible hiv-1 pcr positive samples from south africa’s early infant diagnosis program, 2010–2015. diagn microbiol infect dis. 2018;91(3):248–255. https://doi.org/10.1016/j.diagmicrobio.2018.02.019 haeri mazanderani a, sherman gg. evolving complexities of infant hiv diagnosis within prevention of mother-to-child transmission programs. f1000res. 2019;8:1637. https://doi.org/10.12688/f1000research.19637.1 luo r, boeras d, broyles ln, et al. use of an indeterminate range in hiv early infant diagnosis: a systematic review and meta-analysis. j acquir immune defic syndr. 2019;82(3):281–286. https://doi.org/10.1097/qai.0000000000002104 vojnov l, penazzato m, sherman g, et al. implementing an indeterminate range for more accurate early infant diagnosis. j acquir immune defic syndr. 2019;82(3):e44–e46. https://doi.org/10.1097/qai.0000000000002081 world health organization. who recommendation on postnatal discharge following uncomplicated vaginal birth. the who reproductive health library. geneva: world health organization; 2018. boeke ce, joseph j, wang m, et al. point-of-care testing can achieve same-day diagnosis for infants and rapid art initiation: results from government programmes across six african countries. j int aids soc. 2021;24(3):e25677. odhiambo co, githuka g, bowen n, et al. point-of-care early infant diagnosis improves adherence to the testing algorithm in kenya. j int assoc provid aids care. 2020;19:2325958220906030. https://doi.org/10.1177/2325958220906030 massyn n, pillay y, padarath a. district health barometer 2017/18. durban: health systems trust; 2019. moyo f, haeri mazanderani a, feucht ud, et al. monitoring diagnosis, retention in care and viral load suppression in children testing hiv polymerase chain reaction-positive in two districts in south africa. s afr med j. 2019;109(9):686–692. https://doi.org/10.7196/samj.2019.v109i9.13765 veldsman ka, janse van rensburg a, isaacs s, et al. hiv-1 dna decay is faster in children who initiate art shortly after birth than later. j int aids soc. 2019;22(8):e25368. https://doi.org/10.1002/jia2.25368 kalk e, kroon m, boulle a, et al. neonatal and infant diagnostic hiv-pcr uptake and associations during three sequential policy periods in cape town, south africa: a longitudinal analysis. j int aids soc. 2018;21(11):e25212. https://doi.org/10.1002/jia2.25212 about the author(s) noutin f. michodigni department of molecular biology and biotechnology, pan african university institute for basic sciences technology and innovation (pausti), nairobi, kenya department of reproductive health and biology, institute of primate research (ipr), nairobi, kenya atunga nyachieo department of reproductive health and biology, institute of primate research (ipr), nairobi, kenya juliah k. akhwale department of zoology, school of biological sciences, jomo kenyatta university of agriculture and technology (jkuat), nairobi, kenya gabriel magoma department of molecular biology and biotechnology, pan african university institute for basic sciences technology and innovation (pausti), nairobi, kenya department of biochemistry, college of health sciences, jomo kenyatta university of agriculture and technology (jkuat), nairobi, kenya abdoul-salam ouédraogo department of medical microbiology laboratories, souro-sanou teaching hospital, bobo-dioulasso, burkina faso andrew n. kimang’a department of medical microbiology, college of health sciences, jomo kenyatta university of agriculture and technology (jkuat), nairobi, kenya citation michodigni nf, nyachieo a, akhwale jk, magoma g, ouédraogo a-s, kimang’a an. corrigendum: formulation of phage cocktails and evaluation of their interaction with antibiotics in inhibiting carbapenemase-producing klebsiella pneumoniae in vitro in kenya. afr j lab med. 2023;12(1), a2028. https://doi.org/10.4102/ajlm.v12i1.2028 note: doi of original article published: https://doi.org/10.4102/ajlm.v11i1.1803 correction corrigendum: formulation of phage cocktails and evaluation of their interaction with antibiotics in inhibiting carbapenemase-producing klebsiella pneumoniae in vitro in kenya noutin f. michodigni, atunga nyachieo, juliah k. akhwale, gabriel magoma, abdoul-salam ouédraogo, andrew n. kimang’a published: 09 may 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. in the published article, michodigni nf, nyachieo a, akhwale jk, magoma g, ouédraogo a-s, kimang’a an. formulation of phage cocktails and evaluation of their interaction with antibiotics in inhibiting carbapenemase-producing klebsiella pneumoniae in vitro in kenya. afr j lab med. 2022;11(1), a1803. https://doi.org/10.4102/ajlm.v11i1.1803, on page 1 the following paragraph is updated as it was incorrectly formulated: the original incorrect wording: the precipitated bacteriophages were members of myoviridae, siphoviridae and podoviridae. the revised and updated wording: the precipitated bacteriophages were members of myoviridae and podoviridae. in addition, on page 7 the following paragraph is updated as it was incorrectly formulated: the original incorrect wording: this current study revealed the presence of lytic tailed klebsiella phages belonging to the family myoviridae, siphoviridae and podoviridae in nairobi sewage systems with relatively short latent periods and optimal burst sizes, indicating their therapeutic potential in composing phage cocktails and synergistic interaction in combination with non-sensitive antibiotic (imipenem) against carbapenem-resistant k. pneumoniae clinical isolate in vitro. the revised and updated wording: this current study revealed the presence of lytic tailed klebsiella phages belonging to the family myoviridae and podoviridae in nairobi sewage systems with relatively short latent periods and optimal burst sizes, indicating their therapeutic potential in composing phage cocktails and synergistic interaction in combination with non-sensitive antibiotic (imipenem) against carbapenem-resistant k. pneumoniae clinical isolate in vitro. the authors apologise for these errors. the corrections do not change the study’s findings of significance or overall interpretation of the study’s results or the scientific conclusions of the article in any way. abstract background description of the intervention lessons learnt recommendations acknowledgements references about the author(s) john n. waitumbi kenya medical research institute (kemri)/united states army medical research directorate-africa, basic science laboratory, kisumu field station, kisumu, kenya esther omuseni kenya medical research institute (kemri)/united states army medical research directorate-africa, basic science laboratory, kisumu field station, kisumu, kenya josphat nyataya kenya medical research institute (kemri)/united states army medical research directorate-africa, basic science laboratory, kisumu field station, kisumu, kenya clement masakhwe kenya medical research institute (kemri)/united states army medical research directorate-africa, basic science laboratory, kisumu field station, kisumu, kenya faith sigei kenya medical research institute (kemri)/united states army medical research directorate-africa, basic science laboratory, kisumu field station, kisumu, kenya allan lemtudo kenya medical research institute (kemri)/united states army medical research directorate-africa, basic science laboratory, kisumu field station, kisumu, kenya george awinda kenya medical research institute (kemri)/united states army medical research directorate-africa, basic science laboratory, kisumu field station, kisumu, kenya eric muthanje department of biological sciences, university of embu, embu, kenya brian andika department of molecular biology and bioinformatics, jomo kenyatta, university of agriculture and technology, juja, kenya rachel githii kenya medical research institute (kemri)/united states army medical research directorate-africa, basic science laboratory, kisumu field station, kisumu, kenya rehema liyai kenya medical research institute (kemri)/united states army medical research directorate-africa, basic science laboratory, kisumu field station, kisumu, kenya gathii kimita kenya medical research institute (kemri)/united states army medical research directorate-africa, basic science laboratory, kisumu field station, kisumu, kenya beth mutai kenya medical research institute (kemri)/united states army medical research directorate-africa, basic science laboratory, kisumu field station, kisumu, kenya citation waitumbi jn, omuseni e, nyataya j, et al. covid-19 mass testing and sequencing: experiences from a laboratory in western kenya. afr j lab med. 2022;11(1), a1737. https://doi.org/10.4102/ajlm.v11i1.1737 lessons from the field covid-19 mass testing and sequencing: experiences from a laboratory in western kenya john n. waitumbi, esther omuseni, josphat nyataya, clement masakhwe, faith sigei, allan lemtudo, george awinda, eric muthanje, brian andika, rachel githii, rehema liyai, gathii kimita, beth mutai received: 21 sept. 2021; accepted: 21 apr. 2022; published: 22 july 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the basic science laboratory (bsl) of the kenya medical research institute/walter reed project in kisumu, kenya addressed mass testing challenges posed by the emergent coronavirus disease 2019 (covid-19) in an environment of global supply shortages. before covid-19, the bsl had adequate resources for disease surveillance and was therefore designated as one of the testing centres for covid-19. intervention: by april 2020, the bsl had developed stringent safety procedures for receiving and mass testing potentially infectious nasal specimens. to accommodate increased demand, bsl personnel worked in units: nucleic acid extraction, polymerase chain reaction, and data and quality assurance checks. the bsl adopted procedures for tracking sample integrity and minimising cross-contamination. lessons learnt: between may 2020 and january 2022, the bsl tested 63 542 samples, of which 5375 (8.59%) were positive for covid-19; 1034 genomes were generated by whole genome sequencing and deposited in the global initiative on sharing all influenza data database to aid global tracking of viral lineages. at the height of the pandemic (august and november 2020, april and august 2021 and january 2022), the bsl was testing more than 500 samples daily, compared to 150 per month prior to covid-19. an important lesson from the covid-19 pandemic was the discovery of untapped resilience within bsl personnel that allowed adaptability when the situation demanded. strict safety procedures and quality management that are often difficult to maintain became routine. recommendations: a fundamental lesson to embrace is that there is no ‘one-size-fits-all’ approach and adaptability is the key to success. keywords: covid-19; coronavirus; sars-cov-2; nasal swab; nasopharyngeal swabs; mass testing; genome sequencing. background the first case of coronavirus disease 2019 (covid-19) was traced to a kenyan citizen who arrived in nairobi from the united states on 5th march 2020.1 severe acute respiratory syndrome coronavirus 2 (sars-cov-2) was confirmed in the index case on friday the 13th of march. kenyans are not known to have paraskevidekatriaphobia (“friday the 13th” superstition), but the coincidence did not escape notice. through active contact tracing, two contacts who sat next to the index case enroute to nairobi tested positive 2 days later. since then, as in the rest of the world, covid-19 infections have risen dramatically, appearing in waves,2 to date at a count of five (figure 1). surveillance for the virus and containment measures evolved over time, from original contact tracing, isolation and obligatory testing into mandatory masking, social distancing, lockdown, curfew, social gathering ban, restrictions on local and international flights, and closing of country borders. these strict measures were eventually relaxed and at the time of writing (april 2022), the kenyan ministry of health (moh) had removed the masking requirement. figure 1: daily new confirmed covid-19 cases in kenya from march 2020 to march 2022. by early may 2020, it was clear that long-distance truck drivers were transporting the virus across the borders.3 mandatory mass testing and certification for absence of the sars-cov-2 became a requirement for all cross-border truck drivers. testing could not cope with the demand and long queues of trucks held at the border crossing points were common (figure 2). figure 2: long queues of trucks at malaba, kenya–uganda border on 29 april 2020 as drivers waited to be certified free of covid-19. prior to covid-19, the kenya medical research institute (kemri)/walter reed project, kisumu field station laboratory conducted surveillance for pathogens associated with acute febrile illness and outbreak response, and provided basic science support for clinical trials in western kenya. the kenyan government, through the moh, responded to the covid-19 pandemic by designating multiple laboratories, including the basic science laboratory (bsl) of the kenya medical research institute/walter reed project in kisumu as covid-19 testing centres that would provide public health support in combatting the pandemic. this report covers may 2020 to january 2022 and aims to document how the bsl rose to the challenges of sars-cov-2 diagnostics dictated by increased testing demand amidst global supply shortages. description of the intervention ethical considerations the work reported here was conducted as part of public health support to combat the covid-19 outbreak in kenya. a human subject research protocol review was therefore not required. specimen reception, decontamination, anonymization, and testing upon designation in may 2020 as a covid-19 testing centre, the bsl immediately embarked on developing standard operating procedures to guide specimen reception, decontamination of primary and secondary sample containers, anonymisation, risk mitigation, testing and quality assurance procedures, including algorithms of test result interpretation, re-testing of all inconclusive results and reporting of validated results to the moh. an important part of the standard operating procedure was daily decontamination of workspaces with 5% chemgene (medimark scientific limited, kent, england), before and after use and in case of spillage. surfaces decontaminated included workbenches, thermocyclers, nucleic acid extraction robots, centrifuges and vortexes), pipettes, bio-safety cabinets and door handles. to track down potential contamination, all surfaces listed above were swabbed every monday with a disposable sampler (qingdao yongqiang huashang medical technology co., ltd., danyang, china) and tested for sars-cov-2. all surfaces had to have a negative test before nasal sample testing commenced. this procedure was repeated whenever contamination was identified or suspected. semi-automated extractions of rna from nasal and oropharyngeal samples were performed using either the kingfisher flex (thermo fisher scientific inc., waltham, ma, united states) that can perform 96 extractions per hour and or magpurix evo® 24 (zinexts life science, taipei, taiwan) that performs 24 extractions per hour. we also braced for manual extractions, in case the semi-automated kits ran out. for polymerase chain reaction (pcr) testing, the laboratory had the abi 7300 and 7500 systems (applied biosystems, foster city, california, united states) that can perform 96 tests per 2-h run and the magnetic induction cycler (bio molecular systems, queensland, australia) that performs 48 tests in a 2-h run. samples came from different parts of the country and were transported in cool boxes. our estimated maximum output per 24 h was 750 tests. supplies for rna extractions and testing were provided by the moh. because of global shortages of sequencing reagents, the sequencing efforts at the bsl was supported by the united states department of defense global emerging infectious surveillance programme. all kits (extractions and pcr) were validated against known positive samples before use. for quality assurance, external quality assessment samples were provided by the kemri (nairobi, kenya), national public health laboratory (nairobi, kenya), one world accuracy (vancouver, canada) and thistle, johannesburg (south africa). strict procedures were implemented for entry into the laboratory and weekly testing of personnel for sars-cov-2 was required. entry to the laboratory required a covid-19 wellness self-assessment. personnel were asked not to report to work if they had any of the following symptoms: fever ≥ 37.8 °c, sore throat, flu-like symptoms, direct contact or taking care of a covid-19 patient, direct or accidental unprotected contact with samples suspected to contain sars-cov-2. to encourage compliance, absence of work on suspicion of having contracted covid-19 was considered administrative leave and was not deducted from employees’ leave days. lessons learnt strict personnel procedures prevented disease spread in the laboratory over the 21-month testing period (may 2020 to january 2022), seven out of 11 laboratory personnel tested positive for covid-19. these mitigating interventions were considered successful and considering the slow spread of the infection over the testing period, it is unlikely that the infections originated from inside the bsl. initial travel restrictions and lockdowns slowed virus spread, but impacted sars-cov-2 supplies because initial sample accrual was based on active case detection in each county, zero samples were brought to the laboratory in march 2020 or april 2020. in the 4th week of may 2020, we received a batch of 452 respiratory samples collected at the port of busia from truck drivers. of the 452, only seven (1.6%) were positive for sars-cov-2. from june 2020 onwards, the sample sources diversified to include the community, truck drivers, hospitals and military personnel. from may 2020 to january 2022, five waves of covid-19 were discernible (figure 3). the first wave started picking up steam in the first week of june 2020 at 3.0%, reached peak level by mid-july 2020 at 13.2% and thereafter declined steadily, remaining at 3.0% till end of september 2020. the second wave was discernable from the first week of october 2020 at 9.0%, reached peak level of 32.8% by end of that month, and thereafter declined steadily to 1.0% by the end of january 2021. probably fueled by the christmas and new year festivities, wave three emerged suddenly in nairobi and its environs from the second week of february 2021 at 14.2%. although the wave had burned out in nairobi by june 2021, it was still going on in western kenya. wave four emerged while wave three was still in progress. therefore, there was no clear separation between these two waves and by the time wave four started in july 2021, infection rates from wave three were above 5%. wave four did not die off until november 2021, making it the longest wave in kenya. unlike wave four, wave five emerged stealthily in december 2021, spread quickly, and died off as quickly as it had emerged by january 2022, making it the shortest covid-19 wave in kenya. since then, infection rates have been minimal, forcing the moh to reconsider the mandatory requirement for masking. figure 3: five waves of covid-19 in the samples tested at the basic science laboratory, kenya medical research institute/walter reed project, kisumu field station, may 2020 – january 2022. waves caused by variants of concern had a higher viral load real-time pcr cycle threshold (ct) values for the sars-cov-2 orf1ab gene in samples obtained during the five waves were used as surrogates for tracking viral load (figure 4). samples tested during the third, fourth and fifth waves had significantly higher lower mean ct values (28.439 ± 0.304, 28.013 ± 0.251 and 26.033 ± 0.225 standard error of the mean, respectively) compared to wave one (30.303 ± 0.256 standard error of the mean) and wave two (30.890 ± 0.296 standard error of the mean, p < 0.05), indicating a higher viral load. from our sequence data, wave three was dominated by the alpha variant of concern (originally identified in the united kingdom), four by delta (originally identified in india) and five by omicron (originally identified in south africa).4 these variants of concern have been associated with higher viral loads and/or doubling time.5,6,7 we recognise that there are caveats in relying on ct values, but as suggested by hay et al., at a population level, ct values can be used to explain the trajectory of the covid-19 epidemic.8 figure 4: scatter plot showing the viral load as determined from cycle threshold values for the five waves tested at the basic science laboratory, kenya medical research institute/walter reed project, kisumu field station, may 2020 – january 2022. increased test demand put a huge strain on testing reagents and supplies the automated extraction kits were the first to be depleted, and resupply was erratic due to banning of flights in and out of the country, coupled with increased global demand and competition. in addition, because infection rates in kenya, and indeed in africa, were still very low compared to global rates, kenya was at the bottom of the ladder on supply priorities. the laboratory resorted to manual extraction using kits supplied by moh, philanthropists and different embassies. because of the increased testing demands and to save on resources and time, a sample pooling strategy was adopted. this was done when the infection rates were not more than 5%. creation of process flow units reduced errors and increased efficiency at the height of testing, the laboratory was receiving over 500 respiratory samples per day. laboratory personnel organised themselves into work units consisting of extraction, pcr, and data with quality control (qc) checks at various stages (figure 5). the extraction team was located in the ‘dirty room’ and donned complete body suits and facemasks. they were responsible for sample receipt and disinfection, sorting and assigning processing ids, sample pooling and the nucleic acid extractions. work units helped in two main areas. first, they reduced error rates, because teammates could qc each other. second, because of increased workload that required working late and during weekends, shifts were easier to organise without too much disruption of sample processing and analysis. figure 5: laboratory personnel work units consisting of extraction, pcr, and data with quality control checks at various stages of sample processing at the basic science laboratory, kenya medical research institute/walter reed project, kisumu field station, may 2020 – january 2022. the pcr team worked in the ‘clean room’ equipped with a level 2 biosafety cabinet where pcr master mixes and extracted nucleic acids were added before being loaded in thermocyclers that were located in a separate room. this separation further reduced the chances of amplicon cross contamination. three personnel were designated to perform the pcr, with at least two working together at any given time. the pcr teams anticipated completion of rna extractions to allow the pcr process to commence immediately. real-time pcr amplification took 2 h. once complete, amplification output was analysed, quality assurance checked and interpreted. samples in the negative pools were reported as negative. samples in the positive pools were re-extracted individually and re-tested. each real-time pcr reaction included the human rnase p gene (rnase p) to check for sample integrity. a total of 64 643 samples were tested in the reporting period, of which 5376 (8.3%) were positive for sars-cov-2. resampling was requested in 668 (1.4%) samples because of poor sample quality. of the 5376 positive samples, 1034 with ct values ≤ 33 were sequenced on an illumina miseq (illumina, san diego, california, united states). recommendations a great source of pride for any laboratory contemplating sars-cov-2 testing in support of the covid-19 pandemic control is the realisation that the effort is part of what the world health organization refers to as critical preparedness, readiness and response actions that save lives.9 a fundamental lesson to embrace is that there is no ‘one-size-fits-all’ approach, and that adaptation of traditional workflows and processes are crucial. given the covid-19 diagnostic demand amidst the global shortfall in supplies, the bsl would not have been able to meet the testing requests without adopting the specimen pooling strategy. with these realisations, the bsl was able to quickly adapt to increased testing demand dictated by an emergent new disease. of the 323 272 covid-19 confirmed cases in kenya, bsl contributed 5376 (1.7%) positive samples. the nasopharyngeal swab collection centres did a commendable job of ensuring sample integrity, despite the long distance to the testing laboratory. acknowledgements the kenyan ministry of health provided extraction and pcr kits. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions j.w. conceptualised, wrote the first draft of the manuscript, provided the resources, and obtained funding; e.o. performed the assays, analysed the data, organised the assay workflow for qualitative polymerase chain reaction, was in charge of quality assurance/quality control, and reviewed and edited the manuscript; j.n. performed the assays, analysed the data, was in charge of assay workflow, assisted in plotting graphs, and reviewed and edited the manuscript; c.m. performed the assays, analysed the data, assisted with the qualitative polymerase chain reaction assay workflow, assisted in plotting graphs, and reviewed and edited the manuscript; f.s. performed the assays, analysed the data, assisted in quality assurance and quality control, and reviewed and edited the manuscript; a.l. performed the assays, analysed the data, supervised the nucleic acid extractions workflow, and reviewed and edited the manuscript; g.a. performed the assays, assisted in the nucleic acid extractions workflow, and reviewed and edited the manuscript; e.m., b.a. and r.l. performed the assays, assisted in nucleic acid extractions, and analysed the data; r.g. was in charge of data curation and capture of associated metadata; g.k. analysed the data, reviewed and edited the manuscript, and assisted in assay validation; b.m. ensured that all staff had appropriate personal protective equipment and that all biosafety cabinets were calibrated and safe to use, supervised all aspects of the assays, and revised and edited the manuscript. sources of support sars-cov-2 sequencing and personnel costs were funded by the armed forces health surveillance division (afhsd) and its global emerging infections surveillance and research branch (promis p0095_21_ky, 2021) (silver springs, maryland, united states). the funders had no role in data collection and analysis, the decision to publish, or preparation of the manuscript. data availability all data are included in the manuscript. disclaimer material has been reviewed by the walter reed army institute of research. there is no objection to its publication. the opinions or assertions contained herein are the private views of the authors, and they are not to be construed as official, or as reflecting true views of the department of the army or the department of defense. references ministry of health. first case of coronavirus disease confirmed in kenya [homepage on the internet]. 2020 [cited 13 march 2020]. available from: https://www.health.go.ke/first-case-of-coronavirus-disease-confirmed-in-kenya/ hasell j, mathieu e, beltekian d, et al. a cross-country database of covid-19 testing. sci data. 2020;7:345. https://doi.org/10.1038/s41597-020-00688-8 bugembe d, kayiwa j, phan m, et al. main routes of entry and genomic diversity of sars-cov-2, uganda. emerg infect dis. 2020;26(10):2411–2415. https://doi.org/10.3201/eid2610.202575 who. tracking sars-cov-2 [homepage on the internet]. 2022 [cited 2022 june]. available from: https://www.who.int/activities/tracking-sars-cov-2-variants?msclkid=c2729b29d10811eca952b5e97b4da00a kidd m, richter a, best a, et al. s-variant sars-cov-2 lineage b1.1.7 is associated with significantly higher viral loads in samples tested by thermo fisher taqpath rt-qpcr. j infect dis. 2021;223(10):1666–1670. https://doi.org/10.1093/infdis/jiab082 hill kj, dewar r, templeton k. a multiregional evaluation of ct values in sars-cov-2 voc-20dec-01 variant. j infect dis. 2021;224(5):927–928. https://doi.org/10.1093/infdis/jiab303 karim ssa, karim qa. omicron sars-cov-2 variant: a new chapter in the covid-19 pandemic. lancet. 2021;398(10317):2126–2128. https://doi.org/10.1016/s0140-6736(21)02758-6 hay ja, kennedy-shaffer l, kanjilal s, et al. estimating epidemiologic dynamics from cross-sectional viral load distributions. sci. 2021;373(6552):eabh0635. https://doi.org/10.1126/science.abh0635 critical preparedness, readiness and response actions for covid-19 [homepage on the internet]. world health organization; 2021 [cited 27 may 2021]. available from: https://www.who.int/emergencies/diseases/novel-coronavirus-2019/technical-guidance/critical-preparedness-readiness-and-response-actions-for-covid-19 article information authors: nestor bangoura1 abou a.m. diouara2 mohamed cissé1 halimatou d. ndiaye2 souleymame mboup2 ahidjo ayouba3 coumba t. kane2 affiliations: 1service de dermatologie chu donka, cta, conakry, guinée 2laboratoire de bactériologie virologie chu aristide le dantec, université cheikh anta diop de dakar, sénégal 3umi 233 ird de montpellier, france correspondence to: coumba kane email: ctourekane@yahoo.co.uk postal address: 30 avenue pasteur, bp: 7325 dakar, sénégal dates: received: 24 jan. 2014 accepted: 12 dec. 2014 published: 26 june 2015 how to cite this article: bangoura n, diouara aam, cissé m. quantification de la charge virale et tests de résistance du vih-1 aux arv à partir d’échantillons dbs (dried blood spots) chez des patients guinéens sous traitement antiretroviral. afr j lab med. 2015;4(1), art. #168, 7 pages. http://dx.doi.org/10.4102/ajlm.v4i1.168 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. quantification de la charge virale et tests de résistance du vih-1 aux arv à partir d’échantillons dbs (dried blood spots) chez des patients guinéens sous traitement antirétroviral in this original research... open access • abstrait • abstract • introduction • conception et méthode d’étude    • patients et sites de collecte des échantillons    • echantillonnage et conservation des prélèvements dbs    • l’extraction des acides nucléiques    • la quantification de la charge virale (cv)    • génotypage et analyse phylogénétique • considérations éthiques • résultats    • caractéristiques de la population d’étude    • charge virale (cv) et tests de résistance    • phylogénie et caractérisation moléculaires des souches virale • discussion • remerciements    • conflit d’intérêt    • contributions des auteurs • références abstrait top ↑ problématique: comme dans plusieurs pays du sud, le suivi virologique des patients sous traitement antirétroviral (tarv) en guinée est timide voire inexistant dans certaines localités. le but de cette étude était d’évaluer la faisabilité technique et logistique de l’utilisation des dbs dans les tests de charge virale (cv) et de génotypage. méthode: de septembre à octobre 2010, les dbs ont été préparés à partir de prélèvements sanguins de patients adultes sous tarv. le délai d’envoi des échantillons au laboratoire de référence était de 30 jours maximum après le prélèvement et se faisait à température ambiante. la cv a été quantifiée et les échantillons de patients en échec virologique (cv ≥ 3 log10 copies/ml) ont été génotypés selon le protocole de l’anrs. l’algorithme de stanford version 6.0.8 a été utilisé pour l’analyse et l’interprétation des mutations de résistance. résultats: parmi les 136 patients inclus, 129 et 7 étaient respectivement sous première et deuxième ligne de traitement avec une médiane de suivi de 35 mois [iqr: 6-108]. l’échec virologique a été noté chez 33 patients. parmi eux, 84.8% (n = 28/33) ont bénéficié d’un génotypage. le taux de résistance global était de 14% (n = 19/136). le crf02_ag était le sous type viral le plus prévalent (82%; n = 23) conclusion: en plus de montrer la faisabilité technique et logistique des tests de cv et de génotypage à partir des dbs, ces résultats montrent l’intérêt de leurs utilisations dans le suivi virologique des patients sous tarv. cette étude a permis également de documenter l’échec virologique, la résistance aux arv et la diversité génétique du vih-1 en guinée. mots clés: vih-1, résistance aux arv, dbs (dried blood spots), guinée conakry, génotypage, charge virale. abstract top ↑ quantification of viral load and resistance tests of hiv-1 to arvs from dried blood spots samples in guinean patients undergoing antiretroviral treatment. problem: as in several countries of the south, the virological monitoring of patients undergoing antiretroviral treatment (arvt) in guinea is low or non-existent in some locations. the aim of this study was to assess the technical and logistical feasibility of the use of (dried blood spots) dbss in viral load (vl) and genotyping tests. method: from september 2010 to october 2010, dbs were prepared from blood samples of adult patients under arvt. the samples had to be sent to the reference laboratory within 30 days after the sample had been done at ambient temperature. the vl was quantified and the samples of patients with virological failure (cv ≥ 3 log10 copies/ml) were genotyped according to the anrs protocol. the stanford algorithm, version 6.0.8, was used to analyse and interpret the resistance mutations. results: amongst the 136 included patients, 129 and 7 were under first and second line treatment respectively, and monitored for an average of 35 months [iqr: 6-108]. virological failure was noticed among 33 patients. among them, 84.8% (n = 28/33) benefited from genotyping. the global resistance rate was 14% (n = 19/136). crf02_ag was the most prevalent viral subtype (82%; n = 23) conclusion: in addition to demonstrating the technical and logistic feasibility of vl and genotyping tests from dbss, these results show the relevance of their use in the virological monitoring of patients under arvt. also, this study made it possible to provide information on virological failure, arv resistance and the hiv-1 genetic diversity in guinea. introduction top ↑ en guinée, la prévalence du vih chez les adultes (15-49ans) est estimée à 1.4%.1 la mise sous traitement antirétroviral (tarv) des patients vivant avec le vih/sida a débuté en 1999. depuis, le nombre de sites de prise en charge (pec) opérationnels des patients répartis dans le pays a augmenté et est passé de 46 en 2012 à 51 en fin 2013. la couverture nationale en centres de conseils et dépistage volontaire (cdv) était de 66 sites et 131 sites de prévention de la transmission mère-enfant (ptme) pour 464 structures offrant les services de consultations prénatales (cpn) en 2013.2 de plus, la couverture en tarv a connu une hausse au cours de ces années et est passée de 22.50% en 2007 à 56.91% en fin 2011, pour 40 258 personnes éligibles au tarv selon les lignes directrices de l’oms.3 a l’image de plusieurs pays à ressources limitées, du fait d’un manque d’infrastructures, d’équipements biomédicaux et de personnels qualifiés, les tests moléculaires, notamment la charge virale (cv) et le génotypage, ne sont pas tout le temps disponibles. par conséquent, le suivi des patients sous tarv en guinée se fait essentiellement en se basant sur des critères clinico-immunologiques.4 l’utilisation du papier buvard comme support de prélèvement sanguin alternatif au plasma permettrait la collecte et le transport des échantillons des sites périphériques vers le centre de référence national ou international. le papier buvard (dbs) a été largement utilisé dans le diagnostic sérologique et moléculaire de l’infection à vih.5,6 de plus, les dbs ont été utilisés dans la détermination de la cv et des tests de résistance du vih-1 aux arv.7,8,9 plusieurs études ont évalué et validé le dbs en le comparant au plasma10,11,12,13, qui est le type d’échantillon de référence pour les tests de cv et de génotypage. le dbs est un support facile d’utilisation, moins exigeant que le plasma et le sérum car ne nécessitant pas une chaine de froid pour la conservation, le stockage et le transport des échantillons. c’est dans ce contexte que s’inscrit cette étude pionnière en guinée dont l’objectif était d’évaluer la faisabilité technique et logistique des tests de cv et de génotypage du vih-1 à partir d’échantillons dbs de patients sous tarv collectés dans des conditions de terrain. conception et méthode d’étude top ↑ patients et sites de collecte des échantillons cette étude a porté sur des patients adultes (≥ 18 ans) sous tarv depuis au moins 6 mois, suivis dans le cadre du programme national. les patients inclus dans cette étude ont été recrutés consécutivement sur la période allant de septembre à octobre 2010 au niveau de 4 sites de prise en charge (pec). les individus infectés par le vih-2 ou coinfectés par le vih-1 et vih-2, de même que les femmes ayant bénéficiés d’une prévention de la transmission mère-enfant du vih (ptme), n’étaient pas inclus. le choix de ces sites a été fait de façon aléatoire et sur la base de l’existence d’association de personnes vivant avec le vih pouvant faciliter la collecte des échantillons. un des sites est situé dans la capitale, le centre de traitement ambulatoire de conakry, et les 3 autres sont localisés dans les régions de boké, mamou et labé, distants respectivement de 300, 350 et 600 km de la capitale (figure 1). figure 1: répartition des sites de collecte des échantillons. echantillonnage et conservation des prélèvements dbs pour chaque patient, 5 ml de sang total ont été recueillis dans un tube edta par ponction veineuse au niveau du pli du coude pour servir à la préparation de 2 cartes de papier filtre whatman 903®, conformément aux règles d’hygiène et de sécurité , et 50 µl de sang total ont été déposés sur chacun des 5 spots à raison d’une carte dbs, préalablement identifiée et datée. les échantillons ont été séchés pendant la nuit à température ambiante (30 à 37 °c). chaque échantillon dbs a été placé dans un sachet plastique individuel hermétiquement fermé en présence de dessiccateurs. puis, ils ont été conservés et stockés sur site à température ambiante. l’envoi des dbs vers le laboratoire de bactériologie virologie de l’hôpital aristide le dantec de dakar au sénégal pour les tests de cv et de génotypage s’est fait par voie terrestre dans les 30 jours suivant le prélèvement. l’extraction des acides nucléiques a partir de 2 spots de chaque échantillon dbs/patients et à l’aide de l’appareil nuclisens minimag (biomérieux, craponne, france), les acides nucléiques totaux ont été obtenus par extraction magnétique et manuelle selon la chimie de boom.14 en effet, les spots découpés à l’aide d’un « puncher » dédié ont été trempés dans un tube contenant 2 ml de tampon de lyse et agité pendant 30 minutes à température ambiante. les acides nucléiques ont été extraits et élués dans 25 µl de tampon d’élution après une série de centrifugation et des lavages avec des tampons 1, 2 et 3 comme précédemment décrit.10 la quantification de la charge virale (cv) la détermination de la cv a été effectuée à partir de l’extrait obtenu et ceci en utilisant kit nuclisens easyq hiv-1 v2.0 (biomérieux, craponne,france) conformément aux instructions du fabriquant. la plateforme utilisée était nuclisens® easyq (biomérieux, lyon, france) et le principe est une amplification de type nasba. le seuil de détectabilité de la technique est de 800 copies/ml.15 dans cette présente étude, le seuil de l’échec virologique a été fixé à 3 log10 copies/ml génotypage et analyse phylogénétique le génotypage a été effectué selon le protocole de l’anrs (http://www.hivfrenchresistance.org/) qui consiste à faire des amplifications séparées par pcr des fragments de la protéase en entier et des 240 premiers codons de la reverse transcriptase (rt) du gène pol en utilisant respectivement les couples d’amorces 5’prot1/3’prot1 et mj3/mj4 comme amorces externes et 5’prot2/3’prot2 et a35/ne35 comme amorces internes. les produits de pcr de 2ème tour ont été purifiés avec le kit qiaquick gel extraction kit®, (qiagen, courtaboeuf, france) conformément aux indications du fabriquant. l’adn purifié a été directement séquencé sur la plateforme abi prism 3100 avant (genetic analyzer applied biosystem) selon la technologie du big dye terminator technology®v3.1 (applied biosystems, courtaboeuf, france). les séquences obtenues ont été assemblées et manuellement éditées sur le logiciel seqmantm ii 5.08 de la suite de dnastar® software (lasergene, konstanz, germany). l’analyse et l’interprétation des mutations de résistance ont été réalisées sur l’algorithme de l’université de stanford version 6.0.8 (http://hivdb.stanford.edu/). les séquences générées ont été alignées avec des séquences références du vih-1 groupe m et l’ensemble des formes recombinant les crfs disponibles sur los alamos hiv database (http://www.hiv.lanl.gov/content/index). trois séquences de chaque sous-type pur ont été incluses dans l’alignement. et les séquences ont été alignées avec l’algorithme de muscle puis l’alignement dégapé obtenu a été avec le programme de gblocks du logiciel seaview v4.4.1. l’arbre phylogénétique de maximun de vraisemblance (phyml) a été également généré sur seaview v4.4.1 avec comme paramètres supports de branche déterminés par la méthode approximate likelihood ratio test (alrt), option sh-like. l’analyse de similarité et de bootscanning pour la confirmation des formes recombinantes (crfs, urfs) a été effectuée sur le logiciel simplot v3.5.1.16,17 l’analyse et les calculs statistiques des données ont été réalisés avec les logiciels epi info v3.5.4 et microsoft excel. considérations éthiques top ↑ cette étude est une sous-étude d’un projet multicentrique impliquant 3 pays de l’afrique de l’ouest (sénégal, mali et la république de guinée) et a été approuvée par les comités éthiques nationaux de ces pays. les patients ont été recrutés consécutivement sur base volontaire, sur une période allant de septembre à octobre 2010 après signature d’un formulaire de consentement libre et éclairé. pour garder confidentielles les données des participants, un code unique a été attribué à chaque prélèvement. résultats top ↑ caractéristiques de la population d’étude au total 136 patients infectés par le vih-1 sous tarv ont participé à cette étude. parmi eux, 129 étaient sous traitement de première ligne (2inti + 1innrt) et 7 sous deuxième ligne (2inti + 1ip/r), avec une médiane de suivi thérapeutique de 35 mois [iqr: 6-108 mois]. le sexe ratio homme/femme était de 0,64 et l’âge médian était de 38 ans [iqr: 18-61 ans] boîte 1. charge virale (cv) et tests de résistance l’échec virologique (cv ≥ 3 log10 copies/ml) a été observé chez 33 patients soit un taux de 24.26% (n = 33/136). parmi eux, 4 étaient sous deuxième ligne de tarv et 13/29 patients ont eu des changements de molécules de première ligne (exemple: d4t par azt) pour des raisons cliniques ou de tolérance. dans le tableau 1, figurent entre autre l’historique du traitement et les données virologiques des patients en échec virologique. selon la durée du traitement, 4/13, 7/31 et 22/92 patients étaient en échec virologiques respectivement à des intervalles 6-12, 13-24 et > 24 mois. tableau 1: données liées aux patients en échec virologique (cv ≥ 3 log10 copies/ml). boîte 1: caractéristiques de la population d’étude et nombres de participants par sites. au total, 28/33 échantillons de patients en échec virologique ont pu être génotypés soit un taux d’amplification réussi de 84.84%. la médiane de cv de ces échantillons de patients en échec virologique génotypés (n = 28) était de 4 log10 copies/ml [iqr: 3-6.7] et celui des échantillons non amplifiés (n = 5) était de 3.1 log10 copies/ml [iqr: 3-3.6] pour une p-value = 0.02. au moins une mutation conférant une résistance à une molécule antirétrovirale a été observée chez 19 patients, soit un taux de résistance globale de 14%. la mutation m184v (n = 13) et les tams (thymidine analogue-associated mutations) (n = 32) étaient les plus fréquemment observées pour les inti et la k103n (n = 11) et y181c (n = 7) pour les innti. leurs survenues étaient d’autant plus marquées que la durée du traitement était élevée (figure 2). l’insertion t69 a également été observée chez 5 patients dont un en deuxième ligne. d’autres mutations comme la v198i, g190a/g et y188a/l ont été également notées chez 4, 3 et 2 patients respectivement (tableau 1). par ailleurs, aucune mutation de résistance majeure aux ip (inhibiteur de protéase) n’a été observée et ceci qu’il s’agisse de patients avec une virémie supérieure à 3 log10 copies/ml ou pas, de patients sous première ligne ou deuxième ligne de traitement. figure 2: fréquences des mutations observées en fonction de la durée du traitement. phylogénie et caractérisation moléculaires des souches virale l’analyse phylogénétique des séquences nucléotides a permis de montrer la prédominance du crf02_ag, 82% (n = 23/28). les sous types d et crf06_cpx ont été observés dans les proportions respectives 7% (n = 2/28) et 11% (n = 3/28) (figure 3). figure 3: arbre phyml montrant la relation phylogénétique entre les séquences requêtes (n=28, en traits pointillés) et les références (en traits pleins) dans la région du gène pol (pr+rt) du vih-1. discussion top ↑ cette étude décrit pour la première fois en guinée les données relatives à l’échec virologique et le taux de résistance du vih-1 chez des patients sous tarv. précédemment, dans des conditions relativement similaires à celles décrites dans cette étude, du fait que le plasma soit le type d’échantillon de référence pour la quantification de la cv et la réalisation des tests de résistance, nous avons effectué des études d’évaluation et de faisabilité des tests virologiques à partir d’échantillons dbs. ce fut, d’une part, des comparaisons de valeurs de cv et de profils de résistance entre échantillons pairs plasma et dbs8 et, d’autres part, la faisabilité des tests de résistance à partir de dbs collectés et acheminés dans des conditions réelles de vie.10 le but de cette présente étude était d’évaluer la faisabilité technique et logistique des tests de cv et de génotypage à partir d’échantillons dbs de patients sous tarv collectés dans des conditions de terrain. l’échec virologique a été observé dans 24.26% (n = 33/136) des cas et parmi eux 2/3 des patients étaient à plus de 24 mois de traitement (tableau 1). le taux de suppression virologique (75.7%) semble être satisfaisant pour une médiane de suivi de 35 mois.18 garrido et al en 2008 ont rapporté un taux de suppression virologique similaire mais avec une médiane de suivi thérapeutique de 12 mois.7 dans cette étude, nous avons obtenu un taux d’amplification réussi de 84.84% comparable à ceux obtenus en tanzanie,19 en espagne20 et légèrement inférieur à ceux obtenus précédemment au sénégal10 et en guinée conakry (94%).21 cinq échantillons de patients en rebond virologique, dont la médiane de cv était faible (3.13 log10 copies/ml [3.06-3.63]), n’ont pas pu être génotypés, malgré plusieurs tentatives. ceci pourrait également être dû à une dégradation des acides nucléiques durant les processus de conservation et de stockage des prélèvements dbs à température ambiante. d’ailleurs, plusieurs études ont rapporté un faible taux d’amplification réussi pour des échantillons à cv < 5000 (3.69 log10) copies/ml contrairement à ceux ayant des cv élevées (> 10000 [4 log10] copies/ml).20,22,23,24 les mutations de résistances sélectionnées chez les patients en échec virologique dans cette étude (tableau 1) étaient en accord avec les schémas thérapeutiques en cours ou antérieurs. la proportion de patients ayant ces mutations augmente avec la durée du traitement (14/19 ; supérieure à 24 mois). par ailleurs, la mutation m184v conférant une résistance au 3tc/ftc et les tams pour les inti et les mutations k103n et y181c pour les innti (figure 2), les plus prédominantes ici, ont été également celles observées dans plusieurs études conduites en afrique subsaharienne.7,25,26 l’insertion t69, conférant une multi-résistance aux inti27 et retrouvée chez certains patients, reflète la composition de leur régime thérapeutique, qui inclut soit la didanosine (ddi), soit la stavudine (d4t) ou encore la zidovudine (azt). ces molécules sont connues pour sélectionner la mutation t69.28,29,30 par ailleurs, les résultats de génotypage ont montré que 27.2% (n = 9/28) des patients en échec virologique étaient porteurs de virus sauvages (i.e. encore sensibles aux arv). cet échec virologique serait probablement lié à une mauvaise observance ou encore aux variants minoritaires que le ‘bulk sequencing’ utilisé dans cette étude ne peut pas détecter. des observations similaires ont été rapportées à abidjan (côte d’ivoire) et à bangui (république centrafricaine).31,32 une virémie élevée sous tarv est associée à un risque d’émergence de la résistance du vih aux médicaments. ainsi, cette étude met en évidence la nécessité d’améliorer l’observance au traitement. l’analyse phylogénétique des souches virales étudiées montre une forte prédominance du crf02_ag (81%, n = 26), comme précédemment rapporté dans une étude de résistance primaire du vih-1 chez des patients nouvellement infectés à conakry.21 ces résultats montrent la faisabilité technique et logistique des tests de cv et de génotypage à partir de prélèvements dbs. d’un autre côté, cette étude montre l’intérêt de l’utilisation des dbs comme support de prélèvement dans le monitoring virologique des patients sous tarv en guinée, pays où le suivi virologique est encore peu structuré. de plus elle a permis de documenter le taux de patients en échec virologique, la résistance du vih-1 aux arv et la diversité génétique en guinée. remerciements top ↑ nous remercions l’organisation ouest africaine de la santé (ooas) pour avoir financé cette étude, solthis guinée (solidarité thérapeutique et initiative contre le sida) pour son assistance, toutes les équipes de recherches ayant contribué à ce travail, l’ensemble du personnel des sites de pec du guinée, les différentes organisation des pvvih/sida et les patients qui ont bien voulu participer à cette étude. conflit d’intérêt les auteurs déclarent qu’ils n’ont aucun lien financier ou personnel qui pourrait les influencer de façon inappropriée en écrivant cet article. contributions des auteurs c.t.k. (université cheikh anta diop) était le chef de projet. c.t.k, h.d.n., a.a.m.d. (université cheikh anta diop) et m.c. (service de dermatologie chu donka, cta, conakry) étaient responsables de la conception expérimentale et de celle du projet. a.a.m.d. (université cheikh anta diop) et n.b. (service de dermatologie chu donka, cta, conakry) ont effectué les analyses virologiques et écrit le manuscrit initial. tous les auteurs ont participé à sa rédaction et édition finales. tous les auteurs ont lu et approuvé le manuscrit final. références top ↑ unaids. unaids report on the global aids epidemic. 2012:pp:1–212 [unaids global aids report web site]. disponible sur: http://www.unaids.org/en/media/unaids/contentassets/documents/epidemiology/2012/gr2012jc2434_worldaidsday_results_en.pdf. [consulté le 16 mai 2013]. cnls-guinée. revue des progrès vers la réalisation des cibles de la déclaration 2011 de l’onu sur le vih et le sida. rapport narratif. 2014:pp:1–36. http://www.unaids.org/fr/regionscountries/countries/guinea/ [consulté le 05 juin 2014]. cnls-guinée. rapport ungass 2012 _guinée. 2012:pp:1-71. [unaids global aids report web site]. disponible sur http://www.unaids.org/en/dataanalysis/knowyourresponse/countryprogressreports/2012countries/ce_gn_narrative_report[2011].pdf. [consulté le 26 juin 2013] pnpcsp-ist/sida. normes et protocoles de prise en charge de l’infection par le vih chez l’adulte et l’enfant en guinee. 2012:pp:1–103. [who web site]. disponible sur http://www.who.int/hiv/pub/guidelines/guinea_art.pdf. [consulté le 26 juin 2013] kebe k, ndiaye o, ndiaye hd, et al. rna versus dna (nuclisens easyq hiv-1 v1.2 versus amplicor hiv-1 dna test v1.5) for early diagnosis of hiv-1 infection in infants in senegal. j clin microbiol. 2011 jul;49(7):2590–2593. http://dx.doi.org/10.1128/jcm.02402-10 castro ac, borges lg, souza rda s, grudzinski m, d’azevedo pa. evaluation of the human immunodeficiency virus type 1 and 2 antibodies detection in dried whole blood spots (dbs) samples. rev inst med trop sao paulo. 2008 may-jun;50(3):151–156. http://dx.doi.org/10.1590/s0036-46652008000300004 garrido c, zahonero n, fernandes d, et al. subtype variability, virological response and drug resistance assessed on dried blood spots collected from hiv patients on antiretroviral therapy in angola. j antimicrob chemother. 2008 mar;61(3): 694–698. http://dx.doi.org/10.1093/jac/dkm515 kane ct, ndiaye hd, diallo s, et al. quantitation of hiv-1 rna in dried blood spots by the real-time nuclisens easyq hiv-1 assay in senegal. j virol methods. 2008 mar;148(1–2):291–295. http://dx.doi.org/10.1016/j.jviromet.2007.11.011 johannessen a, garrido c, zahonero n, naman e, de mendoza c. hiv-1 drug resistance testing from dried blood spots collected in rural tanzania using the viroseq hiv-1 genotyping system. j antimicrob chemother. 2011 feb;66(2): 260–264. http://dx.doi.org/10.1093/jac/dkq433 diouara aa, diop-ndiaye h, kebe-fall k, et al. dried blood spots for hiv-1 drug resistance genotyping in decentralized settings in senegal. j med virol. 2014 jan;86(1):45–51. http://dx.doi.org/10.1002/jmv.23778 diouara aam, sow a, leye n, et al. comparaison des charges virales et des mutations de résistance entre plasma et dbs au sénégal. abstract book: 6éme conférence francophone vih/sida, 25–28 mars genève suisse. 2012;abstract numéro 82. monleau m, aghokeng af, eymard-duvernay s, et al. field evaluation of dried blood spots for routine hiv-1 viral load and drug resistance monitoring in patients receiving antiretroviral therapy in africa and asia. j clin microbiol. 2014 feb;52(2):578–586. http://dx.doi.org/10.1128/jcm.02860-13 bertagnolio s, parkin nt, jordan m, brooks j, garcia-lerma jg. dried blood spots for hiv-1 drug resistance and viral load testing: a review of current knowledge and who efforts for global hiv drug resistance surveillance. aids rev. 2010 oct-dec;12(4):195–208. boom r, sol cj, salimans mm, jansen cl, wertheim-van dillen pm, van der noordaa j. rapid and simple method for purification of nucleic acids. j clin microbiol. 1990 mar;28(3):495–503. van deursen p, oosterlaken t, andre p, et al. measuring human immunodeficiency virus type 1 rna loads in dried blood spot specimens using nuclisens easyq hiv-1 v2.0. j clin virol. 2010 feb;47(2):120–125. http://dx.doi.org/10.1016/j.jcv.2009.11.021 lole ks, bollinger rc, paranjape rs, et al. full-length human immunodeficiency virus type 1 genomes from subtype c-infected seroconverters in india, with evidence of intersubtype recombination. j virol. 1999 jan;73(1):152–160. gouy m, guindon s, gascuel o. seaview version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building. mol biol evol. 2010 feb;27(2):221–224. http://dx.doi.org/10.1093/molbev/msp259 barth re, van der loeff mf, schuurman r, hoepelman ai, wensing am. virological follow-up of adult patients in antiretroviral treatment programmes in sub-saharan africa: a systematic review. lancet infect dis. 2010 mar;10(3):155–166. http://dx.doi.org/10.1016/s1473-3099(09)70328-7 johannessen a, holberg-petersen m, lovgaarden g, et al. hiv type-1 drug resistance testing on dried blood spots is feasible and reliable in patients who fail antiretroviral therapy in rural tanzania. antivir ther. 2010;15(7):1003–1009. http://dx.doi.org/10.3851/imp1660 masciotra s, garrido c, youngpairoj as, et al. high concordance between hiv-1 drug resistance genotypes generated from plasma and dried blood spots in antiretroviral-experienced patients. aids. 2007 nov 30;21(18):2503–2511. http://dx.doi.org/10.1097/qad.0b013e3281c618db charpentier c, bellecave p, cisse m, et al. high prevalence of antiretroviral drug resistance among hiv-1-untreated patients in guinea-conakry and in niger. antivir ther. 2011;16(3):429–433. http://dx.doi.org/10.3851/imp1754 youngpairoj as, masciotra s, garrido c, zahonero n, de mendoza c, garcia-lerma jg. hiv-1 drug resistance genotyping from dried blood spots stored for 1 year at 4 degrees c. j antimicrob chemother. 2008 jun;61(6):1217–1220. http://dx.doi.org/10.1093/jac/dkn100 rottinghaus ek, ugbena r, diallo k, et al. dried blood spot specimens are a suitable alternative sample type for hiv-1 viral load measurement and drug resistance genotyping in patients receiving first-line antiretroviral therapy. clin infect dis. 2012 apr;54(8):1187–1195. http://dx.doi.org/10.1093/cid/cis015 monleau m, butel c, delaporte e, boillot f, peeters m. effect of storage conditions of dried plasma and blood spots on hiv-1 rna quantification and pcr amplification for drug resistance genotyping. j antimicrob chemother. 2010 aug;65(8):1562–1566. http://dx.doi.org/10.1093/jac/dkq205 liegeois f, vella c, eymard-duvernay s, et al. virological failure rates and hiv-1 drug resistance patterns in patients on first-line antiretroviral treatment in semirural and rural gabon. j int aids soc. 2012;15(2):17985. http://dx.doi.org/10.7448/ias.15.2.17985 dagnra ay, vidal n, mensah a, et al. high prevalence of hiv-1 drug resistance among patients on first-line antiretroviral treatment in lome, togo. j int aids soc. 2011;14:30. http://dx.doi.org/10.1186/1758-2652-14-30 johnson va, calvez v, gunthard hf, et al. 2011 update of the drug resistance mutations in hiv-1. top antivir med. 2011 nov;19(4):156–164. de antoni a, foli a, lisziewicz j, lori f. mutations in the pol gene of human immunodeficiency virus type 1 in infected patients receiving didanosine and hydroxyurea combination therapy. j infect dis. 1997 oct;176(4):899–903. http://dx.doi.org/10.1086/516511 winters ma, coolley kl, girard ya, et al. a 6-basepair insert in the reverse transcriptase gene of human immunodeficiency virus type 1 confers resistance to multiple nucleoside inhibitors. j clin invest. 1998 nov 15;102(10):1769–1775. http://dx.doi.org/10.1172/jci4948 scherrer au, von wyl v, joos b, et al. predictors for the emergence of the 2 multi-nucleoside/nucleotide resistance mutations 69 insertion and q151m and their impact on clinical outcome in the swiss hiv cohort study. j infect dis. 2011 mar 15;203(6):791–797. http://dx.doi.org/10.1093/infdis/jiq130 messou e, chaix ml, gabillard d, et al. increasing rate of tams and etravirine resistance in hiv-1-infected adults between 12 and 24 months of treatment: the voltart cohort study in cote d’ivoire, west africa. j acquir immune defic syndr. 2013 jun 21. http://dx.doi.org/10.1097/qai.0b013e3182a009e4 pere h, charpentier c, mbelesso p, et al. virological response and resistance profiles after 24 months of first-line antiretroviral treatment in adults living in bangui, central african republic. aids res hum retroviruses. 2012 apr;28(4): 315–323. http://dx.doi.org/10.1089/aid.2011.0127 results about the author(s) asmaa m. zahran department of clinical pathology, south egypt cancer institute, assiut university, assiut, egypt hanaa nafady-hego department of microbiology and immunology, faculty of medicine, assiut university, assiut, egypt sawsan m. moeen department of internal medicine, clinical haematology unit, faculty of medicine, assiut university, assiut, egypt hanan a. eltyb department of medical oncology, south egypt cancer institute, assiut university, assiut, egypt mohammed m. wahman department of clinical oncology, south valley university, qena, egypt asmaa nafady department of clinical and chemical pathology, qena faculty of medicine, south valley university, qena, egypt citation zahran am, nafady-hego h, moeen sm, eltyb ha, wahman mm, nafady a. corrigendum: higher proportion of non-classical and intermediate monocytes in newly diagnosed multiple myeloma patients in egypt: a possible prognostic marker. afr j lab med. 2022;11(1), a1713. https://doi.org/10.4102/ajlm.v11i1.1713 note: doi of original article published: https://doi.org/10.4102/ajlm.v10i1.1296. correction corrigendum: higher proportion of non-classical and intermediate monocytes in newly diagnosed multiple myeloma patients in egypt: a possible prognostic marker asmaa m. zahran, hanaa nafady-hego, sawsan m. moeen, hanan a. eltyb, mohammed m. wahman, asmaa nafady published: 25 mar. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. in the published article, zahran am, nafady-hego h, moeen sm, eltyb ha, wahman mm, nafady a. higher proportion of non-classical and intermediate monocytes in newly diagnosed multiple myeloma patients in egypt: a possible prognostic marker. afr j lab med. 2021;10(1), a1296. https://doi.org/10.4102/ajlm.v10i1.1296, there was an error on page 3 under the ‘results’ section. the mean age of the mm patients should be 63.5 as per table 1 instead of 6.35. the first paragraph under the ‘results’ section is updated to: results baseline characteristics of newly diagnosed multiple myeloma patients and healthy controls the mean age of the mm patients was 63.5 ± 4.07 years, and the number of men (13) was double that of women (7) (table 1). the proportion of plasma cells in bone marrow was 39.7% ± 3.7% and that of the monoclonal band (m-protein) was 4.44 g/dl ± 2.8 g/dl. the authors apologise for this error. the correction does not change the study’s findings of significance or overall interpretation of the study’s results or the scientific conclusions of the article in any way. abstract background description of the intervention lessons learnt recommendations acknowledgements references about the author(s) valerie f. donkeng-donfack mycobacteriology unit, national tuberculosis reference laboratory, centre pasteur du cameroun, yaoundé, cameroon suzanne m. ongoulal mycobacteriology unit, national tuberculosis reference laboratory, centre pasteur du cameroun, yaoundé, cameroon yvonne j. djieugoue mycobacteriology unit, national tuberculosis reference laboratory, centre pasteur du cameroun, yaoundé, cameroon yannick kamdem simo mycobacteriology unit, national tuberculosis reference laboratory, centre pasteur du cameroun, yaoundé, cameroon henri manga national tuberculosis control program, yaoundé, cameroon danielle a.d. tollo national tuberculosis control program, yaoundé, cameroon edwige m.a. belinga national tuberculosis control program, yaoundé, cameroon vincent mbassa national tuberculosis control program, yaoundé, cameroon jean l. abena national tuberculosis control program, yaoundé, cameroon sara eyangoh centre pasteur du cameroun, yaoundé, cameroon citation donkeng-donfack vf, ongoulal sm, djieugoue yj, et al. tuberculosis-loop-mediated isothermal amplification implementation in cameroon: challenges, lessons learned and recommendations. afr j lab med. 2022;11(1), a1792. https://doi.org/10.4102/ajlm.v11i1.1792 lessons from the field tuberculosis-loop-mediated isothermal amplification implementation in cameroon: challenges, lessons learned and recommendations valerie f. donkeng-donfack, suzanne m. ongoulal, yvonne j. djieugoue, yannick kamdem simo, henri manga, danielle a.d. tollo, edwige m.a. belinga, vincent mbassa, jean l. abena, sara eyangoh received: 23 nov. 2021; accepted: 20 apr. 2022; published: 26 aug. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: until 2016, microscopy was the main tool for the early detection of pulmonary tuberculosis in cameroon, especially in remote settings. due to the poor sensitivity of microscopy, there was a need to implement a molecular assay in order to improve tuberculosis case detection. intervention: in 2017, tuberculosis loop-mediated isothermal amplification (tb-lamp), a molecular rapid diagnostic test recommended by the world health organization, was implemented in cameroon as a replacement test of microscopy for initial diagnosis of pulmonary tuberculosis and also as a follow-on test to microscopy for smear-negative sputum specimens. a roll out plan for tb-lamp implementation in cameroon had been developed from january 2017 to april 2017, followed by initial implementation at four sites in may 2017. additional sites were added progressively. lessons learnt: the use of tb-lamp as a follow-on test to microscopy for smear-negative sputum specimens helped in the detection of tuberculosis in 14.77% of those who were sputum-smear negative in 2019. tuberculosis-loop-mediated isothermal amplification usage as an initial test, followed by testing with xpert mtb/rif for rapid tuberculosis and rifampicin resistance detection during tuberculosis mass screening campaigns, reduced the turn-around time by 73.23% as compared to when the gene xpert instrument was used alone. recommendations: the implementation and scaling up of tb-lamp in cameroon contributed to increase access to tuberculosis molecular diagnosis in remote settings and as such improved tuberculosis case notification. however, to better enhance this notification and optimise the use of a tb-lamp instrument, a suitable sample transport system is recommended. keywords: tb-lamp; molecular test; implementation; roll out; notification. background tuberculosis remains a major public health problem worldwide and tuberculosis detection cases remain a challenge and a priority for tuberculosis care and control.1,2 however, microscopy which is the main tool for the early detection of tuberculosis, especially in resource-limited countries with a higher burden of tuberculosis, has a sensitivity of about 50% – 60%.3 this sensitivity is even lower in children, people living with hiv and people with extrapulmonary tuberculosis.4 in addition, this sensitivity is lower compared to molecular tests such as tuberculosis loop-mediated isothermal amplification (tb-lamp) and genexpert.5 culture, which is the gold standard method for mycobacterium tuberculosis complex diagnosis, is time consuming due to the slow growth rate of m. tuberculosis complex and requires the use of sophisticated instruments such as the biosafety cabinet which is expensive, requires a lot of training and has a high cost of maintenance.6 accordingly, to rapidly detect tuberculosis cases as well as improve case detection including negative smear cases, the implementation of molecular world health organization (who)-recommended rapid diagnostic assays as tuberculosis initial diagnostic test is recommended to improve tuberculosis testing efficiency and increase tuberculosis bacteriologically confirmed cases.7 in 2012, the national tuberculosis control program (ntcp) of cameroon introduced xpert® mtb/rif (cepheid, sunnyvale, california, united states), an automated semi-quantitative nested real-time polymerase chain reaction for the rapid and simultaneous detection of m. tuberculosis complex and rifampicin resistance.8 however, xpert® mtb/rif equipment was placed at the regional laboratories and recommended for tuberculosis diagnosis in specific categories of presumptive tuberculosis cases including: children (0–5 years), people living with hiv, contact patients with multidrugor rifampicin-resistant tuberculosis, prisoners, refugees, patients previously treated for tuberculosis or those with treatment failures. in 2016, tb-lamp was endorsed by the who to be used as a replacement test for sputum-smear microscopy. the who recommended to use tb-lamp as the initial diagnosis test for the detection of pulmonary tuberculosis (ptb) in adults who present signs and symptoms consistent with ptb, and also as an add-on test following negative smear microscopy, particularly when additional testing of smear-negative sputum specimens is necessary.9 cameroon is a tuberculosis burden country10 with an estimated annual incidence of 103 new cases per 100 000 inhabitants in 2017. the expected number of new cases each year in cameroon is 47 000. however, only around 50% of tuberculosis cases are reported annually, meaning that around 50% of tuberculosis cases are still missing.11 accordingly, the ministry of public health of cameroon, through the ntcp, decided to implement tb-lamp (eiken chemical, tokyo, japan) in 2017, with the goal to increase access to molecular diagnosis and ultimately improve tuberculosis case findings.9 tuberculosis-lamp is the only molecular test recommended by the who to be used in primary health care for the diagnosis of active tuberculosis.12 this test is a manual assay that does not need an air-conditioned room or sophisticated equipment and is not affected by short power intervals or variations. up to 14 samples can be tested at once using tb-lamp equipment, up to 70 tests can be performed in 8 h,9 and the results can be read with the naked eye under ultraviolet light.9 implementation of the tb-lamp assay requires minimal laboratory infrastructure and few biosafety requirements. the tb-lamp test is a less expensive test,13 with high sensitivity compared to microscopy and a similar sensitivity and specificity to xpert mtb/rif.5,14,15,16 before cameroon, only countries from asia, including the philippines, vietnam, cambodia and myanmar, performed pilot implementation of tb-lamp with no big issues and they received project funding. since the endorsement of tb-lamp by the who, some african countries other than cameroon, such as nigeria, zambia, kenya, uganda, angola, ivory coast, uganda, gambia and senegal, performed pilot implementation of tb-lamp and they would be preparing for the extension phase. however, amongst african countries, cameroon is the country where the implementation process started, in 2017. this paper describes the challenges, lessons learned and recommendations for tb-lamp implementation in cameroon. description of the intervention ethical considerations the ministry of health through the national tuberculosis control program conducted the implementation of tb-lamp in cameroon. as such, no ethical approval was required. policy development in 2014, with the support of the foundation for innovative new diagnostics, cameroon participated in the evaluation of tb-lamp. the centre pasteur du cameroun, through the national reference laboratory, conducted this evaluation at jamot hospital in yaoundé. the results showed that tb-lamp displayed higher sensitivity (82.6%; 95% confidence interval [ci]: 76.9–87.2) compared to smear microscopy with 53.6% (95% ci: 46.8–60.3). tuberculosis-lamp showed a specificity of 96.0% (95% ci: 93.2–97.7) and microscopy, 99.0% (95% ci: 97.1–99.7). meanwhile, the sensitivity and specificity of tb‑lamp were similar to genexpert® (89.9%; 95% ci: 85.0–93.3 and 97.0%; 95% ci: 94.4–98.4, respectively).16 the overall results of this evaluation was used by the who to approve the tb-lamp assay in 2016 as a follow-on and replacement test for sputum-smear microscopy in the diagnosis of ptb in adults with signs and symptoms consistent with tuberculosis.9 therefore, the ministry of health, through the ntcp, reviewed the policy about to be implemented and decided to introduce this new molecular diagnostic technique in cameroon. evidence base tuberculosis-lamp implementation in cameroon started in may 2017 with a 2-month pilot phase. four laboratories in two regions (centre and south) were selected for this phase based on their workload. the centre and the south regions represent, respectively, about 18.0% and 3.5% of the total population of cameroon, with a tuberculosis prevalence of about 229/100 000 and 129/100 000 inhabitants in 2017. the centre region is amongst the three regions with the highest number of tuberculosis case notifications in cameroon. the four laboratories involved during this pilot phase include jamot hospital, mbalmayo and bafia district hospital in the centre region and ebolowa regional hospital in the south region. these laboratories were selected because of their higher workload during the first semester of 2017, compared to other laboratories in their respective regions. in addition, theses laboratories were not far from the national reference laboratory that oversaw training and monthly monitoring during this pilot phase. nine laboratory technicians were trained, and each laboratory was equipped with a tb-lamp machine and an uninterruptible power supply. in these four laboratories, only the tb-lamp assay was used for routine diagnosis of ptb in adults. data obtained after two months of implementation (june and july 2017) at the four pilot sites were compared to the results obtained at the same sites over the same period of the previous year (june 2016 and july 2016), where only microscopy was used for initial ptb diagnosis. the compared data showed a 30.0% increase in positivity rate between 2016 and 2017. table 1 shows the number of patients who accessed the tests from the four pilot sites during the months of june and july 2017 and the proportion of positive to negative cases. these results served as an evidence base to guide and reinforce cameroon’s decision to go ahead with the scale up of tb-lamp in the country. table 1: number of patients tested with tuberculosis loop-mediated isothermal amplification as initial tuberculosis diagnosis test in june 2017 and july 2017 in four diagnostic and treatment centres in cameroon. search for funds in order to scale up the implementation of tb-lamp in cameroon, the results obtained during the pilot phase were used by the ntcp to address cameroon’s 2018–2020 request for global fund (gf) support. in 2018, this request was validated by gf to support tb-lamp implementation in cameroon. in addition to the equipment and reagents, the gf support also covered training of laboratory technicians and supervisors of the tb-lamp sites. however, the allocated funds had neither fees for humaloop t instrument installations nor fees to conduct related activities such as meetings with clinicians. furthermore, these funds did not consider additional materials not provided by the kits, including scissors, gloves, marker pens and tissue paper. fortunately, these additional fees were covered by the government through the tuberculosis national reference laboratory (tb-nrl) hosted at centre pasteur du cameroun for an optimal implementation of the new technique. in addition, the government also supported sample transportation. selection of settings twenty-one tuberculosis diagnostic and treatment centers (dtcs) out of the 260 dtcs present in the country were selected in the 10 regions for the scaling up of tb-lamp. many reasons guided the selection of these dtcs. nineteen of the dtcs are at remote settings and had only microscopy as a tuberculosis diagnostic tool. two of the dtcs are tuberculosis regional reference laboratories. the workload of the selected dtcs was higher compared to the others. all 21 dtcs have electricity and easy access for sample transportation. this selection of sites for tb-lamp extension was performed prior to the training of staff. procurement, revision of tuberculosis national guidelines and tuberculosis diagnosis algorithms the procurement of reagents and equipment (humaloop t and uninterruptible power supply) was done through the global drugs facility (gdf). during the procurement period, the ntcp revised the tuberculosis national guidelines and tuberculosis diagnosis algorithms in may 2018 to include tb-lamp assay. on the revised tuberculosis national guidelines, the ntcp recommended the use of tb-lamp at the dtcs where it was implemented as a replacement test for sputum-smear microscopy for initial diagnosis of ptb in adults with signs and symptoms consistent with tuberculosis. however, at the microscopy testing centres, tb-lamp was recommended to be used as a follow-on test to smear microscopy for smear-negative sputum specimens in adults with signs and symptoms consistent with ptb. steps for tuberculosis-loop-mediated isothermal amplification scaling up in cameroon the scaling up of tb-lamp in cameroon was performed in four steps including trainings, equipment installation, stakeholder engagement and sample transportation. training trainings started with the training of trainers organised by the tb-nrl at the centre pasteur du cameroun. five trainers, including three from the tb-nrl and two from the pilot sites (jamot hospital and ebolowa regional hospital), were trained over five days. they were then involved in the training of about 50 laboratory technicians. trainings included theoretical and practical sessions (figure 1). the objective of these trainings was to improve the technical capacities of laboratory technicians for better diagnosis of tuberculosis in the laboratory using humaloop t equipment. due to budget constraints, only four sessions of three days each were organised for the training in the country. a minimum of 12 personnel were trained per session by two trainers with one humaloop t instrument. the training was laborious for the trainers because of the high number of trainees per session. figure 1: tuberculosis loop-mediated isothermal amplification trainings during scaling up in cameroon, june 2019 and august 2019. (a) transfer of sputum sample to heating tube. (b) removal of heating tube from the heating block of the humaloop t instrument. (c) dna extraction. (d) labelling of reaction tubes. douala tb regional reference laboratory 08-08-2019 (a, b) and garoua tb regional reference laboratory 20-06-2019 (c, d). installation of humaloop t instrument at selected sites after training sessions, humaloop t instruments with uninterruptible power supplies were installed at the 21 selected sites. the role of the uninterruptible power supply is to allow the equipment to keep running for at least a period when the primary power source is lost. the installations were performed in the week following the training to enable technicians to quickly start tb-lamp testing in their respective settings, with a fresh memory of the training. the activity of humaloop t instruments installation was not funded by the gf. the government covered this activity through the tb-nrl. stakeholder engagement in order to ensure a successful implementation of tb-lamp in cameroon, the ministry of health, through the ntcp, had organised a sensitisation meeting in march 2018, on world tb day, on the role of tb-lamp in improving tuberculosis diagnosis in cameroon. the meeting was chaired by the minister of health, together with the head of the ntcp and the general manager of the centre pasteur du cameroun. this meeting involved community leaders from high burden communities, tuberculosis active case finders, physicians, laboratory technicians, directors of tb-dtcs, the ministry of health, other stakeholders, and both national and international media. in addition to this meeting, regional sensitisation meetings were also organised. attendees included physicians, nurses, bikers involved in sample transportation and other health staff involved in tuberculosis patient care. during the meetings, participants were informed about the introduction of the new tuberculosis diagnosis assay at the dtc and regional level, as well the role and the advantages of the new technology. also, tb-lamp algorithms were presented and discussed. the government funded these national and regional sensitisation meetings through the tb-nrl. sample transportation the sample transportation system of smear-negative sputum specimens from microscopy centres to tb-lamp centres for further testing was organised in all regions in 2019 in order to give all ptb presumptive cases the benefit of the tb-lamp assay. according to the number of tb-lamp laboratories in a region, microscopy laboratories were organised into pools, and with a tb-lamp laboratory. this activity of smear-negative sputum specimen transportation was funded by the government. bikers and public transport were used for transportation. city transportation used bikers whereas intercity transportation used intercity public transportation. furthermore, laboratories were equipped with phones and communication credits to ensure communication between them and the bikers for sample collections and results deliveries, to reduce the turn-around time. the government funded this activity. the challenges were insufficient funds as well as the small number of bikers. challenges the implementation of tb-lamp in cameroon took two years (from may 2017 to may 2019). during these two years, 27 dtcs were gradually equipped with a humaloop t instrument. the implementation started with four machines in 2017, followed by two machines in 2018 and 21 machines in 2019. the government funded the first six machines and the gf the other 21. from the four pilot sites in 2017, the ntcp established a 3-year extension plan (2018–2020), supported by the gf. many challenges accompanied this implementation. make pipette-60 set material available on the global drugs facility catalogue the procurement of reagents and equipment (humaloop t and uninterruptible power supply) through the gdf took about 10 months between placement of order and delivery. the issue was due to the absence of the pipette set on the gdf catalogue. therefore, human and eiken companies negotiated with the gdf to include pipette-60 set material on the gdf catalogue, to facilitate procurement. getting additional funds for tuberculosis-loop-mediated isothermal amplification implementation-related activities and for additional materials for an efficient implementation of tb-lamp, we requested additional funds from the government to conduct related activities, such as meetings with clinicians, and to purchase supplementary materials such as scissors, gloves, marker pens and tissue paper needed for tb-lamp assays, but not provided with the kits. trainings of laboratory technicians organising a 3-day training session for a minimum of 12 technicians with only one humaloop t instrument was very challenging because of the reduced number of days and the high number of trainees. accordingly, the tb-nrl proposed to the ntcp to reduce the number of trainees per session for the next year and to train technicians directly in their laboratory. increasing tuberculosis case notification to increase tuberculosis case notification using a humaloop t instrument, the ntcp implemented the use of tb-lamp in 2019 as a follow-on test to smear microscopy for smear-negative sputum specimens in adults with signs and symptoms consistent with ptb. this activity was done through an organised sample transportation system for transportation of smear-negative sputum specimens to tb-lamp sites. this activity helped to detect up to 388 (14.77%) tuberculosis patients out of 3061 smear-negative sputum specimens transferred to tb-lamp sites. these patients could have been released into the community and, as such, could have continued to spread the disease. in addition, the ntcp organised tuberculosis mass screening campaigns at 34 prisons in 2019 and the tb-lamp assay was used as initial tuberculosis diagnosis followed by testing with xpert mtb/rif for tuberculosis and rifampicin resistance detection. tuberculosis mass screening campaigns helped in the detection of 123 tuberculosis cases out of 3672 presumptive tuberculosis cases at the 34 prisons, and three rifampicin resistance cases. table 2 presents the summary stats, by year, for the four initial sites and the number of positive cases picked up by tb-lamp. table 2: summary statistics for the four initial sites by year and number of positive cases picked up by tuberculosis loop-mediated isothermal amplification, cameroon, 2017–2019. lessons learnt the implementation of tb-lamp in cameroon offered an opportunity for us to draw several lessons for an effective implementation. these lessons learned will be useful for countries that will start with tb-lamp implementation after us. pulmonary tuberculosis-loop-mediated isothermal amplification materials and its use the tb-lamp assay needs additional materials such as scissors, gloves, marker pens and tissue paper, that are not provided with the kits. moreover, tb-lamp assays required a positive and a negative control for each run. these controls provided by the kits include one tube with 0.4 ml of positive control and three tubes of 0.5 ml of negative control. the available volume of positive control is necessary for 12 runs of six samples/run. however, when a laboratory performs less than six samples/run, they will perform more than 12 runs and the 0.4 ml of positive control will not be sufficient. also, using less than six samples/run leads to the consumption of more extraction and detection kits for controls. this situation could therefore lead to a stock shortage if the estimations did not consider it. education of tuberculosis-loop-mediated isothermal amplification users the use of tb-lamp does not require a background in molecular biology. during tb-lamp implementation in cameroon, most of the laboratory technicians trained at the remote setting on the use of tb-lamp for tuberculosis diagnosis were the ones performing microscopy with no background in molecular biology. infrastructure tuberculosis-lamp has been implemented in the laboratories performing microscopy as a routine test for tuberculosis diagnosis. therefore, the same infrastructure used for microscopy was applied. there was no need to adjust or to modify infrastructure. reduction of turn-around time during tuberculosis mass screening campaigns in 2019, a diagnostic algorithm based on an initial testing with tb-lamp, followed by testing with xpert mtb/rif to diagnose tuberculosis, reduced the turn-around time by 73.23% during mass campaigns in 34 prisons compared to xpert mtb/rif when used alone. recommendations search for funding implementation of tb-lamp requires consideration, during forecasting, fees of related activities such as meetings and sample transportation, as well as additional materials not provided by the kits. training trainings should include a maximum of four trainees for a 3-day session with one humaloop t instrument, and sufficient financial resources should be allocated for these training activities, so as to train technicians in their various laboratories with the use of their instrument. pulmonary tuberculosis-loop-mediated isothermal amplification settings the robustness of the humaloop t instrument makes it good for peripheral laboratories receiving large numbers of samples. this will avoid the use of less than six samples/run. with the help of solar panels, a humaloop t instrument can be used for tuberculosis active case finding in communities where there is no electricity. implementation of a diagnostic algorithm based on an initial testing with tuberculosis-loop-mediated isothermal amplification followed by testing with xpert mtb/rif to diagnose tuberculosis this approach improved early and rapid tuberculosis detection with an added advantage of providing rifampicin resistance status. data management datatocare, a simple, customisable, and patient-centric application developed by savics srl (brussels, belgium), could help to record patient, physician and laboratory operator information, send results directly to the physician via sms or email, and easily generate weekly, quarterly and annual reports. conclusion despite some difficulties, the implementation of tb-lamp in cameroon was well conducted, and the tb-lamp test is used as a who-recommended initial rapid diagnostic test for all people with signs and symptoms of tuberculosis. the challenges, lessons learned and recommendations resulting from this implementation will help other countries to have a more efficient implementation. acknowledgements the authors would like to thank: the ministry of public health and the national tuberculosis control program for fundraising and the revision of national tuberculosis guideline as well as tuberculosis diagnosis algorithm; the global fund for providing necessary funds for the implementation; centre pasteur du cameroun who conducted this implementation and provided additional funds to support tb-lamp equipment installation as well as organisation of meetings with clinicians; all the national tuberculosis reference laboratory staff for their active participation on the training of laboratory technicians. all the national tuberculosis control program staff for their participation at all the implementation steps; all the laboratory technicians for using tb-lamp test for tuberculosis diagnosis; and all the physicians for requesting tb-lamp as an initial test for ptb diagnosis. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions v.f.d.-d., v.m., e.m.a.b., h.m., j.l.a. and s.e. contributed to the design and implementation of the study. v.f.d.-d. and s.e. contributed to the analysis of the results, and to the writing of the manuscript with input from all authors. v.f.d.-d., s.m.o., y.j.d. and y.k.s. contributed to the training of laboratory technicians and installation of equipment. v.f.d.-d. and d.a.d.t. contributed to quantifying of the reagents, consumables and equipment needed for the implementation and scaling up of the technique. sources of support the global fund through new funding model 2018–2020 (nfm3 2018–2020) supported this work. data availability the authors confirm that the data supporting the findings of this study are available within the article. disclaimer the views expressed in the submitted article are the authors’ own, not an official position of the institution or funder. references world health organization. global tuberculosis report [homepage on the internet]. 2018 [cited 2019 may 15]. available from: http://apps.who.int/iris/bitstream/handle/10665/274453/9789241565646-eng.pdf?sequence=1&isallowed=y world health organization. xpert mtb/rif implementation manual – technical and operational ‘how-to’: practical considerations [homepage on the internet]. 2014 [cited 2015 jan 12]. available from: https://apps.who.int/iris/bitstream/handle/10665/112469/9789241506700_eng.pdf?sequence=1&isallowed=y zijenah ls. the world health organization recommended tb diagnostic tool [homepage on the internet]. 2018 [cited 2019 mar 18]. available from: https://www.researchgate.net/publication/327984940 nahid p, kim sp, evans ca, et al. clinical research and development of tuberculosis diagnostics: moving from silos to synergy. j infect dis. 2012;205(2):159–168. https://doi.org/10.1093/infdis/jis194 donfack vf, ngando l, pefura ew, et al. comparative study of loopamp™ mycobacterium tuberculosis complex kit for rapid detection of mycobacterium tuberculosis complex in cameroon. biomed biotechnol res j. 2018;2(1):46–52. https://doi.org/10.4103/bbrj.bbrj_86_17 khairunisa s, erica l. an activist’s guide to tuberculosis diagnostic tools [homepage on the internet]. 2017 [cited 2020 mar 20]. available from: https://www.treatmentactiongroup.org/wp-content/uploads/2017/04/tb-diagnostics-guide.pdf world health organization. framework of indicators and targets for laboratory strengthening under the end tb strategy [homepage on the internet]. 2016 [cited 2017 jun 25]. available from: https://www.who.int/publications/i/item/9789241511438 boehme cc, nabeta p, hillemann d, et al. rapid molecular detection of tuberculosis and rifampin resistance. n engl j med. 2010;363(11):1005–1015. https://doi.org/10.1056/nejmoa0907847 world health organization. the use of loop-mediated isothermal amplification (tb-lamp) for the diagnosis of pulmonary tuberculosis: policy guidance. world health organization [homepage on the internet]. 2016 [cited 2017 jan 15]. available from: https://apps.who.int/iris/bitstream/handle/10665/249154/9789241511186-eng.pdf?sequence=1&isallowed=y world health organization. global lists of high burden countries for tb, multidrug/rifampicin-resistant tb (mdr/rr-tb) and tb/hiv, 2021–2025 [homepage on the internet]. geneva: world health organization; 2021 [cited 2021 sept 18]. available from: https://cdn.who.int/media/docs/default-source/hq-tuberculosis/who_globalhbcliststb_2021-2025_backgrounddocument.pdf world health organization. global tb report 2017. [homepage on the internet]. 2017 [cited 2018 nov 18]. available from: https://www.who.int/publications/i/item/9789241565516 world health organization. model list of essential in vitro diagnostics [homepage on the internet]. 2018 [cited 2020 apr 18]. available from: https://aslm.org/wp-content/uploads/2018/05/who_edl_2018.pdf tb-lamp test now available for $6 through global drug facility [homepage on the internet]. 2020 [cited 2020 apr 18]. available from: https://www.stoptb.org/news/tb-lamp-test-now-available-6-through-global-drug-facility shete pb, farr k, strnad l, gray cm, cattamanchi a. diagnostic accuracy of tb-lamp for pulmonary tuberculosis: a systematic review and meta-analysis. bmc infect dis. 2019;19:268. https://doi.org/10.1186/s12879-019-3881-y pham th, peter j, mello fcq, et al. performance of the tb-lamp diagnostic assay in reference laboratories: results from a multicentre study. int j infect dis. 2018;68:44–49. https://doi.org/10.1016/j.ijid.2018.01.005 yadav r, sharma n, khaneja r, et al. evaluation of the tb-lamp assay for the rapid diagnosis of pulmonary tuberculosis in northern india. int j tuberc lung dis. 2017;21(10):1150–1153. https://doi.org/10.5588/ijtld.17.0035 article information authors: ruth mcnerney1 kimberly sollis1 rosanna w. peeling1 affiliations: 1london school of hygiene & tropical medicine, united kingdom correspondence to: ruth mcnerney postal address: london school of hygiene & tropical medicine, keppel street, london wc1e 7ht, united kingdom dates: received: 25 june 2013 accepted: 15 nov. 2013 published: 04 apr. 2014 how to cite this article: mcnerney r, sollis k, peeling rw. improving access to new diagnostics through harmonised regulation: priorities for action. afr j lab med. 2014;3(1), art. #123, 7 pages. http://dx.doi.org/10.4102/ ajlm.v3i1.123 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. improving access to new diagnostics through harmonised regulation: priorities for action in this improving access to new diagnostics through harmonised regulation: priorities for action ... open access • abstract • introduction    • regulating diagnostic devices    • risk classification of in vitro diagnostic medical devices    • harmonising regulation of diagnostic devices • a common submission dossier    • status quo    • action going forward • convergence in the auditing of quality systems    • status quo    • action going forward • reducing duplication in studies of clinical performance    • status quo    • action going forward • convergence in post-market surveillance    • status quo    • action going forward • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ a new generation of diagnostic tests is being developed for use at the point of care that could save lives and reduce the spread of infectious diseases through early detection and treatment. it is important that patients in developing countries have access to these products at affordable prices and without delay. regulation of medical products is intended to ensure safety and quality whilst balancing the need for timely access to beneficial new products. current regulatory oversight of diagnostic tests in developing countries is highly variable and weak regulation allows poor-quality tests to enter the market. however, inefficient or overzealous regulation results in unnecessary delays, increases costs and acts as a barrier to innovation and market entry. setting international standards and streamlining the regulatory process could reduce these barriers. four priority activities have been identified where convergence of standards and protocols or joint review of data would be advantageous: (1) adoption of a common registration file for pre-market approval; (2) convergence of quality standards for manufacturing site inspections; (3) use of common evaluation protocols, as well as joint review of data, to reduce unnecessary duplication of lengthy and costly clinical performance studies; and (4) use of networks of laboratories for post-market surveillance in order to monitor ongoing quality of diagnostic devices. the adoption and implementation of such measures in developing countries could accelerate access to new diagnostic tests that are safe and affordable. introduction top ↑ diagnostic tests make a major contribution to global health. they are needed in order to guide treatment decisions and ensure the appropriate use of medicines. they are also vital for screening for infections, such as hiv infection or syphilis, in asymptomatic individuals.1 such tests are often lifesaving, where delay or lack of access can result in deterioration of the patient’s health and lead to further complications. diagnostics are particularly important for the control of infectious diseases, where early detection allows intervention in order to prevent onward transmission.2 they may also be used to monitor the outcome of treatment. most diagnostic interventions utilise in vitro diagnostic medical devices (ivds) which test specimens obtained from a patient, such as blood or urine. ivds include a wide range of technologies, from rapid dip-stick strips for use at the point of care (poc) to sophisticated instrumentation for use in referral laboratories. access to diagnostic tests in developing countries is often limited by their availability, as patients frequently do not live within easy travelling distance of a well-equipped and functional laboratory.2,3 a new generation of diagnostic tests is being developed for use at the point of care that will not need a laboratory.4,5 prompt access to the new tests should be encouraged; if they are made available in developing countries at affordable prices, such tests could increase access to appropriate healthcare, thereby saving lives and reducing the spread of infectious diseases.4,5,6 regulating diagnostic devices regulation of medical products is intended to ensure safety and quality whilst ensuring that the public has timely access to beneficial new products. national regulatory authorities (nras) are usually mandated by law and, for a regulatory system to be effective, it should be enforceable in both the public and private healthcare sector. following impartial review, new tests that are considered substandard or unsafe are refused entry to the market, whereas satisfactory products are approved and registered. for regulatory purposes, ivds are classified as medical devices. in this context, ‘medical device’ means any instrument, apparatus, implement, machine, appliance, implant, reagent for in vitro use, software, material, or any other similar or related article intended by the manufacturer to be used alone or in combination, for human beings for one or more of the specific medical purpose(s): diagnosis, prevention, monitoring, treatment or alleviation of disease; diagnosis, monitoring, treatment, alleviation of or compensation for an injury; investigation, replacement, modification or support of the anatomy or of a physiological process; support or sustenance of life; control of conception; disinfection of medical devices; provision of information by means of in vitro examination of specimens derived from the human body.7 an ivd is defined as:medical device, whether used alone or in combination, intended by the manufacturer for the in vitro examination of specimens derived from the human body solely or principally to provide information for diagnostic, monitoring or compatibility purposes.7 most countries have a legal framework and a nominated body to regulate medicines, but regulation of medical devices in developing countries is less common.8 the demands on regulatory authorities for ivds differ from that of other medical products in that there are a very large number of ivds and, compared with drugs or vaccines, their market life is often short given the rapid pace of technology development.9 current regulatory oversight of ivds is variable8 and, in countries where there is no regulation, substandard and counterfeit tests may be sold openly.10 in countries that do regulate, approval for ivds is often costly, lengthy and, on occasion, lacking in transparency, thus regulation of diagnostics is currently seen as a barrier to innovation and access.9,11,12 in 2001, the pan american health organisation (paho), the regional office for the americas of the world health organization (who) and the united states food and drug administration (usfda) published a model regulatory programme for medical devices, with a set of guiding principles.13 these principles include the statement that: regulatory system should ensure that valuable new technologies are made available to the clinical community and to patients and consumers expeditiously while preventing unsafe or ineffective devices from reaching the market.13 they also state that regulatory decisions must be based on strong and clear science, free of external influences and, in addition, that countries instituting regulatory programmes should be cognisant of ongoing international efforts to harmonise activities. regulatory control of ivd has three components: • pre-market evaluation: to assess safety, performance, benefits and risks prior to approval to market the device. • marketing controls: to stipulate conditions under which devices can be offered for sale; identify who may use the device and under what conditions; and to avoid inappropriate marketing or misleading claims regarding test effectiveness. • post-marketing controls: to maintain vigilance and continued safety and quality of approved products that entails a method for information-sharing amongst users within the country and across national health authorities. there should be a means by which corrections can be made as well as a mechanism for removing substandard products that pose a risk to public health. risk classification of in vitro diagnostic medical devices the degree of regulatory control for a medical device should be proportionate to the risk posed by the product. unlike medicines or vaccines, ivds are not ingested by the patient, greatly reducing the potential for harm. thus the stringency of regulatory oversight required is related to the harm that a false positive or false negative test result may cause to either individual or public health. reagents used in diagnostics tests, such as microbiological stains or culture media, by themselves pose little risk to human or public health and are classed as low risk. high risk tests include those used to screen for infections such as hiv, where a false negative test result could lead to the individual not being given life-saving drugs and continuing to transmit the infection within the community. should the test be used to screen blood products, then recipients of that donation would be at risk of acquiring a life-threatening disease. such tests require more stringent control, including evidence of their performance as obtained through clinical studies. to guide the regulatory process, products are grouped or classified according to their risk of causing harm. in 2006, the global harmonization task force (ghtf), a voluntary partnership of stringent regulatory authorities and diagnostic companies, published recommendations regarding classification of medical devices. they suggested that each medical device be assigned to one of four classes based upon its intended use (table 1).14 classification provides a mechanism by which the cost and delay of registration are moderated according to the potential of a product to cause harm. manufacturers are required to provide less-substantial submission dossiers for products in risk group class a; whereas class d products require stringent conformity assessment, including evidence of performance in a clinical setting that is representative of the intended use. for devices to be used at the poc, studies should be conducted in the settings of intended use (clinic or outreach settings) with testing performed by local health providers. such studies are costly and, for some diseases, may require years to plan and execute. the classification system requires a shorter and less-costly route to pre-market approval for low-risk products. table 1: classification of medical devices with examples of diagnostic products. harmonising regulation of diagnostic devices whilst regulation of medical products is required in order to ensure their safety, it is essential that regulatory review processes do not obstruct or unnecessarily delay access to beneficial new products. it is recognised that the current lack of standardisation across national regulatory authorities and the lack of clarity surrounding the regulatory pathways presents an unnecessary burden on manufacturers and acts as a deterrent to marketing in countries where the financial returns may be modest.9 with the exception of the countries of the european union, where a harmonised system has been adopted, countries each have their own set of requirements. in some countries, transparency is lacking, with little information available about the regulatory process or the fees charged. several transnational initiatives are striving toward improved harmonisation of regulation of medical devices, including ivds (table 2). harmonisation requires the use of standardised terminology and definitions which are employed to classify the products under regulation. guidance on this topic was issued by the ghtf, which transitioned to the international medical device regulators forum (imdrf) in 2012. for ivds used in developing countries there are opportunities to streamline and harmonise activities where convergence of protocols and mutual recognition of other regulatory bodies could improve their safety and quality, accelerating access to new tests while simultaneously minimising the costs incurred. setting international standards and streamlining the regulatory process could reduce the regulatory burden and lower costs of new products. four priority areas for harmonisation have been recognised: (1) adoption of common registration requirements and submission dossiers for pre-market approval; (2) convergence of quality standards and mutual, or third party, recognition of audit activities; (3) rationalisation of clinical performance studies in order to avoid unnecessary duplication; and (4) building of transnational networks for post-market surveillance. each of the aforementioned areas of harmonisation is discussed in the following sections. the status quo and need for change are described, along with recommendations made for the way forward. the impact of the proposed harmonisation activities are summarised in table 3. table 2: organisations promoting regulatory harmonisation of medical devices or in vitro diagnostics. table 3: expected impact of harmonisation activities. a common submission dossier top ↑ companies seeking approval to market an ivd are required to supply a dossier to the appropriate nra describing the device and documenting evidence relating to the quality of manufacture, as well as the safety and stability of the components. in addition, those devices considered to be at risk of causing harm to either individual or public health also require evidence regarding the clinical performance of the device. the adoption of a standardised submission dossier template would promote efficiency within the regulatory review process and facilitate standardised training on good review practice using a common set of teaching materials. nras would retain independent review of the data, along with any decisions for approval based on national requirements as they pertain to their local population needs. the need for a common submission dossier was one of the top priorities recognised by the ghtf and was an activity already adopted by the asian harmonisation working party (ahwp).15 status quo with the exception of the european union, submission dossiers are unique to the country, with each nra utilising its own indicators, nomenclature and format in its own language.9,16 preparation of a submission dossier for regulatory approval is a substantial undertaking and the necessity of preparing individual dossiers and reformulating data for each nra is an unnecessary burden on diagnostic companies, causing overall increase to the cost of marketing a new product. in countries with weak economies, small populations or low prevalence of the condition or disease to be tested, the anticipated market may be insufficient to warrant the cost of registering the product. the problem is particularly acute for small companies with limited regulatory expertise or capacity. large companies are also not exempt from the burden that a submission dossier carries, as it represents a significant commitment of resources and time. a standardised template for submission dossiers would decrease the time and effort required of companies, improve the efficiency of regulatory reviews and reduce the cost of goods for diagnostic companies, resulting in a more affordable product and overall reduced time to market. action going forward following principles established by the ghtf, the ahwp has developed a common submission dossier template (csdt) for premarket submission of ivds. the dossier incorporates: (1) marketing history and, where appropriate, a risk/benefit assessment, any prior approvals and current regulatory status, as well as documentation to demonstrate conformity to the essential principles of the ghtf; (2) a product description which includes intended use and any warnings or precautions; (3) a summary of design verification and validation documents – these may include sensitivity, specificity, precision, stability, storage and controls – and, depending on the product classification, it may also include evidence from clinical performance studies; (4) device labelling, with instructions for use (including operating manual and user manual), patient information leaflet and promotional materials; (5) a summary of the risks identified and a description of how these risks have been controlled to an acceptable level; and (6) manufacturer information in order to identify manufacturing sites and to provide quality management system certification such as (iso) 13485:2003 and a description of the manufacturing process.15 a common dossier is to be piloted in africa and other regions using a poc test as an example. transparency of regulatory requirements and processes shall be promoted, preferably with documents available on line. good review practice will include the monitoring and publication of the time required for review. fees charged to companies should reflect the costs of the approval process and should not be seen as an opportunity for profit. harmonisation of submission dossier requirements and adoption of a common template will: (1) reduce the cost to manufacturers of registering a product, a cost that would ultimately be passed to the consumers of the test; (2) reduce delays in test registration; (3) reduce barriers to marketing in small economies; and (4) facilitate harmonised approval of diagnostic devices, ultimately reducing the burden on nras and the cost of national regulatory approval. harmonisation will require consensus on the fundamental principles of regulating diagnostic devices for health, but independent decision making shall be retained by the nra. convergence in the auditing of quality systems top ↑ regulatory oversight requires assurance that the manufacturer of the device conforms to a satisfactory quality management system. these quality management standards are universal and should not differ from country to country. the quality management system used by a manufacturer to control the quality of their product should be audited in order to provide assurance that safe and effective devices will be manufactured. the audit should make certain that specified minimum standards are met during manufacturing and provide impartial, reliable and objective evaluation of compliance with regulatory requirements. iso 13485:2003 is an international standard established for the manufacture of ivds.17 if satisfactory quality management is not practised by the manufacturers, corrective measures may be recommended. for ivds considered to have a risk of causing harm (to potential patients or users), assessment of adherence to satisfactory quality systems will include visiting the site of manufacture. audit teams must encompass a range of skills and expertise enabling them to assess the quality management system and to determine the effectiveness of its implementation. the range of skills includes understanding the regulations and standards applicable to the specific ivd submitted for approval, the intended use and associated risks of the device, as well as the knowledge to assess the design, manufacturing processes and technologies involved. convergence of quality standards and mutual or third-party recognition of inspection results could reduce duplication of efforts where teams representing different nras undertake independent audit of the same site, saving expenditure and reducing delays in regulatory approval. status quo regulatory audits and site visits are costly for companies, with each audit costing as much as usd 200 000 (personal communication). although costs are met in the first instance by the companies, they will ultimately be reflected in the price of the product and recouped from the end users. duplication of audits inflates the cost of bringing a product to market and results in unnecessary delays. nras that require manufacturing site inspections for every medical device sold often have long delays in approving products for the market. in their 2012 annual report, the brazilian national health surveillance agency (anvisa), reported a backlog of over 1000 products awaiting evaluation.18 nras in the developing world lack the expertise and capacity required in order to undertake audits, which ultimately results in delayed approval. action going forward minimum standards should be identified for quality management in the manufacture of poc diagnostics in addition to iso 13485:2003.17 to reduce duplication, competent authorities or organisations capable of undertaking inspections should be identified and mutual recognition or recognition of competent third-party quality management audits adopted. in addition, mechanisms for sharing information should be established. harmonisation of quality systems audits through convergence of standards and/or mutual recognition will make more efficient use of auditing resources and reduce the number of audits by different regulatory bodies for the same product, saving costs and reducing delays in new products reaching the market. use of standardised audit protocols and expert bodies will streamline auditing, leading to improved quality management and product quality. this will provide greater consistency and increased confidence in audits. enhanced consistency in audit practices and feedback provided to manufacturers regarding quality management will facilitate improvements in manufacturing practice. the ultimate beneficiaries will be patients and users of diagnostic devices, who will gain an increased assurance that medical devices placed on the market are safe and effective whilst simultaneously ensuring that the costs of implementing an effective system do not unnecessarily inflate the price. reducing duplication in studies of clinical performance top ↑ pre-market approval of those ivds considered to be high risk to individual or public health requires supporting evidence of the performance and operational characteristics of the device. for laboratory-based diagnostic tests, such evaluations are often conducted using well-characterised archived samples. if the new product is a poc test intended for use in decentralised health centres or dispensaries, evaluations of sensitivity, specificity, precision and ease of use should be conducted in the settings of intended use, with the test performed by the proposed end users.19 such studies need to be based on sufficient sample size with assurance of quality so as to allow informed decisions on the performance and utility of the device as to whether the probable benefits of the device outweigh the risks. data generated in clinical studies are presented to nras as part of the submission dossier for pre-market approval. the high cost of clinical trials and the length of time they require may result in higher cost of goods and significant delay in gaining access to new products that could potentially save lives.20 status quo the requirement of nras to have national trial data for approval has resulted in unnecessary duplication, where further studies provide little or no added scientific benefit.21,22 high costs and delays incurred during clinical studies are a deterrent for companies entering the market, particularly in smaller countries where financial returns may be modest.9,12 costs borne by the manufacturer are ultimately passed on to the consumer, making devices less affordable. since not every country has the capacity to conduct high quality studies in a timely manner, resulting from a lack of specialist facilities or limited access to appropriate patient groups, multi-country evaluations and nras coming together to review trial data and then making their own decisions for approval would be a more efficient approach. action going forward multi-country studies using a standardised protocol and joint review of trial data should be encouraged in order to shorten trial duration and reduce duplication. a clinical trials registry should be established to reduce unintentional duplication and promote maximal use of resources. clinical performance studies should be conducted by accredited laboratories and clinics with external oversight, so as to ensure conformity with international standards, including good laboratory and clinical practice.23,24,25 prior joint approval of clinical and laboratory protocols by institutional review boards will ensure quality and acceptance by representatives of regulatory authorities. reducing duplication of clinical trials will: (1) reduce the cost of accessing the market, making tests more affordable; (2) reduce delays in regulatory approval, accelerating access to new products; (3) reduce barriers to marketing in small economies; and (4) allow nras to maintain independent decision making for new products without requiring local trials. convergence in post-market surveillance top ↑ post-market surveillance ensures that products continue to meet expected safety and quality standards following approval by nras and an important component of regulatory oversight of diagnostic products for health.26 proactive post-market surveillance requires the systematic collection of data from laboratory studies in order to monitor test performance using a panel of appropriate reagents or, alternatively, through field testing using a panel of well-characterised samples. reactive surveillance requires manufacturers, or testers, to report problems voluntarily through an established reporting system. such activities are applicable to manufacturers, importers and distributors. there is a need for cross-border, regional and global sharing of adverse events that threaten safety of individuals or public health so as to accelerate information gathering and enable substandard products to be withdrawn more quickly. the national competent authorities report (ncar) is a membership-based system open to those countries with stringent regulatory authorities.27 the system incorporates post-market surveillance, vigilance and reporting of adverse events and is aimed at improving safety by reducing the likelihood of repeated adverse events. there are two levels of ncar participation. full participation involves national competent authorities with established national adverse event reporting programmes. being a full participant, a national authority receives both public and confidential or highly-sensitive information from other ncar participants. associate participants are a second tier of membership that receive public information, such as recall notices, safety and hazard alerts. status quo currently, there is limited capacity for post-market surveillance in much of the developing world. systems for post-market surveillance for diagnostic devices are not implemented globally26 and, in most developing countries, reporting and information sharing occurs on an ad hoc basis. in many countries, random quality checks, such as lot testing, are not performed and tests can enter the market without checks on their quality.21,28 most developing countries lack a feedback mechanism to provide manufacturers with information regarding the need for corrective action as well as a mechanism for removal of substandard tests that present a risk to public health. disruption of services for some priority diseases, such as hiv, has occurred where publicity regarding quality problems has led, in some countries, to discontinuation of use without replacement devices being available.29 action going forward a mix of proactive and reactive post-market surveillance activities should be encouraged. reporting should be mandatory in the case of a death, a serious injury that is life threatening or results in permanent damage or impairment of a body function, or a malfunction. to implement post-market surveillance for ivd medical devices in developing countries, regional networks of accredited laboratories should be established in order to undertake batch testing and monitor performance and safety. standardised protocols should be established and reference quality assurance materials shared. these practices would promote good practice and instil confidence in the system, in addition to facilitating the exchange of data. global and regional mechanisms should be established so as to facilitate investigation and procedural corrective actions or recalls for products found to be unsatisfactory. harmonisation of proactive activities, such as batch monitoring, would enhance safety by reducing reporting delays, allowing prompt action to be taken should unsafe products be reported. standardisation of protocols, test reagents and sample panels across geographic regions would assist surveillance whilst simultaneously minimising costs. national and transnational communication platforms are needed to simplify current procedures. networks of accredited laboratories should be established to facilitate mutual recognition of surveillance data. convergence of post-market surveillance activities will: (1) ensure that products continue to meet expected safety, performance and efficacy requirements following pre-market approval; (2) prevent substandard tests, or batches of tests, from entering the market; (3) enable corrective actions to be taken by manufacturers; and (4) facilitate removal of substandard tests from the market, ultimately reducing costs and maximising efficient use of resources by nras. conclusion top ↑ diagnostic tests can play a vital role in improving access to effective healthcare, but the current regulatory landscape in developing countries creates significant barriers to market entry and has become a deterrent to innovation. setting international standards and streamlining the regulatory process for in vitro diagnostic devices could diminish the regulatory burden. four areas where action could lower costs, reduce delays, and accelerate access to quality diagnostic products for health are: (1) adoption of common registration requirements and submission dossier for pre-market approval; (2) convergence of quality standards and mutual, or third-party, recognition of audit activities; (3) rationalisation of clinical performance studies in order to avoid unnecessary duplication; and (4) building transnational networks for post-market surveillance. acknowledgements top ↑ the authors received financial support from grand challenges canada. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions r.w.p. (london school of hygiene & tropical medicine) conceived the article; r.m. (london school of hygiene & tropical medicine) developed the first draft and all authors contributed to writing the article. references top ↑ 1. peeling rw, mabey d, herring a, et al. why do we need quality-assured diagnostic tests for sexually transmitted infections? nat rev microbiol. 2006;4(12):909–921. http://dx.doi.org/10.1038/nrmicro1555, pubmed pmid: 171090302. mabey d, peeling rw, ustianowski a, et al. diagnostics for the developing world. nat rev microbiol. 2004;2(3):231–240. http://dx.doi.org/10.1038/nrmicro841, pubmed pmid: 15083158 3. storla dg, yimer s, bjune ga. a systematic review of delay in the diagnosis and treatment of tuberculosis. bmc public health. 2008;8:15. http://dx.doi.org/10.1186/1471-2458-8-15, pubmed pmid: 18194573, pubmed central pmcid: 2265684 4. peeling rw, mabey d. point-of-care tests for diagnosing infections in the developing world. clin microbiol infect. 2010;16(8):1062–1069. http://dx.doi.org/10.1111/j.1469-0691.2010.03279.x, pubmed pmid: 20670288, 5. mcnerney r, daley p. towards a point-of-care test for active tuberculosis: obstacles and opportunities. nat rev microbiol. 2011;9(3):204–213. http://dx.doi.org/10.1038/nrmicro2521, pubmed pmid: 21326275 6. mabey d, peeling r, perkins m. rapid and simple point of care diagnostics for stis. sex transm infect. 2001;77(6):397–398. http://dx.doi.org/10.1136/sti.77.6.397, pubmed pmid: 11714933, pubmed central pmcid: 1744415 7. global harmonization task force. definition of the terms ‘medical device’ and ‘in vitro diagnostic (ivd) medical device’. ghtf/sg1/n071:2012; 2012. 8. special programme for research & training in tropical diseases (tdr). regulation of vitro diagnostics: a global perspective. find sa/tdr: geneva; 2002 [reproduced as annex to diagnostics for tuberculosis: global demand and market potential. geneva: world health organization; 2006]. 9. peeling r, mcnerney r. increasing access to diagnostics through technology transfer and local production. geneva: who; 2011. 10. dowdy dw, steingart kr, pai m. serological testing versus other strategies for diagnosis of active tuberculosis in india: a cost-effectiveness analysis. plos med. 2011;8(8):e1001074. http://dx.doi.org/10.1371/journal.pmed.1001074, pubmed pmid: 21857810, pubmed central pmcid: 3153451 11. macarthur g. global health diagnostics: research, development and regulation. london: the academy of medical sciences; 2009. isbn no: 1-903401-20-8 12. hoerr ra. regulatory uncertainty and the associated business risk for emerging technologies. j nanopart res. 2011;13(4):1513–1520. http://dx.doi.org/10.1007/s11051-011-0260-z 13. eccleston rc. a model regulatory program for medical devices: an international guide. washington, dc: world health organization and united states food and drug administration; 2001. 14. global harmonization task force. principles of in vitro diagnostic medical devices classification. ghtf/sg1/n452008. [document on the internet]. [c2012] [cited 22 january 2014]. available from: http://www.imdrf.org/docs/ghtf/final/sg1/technical-docs/ghtf-sg1-n77-2012-principles-medical-devices-classification-121102.pdf 15. global harmonization task force. summary technical documentation (sted) for demonstrating conformity to the essential principles of safety and performance of in vitro diagnostic medical devices. ghtf/sg1/n063:2011; 2011. 16. kramer db, xu s, kesselheim as. how does medical device regulation perform in the united states and the european union? a systematic review. plos med. 2012;9(7):e1001276. http://dx.doi.org/10.1371/journal.pmed.1001276, pubmed pmid: 22912563 17. praxiom research group limited. iso 13485 medical device standard translated into plain english. [page on the internet]. [c2004] [updated 2013 may 15; cited 2013 sep 03]. available from: http://www.praxiom.com/iso-13485.htm 18. agência nacional de vigilância sanitária. anvisa. anvisa annual report brazil; 2012. 19. banoo s, bell d, bossuyt p, et al. evaluation of diagnostic tests for infectious diseases: general principles. nat rev microbiol. 2010;8(12 suppl):s17–29. pubmed pmid: 21548184 20. cohen p. the changing regulatory environment of in vitro diagnostics: implications for sponsors, manufacturers and laboratory users. aus j med sci 2010;31(1):9–15. 21. peeling rw, smith pg, bossuyt pm. a guide for diagnostic evaluations. nat rev microbiol. 2006;4(9 suppl):s2–6. http://dx.doi.org/10.1038/nrmicro1568, pubmed pmid: 17003769 22. small pm, perkins md. more rigour needed in trials of new diagnostic agents for tuberculosis. lancet. 2000;356(9235):1048–1049. http://dx.doi.org/10.1016/s0140-6736(00)02724-0, pubmed pmid: 11009137 23. world health organization. handbook: good laboratory practice (glp): quality practices for regulated non-clinical research and development. geneva: world health organization; 2009. 24. world health organization. guidelines for good clinical practice (gcp) for trials on pharmaceutical products. geneva: world health organization; 1995. 25. bossuyt pm, reitsma jb, bruns de, et al. towards complete and accurate reporting of studies of diagnostic accuracy: the stard initiative. clin radiol. 2003;58(8):575–580. http://dx.doi.org/10.1016/s0009-9260(03)00258-7, pubmed pmid: 12887949 26. kramer db, tan yt, sato c, et al. postmarket surveillance of medical devices: a comparison of strategies in the us, eu, japan, and china. plos med. 2013;10(9):e1001519. http://dx.doi.org/10.1371/journal.pmed.1001519 27. global harmonization task force. application requirements for participation in the ghtf national competent authority report exchange program. ghtf/sg2/ n38r19:2009; 2009. 28. bell d, wongsrichanalai c, barnwell jw. ensuring quality and access for malaria diagnosis: how can it be achieved? nat rev microbiol. 2006;4(9 suppl):s7–20. http://dx.doi.org/10.1038/nrmicro1525, pubmed pmid: 17003770 29. africomnet. tanzania: search on for new supplier of hiv test kits. [page on the internet]. [c2012] [cited 2013 oct 28]. available from: http://www.africomnet.org/communication-resources/highlights/1904-tanzania-search-on-for-new-supplier-of-hiv-test-kits.html article information authors: rosemary a. audu1 ugochukwu sylvester-ikondu1 chika k. onwuamah1 olumuyiwa b. salu1 fehintola a. ige1 emily meshack1 maureen aniedobe1 olufemi s. amoo1 azuka p. okwuraiwe1 florence okhiku1 chika l. okoli1 emmanuel o. fasela1 ebenezer. o. odewale1 roseline o. aleshinloye1 micheal olatunji1 emmanuel o. idigbe1 affiliations: 1human virology laboratory, nigerian institute of medical research, lagos, nigeria correspondence to: rosemary audu postal address: nigerian institute of medical research, 6, edmond crescent, p.m.b. 2013, yaba, lagos, nigeria dates: received: 09 dec. 2011 accepted: 05 sept. 2012 published: 29 oct. 2012 how to cite this article: audu ra, sylvester-ikondu u , onwuamah ck, et al. experience of quality management system in a clinical laboratory in nigeria. afr j lab med. 2012;1(1), art. #18, 5 pages. http://dx.doi.org/10.4102/ ajlm.v1i1.18 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. experience of quality management system in a clinical laboratory in nigeria in this lessons from the field... open access • abstract • introduction • description • lessons learned • recommendation • acknowledgement    • competing interests    • authors’ contributions • references abstract top ↑ issues: quality-management systems (qms) are uncommon in clinical laboratories in nigeria, and until recently, none of the nation’s 5 349 clinical laboratories have been able to attain the certifications necessary to begin the process of attaining international accreditation. nigeria’s human virology laboratory (hvl), however, began implementation of a qms in 2006, and in 2008 it was determined that the laboratory conformed to the requirements of iso 9001:2000 (now 2008), making it the first diagnostic laboratory to be certified in nigeria. the hvl has now applied for the world health organization (who) accreditation preparedness scheme. the experience of the qms implementation process and the lessons learned therein are shared here.description: in 2005, two personnel from the hvl spent time studying quality systems in a certified clinical laboratory in dakar, senegal. following this peer-to-peer technical assistance, several training sessions were undertaken by hvl staff, a baseline assessment was conducted, and processes were established. the hvl has monitored its quality indicators and conducted internal and external audits; these analyses (from 2007 to 2009) are presented herein. lessons learned: although there was improvement in the pre-analytical and analytical indicators analysed and although data-entry errors decreased in the post-analytical process, the delay in returning laboratory test results increased significantly. there were several factors identified as causes for this delay and all of these have now been addressed except for an identified need for automation of some high-volume assays (currently being negotiated). internal and external audits showed a trend of increasing non-conformities which could be the result of personnel simply becoming lax over time. application for laboratory accreditation, however, could provide the renewed vigour needed to correct these non-conformities. recommendation: this experience shows that sustainability of the qms at present is a cause for concern. however, the tiered system of accreditation being developed by who–afro may act as a driving force to preserve the spirit of continual improvement. introduction top ↑ laboratories play a pivotal role in all disease control and prevention programmes by providing timely and accurate information for patient management and disease surveillance.1 clinical laboratories specifically, provide accurate diagnosis of present, recent or past infections for appropriate case management. 1 though laboratories form the backbone of health systems, providing health care workers with critical test results for numerous deadly diseases, yet in sub-saharan africa, which carries a huge disease burden, laboratories are among the world’s most ill-equipped and poorly resourced facilities. as of 2009, there were 340 diagnostic laboratories in sub-saharan africa that were accredited and 312 of these (92%) were found in south africa.2 in nigeria, there are 5349 diagnostic laboratories, of which only the human virology laboratory (hvl) has iso certification.in 2000, the ford foundation provided a grant to the nigerian institute of medical research for the establishment of a national reference laboratory with capacities for basic, applied and operational research on hiv and aids in the country. the grant, which was worth $250 000, was meant for the construction of a laboratory facility, the purchase of equipment, and training of personnel. subsequently, an additional grant of $150 000 was awarded for two years for sustainability activities, such as revolving funds for purchase of kits and reagents. prior to this, there was no laboratory with facilities for hiv molecular diagnostics in nigeria, requiring clinical samples from people living with hiv to be sent out of the country to laboratories abroad for relevant tests and analyses. with the grant funding, two new large laboratory facilities were built and fully equipped with state-of-the-art equipment for hiv and aids research and services. setting up the quality management system at the hvl involved two major stages, namely, planning and implementation. the planning stage involved identifying and mapping out processes, risk assessment of the processes and appropriate management, as well as identifying quality indicators and setting targets for monitoring. implementing this process approach to management facilitated bottom-up communication, ownership and participation, thereby improving the quality of services provided. in order to meet international standards, the laboratory worked to conform to the requirement of nis iso 9001:2000 and has in fact converted to the iso 9001:2008 standard, making it the first diagnostic laboratory to be so certified in nigeria. the major difference between the two standards is that the iso 9001:2008 standard has a broader interpretation and application of its requirements. because iso 15890 requirements are more relevant to diagnostic laboratories, the hvl has applied for the who accreditation scheme through the efforts of the president’s emergency plan for aids relief (pepfar), which uses this standard. the effort of obtaining certification originated with the then-director general, a fact which facilitated securing resources and approvals. despite the high pre-assessment non-conformities, this ‘buy-in’ from staff at the highest levels of management was key to the success of this endeavour. implementing the qms was not without its challenges, however. one major challenge was getting the other staff to buy into this vision. this was achieved through involving every staff member, regular awareness creation at staff meetings, ensuring good staff morale and recognition of staff for exemplary performance. there is a clearly defined need for attracting and retaining qualified, motivated personnel within the public health sector. in the hvl, the majority of laboratory personnel were either recent graduates completing compulsory service to the nation or on internships with the laboratory and, as such, they typically maintained a high level of performance. a few of the longer-serving staff were determined to be performing at an unsatisfactory level and were posted out of the unit. this reduced the staff to 7 research fellows, 12 laboratory scientists and 12 support staff of varying cadres. an important aspect of addressing shortcomings in staff performance was the requirement that all personnel undergo basic training in performance standards. initially, documentation of non-conformities among staff was challenging as this was frequently misunderstood as a means of ‘reporting an individual’ for an infraction. staff members were informed that this documentation was not for any punitive purpose but was intended as a means of tracking and improving the system. the need to address this misperception among staff has been ongoing. another factor related to laboratory staffing had to be addressed as well. though nigeria, unlike many african nations, is not facing a crisis with respect to shortage of health workers, ‘brain circulation syndrome’ is quite high, with personnel frequently moving from one job to another in search of better wages or conditions of service. the hvl employed a group of highly skilled and dedicated staff who were committed to the vision of adopting the qms and working toward accreditation after having undergone several training sessions on qms. however, because of the hvl’s unique status as a self-sustaining laboratory within the government establishment, personnel were not guaranteed job security and this fact adversely affected the sustainability of the laboratory. moreover, the who recommends training of health workers to ensure quality service delivery and the retention of these trained workers because essential health services cannot be provided by people working on a voluntary basis if the service is to be sustainable. it is also recommended that trained health workers who are providing essential health services receive adequate wages and appropriate and commensurate incentives.3 hence, the need to ‘absorb’ hvl’s staff as permanent ministry of health employees was identified as a solution to this issue. in july 2008, 20 project staff of the hvl were absorbed as permanent government staff and the laboratory was made a unit in the microbiology division of the nigerian institute of medical research (nimr), which is a parastatal institution under the ministry of health. an additional challenge was the lack of a basic quality manual to establish guidelines for the hvl that were codified and easily accessible for personnel. with assistance from a trainer from the standard organisation of nigeria (son), this quality manual was eventually developed and adopted by the laboratory. the quality manual, which was written in line with the requirements of the standard, contained the structure of the quality system and its policies. but because son was not typically involved with developing capacity for qms in clinical settings, this made the overall expenditure on training higher, because assistance had to be sought first from senegal. similarly, the laboratory found it necessary to adopt a quality policy which drives its vision as well as to establish a top management committee, in line with the iso requirement, which serves as the management advisory committee to the laboratory. description top ↑ in november 2005, two personnel from the hvl were sent to the bio 24 laboratory in dakar, senegal, which was an iso 9001:2000 certified clinical laboratory, to study the implementation of a qms there. by july 2006, a consultant from senegal and the laboratory manager from the certified laboratory had trained the hvl staff on a basic implementation course, and a baseline assessment had been conducted. the laboratory then defined its processes and began to implement some basic qms structures such as developing of policies, proper archiving of records, and selection of quality indicators for monitoring the system. we also introduced regular and quality-focused staff meetings, which included training and retraining. by june 2007, the son was identified as an appropriate organisation to train staff on the iso 9000 basic and auditors’ course; 13 staff members qualified as internal auditors as a result of this training. twenty staff members were again trained on iso/iec 17025 in august 2008 and later undertook the iso 15189:2007 laboratory accreditation and auditors’ course in march 2009. both of these training courses were also conducted by the son. the laboratory also successfully completed a revalidation course for iso 9001:2008. though the process of obtaining certification and ultimately accreditation is an expensive one, the hvl project was able to address some of the costs through income generated from the services rendered by the laboratory. because nimr management saw the value of the improvements in laboratory personnel performance, the resources required have been perceived as a worthy investment and provided to continue the process. since 2007, the hvl’s quality indicators have been monitored, customer surveys have been performed, and internal and external audits have been conducted as part of the ongoing process. various methods were used for the customer survey conducted twice a year. the commonest method was the use of questionnaires administered to patients, clinicians and other laboratories that patronised the hvl. these questionnaires were designed and analysed by staff members. other occasional methods used included in-depth interviews with clinicians, focused group discussion with patients and the use of questionnaires administered to suppliers. suggestions from these surveys were used to improve the system. some staff of the laboratory who had qualified as internal auditors carried out internal audits twice a year. they prepared checklists using the iso 9001:2000 standards and the laboratory quality manual; corrective actions were taken for all reported non-conformities. external audits were however carried out twice a year by the standards organisation of nigeria to ensure the qms was still functional. this is a mandatory requirement, if the certification was to be maintained. reports of all of these activities were presented to the management during annual reviews. lessons learned top ↑ by december 2007, the laboratory was deemed to be in compliance with the iso 9001:2000 standard and was awarded its certificate in february 2008. by 2010, the hvl had conformed to iso 9001:2008. the quality indicators for the operational processes and the audit analyses from 2007 to 2009 are presented below. table 1: performance of some indicators in the operational system between 2007 and 2009 for all assays in hvl. table 1 shows the performance of some indicators in the operational system for all assays within the period reported. there was an improvement in the indicators for the pre-analytical and analytical processes over the years. it was observed earlier that there was a gap in awareness on how to collect samples for the various tests that were rendered in the laboratory. continuous education is needed for all stakeholders involved in laboratory testing to improve the quality of the pre-analytical phase of the total-testing process.4 the laboratory prepared a clients’ handbook which contained information on time of operation, test offered, specimen type, test turnaround time, packaging, storage and transportation of samples. this was used to educate clinicians and other laboratory scientists who send samples to hvl. these clients were also invited to the laboratory and were further trained on proper sample collection, storage and transportation. there was a continuous decline in the number of unanalysed samples and external controls exceeding specification, which is a positive development for the qms. westgard rules applied to a levey-jennings chart as practiced in the hvl are programmed to determine when an analytical run should be rejected. these rules are applied carefully so that true errors are detected while false rejections are minimised. the rules applied to high volume chemistry and hematology instruments produce low false rejection rates.5 this was achieved in the hvl; however, values recorded for the external controls were higher than values reported in other studies,6 hence more rigorous monitoring is required. during the monitoring, it was observed that a few personnel still had challenges in using the tool while another staff member had not fully understood the benefit of complying strictly with the rules, hence training and retraining on application and benefits of the westgard rule is now a regular practice in the hvl. there was an improvement in maintaining environmental temperatures within acceptable ranges. scores obtained from participation in external quality assessment were within an acceptable range.an improvement was also observed in the reduction of errors in data entry in the post-analytical process (table 1). the delay in reporting results became significantly higher in 2009 in spite of the fact that implementing the qms is expected to improve turnaround time.7 determining the root cause of this delay in order to eliminate it is a necessary approach.8 several factors contributed to the delay of results such as issues in supply chain management (unavailability of test kits provided by partners), failure of quality control for two assays (westgard rules), network problems (support infrastructure), and staff rotation. another major challenge was the daily manual testing of large numbers of samples for some of the assays. because the unavailability of kits was beyond the control of the laboratory, discussions were held with the supporting partner providing the kits to correct the challenges faced with supplies. there was also a change required in the brand of test kit because of repeated failure of the internal quality control, and an agreement for the maintenance of the database network was signed. in terms of staff rotation, an improved competency evaluation was established. appeals are still being made to the supporting partner for automation of these assays particularly in a reference laboratory such as the hvl. comparison of internal audits by staff and external audits by son over the three years showed an increase in the number of non-conformities, from 157 to 192 for internal audit and 39 to 55 for external audits – perhaps an indication that laboratory personnel were becoming lax in implementing the system. it is presumed that the process of applying for laboratory accreditation with its stringent technical demands could stimulate staff to sustain and even improve the system.9 this is most likely because of the stepwise accreditation preparedness approach of the world health organisation regional office for africa (who-afro). this programme is intended to provide an interim pathway for measuring, monitoring and recognising improvement toward the realization of international laboratory standards and subsequent application to full iso 15189 accreditation.2 recommendation top ↑ for qms to be implemented effectively there must be support from the highest levels of management; therefore strong advocacy to management is a necessary starting point. for laboratories planning to obtain accreditation, considerable training is required and this must be taken into account. automation of laboratory systems has advantages such as reduction in staff time and supply costs, reduced turnaround times, as well as reduction in operational errors .6 therefore it is recommended that clinical laboratories performing high volume testing in africa adopt automation of their systems as they prepare for possible who assisted approach for laboratory accreditation.the hvl audit experience shows there was an improvement in 2008 – the year that certification was obtained – but that, soon after, these gains diminished, suggesting that sustainability will continue to be a major challenge. the who’s accreditation preparedness scheme for african medical laboratories utilises a tiered system under which laboratories complying with the iso 15189 standards are audited and awarded recognition from one to five stars (in the form of a certificate). this may serve to provide the necessary impetus for continual quality improvement for african laboratories. under this program, audits are conducted annually and laboratories that fall behind will have their certificate of recognition withdrawn. only a few of africa’s laboratories are currently accredited to international standards, but, through this five-star accreditation preparedness process, laboratories will be able to be recognised for their improvement efforts using the who afro slipta checklist – and eventually apply for accreditation by a nationally-, regionally-, or internationally-recognised accreditation body. this is critical because good laboratory support leads to better health care delivery through correct diagnoses of diseases and monitoring of drug resistance. this is crucially important in nigeria (and across africa) to monitor patients infected with hiv, tb, malaria, and a host of other diseases. in addition, an efficient laboratory also can dramatically reduce waiting time to get results – allowing patients who often travel a day or more for testing to receive the laboratory results sooner. studies have shown that when patients need to return for a second visit to a hospital or clinic for test results, significant percentages fail to do so.10 in spite of the challenges inherent in this process and the continued need for improvement, the hvl can attest that implementing qms has brought about reliability and reproducibility of results as confirmed by its numerous clients. also there has been a widespread recognition of the laboratory such that corporate and private clients seek services, which has resulted in improved profit. consequently, most of the assays have now been automated. the introduction of the qms in nigerian medical laboratories – and in africa generally – truly represents a major step forward and the beginning of a whole new era for the continent’s public health systems. acknowledgement top ↑ the authors wish to acknowledge the commitment of the management of the nigerian institute of medical research, particularly prof oni idigbe, the immediate past director general, for nursing the idea and bringing it to reality. the entire management have supported this vision. the staff of the human virology laboratory, who have worked tirelessly, are highly appreciated. we also wish to acknowledge our implementing partners (aids prevention initiative in nigeria, apin) as well as our clients and suppliers who have supported and cooperated with us respectively. finally, we wish to acknowledge erik friedly, who assisted with the preparation of this manuscript. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. authors’ contributions r.a.a. was the project leader, responsible for the project design and also wrote the manuscript. u.s. and c.k.o. performed internal audits and data management, they also monitored processes. o.b.s., f.a.i., e.m., m.a., o.s.a., a.p.o., e.o.f., e.o.o. and r.o.a. performed internal audits and monitored processes. f.o., c.l.o. and m.o. monitored processes, while e.o.i. made conceptual contributions. references top ↑ 1. strengthening public health laboratories in the who african region: a critical need for disease control. in: 58th session of the who regional committee for africa (afr/rc58/r2). yaounde: world health organization regional office for africa; 2008. 2. gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accredited process: improving the quality of laboratory systems in the african region. am. j. clin. pathol. 2010;134:393–400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm 3. who/pepfar/unaids. task shifting: global recommendations and guidelines; 2008, isbn 978 92 4 159631 2. 4. simundic a, nikolac n. the prevalence of preanalytical errors in a croatian iso 15189 accredited laboratory. clin. chem. lab. med. 2010;48(7):1009–1014, issn (online) 1437-4331, issn (print) 1434-6621, doi: 10.1515/cclm.2010.221 5. westgard, jo, barry pl. cost-effective quality control: managing the quality and productivity of analytical processes. aacc press; 1986. 6. llopis ma, trujillo g, llovet mi, et al. quality indicators and specifications for key analytical-extranalytical processes in the clinical laboratory. five years’ experience using the six sigma concept. clin chem lab med. 2011 jan 31; [epub ahead of print]. http://dx.doi.org/10.1515/cclm.2011.067 7. markin rs, what a. laboratory automation: trajectory, technology and tactics. clin. chem. 2000; 46: 764–771. 8. howanitz jh, howanitz pj. laboratory results. timeliness as a quality attribute and strategy. am. soc. clin. path. 2001;116:311–5. http://dx.doi.org/10.1309/h0dy-6vtw-nb36-u3l6 9. aoyagi t. iso 15189 medical laboratory accreditation. rinsho byori. 2004 oct;52(10):860–865. 10. cdc laboratory accreditation success stories, global hiv/aids; 2009. http://www.cdc.gov/globalaids/success-stories/lab-accreditation.html abstract introduction methods results discussion acknowledgements references about the author(s) sarishna singh national health laboratory service tygerberg academic laboratory, division of medical microbiology, tygerberg hospital, tygerberg, south africa division of medical microbiology, department of pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa mae newton-foot national health laboratory service tygerberg academic laboratory, division of medical microbiology, tygerberg hospital, tygerberg, south africa division of medical microbiology, department of pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa pieter nel national health laboratory service tygerberg academic laboratory, division of medical microbiology, tygerberg hospital, tygerberg, south africa division of medical microbiology, department of pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa colette pienaar national health laboratory service tygerberg academic laboratory, division of medical microbiology, tygerberg hospital, tygerberg, south africa division of medical microbiology, department of pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa citation singh s, newton-foot m, nel p, pienaar c. comparison of commercial assays and two-step approach to detect clostridioides difficile in south africa. afr j lab med. 2022;11(1), a1809. https://doi.org/10.4102/ajlm.v11i1.1809 note: additional supporting information may be found in the online version of this article as online supplementary material. original research comparison of commercial assays and two-step approach to detect clostridioides difficile in south africa sarishna singh, mae newton-foot, pieter nel, colette pienaar received: 03 dec. 2021; accepted: 26 may 2022; published: 29 sept. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: clostridioides difficile is the number one cause of hospital-acquired diarrhoea. accurate diagnosis of c. difficile is of utmost importance as it guides patient management and infection control practices. studies evaluating the performance of commercially available nucleic acid amplification tests (naats) versus algorithms are lacking in resource-limited settings. objective: this study assessed the performance of three commercially available tests and a two-step approach for the diagnosis of c. difficile infection using toxigenic culture (tc) as the gold standard. methods: two hundred and twenty-three non-duplicate loose stool samples were submitted to the national health laboratory service microbiology laboratory at tygerberg hospital, cape town, south africa, from october 2017 to october 2018. the samples were tested in parallel using the c. diff quik chek complete enzyme immunoassay (eia) and two naats (xpert c. difficile and bd max cdiff), and the results were compared to tc. the performance of a two-step approach consisting of the c. diff quik chek complete followed by the xpert c. difficile was also determined. results: of 223 faecal specimens tested, 37 (16.6%) were tc-positive. the sensitivity and specificity of the c. diff quik chek complete were 54.1% and 98.9%; xpert c. difficile, 86.4% and 96.8%; bd max cdiff, 89.2% and 96.8%; and two-step approach, 89.2% and 96.2%. conclusion: the c. diff quik chek complete, in a two-step approach with the xpert c. difficile, performed similarly to the naats on their own and offer advantages in terms of cost and workflow in low-resource settings. keywords: clostridioides difficile; clostridium difficile; xpert; bd max; quik chek; toxigenic culture. introduction clostridioides (clostridium) difficile is an anaerobic gram-positive rod capable of forming endospores and producing toxins. c. difficile infection is associated with an asymptomatic carrier state, self-limiting diarrhoea, pseudomembranous colitis, and toxic megacolon which can be fatal.1 c. difficile infection accounts for up to 20% of nosocomial diarrhoea cases worldwide.2 in south africa, there is very little published data regarding the incidence and prevalence of c. difficile; a previous study in patients with diarrhoea reported the prevalence to be 16%.3 in the past two decades, c. difficile has also emerged as a cause of community-acquired diarrhoea.2,4 most c. difficile strains produce two major toxins, namely toxin a, an enterotoxin, encoded by the tcda gene, and toxin b, a cytotoxin, encoded by the tcdb gene. c. difficile infection associated with increased severity and mortality has been reported in north america and europe.5 the c. difficile strain responsible for these outbreaks, the hypervirulent 027/nap1/bi, is characterised by a deletion in the tcdc gene (a negative regulator of tcda and tcdb expression), resulting in increased production of toxin a and toxin b.6 certain strains of c. difficile may produce a binary toxin, c. difficile transferase, encoded by cdta and cdtb. data regarding binary toxin conflict, with some studies suggesting its significance is unclear whereas others have shown that it may be associated with a higher mortality rate.7,8,9 accurate diagnosis of c. difficile infection is essential as it guides patient management and infection control practices. the two diagnostic reference standards for the diagnosis of c. difficile are toxigenic culture (tc) and the cell culture neutralisation assay; however, these are labour-intensive and have extended turnaround times of 2–3 days.10 current guidelines recommend a two-step approach: an enzyme immunoassay (eia), followed by a molecular test to increase the diagnostic yield.11 this approach, compared to toxin eia only, is much more sensitive. algorithm-based testing is recommended to optimise the positive predicative value of laboratory results.11 commercial eias utilise monoclonal antibodies to detect glutamate dehydrogenase (gdh), an antigen common to all c. difficile strains irrespective of toxin production, and toxin a and/or toxin b (tox a/b). the c. diff quik chek complete eia detects gdh as well as tox a/b. commercial nucleic acid amplification tests (naats) are usually real-time polymerase chain reaction (pcr) assays which target tcdb. in this study, the performance of c. diff quik chek complete (quik chek) (alere techlab, blacksburg, virginia, united states) and two commercial naats, the xpert c. difficile (xpert) (cepheid, sunnyvale, california, united states) and bd max cdiff (bdm) (becton dickinson, san jose, california, united states) were compared to tc. the performance of a two-step approach consisting of the quik chek and xpert, that is, the current standard of care (soc) at our institution, was also evaluated. methods ethical considerations ethics approval was obtained from the health research ethics committee of stellenbosch university, cape town, south africa (reference number s17/03/064). a waiver of informed consent was obtained as no patient identifiers were published and no invasive procedures were performed as a result of this study. only the results of the current soc tests were reported to physicians. after the results were recorded, a study number was assigned to the specimen and all patient identifiers were removed. study design this was a prospective diagnostic test accuracy study comparing three different assays, namely the quik chek, xpert and bdm, to tc (reference method) for the detection of toxigenic c. difficile in faecal samples. the performance of the current soc (a two-step approach consisting of the quik chek and xpert), at our institution was also compared to tc. a composite reference standard (crs) analysis was performed to account for limitations in tc as a reference method (supplementary material).12 the crs composite positive was defined as tc-positive or positive by two commercial assays in a tc-negative sample. sample size tygerberg hospital near cape town, south africa, is a 1380-bed tertiary hospital which delivers specialist services to approximately half the population of the western cape province (total population 6.2 million). the microbiology laboratory of the national health laboratory service at tygerberg hospital performs c. difficile testing on patient samples from tygerberg hospital as well as peripheral hospitals and clinics within the tygerberg drainage area. assuming a c. difficile prevalence of 16% based on previous studies, and using a 95% confidence interval with a 5% error rate on both sides, a sample size of 207 was calculated using the open epi sample size calculator (g dean & km sullivan, atlanta, georgia, united states).13 sample processing non-duplicate loose stool samples (defined as taking the shape of the container) from adult and paediatric patients older than two years of age submitted to the national health laboratory service microbiology laboratory at tygerberg hospital from october 2017 to october 2018 for routine c. difficile testing were tested in parallel with the four assays. none of the samples was frozen and thawed prior to testing. in the rare event that samples could not be tested on the day of collection, they were kept at 2 °c – 8 °c and processed within 48 h of collection. the quik chek eia was performed as per instructions provided by the manufacturer and read by multiple laboratory technologists as part of their routine daily work. the results were interpreted as follows: gdh-positive and tox a/b-positive samples were regarded as positive, gdh-negative and tox a/b-negative samples were regarded as negative, and gdh-positive and tox a/b-negative samples were regarded as negative. the xpert naat was performed as per the instructions of the manufacturer. the test was interpreted as positive for toxigenic c. difficile if the cytotoxin gene (tcdb) was detected within the valid cycle threshold range and above the minimum endpoint setting, and as toxigenic c. difficile negative if the tcdb gene was not detected, provided the sample processing control and probe check controls met the manufacturer’s requirements. testing was repeated on any samples with invalid or error results due to failure of the sample processing control or probe check controls. the bdm naat was performed by following the instructions of the manufacturer. test results were automatically interpreted by the bdm instrument. positive, negative and unresolved results were based on the target’s and sample processing control’s amplification status. the test was interpreted as toxigenic c. difficile positive if the cytotoxin gene (tcdb) was detected within the valid cycle threshold range and above the minimum endpoint setting, and as toxigenic c. difficile negative if the tcdb gene was not detected, provided the sample processing and probe check controls met the manufacturer’s requirements. indeterminate and incomplete results are obtained when the bdm system fails. any unresolved, indeterminate and incomplete samples were repeated. the two-step algorithm was performed as follows: the quik chek was done and samples that were gdh-positive and tox a/b-positive were regarded as positive; gdh-negative and tox a/b-negative samples were regarded as negative. samples that were gdh-positive and tox a/b-negative were interpreted in conjunction with the xpert results to establish if toxin genes were present or absent. toxigenic culture was used as the reference method and performed by culturing c. difficile from stool samples on chromid c.diff agar (biomérieux, marcy l’etoile, france), a differential and selective medium, followed by testing for the organism’s ability to produce toxin by pcr.14,15,16 a swab was dipped into the stool sample and inoculated onto the agar medium. the inoculum was streaked to enhance the recovery of single colonies. the plates were placed in a jar with an anaerobic sachet, anaeropack-anaero (mitsubishi gas chemical company, inc., tokyo, japan) and incubated at 35 ºc. after 48 h of incubation, the plates were examined for grey and black colonies; if none were present, the culture was considered negative for c. difficile. a multiplex pcr was performed on a streak of grey and black colonies to detect the c. difficile-specific tpi gene, as well as the tcda and tcdb genes.17 cultures that were pcr-positive for the tpi and tcdb genes were considered tc-positive, while those that were pcr-negative for the tpi or tcdb genes were considered tc-negative. in humans, the tcda gene has been reported in tcdb-positive c. difficile strains only.18 statistical analysis sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) were calculated for each assay against both tc and the crs using epicalc 2000 version 1.00 software (brixton books, london, england). results a total of 223 samples were included in the study. of these, 37 (16.6%) were tc-positive and 19/37 (51.4%) were also positive from all three commercial assays. the soc positivity rate was 17.9% (40/223). quik chek was gdh-pos in 45/223 (20.2%) samples. twenty-two of the 45 samples (48.9%) were tox a/b-pos, of which 20/22 (91%) were also tc-positive. of the 23 gdh-pos samples that were tox a/b-neg, 11 (47.8%) were tc-positive (table 1). table 1: performance characteristics of diagnostic assays in comparison with toxigenic culture on stool samples submitted to the national health laboratory service microbiology laboratory at tygerberg hospital, cape town, south africa, from october 2017 to october 2018.1 thirty-eight of the 223 stools (17%) tested positive by xpert and 32/38 (84.2%) were confirmed as positive by tc. none of the samples was positive for the binary toxin or epidemic 027/nap1/bi strain. of the 185 xpert-negative stools, 180 (97.3%) were confirmed as negative by tc, while 5/185 (2.7%) xpert-negative samples were tc-positive (table 1). the bdm detected 39/223 (17.5%) positive stool samples; 33/39 (84.6%) were confirmed by tc. one hundred and eighty of the 184 bdm-negative stools (97.8%) were confirmed as negative using tc, while the other 4/184 (2.2%) were tc-positive. the soc two-step approach detected 40 positive stool samples, 33 (82.5%) of which were confirmed by tc. of the 183 soc-negative stool samples, 179 (97.8%) were confirmed as negative by tc (table 1). the quik chek performed poorly while the xpert and bdm and the two-step approach had similar sensitivities, specificities, ppv and npv when compared to tc. results were also compared to a crs (supplementary table 1). the xpert, bdm and two-step approach showed higher sensitivities, specificities and ppv in comparison with the crs, but the npv of all the assays were similar. discussion there is a lack of consensus regarding the optimal diagnostic c. difficile laboratory assays. high-quality evidence for the best diagnostic testing strategy is scarce and researchers rarely use either of the two accepted reference standards, that is, cell culture neutralisation assay or tc, for assessment of diagnostic accuracy, but rather their own laboratory-defined crs criteria. in this study, we determined the diagnostic accuracy of three different commercial assays for the detection of toxigenic c. difficile in stools compared to both tc and a crs to account for any limitations of the tc. in this study, the sensivitity of the quik chek was 54.1%, xpert was 86.4%, bdm was 89.2% and the two-step approach was 89.2%. the specificity for quik chek was 98.9%, for xpert was 96.8%, for bdm was 96.8% and for the two-step approach was 96.2%. the ppv of the quik chek was 90.9%, xpert was 84.2%, bdm was 84.6% and the two-step approach was 82.5%. the npv for quik chek was 91.5%, xpert was 97.3%, bdm was 97.8%, and the two-step approach was 97.2%. clostridioides difficile toxin eias lack sensitivity. all c. difficile contain the gdh antigen, whether toxin genes are present or absent. glutamate dehydrogenase eias have high sensitivities but poor specificities; therefore, an additional test (most commonly a toxin assay) must be performed. the gdh eia is the first test performed in a two-step or three-step approach where a gdh-positive result is followed by a toxin assay or a naat to detect toxin genes.11 our findings for the quik chek showed a sensitivity, specificity, ppv and npv of 54.1%, 98.9%, 90.9% and 91.5%, respectively. these findings were similar to a study conducted in 2012 in kuwait comparing the xpert, quik chek and tc, which found the sensitivity, specificity, ppv and npv of the quik chek to be 53.85%, 100%, 100% and 98.51%, respectively, when using a crs defined as two tests being in agreement.19 a 2009–2018 study conducted by chung and lee in korea compared the diagnostic performance of the quik chek to xpert as a reference test, with sensitivity, specificity, ppv and npv of 55.4%, 100.0%, 100.0% and 80.0%, respectively.20 however, they had a higher prevalence of c. difficile infection (35.9%) than our study population (16.6%), which could explain the difference in ppv (100.0% vs 90.9%).20 in contrast, a study conducted in 2013–2014 by seo et al. in korea, found a lower sensitivity of 45.7% when using either tc or the combination of quik chek and xpert as a reference standard.21 nucleic acid amplification tests that target chromosomal toxin genes show high sensitivity and specificity, provide rapid results, and are amenable to both batch and on-demand testing.22 reported estimates of sensitivity for naats range from 77% to 100% when compared to tc. specificity ranges from 83% to 100% in comparison to tc.23 nucleic acid amplification tests may detect asymptomatic carriage due to possible non-expression of the toxin encoding gene and therefore the clinical specificity may be lower than reported.24 in their meta-analysis conducted in 2019, kraft et al. reported an estimated sensitivity of 94% and a specificity of 97% when naats were compared to either cell culture neutralisation assay or tc or both in studies where it was specifically stated that stools were only included if conforming to the shape of the container.10 in this study, the xpert had a sensitivity, specificity, ppv and npv of 86.4%, 96.8%, 84.2% and 97.3% respectively, and the bdm performed similarly showing a sensitivity, specificity, ppv and npv of 89.2%, 96.8%, 84.6% and 97.8%, respectively. a study by yoo et al. in korea found a sensitivity of 82.8% for xpert and 81.6% for bdm when compared to tc. they attributed the difference in sensitivity between the two tests to a freeze-thaw cycle before the bdm testing.25 in a study conducted in germany, dalpke et al. found a sensitivity, specificity, ppv and npv of 97.3%, 97.9%, 90.0% and 99.5% for the xpert and 90.5%, 97.9%, 89.3% and 98.1% for the bdm.26 in a study conducted 2014–2017 in south africa demonstrated the impact of diagnostic methods on the diagnosis of c. difficile infection, nomlomo et al. found a 15.9% positivity rate when using an algorithm approach (consisting of eia followed by pcr) versus 11.4% and 21.1% when using a toxin eia and pcr.27 however, neither of the two accepted references tests was performed as the comparator test in this study. we showed a 16.6% tc positivity rate which is very similar to nomlomo et al.’s finding using the algorithm approach.27 similar to the sensitivity of 89.2% and specificity of 96.2% for the two-step algorithm approach in our study, kraft et al.’s meta-analysis found a sensitivity of 89.0% and specificity of 99.0% when comparing gdh/toxin/naat algorithms to the tc or cell culture neutralisation assay.10 in contrast, the seo et al. study found a higher sensitivity, specificity, ppv and npv of 94.0%, 100.0%, 100.0% and 100.0% for the two-step approach, which could be attributed to the crs in this study being either tc-positive or a combination of xpertand quik chek-positive.21 our algorithm performed similarly to the xpert or the bdm alone in terms of sensitivity, specificity, ppv and npv. the xpert, bdm and two-step algorithm performed similarly, with overlapping confidence intervals when compared to tc and crs. from these findings it is evident that tc is a robust reference test for the statistical measures of sensitivity, specificity, ppv and npv for the xpert, bdm and two-step approach. limitations limitations of our study include the use of stool sampless conforming to the shape of the container as a surrogate for diarrhoea, as well as not excluding other causes of diarrhoea. in addition, we did not collect pre-analytical data such as prior or current antibiotic use or determine any other risk factors for c. difficile. post-analytical patient outcome data was also not collected. the sensitivity of lateral flow assays is limited by the dissociation constant of the antibody–antigen conjugate and by user interpretation of the colorimetric read-out. the tc method used in this study did not include heat-shock treatment prior to the inoculation of samples onto media, which may have improved the detection of c. difficile. conclusion the xpert and bdm assays and two-step approach performed similarly in detecting toxigenic c. difficile in faecal samples. quik chek cannot be used on its own for the diagnosis of c. difficile due to its poor sensitivity but the soc two-step approach using quik chek followed by xpert showed a similar sensitivity and ppv compared to molecular testing alone. the continued use of the current two-step approach in a resource-limited setting such as south africa is recommended as it is rapid, easy to perform and reduces cost without compromising diagnostic accuracy. acknowledgements the authors would like to thank bd diagnostics for partly funding the bdm kits and placement of the bdm instrument, dr motlatji maloba from the division of medical microbiology, university of the free state and national health laboratory service, bloemfontein, south africa, for her guidance with the study protocol design, brian kullin from the department of molecular and cell biology, university of cape town, south africa, for providing the toxigenic c. difficile control strains, and the biostatistics unit, division of epidemiology and biostatistics, faculty of medicine and health sciences, stellenbosch university, south africa, for their assistance with the statistical analysis of data. competing interests bd diagnostics provided the bdm instrument for the duration of the study and partially sponsored the bdm kits used in the study; however, bd diagnostics was not involved in the study design, collection, analysis and interpretation of data, writing of the article, or in the decision to submit the article for publication. authors’ contributions s.s. was involved in the conceptualisation, methodology, validation, investigation, data curation, original draft preparation and funding acquisition. m.n.-f. contributed to the conceptualisation, methodology, software, validation, data curation, reviewing and editing, supervision and project administration. p.n. and c.p. assisted with the methodology, reviewing and editing, supervision and project administration while c.p. also contributed to the visualisation. sources of support this research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. data availability raw data were generated at the national health laboratory service. derived data supporting the findings of this study are available from the corresponding author, s.s., on request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references burnham ca, carroll kc. diagnosis of clostridium difficile infection: an ongoing conundrum for clinicians and for clinical laboratories. clin microbiol rev. 2013;26(3):604–630. https://doi.org/10.1128/cmr.00016-13 depestel dd, aronoff dm. epidemiology of clostridium difficile infection. j pharm pract. 2013;26(5):464–475. https://doi.org/10.1177/0897190013499521 kullin b, meggersee r, d’alton j, et al. prevalence of gastrointestinal pathogenic bacteria in patients with diarrhoea attending groote schuur hospital, cape town, south africa. s afr med j. 2015;105(2):121–125. https://doi.org/10.7196/samj.8654 abdullatif vn, noymer a. clostridium difficile infection: an emerging cause of death in the twenty-first century. biodemography soc biol. 2016;62(2):198–207. https://doi.org/10.1080/19485565.2016.1172957 warny m, pepin j, fang a, et al. toxin production by an emerging strain of clostridium difficile associated with outbreaks of severe disease in north america and europe. lancet. 2005;366(9491):1079–1084. https://doi.org/10.1016/s0140-6736(05)67420-x kuijper ej, coignard b, tüll p, et al. emergence of clostridium difficile-associated disease in north america and europe. clin microbiol infect. 2006;12(suppl 6):2–18. https://doi.org/10.1111/j.1469-0691.2006.01580.x di bella s, ascenzi p, siarakas s, petrosillo n, di masi a. clostridium difficile toxins a and b: insights into pathogenic properties and extraintestinal effects. toxins (basel). 2016;8(5):134. https://doi.org/10.3390/toxins8050134 berry ce, davies ka, owens dw, wilcox mh. is there a relationship between the presence of the binary toxin genes in clostridium difficile strains and the severity of c. difficile infection (cdi)? eur j clin microbiol infect dis. 2017;36(12):2405–2415. https://doi.org/10.1007/s10096-017-3075-8 gerding dn, johnson s, rupnik m, aktories k. clostridium difficile binary toxin cdt: mechanism, epidemiology, and potential clinical importance. gut microbes. 2014;5(1):15–27. https://doi.org/10.4161/gmic.26854 kraft cs, parrott js, cornish ne, et al. a laboratory medicine best practices systematic review and meta-analysis of nucleic acid amplification tests (naats) and algorithms including naats for the diagnosis of clostridioides (clostridium) difficile in adults. clin microbiol rev. 2019;32(3):e00032-18. https://doi.org/10.1128/cmr.00032-18 mcdonald lc, gerding dn, johnson s, et al. clinical practice guidelines for clostridium difficile infection in adults and children: 2017 update by the infectious diseases society of america (idsa) and society for healthcare epidemiology of america (shea). clin infect dis. 2018;66(7):987–994. https://doi.org/10.1093/cid/ciy149 tang s, hemyari p, canchola ja, duncan j. dual composite reference standards (dcrs) in molecular diagnostic research: a new approach to reduce bias in the presence of imperfect reference. j biopharm stat. 2018;28(5):951–965. https://doi.org/10.1080/10543406.2018.1428613 soe dask. [homepage on the internet]. open epi: open source epidemiologic statistics for public health, version 3.01. 2013 [cited 2021 mar 29]. available from: https://www.openepi.com/menu/oe_menu.htm planche t, wilcox m. reference assays for clostridium difficile infection: one or two gold standards? j clin pathol. 2011;64(1):1–5. https://doi.org/10.1136/jcp.2010.080135 oldfield ec iv, oldfield ec iii, johnson da. clinical update for the diagnosis and treatment of clostridium difficile infection. world j gastrointest pharmacol ther. 2014;5(1):1–26. https://doi.org/10.4292/wjgpt.v5.i1.1 eckert c, burghoffer b, lalande v, barbut f. evaluation of the chromogenic agar chromid c. difficile. j clin microbiol. 2013;51(3):1002–1004. https://doi.org/10.1128/jcm.02601-12 lemee l, dhalluin a, testelin s, et al. multiplex pcr targeting tpi (triose phosphate isomerase), tcda (toxin a), and tcdb (toxin b) genes for toxigenic culture of clostridium difficile. j clin microbiol. 2004;42(12):5710–5714. https://doi.org/10.1128/jcm.42.12.5710-5714.2004 shen a. clostridium difficile toxins: mediators of inflammation. j innate immun. 2012;4(2):149–158. https://doi.org/10.1159/000332946 jamal w, pauline em, rotimi vo. comparative performance of the genexpert c. difficile pcr assay and c. diff quik chek complete kit assay for detection of clostridium difficile antigen and toxins in symptomatic community-onset infections. int j infect dis. 2014;29:244–248. https://doi.org/10.1016/j.ijid.2014.10.025 chung hs, lee m. evaluation of the performance of c. diff quik chek complete and its usefulness in a hospital setting with a high prevalence of clostridium difficile infection. j investig med. 2017;65(1):88–92. https://doi.org/10.1136/jim-2016-000231 seo jy, jeong jh, kim kh, ahn jy, park pw, seo yh. laboratory diagnosis of clostridium difficile infection: comparison of techlab c. diff quik chek complete, xpert c. difficile, and multistep algorithmic approach. j clin lab anal. 2017;31(6):e22135. https://doi.org/10.1002/jcla.22135 tenover fc, baron ej, peterson lr, persing dh. laboratory diagnosis of clostridium difficile infection can molecular amplification methods move us out of uncertainty? j mol diagn. 2011;13(6):573–582. https://doi.org/10.1016/j.jmoldx.2011.06.001 crobach mjt, baktash a, duszenko n, kuijper ej. diagnostic guidance for c. difficile infections. adv exp med biol. 2018;1050:27–44. https://doi.org/10.1007/978-3-319-72799-8_3 chung hs, park js, shin bm. laboratory diagnostic methods for clostridioides difficile infection: the first systematic review and meta-analysis in korea. ann lab med. 2021;41(2):171–180. https://doi.org/10.3343/alm.2021.41.2.171 yoo j, lee h, park kg, lee gd, park yg, park yj. evaluation of 3 automated real-time pcr (xpert c. difficile assay, bd max cdiff, and imdx c. difficile for abbott m2000 assay) for detecting clostridium difficile toxin gene compared to toxigenic culture in stool specimens. diagn microbiol infect dis. 2015;83(1):7–10. https://doi.org/10.1016/j.diagmicrobio.2015.05.005 dalpke ah, hofko m, zorn m, zimmermann s. evaluation of the fully automated bd max cdiff and xpert c. difficile assays for direct detection of clostridium difficile in stool specimens. j clin microbiol. 2013;51(6):1906–1908. https://doi.org/10.1128/jcm.00344-13 nomlomo e, nana t. the impact of diagnostic methods on the diagnosis of clostridiodes difficile infection. s afr med j. 2020;110(2):135–139. https://doi.org/10.7196/samj.2020.v110i2.13684 introduction human resources technology upgrades and investments funding and financing strengthening policy recommendations conclusion acknowledgements references about the author(s) symon f. nayupe laboratory department, kamuzu university of health sciences private clinic, blantyre, malawi patrick mbulaje center for the development of people, lilongwe, malawi steven munharo montfort hospital, chikwawa diocese, nchalo, chikwawa, malawi parth patel department of health systems and policy, faculty of health systems, kamuzu university of health sciences, blantyre, malawi don e. lucero-prisno iii department of global health and development, london school of hygiene and tropical medicine, london, united kingdom citation nayupe sf, mbulaje p, munharo s, patel p, lucero-prisno de. medical laboratory practice in malawi – current status. afr j lab med. 2023;12(1), a1921. https://doi.org/10.4102/ajlm.v12i1.1921 opinion paper medical laboratory practice in malawi – current status symon f. nayupe, patrick mbulaje, steven munharo, parth patel, don e. lucero-prisno received: 13 apr. 2022; accepted: 26 sept. 2022; published: 06 jan. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction medical practice has evolved over the past years from symptom-based clinical diagnoses to evidence-based diagnoses demanding clinical laboratory investigations. clinical experts at the mayo clinic in the united states estimated that almost 70% of patient management decisions rely on laboratory diagnostic information.1,2 in sub-saharan africa, the need for quality diagnostic services is apparent; nevertheless, access to quality and reliable laboratory services in the region has been a big challenge.3 there has been significant development in medical laboratory practice across the world with the adoption of state-of-the-art technology and methods and subsequent demand for more specialised skills in medical laboratory scientists.4 in sub-saharan africa, medical laboratory practice has also evolved, though significantly slower than in other countries in the west, except countries like the republic of south africa which has many accredited and technologically advanced laboratory institutions.5 since many sub-saharan africa countries are resource limited, financing of laboratory activities has not been of primary concern; hence, laboratory improvement activities have suffered a persistent shortage of funds, dragging the pace of advancement in medical laboratory practice.3 additionally, the slow growth of medical laboratory practice has also been facilitated by continued neglect of the medical laboratory profession leading to inadequately trained personnel in some parts and an unrecognised cadre.6 in malawi, medical laboratory practice has suffered similar setbacks. the laboratory profession has, nonetheless, achieved some milestones as we will discuss. however, some serious reforms, recommendations of, have to be implemented to tackle the challenges that seriously hamper efficient, high-quality, and technologically advanced diagnostic services as are needed not only in malawi but in sub-saharan africa as a region. we hereby present the current status of medical laboratory practice in malawi, from various dimensions, namely human resources, equipment and technology, funding, as well as strengthening policy. as a background, the laboratory is structured in four ascending tiers, namely health centre laboratories, district hospital laboratories, central hospital laboratories and the national reference laboratory.7 a lower laboratory refers to the next level laboratory where advanced tests are required. laboratory falls under the health technical support services, a directorate under the ministry of health. the health technical support services is responsible for ensuring the provision of quality diagnostic capacity, monitoring drug efficacy, and patient management services. human resources malawi is one of the countries in sub-saharan africa where the ratio of healthcare workers to patients is high, reflecting a shortage of healthcare staff. since the 1990s, however, there has been a significant effort from the government to train more healthcare personnel.8 historically, malamulo college of health sciences was the first to offer certificates in medical laboratory sciences in 1968 and later in 1978 started offering diplomas. since then, malawi has progressed to offering laboratory science degrees at three accredited institutions namely the university of malawi, malawi adventist university and mzuzu university today. as per malawi association of medical laboratory scientists (mamls) unpublished records for 2021, there were approximately 2069 trained laboratory personnel registered with the medical council of malawi: 677 laboratory technologists, 1073 laboratory technicians and 320 laboratory assistants. currently, there are over 468 medical laboratory technologists and technicians who are unemployed. a commonly given reason is that there are no posts available as per government establishment despite the country facing a huge gap in laboratory personnel in many facilities. there is no doubt that the laboratory is an essential service, although the practice and utilisation of the service have generally been suboptimal for the past years with an evident need for infrastructural and capacity development.9 good-quality laboratory services are largely dependent on adequate, appropriately trained, and qualified laboratory personnel, yet laboratory professionals are prominently among neglected health cadres in malawi and across most sub-saharan african countries.10 services offered are affected by insufficient staff even though colleges are producing many graduates, as there are not enough formal posts currently. in addition, services are affected by lack of specialist qualifications and almost non-existent career progression opportunities since the laboratory profession appears not to be a primary area of concentration for professional improvement and recognition in the country. this understandably lowers the motivation of laboratory professionals. those employed by the ministry of health are often working in underfunded, poorly equipped facilities with low safety standards and unmotivating environments. technology upgrades and investments the malawi health sector has gradually expanded and improved tests available to patients. this has happened due to the availability and increased coverage of several modern machines such as the genexpert (cepheid, sunnyvale, california, united states) and full blood count machines purchased through the global fund mainly at district and central hospitals.11 in addition, the country has integrated tests on the existing machines to fully utilise the existing technology. for example, targeted viral load testing is now being done on genexpert platforms in most facilities, an upgrade from just running tuberculosis specimens. this is a huge investment that has saved money since the procurement is only focusing on procuring the reagents instead of the new machines. currently, the laboratory system is still struggling to provide high-quality diagnostic services. frequent shortages in supplies and reagents challenge sustainable service provision. additionally, factors such as poor equipment maintenance systems, poor laboratory infrastructure and limited backup testing services exacerbate the operational inefficiencies of testing services. despite the presence of equipment service contracts for government laboratories in malawi, the provision of both emergency and routine services for machine breakdowns and maintenance has been significantly slow. this, in part, is due to the availability of a few professionally trained biomedical engineers locally. consequently, these delays interrupt diagnostic service delivery.9 it has been observed that many clinicians often doubt the laboratory results of their patients. this leads to the repetition of tests, since the clinical presentation of the patients is at times inconsistent with results from laboratory investigations.9 the observations in this study could be attributed to task shifting, the use of non-laboratory trained personnel to perform tests in point-of-care settings failure to calibrate equipment, usage of expired reagents, lack of external quality control and refresher courses as well as total disregard of laboratory quality management systems.10 funding and financing in resource-limited countries, allocation of resources to diagnostic services is barely a priority.3 inadequate funding has downgraded the laboratory profession leading to poor infrastructures failing to meet the demand of its specialty to the growing population. lack of representation in key decision-making bodies is one of the top contributing factors leading to poor laboratory services in malawi. this has led to the underperformance of instrument maintenance services and a zero integrated supply chain for laboratory consumables.11 the position of laboratory manager is not an established one at the district level and hence it is not represented in the district health management team. most of the projects in malawi and sub-saharan africa at large are donor driven and hence are somewhat disease specific. this has led to a lack of cross-sector laboratory capacity, fragmentation of laboratory services and diversion of scarce resources.12 poor salary structures have also aggravated the migration of highly skilled individuals to the private sector and research institutions, further derailing the system. strengthening policy as part of laboratory professional practice improvement, medical laboratory professionals in malawi have revived its previously dormant body, mamls. formed in 1998, mamls was not active until february 2020, when a group of medical laboratory scientists, in collaboration with the international federation of clinical chemistry and laboratory medicine, facilitated the hosting of laboratory professionals from across malawi and guests from canada, the united states, egypt and the united kingdom, to the first ever medical laboratory scientific conference, where a task force dedicated to revamping mamls was formed. in december 2021, a second scientific conference was held in the country’s capital, lilongwe.13 laboratorians have often complained about the lack of a body specifically formulated to have regulatory oversight over ethical conduct, performing objective quality assurance and accreditation of medical laboratories in the country as well as representing professional interests at the policy level.14 the revamping of mamls aims at promoting and safeguarding the interests of professional medical laboratory science practice which ultimately safeguards patients who access laboratory services. continued lobbying by mamls focuses on establishing an independent medical laboratory regulatory body that will ensure a robust diagnostic representation at the policy level. recommendations define clear laboratory networks a strong laboratory organisational infrastructure in sub-saharan africa is necessary to improve access to quality healthcare.10 similarly, in malawi, such a clear definition of function, authority and responsibility of the laboratory system is necessary as a baseline for improving laboratory standards. the ministry of health in liaison with the laboratory leadership in malawi should define these networks. laboratory networks should include a multilevel systematic integration of functions with an enhanced referral system where laboratories with less testing capacity at the bottom of the system can refer advanced tests to laboratories at higher levels with more testing capacity with ease and within acceptable expected turnaround times. additionally, laboratories at all levels should adhere to national and international quality systems.5 the call for a regulatory body specific for laboratory practice in the strengthening policy section serves this purpose as one of the duties of the body. establish regulatory body for medical laboratories to curtail challenges faced by laboratories and laboratory personnel to provide quality service, the malawi government should draft a medical laboratory regulatory act to lead the way in addressing chronic challenges affecting ethical, professional and legal laboratory practice and policy. establishing a regulatory body will ensure that medical laboratories are objectively regulated for laboratory quality assurance, help shape laboratory policy and improve quality service delivery expected by patients and hospitals. the regulatory act and legal mandate will prevent encroachment and imposition from other departmental mandates.15 designate specialised laboratory posts the availability of adequate and trained human resources is one of the key elements to achieving quality diagnostics services. the absence of such, or the presence of staff who have no formally defined roles or positions, compromises the efficiency of achieving such quality as they lack direction and motivation. designating managerial and non-managerial laboratory-based positions in the medical laboratory setup through the directorate of human resources in ministry of health will map out career development prospects as well as equip particular departments with necessary skills depending on individual previous experiences to ensure competence and skills in handling jobs. include medical laboratory professionals in policy boards and regulatory bodies including medical laboratory professionals in boards and regulatory bodies by the appointing authorities will ensure the implementation of policy that promotes the welfare of laboratory personnel as well as promotion of the ever-evolving standard quality and harmonised laboratory practice, reshaping regulation and helping to redefine professionalism. regulation should be done by those vested with the dogma, qualifications, philosophy and understanding of the current problems affecting the profession and trends of medical laboratory science and practice around the globe. conclusion it is evident that there has been significant progress in the laboratory profession in malawi and generally in sub-saharan africa since colleges started training professionals locally. however, with the current health demands in modern medical practice that require efficient and quality diagnostic services, the laboratory profession is facing new challenges.15 our recommendations on defining clear laboratory networks, enacting a medical laboratory regulatory act, designation of administrative and specialised posts for laboratory professionals at the district and central levels, and the inclusion of laboratory professionals in decision-making bodies will contribute to strengthening laboratory practice in malawi. strong laboratory systems will ensure reliable diagnostic services, a contributor to access to quality health services.10 regionally, it is essential to have reliable laboratory systems in sub-saharan africa as this not only serves the individual countries but helps to strengthen regional interdependence as countries will now trust each other’s services, leading to the formation of regional networks for advanced and more specialised tests. acknowledgements competing interests the authors do not have an association with a body, entity or organisation that might pose any conflict of interest. the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions s.f.n., p.m., s.m. and p.p. were involved in the conceptualisation, investigation and formal analysis of findings. they were involved in writing the manuscript draft and reviewing and editing the final version of the manuscript. d.e.l.-p. was involved in the activities listed for all the other authors and also supervision of the whole work. ethical considerations no ethical approval was sought for this opinion paper as there was no requirement. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability data sharing does not apply to this article as no new data were created or analysed in this study. disclaimer the view and opinions expressed in this article are those of the authors and do not in any way represent the official policy or stand of the authors’ affiliated agencies or institutions. references forsman rw. why is the laboratory an afterthought for managed care organizations? clin chem. 1996;42(5):813–816. https://doi.org/10.1093/clinchem/42.5.813 hallworth mj. the ‘70% claim’: what is the evidence base? ann clin biochem. 2011 nov 1;48(6):487–488. https://doi.org/10.1258/acb.2011.011177 petti ca, polage cr, quinn tc, ronald ar, sande ma. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006 feb 1;42(3):377–382. https://doi.org/10.1086/499363 miller wd, kalfoglou a, leroy l. technology trends in the clinical laboratory industry. in: wolman dm, kalfoglou al, leroy l, editors. medicare laboratory payment policy: now and in the future. 1st ed. washington, dc: national academies press (us), 2000; p. 58–71. schroeder lf, amukele t. medical laboratories in sub-saharan africa that meet international quality standards. am j clin pathol. 2014;141(6):791–795. https://doi.org/10.1309/ajcpq5ktkagsscfn schneidman m, dacombe rj, carter j. laboratory professionals in africa: the backbone of quality diagnostics [homepage on the internet]. health, nutrition and population (hnp) discussion paper series. world bank group, 2014 [cited 2022 may 14]; p. 1–52. available from: https://openknowledge.worldbank.org/handle/10986/21115 butao d, felling b, msipa p. malawi: laboratory services and supply chain assessment. task order 1. arlington, va: usaid, 2009; p. 1–104. o’neil m, jarrah z, nkosi l, et al. evaluation of malawi’s emergency human resources programme. cambridge: ehrp; 2010. moyo k, porter c, chilima b, et al. use of laboratory test results in patient management by clinicians in malawi. afr j lab med. 2015 may 13;4(1):277. https://doi.org/10.4102/ajlm.v4i1.277 davies j, abimiku a, alobo m, et al. sustainable clinical laboratory capacity for health in africa. lancet glob health. 2017 mar 1;5(3):e248–e249. https://doi.org/10.1016/s2214-109x(17)30024-4 mtonya b, chizimbi s. systemwide effects of the global fund in malawi: final report. washington, dc: global fund; 2006. zere e, walker o, kirigia j, zawaira f, magombo f, kataika e. health financing in malawi: evidence from national health accounts. bmc int health hum rights. 2010 nov 10;10(1):1–11. https://doi.org/10.1186/1472-698x-10-27 chipofya e. international federation of clinical chemistry and laboratory medicine. n° 1/2 – january/february 2021 [cited 2022 may 14]. 2021; p. 33–35. available from: https://www.ifcc.org/ifcc-news/2010-2021-archive/2021-archive/2021-02-10-ifcc-enews-no-1-2-january-february-2021/ chidzaye rw. assessing barriers to medical laboratory diagnostic service delivery in mzuzu city. int j biomed sci [cited 2022 may 14]. 2019;15(1):32–56. available from: http://www.embase.com/search/results?subaction=viewrecord&from=export&id=l2001804557 bossuyt x, verweire k, blanckaert n. laboratory medicine: challenges and opportunities. clin chem. 2007 oct 1;53(10):1730–1733. https://doi.org/10.1373/clinchem.2007.093989 abstract introduction results and discussion acknowledgements references about the author(s) passoret vounba economic community of central african states (eccas) commission/fourth phase of the regional disease surveillance systems enhancement project (redisse iv), libreville, gabon severin loul economic community of central african states (eccas) commission/fourth phase of the regional disease surveillance systems enhancement project (redisse iv), libreville, gabon ludovic f. tamadea economic community of central african states (eccas) commission/fourth phase of the regional disease surveillance systems enhancement project (redisse iv), libreville, gabon joël f.d. siawaya department of laboratory services, chu mère-enfant fondation jeanne ebori, libreville, gabon regional integrated surveillance and laboratory network (rislnet) for central africa, libreville, gabon citation vounba p, loul s, tamadea lf, siawaya jfd. microbiology laboratories involved in disease and antimicrobial resistance surveillance: strengths and challenges of the central african states. afr j lab med. 2022;11(1), a1570. https://doi.org/10.4102/ajlm.v11i1.1570 note: additional supporting information may be found in the online version of this article as online supplementary document 1. review article microbiology laboratories involved in disease and antimicrobial resistance surveillance: strengths and challenges of the central african states passoret vounba, severin loul, ludovic f. tamadea, joël f.d. siawaya received: 27 feb. 2021; accepted: 10 jan. 2022; published: 31 mar. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract laboratory systems have been largely neglected on the margins of health systems in africa. however, since the 2000s, many african countries have benefited from massive investments to strengthen laboratory capacities through projects fighting priority diseases (hiv/aids, tuberculosis, malaria). this review examined the laboratory capacities of the economic community of central african states (eccas). online research using specific terms was carried out. studies published between 2000 and 2021 on the role of the laboratory in disease and antimicrobial resistance surveillance in the 11 eccas countries were considered. the number of human and animal health laboratories meeting international standards was very low in the sub-region. there were only seven international organization for standardization (iso) 15189-accredited human health laboratories, with five in cameroon and two in rwanda. there were five high biosafety level (bsl) laboratories (one bsl3 laboratory each in cameroon, the central african republic, democratic republic of congo and the republic of congo, and one bsl4 laboratory in gabon) and three iso 17025-accredited laboratories in the eccas sub-region. only six countries currently have whole-genome sequencing devices, which is insufficient for a sub-region as large and populous as eccas. yet, a plethora of pathogens, particularly haemorrhagic viruses, are endemic in these countries. the need for laboratory capacity strengthening following a one health approach is imperative. since emerging and re-emerging zoonotic infectious diseases are projected to triple in frequency over the next 50 years and given the inextricable link between human and animal health, actors in the two health sectors must collaborate to preserve world health. keywords: laboratory capacity; economic community of central african states (eccas); laboratory strengthening; one health; epidemics; antimicrobial resistance. introduction laboratory results guide evidence-based clinical decisions. in 1923, louis pasteur declared that ‘without laboratories, men of science are soldiers without arms’.1 a standard medical laboratory is essential for patient care and communicable disease surveillance. the laboratory is indispensable, both for routine diagnosis of infections and for the rapid identification of epidemic outbreaks. in addition, antimicrobial resistance (amr) surveillance, food safety and water quality assessment, analysis of environmental samples, etc. are also laboratory dependent. in sub-saharan africa, unfortunately, laboratory service has been a sidelined health service that receives very little government budgetary allocation. in 2008, representatives of african governments recognised that: in resource-limited settings, several challenges have resulted in inadequate laboratory systems to support the scale-up of programs. these include a lack of leadership and advocacy, human resources, career path and retention of staff, national laboratory policy, strategic planning (budgetary concerns), sufficient physical infrastructure, supply chain management, and quality management systems (quality assurance). (p. 1)2 the limited investment in laboratory systems impairs the quality of laboratory services. for instance, sub-saharan african laboratories are deficient in terms of qualified staff, modern equipment, a regular supply of quality reagents, water, or electricity, standard operating procedures, and quality assurance systems.3,4 this review aimed to highlight the roles, strengths, and challenges of the human and animal laboratories in the economic community of central african states (eccas). the eccas is an integrated african space created in 1983 and includes 11 countries: angola, burundi, cameroon, central african republic, chad, democratic republic of congo, equatorial guinea, gabon, republic of congo, rwanda, and são tomé and príncipe. according to the 2014 estimates, the population of the eccas is about 161 million inhabitants, spread over an area of 6 640 490 km2.5 a systematic search was carried out using terms ‘role of the laboratory in africa’, ‘role of laboratory in disease surveillance in africa’, ‘role of veterinary laboratories in africa’, ‘role of the laboratory in amr surveillance in africa’, ‘iso [international standardization organization] 15189-accredited laboratory in africa’, ‘iso 17025-accredited laboratory in africa’, ‘health reference laboratories in africa’, ‘veterinary reference laboratories in africa’, and each term accompanied by the name of each of the 11 eccas countries. these terms were searched on google, google scholar, pubmed, african journals online and international organisations’ websites: (world health organization [who], world organization for animal health [oie], food and agriculture organisation of the united nations, africa centres for disease control and prevention, african society for laboratory medicine [aslm]). all studies published between 2000 and 2021 in the four official languages used by the eccas (english, french, spanish and portuguese) on the role of the laboratory in the surveillance of diseases and amr in the 11 countries were included. results and discussion role of human health laboratory in patient management medical laboratories provide a precise diagnosis for patient management. early diagnosis and treatment reduce the risk of long-term complications in patients and prevent further transmission.6 thus, proper patient management requires close tripartite collaboration between the patient, clinicians and laboratory staff.7 in sub-saharan africa, access to reliable diagnostic tests is very limited and misdiagnosis occurs frequently, leading to physicians’ distrust of laboratory results.6 limited access to reliable diagnosis results in no or inadequate treatment, increased mortality, and an inability to determine the true prevalence of diseases.8 in the democratic republic of congo, the laboratories were unable to diagnose the diseases frequently encountered in this country.4 the lack of laboratory infrastructure may delay patients’ recovery. delay may occur due to referrals to other laboratories that are further away and often more expensive, or due to inappropriate treatment that lacks laboratory diagnosis. arsuaga et al.9 reported an example of the latter: the case of a missionary symptomatically diagnosed and unsuccessfully treated for malaria in cameroon and equatorial guinea, but was laboratory diagnosed and successfully treated for babesiosis, a malaria-like illness, eight months later in spain. in rwanda, a strong laboratory network was built in the early 2000s. internal and external laboratory control and assurance activities are regularly conducted at all levels of the network by the national reference laboratory (nrl).10 the external quality assessment focuses on enteric and meningitis pathogens, tuberculosis, malaria, and hiv/aids. in 2003, a concordance of 100% was reported for the unlinked, anonymous hiv/aids testing of all 288 samples sent by the rwandan nrl to the united states centre for disease control and prevention.10 nevertheless, rwandan laboratories are not exempt from cross-cutting problems, such as service interruptions due to reagent stock-out and equipment breakdown.11 the positive impact of laboratory capacity building on reducing infections and improving patient management has been reported in cameroon. eleven months after implementing capacity strengthening activities, the regional hospital of buea reported a reduced patient wait time at the reception from 3 h to less than 30 min.12 similarly, laboratory improvement capacities in the bamenda regional hospital laboratory resulted in fewer specimen recalls, improved test reliability, and the provision of feedback channels on services offered.13 from the eccas sub-region, gabon, cameroon, the democratic republic of congo, and the central african republic are part of the who’s emerging dangerous pathogens laboratory network.14 all four countries have national viral haemorrhagic fever (vhf) testing capacity, while gabon hosts the who afro eccas regional vhf reference laboratory.15 all eccas countries have influenza laboratories except chad, equatorial guinea, and são tomé and príncipe. however, the influenza laboratory network in the republic of congo, angola, and rwanda can easily be upgraded to include vhf testing capacities.15 in cameroon, the centre pasteur du cameroun is a reference centre for the network of quantitative polymerase chain reaction diagnostic laboratories for buruli ulcer. this network brings together 11 laboratories located in nine west and central african countries where buruli ulcer is endemic.16 ultimately, other neglected tropical diseases such as leprosy and cutaneous leishmaniasis will be integrated into the buruli ulcer platform. the network also plans to implement activities such as clinical trials evaluating new treatments, assays validating new molecular diagnostic tools, and surveillance of amr. role of laboratories in disease surveillance in the eccas sub-region the information provided by the laboratory is critically important for disease surveillance and response programmes. for efficient management of an epidemic and its containment, a strong laboratory system should be operational before, during, and after the epidemic.17,18 before an epidemic, the laboratory collects early warning signals and identifies the aetiological agent. during the outbreak, the laboratory is involved in the response and management measures for the containment of the epidemic, and after the outbreak, the laboratory monitors disease trends, evaluates interventions, and monitors progress towards control objectives. apart from disease outbreaks, laboratories monitor microbial genetic changes of public health concerns, such as changes that confer amr in bacteria or changes in rna viruses (such as ebola or coronavirus) that lead to the emergence of genetically diverse strains (variants) with high pathogenicity or transmissibility.19 thus, during certain outbreaks, such as the ebola or coronavirus outbreaks, the aetiological agent must be laboratory-characterised to detect the emergence of variants and guide response decisions.18 detection of extremely dangerous pathogens, such as the ebola virus, requires higher biosafety level (bsl3 or 4) laboratories. bsl4 laboratories are built to ensure biosafety and biosecurity when studying class 4 pathogens; pathogens transmitted via aerosols or unknown mechanisms and are often lethal, without known treatment or vaccine to fight them. however, in the absence of a bsl4 laboratory such as in the democratic republic of congo, bsl3 laboratories, for studying class 3 pathogens, usually, viruses or bacteria that infect humans or animals through inhalation and could be lethal, with reinforced biosafety and biosecurity, have been used to diagnose ebola cases. africa experiences approximately 100 public health events every year, of which 80% are caused by infectious agents.14 many of these events involve extremely dangerous pathogens. for example, since 1994, the eccas region has regularly recorded ebola epidemics, mainly in the democratic republic of congo, the republic of congo, and gabon,20 which due to their ecosystems are at high risk of vhf.21 unfortunately, despite the high health risks evident in african countries, resources for epidemic surveillance and response such as laboratory capacity are mostly lacking. as of 2015, across the entire african continent, only three countries (nigeria, kenya and south africa) had fixed bsl3 laboratories, while only two countries had bsl4 laboratories22: one in gabon, centre interdisciplinaire de recherches médicales de franceville (cirmf), and the other in south africa. as at march 2021, the number of operational bsl4 laboratories on the continent has not much changed: two are under construction in south africa and côte d’ivoire23; the number of bsl3 laboratories has increased, particularly in the eccas sub-region. bsl3 laboratories were recently built, including the institut national de recherches biomédicales in the democratic republic of congo, the centre pasteur du cameroun in cameroon, the institut pasteur de bangui in the central african republic,14 and the bsl3 laboratory dedicated to the management of multidrug-resistant tuberculosis in the republic of congo.24 the presence of the bsl3 and bsl4 laboratories in some eccas countries is a great asset for the sub-region to effectively monitor and respond to epidemics. for instance, the bsl4 laboratory in the cirmf, gabon, actively surveils emerging and re-emerging diseases not only in gabon but also in the other eccas countries.25 the objectives assigned to the cirmf includes diagnosing suspected vhf cases, developing new diagnostic methods, monitoring deaths in animal reservoir hosts, and conducting laboratory techniques training at national, regional and international levels. the cirmf has established a research partnership with the national public health laboratory in brazzaville, republic of congo, and the institut national de recherches biomédicales in kinshasa, democratic republic of congo, to study infectious diseases transmitted by animals in the tropical rainforest regions of equatorial africa.25 capacities of the human health laboratories in eccas sub-region to comply with international health regulations in 2015, the who recommended that member states annually report their progress in implementing the revised international health regulations (2005 ihr)26 and conduct a self-assessment of their capacity, followed by a joint external evaluation (jee).26 the jee tool is developed using the who instruments as well as different strategies and initiatives including the global health security action programme and the oie tool for the evaluation of the performance of veterinary services (pvs).26 the jee assesses ihr capacities in 19 technical areas, grouped into four main themes: ‘prevention’, ‘detection’, ‘response’, and ‘entry points and other ihr risks’ (chemicals and radiation). a national laboratory system is one of the four technical areas of the domain ‘detection’. the 2005 ihr capacity scores are classified from level 1 (no capacity) to level 5 (sustainable capacity). in the eccas region, except angola and equatorial guinea, all countries have completed the jee of their public health capacity to meet the requirements of the 2005 ihr. as per figure 1, in the laboratory area, most eccas countries scored low in several indicators both in terms of disease and amr surveillance capacities.24,27,28,29,30,31,32,33,34 figure 1: country scores for laboratory capacity assessment in the eccas countries. capacities of the animal health and food laboratories in the eccas sub-region to comply with the international standards in animal health and food safety, the performance evaluation of the veterinary services has shown that laboratory reliability and quality assurance are major issues in most african countries.35 for eccas countries, evaluation or gap reports reveal that laboratory capacities are very low for disease and amr surveillance. these weaknesses are both qualitative (very low performance scores, around 1–2 for the majority of countries) and quantitative (often only one regional or national veterinary laboratory per country, rarely two) (supplementary table 1). table 1: role of the laboratory system in achieving the goals of the who’s global plan against amr. to achieve the goal of eliminating dog-mediated human rabies deaths by 2030, many african veterinary laboratories, including the laboratoire national vétérinaire (lanavet) in cameroon and the veterinary laboratory of kinshasa, democratic republic of congo, recently benefited from increased capabilities for rabies diagnosis. the staff were trained to diagnose rabies using the direct fluorescence antibody test and conventional real-time polymerase chain reaction at the food and agriculture organisation of the united nations rabies reference centre in italy.36 mobile laboratories as a solution to the lack of fixed laboratory infrastructures in sub-saharan africa, particularly eccas countries, the few bsl3 and bsl4 laboratories are located in large urban centres. thus, they could be located far from epidemic outbreaks or areas at risk of emergence or re-emergence of epidemics. in the case of an epidemic such as ebola, the safe delivery of samples to the laboratory, reliable diagnosis, and prompt communication of results are crucial for a successful response.37 a mobile laboratory can alleviate this problem by shortening the time required to obtain results. mobile laboratories circumvent fixed laboratory construction delays, particularly in times of emergencies, as they can be deployed almost immediately. this has been demonstrated in some countries in the sub-region. during the 2005 ebola epidemics in the democratic republic of congo and the 2007 marburg fever epidemic in angola, mobile laboratories confirmed suspected cases within 4 h, consequently facilitating the work of the contact-tracing team.38 more recently, a portable sequencer in one of the mobile laboratories was used to investigate the date of introduction and geographical origin of the zika virus in angola.39 however, field mobile laboratories are capitaland logistics-intensive, and are thus best suited for providing limited services for brief periods.18 therefore, it is necessary to develop additional fixed and sustainable bsl3 and bsl4 laboratories in the eccas. role of laboratories in amr surveillance overview of the challenges of amr surveillance the advent of antibiotic therapy, which began with the discovery by 1928 of penicillin, completely revolutionised medicine and significantly reduced infectious disease mortality and disability. the use of antimicrobials has also increased animal production by improving animal welfare. unfortunately, amr seriously undermines the hopes raised by the discovery of antimicrobials. antimicrobial resistance is considered one of the most significant threats to human, animal and ecosystems health. antimicrobial resistance is exacerbated by the overuse and misuse of antimicrobials; 30% – 50% of antimicrobial prescriptions in human medicine are unnecessary.40,41,42 in animal health, the irrational use of antimicrobials is compounded by the use of growth promoters in animals or the use of antimicrobials for metaphylaxis. some growth promoters contain antimicrobials of critical importance to human health.43 in addition, in veterinary medicine metaphylaxis is rampant; metaphylaxis is the administration of antimicrobials to a herd of animals to treat sick individuals and prevent the disease in healthy individuals. antimicrobial resistant microorganisms in animals can subsequently be transmitted to humans through direct or indirect contact.44 indeed, there is a much higher risk of human colonisation through cattle, pigs and poultry infected with methicillin-resistant staphylococcus aureus.45 furthermore, genetic determinants of amr can be transferred from commensal or pathogenic animal bacteria to pathogenic human bacteria. last resort antimicrobials used to fight multidrug-resistant infections such as fluoroquinolones, third generation cephalosporins or colistin are becoming ineffective globally. in parallel to the extensive misuse of antimicrobials, the discovery of new antimicrobials has become increasingly seldom. as a result, the feared therapeutic impasse is becoming increasingly real.46 according to projections, by the year 2050, amr will be the world’s leading cause of annual death, with 10 million deaths per year, ahead of cancers (8.2 million) or diabetes (1.5 million).47 also, africa and asia will likely be the most affected continents. this is why, on the margins of the 71st session of the united nations general assembly in 2016, the alarm bell was sounded on amr.48 on this occasion and for the first time, heads of states and governments came together to adopt a common approach to fight the causes of amr in human and animal health, as well as in the environment. in this noble fight, the laboratory has a prominent part to play. role of the laboratory in fighting amr the early symptoms of an infectious disease may be non-specific and may combine clinical signs of several infectious diseases. for example, the first manifestations of an ebola virus infection include fever, headache, myalgia, and gastrointestinal disorders.49 to increase the chance of effective antibiotic therapy, a common approach is to use broad-spectrum antimicrobials while waiting for antimicrobial susceptibility test results, which are not usually available before 72 h.50 this practice runs contrary to the goal of the who global plan to optimise antimicrobial use.51 this empirical usage selects for antimicrobial-resistant microorganisms. therefore, medication before laboratory diagnosis should only be used when the disease is life-threatening and, even so, microbiological sampling should be performed before treatment is initiated.52 the focus should be on the development of innovative rapid diagnostic techniques that allow clinicians to identify the pathogen in minutes rather than days.53 antibiotic susceptibility testing is a key indicator for the design of effective interventions and rational use of antibiotics.54,55 reporting of antibiotic susceptibility results by medical laboratories is necessary to monitor emerging resistances and develop appropriate antimicrobial stewardship guidelines.54,56 therefore, antimicrobial susceptibility testing capacity is essential.57 in the 2018–2023 amr framework,58 the africa centre for disease control and prevention, in collaboration with existing partners, aimed to increase laboratory capacity for the detection of resistant microorganisms in humans and animals. the who’s global plan of action defines five strategic objectives for amr containment.45 as per table 1, the laboratory system has a key role to play in achieving these goals, both in terms of clinical and public health activities.45,51,59,60,61,62 in the eccas countries, probably due to weak laboratory capabilities, there is a huge amr data gap from the human,63 animal and environmental health sectors.64 however, the literature suggests that substantial effort is being made to achieve some of the objectives of the who’s global plan against amr. scientific articles contribute to public awareness, understanding, and knowledge building on amr. in belgium, for example, as a result of national awareness campaigns, streptococcus pneumoniae penicillin resistance decreased from 18% in 2000 to 7% in 2009.65 thus, in the eccas zone, research and public awareness should be at the heart of global amr control strategies. in animal health, the lanavet in cameroon and the institut de recherche en elevage pour le développement in chad are major vaccine production laboratories in africa66 aimed at preventing infections, thereby reducing antimicrobial use in animals. next-generation sequencing capacity in the eccas region next-generation sequencing (ngs) offers the potential to provide more accurate and timely information, thereby increasing the likelihood of meeting the 2005 ihr recommendations. the ihr recommends that urgent events are reported within 48 h to determine whether an event is ‘notifiable’. this information will rapidly inform the necessary control measures to prevent national and international transmission. diagnosis and surveillance of pathogens are the core capacity of public health systems.67 the whole-genome sequencing is a leading technique in the response, not only to the ongoing coronavirus disease 2019 pandemic but to future emerging and re-emerging infections. in the eccas region, countries with ngs devices are gabon (four devices), the democratic republic of congo and rwanda (two devices each), angola and cameroon (one each),68 and the republic of congo and equatorial guinea (unknown number each).69 the illumina platform is by far the most used in these countries, followed by ion torrent and nanopore. iso 15189or iso 17025-accredited laboratories in the eccas region the who afro through the african society of laboratory medicine (aslm), implemented the stepwise laboratory management towards accreditation (slmta), to improve medical laboratories in africa.70 the slmta was launched in 2009 to improve the quality of public and private health laboratories in african countries to achieve iso 15189 standards accreditation. the framework to audit the implementation of the slmta in laboratories is the stepwise laboratory improvement process towards accreditation (slipta).70 until october 2021, there were only two eccas countries with iso 15189-accredited laboratories: cameroon had five accredited laboratories while rwanda had two.71 although cameroon scored very low on most capacity attributes in the jee (figure 1), it has the largest number of accredited laboratories in the eccas region. the likely explanation could be that these currently accredited laboratories were not included in the jee cohort or that they rigorously accelerated their certification process after the jee. the last decade has seen the emergence of several projects supporting laboratory systems in low-income countries, notably as part of the fight against priority diseases (malaria, hiv/aids and tuberculosis). these projects have positively impacted laboratory services. the accreditation of veterinary laboratories is subject to iso 17025 standards. by the end of the european union-funded central african quality infrastructure project (piqac) in 2019, two food safety laboratories had been audited and were in the process of iso 17025 accreditation.72 six others were in the process of capacity building for accreditation, including the microbiology laboratory in the centre de contrôle de la qualité des denrées alimentaires (cecoqda) in n’djamena, chad.72 as of march 2021, the cecoqda’s microbiology laboratory with the congolese office control laboratory in the democratic republic of congo and the africa improved food laboratory in rwanda were the few iso 17025-accredited laboratories in the eccas region. in addition, the lanavet in cameroon is considered a centre of laboratory excellence by the food and agriculture organisation of the united nations.73 this laboratory organises training sessions on animal disease diagnosis for technicians in the sub-region and provides african swine fever diagnostic services for chad. capacity building needs of laboratories in the eccas zone the eccas countries need to incite both medical and veterinary laboratories to register in iso 15189 or iso 17025 accreditation processes. accreditation assessments are snapshot measurements of laboratory compliance, creating the risk that the efforts may weaken after an assessment.74 therefore, for already accredited laboratories, the most important challenge is maintaining their status. according to 2017 forecasts, the world’s population is expected to increase by 2.2 billion by 2050, with 1.3 billion of this growth occurring in africa.75 predictions also suggest that health risks will increase dramatically in africa, with the endemic rate of zoonotic viruses more than tripling by 2070.76 consequently, health services, particularly laboratory services, will have to support this demographic growth and high risk of emerging infectious diseases by providing services at low cost while maintaining quality. given the low scores recorded and the few numbers of accredited and bsl3 and bsl4 laboratories, it appears that the eccas countries have weak laboratory capacities in both human and veterinary medicine and must be strengthened. building the laboratory workforce is another challenge that eccas countries face since laboratory work has long been down on the lists of priorities of most health ministries in the eccas region. the central africa’s regional integrated surveillance and laboratory network (rislnet) meeting, which took place in malabo in march 2019,77 revealed that only a few countries including burundi, the democratic republic of congo and cameroon have their laboratory policies and strategic plans drawn up and validated. the republic of congo and são tomé and príncipe laboratory strategic plans remain to be validated. the central african republic, chad, gabon and equatorial guinea have no laboratory policies and no laboratory strategic plans. for those with laboratory policies and strategic plans, implementing them remain a challenge due to budgetary constraints. capacity strengthening through laboratory quality enhancement the information provided by the laboratory must be accurate, timely and subjected to quality assurance procedures. in other words, laboratory results must be accurate. to this end, all aspects of laboratory activities must be reliable and the reporting of results must be correct to be used for clinical or public health purposes. the iso divides the various laboratory analysis activities into three processes: pre-analytical, analytical, and post-analytical. laboratory errors that may negatively impact patient management or public health policies occur at 32% – 75% in the pre-analytical phase, 13% – 32% in the analytical phase and 9% – 31% in the post-analytical phase.78 therefore, as shown in figure 2, capacity building strategies need to be designed to address all aspects of the laboratory analysis and organisation.79 figure 2: fishbone diagram for medical laboratory analysis showing the focal elements in the capacity strengthening strategy. the best way to strengthen capacity is to enrol laboratories within the who’s slmta process. this process allows a substantial improvement in the quality of laboratories even if they do not reach the end of the accreditation process.11,12,13,80 unfortunately, few countries in the eccas zone have engaged their laboratories in the slmta process. and even for those countries that have signed up, the number of both public and private laboratories involved in the process is very low. for example, between 2012 and 2019, only 16 public and private laboratories in cameroon were enrolled in slmta, but this country has 3279 public laboratories.81 also, only 1.34% of the 1113 public and private laboratories in burundi were engaged in the slmta process.81 to avoid a decline in performance, slmta-enrolled laboratories must continue follow-up performance and apply the lessons learned during the process and, most importantly, attract national political commitment.82 moving towards integrated laboratory systems and networks the maputo declaration called for the development of national laboratory policies and national strategic laboratory plans. the call prioritises laboratory systems in the national health development plan.2 an integrated laboratory network can provide all primary diagnostic services needed for the care and treatment of patients without requiring them to go to different laboratories for specific tests.83 in resource-limited settings, such as in some african countries, the who recommends four operational levels of laboratories to better provide services in a national laboratory network84: level i or primary (health post and health centre laboratories that primarily serve outpatients), level ii or district level (laboratories in intermediate referral facilities), level iii or regional or provincial level (laboratories in a regional or provincial referral hospital that may be part of a regional or provincial health bureau), and level iv or national or multi-country reference laboratory (reference laboratory for one or more countries). thus, laboratory levels are determined by their diagnostic platforms as well as their functions. the national reference level carries out the most complex tests. a tiered, integrated laboratory network should meet the following criteria83: (1) provide quality-assured basic laboratory testing, (2) collect the common specimens, report results timeously, and use diagnostic platforms to detect different diseases within the same facility, and (3) increase capacity for introducing and using new and more complex technologies. an integrated laboratory should have the capacity to adequately monitor people with hiv/aids for tuberculosis, malaria or other opportunistic infections. it should also provide rapid molecular tests for multidrug-resistant tuberculosis in patients co-infected with hiv and tuberculosis to improve infection control and treatment outcomes. therefore, the integrated network avoids wastage of already limited resources and the referral of patients outside the network for certain laboratory tests.83 in 1993, the who afro established an integrated laboratory network in 15 african countries, including cameroon, the central african republic and the democratic republic of congo, to support the global polio eradication initiative.15 the polio laboratories of these three eccas countries have also integrated measles, yellow fever, and rotavirus programmes. more recently, the africa centre for disease control and prevention established rislnet within defined geographic regions of africa including central africa.77 the rislnet aims to effectively support prevention, rapid detection, and response to current and emerging public health threats. the rislnet operates under the one health concept, integrating human and animal health laboratories and surveillance assets. the materialisation of the one health concept is critical to efficiently prevent and respond to public health threats. as 65% of the recent major epidemics in the world have a zoonotic origin,85 there should be no division between disciplines in the human and animal health sectors. the one health approach will enable the early identification of emergent zoonosis. this can be achieved through the simultaneous surveillance of both human and animal disease in integrated surveillance and laboratory systems or networks. the one health approach, thus, mutualises resources and cuts costs. this was illustrated in uganda and nigeria, where during avian influenza outbreaks, hiv/aids diagnostic laboratories provided diagnostic support for avian influenza cases.83 in the fight against emerging amr threats, a stronger laboratory system will allow the detection of resistance and provide data for better trend tracking and infection control. also, standardised isolates banks, which would result from such a system, would support the research for better diagnostics and treatment. limitations in response to the coronavirus disease 2019 pandemic, several countries must have increased their response capacity by improving their laboratory capacity and biosafety levels. the laboratory capacities reported here may not have considered all the newly acquired capacities. this study was based on data available online; thus, a country’s laboratory capacity may not necessarily have been the subject of a study published on the internet. within the framework of the regional disease surveillance systems enhancement project (redisse iv) currently underway in the eccas countries, an inventory of laboratory capacities is being conducted and will provide exhaustive data on laboratory capacities. conclusion laboratory services are an essential component of a health system. in africa, particularly in the eccas sub-region, the need for reliable laboratory systems is greatest due to the higher risk of vhf. unfortunately, from this review, it is evident that laboratory capacity for disease and amr surveillance and response is weak. indeed, the capacities of laboratories in eccas countries are weak given the who’s jee scores and the very limited number of high biosafety levels (bsl3 and bsl4) and accredited laboratories. there is, therefore, a pressing need to strengthen the laboratory capacities in the sub-region to cope with the risk of disease emergence, which is predicted to triple in the coming decades. acknowledgements we sincerely thank dr skander hathroubi (from humboldt university of berlin, germany) for his contribution by reading and correcting the first draft of the manuscript. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions p.v. was responsible for conceptualisation, methodology, formal analysis, investigation, writing of the original draft, visualisation, project administration, validation, resources, writing of the review, and editing and supervision. s.l. was involved in the investigation, visualisation, validation, writing, review and editing. l.f.t. contributed to the methodology, investigation, validation, writing, review and editing. j.f.d.s. was involved in the validation, data curation, writing, review and editing. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references leng ll, luke lsg. ‘without laboratories, men of science are soldiers without arms’. singapore fam physician. 2017;43(4):3–4. world health organization-regional office for africa. the maputo declaration on strengthening of laboratory systems. 2008; [homepage on the internet]. [cited 2021 march 23]. available from: https://www.who.int/diagnostics_laboratory/maputo-declaration_2008.pdf frean j, perovic o, fensham v, et al. external quality assessment of national public health laboratories in africa, 2002–2009. bull world health org. 2012;90:191–199. https://doi.org/10.2471/blt.11.091876 linsuke s, nabazungu g, ilombe g, ahuka s, muyembe j-j, lutumba p. medical laboratories and quality of care: the most neglected components of rural hospitals in the democratic republic of the congo. pan afr med j. 2020;35:22. https://doi.org/10.11604/pamj.2020.35.22.18755 moutou-nkounkou s-m. etude comparative de deux processus d’intégration régionale économique en afrique subsaharienne: le cas de la ceeac et de la cedeao [homepage on the internet]. master thesis. université du québec à montréal; 2019 [cited 2020 dec 29]. available from: https://archipel.uqam.ca/12540/1/m15983.pdf boeras di, peeling rw. external quality assurance for hiv point-of-care testing in africa: a collaborative country-partner approach to strengthen diagnostic services. afr j lab med. 2016;5(2):a556. https://doi.org/10.4102/ajlm.v5i2.556 watson id, wilkie p, hannan a, beastall gh. role of laboratory medicine in collaborative healthcare. clin chemistr lab med (cclm). 2019;57(1):134–142. https://doi.org/10.1515/cclm-2017-0853 petti ca, polage cr, quinn tc, ronald ar, sande ma. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377–382. https://doi.org/10.1086/499363 arsuaga m, gonzález lm, padial es, et al. misdiagnosis of babesiosis as malaria, equatorial guinea, 2014. emerg infect dis. 2018;24(8):1588. https://doi.org/10.3201/eid2408.180180 kebede s, gatabazi jb, rugimbanya p, et al. strengthening systems for communicable disease surveillance: creating a laboratory network in rwanda. health res policy syst. 2011;9:27. https://doi.org/10.1186/1478-4505-9-27 nzabahimana i, sebasirimu s, gatabazi jb, et al. innovative strategies for a successful slmta country programme: the rwanda story. afr j lab med. 2014;3(2):6. https://doi.org/10.4102/ajlm.v3i2.217 eno lt, asong t, ngale e, et al. driving hospital transformation with slmta in a regional hospital in cameroon. afr j lab med. 2014;3(2):5. https://doi.org/10.4102/ajlm.v3i2.221 nkwawir sc, batumani nn, maruta t, awasom cn. from grass to grace: how slmta revolutionised the bamenda regional hospital laboratory in cameroon. afr j lab med. 2014;3(2):203. https://doi.org/10.4102/ajlm.v3i2.203 who afro. report on the status of emerging and dangerous pathogen laboratory network biosecurity level-3 in select countries in the african region [homepage on the internet]. 2016 [cited 2021 feb 20], 52 p. available from: https://www.afro.who.int/sites/default/files/2017-08/report%20on%20the%20status%20of%20edpln%20bsl-3%20in%20select%20countries%20in%20the%20african%20region.pdf boeras di, peeling rw, onyebujoh p, yahaya aa, gumede-moeletsi hn, ndihokubwayo jb. the who afro external quality assessment programme (eqap): linking laboratory networks through eqa programmes. afr j lab med. 2016;5(2):1–6. https://doi.org/10.4102/ajlm.v5i2.560 world health organization. first meeting of the network on buruli ulcer pcr laboratories in the who african region, centre pasteur du cameroon, yaoundé, cameroon, 21–24 october 2019 [homepage on the internet]. geneva: world health organization, 2020 [cited 2021 feb 22]; 22p. available from: https://www.who.int/publications/i/item/9789240007222 mérieux f. opération de réponse à l’épidémie d’ebola en rdc [homepage on the internet]. dossier presse, 2019 [cited 2020 oct 19]; 12 p. available from: https://www.fondation-merieux.org/wp-content/uploads/2019/10/operation-de-reponse-al-epidemie-2019-dossier-de-presse.pdf kay ba. the role of the laboratory in disease surveillance. rev santé méditerr orient. 1996;2(1):68–72. https://doi.org/10.26719/1996.2.1.68 carrasco-hernandez r, jácome r, lópez vidal y, ponce de león s. are rna viruses candidate agents for the next global pandemic? a review. ilar j. 2017;58(3):343–358. https://doi.org/10.1093/ilar/ilx026 munster vj, bausch dg, de wit e, et al. outbreaks in a rapidly changing central africa – lessons from ebola. new engl j med. 2018;379(13):1198–1201. https://doi.org/10.1056/nejmp1807691 world health organization. mapping the risk and distribution of epidemics in the who african region: a technical report [homepage on the internet]. 2016 [cited 2020 oct 29]. available from: https://apps.who.int/iris/handle/10665/206560 ahmad a, ashraf s, komai s. are developing countries prepared to face ebola-like outbreaks? virol sin. 2015;30(3):234–237. https://doi.org/10.1007/s12250-015-3564-9 world health organization. report of the who consultative meeting on high/maximum containment (biosafety level 4) laboratories networking [homepage on the internet]. lyon, france, 13–15 december 2017. geneva: world health organization; 2018 [cited 2020 nov 3]. available from: https://www.who.int/ihr/publications/who-whe-cpi-2018.40/en/ world health organization. evaluation externe conjointe des principales capacités rsi en république du congo. rapport de mission d‘évalution externe conjointe [homepage on the internet]. 2019 [cited 2020 nov 8]. available from: https://www.who.int/ihr/publications/who-whe-cpi-2019.60/fr/ leroy e, gonzalez jp. filovirus research in gabon and equatorial africa: the experience of a research centre in the heart of africa. viruses. 2012;4(9):1592–1604. https://doi.org/10.3390/v4091592 world health organization. joint external evaluation tool: international health regulations (2005) [homepage on the internet]. 2nd ed. geneva: world health organization; 2018 [cited 2021 jan 5]. available from: https://www.who.int/ihr/publications/who_hse_gcr_2018_2/en/ world health organization. evaluation externe conjointe des principales capacités rsi de la république du burundi [homepage on the internet]. genève: organisation mondiale de la santé; 2018 [cited 2021 jan 5]. available from: https://apps.who.int/iris/handle/10665/273156?show=full world health organization. evaluation externe conjointe des principales capacités rsi de la république du cameroun [homepage on the internet]. genève: organisation mondiale de la santé, 2017 [cited 2021 jan 18]; p. 26. available from: https://appswhoint/iris/bitstream/handle/10665/259676/who-whe-cpi-rep-201760-frepdf?sequence=1 world health organization. evaluation externe conjointe des principales capacités rsi en république gabonaise [homepage on the internet]. 2019 [cited 2020 nov 22]. available from: https://appswhoint/iris/bitstream/handle/10665/329977/who-whe-cpi-201918-frepdf?ua=1 world health organization. evaluation externe conjointe des principales capacités rsi de la république centrafricaine [homepage on the internet]. 2018 [cited 2020 nov 24]. available from: https://appswhoint/iris/bitstream/handle/10665/280124/who-whe-cpi-201930-frepdf?sequence=1 world health organization. evaluation externe conjointe des principales capacités rsi de la république démocratique du congo [homepage on the internet]. 2018 [cited 2020 nov 24]. available from: https://appswhoint/iris/bitstream/handle/10665/274352/who-whe-cpi-201828-frepdf?ua=1 world health organization. joint external evaluation of ihr core capacities of the republic of rwanda [homepage on the internet]. 2018 [cited 2020 nov 25]. available from: https://appswhoint/iris/bitstream/handle/10665/274353/who-whe-cpi-rep-201822-engpdf world health organization. évaluation externe conjointe des principales capacités rsi de la république du tchad [homepage on the internet]. 2017 [cited 2020 nov 25]. available from: https://appswhoint/iris/bitstream/handle/10665/260441/who-whe-cpi-rep-20183-frepdf?sequence=1 world health organization. avaliação externa conjunta das principais capacidades do rsi república democrática de são tomé e príncipe [homepage on the internet]. relatório da missão. 2019 [cited 2020 nov 25]. available from: https://apps.who.int/iris/handle/10665/330004 world organization for animal health (oie). independent review of pvs pathway reports from african member countries: final report – august 2019 [homepage on the internet]. 2019 [cited 2020 dec 21]; p. 121. available from: https://rr-africa.oie.int/wp-content/uploads/2020/01/oie_pvs_africa_evaluation-report_final_revised.pdf gourlaouen m, angot a, mancin m, et al. an inter-laboratory trial as a tool to increase rabies diagnostic capabilities of sub-saharan african veterinary laboratories. plos negl trop dis. 2020;14(2):e0008010. https://doi.org/10.1371/journal.pntd.0008010 dhillon rs, srikrishna d, sachs j. controlling ebola: next steps. lancet. 2014;384(9952):1409–1411. https://doi.org/10.1016/s0140-6736(14)61696-2 grolla a, jones sm, fernando l, et al. the use of a mobile laboratory unit in support of patient management and epidemiological surveillance during the 2005 marburg outbreak in angola. plos negl trop dis. 2011;5(5):e1183. https://doi.org/10.1371/journal.pntd.0001183 hill sc, vasconcelos j, neto z, et al. emergence of the asian lineage of zika virus in angola: an outbreak investigation. lancet infect dis. 2019;19(10):19:1138–1147. https://doi.org/10.1016/s1473-3099(19)30293-2 hecker mt, aron dc, patel np, lehmann mk, donskey cj. unnecessary use of antimicrobials in hospitalized patients: current patterns of misuse with an emphasis on the antianaerobic spectrum of activity. arch intern med. 2003;163(8):972–978. https://doi.org/10.1001/archinte.163.8.972 werner nl, hecker mt, sethi ak, donskey cj. unnecessary use of fluoroquinolone antibiotics in hospitalized patients. bmc infect dis. 2011;11(1):187. https://doi.org/10.1186/1471-2334-11-187 john jf, jr., fishman no. programmatic role of the infectious diseases physician in controlling antimicrobial costs in the hospital. clin infect dis. 1997;24(3):471–485. https://doi.org/10.1093/clinids/24.3.471 vounba p, arsenault j, bada-alambédji r, fairbrother jm. prevalence of antimicrobial resistance and potential pathogenicity, and possible spread of third-generation cephalosporin resistance, in escherichia coli isolated from healthy chicken farms in the region of dakar, senegal. plos one. 2019;14(3):e0214304. https://doi.org/10.1371/journal.pone.0214304 economou v, gousia p. agriculture and food animals as a source of antimicrobial-resistant bacteria. infect drug resist. 2015;8:49–61. https://doi.org/10.2147/idr.s55778 world health organization. plan d’action mondial pour combattre la résistance aux antimicrobiens [homepage on the internet]. genève: oms, 2015 [cited 2020 dec 19]; 32 p. available from: https://www.who.int/antimicrobial-resistance/global-action-plan/fr/ vernhet a, licznar-fajardo p, jumas-bilak e. antibiorésistance, quels rôles pour le pharmacien d’officine? actual pharm. 2016;55(556):37–40. https://doi.org/10.1016/j.actpha.2016.03.009 dadgostar p. antimicrobial resistance: implications and costs. infect drug resist. 2019;12:3903–3910. https://doi.org/10.2147/idr.s234610 united nations. draft political declaration of the high-level meeting of the general assembly on antimicrobial resistance [homepage on the internet]. new york, ny: united nations; 2016 [cited 2021 may 22]. available from: https://www.un.org/pga/71/wp-content/uploads/sites/40/2016/09/dgacm_gaead_escab-amr-draft-politicaldeclaration-1616108e.pdf beeching nj, fenech m, houlihan cf. ebola virus disease. bmj. 2014;349:g7348. https://doi.org/10.1136/bmj.g7348 leekha s, terrell cl, edson rs. general principles of antimicrobial therapy. mayo clin proc. 2011;86(2):156–167. https://doi.org/10.4065/mcp.2010.0639 okeke in. laboratory systems as an antibacterial resistance containment tool in africa. afr j lab med. 2016;5(3):497. https://doi.org/10.4102/ajlm.v5i3.497 world health organization. diagnostic stewardship: a guide to implementation in antimicrobial resistance surveillance sites [homepage on the internet]. 2016 [cited 2020 dec 21]; 27p. available from: https://apps.who.int/iris/bitstream/handle/10665/251553/who-dgo-amr-2016.3-eng.pdf?sequence=1&isallowed=y o’neill j. rapid diagnostics: stopping unnecessary use of antibiotics: review on antimicrobial resistance [homepage on the internet]. 2015 [cited 2020 dec 21]. available from: https://amr-review.org/sites/default/files/paper-rapid-diagnostics-stopping-unnecessary-prescription.pdf file tm. the science of selecting antimicrobials for community-acquired pneumonia (cap). j manag care pharm. 2009;15(2 supp a):5–11. https://doi.org/10.18553/jmcp.2009.15.s2.5 fuller j, mcgeer a, low d. drug-resistant pneumococcal pneumonia: clinical relevance and approach to management. eur j clin microbiol infect dis. 2005;24(12):780–788. https://doi.org/10.1007/s10096-005-0059-x roberts rr, hota b, ahmad i, et al. hospital and societal costs of antimicrobial-resistant infections in a chicago teaching hospital: implications for antibiotic stewardship. clin infect dis. 2009;49(8):1175–1184. https://doi.org/10.1086/605630 ayukekbong ja, ntemgwa m, atabe an. the threat of antimicrobial resistance in developing countries: causes and control strategies. antimicrob resist infect control. 2017;6(1):1–8. https://doi.org/10.1186/s13756-017-0208-x varma jk, oppong-otoo j, ondoa p, et al. africa centres for disease control and prevention’s framework for antimicrobial resistance control in africa. afr j lab med. 2018;7(2):1–4. https://doi.org/10.4102/ajlm.v7i2.830 avery bp, parmley ej, reid-smith rj, daignault d, finley rl, irwin rj. canadian integrated program for antimicrobial resistance surveillance: retail food highlights, 2003–2012. can commun dis rep. 2014;40(suppl 2):29–35. https://doi.org/10.14745/ccdr.v40is2a05 kelley p. antimicrobial stewardship: the role of the clinical microbiology service. pathol. 2014;46(suppl 1):s45. https://doi.org/10.1097/01.pat.0000443495.55603.fb avdic e, carroll kc. the role of the microbiology laboratory in antimicrobial stewardship programs. infect dis clin north am. 2014;28(2):215–235. https://doi.org/10.1016/j.idc.2014.01.002 bouza e, muñoz p, burillo a. role of the clinical microbiology laboratory in antimicrobial stewardship. med clin north am. 2018;102(5):883–898. https://doi.org/10.1016/j.mcna.2018.05.003 njukeng pa, ako-arrey de, amin et, njumkeng c, wirsiy fs. antimicrobial resistance in the central african region: a review. j environ sci pub health. 2019;3(3):358–378. kimera zi, mshana se, rweyemamu mm, mboera leg, matee min. antimicrobial use and resistance in food-producing animals and the environment: an african perspective. antimicrob resist infect control. 2020;9(1):37. https://doi.org/10.1186/s13756-020-0697-x harbarth s, balkhy hh, goossens h, et al. antimicrobial resistance: one world, one fight! antimicrob resist infect control. 2015;4:49. https://doi.org/10.1186/s13756-015-0091-2 el harrak m. biorisk: african experience [homepage on the internet]. 12th oie seminar. 2017 [cited 2021 dec 29]; sorrento, italy, 7–10 june 2017. available from: https://www.oie.int/eng/wavld2017/presentations.htm the lancet. genomic sequencing in pandemics. lancet. 2021;397(10273):445. https://doi.org/10.1016/s0140-6736(21)00257-9 inzaule sc, tessema sk, kebede y, ouma aeo, nkengasong jn. genomic-informed pathogen surveillance in africa: opportunities and challenges. lancet infect dis. 2021;21(9):e281–e289. https://doi.org/10.1016/s1473-3099(20)30939-7 ntoumi f, mapanguy ccm, tomazatos a, et al. genomic surveillance of sars-cov-2 in the republic of congo. int j infect dis. 2021;105:735–738. https://doi.org/10.1016/j.ijid.2021.03.036 who. guide for the stepwise laboratory improvement process towards accreditation in the african region (slipta) [homepage on the internet]. 2015 [cited 2020 nov 20]. available from: https://wwwafrowhoint/publications/who-guide-stepwise-laboratory-improvement-process-towards-accreditation-slipta-african strengthening laboratory management toward accreditation (slmta). slmta laboratories that have achieved accreditation [homepage on the internet]. [cited 2021 oct 31]. available from: https://slmta.org/accredited-labs/ programme infrastructure qualité de l’afrique centrale (piqac). brochure des résultats de mise en œuvre [homepage on the internet]. 2019 [cited 2021 dec 21]; 66 p. available from: https://www.unido.org/sites/default/files/files/2019-02/piqac_brochure.pdf food and agriculture organization (fao). building veterinary laboratory diagnostic capacity in africa: the vetlab network [homepage on the internet]. fcc-empres information sheets no 6. 2015 [cited 2021 dec 31]. available on: http://www.fao.org/resilience/resources/ressources-detail/fr/c/295768/ datema ta, oskam l, van beers sm, klatser pr. critical review of the stepwise laboratory improvement process towards accreditation (slipta): suggestions for harmonization, implementation and improvement. trop med int health. 2012;17(3):361–367. https://doi.org/10.1111/j.1365-3156.2011.02917.x greaves rf, bernardini s, ferrari m, et al. key questions about the future of laboratory medicine in the next decade of the 21st century: a report from the ifcc-emerging technologies division. clinica chimica acta. 2019;495:570–589. https://doi.org/10.1016/j.cca.2019.05.021 redding dw, atkinson pm, cunningham aa, et al. impacts of environmental and socio-economic factors on emergence and epidemic potential of ebola in africa. nat commun. 2019;10(1):4531. https://doi.org/10.1038/s41467-019-12499-6 africa cdc. implementation of laboratory systems and network in central africa region. activity report for 2018/2019 project year [homepage on the internet]. 2019 [cited 2021 feb 25]. available from: https://ghsscmorg/wp-content/uploads/2020/01/implementation-of-laboratory-systems-and-network-in-central-africa-regionpdf bonini p, plebani m, ceriotti f, rubboli f. errors in laboratory medicine. clin chem. 2002;48(5):691–698. https://doi.org/10.1093/clinchem/48.5.691 clinical and laboratory standards institute (clsi). a quality management system model for laboratory services. 5th ed. clsi guideline qms01. wayne, pa: clinical and laboratory standards institute; 2019. ndasi j, dimite l, mbome v, et al. decentralised facility-based training as an alternative model for slmta implementation: the cameroon experience. afr j lab med. 2014;3(2):231. https://doi.org/10.4102/ajlm.v3i2.231 ondoa p, ndlovu n, keita m-s, et al. preparing national tiered laboratory systems and networks to advance diagnostics in africa and meet the continent’s health agenda: insights into priority areas for improvement. afr j lab med. 2020;9(2):1103. https://doi.org/10.4102/ajlm.v9i2.1103 rusanganwa v, gahutu jb, nzabahimana i, ngendakabaniga jmv, hurtig a-k, evander m. clinical referral laboratories in rwanda: the status of quality improvement after 7 years of the slmta program. am j clin pathol. 2018;150(3):240–245. https://doi.org/10.1093/ajcp/aqy047 parsons lm, somoskovi a, lee e, et al. global health: integrating national laboratory health systems and services in resource-limited settings. afr j lab med. 2012;1(1):11. https://doi.org/10.4102/ajlm.v1i1.11 world health organization. consultation on technical and operational recommendations for clinical laboratory testing harmonization and standardization; helping to expand sustainable quality testing to improve the care and treatment of people infected with and affected by hiv/aids, tb and malaria, 22–24 january 2008, maputo, mozambique [homepage on the internet]. [cited 2021 feb 26]. available from: https://pdf4pro.com/amp/download?data_id=4b9409&slug=consultation-on-technical-and-operational-recommendations wendt a, kreienbrock l, campe a. zoonotic disease surveillance – inventory of systems integrating human and animal disease information. zoonoses pub health. 2015;62(1):61–74. https://doi.org/10.1111/zph.12120 abstract early infant diagnosis of hiv and virological monitoring of patients on antiretroviral therapy – ongoing challenges for sub-saharan africa the concept of pooled testing (‘pooling’) – some important considerations perspectives and outlook acknowledgements references about the author(s) wolfgang preiser division of medical virology, department of pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa national health laboratory service (nhls) tygerberg, cape town, south africa gert u. van zyl division of medical virology, department of pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa national health laboratory service (nhls) tygerberg, cape town, south africa citation preiser w, van zyl gu. pooled testing: a tool to increase efficiency of infant hiv diagnosis and virological monitoring. afr j lab med. 2020;9(2), a1035. https://doi.org/10.4102/ajlm.v9i2.1035 review article pooled testing: a tool to increase efficiency of infant hiv diagnosis and virological monitoring wolfgang preiser, gert u. van zyl received: 24 apr. 2019; accepted: 15 apr. 2020; published: 11 aug. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: pooled testing, or pooling, has been used for decades to efficiently diagnose relatively rare conditions, such as infection in blood donors. programmes for the prevention of mother-to-child transmission of hiv and for antiretroviral therapy (art) are being rolled out in much of africa and are largely successful. this increases the need for early infant diagnosis (eid) of hiv using qualitative nucleic acid testing and for virological monitoring of patients on art using viral load testing. while numbers of patients needing testing are increasing, infant hiv infections and art failures are becoming rarer, opening an opportunity for pooled testing approaches. aim: this review highlights the need for universal eid and viral load coverage as well as the challenges faced. we introduce the concept of pooled testing and highlight some important considerations before giving an overview of studies exploring pooled testing for eid and virological monitoring. results: for art monitoring, pooling has been shown to be accurate and efficient; for eid it has not been tried although modelling shows it to be promising. the final part attempts to place pooling into the context of current mother-to-child transmission of hiv and art programmes and their expected trajectories over the next years. conclusion: several points warrant consideration: pre-selection to exclude samples with an elevated pre-test probability of positivity from pooled testing, the use of dried blood or plasma spots, and choosing a pooling strategy that is both practically feasible and economical. finally, novel ideas are suggested to make pooling even more attractive. keywords: hiv; antiretroviral treatment; early infant diagnosis; pooling; pooled testing. early infant diagnosis of hiv and virological monitoring of patients on antiretroviral therapy – ongoing challenges for sub-saharan africa the prevention of mother-to-child transmission (pmtct) of hiv has made enormous progress over the past 15 years, with much-increased coverage and greatly improved programme components. according to estimates for 2017, 80% of hiv-positive pregnant women globally – and as many as 93% in eastern and southern africa – received antiretroviral drugs for pmtct. while 180 000 children aged up to 14 years were newly infected with hiv globally, 210 000 infant infections were averted due to pmtct.1 globally, 14.8 million hiv-exposed children remained uninfected.1 in south africa, the national pmtct programme has followed the world health organization option b+ policy since 2015: provision of lifetime combination antiretroviral therapy (art) for all hiv-positive pregnant women, regardless of their immunological or clinical stage.2 this has resulted in the reduction of the mother-to-child transmission rate to an estimated 4.6% overall in 2016.3 despite these successes, all hiv-exposed infants continue to need early infant diagnosis (eid). maternal antibodies persist up to age 18 months. therefore, eid relies on virological assays that detect either viral nucleic acid or viral proteins at different ages to diagnose cases of prenatal, perinatal and postnatal transmission.4 the prompt diagnosis of hiv infection is a prerequisite for starting infected babies on art early on, thus improving their prognosis.5 while infant hiv infection has now become or will soon become a relatively rare event,6 the sheer number of exposed infants in need of testing at different time points will continue to pose a major challenge to laboratory systems. another area that has seen enormous progress over the past two decades is art programmes, which have been implemented in much of africa and have been generally successful. of the 20 million people estimated to be living with hiv infection in eastern and southern africa in 2017, about 13 million or 66% were on art, of whom more than 10 million were estimated to have suppressed viral load (vl).1 for south africa, according to 2017 estimates, 90% of hiv-positive individuals know their status; 68% of these are on art and 78% of those on art achieve viral suppression.1 previously regarded as optional,7 routine virological monitoring of patients on art by hiv vl testing to detect art failure allows optimal patient management. addressing either adherence or presumed drug resistance (when there is no response to adherence interventions) secures long-term viability of art programmes by limiting emergence and transmission of antiretroviral drug resistance.8,9 optimal virological suppression is essential to prevent onward transmission; therefore, achieving an undetectable vl in 90% of those on art has been included as the last component of the joint united nations programme on hiv/aids (unaids) 90-90-90 targets to help end the hiv/aids epidemic.10 the need for virological monitoring is increasingly reflected in art guidelines.4 yet significant barriers remain: currently available vl tests are expensive, technically demanding and thus not widely available in many resource-limited settings (rls).11 while point-of-care vl tests may be an ideal solution and several systems are approaching clinical usability, their relatively high cost and other challenges remain unresolved.12 a recent forecast study underlines the need for a substantial rollout of both eid and vl testing.13 the concept of pooled testing (‘pooling’) – some important considerations pooled testing or ‘pooling’, also known as ‘group testing’, refers to mixing several samples together prior to performing a laboratory test. individual samples contained in a negative pool can be classified as negative without further testing, whereas samples contained in pools testing positive need to be re-tested individually. while pooling may seem rather crude and does not apply to measurements of analytes that are present in both physiological and pathological states, albeit, at different concentrations, pooling has a long tradition for infectious disease markers, for example for the screening of blood donations.14 here the physiological state is ‘negative’ (which means the laboratory marker, for example specific antibodies or pathogen-specific nucleic acid sequences, are undetectable), thus, the prevalence of positive samples among donors is low, thanks to the application of stringent donor selection criteria. used for antibody-based screening (e.g. syphilis) for decades, pooled testing was later adopted for nucleic acid testing (nat), too.15 the advantage of pooling lies in its ability to save on the number of tests needed and, thus, costs. as long as the condition to be detected occurs at low prevalence, most pools will test as negative. then, all samples contained in a negative pool can have a negative result reported while having used just one test. if a pool tests positive, it needs to be determined which of the constituent samples is responsible. this is achieved by re-testing the constituent samples individually (a process termed ‘deconvolution’ or ‘resolution’) which thus negates any saving for the samples concerned, in that the constituent samples together will, in the end, have used a number of tests equal to their number plus one (the test used for the pool). alternative strategies have been proposed that are more efficient than using straightforward pooling and deconvolution by re-testing constituent samples simultaneously. in two-dimensional or multidimensional matrix approaches, each sample is contained in a unique combination of different pools; the combination of pools that test positive resolves the sample responsible without re-testing.16 multistage or pyramid-type pooling strategies may likewise be more efficient than simple pooling.17 when a pooling approach is used for quantitative tests, a strategy of individually re-testing the constituent samples one after the other may be designed; when an individual sample’s quantitative test result can explain the quantitative result for the pool, the remaining samples may be regarded as negative.18 such an approach is, however, not without challenges, especially given the test-to-test variation of quantitative results. the main factors determining whether pooling may be advantageous are the prevalence of the condition to be tested for, and the pool size. pooling is predicated on a relatively low prevalence condition being sought. if a condition is highly prevalent, pooling will not save tests, due to frequent deconvolution and individual re-testing. the larger the pool size (i.e. the number of samples mixed together to form one pooled specimen for testing), the greater the potential saving. however, pool size is not only limited by practical considerations – test volumes and dilution factors, the latter linked to test sensitivity; but by the tested condition’s prevalence – a low prevalence ensures that a substantial proportion of test pools are negative. a simple example illustrates this: assuming a prevalence of 20%, a pool size of five specimens will yield almost no savings and a pool size of four, only marginal savings.19 by excluding patient samples with a high probability of positivity from pooling, the prevalence of the condition tested in the pooled samples is reduced invariably improving pooling efficiency.20 examples of such patients are children whose mothers did not receive pmtct, those who failed art previously or are clinically unwell, and those sent for confirmation of a previous positive result. several aspects need to be taken into account when considering pooled testing. the first aspect is the test sensitivity. pooled test sensitivity is often somewhat lower than the sensitivity of individual tests because of the required dilution of pooled specimens. however, this lowered may not be clinically relevant and counteracted to some degree by modifying the testing process (e.g. substituting some of the diluent or sample buffer with specimen). the sensitivity of pooled testing and any measures to improve it has to be considered and validated carefully before using pooling for diagnostic purposes. currently, available commercial vl assays have lower limits of detection below 50 copies/ml. this redundant sensitivity can be exploited by testing pools consisting of several mixed samples, if the world health organization’s recommended threshold is used for clinical decision-making. sample throughput is another important consideration. there needs to be a balance between volumes of samples (requests) received per day and the pool sizes (to allow for efficient pooling) without increasing test turn-around time while waiting for enough specimens to be received to allow for pooling. finally, the processes of pooling and deconvolution (of positive pools) add to the operator workload. on the other hand, operator workload is reduced through efficient pooling, as fewer tests need to be performed. however, performing pooling and deconvolution may also introduce clerical errors; the approach must therefore be designed well and conducted carefully. these factors need to be factored into calculations of pooling efficiency and assessing practicability. the interaction between the above-listed factors is rather complex. it is highly unlikely that anyone or a limited choice of pooling solutions, would be suitable for different scenarios. pooled testing for early infant diagnosis of hiv the earliest possible diagnosis of hiv infection in hiv-exposed infants is a prerequisite for early initiation of art, which markedly improves the prognosis of infants with hiv infection.5 this requires testing uncoagulated whole blood or dried blood spot (dbs) samples for viral genome (proviral dna, viral rna, or both) by means of polymerase chain reaction (pcr) or similar genome amplification technologies.4 pcr is technically demanding and costly, limiting access to it in many rls. dbs are widely used in rls, as they are easier to obtain, to store and to transport while performing generally well.21 recommendations about the optimal time points for eid testing are evolving, due to programmatic improvements and new scientific findings. the optimum would be three eid tests for each exposed infant: the first at or soon after birth (to detect infection acquired in utero), the second at around 6–10 weeks of age (to detect perinatal transmission; the preferred timing depends on the duration of postnatal post-exposure prophylaxis), and a third one 6 weeks after cessation of breastfeeding (to detect postnatal transmission, which would usually be via breast milk).2 not only is eid coverage across most of sub-saharan africa poor and the results commonly delayed,1 performing three tests for each exposed infant also places an enormous burden on health and laboratory services. measures to increase eid capacity and reduce costs are therefore sorely needed. point-of-care assays are increasingly being made available and have been piloted in several studies, but several drawbacks remain unresolved: their relatively high complexity requires trained staff; their relatively low throughput requires multiple instruments at busy clinics; and their relatively high cost which is and will likely remain higher than testing at centralised laboratories.22 interestingly, there does not seem to be much interest in pooled testing for eid. the first and so far only known study on this topic, published in february 2019, determined the clinical accuracy of pooled testing of infant dbs and developed a model to simulate the saving of reagent and consumable costs based on real data from a south african public health laboratory.23 the study found a high sensitivity (98.8%) for pooled testing. the one observed false-positive pool test result was confirmed through re-testing of the constituent patient samples. the savings of laboratory costs – 64% based on 2015 prices – were substantial. the article concludes that pooling eid pcr tests retains accuracy while substantially reducing costs, provided a laboratory receives sufficient numbers of samples and has a low to moderate infant hiv prevalence. the authors have developed and made available publicly an r-based web tool to assess the cost-efficiency of pooled pcr testing for infant hiv diagnosis for varying parameters (https://carivs.shinyapps.io/calculator). users will enter the average number of samples tested per day, the estimated positivity rate (maximum allowed is 10% as above that pooling would not be viable), the sample type (dbs or whole blood), the cost of reagents (if unknown, the model provides an estimated percentage saving) and the minimum number of samples required to run a batch for pooling and for individual testing. the model will then do a calculation, based on the above, and report the optimal pool size, the cost saving, and the reduction in daily batch runs both as a sentence (e.g. based on your input values, at the optimal pool size of 5, you will save 62% of costs and daily batched runs will decrease from 2 to 1) and graphically as a three-dimensional plot with axes for positivity (%), pool size and cost as a percentage of cost for testing individual samples. pooled testing for virological monitoring the sustained suppression of hiv replication in patients on art is the goal of hiv treatment. halting viral replication provides individual benefit as it allows immune reconstitution and reduces inflammation, thereby lowering the risk for hiv-associated infectious and non-communicable complications. moreover, on a population level, sustained virological suppression renders treated patients virtually non-infectious and thus prevents onward transmission.24 conversely, failing to achieve virological suppression puts the patient and the population at risk; this is not only through hiv disease and the transmission of hiv infection, but also through the emergence of antiretroviral drug-resistant hiv strains which may jeopardise future art options for the individual and, through the transmission of resistant virus strains could put the whole art programme at risk of failure.25 monitoring of patients on art by means of virological assays is therefore of paramount importance and after years of advocacy, this is now widely acknowledged and reflected in the third ‘90’ of the unaids’s 90-90-90 goals.26,10 in 2017, the proportion of people of all age groups on art who achieved virological suppression was estimated at 81% globally, at 79% for eastern and southern africa and at 73% for west and central africa. for botswana, the estimate is > 95% and for south africa, 78%. virological non-suppression appears to be increasingly rare in many settings and may have become a low prevalence condition in some places already.1 virological monitoring is usually done through hiv vl testing, that is, using assays that quantify the concentration of hiv rna in patient plasma. a vl result of less than 50 copies of hiv rna per millilitre of plasma is usually regarded as full suppression, which is the aim of art in industrialised countries. in rls, vl of 1000 copies/ml in patients on art for at least 6 months or a vl above 1000 copies/ml in patients after two consecutive measurements 3 months apart with adherence support defines virological failure (vf) and should trigger clinical action.4 the rationale for using 1000 copies/ml as the widely accepted public health threshold is twofold: below this level, there is a limited risk of both disease progression and hiv transmission.4 additionally, it avoids the irrelevant detection of viral ‘blips’, short episodes of low-level viraemia that may occur during effective art and are not associated with an increased risk of art failure.27 smith et al.28 was the first study to report the use of pooled vl testing to identify art failure. it was conducted on a small number of art patient samples in the united states and compared two different pooling strategies, namely mini pools of five samples each and 10 × 10 matrix pools, with individual vl testing as the gold standard. in a matrix pooling approach, each sample is contained in more than one pool and the combination of pools testing positive allows direct identification of the sample responsible in most instances when the prevalence of failure is low, making deconvolution unnecessary. even though 23% of samples had a vl of > 50 copies/ml, both pooling strategies combined with a search and retest algorithm, led to savings of > 50% compared with individual testing, with only a minimal decrease in accuracy. a subsequent modelling study assessed various pooling strategies for monitoring art patients with vf prevalences between 1% and 25% in terms of efficiency and accuracy.19 it also compared three different pooling approaches – mini pools, mini pools with algorithm-based deconvolution, and matrix pooling to individual testing. using the quantitative information, that is, vl measurements available, this study showed pooling efficiency at higher prevalences than for which a purely binary approach (vl either detectable or undetectable) would have been efficient. however, such relatively complicated approaches may not prove feasible in many rls. a third study, conducted in south africa, investigated three aspects. firstly, whether the use of basic data recorded on routine laboratory request forms would allow selection of art patients with a low probability of art failure for pooled testing. secondly, whether mini pool or matrix pooling strategies were more suitable, comparing them against individual vl testing in a laboratory study. thirdly, the use of dbs and dried plasma spots instead of liquid plasma samples for preparing mini pools.20 in the retrospective cohort studied, the vf rate was 14.2%.20 after applying age > 15 years and being on a first-line art regimen as selection criteria (information easily obtained from test request forms), this dropped to 8.7% making pooling of such pre-selected samples more feasible. four hundred plasma specimens were tested in 80 mini pools of five each, and 300 of these samples also with a 10 × 10 matrix strategy. pooled testing reduced the number of tests needed by 30.5% – 60.0%. even though the matrix pooling strategy was more efficient than mini pooling, the latter was deemed overall more suitable as it proved much easier to perform, taking less operator time and requiring less expertise for constitution and deconvolution of pools. in addition, waiting for 100 samples to be available for testing before a 10 × 10 matrix can be constructed may increase turn-around times in many laboratories; this is generally not a problem with mini pools of five samples. the study found a poorer specificity and efficiency of pooled dbs testing. factors such as the contribution of cell-associated hiv rna and the variable plasma fraction (depending on patients’ haematocrit values) could cause falsely high vl results, while the presence of haemoglobin and other inhibitory substances in dbs could lead to falsely low results. pooled dried plasma spot testing, however, showed less variability and excellent accuracy and an efficiency of 60% for a pool threshold of 200 hiv rna copies/ml. a large field study in malawi subsequently addressed two issues relevant to rls: a higher vf rate and the problems inherent in testing plasma samples.29 testing 350 patient samples, the study found that pooled testing of plasma samples had excellent sensitivity (96.4%) and reduced the number of tests required by 44.3%. using dbs, prepared either directly by finger prick (which would be the preferred option in real-life use of pooled testing in rls) or from venous blood, sensitivity was 78.6% and 89.3%, saving 28.6% – 32.9% of tests. it concluded that when using the nuclisens assay – which does not amplify the cell-associated hiv dna contained in dbs, in contrast to most other hiv vl assays – testing dbs is a feasible option. pooling for virological monitoring of art patients has been assessed in other settings: those with a low prevalence of vf such as south korea30 and in rls with typically much higher failure rates such as mexico,31 kenya32 and mozambique.33 even under rls conditions pooled testing was generally efficient with acceptable accuracy, although not as efficient as in settings with low failure rates. however, el bouzidi et al.34 reported that testing in mini pools of four samples could not reliably identify vf in a london, united kingdom, art population, due to insufficient diagnostic sensitivity. while almost one in three failing patients was missed, the vl values of failing patients were low and mostly irrelevant for rls. perspectives and outlook pooled testing has a long history of successful use in the field of infectious disease testing, the most notable application being for screening blood donors both by serology and since the 1990s by nat.15 more recently, a multitude of studies have investigated pooled nat as a tool to detect early hiv infection that would be missed by routine hiv screening tests. in africa, karim et al.35 were the first to propose such a strategy; yet even in a high risk setting such as described by gous et al.,36 pooled nat would still be relatively expensive per additional case detected, as very few patients will be in the nat-positive antibody-negative stage of recent infection.37 more recently, a study by dowling et al.38 demonstrated that dbs can be used for this purpose, which would facilitate its wide implementation in rls. with ongoing major advances in both eid and art programmes, it is surprising that the use of pooled testing has hardly been explored for the purposes of eid, while there is ample literature on pooled testing for monitoring patients on art in a variety of settings. the need for eid testing will remain unchanged even as mother-to-child transmission rates are in decline. point-of-care testing might appear as an ideal solution for a variety of reasons,39 yet it is questionable whether it will be the most affordable solution against the background of an enormous, only partially met need. pooled eid testing has been modelled to be efficient,23 and infant hiv infection is becoming less prevalent in most places. however, ongoing vigilance is required as the evolving nature of the pmtct programme makes establishing a definite diagnosis increasingly challenging.40,41 the need for art monitoring will increase in the foreseeable future. in principle, when the prevalence of vf is low, pooled testing is very efficient, saves costs and may even reduce the average turn-around time (time from obtaining sample to receipt of test result). it is expected that with the rollout of newer therapy regimens the prevalence of vf may drop further, to below 7%.42 this might cause pressure to decrease the frequency of vl testing further, when in actual fact failure needs to be identified more efficiently. the performance characteristics of modern laboratory-based vl assays are redundant for widely accepted vl thresholds and thus provide comfortable room for pooling; highly sensitive assays offer leeway when a threshold of 1000 copies/ml is used to define art failure. pooling would potentially increase throughput and reduce turn-around time. pooled hiv vl testing has the potential to reduce the reagent cost of vl testing. early initiation of art and the use of more tolerable high genetic barrier regimens for first-line art have the potential to further drive down the prevalence of vf.42 pooled vl testing is therefore likely to become even more efficient in the near future. nevertheless, large scale implementation of pooled testing requires overcoming several hurdles. the first is susceptibility to human error, when pooling and deconvolution are performed manually. the second is that pooling and deconvolution add to hands-on time and therefore increase labour costs. the third is that the efficiency of pooled testing is highly susceptible to test failure. important considerations as regards pooling for eid and monitoring art, a number of pivotal points are emerging from the published studies, and these issues need to be taken into account when considering the introduction of pooled testing. firstly, to ensure the highest possible efficiency of pooling, a pre-selection step should be considered to identify samples from patients with a high pre-test probability of vf (i.e. hiv-positive patients). this could be achieved by interrogating the information contained on most test request forms or the information contained in the laboratory information system, where possible. the purpose of this would be to exclude ‘high risk’ specimens from being pooled lest they ‘poison’ pools. secondly, to maximise the usefulness of pooled testing in rls, the use of dbs or dried plasma spot would be ideal. once dried onto filter paper, such samples are non-infectious and stable for prolonged periods, as long as they are not subjected to extreme temperatures and excessive humidity; they can therefore be stored and transported even over long distances easily and cheaply.43 dried blood spots are relatively easy to obtain using finger (or toe or heel, in infants) pricks and are already widely used for eid in rls. for eid, their sensitivity may need consideration as the proportion of infected infants with low vl values increases.44 regarding vl testing, dbs have certain intrinsic disadvantages that are unlikely to be overcame, such as the detection of intracellular viral genomes. on the other hand, dried plasma spots negate part of the attractiveness of dried samples as they require expert sample handling and manipulation at the clinical site. this expertise is often lacking in many peripheral clinics and also increases operator time, which in turn increases the risk of sample mix-ups, mislabelling, and the like. how much solution simple-to-use plasma separation devices will offer for this issue remains to be ascertained.45 thirdly, regarding the preferable pooling strategy, it appears that in rls straightforward mini pools and parallel deconvolution probably offer the best efficiency with ease of handling. in many rls it will be necessary to avoid placing too much extra strain on scarce laboratory staff in terms of work time and effort. a misconceived, over-ambitious approach may lead to an increase in clerical errors and other problems that could easily negate the efficiency gained through pooling. limitations apart from the factors outlined above, a clear limitation of pooling is that it needs to be done in a centralised laboratory in order to have access to preferably automated equipment and skilled staff to operate it and to assemble large enough numbers of specimens for testing. this necessitates sample transport, which is often challenging and increases turn-around times. point-of-care testing obviously has a number of advantages over any centralised testing, including pooled testing. however, it also poses a number of challenges such as price, operator training and supervision, quality control, etc., which still have to be resolved. ultimately, both will have a role to play: point-of-care testing for remote rural clinics (to avoid expensive shipping and lengthy delays) and pooling for urban areas and clinics with good transport links to major laboratories. suggestions for future research two opportunities exist that could make pooling even more attractive for rls. the first can be summarised by the question whether the ‘load’ in ‘viral load’ is actually needed. using a qualitative hiv pcr assay calibrated to detect down to approximately 1000 copies/ml per constituent sample and which generates a product covering most of the hiv pol gene for sanger sequencing, pooled testing could inform about vf (albeit without assigning a vl value) and the presence of the most relevant drug resistance mutations. this unconventional approach seems to work well in both developed countries and in rls,46,47,48 while addressing the urgent and largely unmet need for resistance testing in africa.49 the second opportunity addresses a major challenge for most african laboratories: the scarcity of well-qualified and trained staff. pooled testing has one principal disadvantage: it requires additional handling and, thus, increases hands-on time for qualified staff. this is necessary to perform the additional steps involved, namely the preparation of the pools and then the deconvolution of positive pools, involving retrieval of the samples that comprised a positive pool and re-testing them. unfortunately, both of these tasks are prone to human errors, which can cause sample mix-ups and other mistakes. automated pooling may help to overcome the important implementation hurdle posed by the shortage of trained laboratory staff. a pre-analytic sample robot could conduct the actual pooling process, then store samples (preferably cooled) until the pooled test result is available and lastly retrieve samples from positive pools for deconvolution. additionally, such a system could be integrated with the laboratory information system and also pre-screen patient samples with a low probability for failure, before preparing the pools educing the need for deconvolution. to conclude, pooling offers attractive prospects for african countries, not just for monitoring art but also for conducting eid testing. the time has come to conduct large-scale pilot studies, first on a limited scale, in parallel to individual testing to demonstrate non-inferiority of pooled testing in everyday use and subsequently in settings with suboptimal eid availability or which do not currently offer routine regular virological monitoring. acknowledgements competing interests the authors declare that no competing interests exist. authors’ contributions both authors shared all aspects equally, from developing the concept to writing and revising the review paper. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references unaids (joint united nations programme on hiv/aids). unaids data 2018. 2018 reference. unaids/jc2929e [homepage on the internet]. c2018 [cited 2019 apr 23]. available from: https://www.unaids.org/en/resources/documents/2018/unaids-data-2018 national department of health, south africa. national consolidated guidelines for the prevention of mother-to-child transmission of hiv (pmtct) and the management of hiv in children, adolescents and adults [homepage on the internet]. pretoria: national department of health; c2015 [cited 2019 apr 23]. available from: https://sahivsoc.org/files/art%20guidelines%2015052015.pdf world health organization. south africa hiv country profile: 2016. who/hiv/2017.59 [homepage on the internet]. c2016 [cited 2019 apr 23]. available from: https://www.who.int/hiv/data/profiles/en/ world health organization. consolidated guidelines on the use of antiretroviral drugs for treating and preventing hiv infection. recommendations for a public health approach. second edition, june 2016. who/cds/hiv/18.51 [homepage on the internet]. c2016 [cited 2019 apr 23]. available from: https://www.who.int/hiv/pub/arv/arv-2016/en/ violari a, cotton mf, gibb dm, et al. early antiretroviral therapy and mortality among hiv-infected infants. n engl j med 2008;359(21):2233–2244. https://doi.org/10.1056/nejmoa0800971 sherman gg, mazanderani ah, barron p, et al. toward elimination of mother-to-child transmission of hiv in south africa: how best to monitor early infant infections within the prevention of mother-to-child transmission program. j glob health. 2017 jun;7(1):010701. https://doi.org/10.7189/jogh.07.010701 gilks cf, crowley s, ekpini r, et al. the who public-health approach to antiretroviral treatment against hiv in resource-limited settings. lancet. 2006 aug;368(9534):505–510. https://doi.org/10.1016/s0140-6736(06)69158-7 calmy a, ford n, hirschel b, et al. hiv viral load monitoring in resource-limited regions: optional or necessary? clin infect dis. 2007 jan;44(1):128–134. https://doi.org/10.1086/510073 smith dm, schooley rt. running with scissors: using antiretroviral therapy without monitoring viral load. clin infect dis. 2008;46(10):1598–1600. https://doi.org/10.1086/587110 unaids (joint united nations programme on hiv/aids). 90-90-90: an ambitious treatment target to help end the aids epidemic. unaids / jc2684 [homepage on the internet]. c2014 [cited 2019 apr 23]. available from: http://www.unaids.org/sites/default/files/media_asset/90-90-90_en_0.pdf roberts t, bygrave h, fajardo e, ford n. challenges and opportunities for the implementation of virological testing in resource-limited settings. j int aids soc. 2012 oct 09;15(2):17324. https://doi.org/10.7448/ias.15.2.17324 stevens w, gous n, ford n, scott le. feasibility of hiv point-of-care tests for resource-limited settings: challenges and solutions. bmc med. 2014 sep;12:173. https://doi.org/10.1186/s12916-014-0173-7 habiyambere v, dongmo nguimfack b, vojnov l, et al. forecasting the global demand for hiv monitoring and diagnostic tests: a 2016-2021 analysis. plos one. 2018 sep 19;13(9):e0201341. https://doi.org/10.1371/journal.pone.0201341 dorfman r. the detection of defective members of large populations. ann math stat. 1943;14:436–440. https://doi.org/10.1214/aoms/1177731363 roth wk, weber m, buhr s, et al. yield of hcv and hiv-1 nat after screening of 3.6 million blood donations in central europe. transfusion. 2002 jul;42(7):862–868. https://doi.org/10.1046/j.1537-2995.2002.00129.x westreich dj, hudgens mg, fiscus sa, pilcher cd. optimizing screening for acute human immunodeficiency virus infection with pooled nucleic acid amplification tests. j clin microbiol. 2008 may;46(5):1785–1792. https://doi.org/10.1128/jcm.00787-07 quinn tc, brookmeyer r, kline r, et al. feasibility of pooling sera for hiv-1 viral rna to diagnose acute primary hiv-1 infection and estimate hiv incidence. aids. 2000 dec; 14(17):2751–2757. https://doi.org/10.1097/00002030-200012010-00015 smith dm, may sj, pérez-santiago j, et al. the use of pooled viral load testing to identify antiretroviral treatment failure. aids. 2009 oct;23(16):2151–2158. may s, gamst a, haubrich r, benson c, smith dm. pooled nucleic acid testing to identify antiretroviral treatment failure during hiv infection. j acquir immune defic syndr. 2010 feb;53(2):194–201. https://doi.org/10.1097/qai.0b013e3181ba37a7 van zyl gu, preiser w, potschka s, lundershausen at, haubrich r, smith d. pooling strategies to reduce the cost of hiv-1 rna load monitoring in a resource-limited setting. clin infect dis. 2011 jan;52(2):264–270. https://doi.org/10.1093/cid/ciq084 smit pw, sollis ka, fiscus s, et al. systematic review of the use of dried blood spots for monitoring hiv viral load and for early infant diagnosis. plos one. 2014 mar;9(3):e86461. https://doi.org/10.1371/journal.pone.0086461 celletti f, sherman g, mazanderani ah. early infant diagnosis of hiv: review of current and innovative practices. curr opin hiv aids. 2017 mar;12(2):112–116. https://doi.org/10.1097/coh.0000000000000343 van schalkwyk c, maritz j, van zyl gu, preiser w, welte a. pooled pcr testing of dried blood spots for infant hiv diagnosis is cost efficient and accurate. bmc infect dis. 2019 feb;19(1):136. https://doi.org/10.1186/s12879-019-3767-z cohen ms, chen yq, mccauley m, et al. prevention of hiv-1 infection with early antiretroviral therapy. n engl j med. 2011 aug;365(6):493–505. sigaloff kc, hamers rl, menke j, et al. early warning indicators for population-based monitoring of hiv drug resistance in 6 african countries. clin infect dis. 2012 may;54(suppl 4):s294–s299. https://doi.org/10.1093/cid/cir1015 msf access campaign. undetectable: how viral load monitoring can improve hiv treatment in developing countries. report [homepage on the internet]. geneva: msf; c2012 [cited 2019 apr 23]. available from: https://msfaccess.org/undetectable ellman tm, alemayehu b, abrams ej, arpadi s, howard aa, el-sadr wm. selecting a viral load threshold for routine monitoring in resource-limited settings: optimizing individual health and population impact. j int aids soc. 2017 nov;20(suppl 7):16–18. https://doi.org/10.1002/jia2.25007 smith dm, may sj, pérez-santiago j, et al. the use of pooled viral load testing to identify antiretroviral treatment failure. aids. 2009 oct;23(16):2151–2158. pannus p, fajardo e, metcalf c, et al. pooled hiv-1 viral load testing using dried blood spots to reduce the cost of monitoring antiretroviral treatment in a resource-limited setting. j acquir immune defic syndr. 2013 oct;64(2):134–137. https://doi.org/10.1097/qai.0b013e3182a61e63 kim sb, kim hw, kim hs, et al. pooled nucleic acid testing to identify antiretroviral treatment failure during hiv infection in seoul, south korea. scand j infect dis. 2014 feb;46(2):136–140. tilghman mw, guerena dd, licea a, et al. pooled nucleic acid testing to detect antiretroviral treatment failure in mexico. j acquir immune defic syndr. 2011;56(3):e704. https://doi.org/10.1097/qai.0b013e3181ff63d7 chohan bh, tapia k, merkel m, et al. pooled hiv-1 rna viral load testing for detection of antiretroviral treatment failure in kenyan children. j acquir immune defic syndr. 2013 jul;63(3):e87–e93. https://doi.org/10.1097/qai.0b013e318292f9cd tilghman m, tsai d, buene tp, et al. pooled nucleic acid testing to detect antiretroviral treatment failure in hiv-infected patients in mozambique. j acquir immune defic syndr. 2015 nov;70(3):256–261. https://doi.org/10.1097/qai.0000000000000724 el bouzidi k, grant p, edwards s, et al. pooled specimens for hiv rna monitoring: cheaper, but is it reliable? clin infect dis. 2014 nov;59(9):1346–1347. https://doi.org/10.1093/cid/ciu562 karim ss, mlisana k, kharsany ab, williamson c, baxter c, karim qa. utilizing nucleic acid amplification to identify acute hiv infection. aids. 2007 mar;21(5):653–655. https://doi.org/10.1097/qad.0b013e3280327923 gous n, scott l, perovic o, venter f, stevens w. should south africa be performing nucleic acid testing on hiv enzyme-linked immunosorbent assay-negative samples? j clin microbiol. 2010 sep;48(9):3407–3409. https://doi.org/10.1128/jcm.00702-10 maritz j, preiser w. cost-effectiveness of nucleic acid amplification tests for identifying acute hiv infections. j clin microbiol. 2011 apr;49(4):1704. https://doi.org/10.1128/jcm.02497-10 dowling w, veldsman k, katusiime mg, et al. hiv-1 rna testing of pooled dried blood spots is feasible to diagnose acute hiv infection in resource limited settings. s afr j infect dis. 2018;33(2):50–53. https://doi.org/10.4102/sajid.v33i2.6 jani iv, de schacht c. innovations and challenges in early infant diagnosis of hiv. curr opin hiv aids. 2019 jan;14(1):55–59. https://doi.org/10.1097/coh.0000000000000511 mazanderani ah, du plessis nm, thomas wn, venter e, avenant t. loss of detectability and indeterminate results: challenges facing hiv infant diagnosis in south africa’s expanding art programme. s afr med j. 2014 jun;104(8):574–577. https://doi.org/10.7196/samj.8322 veldsman ka, maritz j, isaacs s, et al. rapid decline of hiv-1 dna and rna in infants starting very early antiretroviral therapy may pose a diagnostic challenge. aids. 2018 mar;32(5):629–634. https://doi.org/10.1097/qad.0000000000001739 dorward j, lessells r, drain pk, et al. dolutegravir for first-line antiretroviral therapy in low-income and middle-income countries: uncertainties and opportunities for implementation and research. lancet hiv. 2018 jul;5(7):e400–e404. https://doi.org/10.1016/s2352-3018(18)30093-6 cassol s, salas t, gill mj, et al. stability of dried blood spot specimens for detection of human immunodeficiency virus dna by polymerase chain reaction. j clin microbiol. 1992 dec;30(12):3039–3042. https://doi.org/10.1128/jcm.30.12.3039-3042.1992 mazanderani ah, moyo f, kufa t, sherman gg. brief report: declining baseline viremia and escalating discordant hiv-1 confirmatory results within south africa’s early infant diagnosis program, 2010-2016. j acquir immune defic syndr. 2018 feb;77(2):212–216. https://doi.org/10.1097/qai.0000000000001581 carmona s, seiverth b, magubane d, hans l, hoppler m. separation of plasma from whole blood by use of the cobas plasma separation card: a compelling alternative to dried blood spots for quantification of hiv-1 viral load. j clin microbiol. 2019 mar;57(4):e01336. https://doi.org/10.1128/jcm.01336-18 tilghman mw, may s, pérez-santiago j, et al. a combined screening platform for hiv treatment failure and resistance. plos one. 2012;7(4):e35401. https://doi.org/10.1371/journal.pone.0035401 newman h, breunig l, van zyl g, stich a, preiser w. a qualitative pcr minipool strategy to screen for virologic failure and antiretroviral drug resistance in south african patients on first-line antiretroviral therapy. j clin virol. 2014 aug;60(4):387–391. https://doi.org/10.1016/j.jcv.2014.05.011 boobalan j, dinesha tr, gomathi s, et al. pooled nucleic acid testing strategy for monitoring hiv-1 treatment in resource limited settings. j clin virol. 2019 aug;117:56–60. https://doi.org/10.1016/j.jcv.2019.05.012 van zyl gu, frenkel lm, chung mh, preiser w, mellors jw, nachega jb. emerging antiretroviral drug resistance in sub-saharan africa: novel affordable technologies are needed to provide resistance testing for individual and public health benefits. aids. 2014 nov;28(18):2643–2648. https://doi.org/10.1097/qad.0000000000000502 page 1 of 1 reviewer acknowledgement http://www.ajlmonline.org open access read online: scan this qr code with your smart phone or mobile device to read online. acknowledgement to reviewers in an effort to facilitate the selection of appropriate peer reviewers for the african journal of laboratory medicine, we ask that you take a moment to update your electronic portfolio on https://ajlmonline.org for our files, allowing us better access to your areas of interest and expertise, in order to match reviewers with submitted manuscripts. if you would like to become a reviewer, please visit the journal website and register as a user. in order to be considered, please email submissions@ajlmonline. org indicating your intention to register as a reviewer for the journal. to access your details on the website, you will need to follow these steps: 1. log into the online journal at https:// ajlmonline.org 2. in your ‘user home’ [https://ajlmonline.org/ index.php/ajlm/user] select ‘edit my profile’ under the heading ‘my account’ and insert all relevant details, bio statement and reviewing interest(s). 3. it is 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following reviewers for their precious time and dedication, regardless of whether the papers they reviewed were finally published. we apologise for any names that have been inadvertently left out. these individuals provided their services to the journal as a reviewer from 01 october 2021 to 30 september 2022. moses adoga moses o. akiibinu hazim a. alhiti george asare ojor r. ayemoba emmanuel o. babafemi marinus barnard ebi c. bile naseem cassim macdonald m. chaava bettina chale-matsau modibo coulibaly anicet dahourou shewaneh damite tjeerd datema glenda m. davison alpha a. diallo mustapha dibbasey yakhya dieye titilope m. dokunmu peter drotman beverly egyir adetoun ejilemele okechukwu ekenna mathias emokpae kathleen england nicolas epie folorunso o. fasina foluke a. fasola tusabe fred lisa gerwing-adima verena gounden eshetu l. haile samson m. haumba rena hoffman fatih hunç prenika jaglal thumeka jalavu frantz jean louis feyisayo e. jegede sudhakar kalvatala celso khosa byung s. kim 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williamson sheila woodcock raj n. yadav annalise e. zemlin mashayed m. ziadi alban g.c. zohoun http://www.ajlmonline.org� https://ajlmonline.org mailto:submissions@ajlmonline.org mailto:submissions@ajlmonline.org https://ajlmonline.org https://ajlmonline.org https://ajlmonline.org/index.php/ajlm/user https://ajlmonline.org/index.php/ajlm/user mailto:publishing@aosis.co.za acknowledgement to reviewers acknowledgements references about the author(s) christoffel j. opperman green point tb laboratory, national health laboratory service, cape town, south africa sarishna singh green point tb laboratory, national health laboratory service, cape town, south africa francois barton green point tb laboratory, national health laboratory service, cape town, south africa citation opperman cj, singh s, barton f. appropriate disposal of waste in the laboratory: neglected but not forgotten. afr j lab med. 2022;11(1), a1786. https://doi.org/10.4102/ajlm.v11i1.1786 scientific letter appropriate disposal of waste in the laboratory: neglected but not forgotten christoffel j. opperman, sarishna singh, francois barton received: 12 nov. 2021; accepted: 04 apr. 2022; published: 14 july 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. laboratory waste management should focus on environmental and worker safety in a cost-effective manner to ensure ongoing diagnostic testing in accredited laboratories, especially in lowand middle-income countries with limited resources.1 for example, facilities focusing on mycobacterium tuberculosis generate biosafety level three infectious material that must be decontaminated and disposed of correctly to maintain good laboratory practice within a legislative framework.2 therefore, a holistic outline to support sustainable waste management in the laboratory is essential. this may include various components, such as waste disposal awareness campaigns, keeping abreast of technological advances,3 or implementing managerial policies. in this letter, we discuss practical suggestions for appropriately disposing of different waste types generated in most laboratories, with specific reference to a high-throughput, public, m. tuberculosis diagnostic laboratory. a technical brief published in 2020 by the global fund on sustainable healthcare management highlighted strategic waste management and best practice principles to limit hazardous infectious waste.4 their recommendations include: waste avoidance, reduction, and minimisation.4 in our laboratory setting, the disposal of hazardous biological material is not weight dependent. therefore, switching from single-use items to reusable equipment in low-risk laboratory areas that are not dedicated to processing or culturing and reducing ‘space-occupying’ objects, such as disposable laboratory coats (figure 1, number 1), effectively reduces waste and cost. digital platforms can limit paperwork and, thereby, paper waste. it is often noticed that forms and labels are discarded in biological waste containers within a busy laboratory. disposal of these and other reusable materials in bins designated for recyclables is not only a cost-saving initiative but should be a moral obligation on our journey to a ‘green’ and sustainable environment. implementing local guidelines with testing algorithms is essential to limit unnecessary investigations that generate extensive ‘routine diagnostic’ waste (figure 1, number 2). waste created by high sample rejection rates secondary to leaked specimens, insufficient volumes of poor quality, inappropriately submitted sample types, unlabelled containers, mismatched samples with the laboratory request forms, samples unsuitable due to contamination, et cetera, and should form part of quality control procedures. gatekeeping (reducing tests that can be avoided without negatively impacting patient management) and letting local healthcare facilities know the reason when they have a sample rejected from a laboratory can positively reinforce national guidelines and reduce sample rejection.5 figure 1: open-lid image from a biological infectious waste disposal container in a reference tuberculosis laboratory at the end of a specimen processing shift in cape town, south africa, 2022. 1, disposable laboratory coat; 2, appears to be a rejected specimen; 3, sterile cepheid xpert® mtb/rif ultra packaging material; 4, extensive plastic packaging from a tuberculosis specimen; 5, large amount of absorbable cleaning paper; 6, disposable latex gloves. laboratory waste should be classified according to the category that will dictate the waste management approach.6 laboratory products and kits containing nontoxic materials that can be discarded in general waste should be sought during the procurement process. for example, sterile cepheid xpert® mtb/rif ultra (solna, stockholm, sweden) packaging material (figure 1, number 3) does not require a biological infectious material container for discard. in addition, large amounts of packaging (figure 1, number 4) and large containers should be avoided during transportation from a local healthcare facility to the laboratory. such packaging creates a financial burden and consumes space in waste bins. although it is tempting to use recyclable products in the laboratory (plastics), laboratory professionals should be careful not to contaminate new products when using recycled instruments, as partial decontamination could cause erroneous results and impact patient management. unless being used to absorb spilled liquids, surface cleaning materials, such as paper towels, should be kept to a minimum (figure 1, number 5). care should be taken to maximise the use of personal protective equipment in the laboratory to preserve the supply chain, particularly during the coronavirus disease 2019 pandemic.7,8 to our knowledge, no guideline has been published on how many times gloves should be changed without obvious contamination between laboratory samples; discretion must be used in this regard (figure 1, number 6). a ‘just in time’ approach should be utilised when purchasing inventory. this could limit waste generated by reagents or diagnostic tests lost to expiration. however, we acknowledge that many african laboratories have constrained resource allocations and challenges maintaining sustainable budgets. this correspondence is not intended to be an all-inclusive guideline on the management of waste in the laboratory. instead, we looked critically into the waste bins to remind all laboratory workers about their responsibilities and opportunities for disposing of waste diligently and correctly. staff should be trained and updated regularly on correct waste disposal procedures. in addition, waste auditing systems should be implemented to gather robust data, guide planning, and assist laboratory managers with decision-making.4 auditing reviews on the amount, type, and laboratory area of waste generated ought to form the baseline for waste management initiatives. after all, correct waste disposal remains a ‘low-hanging fruit’ for saving money in every laboratory. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions c.j.o. conceptualised and wrote the original draft, then reviewed and edited the manuscript. s.s. and f.b. reviewed and edited the manuscript. ethical considerations this article does not contain any studies involving human participants performed by any of the authors. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability data sharing is not applicable to this article, as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect official policy or position of any affiliated agency of the authors. this article does not contain any studies involving human participants performed by any of the authors. references tuberculosis laboratory biosafety manual. in: essential biosafety measures for tb laboratories. geneva: world health organization; 2012. available from: https://www.ncbi.nlm.nih.gov/books/nbk179129/ johnson kr, braden cr, cairns kl, et al. transmission of mycobacterium tuberculosis from medical waste. jama. 2000;284(13):1683–1688. https://doi.org/10.1001/jama.284.13.1683 myneedu vp, aggarwal a. disposal of the large volume of sputum positive for mycobacterium tuberculosis by using microwave sterilisation technology as an alternative to traditional autoclaving in a tertiary respiratory care hospital in delhi, india. infect prev pract. 2020;2(3):100072. https://doi.org/10.1016/j.infpip.2020.100072 technical brief: sustainable health care waste management. geneva: the global fund; 2020. rooper l, carter j, hargrove j, hoffmann s, riedel s. targeting rejection: analysis of specimen acceptability and rejection, and framework for identifying interventions in a single tertiary healthcare facility. j clin lab anal. 2017;31(3):e22060. https://doi.org/10.1002/jcla.22060 ferronato n, torretta v. waste mismanagement in developing countries: a review of global issues. int j environ res public health. 2019;16(6):1060. https://doi.org/10.3390/ijerph16061060 yuan x, wang x, sarkar b, sik ok y. the covid-19 pandemic necessitates a shift to a plastic circular economy. nat rev earth environ. 2021;2:659–660. https://doi.org/10.1038/s43017-021-00223-2 bauchner h, fontanarosa pb, livingston eh. conserving supply of personal protective equipment-a call for ideas. jama. 2020;323(19):1911. https://doi.org/10.1001/jama.2020.4770 abstract introduction methods results discussion acknowledgements references about the author(s) robert k. langat kenya aids vaccine initiative, institute of clinical research, university of nairobi, nairobi, kenyainternational aids vaccine initiative (iavi), human immunology laboratory, imperial college, london, united kingdom bashir farah kenya aids vaccine initiative, institute of clinical research, university of nairobi, nairobi, kenya jackton indangasi kenya aids vaccine initiative, institute of clinical research, university of nairobi, nairobi, kenya simon ogola kenya aids vaccine initiative, institute of clinical research, university of nairobi, nairobi, kenya gloria omosa-manyonyi kenya aids vaccine initiative, institute of clinical research, university of nairobi, nairobi, kenya omu anzala kenya aids vaccine initiative, institute of clinical research, university of nairobi, nairobi, kenya jean bizimana projet san francisco, kigali, rwanda emmanuel tekirya projet san francisco, kigali, rwanda caroline ngetsa kenya medical research institute centre for geographical medicine research coast, mombasa, kenya moses silwamba zambia emory hiv research project, lusaka, zambia enoch muyanja ugandan virus research institute-iavi, entebbe, uganda paramesh chetty international aids vaccine initiative, johannesburg, south africa maureen jangano clinical laboratory services, johannesburg, south africa nancy hills school of medicine, university of california, san francisco, california, united states jill gilmour international aids vaccine initiative (iavi), human immunology laboratory, imperial college, london, united kingdom len dally emmes corporation, rockville, maryland, united states josephine h. cox clinical trials program, vaccine research center, national institutes of health, bethesda, maryland, united states peter hayes international aids vaccine initiative (iavi), human immunology laboratory, imperial college, london, united kingdom citation langat rk, farah b, indangasi j, et al. performance of international aids vaccine initiative african clinical research laboratories in standardised elispot and peripheral blood mononuclear cell processing in support of hiv vaccine clinical trials. afr j lab med. 2021;10(1), a1056. https://doi.org/10.4102/ajlm.v10i1.1056 note: additional supporting information may be found in the online version of this article as supplementary document 1. original research performance of international aids vaccine initiative african clinical research laboratories in standardised elispot and peripheral blood mononuclear cell processing in support of hiv vaccine clinical trials robert k. langat, bashir farah, jackton indangasi, simon ogola, gloria omosa-manyonyi, omu anzala, jean bizimana, emmanuel tekirya, caroline ngetsa, moses silwamba, enoch muyanja, paramesh chetty, maureen jangano, nancy hills, jill gilmour, len dally, josephine h. cox, peter hayes received: 06 june 2019; accepted: 23 sept. 2020; published: 17 feb. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: standardisation of procedures for performing cellular functional assays across laboratories participating in multicentre clinical trials is key for generating comparable and reliable data. objective: this article describes the performance of accredited laboratories in africa and europe on testing done in support of clinical trials. methods: for enzyme-linked immunospot assay (elispot) proficiency, characterised peripheral blood mononuclear cells (pbmcs) obtained from 48 hiv-negative blood donors in johannesburg, south africa, were sent to participating laboratories between february 2010 and february 2014. the pbmcs were tested for responses against cytomegalovirus, epstein barr and influenza peptide pools in a total of 1751 assays. in a separate study, a total of 1297 pbmc samples isolated from healthy hiv-negative participants in clinical trials of two prophylactic hiv vaccine candidates in kenya, uganda, rwanda and zambia were analysed for cell viability, cell yield and cell recovery from frozen pbmcs. results: most (99%) of the 1751 elispot proficiency assays had data within acceptable ranges with low responses to mock stimuli. no significant statistical difference were observed in elispot responses at the five laboratories actively conducting immunological analyses. of the 1297 clinical trial pbmcs processed, 94% had cell viability above 90% and 96% had cell yield above 0.7 million per ml of blood in freshly isolated cells. all parameters were within the predefined acceptance criteria. conclusion: we demonstrate that multiple laboratories can generate reliable, accurate and comparable data by using standardised procedures, having regular training, having regular equipment maintenance and using centrally sourced reagents. keywords: pbmc processing; peripheral blood mononuclear cells; elispot; clinical trials; good clinical laboratory practice; proficiency testing. introduction clinical trials related to hiv, malaria and tuberculosis have been conducted in africa for many years.1,2,3,4,5,6 to harmonise the immunological data generated from these trials, laboratories responsible for clinical sample processing must establish standardised procedures to meet international conference on harmonization good clinical laboratory practice (gclp) and the world health organization guidelines for collecting, processing, storing and performing of functional assays of samples such as enzyme-linked immunospot assay (elispot), which is used to assess the immunogenicity of vaccine candidates. the international aids vaccine initiative (iavi) has partnered with local institutions and established good clinical laboratory practice (gclp)-compliant laboratories across africa, europe and india to conduct safety and immunogenicity assessments in support of clinical trials of hiv vaccine candidates.7,8 these laboratories are equipped to process and store samples for later testing and can perform elispot and flow cytometry immunological assays. to-date iavi has conducted over 20 phase 1 hiv vaccine trials (https://www.iavi.org/our-work/clinical-epidemiology-research/clinical-research-centres), most of them in africa.9.10,11,12 to ensure uniformity of data, iavi sponsored a central laboratory – the iavi human immunology laboratory (hil) – based at imperial college london to provide standardised operating procedures, technical training, critical assay reagents and long-term storage of samples. additionally, iavi and partners have built the capacity of local personnel both professionally and academically, through technical training, mentoring and funding for investigator-initiated research projects within sites. the iavi gclp-accredited laboratories have been used as reference laboratories by local and international research organisations for training and storage of samples on a short-term basis. cellular functional assays have been used for assessing the immune response to many vaccines.13 in this article, we focus on the interferon-gamma (ifn-γ) elispot assay and report on the results from the elispot proficiency scheme. ifn-γ elispot is a standard assay for measuring the immunogenicity of vaccine candidates such as for hiv and tuberculosis13 and has been utilised by many research groups. although the elispot assay has been used in many clinical trials, it is prone to inherent variability within samples and between operators within and across laboratories.14 these discrepancies could be attributed to different reagents and equipment being used, inadequate training of personnel, lack of proficiency testing schemes and lack of quality management systems. therefore, there is an urgent need to standardise this assay to generate accurate and reliable data across multiple laboratories. previously, it was shown that multiple laboratories reported varied responses in elispot proficiency testing.15 there have been measures put in place to address this shortcoming and recent reports on elispot proficiency have shown incredibly improved results with concordant results between multiple gclp-accredited laboratories.16,17,18 the assessment of ifn-γ producing t-cells in vaccinees by elispot is a standard measure for determining a vaccinee’s immune response to hiv-1 vaccine candidates. in the field of human t-cell immunology, ifn-γ has been widely used as a measure of cd4 and cd8 activation after stimulation with various peptides and ifn-γ is easily detected by elispot. samples showing ifn-γ t-cell responses to test peptides are further assayed for other cytokine responses and immunological functions. it is worth noting that most of the laboratories performing end-point ifn-γ elispot either for proficiency testing or clinical trial schemes are based either in europe or the united states,16,19 which is disproportionate to where the burden of hiv/aids, infectious diseases and re-emerging ‘orphan’ tropical infectious diseases are predominant.20 there is now an increased focus on conducting vaccine trials where the pandemic is most severe; thus, networks of laboratories to support large clinical trials need to be developed in sub-saharan africa. on this front, iavi with its partners pioneered the gclp accreditation of laboratories in africa. for external quality assurance, these gclp-accredited laboratories were also enrolled in an elispot proficiency scheme. the gclp-accredited laboratories now conduct safety and immunogenicity assays such as elispot and flow cytometry for the assessment of hiv vaccine candidates and are fully equipped to collect, process and store samples for later testing. methods ethical considerations the study protocol was approved by the ethics committees of kenyatta national hospital, university of nairobi, kenya (reference numbers p81/3/2010 & p298/7/2011), the uganda virus research institute, entebbe, uganda (gc/127/10/08/31 & gc/127/11/09/12), projet san francisco (psf), kigali, rwanda (006/rnec/2011), zambia emory hiv research project, university of zambia (008-03-10), emory university (rec-270606-013) and south african national blood service (irb00041163) and was reviewed by the responsible regulatory authorities in each country. each participant provided written informed consent before undertaking any study procedures. at the south african national blood service, blood donors signed informed consent after the procedure was explained to them. blood collected from these donors was not labelled with donor names, but blood bag identifiers. the blood bag was further labelled at clinical laboratory services (cls) with a project number linked to the blood bag on the laboratory information management system. each participant in the hiv-1 clinical trial study provided written informed consent before blood samples were collected. the samples were de-identified and labelled with a study number. participating laboratories six of the seven participating laboratories were iavi-supported laboratories: (1) kenya aids vaccine initiative-institute of clinical research (kavi-icr) university of nairobi, nairobi, kenya, (2) iavi human immunology laboratory, imperial college london, united kingdom, (3) uganda virus research institute (uvri), entebbe, uganda, (4) kenya medical research institute centre for geographical medicine research coast (kemri-cgmrc), kilifi, kenya, (5) zambia emory hiv research project (zehrp), lusaka, zambia, and (6) projet san francisco (psf), kigali, rwanda. clinical laboratory services, witwatersrand university, johannesburg, south africa, is the only laboratory not supported by iavi but contracted to coordinate the sourcing, testing and shipping of peripheral blood mononuclear cells (pbmcs) for elispot proficiency testing. clinical laboratory services is accredited for both iso 15189 and gclp and followed the same operating procedures as the iavi-sponsored laboratories. clinical laboratory services also participated in elispot proficiency testing and is included in this report. laboratory preparation process of establishing clinical trial laboratories under good clinical laboratory practice guidelines comprehensive training programmes, calibrated and maintained equipment and quality control measures were integral in establishing iavi’s sponsored laboratories (table 1) and are described in detail in supplementary document 1. table 1: summary of the process of establishing a clinical trial laboratory under good clinical laboratory practice guidelines. to minimise potential failures in the iavi laboratories, quality control systems and operating procedures were put in place and corrective actions were instituted whenever a laboratory encountered a technical or assay failure to prevent future re-occurrence. two laboratories (hil and cls) were designated to provide laboratory support and quality control management as these two laboratories were based in ideal locations to support a global clinical trial programme. both locations are major international hubs in europe and africa, with direct flights to and from the iavi-supported laboratories, thereby reducing time and cost to transport samples as well as reduce damage risk to samples while in transit. elispot proficiency panel design international aids vaccine initiative gclp laboratories were enrolled in an ifn-γ elispot proficiency scheme coordinated by cls, south africa. clinical laboratory services sourced buffy coat blood pack samples from the south african national blood service, then isolated and cryopreserved pbmcs. frozen pbmc samples were thawed and assessed for cell viability, cell recovery and performance in elispot. samples with poor viable cell recovery or performance in elispot were excluded. pbmc samples with 50 to 100 vials were selected to provide laboratories with identical sets of pbmc samples (six pbmc samples per set) to allow testing of the same pbmc set over 6 to 12 months (that is 7 labs over 6 months would require 42 vials of each pbmc sample; 12 months would require 84 vials). frozen pbmc were maintained within a liquid nitrogen vapour phase freezer repository at cls until distribution to the laboratories participating in elispot proficiency. four laboratories – actively involved in clinical trials and performing cellular functional assays – conducted elispot monthly (kavi-icr, uvri, psf-kigali and hil), while three laboratories – not involved in clinical trials – conducted elispot quarterly (kemri-cgmrc, zehrp and cls). pbmcs were tested against two peptide pools; (1) 32 8–10-mer peptides representing an immunodominant cluster of differentiation 8+ (cd8+) t-cell epitopes from cytomegalovirus, epstein barr virus and influenza virus (anaspec inc., california, united states),21 and (2) 138 15-mer peptides overlapping by 11 amino acids spanning the human cytomegalovirus pp65 protein. these peptides were chosen because the majority of the population have pre-exposed immunity against these viruses and will have detectable t-cell response to these peptides. phytohaemagglutinin (pha-l; sigma l4144, sigma, poole, dorset, united kingdom) and dimethyl sulfoxide (diluted in culture medium) were used as positive and negative controls respectively. sample processing and elispot assay were performed according to the minimal information about t-cell assays guidelines.22 source of proficiency peripheral blood mononuclear cells and clinical trial samples peripheral blood mononuclear cells used in elispot proficiency testing were obtained from the buffy coat (south african national blood service, johannesburg, south africa) by ficoll-paque gradient centrifugation. blood donors were screened for hiv, hepatitis b, and syphilis. briefly, whole blood was diluted with sterile phosphate-buffered saline (pbs, sigma-aldrich. st. louis, missouri, united states) at a ratio of 1:1 and layered gently over 20 ml ficoll density gradient (ficoll-hypaque premium; ge healthcare, uppsala, sweden) at a ratio of 2:1 in a 50 ml falcon tube (greiner bio-one, stonehouse, united kingdom). the tubes were centrifuged at 750 × g for 25 min at room temperature with the brakes off. after centrifugation, the plasma component was aspirated using a sterile pasteur pipette (alpha laboratories, eastleigh, united kingdom) into a waste container containing bleach. the pbmc component was then removed into a new sterile 50 ml tube and washed two times with 20 ml pbs by centrifugation at 400 × g for 10 min with the brakes on. after washing, the cells were resuspended in 10 ml of roswell park memorial institute (rpmi) 1640 medium supplemented with 10% calf serum, 1 u/ml penicillin, 1 μg/ml streptomycin, and 300 µg/ml l-glutamine. peripheral blood mononuclear cells obtained from clinical trial participants was isolated from heparinised blood by density gradient centrifugation using histopaque 1077 (h8889, sigma). briefly, 20 ml of blood was layered onto 20 ml of histopaque in a 50 ml tube (falcon 357522, sarstedt, nümbrecht, germany) using a sterile serological pipette (falcon, sarstedt). the blood was then centrifuged at 400 × g for 40 min at room temperature with brakes off. after centrifugation, the upper plasma fraction was aspirated to within 1.5 to 2 cm of the pbmc band located at the interface between the yellow plasma fraction and the clear fluid below the pbmc band. the cells were washed in hank’s balanced salt solution without ca+ or mg+ with phenol red (sigma-aldrich) by centrifugation at 500 × g for 10 min at room temperature (1st wash) with brakes on. after centrifugation, the supernatant fluids were discarded into the waste container, the cells were resuspended in hank’s balanced salt solution and the tubes were centrifuged again at 400 × g for 10 min (second wash). after centrifugation, the supernatant fluids were discarded into the waste container and cells were resuspended in hank’s balanced salt solution and centrifuged at 400 × g cells for 10 min at room temperature with brakes on (third wash). after centrifugation, the supernatant fluids were discarded, and cells were resuspended in hank’s balanced salt solution for counting. in a separate study, pbmc from clinical trial samples were obtained from heparinised blood from a healthy hiv-negative placebo and vaccine recipients participating in clinical trials of two prophylactic hiv vaccine candidates at kavi-icr, uvri-iavi, psf and zehrp.10,11 these four laboratories participated in hiv clinical trials and the pbmc data were available for analysis. these pbmcs were processed as described above and had to meet the following predefined acceptance criteria: (1) processing of pbmc within 6 h from blood draw to cryopreservation, (2) cell viability above 90% and cell yield greater than 0.7 × 106 pbmc per ml of blood for freshly isolated pbmc, (3) cell viability above 80% for frozen, thawed and overnight rested pbmc. after isolation, cells were counted using a vi-cell xr automated cell counter (beckman coulter, united kingdom) and cryopreserved in 1 ml foetal calf serum containing 10% dimethyl sulfoxide in nalgene system 100tm cryogenic tubes (thermofisher scientific, new york, united states) at a final concentration of 10–15 ×106 pbmc per ml and transferred to a rate-controlled freezer (planer, sunbury-on-thames, united kingdom). this system cools the cells by a temperature decrease of 1°c per min from +4 °c to –80 °c followed by a 10 °c per min decrease until the temperature of the pbmcs was –120 °c after which the cells were transferred to vapour phase liquid nitrogen (ln) for long-term storage. pbmcs for elispot proficiency testing were cryopreserved in the same manner. shipment of proficiency peripheral blood mononuclear cells cryopreserved pbmcs were shipped to the participating laboratories as non-infectious human specimens –biological substances, category b and un337, packed in compliance with the international air transport association (iata) packing instruction 650. before shipping, cls notified the receiving laboratories of the pbmc proficiency panels’ shipping itinerary so that they can be ready to appropriately store the samples upon receipt. the proficiency pbmcs were shipped on a temperature-controlled dry shipper (mve jencons, united kingdom). dry shippers were calibrated for 7 days to ensure they are in good condition for shipment of pbmcs. briefly, on day 1, empty dry shippers were weighed and filled with ln to the brim and left overnight to adsorb. the next day, excess ln was decanted; the weight and temperature of the shipper were recorded. for the next 5 days and at the same time as the second day, the weight and temperature of the dry shipper were measured and recorded to determine the weight and temperature loss. for each dry shipper to pass calibration, its average weight and temperature loss in 24 h over the 5 days should be no more than 0.66kg (manufactureres specification is 0.6kg + 10%) and less than –190 °c. a day before shipment, the calibrated dry shippers were filled with ln and left overnight. the next day, the excess ln was decanted after which the weight and temperature of the shipper were recorded. the pbmcs were loaded onto a pre-cooled canister and placed into the shipper. the shippers were fitted with temperature loggers which were activated only after the samples were loaded to monitor the temperature of pbmcs while in transit. once the paperwork was completed the dry shippers were collected by the iata certified shippers (world courier). upon arrival at the laboratory, the loggers were removed and temperature data was downloaded and recorded. quality checks were then done for the pbmcs against the shipment manifest that accompanied the samples and samples cryopreserved in the ln tank until use. ifn-γ elispot assay setup of ifn-γ elispot assay: to minimise variations and allow investigation of any technical issue resulting from reagent use, although no such reagent issues were noted, the following measures were taken. all laboratories used the same batch of calf serum, which was pre-tested for performance in pbmc isolation, cryopreservation, cell recovery from frozen pbmc, and pha-l and cytomegalovirus acceptable response elispot assay. also, the catalogue number, lot number and expiry date of all reagents used in each assay batch were recorded, including elispot plates, which were obtained in batches of at least 50 plates. thawing and recovery of pbmcs: the cryopreserved pbmc panel was thawed and rested overnight in culture media. briefly, pbmcs were removed from the ln tank and transported to the laboratory in dry ice. they were immediately immersed in a 37 °c water bath and left until a small amount of ice of the freezing media remained. the cells were aspirated into a 10 ml rpmi medium supplemented with 10% calf serum, 1 mm l-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 1 mm sodium pyruvate and 0.5 mm hepes (all supplements were diluted in rpmi and purchased from sigma-aldrich). the tube was centrifuged at 250 × g for 10 min room temperature, after which the cell supernatant was discarded. the cells were resuspended in 4 ml rpmi medium supplemented with 20% calf serum and plated in two wells of a 24-well plate at a concentration of 2.5 × 106 pbmcs per ml. the inoculated plates were incubated overnight. the next day, cells were washed in culture media and counted. plate treatment: the 96-well filter plates (multiscreen hts ip msip4510; millipore, united kingdom) to be used for the assay were treated with ethanol. briefly, 50 µl of 70% ethanol was added to the wells and let to stand for 2 min. the wells were then washed three times with 200 µl sterile pbs. plate coating: after the ethanol treatment, each well was coated with 100 µl of 10 µg/ml pbs-diluted anti-human ifn-γ antibody (clone 1-d1k; 1 mg/ml; mabtech, sweden). the plates were then incubated at 4 oc overnight. plate blocking: the next day, plates were washed three times with 200 μl pbs and blocked with 200 μl rpmi medium supplemented with 10% calf serum per well at 37 °c for at least 2 h. after 2 h of incubation, the plates were removed and the medium was aspirated and discarded. addition of antigen and inoculation of cells: 100 µl of: (1) cell medium containing 2.25 µg/ml each of cytomegalovirus, epstein-barr virus and influenza virus (cef) and cytomegalovirus peptide was added to the reaction; (2) mock culture (dimethyl sulfoxide + culture medium) was added to the mock wells (medium negative control); (3) cell medium containing 15 µg/ml pha-l was added to the assay positive control wells and (4) cell medium containing 2.25 µg/ml cytomegalovirus peptide into the cell-free (peptide negative control) wells. pbmcs were added to all the wells except the cytomegalovirus cell-free control well at a density of 200 000 cells per 50 µl culture media. assay was done in quadruplicate. the plates were then moved to the incubator for 16 h – 24 h. addition of detection antibody: the next day, the plates were washed six times with 200 µl pbs supplemented with 0.05% tween (pbs/t) using an automated washer (bio-tek instruments inc., winooski, vermont, united states). detection was done by adding 100 μl 1 μg/ml pbs-diluted biotinylated anti-human ifn-γ antibody (clone 7-b6-1, 1 mg/ml) (mabtech, sweden) to the plates. the plates were then incubated at room temperature for 2 h. the plates were then washed six times as above after which 100 µl of peroxidase avidin-biotin complex (vector laboratories, burlingame, california, united states) was added to each well and incubated at room temperature for an hour. after incubation, plates were washed three times with 200 µl pbs/t followed by another triple wash with 200 µl pbs. spot development: 100 µl of 3-amino-9-ethyl carbazole substrate solution (vector laboratories, burlingame, california, united states) was added to the plates. the 3-amino-9-ethyl carbazole substrate solution was obtained by dissolving 3-amino-9-ethyl carbazole tablets in 2.5 ml dmf (vwr international, united states) after which the solution was diluted in 47 ml sterile tissue culture water supplemented with 280 µl 2m acetic acid, 180 µl 2m sodium acetate and 25 µl hydrogen peroxide (all from sigma-aldrich, st. louis, missouri, united states). the plates were then incubated at room temperature for 4 min. the reaction was stopped by running the plates over gentle-running tap water. the underdrain of the plates was removed, and plates left to dry overnight in the dark. imaging and analysis of elispot plates: all of the participating laboratories in this study used the same elispot reader system (autoimmun diagnostika, germany) with software version 4.0. it is a computer-based system for the semi-automatic interpretation of 96-well elispot plates. the system had the following settings for ifn-γ: well(s): a1 h12; count settings: ifn-γ; algorithm: v.3.2.x; intensity min: 15; size min: 72; and emphasis: small. this plate reader system is regularly maintained by use of a master lot plate supplied alongside the reader system, which contains artificial spots designed to test the performance of the reader system. as part of the internal quality control programme, each laboratory read this control plate once a week to assess the performance of the elispot reader using predefined spot parameters. the developed plates were imaged on the aid elispot reader system. the elispot data were expressed as the numbers of spot-forming cells (sfc) per million pbmc (supplementary document 1 figure 2). the acceptance criteria for each assay are: (1) the average sfc in the mock wells (peptide and pha free) must be less than 50 sfc per 106 pbmc; (2) the average sfc in the negative antigen control wells (cell free) must be less than 5 sfc per well; (3) the average sfc in the assay positive control wells (peptide free + pha) must be more than 50 sfc per 106 pbmc in the positive control pha. the plates were read and raw sfc data were submitted to hil for evaluation by a senior scientist and results shared on the access restricted cls website. statistical analyses this study aimed to compare the assay results obtained from the analysis of the same (frozen) pbmc samples provided to seven laboratories. we analysed the inter-lab and inter-operator variability and investigated cell recovery, viability and processing time. outcome measures include the recovery and viability rates of frozen pbmcs, and elispot counts for mock, cytomegalovirus and cef stimuli. for uniformity of data, cell recovery data after thawing and resting overnight from one clinical trial with pbmcs frozen at 15 million cells per ml/vial were normalised to 10 m cells per ml. for each sample, four replicate elispot plate wells per peptide pool were assayed and the arithmetic mean was used for analysis. results based on fewer than 4 replicate counts were assumed to be less accurate and excluded from the analysis. a total of 1751 assays were performed of which 50 were excluded (i.e. about 2.9%). for peptide pool repeated measures, the poisson regression model was fit to background-subtracted (except mock) elispot counts, with counts from the same volunteer assumed to be correlated. the resulting least-squares parameter estimates are presented together with their 95% confidence intervals adjusted for multiple comparisons using the bonferroni method. each model included volunteer, laboratory and month as covariates. pair-wise comparisons between laboratories are shown as the ratio of the least-squares estimates of the mean count with corresponding adjusted (bonferroni) 95% confidence intervals. statistical significance was defined as a 95% confidence interval for the ratio that excludes unity (i.e. entirely above or below the value 1). figures 1, 4 and 5, and supplementary document 1 figure 1 were generated by graph pad prism software version 8.3.0 (graphpad software, san diego, california, united states), while the rest of the figures and statistical analyses were performed using sas version 9.3 (sas institute, cary, north carolina, united states). results elispot testing performance across seven laboratories almost all (1733/1751; 99%) of the elispot proficiency assays performed had data within acceptable ranges with low responses to mock stimuli within the acceptance criteria of less than 50 sfc per million cells across the seven laboratories over time (figure 1). a small fraction of these (18/1751; 0.01%) pbmcs had mock responses over 50 sfc per million cells; however, on average replicates per donor, all mock responses were below 50 sfc per million cells in all the panels. all the five laboratories actively conducting immunological analyses in support of iavi-sponsored clinical trials performed similarly in elispot testing with comparable data in elispot counts against cytomegalovirus and cef peptides (figure 1). the elispot counts were background-subtracted (except mock). the covariates in the model were sample, laboratory and month. across the seven laboratories, the geometric mean elispot counts (sfc per 106 pbmc) for mock stimuli were 6–10 (supplementary document 1 table 1), 289–438 for cef stimuli (supplementary document 1 table 2) and 172–266 for cytomegalovirus (supplementary document 1 table 3). we observed significant differences in mock counts across the laboratories with cls mock counts estimated to be 1.73 times higher than zehrp and at psf which were 0.78 times lower than uvri. zambia emory hiv research project tends to have lower mock counts than all other laboratories (supplementary document 1 table 1; p < 0.001). figure 1: distribution of elispot responses across laboratories in kenya, uganda, rwanda, zambia, south africa and the united kingdom, 2010–2014. responses against mock, cytomegalovirus and cytomegalovirus, epstein-barr virus, and influenza virus stimuli from a panel of six peripheral blood mononuclear cells tested over 6 months, (a) first 24 months and (b) second 24 months. box plots represent the quartiles, horizontal line the median and whiskers the maximum and minimum values. each point represents average spot-forming cells per 106 peripheral blood mononuclear cells from replicates per donor at each laboratory. the laboratories are color-coded as follows: kenya aids vaccine initiative-institute of clinical research (red); uganda virus research institute (blue); projet san francisco (green); zambia emory hiv research project (purple); kenya medical research institute centre for geographical medicine research coast (yellow); clinical laboratory services (cyan); human immunology laboratory (black). when we compared the elispot responses against cef peptide pools across laboratories, kemri-cgmrc had significantly higher counts than other laboratories (p = 0.004, figure 2 and supplementary document 1 table 2). when data from kemri-cgmrc are excluded, the overall difference between laboratories is not statistically significant (p = 0.11, supplementary document 1 table 2). the same trend was seen in elispot responses against the cytomegalovirus stimulus where kemri-cgmrc again had significantly higher counts than other laboratories (p = 0.012, figure 2 and supplementary document 1 table 3). even after excluding data from kemri-cgmrc, the overall difference between laboratories is still statistically significant because of lower counts in zehrp than in cls and kavi-icr (p = 0.033, supplementary document 1 table 3). figure 2: comparison of elispot responses across laboratories in kenya, uganda, rwanda, zambia, south africa and the united kingdom, 2010–2014. the graphs show which site pairs are significantly different (blue lines) and which are not (red lines). for each comparison, a line segment, centred at the least-squares-means in the pair, is drawn. the segment length corresponds to the projected width of a confidence interval for the least-squares mean difference. each line corresponds to the pair of labs with reference lines that cross at the midpoint. shown here are the pair-wise least-squares means and their statistical significance, on a natural log scale, for mock, cytomegalovirus, epstein-barr virus, and influenza virus and cytomegalovirus stimuli. differences for alpha = 0.05 (bonferroni adjustment); red line denotes not significant while blue line denotes significant. (a) mock, (b) cytomegalovirus, epstein-barr virus, and influenza virus and (c) cytomegalovirus. inter-operator analysis the performance of three operators from kavi-icr in elispot testing during the study period was analysed. pbmcs from 12 volunteers were analysed by the three operators on a rotational basis with each operator conducting the same set of samples at monthly time points. elispot counts were obtained for mock and background-subtracted cytomegalovirus and cef responses and the data analysed using the repeated measures poisson regression model and comparison-adjusted against the sample, operator and month. the geometric mean elispot counts for mock were 9–12, 368–393 for cef and 538–598 for cytomegalovirus stimuli (supplementary document 1 table 4). we found no significant difference in elispot performance between the three operators (figure 3 and supplementary document 1 table 4). figure 3: comparison of inter-operator elispot responses from three operators at kenya aids vaccine initiative-institute of clinical research, kenya, 2010–2014. the graphs show which operators are significantly different (blue lines) and which are not (red lines). shown here are the pair-wise least-squares means and their statistical significance, on a natural log scale, for mock, cytomegalovirus, epstein-barr virus, and influenza virus and cytomegalovirus stimuli. for each comparison, a line segment, centred at the least-squares means in the pair, is drawn. the length of the segment corresponds to the projected width of a confidence interval for the least-squares mean difference. segments that fail to cross the 45° reference line correspond to significant least-squares mean differences. none of the pairs of operators is significantly different (all lines cross the 45° reference line). differences for alpha = 0.05 (bonferroni adjustment); red line denotes not significant while blue line denotes significant. (a) mock, (b) cytomegalovirus, epstein-barr virus, and influenza virus and (c) cytomegalovirus. performance of four laboratories in pbmc processing a total of 1297 pbmcs isolated from clinical trial samples at the four aforementioned laboratories supporting two iavi-sponsored hiv clinical trials were analysed for cell viability, recovery and cell yield per ml of blood. of the 1297 pbmcs processed, 1220 (94%) freshly isolated pbmcs had viability above 90% with a median of 95% (range 81% – 100%) and those with viability below 90% had a median of 88% (range 81% – 90%) (figure 4a). over 96% (1249/1297) of these samples had cell yields greater than 0.7 × 106 pbmc per ml blood, all within the predefined acceptability criteria (figure 4b). there were a few samples that had low cell yield ranging from 0.13 to 0.56 × 106 pbmc per ml blood (figure 4b). a fraction of these samples (1205/1297; 93%) including those with cell yield below 0.7 × 106 pbmc per ml blood were thawed and tested for elispot performance and almost all (1196/1205; 99%) pbmcs had viability above 80% following thaw and overnight rest (within acceptability criteria) and only 9 pbmcs were below 80% (range 66% – 78%) (figure 4c). the cell recovery for these samples was above 6.0 × 106 pbmc per vial (data were normalised to 10 m cells (figure 4d); samples from the clinical trials analysed in this study were frozen at 10 m cells per ml except one trial where samples were frozen at 15 m cells per ml per vial at which the data were normalised to 10 m cells per ml for uniformity of data. in elispot testing, all the samples were functional with over 95% having mock responses under 50 sfc per 106 pbmc, pha over 1000 sfc per 106 pbmc and a range of cytomegalovirus responses. figure 4: performance of laboratories in peripheral blood mononuclear cell (pbmc) processing, kenya, uganda, rwanda, zambia, 2010–2014. (a) the percentage cell viability of freshly isolated pbmc, (b) cell yield per ml of blood, (c) percentage cell viability from frozen pbmcs, (d) cell recovery of frozen pbmc (pbmcs were cryopreserved at a final concentration of 10–15 m cells per ml; here data were normalised to 10 m cells), and (e) duration of pbmc processing (in hours). each dot represents a sample and the horizontal line represents the median with interquartile range. the long horizontal line shows the acceptance cut-off. the duration of pbmc processing from blood draw to cryopreservation has been shown to affect the integrity of cells.23,24,25 in this study, we analysed the processing time of pbmc (in hours) and report that nearly all the samples were processed within 6 h with only 6% (81/1297) processed beyond 6 h (range 6.1–9.5 h) (figure 4e and supplementary document 1 figure 1). to assess the integrity of samples processed past 6 h, we analysed the pbmc viability and cell yield from freshly isolated pbmcs and later analysed the viability and cell recovery from thawed frozen pbmcs. we found the delays did not affect the pbmcs’ integrity in all samples; one had cell viability above 90% and a cell yield greater than 0.7 × 106 pbmc per ml blood (both parameters within acceptable range) (supplementary document 1 figure 1). only one sample had a slightly lower cell yield of 0.57 × 106 per ml blood and cell viability of 98%. post pbmc freezing cell viability ranged from 93% to 100% and cell recovery was above 6 × 106 pbmc per vial in 71 of 81 (87%) samples (supplementary document 1 figure 1). we further tested these samples in an elispot assay to assess their cell functionality and all samples performed well with the mock responses under 50 sfc per 106 pbmc, pha responses greater than 1000 sfc per 106 pbmc and a typical range of cytomegalovirus responses. these data are similar to what was seen from samples processed within 6 h (supplementary document 1 figure 1). discussion we compared data generated in multiple laboratories over time to assess their performance in the processing of pbmc samples and elispot testing. some of these laboratories supported clinical trials of hiv prophylactic vaccine candidates and to standardise their cell functionality assays and harmonise procedures to achieve reliable and accurate data, they were enrolled in an elispot proficiency scheme as part of external quality assurance. all laboratories performed well in elispot proficiency testing with data comparable across laboratories; however, there were a few sporadic outliers in the data. these outliers were expected considering the large number of data points analysed: sporadic outliers would be expected even from experienced and competent laboratories. in this study, we saw comparable elispot data from five out of seven laboratories. these five laboratories all supported clinical trials except one: cls which performed elispot regularly. since cls was the laboratory responsible for sourcing and qualifying the pbmcs for the elispot proficiency scheme, they were expected to perform well in elispot testing. in the two laboratories that did not support clinical trials and routine elispot testing, we saw significant differences in elispot proficiency data compared to other laboratories. it is worth mentioning that these laboratories performed elispot testing quarterly and would be less experienced compared to other laboratories. to identify the root cause of this data discrepancy, corrective action was initiated at these two laboratories. the staff were retrained and assessed for competency. also, improvement measures were put in place such as continuous training and monitoring of their performance in elispot testing in subsequent rounds of proficiency testing panels. as a requirement of the gclp programme, at any given time, there should be more than one person processing the clinical trial samples to ensure accuracy and reliability of data. likewise, in elispot proficiency testing, different operators at each laboratory fully trained in the required procedures conducted elispot proficiency assays on a rotational basis as determined by laboratory management. the influence of the operator on the variability of results is a known factor as shown by janetzki and colleagues;14 here we analysed the performance of three operators in elispot proficiency testing in one laboratory which maintained the same staff throughout the study period. it was not possible to analyse inter-operator variability in all laboratories as many sites experienced frequent staff turnover during the study period. from the analysis, there was no significant difference in data generated by the three operators over the study period. this demonstrates that with regular training and competency assessment, reliable, accurate and comparable data can be obtained notwithstanding the inherent operator variability. sample integrity is critical for achieving accurate and reliable results in clinical trials. in a multicentre trial, processes for sample processing need to be harmonised to generate comparable data. we assessed the processing of pbmcs in four of seven laboratories, focusing on five areas: sample collection, isolation of pbmc, cryopreservation, thawing of frozen pbmc, and performance in elispot testing of clinical trial samples. first, all laboratories were required to process samples to cryopreservation within 6 h of a blood draw. this is to ensure that pbmcs obtained are of good quality as it has been documented by olson and colleagues that a delay in the processing of pbmc of more than 8 h may reduce cell viability and compromise cell functionality.26 we found most samples were processed within 6 h with few samples outside of this time frame. the few samples were processed outside this time frame due to reasons such as a delay in delivery to the laboratory, as some clinics were some distance from the laboratory, batching of sample processing and a backlog of samples. to mitigate these, corrective and preventative actions were initiated to prevent such recurrence which included prioritising the delivery of samples from the clinics, processing samples promptly as they arrive in the laboratory and cryopreserving pbmc immediately after processing to reduce any backlog that may have arisen from batching of cryopreservation. these measures initiated were monitored monthly. although some samples were processed beyond the expected time frame, their integrity was not compromised as cell viability, cell yield and recovery after thawing were indistinguishable from other samples processed within the stipulated time. additionally, these samples were tested for functionality in downstream assays such as elispot and they performed well, again with indistinguishable results compared with samples processed within the stipulated time. in conclusion, we have shown that samples processed beyond 6 h and up to 9 h from blood draw to cryopreservation are still viable and functional, similar to findings from other groups.24,25 in assessments of pbmc isolation, our focus was on cell viability and cell yield. we found that the majority of pbmc samples isolated in all four laboratories supporting clinical trials met the predefined acceptance criteria for viability and cell yield with few outliers seen across the laboratories. in most cases, samples obtained in a multicentre clinical trial are shipped to a central laboratory either for long-term storage or cellular functionality testing. therefore, proper cryopreservation after pbmc isolation and shipping is critical in preserving cell integrity and functionality.27 clinical trial samples isolated at four laboratories were cryopreserved at the local freezing repository until shipped to a central laboratory. upon request, the samples were shipped to the central laboratory according to the iata guidelines for long-term storage. the majority of thawed samples were viable with cell viability and recovery within the acceptable limits and thus demonstrated the competency of the laboratories in cryopreservation and shipping of samples. additionally, thawed samples performed well in elispot testing with responses within the expected ranges. to maintain high standards and produce reliable and comparable data, these laboratories were audited regularly for gclp compliance either by internal or external independent auditors. the audit covers areas such as operating procedure development and documentation, elispot and flow cytometry testing proficiency schemes and the data management system. all laboratories were audited annually by an external auditor for gclp accreditation. limitations the main limitation of this study was the variation in the operators who performed elispot testing at the laboratories. there was a frequent turnover of personnel in most of the laboratories during the study period which made it difficult to assess the inter-operator variability within sites. to assess the operator effect on data variability, data were analysed from only one laboratory which had three operators who consistently performed elispot testing over the 4 years on a rotational basis. though we saw no significant difference in data generated between these three operators, this result does not entirely represent what could be seen across all seven laboratories. conclusion in conclusion, we have demonstrated the capabilities of multiple laboratories in africa and europe in processing clinical trial samples to high standards and performing cell functionality assays. furthermore, we have shown that multiple laboratories can generate reliable, accurate and comparable data by using standardised procedures, having regular training, regular equipment maintenance and using centrally sourced reagents. these efforts and the elispot proficiency programme continue across the network of iavi-sponsored laboratories supporting clinical trials. therefore, we highly recommend the approach taken by the iavi gclp-accredited laboratories to produce such data to any donor, sponsor or research institution who may plan to conduct clinical trials in the region. acknowledgements we wish to acknowledge the support from the university of california, san francisco’s international traineeships in aids prevention studies, us nimh r25 mh064712, under which this manuscript was written. we thank university of california san francisco faculty staff for helpful discussions, matt price and kathy crisafi of iavi for manuscript review and laboratory technicians for experimental assistance. we also thank all iavi clinical research centres’ principal investigators for overseeing the laboratories. the contents of this manuscript are the responsibility of the authors and do not necessarily reflect the views of united states agency for international development or the united states government. competing interests the authors have declared that no competing interests exist. authors’ contributions r.k.l. performed the experiments and wrote the manuscript. j.i., s.o., e.t., c.n., m.s. and e.m., performed the laboratory experiments. m.j. isolated and qualified the proficiency pbmcs used in this study and also performed the laboratory experiments. n.h. and l.d. performed the statistical analysis. b.f., j.b., o.a., p.c., g.o.-m. and j.g. designed the study. j.h.c. and p.h. designed the experiments, interpreted results and critically reviewed the manuscript. sources of support this work was funded in part by iavi and made possible by the support of the united states agency for international development and other donors. the full list of iavi donors is available at www.iavi.org. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references gilmour jw, stevens ws, gray c, de souza m. laboratory expansion to large-scale international hiv preventive vaccine trials. curr opin hiv aids. 2007;2(3):201–206. https://doi.org/10.1097/coh.0b013e3280eec77a kublin jg, morgan ca, day ta, et al. hiv vaccine trials network: activities and achievements of the first decade and beyond. clin investig. 2012;2(3):245–254. https://doi.org/10.4155/cli.12.8 muyanja e, ssemaganda a, ngauv p, et al. immune activation alters cellular and humoral responses to yellow fever 17d vaccine. j clin investig. 2014;124(7):3147–3158. https://doi.org/10.1172/jci75429 schmidt c, jaoko w, omosa-manyonyi g, et al. long-term follow-up of study participants from prophylactic hiv vaccine clinical trials in africa. hum vaccines immunother. 2014;10(3):714–723. https://doi.org/10.4161/hv.27559 mwangoka g, ogutu b, msambichaka b, et al. experience and challenges from clinical trials with malaria vaccines in africa. malar j. 2013;12(1):86. https://doi.org/10.1186/1475-2875-12-86 kent dm, mwamburi dm, bennish ml, kupelnick b, ioannidis jp. clinical trials in sub-saharan africa and established standards of care: a systematic review of hiv, tuberculosis, and malaria trials. jama. 2004;292(2):237–242. stiles t, grant v. good clinical laboratory practice (gclp): an international quality system for laboratories which undertake the analysis of samples from clinical trials. ipswich: research quality association (rqa); 2012. sarzotti-kelsoe m, cox j, cleland n, et al. evaluation and recommendations on good clinical laboratory practice guidelines for phase i–iii clinical trials. plos med. 2009;6(5):e1000067. https://doi.org/10.1371/journal.pmed.1000067 baden lr, karita e, mutua g, et al. assessment of the safety and immunogenicity of 2 novel vaccine platforms for hiv-1 prevention: a randomized trial. ann intern med. 2016;164(5):313–322. omosa-manyonyi g, mpendo j, ruzagira e, et al. a phase i double blind, placebo-controlled, randomized study of the safety and immunogenicity of an adjuvanted hiv-1 gag-pol-nef fusion protein and adenovirus 35 gag-rt-int-nef vaccine in healthy hiv-uninfected african adults. plos one. 2015;10(5):e0125954. https://doi.org/10.1371/journal.pone.0125954 mpendo j, mutua g, nyombayire j, et al. a phase i double blind, placebo-controlled, randomized study of the safety and immunogenicity of electroporated hiv dna with or without interleukin 12 in prime-boost combinations with an ad35 hiv vaccine in healthy hiv-seronegative african adults. plos one. 2015;10(8):e0134287. https://doi.org/10.1371/journal.pone.0134287 hanke t, mutua g, farah b, et al. broad hiv-1 inhibition in vitro by vaccine-elicited cd8(+) t cells in african adults. mol ther methods clin dev. 2016;3:16061. https://doi.org/10.1038/mtm.2016.61 klausner rd, fauci as, corey l, et al. medicine. the need for a global hiv vaccine enterprise. science (new york, ny). 2003;300(5628):2036–2039. https://doi.org/10.1126/science.1086916 janetzki s, schaed s, blachere ne, ben-porat l, houghton an, panageas ks. evaluation of elispot assays: influence of method and operator on variability of results. j immunol methods. 2004;291(1–2):175–183. https://doi.org/10.1016/j.jim.2004.06.008 cox jh, ferrari g, kalams sa, lopaczynski w, oden n, d’souza mp. results of an elispot proficiency panel conducted in 11 laboratories participating in international human immunodeficiency virus type 1 vaccine trials. aids res hum retroviruses. 2005;21(1):68–81. https://doi.org/10.1089/aid.2005.21.68 sanchez am, rountree w, berrong m, et al. the external quality assurance oversight laboratory (eqapol) proficiency program for ifn-gamma enzyme-linked immunospot (ifn-γ elispot) assay. j immunol methods. 2014;409:31–43. https://doi.org/10.1016/j.jim.2014.03.017 gill dk, huang y, levine gl, et al. equivalence of elispot assays demonstrated between major hiv network laboratories. plos one. 2010;5(12):e14330. https://doi.org/10.1371/journal.pone.0014330 boaz mj, hayes p, tarragona t, et al. concordant proficiency in measurement of t-cell immunity in human immunodeficiency virus vaccine clinical trials by peripheral blood mononuclear cell and enzyme-linked immunospot assays in laboratories from three continents. clin vaccine immunol. 2009;16(2):147–155. https://doi.org/10.1128/cvi.00326-08 rountree w, vandergrift n, bainbridge j, sanchez am, denny tn. statistical methods for the assessment of eqapol proficiency testing: elispot, luminex, and flow cytometry. j immunol methods. 2014;409:72–81. https://doi.org/10.1016/j.jim.2014.01.007 liese bh, houghton n, teplitskaya l. development assistance for neglected tropical diseases: progress since 2009. int health. 2014;6(3):162–171. https://doi.org/10.1093/inthealth/ihu052 currier jr, kuta eg, turk e, et al. a panel of mhc class i restricted viral peptides for use as a quality control for vaccine trial elispot assays. j immunol methods. 2002;260(1–2):157–172. https://doi.org/10.1016/s0022-1759(01)00535-x britten cm, janetzki s, butterfield lh, et al. t cell assays and miata: the essential minimum for maximum impact. immunity. 2012;37(1):1–2. https://doi.org/10.1016/j.immuni.2012.07.010 mallone r, mannering si, brooks-worrell bm, et al. isolation and preservation of peripheral blood mononuclear cells for analysis of islet antigen-reactive t cell responses: position statement of the t-cell workshop committee of the immunology of diabetes society. clin exp immunol. 2011;163(1):33–49. https://doi.org/10.1111/j.1365-2249.2010.04272.x kierstead ls, dubey s, meyer b, et al. enhanced rates and magnitude of immune responses detected against an hiv vaccine: effect of using an optimized process for isolating pbmc. aids res hum retroviruses. 2007;23(1):86–92. https://doi.org/10.1089/aid.2006.0129 bull m, lee d, stucky j, et al. defining blood processing parameters for optimal detection of cryopreserved antigen-specific responses for hiv vaccine trials. j immunol methods. 2007;322(1–2):57–69. https://doi.org/10.1016/j.jim.2007.02.003 olson wc, smolkin me, farris em, et al. shipping blood to a central laboratory in multicenter clinical trials: effect of ambient temperature on specimen temperature, and effects of temperature on mononuclear cell yield, viability and immunologic function. j transl med. 2011;9:26. https://doi.org/10.1186/1479-5876-9-26 smith jg, joseph hr, green t, et al. establishing acceptance criteria for cell-mediated-immunity assays using frozen peripheral blood mononuclear cells stored under optimal and suboptimal conditions. clin vaccine immunol. 2007;14(5):527–537. https://doi.org/10.1128/cvi.00435-06 abstract introduction methods results discussion acknowledgements references about the author(s) katherine klein centers for disease control and prevention, national center for hiv/aids, viral hepatitis, std and tb prevention, division of tb elimination, atlanta, georgia, united states kyle degruy centers for disease control and prevention, division of global hiv and tb, international laboratory branch, atlanta, georgia, united states zilma rey centers for disease control and prevention, division of global hiv and tb, international laboratory branch, atlanta, georgia, united states patricia hall centers for disease control and prevention, division of global hiv and tb, international laboratory branch, atlanta, georgia, united states andrea kim centers for disease control and prevention, division of global hiv and tb, epidemiology and surveillance branch, atlanta, georgia, united states steve gutreuter centers for disease control and prevention, division of global hiv and tb, health informatics, data management and statistics branch, atlanta, georgia, united states heather alexander centers for disease control and prevention, division of global hiv and tb, international laboratory branch, atlanta, georgia, united states citation klein k, degruy k, rey z, et al. a global proficiency testing programme for xpert® mtb/rif using dried tube specimens, 2013–2015. afr j lab med. 2020;9(1), a1167. https://doi.org/10.4102/ajlm.v9i1.1167 original research a global proficiency testing programme for xpert® mtb/rif using dried tube specimens, 2013–2015 katherine klein, kyle degruy, zilma rey, patricia hall, andrea kim, steve gutreuter, heather alexander received: 14 jan. 2020; accepted: 12 aug. 2020; published: 27 nov. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: proficiency testing (pt) is an important quality assurance measure toward ensuring accurate and reliable diagnostic test results from clinical and public health laboratories. despite the rapid expansion of the xpert® mtb/rif assay for the detection of tuberculosis in resource-limited settings (rls), low-cost pt materials for xpert mtb/rif external quality assessment (eqa) are not widely available. objective: we sought to determine whether a dried tube specimen (dts)-based pt programme would be a feasible option to support xpert mtb/rif eqa in rls. methods: between 2013 and 2015, the united states centers for disease control and prevention developed and conducted a voluntary eqa programme using dts-based pt material. eight rounds of pt, each comprising five dts samples, were provided to enrolled testing sites. after each round, participant results were compared to expected results, scored as satisfactory or unsatisfactory, and sites were provided with performance reports. results: programme enrolment increased from 102 testing sites in seven countries to 441 testing sites in 14 countries over the course of three years. in each pt round, approximately 90% of participating sites demonstrated satisfactory performance. in seven of the 14 enrolled countries, the proportion of sites with a satisfactory score increased between the first round of participation and the most recent round of participation. conclusion: this programme demonstrated that it is possible to implement an xpert mtb/rif pt programme for rls using dts, that substantial demand for xpert mtb/rif pt material exists in rls, and that country performance can improve in a dts-based pt programme. keywords: external quality assessment; xpert® mtb/rif; proficiency testing; dried tube specimens; resource-limited settings. introduction the world health organization recommends the xpert® mtb/rif (mycobacterium tuberculosis/rifampicin) assay (cepheid, sunnyvale, california, united states) as one of the initial diagnostic tests for all individuals evaluated for tuberculosis.1,2 the xpert mtb/rif assay is an integrated sputum processing and real-time polymerase chain reaction system that rapidly and simultaneously detects m. tuberculosis and the presence of mutations conferring resistance to rifampicin.3 the roll-out of the xpert mtb/rif assay to resource-limited settings (rls) with a high tuberculosis burden has been remarkable. by the end of 2016, 6659 genexpert instruments had been purchased at concessional prices for use at multiple levels of the tiered laboratory network.4 external quality assessment is a critical component of a laboratory quality management system. proficiency testing (pt) is one means of conducting external quality assessment for tests in clinical laboratories. proficiency testing provides objective evidence of accurate testing and may identify areas in the diagnostic process, including pre-analytic, analytic, and post-analytic phases, where quality improvement is needed.5,6 the rapid scale-up of xpert mtb/rif testing in rls has not coincided with the same investment in quality assurance activities for the assay. few pt providers for the xpert mtb/rif assay offer materials to sites in rls, leaving a substantial gap in external quality assessment implementation that could otherwise help to confirm site competency in assay administration and accuracy of test results for limited-resource countries.7,8,9 the united states centers for disease control and prevention (cdc) developed a pt panel for xpert mtb/rif, using a dried tube specimen (dts)-based method.10 the dts technique has successfully been used to develop pt panels for hiv, malaria and syphilis diagnostic assays in rls.11,12,13 our goal in this evaluation was to assess the performance and utility of cdc-prepared dts panels in an xpert mtb/rif pt programme. methods ethical considerations no patient specimen collection was required and no patient identifiers were recorded on quality assurance tools. participation in the xpert mtb/rif quality assurance and pt programme was voluntary and free of charge. no incentives were provided. this study was approved by the cdc center for global health division of human research protection (cgh hsr tracking number 2014-082). dried tube specimen production the method for producing dts was described previously by degruy et al.10 briefly, dts were produced by chemically inactivating liquid cultures of well-characterised mycobacterial strains using equipment and supplies commonly found in laboratories conducting tuberculosis liquid culture.14 mycobacteria were grown in bactec® mgit 7 ml tubes (becton, dickinson and company, sparks, maryland, united states) and inactivated using a 2:1 ratio of xpert mtb/rif sample reagent (cepheid, sunnyvale, california, united states) to mgit culture incubated for 2 hours with intermittent vortexing. inactivated cultures were washed, concentrated, resuspended, homogenised and diluted, before being dispensed into cryovials and air-dried in a class ii biological safety cabinet within a biosafety level iii tuberculosis containment laboratory. inactivation verification was performed by inoculating 0.5 ml of undiluted stock solution into mgit tubes supplemented with polymyxin b, amphotericin b, nalidixic acid, trimethoprim, and azlocillin (panta) (becton, dickinson and company, sparks, maryland, united states) and incubating for a total of 84 days over two incubation cycles in the bactec® mgit 960® instrument (becton, dickinson and company, sparks, maryland, united states) to confirm a lack of growth. dried tube specimens were validated prior to distribution by testing 10% of dts produced with the xpert mtb/rif assay (cepheid, sunnyvale, california, united states), as described previously.10 to test dts, the dts were rehydrated with 2.5 ml of sample reagent, shaken vigorously 20 times and incubated at room temperature for 15 minutes with additional shaking repeated after 10 min. the xpert mtb/rif cartridges were inoculated with approximately 2.0 ml of rehydrated samples using the transfer pipette provided with the kit, and tested immediately on a genexpert iv or genexpert viii using genexpert dx software version 4.0 (cepheid, sunnyvale, california, united states) according to manufacturer instructions.15 proficiency testing programme in 2013, select countries in sub-saharan africa, southeast asia, and the caribbean, supported by the united states president’s emergency plan for aids relief were invited to participate in cdc-atlanta’s pilot dts xpert mtb/rif pt programme at no cost. seven countries elected to participate in the pilot, designated a pt country coordinator, and enrolled testing sites that routinely conducted xpert mtb/rif testing on patient specimens. these countries continued to enrol additional sites throughout 2013, and four additional countries enrolled in subsequent 2013 pt rounds. all countries remained enrolled throughout 2014. in 2015, enrolment was extended to additional sites in all participating countries plus three new countries in africa. the pt kits included a panel of five dts samples, five transfer pipettes, testing instructions, and a paper form for recording results. four pt rounds were distributed quarterly in 2013 (2013-a, 2013-b, 2013-c and 2013-d). only one pt round was distributed at the end of 2014 (2014-a) to allow for evaluation of 2013 data. three pt rounds were distributed in 2015 (2015-a, 2015-b and 2015-c). country coordinators were responsible for distributing the panels to enrolled testing sites, collating results (including transferring data from paper forms to a template microsoft excel file (microsoft corp, redmond, washington, united states) and submitting data to cdc-atlanta via email within nine weeks of panel receipt. a sufficient number of pt kits were sent to enrolled countries to allow for distribution of one pt kit to each enrolled site; enrolled sites that did not submit results were categorised as enrolled but not participating in a given pt round. participant data were analysed by cdc-atlanta using microsoft excel. a report was generated for each participating site; this compared the site’s reported results to expected results (cdc-atlanta validation results) and included the consensus results from all the participating testing sites in all the ountries. consensus results detailed the number of testing sites detecting and not detecting m. tuberculosis complex, detecting and not detecting rifampicin resistance, reporting indeterminate rifampicin resistance, reporting uninterpretable tests (error, invalid, or no result), and not reporting m. tuberculosis complex and/or rifampicin results for each sample in the panel. to establish a consensus result for each pt sample in the round, we required at least 80% of participant results for that sample to match the expected result. in the 2013 pilot phase, participating sites were provided with qualitative reports indicating overall concordance of submitted results with the expected results. beginning in 2014, participating sites were assigned a quantitative score for each pt round. scores were calculated by assigning a value of 20 points to each of the five dts, for a total of 100 possible points. if the site’s qualitative m. tuberculosis complex and rifampicin resistance detection results matched the expected results for a given sample, 20 points were awarded. if either the m. tuberculosis complex detection result or the rifampicin resistance detection result did not match the expected results, zero points were awarded. if the m. tuberculosis complex detection result matched the expected result but the site’s rifampicin resistance detection result was indeterminate, 10 points were awarded. if a site reported an uninterpretable test, five points were awarded. if a site did not report a result, zero points were awarded. a total score of greater than, or equal to, 80 points was considered satisfactory for the pt round. for this evaluation, 2013 results were retrospectively scored to compare performance across all years of the programme. to further investigate concordance and discordance of site results with expected results, additional analyses were conducted in sas version 9.3 (sas institute, cary, north carolina, united states). results were categorised as concordant if both the qualitative m. tuberculosis complex and rifampicin results matched the expected results. if discordant, results were categorised according to the type of discordance (false-positive m. tuberculosis complex detection, unsuccessful run, etc.). tests were considered unsuccessful when the test was started, but a definitive result was not obtained due to an error, invalid result, power failure during testing, or similar problem. tests were also considered unsuccessful if the site chose not to test the sample, was unable to test the sample due to unavailability of xpert mtb/rif kits, or failed to report either the m. tuberculosis complex or rifampicin detection result. frequencies for results in each category were calculated by pt sample and in aggregate for all samples. the mycobacterial strain used to produce the pt sample and the mean cycle threshold (ct) of probe a during validation of the pt sample were included in the analysis to look for trends between these variables and the amount and type of discordance observed. in line with previous studies, probe a was selected for most analyses as it was the first probe to reach the detection threshold.7 we conducted additional statistical analyses to test for an association between the mean ct of probe a during validation and several types of discordance including false-negative m. tuberculosis complex detection, false-positive rifampicin resistance, and indeterminate rifampicin resistance. logistic regression of the type of discordance on mean ct of probe a was conducted using r glm (r foundation for statistical computing, vienna, austria).16 results enrolment in the dts-based xpert mtb/rif pt programme increased steadily from round 2013-a to round 2015-c, with 102 sites in seven countries enrolled in round 2013-a and 441 sites in 14 countries enrolled in round 2015-c (figure 1). participation by enrolled sites increased from 68 sites in six countries in round 2013-a to 342 sites in 13 countries in round 2015-c. the participation among sites ranged from a minimum of 66.7% in round 2013-a to a maximum of 83.8% in round 2015-b. figure 1: number of sites enrolled, participating, and achieving a passing score in dried tube specimen-based xpert® mtb/rif proficiency testing programme, 2013–2015. the proportion of participating sites with a satisfactory score ranged from a minimum of 88.1% in round 2015-b to a maximum of 93.1% in round 2014-a (table 1). individual countries had as few as 50% and as many as 100% of their participating sites achieve satisfactory scores. in seven of the 14 enrolled countries, the proportion of sites with a satisfactory score increased between the first round of participation and the most recent round of participation. of the remaining seven countries, the proportion of sites with a satisfactory score remained constant in three countries but decreased in four countries (table 1). table 1a: number and percentage of sites participating and achieving passing scores in dried tube specimen-based xpert® mtb/rif proficiency testing programme for participating countries, 2013–2015. table 1b: number and percentage of sites participating and achieving passing scores in dried tube specimen-based xpert® mtb/rif proficiency testing programme for participating countries, 2013–2015. all 40 pt samples met the criteria for establishing a consensus result except 2015-b-1. 2015-b-1 was below 80% concordance with expected results, primarily due to a high rate of false-negative m. tuberculosis complex detection results (19.9%) (table 2). this sample is further discussed in the limitations section. for 29 of the 39 pt samples with a consensus result, over 90% of dts results received were concordant with the expected result. five of the 10 pt samples with < 90% concordance had false-negative m. tuberculosis complex detection as the most frequently observed cause for discordance, four of the 10 samples had unsuccessful tests (errors, invalids, and other causes) as the most frequently observed cause for discordance, while one sample had an equal number of unsuccessful tests and indeterminate rifampicin results. error rates ranged from 0% to 5.9% across pt samples. table 2: comparison of dried tube specimen results with expected results for xpert® mtb/rif proficiency testing programme, 2013–2015. there were 765 (9.4%) discordant results out of 8150 dts results received throughout the eight pt rounds. of those, 350 (46%) discordant results were unsuccessful tests (figure 2). false-negative m. tuberculosis complex results were the second-largest contributor to discordant results 224 (29%), followed by indeterminate rifampicin results 79 (10%). the remaining discordant results consisted of: false-negative rifampicin results 45 (6%), false-positive rifampicin results 34 (4%), and false-positive m. tuberculosis complex results 33 (5%). figure 2: distribution of 765 total discordant results in xpert® mtb/rif proficiency testing programme among participating countries, 2013–2015. logistic regression of the proportion of false-negative m. tuberculosis complex detection results on the mean ct of probe a during validation yielded an odds ratio of 1.42 (95% confidence interval [ci] 1.32–1.52). however, sample 2015-b-1’s unusually high mean probe a ct, combined with its unusually high proportion of false-negative m. tuberculosis complex detection results, may have unduly influenced this estimate. when this sample was removed from the calculation, the odds ratio decreased to 1.14 (95% ci 1.04–1.26). logistic regression of the proportion of indeterminate rifampicin results and the proportion of false-positive rifampicin resistance results on the mean ct of probe a during validation yielded odds ratios of 1.14 (95% ci 1.02–1.25) for indeterminates and 0.96 (95% ci 0.79–1.16) for false positives. discussion we have shown that it is feasible to implement an xpert mtb/rif pt programme for rls using dts. over the eight rounds of pt panels provided across a period of three years, the number of countries participating in the programme doubled and the number of participating sites increased five-fold. in each pt round, at least two-thirds of enrolled sites participated in pt panel testing and reported test results. lower participation rates were seen in rounds during which certain countries or their enrolled sites were subsequently unable to participate due to logistical challenges, such as a lack of funding for in-country panel distribution, lack of staff, pt kits being lost in the mail between the country coordinator and the enrolled testing site, non-functioning xpert instruments or computers, and stockouts (inability to procure test cartridges). further work is needed to investigate and address the reasons for non-participation in proficiency testing as part of a comprehensive continuous quality improvement package. most participating sites performed well in the dts-based xpert mtb/rif pt programme. approximately 90% of participating sites in each pt round demonstrated satisfactory performance, and over 90% of returned results during the study period were concordant with expected results. the most common reason for a result that was not concordant with the expected result was an unsuccessful test. some of the reported causes of unsuccessful tests, such as power failures and stockouts of testing kits, are recurring challenges in rls.17 the use of a similar sample type, dried culture spots, for xpert mtb/rif external quality assessment in south africa has been described.7,8 we observed similar error rates to those reported by scott et al. and gous et al.; however, we observed a greater proportion of discordant results.7,8 the average ct of probe a obtained from dried culture spots was lower than that of many dts pt samples. lower cts correlate with greater amounts of m. tuberculosis complex dna present in the sample, indicating that a higher concentration of m. tuberculosis complex dna was present in the dried culture spots. this may account for the smaller proportion of false-negative m. tuberculosis complex detection results from dried culture spots. we observed that false-negative m. tuberculosis complex detection results were often more common among dts samples with higher probe a cts during validation. our analyses using logistic regression confirmed a statistically significant positive association between higher probe a cts and false-negative m. tuberculosis complex detection. for dts samples produced at cdc during the study period, an increase of 1 cycle in the mean probe a ct resulted in an estimated 42% increase in the proportion of false-negative m. tuberculosis complex detection results (the estimate decreased to 14% when sample 2015-b-1 was excluded). similarly, we found a statistically significant positive association between higher probe a cts and indeterminate rifampicin resistance results, with an estimated 14% increase in the proportion of indeterminate rifampicin resistance results for every 1 cycle increase in a sample’s mean probe a ct. no association was found between mean probe a ct and false-positive rifampicin resistance, and we did not observe any trends between the strain used to produce dts and the proportion and types of discordance during the study period. based on these results, we are investigating modifications to the dts preparation method that will increase the concentration of m. tuberculosis complex dna present in dts. however, it is not possible to rule out the presence of or determine the frequency of other site-specific factors, such as adherence to standard operating procedures, transcription errors, instrument maintenance and calibration, and dts and test cartridge storage and shipping conditions that may influence the accuracy and reliability of xpert mtb/rif test results. the large increase in enrolment we observed over the course of three years may have been influenced by several factors. as the world health organization endorsed xpert mtb/rif in 2010 and funds became available for the purchase of instruments, the number of laboratories in rls utilising xpert mtb/rif increased dramatically.4 the world health organization’s expanded recommendations for use of xpert mtb/rif in 2013 also encouraged rapid adoption of the technology.1 over the same time period, the number of laboratories in rls electing to pursue external accreditation also increased, facilitated by such programmes as strengthening laboratory management toward accreditation, and stepwise laboratory improvement program towards accreditation.18,19 thus, an increasing number of laboratories in rls are in need of xpert mtb/rif pt material to demonstrate proficiency in line with recommendations and requirements for continuous quality improvement and accreditation programmes. limitations several challenges arose during operation of the dts-based xpert mtb/rif pt programme. the participant consensus for sample 2015-b-1 was below 80% concordance with expected results, mainly due to false-negative m. tuberculosis complex results (failure to detect m. tuberculosis complex when the expected result was ‘mtb detected’). although this sample was produced using the same method as other dts samples, the probe a cts during validation were on average higher than those of other m. tuberculosis complex samples produced. this indicates 2015-b-1 contained fewer inactivated organisms and thus less dna than other samples, potentially contributing to the higher proportion of false-negative m. tuberculosis complex results received.20 to investigate the low concordance of 2015-b-1, three remaining aliquots were tested and confirmed the lower than average semi-quantitative result. due to the low participant consensus, the decision was made to award all sites that submitted results for 2015-b-1 full credit (20 points). based on this experience, we have implemented additional dts validation criteria to ensure dts contain enough inactivated organism to perform reliably. beginning in 2016, only dts samples with an average ct value of less than 23 for the first probe detected are included in the distributed pt panels. the mean ct value of 23 was selected based on the findings of previous investigators, who observed good concordance with mean ct values up to 23.7 in addition, recording and reporting of results was also a recurring challenge. at times, no explanation was provided for missing or incomplete results, and it was not possible to determine whether a site attempted to test the sample or if the test was unsuccessful and the result not reported. more clearly defined reporting language and procedures were added to report forms in future pt rounds to reduce the reliance of free text data entry and improve our ability to provide assistance on determining the root causes of discordant results and unsuccessful tests. recommendations and next steps the steady increase in enrolment and number of sites participating in this dts-based xpert mtb/rif pt programme demonstrates that there is a substantial demand for routine pt material for the xpert mtb/rif assay. the ease of large-scale batch preparation of dts using readily available supplies and equipment, and the successful establishment of country coordinator roles for in-country management of pt programme operations, indicate that a dts-based xpert mtb/rif pt programme could be a feasible option for countries wanting to manage their own xpert mtb/rif pt programme. future work will focus on piloting the transfer of the dts-based pt package to national and regional programmes as part of a comprehensive continuous quality improvement package, as well as developing a web-based pt data entry and reporting system. conclusion a continuous quality improvement package for xpert mtb/rif in rls that includes routine pt material is an important contributor to ensuring provision of accurate results. a dts-based pt programme for xpert mtb/rif is a useful tool for monitoring and improving xpert mtb/rif performance. the simplicity of producing dts and use of common mycobacteriology laboratory supplies make dts a feasible way for countries to provide xpert mtb/rif pt material to their testing sites. acknowledgements we thank bharat parekh for his inspiration and guidance, and all testing sites for their willingness to participate in the proficiency testing program. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions k.d. and h.a. conceived and designed the experiments, which were performed by k.d. and k.k. also, k.k., k.d. and s.g. analysed the data. k.k., k.d., z.r., p.h. and a.k. wrote the article and all authors reviewed and approved the manuscript. sources of support this research has been supported by the president’s emergency plan for aids relief through the united states centers for disease control and prevention, atlanta georgia. data availability statement data access level is public without identifying information of proficiency testers, testing sites and country. disclaimer the findings and conclusions in this article are those of the authors and do not necessarily represent the official position of the united states centers for disease control and prevention. references in this manuscript to any specific commercial products, process, service, manufacturer, or company do not constitute its endorsement or recommendation by the united states government. references world health organization. rapid communication: molecular assays as initial tests for the diagnosis of tuberculosis and rifampicin [homepage on the internet]. geneva: who press; c2020 [cited 2020 sep 1]. available from: https://www.who.int/publications-detail/rapid-communication-molecular-assays-as-initial-tests-for-the-diagnosis-of-tuberculosis-and-rifampicin-resistance world health organization. global tuberculosis report [homepage on the internet]. geneva: who press; c2019 [cited 1 september 2020]. available from: https://www.who.int/tb/publications/global_report/en/ helb d, jones m, story e, et al. rapid detection of mycobacterium tuberculosis and rifampin resistance by use of on-demand, near-patient technology. j clin microbiol. 2010;48(1):229–237. https://doi.org/10.1128/jcm.01463-09 world health organization. cumulative number of genexpert instrument modules and xpert mtb/rif cartridges procured under concessional pricing [homepage on the internet]. geneva: who press; c2016 [cited 2020 sep 1]. available from: https://www.who.int/tb/areas-of-work/laboratory/status_xpert_rollout_dec_2016.pdf?ua=1 world health organization. laboratory quality management system: handbook [homepage on the internet]. geneva: who press; c2011 [cited 2020 sep 1]. available from: http://apps.who.int/iris/bitstream/10665/44665/1/9789241548274_eng.pdf global laboratory initiative. guide for providing technical support to tb laboratories in lowand middle-income countries [homepage on the internet]. n.d. [cited 2020 may 20]. available from: http://stoptb.org/wg/gli/assets/documents/guideforprovidingtechnicalsupport_gb_web.pdf scott le, gous n, cunningham be, et al. dried culture spots for xpert mtb/rif external quality assessment: results of a phase 1 pilot study in south africa. j clin microbiol. 2011;49(12):4356–4360. https://doi.org/10.1128/jcm.05167-11 gous n, cunningham b, kana b, stevens w, scott le. performance monitoring of mycobacterium tuberculosis dried culture spots for use with the genexpert system within a national program in south africa. j clin microbiol. 2013;51(12):4018–4021. https://doi.org/10.1128/jcm.01715-13 scott l, albert h, gilpin c, alexander h, degruy k, stevens w. multicenter feasibility study to assess external quality assessment panels for xpert mtb/rif assay in south africa. j clin microbiol. 2014;52(7):2493–2499. https://doi.org/10.1128/jcm.03533-13 degruy k, rey z, alexander h. dried tube specimens (dts) for xpert mtb/rif proficiency panels [poster]. presented at: african society for laboratory medicine. 1st international conference; 2012 december 1–7; cape town. benzaken as, bazzo ml, galban e, et al. external quality assurance with dried tube specimens (dts) for point-of-care syphilis and hiv tests: experience in an indigenous populations screening programme in the brazilian amazon. sexually trans inf. 2014;90(1):14–18. http://doi.org/10.1136/sextrans-2013-051181 nguyen s, ramos a, chang j, et al. monitoring the quality of hiv-1 viral load testing through a proficiency testing program using dried tube specimens in resource-limited settings. j clin microbiol. 2015;53(4):1129–1136. https://doi.org/10.1128/jcm.02780-14 parekh b, anyanwu j, patel h, et al. dried tube specimens: a simple and cost-effective method for preparation of hiv proficiency testing panels and quality control materials for use in resource-limited settings. j virol methods. 2010;163(2):295–300. https://doi.org/10.1016/j.jviromet.2009.10.013 morlock gp, plikaytis bb, crawford jt. characterization of spontaneous, in vitro-selected, rifampin-resistant mutants of mycobacterium tuberculosis strain h37rv. antimicrob agents chemother. 2000;44(12):3298–3301. https://doi.org/10.1128/aac.44.12.3298-3301.2000 xpert mtb/rif assay product insert [homepage on the internet]. sunnyvale, ca: cepheid; version 301-1404, rev. f august 2019 [cited 2020 sep 1]. available from: https://www.cepheid.com/package%20insert%20files/xpert-mtb-rif-english-package-insert-301-1404-rev-f.pdf r core team. r: a language and environment for statistical computing [homepage on the internet]. vienna: r foundation for statistical computing; c2019 [cited 2020 sep 1]. available from: http://www.r-project.org/ ondoa p, datema t, keita-sow ms, et al. a new matrix for scoring the functionality of national laboratory networks in africa: introducing the labnet scorecard. afr j lab med. 2016;5(3):a498. https://doi.org/10.4102/ajlm.v5i3.498 yao k, maruta t, luman e, nkengasong j. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(2):a194. https://doi.org/10.4102/ajlm.v3i2.194 datema tam, oskam l, van beers sm, klatser pr. critical review of the stepwise laboratory improvement process towards accreditation (slipta): suggestions for harmonization, implementation and improvement. trop med int health. 2012;17(3):361–367. https://doi.org/10.1111/j.1365-3156.2011.02917.x blakemore r, story e, helb d, et al. evaluation of the analytical performance of the xpert mtb/rif assay. j clin microbiol. 2010;48(7):2495–2501. https://doi.org/10.1128/jcm.00128-10 abstract introduction ethical considerations case presentation management and outcomes discussion acknowledgements references about the author(s) nicolene steyn department of chemical pathology, faculty of health sciences, university of pretoria, pretoria, south africa national health laboratory service, tshwane academic division, pretoria, south africa bettina chale-matsau department of chemical pathology, faculty of health sciences, university of pretoria, pretoria, south africa national health laboratory service, tshwane academic division, pretoria, south africa aron b. abera inqaba biotechnical industries (pty) ltd, pretoria, south africa gertruida van biljon national health laboratory service, tshwane academic division, pretoria, south africa division of paediatric nephrology, department of paediatrics, faculty of health sciences, university of pretoria, pretoria, south africa tahir s. pillay department of chemical pathology, faculty of health sciences, university of pretoria, pretoria, south africa national health laboratory service, tshwane academic division, pretoria, south africa citation steyn n, chale-matsau b, abera ab, van biljon g, pillay ts. neonatal presentation of a patient with liddle syndrome, south africa. afr j lab med. 2023;12(1), a1998. https://doi.org/10.4102/ajlm.v12i1.1998 case study neonatal presentation of a patient with liddle syndrome, south africa nicolene steyn, bettina chale-matsau, aron b. abera, gertruida van biljon, tahir s. pillay received: 29 june 2022; accepted: 03 feb. 2023; published: 14 apr. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract introduction: liddle syndrome is an autosomal dominantly inherited disorder usually arising from single mutations of the genes that encode for the alpha, beta and gamma epithelial sodium channel (enac) subunits. this leads to refractory hypertension, hypokalaemia, metabolic alkalosis, hyporeninaemia and hypoaldosteronism, through over-activation of the enac. case presentation: we describe a 5-day old neonate who presented with severe hypernatraemic dehydration requiring admission to steve biko academic hospital in south africa in 2012. further evaluation revealed features in keeping with liddle syndrome. two compound heterozygous mutations located at different subunits encoding the enac were detected following genetic sequencing done in 2020. the severe clinical phenotype observed here could be attributed to the synergistic effect of these known pathological mutations, but may also indicate that one of the other variants detected has hitherto undocumented pathological effects. management and outcome: this child’s treatment course was complicated by poor adherence to therapy, requiring numerous admissions over the years. adequate blood pressure control was achieved only after the addition of amiloride at the end of 2018, which raised the suspicion of an enac abnormality. conclusion: to our knowledge, this is the first liddle syndrome case where a combined effect from mutations resulted in severe disease. this highlights the importance of early recognition and management of this highly treatable genetic disease to prevent the grave sequelae associated with long-standing hypertension. whole exome sequencing may assist in the detection of known mutations, but may also unveil new potentially pathological variants. what this study adds: this study highlights the importance of developing a high index of suspicion of tubulopathy such as liddle syndrome for any child presenting with persistent hypertension associated with hypokalaemic metabolic alkalosis. keywords: liddle syndrome; epithelial sodium channels; genetic sequencing; hypertension; hyporeninaemia; hypoaldosteronism. introduction liddle syndrome, the most common monogenic cause of hypertension,1 is an autosomal dominantly inherited disorder typified by salt-sensitive hypertension, hyporeninaemia, hypoaldosteronism, metabolic alkalosis and variable hypokalaemia.2 even though symptoms and signs may present in infancy, the diagnosis is often significantly delayed.3 sodium reabsorption in the epithelial cells of the distal renal tubule is regulated by the epithelial sodium channel (enac).4 liddle syndrome arises from activating mutations of the scnn1a, scnn1b and scnn1g genes,5 which encode for the intracellular carboxy-terminal domains of the alpha, beta and gamma enac subunits. this results in an elevated number of channels and markedly increased independent activity with consequent sodium and water retention, hypertension and negative feedback suppression of renin and aldosterone secretion.2,3 ethical considerations written consent was obtained from the child’s parents and ethics approval obtained from the research and ethics committee at the university of pretoria (no. 536/2020). confidentiality was ensured in the preparation of this case study. case presentation a male patient of ethiopian descent, born in november 2012, presented on day five of life with severe hypernatraemic dehydration and acute renal failure requiring admission. on examination, he appeared severely wasted and dehydrated with absent femoral pulses. abdominal ultrasound revealed a thrombus in the aorta, attributed to the hyperviscosity associated with the severe dehydration. the thrombus resolved following heparin therapy and the child was discharged upon resolution of his renal failure with rehydration in december 2012. he was reviewed a week after discharge and found to have hypertension. during subsequent admissions over the years of follow-up, no clotting abnormalities were found and renal ultrasonography revealed no new thrombus nor renal abnormalities. no cardiac abnormalities were detected on sonography. there was no history of consanguinity. laboratory results (analysed on an abbott architect ci8200 (abbott laboratories, chicago, illinois, united states) up to 8 years of age revealed potassium values ranging from 2.0 mmol/l to 3.0 mmol/l (reference interval [ri]: 3.7 mmol/l – 5.9 mmol/l) and sodium levels 159 mmol/l – 171 mmol/l (ri: 136 mmol/l – 145 mmol/l) (table 1). metabolic alkalosis was also present (hco3− = 29 mmol/l to 34 mmol/l [ri: 23 mmol/l – 29 mmol/l]). random urine potassium was elevated (12.0 mmol/l – 22.0 mmol/l [ri: < 10 mmol/l]) despite the low serum potassium on presentation. table 1: laboratory results trends during follow-up at steve biko academic hospital in south africa, 2020. initial investigations in 2019, for the specific r563q mutation and mutations on exon 13 of the beta subunit of the enac (most common causes of liddle syndrome in south africa), were negative. further investigation was undertaken, with the aid of external funding, of all exons and exon-intron boundaries of the alpha (scnn1a [genbank nm_001038.5]), beta (scnn1b [genbank nm_000336.2]) and gamma (scnn1g [genbank nm_001039.3]) subunits encoding for the enac; these were amplified by polymerase chain reaction and sequenced using the nimagen, brilliantdye™ terminator cycle sequencing kit v3.1, brd3-100/1000 (nimagen, nijmegen, the netherlands), according to manufacturer’s instructions, in 2020. two sets of compound heterozygous transition mutations were found in the coding regions of the scnn1a and scnn1b genes (figure 1). in scnn1a, c.1000g>a in exon 5 resulted in ala334thr substitution and c.1987a>g in exon 13 led to thr663ala amino acid change. in the scnn1b gene, there was a c.7g>a mutation in exon 2 leading to a val3met substitution, and a c.1325g>t mutation in exon 9 leading to a gly442val substitution. no mutations were detected in the coding region of the scnn1g gene. figure 1: sanger sequencing electropherogram results of subunits scnn1a and scnn1b of the enac indicating mutations (arrow). (a) sequence electropherogram showing a heterozygous c.1000g>a mutation (chr12:6355415 [grch38.p14]) in exon 5 of scnn1a. (b) sequence electropherogram showing a heterozygous c.1987a>g mutation (chr12:6347896 [grch38.p14]) in exon 13 of scnn1a. (c) sequence electropherogram showing a heterozygous of scnn1a c.7g>a (chr16:23348606 [grch38.p14] in exon 2 of scnn1b. (d) sequence electropherogram showing a heterozygous c1325g>t mutation (chr16:23377219 [grch38.p14]) in exon 9 of scnn1b. management and outcomes the patient was initially managed on multiple antihypertensive drugs and potassium supplementation, but the treatment course was complicated by poor adherence to therapy and follow-up, resulting in numerous re-admissions over the years to achieve blood pressure control. effective blood pressure control was only achieved on commencement of amiloride in 2018, years after the initial presentation. this prompted the investigations for an enac abnormality. discussion in a neonate presenting with hypernatraemia and hypokalaemic metabolic alkalosis, the use of diuretics, persistent vomiting, nasogastric free drainage losses or a tubular disorder such as gitelman or bartter syndrome should be considered. if hypertension is also present, secondary causes such as congenital adrenal hyperplasia, primary hyperaldosteronism, syndrome of apparent mineralocorticoid excess, glucocorticoid-remediable aldosteronism, renal artery stenosis and a deoxycorticosterone-producing tumour should be included in the differential diagnosis.6 the presence of persistent hypertension with hypokalaemic metabolic alkalosis should raise the suspicion of unregulated enac activation.4 sodium reabsorption in the epithelial cells of the distal renal tubule is regulated by the enac, which is activated through the renin-angiotensin-aldosterone system6 (figure 2). it is important to determine if the hypertension is associated with low renin levels. in this instance, both serum aldosterone (< 27.0 pmol/l [ri: 49–643 pmol/l, supine], measured by diagnostic products corporation aldosterone coat-a-count kit, diagnostic products corporation, los angeles, california, united states) and plasma renin concentration (0.5 miu/l [ri: 6.5–36.2 miu/l, supine], measured by cis bio active renin assay, cisbio bioassays, codolet, france) levels were suppressed. figure 2: renin-angiotensin aldosterone system physiology. when the enac is activated independently of aldosterone stimulation, treatment with aldosterone antagonists has no effect. however, the use of amiloride or triamterene can lead to complete resolution of symptoms, as they are direct antagonists of the renal tubular enac and cause natriuresis while being both potassium and magnesium sparing.3 if there is a suspicion of an enac mutation based on the response to these drugs,3 whole exome sequencing should be undertaken. the very rare missense single nucleotide variant (for which our patient is heterozygous), that causes the substitution of glycine to valine (p.gly442val), has been linked to hypertension with increased enac activity.7,8 the effect of this polymorphism has been assessed by measuring the urine-aldosterone to -potassium ratio.7 increased enac activity would decrease this ratio as excess sodium absorption results in reduced aldosterone production and elevated urinary potassium excretion.7 this phenomenon has been confirmed in liddle syndrome patients where the urine-aldosterone to -potassium ratio was lower in subjects with the polymorphism than in normal subjects.9 the association of the alpha enac polymorphism (for which our patient is heterozygous), that causes the substitution of alanine to threonine (pala334thr), has been related to hypertension in certain studies10 and is associated with increased enac activity of 1.6-fold10 in functional studies. these findings in the current patient indicate that the severe clinical phenotype observed could be attributed to the compound heterozygous mutations located at different subunits of the enac and may indicate the presence of a yet unrecognised pathological variant. analogous to disorders caused by mineralocorticoid excess, liddle syndrome classically presents with hypertension, hypokalaemia and metabolic alkalosis. these findings are not always present, which may lead to under-diagnosis of the syndrome.11 identification of this condition is challenging, as the differential diagnosis for secondary hypertension is broad and the syndrome may present atypically. patients may have marked variations in phenotype, even with the same genotype.3 liddle syndrome principally arises from a transport impairment causing increased sodium reabsorption and excretion of potassium and hydrogen ions in the distal renal tubule. invariably, this leads to hypertension due to sodium and fluid retention with consequent hypokalaemic metabolic alkalosis and the suppression of renin and aldosterone through negative feedback. rare variants may, autonomously or cumulatively, cause hereditary disorders. liu and colleagues observed substantial differences in serum potassium levels and symptom onset in rare and non-rare scnn1b and scnn1g variant carriers, suggesting potential pathogenicity of some variants.12 to date, approximately 31 different liddle syndrome-causing alleles have been described in 72 families from four continents.3,10,13 interpretation of results from next-generation sequencing technologies are challenging as they have increased not only the diagnostic sensitivity, but also the number of variants with uncertain clinical significance. it is feasible that additional mutations that increase enac activity and result in phenotypical liddle syndrome will be discovered. in a study including patients with the liddle syndrome phenotype from kenya, nigeria and south africa, most patients had variants of a number of diverse genes that affect the enac channel.13 the authors speculated that certain patients may have combinations of variants that predispose to both increased aldosterone secretion and increased activity of the enac.13 in the assessment of patients with hyporeninaemic hypertension, investigation for a mutation in the enac subunits is recommended since early diagnosis and correct management of these patients may improve outcomes. delayed treatment is associated with failure to thrive and hypertension-related morbidity and mortality from cardiovascular disease, cerebrovascular disease, nephrosclerosis and progressive renal failure. furthermore, mutation studies permit clinicians to advocate for family screening based on the proband to identify carriers.14 this is the first case where a combined effect from mutations resulted in severe disease. genetic testing (including whole exome sequencing) should be performed, when possible, to identify mutations in patients with suspected secondary hypertension and unusual presentation.14 conclusion liddle syndrome is a poorly understood, but treatable genetic disease. misor late diagnosis may lead to unfavourable clinical sequelae. thus, any infant presenting with hypertension and metabolic alkalosis, with or without hypokalaemia, should raise suspicion for liddle syndrome, even in patients without a family history of hypertension. more functional studies are needed to characterise the numerous variants associated with the syndrome and their potential pathogenicity. acknowledgements christiaan labuschagne and erika viljoen of inqaba biotechnical industries for technical assistance with sequencing studies. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions n.s. drafted the original manuscript; b.c.-m. conceptualised and edited the manuscript; a.b.a. performed genetic studies and interpretation; g.v.b. undertook patient examination and investigation; and t.s.p. supervised the study. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability data supporting the findings of this study are available within the article. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated institutions or organisations of the authors. references buyukkaragoz b, yilmaz ac, karcaaltincaba d, ozdemir o, ludwig m. liddle syndrome in a turkish family with heterogeneous phenotypes. pediatr int. 2016;58(8):801–804. https://doi.org/10.1111/ped.12985 bogdanovic r, kuburovic v, stajic n, et al. liddle syndrome in a serbian family and literature review of underlying mutations. eur j pediatr. 2012;171(3):471–478. https://doi.org/10.1007/s00431-011-1581-8 assadi fk, kimura re, subramanian u, patel s. liddle syndrome in a newborn infant. pediatr nephrol. 2002;17(8):609–611. https://doi.org/10.1007/s00467-002-0897-z enslow bt, stockand jd, berman jm. liddle’s syndrome mechanisms, diagnosis and management. integr blood pressure control. 2019;12:13–22. https://doi.org/10.2147/ibpc.s188869 hanukoglu i, hanukoglu a. epithelial sodium channel (enac) family: phylogeny, structure-function, tissue distribution, and associated inherited diseases. gene. 2016;579(2):95–132. https://doi.org/10.1016/j.gene.2015.12.061 rifai n. tietz textbook of clinical chemistry and molecular diagnostics. e-book. elsevier health sciences: st louis, mo; 2017. dong yb, zhu hd, baker eh, et al. t594m and g442v polymorphisms of the sodium channel beta subunit and hypertension in a black population. j hum hypertens. 2001;15(6):425–430. https://doi.org/10.1038/sj.jhh.1001182 baker eh, dong yb, sagnella ga, et al. association of hypertension with t594m mutation in β subunit of epithelial sodium channels in black people resident in london. lancet. 1998;351(9113):1388–1392. https://doi.org/10.1016/s0140-6736(97)07306-6 ambrosius wt, bloem lj, zhou l, et al. genetic variants in the epithelial sodium channel in relation to aldosterone and potassium excretion and risk for hypertension. hypertension. 1999;34(4):631–637. https://doi.org/10.1161/01.hyp.34.4.631 tetti m, monticone s, burrello j, et al. liddle syndrome: review of the literature and description of a new case. int j mol sci. 2018;19(3):812. https://doi.org/10.3390/ijms19030812 rossi e, farnetti e, nicoli d, et al. a clinical phenotype mimicking essential hypertension in a newly discovered family with liddle’s syndrome. am j hypertens. 2011;24(8):930–935. https://doi.org/10.1038/ajh.2011.76 liu k, qin f, sun x, et al. analysis of the genes involved in mendelian forms of low-renin hypertension in chinese early-onset hypertensive patients. j hypertens. 2018;36(3):502–509. https://doi.org/10.1097/hjh.0000000000001556 jones es, rayner bl, spence jd, et al. high frequency of variants of candidate genes in black africans with low renin-resistant hypertension. am j hypertens. 2017;30(5):478–483. https://doi.org/10.1093/ajh/hpw167 polfus lm, boerwinkle e, gibbs ra, et al. whole-exome sequencing reveals an inherited r566x mutation of the epithelial sodium channel beta-subunit in a case of early-onset phenotype of liddle syndrome. cold spring harb mol case stud. 2016;2(6):a001255. https://doi.org/10.1101/mcs.a001255 acknowledgements references about the author(s) nqobile ndlovu african society for laboratory medicine, addis ababa, ethiopia yenew k. tebeje africa centres for disease control and prevention, addis ababa, ethiopia renuka gadde clinton health access initiative, boston, massachusetts, united states trevor peter clinton health access initiative, boston, massachusetts, united states citation ndlovu n, tebeje yk, gadde r, peter t. to break the cycle of covid-19 lockdowns and safely open up economies, we must ensure equitable access to diagnosis and treatment. afr j lab med. 2022;11(1), a1650. https://doi.org/10.4102/ajlm.v11i1.1650 opinion paper to break the cycle of covid-19 lockdowns and safely open up economies, we must ensure equitable access to diagnosis and treatment nqobile ndlovu, yenew k. tebeje, renuka gadde, trevor peter received: 17 june 2021; accepted: 10 feb. 2022; published: 27 may 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. in june 2021, sub-saharan africa was facing the worst crisis from coronavirus disease 2019 (covid-19) since the pandemic began, with cases and deaths rising amidst the emergence of new virulent strains of severe acute respiratory coronavirus 2.1 in the midst of this, countries need to ramp up effective prevention and treatment programmes in a way that permits economies to function. these measures include vaccination, physical distancing and wearing masks to protect others, testing and tracing to identify and isolate infected individuals and those who have been in contact with them, and treatment with emerging drug therapies in order to reduce transmission and save lives.2 there is optimism and hope in the global discussion on vaccination and novel therapies. however, effective covid-19 disease control is likely to require that countries jointly balance their investments in vaccines, personal protection, testing and treatment.3,4 testing programmes which have been a backbone of control efforts since the onset of the pandemic, now need to be integrated effectively with other interventions as well as continually improve their efficacy. for example, vaccine rollout must be supported by testing and surveillance data to improve outcomes. the use of routine testing data can help to identify transmission hot spots and to prioritize locations and populations within vaccination campaigns.5 testing data will be important for monitoring the occurrence of outbreaks within populations post-vaccination and to inform ongoing efforts to close residual gaps in coverage, including informing governments on how to develop targeteted approaches for emerging viral variants.5 testing is also needed for the effective use of emerging drug therapies and medical oxygen. these rely on diagnosis and treatment early in the course of disease to achieve the highest benefits in morbidity and mortality.4 in the near future, the combination of rapid tests and new drug regimens will allow the expansion of test and treat programmes that will make covid-19 a manageable infection. in the meantime, to operate schools and workplaces safely and sustainably, it is important to ensure that rapid testing is available where and when it is needed and that we have the tools in place to comprehensively monitor and track the disease.6 current testing efforts in many sub-saharan countries are well below the threshold of one test per 1000 population per week set by the world health organization.7 this means that not only is it not clear how many infections have occurred, but there is limited insight into the emergence and spread of new outbreaks. there are ongoing efforts to better understand vaccine variants through genomic sequencing but there are limitations. for example, sequencing technologies are still not widely available and they often do not provide real-time information on variant outbreaks to help limit spread.8 without the ability of individuals and public health programmes to understand their infection status and take actions limit disease spread, the current cycle of covid-19-related restrictions on work, school and travel will continue to threaten africa’s economies, increasing poverty and hunger, and reversing gains made in recent decades. that is why it is critical for the global health community, governments, and the private sector to come together to ensure that accurate, rapid antigen diagnostic tests (rdts) are accessible at community level, including self-tests. rapid antigen diagnostic tests must be available in all primary health care facilities and made available through public and private channels for self testing, and must be appropriately used to help reopen borders, schools, and places of work and worship.9,10 examples of this work were presented in a recent series of webinars organised by the african society for laboratory medicine and the africa centers for disease control and focused on expanding access to antigen rdts for covid-19.9 leading scientists and public health experts from africa, europe, the united states and asia presented the latest updates on testing and covid-19 control. in rwanda, covid-19 rdts are being used in public and private health facilities, at weddings, schools, prisons, churches, market places, as well as for refugees and others recommended by rwanda’s ministry of health. this has helped treble the number of testing locations and reduce testing costs. in south africa, rdts have been widely used in open border posts (land, sea, and air) and are being used within the provinces of the country. in nigeria, scaled-up use of rdts within national youth camps has made it possible to quickly detect cases and isolate youth as they entered the camps, thus preventing potential outbreaks.9 schools provide one of the best opportunities for testing and other strategies to prevent transmission. the most important lines of defence in preventing transmission of severe acute respiratory coronavirus 2 include making sure students do not come to school with symptoms, enforcing universal mask adherence and physical distancing, improving ventilation, and swiftly investigating any cases associated with schools, including contact tracing and quarantine and isolation.6 testing is another tool in the prevention arsenal for schools and can provide guidance on how to plan better prevention and increase layers of defense. testing also ensures early identification of cases among students and staff and helps with contact tracing efforts, and can also identify infection among students and staff at high-risk of developing severe disease due to underlying conditions and support investigations and studies in understanding the role children play in disease transmission. testing must reflect the purposes and resources of a community. sweeping guidance that schools should be closed if the positivity rate exceeds a certain threshold needs to be reviewed in the context of a country. for example, if a school is conducting all in-person classes and the prevalence in community increases, it may consider reducing class sizes and having students attend on alternating schedules. in other words, a deeper analysis should guide decision making. there is mounting evidence of the detrimental effects of school closures and the impact it is having on children from learning loss to emotional wellness.10 one can argue that schools should be in the same category as essential services, similar to healthcare facilities, in that they should only close when there is no other option. in september 2020, several organisations, including the world health organization, the global fund, the africa centres for disease control and prevention, find and others, announced an agreement to make available 120 million low-cost, high-quality antigen rdts for covid-19 for lowand middle-income countries.11 while this was necessary to ensure that tests were available, adoption and use of antigen testing was slow, and many countries implemented these professional-use tests widely within health facilities and at the community level (i.e., for symptomatic and high-risk individuals). high-performing self-tests are now more widely available and provide opportunities to expand access to testing, decongest health facilities, and limit the risk of transmission.12 it is important that countries identify the most effective use for both professional use rapid tests and self tests to be deployed within public testing programmes and for private use.13 now is not the time to debate whether we should be spending on vaccines, oxygen, personal protective equipment, drug treatment, or testing. effective programmes need to strategically deploy each of these interventions in an integrated way, just as integrated prevention, testing and treatment control programmes have been developed for other diseases. fortunately, antigen rdts with excellent performance are available and can be used at the point of care or in laboratory settings, and are increasingly available for self testing.12,14 even though much progress has been made in recent years, gaps in access to essential tests remain an issue throughout africa, including covid-19. while robust systems to deliver testing and surveillance for covid-19 are developed, it is important to ensure that they are sustainable into the future and also address other major health issues, including malaria, hiv, tuberculosis, cervical cancer and other diseases. maintaining a joint focus on prevention, testing and treatment will be necessary, building on partnerships between governments, donors, policymakers and industry to use the available tools to solve this global crisis. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions n.n. contributed to the planning and execution of the webinars that generated the basis of the paper. in addition, he contributed to the conceptualisation and the design of the write-up, reviewed and edited the paper. y.k.t. also contributed to the planning and execution of the webinars that generated the basis of the paper. in addition, he contributed in the conceptualisation and the design of the write-up, reviewed and edited the paper. r.g. contributed to the original conceptualisation, writing of the paper and execution of the webinars that generated the basis of the paper. in addition, he contributed to the design of the write-up, reviewed and edited the paper. t.p. contributed to the planning and execution of the webinars that generated the basis of the paper. in addition, he contributed to the design of the write-up, reviewed and edited the paper. ethical considerations this article followed all ethical standards for research. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for profit sectors. data availability data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references burke j. african nations fear more covid-19 deaths before vaccination begins [homepage on the internet]. the guardian, 04 february 2021. [cited 2021 mar 23]. available from: https://www.theguardian.com/global-development/2021/feb/04/african-nations-fear-more-covid-deaths-before-vaccination-begins noorbhai h. a mathematical model to guide the re-opening of economies during the covid-19 pandemic. ann med surg (london). 2020;57:5–6. https://doi.org/10.1016/j.amsu.2020.06.041 majumder j, minko t. recent developments on therapeutic and diagnostic approaches for covid-19. aaps j. 2021;23(1):14. https://doi.org/10.1208/s12248-020-00532-2 world health organization. who recommends two new drugs to treat covid-19 [homepage on the internet]. [cited 2020 jan 30]. available from: https://www.who.int/news/item/14-01-2022-who-recommends-two-new-drugs-to-treat-covid-19 koks s, williams rw, quinn j, et al. covid-19: time for precision epidemiology. exp biol med (maywood). 2020;245(8):677–679. https://doi.org/10.1177/1535370220919349 dzinamarira t, musuka g. the paradox of re-opening schools in zimbabwe in the covid-19 era. public health pract 2021;2:00070. issn 2666-5352. https://doi.org/10.1016/j.puhip.2020.100070 jani iv, peter tf. nucleic acid point-of-care testing to improve diagnostic preparedness. clin infect dis. 2022; ciac013. htttps://doi.org/10.1093/cid/ciac013 otu a, agogo e, ebenso b. africa needs more genome sequencing to tackle new variants of sars-cov-2. nat med. 2021;27:744–745. https://doi.org/10.1038/s41591-021-01327-4 african society for laboratory medicine. special covid-19 echo sessions 37 [homepage on the internet]. 2021 [cited 2021 mar 23]. available from: https://aslm.org/resource/special-covid-19-echo-session-37/ chen ih, chen cy, pakpour ah, griffiths md, lin cy. internet-related behaviors and psychological distress among schoolchildren during covid-19 school suspension. j am acad child adolesc psychiatry. 2020;59(10):1099–1102.e1. https://doi.org/10.1016/j.jaac.2020.06.007 world health organization. global partnership to make available 120 million affordable quality covid-19 rapid tests for low and middle income countries [homepage on the internet]. 2020 [cited 2022 jan 30]. available from: https://www.who.int/news/item/28-09-2020-global-partnership-to-make-available-120-million-affordable-quality-covid-19-rapid-tests-for-low--and-middle-income-countries boum y, eyangoh s, okomo mc. beyond covid-19 – will self-sampling and testing become the norm? lancet infect dis. 2021;21(9):1194–1195. https://doi.org/10.1016/s1473-3099(21)00197-3 africa cdc. new guidance to expand rapid antigen testing for covid-19 response in africa released [homepage on the internet]. 2020 [cited 2022 jan 30]. available from: https://africacdc.org/news-item/new-guidance-to-expand-rapid-antigen-testing-for-covid-19-response-in-africa-released/ peeling rw, olliaro pl, boeras di, fongwen n. scaling up covid-19 rapid antigen tests: promises and challenges. lancet infect dis. 2021;21(9):e290–e295. https://doi.org/10.1016/s1473-3099(21)00048-7 abstract introduction methods results discussion acknowledgements references about the author(s) leanne swart department of molecular medicine and haematology, charlotte maxeke johannesburg academic hospital, national health laboratory service, johannesburg, south africa department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa melanie pretorius department of molecular medicine and haematology, charlotte maxeke johannesburg academic hospital, national health laboratory service, johannesburg, south africa department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa denise lawrie department of molecular medicine and haematology, charlotte maxeke johannesburg academic hospital, national health laboratory service, johannesburg, south africa department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa deborah k. glencross department of molecular medicine and haematology, charlotte maxeke johannesburg academic hospital, national health laboratory service, johannesburg, south africa department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa citation swart l, pretorius m, lawrie d, glencross dk. commercial duraclone panels for extending the repertoire of multicolour immunophenotypic panels in an academic flow cytometry laboratory in south africa. afr j lab med. 2022;11(1), a1720. https://doi.org/10.4102/ajlm.v11i1.1720 original research commercial duraclone panels for extending the repertoire of multicolour immunophenotypic panels in an academic flow cytometry laboratory in south africa leanne swart, melanie pretorius, denise lawrie, deborah k. glencross received: 31 aug. 2021; accepted: 28 july 2022; published: 29 nov. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: commercial multicolour fixed immunophenotyping panels can improve flow cytometric diagnostic immunophenotyping repertoire. objective: this study validated the commercially available, standardised beckman coulter lyophilised duraclone re panels to discriminate specific haematolymphoid subtypes. methods: we compared the diagnostic capability of the duraclone acute leukaemia b (alb), chronic leukaemia b (clb), and plasma cells (pc) panels to the predicate second-line panels in charlotte maxeke johannesburg academic hospital, johannesburg, south africa, from april to august 2020. clinical diagnostic concordance between the in-house second-line immunophenotyping (the predicate method) and duraclone was established. the alb panels tested for precursor b-cell acute lymphoblastic leukaemia (n = 11) or normal bone marrow haematogones (n = 9); clb panels established haematolymphoid subtypes of mature b-cell lymphoproliferative disorders (b-lpd) (n = 20), while pc panels detected plasma cell dyscrasias (pcd) (n = 17). flow cytometer setup and data interpretation to discriminate normal and aberrant immunophenotypes were per manufacturer’s instructions. results: there was 100% clinical diagnostic concordance between the predicate and the test panels for second-line diagnostic investigation of b-all (with additional cd56), mature b-lpd (with additional discernment of cd81, ror-1, cd79b and cd43) and pcd. conclusion: the duraclone clb exceeded the predicate second-line performance, offering extended second-line diagnostic discernment of mature b-lpd subtypes and discernment of cd5+ b-lpd from other non-cd5+ (or cd5–) b-lpd; likewise, the pc panels enabled discovery of pcd. while alb testing offered no additional diagnostic advantage over existing predicate investigation, cd58 did offer additional information to discern haematogones from b-all. keywords: flow cytometry; multicolour; immunophenotyping; lymphoma; leukaemia; plasma cell dyscrasia; acute lymphoblastic leukaemia; b-cell lymphoproliferative disorder; lyophilised reagent; fixed panels. introduction flow cytometry immunophenotypic analysis is a powerful diagnostic and research tool for investigating haemato-lymphoid malignancies, such as leukaemia, lymphoma, and plasma cell dyscrasias (pcd). recently, multicolour flow cytometric immunophenotyping has become invaluable for improving detection and detailed immunophenotyping during diagnosis and detection of low frequencies of abnormal or aberrant cell populations during minimal residual disease assessment.1,2 aberrant cell phenotypes are distinguished from normal developmental counterparts2 and normal background leucocyte populations3 by analysing and comparing several simultaneous cell surface and cytoplasmic antigen expression profiles. multicolour fluorochrome-conjugated antibody panels generally require antibody concentrations and fluorescence intensity optimisation through titration techniques.4,5 these assays can be technically challenging and error-prone due to the addition of multiple individual liquid reagents requiring manual pipetting,4,6 thus requiring laboratory technicians skilled in titration technique and flow cytometry,4,7 including fluorescence-minus-one, colour compensation, and other technical flow cytometry issues. commercially available, standardised, lyophilised, fixed and pre-titrated antibody panels,8,9,10,11,12,13 like the beckman coulter (bc) duraclone inventory, including the identification of normal and abnormal b progenitor cells (re alb),14 various chronic b-cell lymphoproliferative disorders including cd5+ chronic lymphocytic leukaemia (re clb)15 and normal or abnormal plasma cells (re pc) panels, offer an attractive alternative to the locally developed, in-house multicolour panels described. commercial multicolour panels also usually offer the advantage of a standardised instrument and analysis protocols; preset software-guided controls are available to establish optimal standardised instrument settings, verified with abnormal and normal stabilised blood material controls.9 in addition, these commercial panels are typically fixed panels supplied in a stable, dry (lyophilised) format with predetermined antibody combinations in a single tube9; these qualities enhance laboratory efficiency, reduce technical laboratory error and simplify reagent inventory. some commercial panels also offer some flexibility in marker combinations by providing ‘open’ fluorescence channels14,15 so that liquid drop-in markers can be added to the ‘fixed’ panels to accommodate unique and specific diagnostic requirements of the investigating laboratory. our laboratory previously established the positive impact on quality, laboratory workflow and diagnostic usefulness of the commercially available, fixed and standardised bc clearllab 10c16 multicolour panel system (beckman coulter, miami, florida, united states). although this method provides excellent coverage to discover most acute leukaemias,9,16 the clearllab 10c system lacks additional markers to further identify and discern specific haematolymphoid subtypes, especially within the group of mature b-cell lymphoproliferative disorders and pcd. specifically, although the bc clearllab 10c can easily discern normal b-cell precursors or haematogones from similar immunophenotypes present in minimal residual b-cell acute lymphoblastic leukaemia (b-all) disease, the cd58 marker is not included. however, it may be helpful to discriminate disease from normal b-cell precursors during minimal b-all disease assessment.1,2 the clearllab 10c system also lacks markers to discern cd5-positive and cd5-negative mature b-cell lymphoproliferative disorders (lpd), using markers such as cd23, cd43, cd79b, cd81, and ror-1 recommended for classification by rawstron et al.17,18 investigation of pcd is also limited if investigated only with the clearllab 10c system which lacks cd138, an absolute requirement for identifying plasma cells together with cd3819. in our laboratory, we have developed several 2–4-colour in-house, second-line immunophenotyping panels to identify the specific haematolymphoid subtypes mentioned, including markers such as cd23 and fmc-7 for mature b-cell lpd and cd138 with cd56 and cd200, to diagnose pcd. these, however, require a separate repertoire of liquid reagents and necessary flow cytometry technical expertise to set up and run the tests. for a busy site, such as our unit, which processes over 400 samples per month, standardised commercially available panels that can supplement second-line immunophenotypic investigation after clearllab10c investigation could dramatically increase the repertoire of markers tested and also improve workflow efficiency in our site. existing predicate second-line investigation utilises locally established, in-house, non-standardised and manually assembled liquid fluorochrome-conjugated antibody panels. the primary aim of this study was to evaluate the fixed and standardised, pre-titrated commercially available duraclone re alb, re clb and re pc panels (beckman coulter, mumbai, india) as a comprehensive and compact alternative to our existing second-line immunophenotypic investigation of haematological neoplasms. there were two parts to this study. first, we compared the duraclone clb and pc tubes to the laboratory’s in-house predicate 2–4-colour method for the following markers: combinations of cd10, cd23, fmc7 and cd22 to discern mature b-cell lymphoproliferative disorders, or cd19, cd38, cd56, cd200 and cd138 to characterise plasma cells. secondly, we verified manufacturer-described expression for those additional markers that were not included in the older predicate 2–4-colour method, such as verifying the expression of cd43, cd79b, cd81 and ror-1 in the clb tube and cd38 and cd138 with cd27, cd81 and cd56 included in the duraclone pc tube against expected expression in normal leucocyte population counterparts. we also validated the overall diagnostic outcome of the alb tube; here, we asked whether the cd58 included in the alb tubes offered any additional diagnostic advantage in identifying a b-all that had been identified in the first-line clearllab 10c investigation. methods ethical considerations the university of the witwatersrand health research ethics committee approved the study (ethics clearance number m1704129). the study’s objective was to validate the commercially available beckman coulter duraclone pc, clb and pc fixed-tube, pre-titrated panels as an alternative second-line diagnostic workup to our existing in-house second-line immunophenotypic investigation. the interpretation of flow cytometric data and consequent clinical diagnostic outcomes of the existing in-house, second-line, non-standardised panels were compared to the overall clinical diagnostic outcomes of the duraclone analyses. study design and site this study was a prospective observational cohort study conducted according to the standards for reporting diagnostics accuracy.20 the work was undertaken from april 2020 to august 2020 and conducted in the national health laboratory service flow cytometry laboratory at the charlotte maxeke johannesburg academic hospital in johannesburg, south africa. this laboratory performs routine immunophenotypic investigation of leukaemias and lymphomas for both children and adults, referred from charlotte maxeke johannesburg academic hospital and other hospitals within the university of the witwatersrand service precinct, as well as from other centres around south africa. morphological assessment of peripheral blood, bone marrow and trephine samples (from the same patients whose samples are tested in the flow cytometry unit) is performed in the sister haematology laboratory; auxiliary molecular and cytogenetic investigations are also performed on site. the laboratory participates in the united kingdom national external quality assurance scheme (uk neqas, sheffield, united kingdom) leukaemia immunophenotyping part 1 and part 2 (interpretation) proficiency testing scheme.21 testing approach immunophenotypic testing of all samples at the charlotte maxeke johannesburg academic hospital flow cytometry unit is two-tiered. firstly, an initial first-line workup is performed with bc clearllab 10-c (beckman coulter, miami, florida, united states). the first workup identifies most acute myeloid and lymphoid leukaemia subtypes, distinguishes early and mature t-cell or b-cell lymphoproliferative disorders, and hints at the presence of a pcd (if a population of brightly expressing cd38 cells is noted). a limitation of this clearllab 10c system is that further immunophenotypic characterisation of mature b-cell lymphoproliferative disorders17 or pcd19,22 requires second-line testing using markers that are not included in the first-line testing. for this study, in-house second-line testing specifically assessed cd23 and fmc-7 expression on mature b-cells or cd38 and cd138 expression on plasma cells. specifically, in the context of b-lpd and in comparison to our in-house second-line investigation that included cd23 and fmc-7, could duraclone clb testing with ror-1, cd20, cd43, cd79b, and 81 replace cd23 and fmc-7 in discerning cd5 b-cell chronic lymphocytic leukaemia (cll) from other cd5 and non-cd5 expressing b-lpd.17,18 samples after all routine testing with clearllab 10c and in-house second-line testing (table 1), samples having sufficient remnant prepared-cell-concentrate or at least 1 ml remaining of the whole sample were re-tested using one of the test methods (either duraclone re alb, re clb or re pc). the aim was to collect at least 20 clinical specimens for each validation (estimated to be 60 samples). all peripheral blood and bone marrow samples were collected by attending physicians into ethylenediaminetetraacetic acid vacutainer tubes; the single body fluid sample was not collected into an anticoagulant but submitted in a vacutainer tube without anticoagulant. at the end of the study period, a total of 57 anonymised samples were identified for second-line duraclone comparison, including peripheral blood (n = 13), bone marrow (n = 43), and a pleural fluid sample (n = 1) collected from paediatric and adult patients. for duraclone alb testing, 20 bone marrow aspirate samples were tested. eleven of these patients had a diagnosis of b-all (n = 11), while nine had immunophenotypically normal haematogones. one of the nine was a follow-up of a b-all with normal haematogones and no evidence of residual disease. twenty samples diagnosed with a b-cell lymphoproliferative disorder by predicate clearllab 10c and in-house second-line were referred for duraclone clb testing. the clb testing set comprised 12 bone marrow aspirate samples, seven peripheral blood samples, and a single pleural fluid sample. due to the decommissioning of the older facscalibur flow cytometer during the period of study (the instrument used to undertake the predicate in-house second-line testing), only 17 samples with a diagnosis of a pcd by clearllab 10-c and in-house second-line investigation were tested by duraclone pc panels. table 1: possible reagent selection for method comparison according to suspected target population undertaken from april 2020 to august 2020 at an academic pathology service in johannesburg, south africa. instruments the naviostm (bc, miami, florida, united states) and the facscalibur flow cytometer (becton dickinson biosciences, san jose, california, united states) were used during this study. the naviostm flow cytometer was used to analyse the predicate clearllab 10c as well as the test duraclone panels. internal quality control on the naviostm included daily assessment of background contamination, cellular events carryover between tubes, and fluorospheres acquisition to verify the flow cytometer optical alignment and fluidics (flow-check pro, bc, lismeehan, ireland). in addition, clearllab™ normal and abnormal process control cells (bc, lismeehan, ireland) were used to verify sample processing, acquisition, and analysis. clearllab 10c panel acquisition setup was achieved by applying target values set in the manufacturer manual and using bc flowset pro beads to adjust the voltages that enabled optimal detection and separation of dim and bright antigens. after pilot testing (data not shown), the naviostm instrument clearllab 10c settings were deemed appropriate for the duraclone multicolour data acquisition and analysis. the facscalibur flow cytometer was used to acquire the predicate in-house second-line 2–4-colour fluorescence panels using cellquest software (bd, san jose, california, united states). quality control performed on the bd facscalibur included daily assessment of background contamination, carryover, acquisition, and analysis of manufacturer-recommended 3-colour and apc calibrite beads (becton dickinson biosciences, san jose, california, united states). further acquisition and analysis of immunotroltm process control (bc, brea, california, united states) using four monoclonal antibodies, cd14 fitc, cd13 pe, cd45 percp and cd3 apc (all becton dickinson biosciences, san jose, california, united states), was done. sample preparation sample preparation included lysing two or more 0.5 ml sample aliquots (dependent on the initial white cell count) with 14.5 ml isotonic ammonium chloride ph 7.1–7.4 (8.99% nh4cl, 0.84% nahco3 and 0.0372% ethylenediaminetetraacetic acid; merck, darmstadt, germany) in a 15 ml conical centrifuge tube for 15 min at room temperature. after incubation, samples were spun at 3000 g for 3 min, the supernatant was decanted, while the pellet was washed four times with 14 ml phosphate-buffered saline at ph 7.3 ± 2 (oxoid ltd, basingstoke, united kingdom) containing 0.09% sodium azide (nan3) (merck, darmstadt, germany) and 0.2% bovine serum albumin (biowest, nuaille, france). following washing, samples were reconstituted to 0.5 ml with phosphate-buffered saline, and the white cell count was determined. cells were then diluted (or concentrated) to achieve the recommended cellular concentration of ≤ 10 000 cells/µl for monoclonal antibody incubation. subsequently, 100 µl of this cell concentrate, which contained approximately 106 cells, was added to the clearllab 10c and in-house panels (see table 1). if the sample was deemed to be suitable for inclusion in the study (i.e. had an established diagnosis by predicate clearllab 10c), and if there was sufficient remaining material, 100 µl of the remaining cell concentrate was tested with either duraclone alb, clb or pc panels (table 1). details of the markers included in these duraclone panels are outlined in table 2. each duraclone panel has a ‘spare’ capacity for additional markers per the investigator’s need. in our study, in two cases of precursor b-all, the marker cd22 (apc) (bc, miami, florida, united states) was added in liquid format to the alb panel to occupy the free apc channel (see table 2, duraclone re alb panel) to verify cd22 expression in the context of a b-all. in a single case of a suspected pcd, liquid cd117 ecd (bc, miami, florida, united states) was added to the duraclone re pc tube in the ‘free’ ecd channel to demonstrate expression of cd117 in a case of multiple myeloma. after adding the cell aliquot into panels, and, where applicable, the addition of liquid monoclonal reagent, all samples were incubated at room temperature (average 20 °c – 22 °c) in the dark for 15 min. after incubation, all samples were washed once with phosphate-buffered saline and reconstituted to 0.5 ml. table 2: possible reagent selection for method comparison according to suspected target population validated at the charlotte maxeke johannesburg academic hospital flow cytometry laboratory in johannesburg, south africa, april 2020 to august 2020. data acquisition and interpretation of flow cytometric case data all samples were acquired on a flow cytometer to acquire raw flow cytometric listmode data. for the predicate in-house panels acquired on the facscalibur, 5000 (.fcs data) events were collected. paint-a-gate software (becton dickinson biosciences, san jose, california, united states) was used for analysis of raw facscalibur flow cytometric data with a primary gating focus using cd45 and side scatter. for both the clearllab 10c and duraclone panels, at least 50 000 (listmode data) events were acquired on the navios. kaluza c™ version 1.1 (bc, miami, florida, united states) software was used to analyse all raw listmode data to facilitate clinical interpretation. kaluza c™ clearllab b, t, m1 and m2, and duraclone panel analysis protocols were developed according to manufacturer specifications.23 first, doublets, debris and unlysed red blood cells were excluded, and, where possible, a neoplastic target population was identified in a primary gate using cd45 and side scatter. thereafter, secondary gating focused on identifying the same target immunophenotype across each of the t, m1 and m2 panels.9,16 finally, simultaneous identification of normal populations in the background was based on local in-house developed gating strategies to identify granulocytes, monocytes and mature lymphocytes using cd45, side scatter characteristics and specific regular expression of the markers studied (figure 1 and figure 2). figure 1: relative expression of cd81 relative to normal t-cells and granulocytes in the clb tube validated at the charlotte maxeke johannesburg academic hospital flow cytometry laboratory in johannesburg, south africa, april 2020 to august 2020. each plot (a, b and c) shows cd81 expression (x-axis) versus frequency (y-axis). in a, there is relative (weak) under-expression of cd81 in a patient with cll gated on cd19+|cd5+; in b, there is relative over-expression of cd81 in a patient with follicular lymphoma gated on cd19+|cd10+ c is a control experiment of a paediatric sample with 20% haematogones showing bright expression of cd81 on normal precursor b-cells gated on weak cd45+| cd19+. orange represents the b-cells identified with cd19; the blue population represents background granulocytes identified with cd45 and side scatter, and red represents background t-cells gated on cd5 (all markers are included in the clb panel). figure 2: identification of plasma cells using the duraclone pc tube validated at the charlotte maxeke johannesburg academic hospital flow cytometry laboratory in johannesburg, south africa, april 2020 to august 2020. in histogram a, negative cd45 expression of plasma cells is shown (purple), with cd45 positive (background) expression noted amongst granulocytes (blue), monocytes (green) and mature lymphocytes (red). the target plasma cell population were primary-gated on bright cd138 and cd38 to reveal aberrant cd117 expression (b) and under-expression of cd81 (c) and cd27 (d). a similar approach was used for each of the comparative second-line duraclone re alb, re clb, and re pc listmode data analysis but additionally incorporated manufacturer-recommended gating strategies for clinical interpretation of data.23 the relative intensity of expression of cell surface antigens was used to discriminate between normal and aberrant immunophenotypes according to manufacturer specifications. positivity (or no expression) of individual markers was established, and overall haematolymphoid immunophenotypes sub-classified according to the established positive markers outlined in the world health organization (who) 2016 classification of tumours of haemopoietic and lymphoid tissues.24 specifically, expression of cd27, cd43, cd56, cd58, and cd81, which were not included in the predicate clearllab 10c and in-house second-line panel analysis, were verified on background (normal) populations as evidence of a satisfactory internal positive control and proof that the reagents met the manufacturer’s performance specifications. in the alb analyses, typical secondary backbone markers, including co-expression of cd19/cd10/cd34 enabled further characterisation of markers cd20, cd38, and cd58 and established the presence of normal precursor b-cells (haematogones) or abnormal precursor b-cells. likewise, for the duraclone re clb, dual expression of either cd19/cd5 or cd19/cd20 was used to identify the ‘target’ or neoplastic b-cell population before specific characterisation of cd43, cd79b, cd81 and ror1 expression. plasma cells were defined in the duraclone re pc panel by the dual expression of both cd38 and cd138; a normal or malignant plasma cell immunophenotype was subsequently noted following interrogation of markers including cd81, cd27, cd19, cd200, cd56, and cd45. statistical analysis clinical diagnostic outcomes were collated into microsoft excel (redmond, washington, united states) spreadsheets. marker expressions and specific clinical diagnoses noted for the duraclone re alb, re clb, and re pc panels were compared to the clinical diagnostic outcomes from the existing in-house 2–4-colour antibody panels described (table 2). the agreement between methods (comparing the final diagnostic outcome) was assessed using contingency tables to determine sensitivity and specificity. true positives were regarded as clinical diagnostic outcomes matching the predicate clearllab 10-c with in-house panels, and test system outcomes using alb, clb or pc. for example, when a b-cell cll was diagnosed on the initial clearllab investigation with cd23 expression by predicate second-line testing, a diagnosis of a b-cell cll was confirmed in the clb tube with positive ror-1 and cd43 expression. true negatives were those cases where there was no disease noted, either by predicate clearllab and second-line testing or by test method with duraclone panel testing. false positives were defined as those cases where there was no disease noted on predicate clearllab with in-house second-line testing, but the disease was noted by respective duraclone analysis. likewise, false negatives were defined as disease on predicate first-line clearllab and second-line testing, but no disease by respective duraclone panel testing. results normal and abnormal b progenitor cells panel twenty samples were tested with the duraclone alb panel. the duraclone re alb evaluation on both routine diagnostic precursor b-all samples, and patient samples who were being followed up after therapy for precursor b-all, revealed 100% positive and negative agreement to clearllab 10c reported outcomes (table 3). the estimated sensitivity and specificity rates were both 100%. cd58 expression (an additional marker included in the duraclone alb tube, but not clearllab tubes) enabled further confirmation of the diagnostic outcome reported; here, there was consistent under-expression of cd58 noted on haematogones and consistent over-expression seen on abnormal precursor b-cells (blasts), verifying manufacturer-described cd58 expression. cd22 apc expression (that was added as an additional marker panel in two precursor b-all cases to demonstrate that the addition was possible) was verified against the expected disease outcome (positive in two cases of b-all) and met the manufacturer’s specifications in the duraclone re alb (table 3). table 3: comparison of predicate reagents and duraclone re alb reagent during the assessment of normal and abnormal precursor b-cells undertaken at the charlotte maxeke johannesburg academic hospital flow cytometry laboratory in johannesburg, south africa, april 2020 to august 2020. chronic b-cell lymphoproliferative disorder panel the duraclone re clb panel identified the b-cell target population using a combination of cd19 and cd5 or cd20, as well as surface cd81, ror1, cd79b, and cd43 expression. twenty samples were tested with the duraclone clb panel. the calculated sensitivity and specificity were 100%, with full diagnostic concordance noted across all cases evaluated (n = 20). all cll cases (n = 16/20) showed under-expression (weak) of cd81, cd79b and cd20 (table 4). further, surface ror1 expression was detected in 15 of the 16 (93.75%) cd5-expressing cll cases; all cll showed expression of cd43. a single case of follicular lymphoma (n = 1) showed a bright expression of cd79b. three cases (15%) evaluated could not be definitively sub-classified with the in-house second-line panel analysis or the duraclone re clb reagents. an additional control sample (not included in table 2), with confirmed 20% of b-cell precursors, revealed and confirmed bright cd81 on the normal precursor b-cells (figure 1). table 4: comparison of predicate reagents and duraclone re clb reagent during the assessment of mature b-cell lymphoid proliferations undertaken at the charlotte maxeke johannesburg academic hospital flow cytometry laboratory in johannesburg, south africa, april 2020 to august 2020. normal and abnormal plasma cells panel seventeen samples were tested with the duraclone pc panel. a plasma cell population was identified by backbone markers cd138 and cd38 in the duraclone re pc panel, with subsequent determination of expression of cd19, cd27, cd45, cd56 and cd200. there was cross-panel marker equivalency to the existing in-house panels (table 5) and clinical outcomes, with 100% agreement achieved, and estimated sensitivity and specificity rates of 100%. in addition, the assessment of cd27 and cd81 on target plasma cells revealed consistent under-expression of cd81 or cd27 (n = 14/14) (figure 2). a single (n = 1) pcd had positive surface cd117 expression (figure 2) on the aberrant identified plasma cell population, in agreement with cd117 expression noted in the matching clearllab 10c m2 analysis. table 5: comparison of predicate reagents and duraclone re pc reagent during assessment of normal and abnormal plasma cell populations undertaken at the charlotte maxeke johannesburg academic hospital flow cytometry laboratory in johannesburg, south africa, april 2020 to august 2020. discussion the recently evaluated,9,16 fixed-tube, standardised, pre-titrated reagent system, clearllab 10c (including the b-cell, t-cell and m1 and m2 tubes), and clearllab ls11 were recently implemented to replace our outdated facscalibur/paint-a-gate method for routine immunophenotypic investigation of haematological neoplasms at our laboratory. this new system provides flow cytometric workup for haematological neoplasms and enables processing up to 400 samples a month. previously, leukaemia flow cytometry testing was limited to simple 2–4-colour analysis; sample setup was manually intensive and error-prone. systems previously implemented into our laboratory streamlined standardised and automated testing and improved workflow and quality of reporting.25,26,27 similarly, implementing the clearllab 10c and ls system in our laboratory16 has markedly improved workflow and minimised errors while providing standardised and substantially better quality-controlled sample and data acquisition and comprehensive data. the latter allows for improved target population identification, especially for small populations. complementary and supplementary multicolour panel options are published3,4,10,19,28 and could theoretically be assembled as required in the lab to provide additional marker panels needed to complete full patient immunophenotypic investigation. however, setting up quality-controlled multicolour panels in lowand middle-income countries is both challenging and less reliable16. the clearllab system facilitates detailed diagnosis across a broad range of acute leukaemias, and is sufficiently sensitive to detect the presence of a mature b-cell lpd, but it lacks specificity to discern and diagnose different mature b-cell lpd9 or confirm pcds. for example, although the b-cell panel is useful to differentiate precursor b-cell from more mature b-cell lpd, the panel does not accommodate sufficient markers to discern different types of mature lpds, including cd23, cd43, cd79b and fmc-7 markers necessary to distinguish b-cell lpd per the who classification.24 likewise, markers, such as cd58, for diagnosis for subsequent follow-up of precursor b-all, or cd79a and cd81, would be of value in the b-cell panel to discern neoplasm from haematogones. plasma cell dyscrasias are also not easily identified in the clearllab system, which lacks cd138 and other markers useful for diagnoses and follow-up of pcd.22,29,30 although cd38, cd56, and cd45, used to identify plasma cells, are included in the clearllab panels, plasma cell identification is not definitive. specifically, cd56, included in the t-cell panel, is assessed separately from cd38 (included in the b-cell and m2 panels). a pre-titrated-monoclonal panel including all three markers would suitably enhance the clearllab system; the duraclone panels used in this study provided this supplement format. normal and abnormal b progenitor cells panel the expression of cd10, cd19, cd34, cd38, cd20 and cd45 in the duraclone re alb panel showed excellent cross-panel marker expression equivalency and concordant clinical diagnostic outcome when compared to clearllab 10c b-cell panel expression. cd58 together with cd38, cd10, cd19, cd34, cd20 and cd45 has potential for sensitive minimal residual disease assessment31,32 along with cd81 expression.31 cd58 in the index test alb tube proved to be an excellent adjunct for assessing precursor b-cells and was helpful in discerning abnormal precursor b-cells from normal precursor b-cells (haematogones)31. distinct discordant cd123 and cd34 expression patterns have also been described on haematogones and can be used in a strategy to determine b-all minimal residual disease.32 cd123 was not however included in the alb tube. in our predicate method, the clearllab 10c system, co-expression of cd123 with cd34 and cd19 assessment within the clearllab m2 panel, used to identify precursor b-cells, together with cd19, cd10 and cd34 co-expression in the clearllab b-cell tube, therefore offers more diagnostic information than the duraclone alb tube alone for the follow-up of precursor b-all and residual disease assessment. the clearllab 10c system was developed to diagnose early and later b-cell lymphoproliferative disorders; these 10-colour panels are fixed. thus, additional markers cannot be tested for in the panels. however, additional markers can be added to the re alb, fine-tuned toward detecting early b-cell precursors only. cd22 and cd123 could be valuable additions to the alb panel. for example, a liquid reagent drop-in of cd22-apc in the free apc fluorescence channel of the duraclone re alb panel could provide a baseline assessment of cd22 expression on b-all blasts, which would be useful for deciding on anti-cd22 targeted therapy in b-all patients. cd123 could also be added in a second available channel to enable further discrimination of disease (b-all) from normal haematogones (see table 2). chronic b-cell lymphoproliferative disorder panel the expression of cd5, cd19, cd20 and cd45 in the duraclone re clb reagent showed excellent cross-panel marker expression, equivalent to clearllab 10c b panel expression, with 100% (n = 20/20) and clinical diagnostic outcomes agreement. the additional markers in the duraclone re clb panel, including cd81, ror1, cd79b and cd43 which are not included in the laboratory predicate clearllab system, specifically the b-cell tube, met the requirements for intended use and the manufacturer performance specifications. the duraclone re clb panel proved to be an excellent fixed supplementary panel to replace our outdated second-line investigation for the workup of mature b-cell lymphoid proliferations and proved to be especially useful for discerning cd19/cd5 positive haematolymphoid disease subtypes. the surface cd81, cd79b and cd20 showed consistent under-expression in cll, while the presence of ror1 was also helpful in discerning cll over mantle cell lymphoma as described in previous studies,17,18,33 especially when interpreted in conjunction with cd200 expression in the clearllab b-cell panel.34,35 in the event of a cd19/cd10 co-expressing mature b-cell lymphoid proliferation, cd43 in the duraclone re clb panel was shown to be a useful cell surface marker to discriminate follicular lymphoma from a high-grade b-cell lymphoma. similarly, cd79b within the duraclone re clb panel, read together with cd38 expression in the clearllab b-cell tube, identifies a mature b-cell lymphoid proliferation with plasmacytoid differentiation. as for the alb panel, free channels in the panel allow for additional flexibility to investigate related markers that are regarded as useful to discern mature b-cell haematolymphoid subtypes. a liquid reagent drop-in of cd23-ecd in the duraclone re clb panel could further confirm b-cell cll and discern b-cell cll from other cd19/cd5 co-expressing target populations.17,18,33 normal and abnormal plasma cells panel the cd38, cd138, cd45, cd19, cd56, and cd200 expressions in the duraclone re pc panel showed excellent cross-panel marker expression and equivalent diagnostic outcome and facscalibur/pag analysis outcomes, with 100% (n = 17/17) clinical diagnostic concordance (agreement). in addition, as previously described,29 cd27 and cd8130 proved to be valuable markers to discern aberrant plasma cells over normal plasma cells. finally, the usefulness of the re pc panel can be further extended by adding cd117-ecd30 reagents. for one sample, we added cd117-ecd reagents to the pc tube, confirming the presence of malignant plasma cells and minimal residual disease assessment.14,30. limitations firstly, the sample size per panel evaluated is small, and further studies are needed to confirm the outcomes reported here. secondly, the fluorochromes used in the panels are specifically designed for use on a bc navios instrument. therefore, the products could be used on alternative instruments only if the respective filter setups23 (of the particular laboratory’s flow cytometer) can accommodate data collection from the fluorochromes used in the clearllab 10c or duraclone panels. conclusion this study confirms that the bc duraclone alb and clb panels are suitable to provide additional second-line immunophenotypic workup of precursor b-all and mature b-cell lymphoproliferative disorders resepectively, at disease presentation and are suitable to supplement first-line clearllab 10c laboratory predicate method testing. the under-expression of cd81, cd79b, and positive cd43 and ror1 expression, noted in the duraclone clb panel specifically assists in distinguishing b-cell cll from mantle cell lymphoma and other cd5 negative b-cell lpds. the duraclone re pc panel is suitable for the second-line investigation of pcd. cd27, cd56, cd81 and cd200, together with cd19, cd38 and cd138 in a single analysis, were efficient in identifying aberrant plasma cells. although there was diagnostic and individual marker concordance, we did not find the alb more useful for diagnosing b-all over our existing clearllab system (utilising the b-cell and m2 tubes). the inclusion of cd5831 in the re alb tube may however be a valuable adjunct for discerning normal reactive b-cell precursors from the minimal residual disease at follow-up. lastly, the potential benefit of using commercialised multicolour lyophilised fixed panel preparations having stable, standardised antibody reagents reduces the risks of technical error, improves laboratory efficiency, and simplifies reagent inventory. in addition, the duraclone ‘free fluorescence channels’ provide some additional flexibility for a laboratory to use their preferred markers to establish diseases (or not) of their choice. acknowledgements d.k.g. and d.l. thank mrs merriam machaba at charlotte maxeke johannesburg academic hospital flow cytometry. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions d.k.g. conceptualised, led and funded the study, provided project administration and project supervision as well as supervision of all laboratory testing and data analysis. d.l. and d.k.g. oversaw technical aspects and the setup of protocols for flow cytometric data acquisition and data analysis. l.s. and m.p. analysed flow cytometric data and recorded the outcomes in spreadsheets for analysis comparison. d.k.g. undertook final checking of data and compiled the final laboratory validation report. l.s. and d.k.g. translated the validation report into a first draft for publication format. all subsequent drafts were written by d.k.g. sources of support funding for this study was made possible with research incentive funds accrued to d.k.g. through the university of the witwatersrand. data availability all outcomes are anonymised and published in the maunscript. individal raw flow cytometric listmode data files are available on request from the corresponding author, d.k.g. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any of the affiliated agencies of the authors. references borowitz mj, wood bl, devidas m, et al. prognostic significance of minimal residual disease in high risk b-all: a report from children’s oncology group study aall0232. blood. 2015;126(8):964–971. https://doi.org/10.1182/blood-2015-03-633685 theunissen p, mejstrikova e, sedek l, et al. standardized flow cytometry for highly sensitive mrd measurements in b-cell acute lymphoblastic leukemia. blood. 2017;129(3):347–357. https://doi.org/10.1182/blood-2016-07-726307 van dongen jj, lhermitte l, bottcher s, et al. euroflow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes. leukemia. 2012;26(9):1908–1975. https://doi.org/10.1038/leu.2012.120 kalina t, flores-montero j, van der velden vh, et al. euroflow standardization of flow cytometer instrument settings and immunophenotyping protocols. leukemia. 2012;26(9):1986–2010. https://doi.org/10.1038/leu.2012.122 glier h, heijnen i, hauwel m, et al. standardization of 8-color flow cytometry across different flow cytometer instruments: a feasibility study in clinical laboratories in switzerland. j immunol methods. 2019;475:112348. https://doi.org/10.1016/j.jim.2017.07.013 kalina t, brdickova n, glier h, et al. frequent issues and lessons learned from euroflow qa. j immunol methods. 2019;475:112520. https://doi.org/10.1016/j.jim.2018.09.008 davis bh, holden jt, bene mc, et al. 2006 bethesda international consensus recommendations on the flow cytometric immunophenotypic analysis of hematolymphoid neoplasia: medical indications. cytometry b clin cytom. 2007;72 suppl 1:s5–s13. https://doi.org/10.1002/cyto.b.20365 hedley bd, cheng g, luider j, et al. initial flow cytometric evaluation of the clearllab lymphoid screen. cytometry b clin cytom. 2018;94(5):707–713. https://doi.org/10.1002/cyto.b.21603 hedley bd, cheng g, keeney m, et al. a multicenter study evaluation of the clearllab 10c panels. cytometry b clin cytom. 2020. flores-montero j, grigore g, fluxa r, et al. euroflow lymphoid screening tube (lst) data base for automated identification of blood lymphocyte subsets. j immunol methods. 2019;475:112662. https://doi.org/10.1016/j.jim.2019.112662 hedley bd, keeney m, popma j, chin-yee i. novel lymphocyte screening tube using dried monoclonal antibody reagents. cytometry b clin cytom. 2015;88(6):361–370. https://doi.org/10.1002/cyto.b.21251 van der velden vhj, flores-montero j, perez-andres m, et al. optimization and testing of dried antibody tube: the euroflow lst and pidot tubes as examples. j immunol methods. 2019;475:112287. https://doi.org/10.1016/j.jim.2017.03.011 correia rp, rajab a, bento lc, et al. a ten-color tube with dried antibody reagents for the screening of hematological malignancies. int j lab hematol. 2018;40(2):136–143. https://doi.org/10.1111/ijlh.12753 bouriche l, bernot d, nivaggioni v, arnoux i, loosveld m. detection of minimal residual disease in b cell acute lymphoblastic leukemia using an eight-color tube with dried antibody reagents. cytometry b clin cytom. 2019;96(2):158–163. https://doi.org/10.1002/cyto.b.21766 bento l, correia r, de sousa f, et al. performance of eight-color dry antibody reagent in the detection of minimal residual disease in chronic lymphocytic leukemia samples. cytometry b clin cytom. 2020;98(6):529–535. https://doi.org/10.1002/cyto.b.21875 glencross dk, pretorius m, swart l, lawrie d. evaluation of the beckman coulter (bc) clearllab 10c leukaemia/lymphoma immunophenotyping panel system in a busy, high-volume academic laboratory in johannesburg. afr j lab med. 2022;11(1):a1458. https://doi.org/10.4102/ajlm.v11i1.1458 rawstron ac, fazi c, agathangelidis a, et al. a complementary role of multiparameter flow cytometry and high-throughput sequencing for minimal residual disease detection in chronic lymphocytic leukemia: an european research initiative on cll study. leukemia. 2016;30(4):929–936. https://doi.org/10.1038/leu.2015.313 rawstron ac, kreuzer ka, soosapilla a, et al. reproducible diagnosis of chronic lymphocytic leukemia by flow cytometry: an european research initiative on cll (eric) & european society for clinical cell analysis (escca) harmonisation project. cytometry b clin cytom. 2018;94(1):121–128. https://doi.org/10.1002/cyto.b.21595 roshal m, flores-montero ja, gao q, et al. mrd detection in multiple myeloma: comparison between mskcc 10-color single-tube and euroflow 8-color 2-tube methods. blood adv. 2017;1(12):728–732. https://doi.org/10.1182/bloodadvances.2016003715 bossuyt pm, reitsma jb, bruns de, et al. towards complete and accurate reporting of studies of diagnostic accuracy: the stard initiative. clin chem lab med. 2003;41(1):68–73. https://doi.org/10.1515/cclm.2003.012 united kingdom national external quality assessment scheme (u.k. neqas) u. leukaemia immunophenotyping part 1 and part 2 [homepage on the internet]. [cited 2021 aug]. available from: http://www.ukneqasli.co.uk/eqa-pt-programmes/flow-cytometry-programmes/leukaemia-immunophenotyping-part-1-accredited/ wang hw, lin p. flow cytometric immunophenotypic analysis in the diagnosis and prognostication of plasma cell neoplasms. cytometry b clin cytom. 2019;96(5):338–350. https://doi.org/10.1002/cyto.b.21844 beckman coulter sa. duraclone panels 2021 [homepage on the internet]. [cited 2021 aug]. available from: https://www.beckman.co.za/reagents/coulter-flow-cytometry/antibodies-and-kits/duraclone-panels campo e, harris nl, jaffe es, et al. world health organization (who) classification of tumours of haematopoietic and lymphoid tissues (medicine). geneva: who; 2017.. glencross dk, coetzee lm, cassim n. an integrated tiered service delivery model (itsdm) based on local cd4 testing demands can improve turn-around times and save costs whilst ensuring accessible and scalable cd4 services across a national programme. plos one. 2014;9(12):e114727. https://doi.org/10.1371/journal.pone.0114727 glencross dk, janossy g, coetzee lm, et al. large-scale affordable panleucogated cd4+ testing with proactive internal and external quality assessment: in support of the south african national comprehensive care, treatment and management programme for hiv and aids. cytometry b clin cytom. 2008;74 suppl 1:s40–s51. https://doi.org/10.1002/cyto.b.20384 coetzee lm, glencross dk. performance verification of the new fully automated aquios flow cytometer panleucogate (plg) platform for cd4-t-lymphocyte enumeration in south africa. plos one. 2017;12(11):e0187456. https://doi.org/10.1371/journal.pone.0187456 novakova m, glier h, brdickova n, et al. how to make usage of the standardized euroflow 8-color protocols possible for instruments of different manufacturers. j immunol methods. 2019;475:112388. https://doi.org/10.1016/j.jim.2017.11.007 flores-montero j, de tute r, paiva b, et al. immunophenotype of normal vs. myeloma plasma cells: toward antibody panel specifications for mrd detection in multiple myeloma. cytometry b clin cytom. 2016;90(1):61–72. https://doi.org/10.1002/cyto.b.21265 chen f, hu y, wang x, fu s, liu z, zhang j. expression of cd81 and cd117 in plasma cell myeloma and the relationship to prognosis. cancer med. 2018;7(12):5920–5927. https://doi.org/10.1002/cam4.1840 tsitsikov e, harris mh, silverman lb, sallan se, weinberg ok. role of cd81 and cd58 in minimal residual disease detection in pediatric b lymphoblastic leukemia. int j lab hematol. 2018;40(3):343–351. https://doi.org/10.1111/ijlh.12795 zeidan ma, kamal hm, el shabrawy da, esh am, sattar rh. significance of cd34/cd123 expression in detection of minimal residual disease in b-acute lymphoblastic leukemia in children. blood cells mol dis. 2016;59:113–118. https://doi.org/10.1016/j.bcmd.2016.05.005 broome he, rassenti lz, wang hy, meyer lm, kipps tj. ror1 is expressed on hematogones (non-neoplastic human b-lymphocyte precursors) and a minority of precursor-b acute lymphoblastic leukemia. leuk res. 2011;35(10):1390–1394. https://doi.org/10.1016/j.leukres.2011.06.021 kohnke t, wittmann vk, bucklein vl, et al. diagnosis of cll revisited: increased specificity by a modified five-marker scoring system including cd200. br j haematol. 2017;179(3):480–487. https://doi.org/10.1111/bjh.14901 mora a, bosch r, cuellar c, et al. cd200 is a useful marker in the diagnosis of chronic lymphocytic leukemia. cytometry b clin cytom. 2019;96(2):143–148. https://doi.org/10.1002/cyto.b.21722 africa has suffered disproportionately although it has done little to cause the crisis the fight against the climate crisis needs all hands on deck acknowledgements references about the author(s) lukoye atwoli department of internal medicine, medical college east africa, the aga khan university, nairobi, kenya brain and mind institute, the aga khan university, nairobi, kenya gregory e. erhabor department of medicine, college of health sciences, obafemi awolowo university, ile-ife, nigeria chest unit, obafemi awolowo university teaching hospital, ile-ife, nigeria aiah a. gbakima department of technical and higher education, government of sierra leone, freetown, sierra leone abraham haileamlak college of public health and medical sciences, jimma university, jimma, ethiopia jean-marie kayembe ntumba department of pneumology, faculty of medicine, university of kinshasa, kinshasa, the democratic republic of the congo james kigera department of human anatomy, college of health sciences, university of nairobi, nairobi, kenya laurie laybourn-langton department of sustainability accelerator, chatham house, london, united kingdom robert mash division of family medicine and primary care, faculty of medicine and health sciences, stellenbosch university, cape town, south africa joy muhia centre for global mental health, london school of hygiene and tropical medicine, london, united kingdom fhumulani m. mulaudzi department of nursing science, faculty of health sciences, university of pretoria, pretoria, south africa david ofori-adjei department of medicine and therapeutics, university of ghana, accra, ghana friday okonofua department of obstetrics and gynecology, university of medical sciences, ondo, nigeria arash rashidian department of science, information and dissemination, eastern mediterranean regional office, world health organization, cairo, egypt maha el-adawy department of health protection and promotion, eastern mediterranean regional office, world health organization, cairo, egypt siaka sidibé faculty of medicine and odonto-stomatology, university of sciences, techniques and technology of bamako, bamako, mali abdelmadjid snouber faculty of medicine, university of oran 1, es sénia, algeria james tumwine department of paediatrics and child health, school of medicine, kabale university, kabale, uganda sahar yassien mohammad faculty of nursing, ain shams university, cairo, egypt paul yonga ca medlynks clinic and laboratory, nairobi, kenya nairobi fountain projects and research office (fopro), fountain health care hospital, eldoret, kenya lilia zakhama faculty of medicine of tunis, university of tunis el manar, tunis, tunisia department of cardiology, security forces hospital, la marsa, tunisia chris zielinski centre for global health, faculty of health and wellbeing, university of winchester, winchester, united kingdom citation atwoli l, erhabor ge, gbakima aa, et al. cop27 climate change conference: urgent action needed for africa and the world. afr j lab med. 2022;11(1), a2080. https://doi.org/10.4102/ajlm.v11i1.2080 editorial cop27 climate change conference: urgent action needed for africa and the world lukoye atwoli, gregory e. erhabor, aiah a. gbakima, abraham haileamlak, jean-marie kayembe ntumba, james kigera, laurie laybourn-langton, robert mash, joy muhia, fhumulani m. mulaudzi, david ofori-adjei, friday okonofua, arash rashidian, maha el-adawy, siaka sidibé, abdelmadjid snouber, james tumwine, sahar yassien mohammad, paul yonga, lilia zakhama, chris zielinski copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. wealthy nations must step up support for africa and vulnerable countries in addressing past, present and future impacts of climate change the 2022 report of the intergovernmental panel on climate change (ipcc) paints a dark picture of the future of life on earth, characterised by ecosystem collapse, species extinction, and climate hazards such as heatwaves and floods.1 these are all linked to physical and mental health problems, with direct and indirect consequences of increased morbidity and mortality. to avoid these catastrophic health effects across all regions of the globe, there is broad agreement – as 231 health journals argued together in 2021 – that the rise in global temperature must be limited to less than 1.5°c compared with pre-industrial levels. while the paris agreement of 2015 outlines a global action framework that incorporates providing climate finance to developing countries, this support has yet to materialise.2 cop27 is the fifth conference of the parties (cop) to be organised in africa since its inception in 1995. ahead of this meeting, we – as health journal editors from across the continent – call for urgent action to ensure it is the cop that finally delivers climate justice for africa and vulnerable countries. this is essential not just for the health of those countries, but for the health of the whole world. africa has suffered disproportionately although it has done little to cause the crisis the climate crisis has had an impact on the environmental and social determinants of health across africa, leading to devastating health effects.3 impacts on health can result directly from environmental shocks and indirectly through socially mediated effects.4 climate change-related risks in africa include flooding, drought, heatwaves, reduced food production, and reduced labour productivity.5 droughts in sub-saharan africa have tripled between 1970–1979 and 2010–2019.6 in 2018, devastating cyclones impacted 2.2 million people in malawi, mozambique and zimbabwe.6 in west and central africa, severe flooding resulted in mortality and forced migration from loss of shelter, cultivated land, and livestock.7 changes in vector ecology brought about by floods and damage to environmental hygiene has led to increases in diseases across sub-saharan africa, with rises in malaria, dengue fever, lassa fever, rift valley fever, lyme disease, ebola virus, west nile virus and other infections.8,9 rising sea levels reduce water quality, leading to water-borne diseases, including diarrhoeal diseases, a leading cause of mortality in africa.8 extreme weather damages water and food supply, increasing food insecurity and malnutrition, which causes 1.7 million deaths annually in africa.10 according to the food and agriculture organization of the united nations, malnutrition has increased by almost 50% since 2012, owing to the central role agriculture plays in african economies.11 environmental shocks and their knock-on effects also cause severe harm to mental health.12 in all, it is estimated that the climate crisis has destroyed a fifth of the gross domestic product (gdp) of the countries most vulnerable to climate shocks.13 the damage to africa should be of supreme concern to all nations. this is partly for moral reasons. it is highly unjust that the most impacted nations have contributed the least to global cumulative emissions, which are driving the climate crisis and its increasingly severe effects. north america and europe have contributed 62% of carbon dioxide emissions since the industrial revolution, whereas africa has contributed only 3%.14 the fight against the climate crisis needs all hands on deck yet it is not just for moral reasons that all nations should be concerned for africa. the acute and chronic impacts of the climate crisis create problems like poverty, infectious disease, forced migration, and conflict that spread through globalised systems.6,15 these knock-on impacts affect all nations. covid-19 served as a wake-up call to these global dynamics and it is no coincidence that health professionals have been active in identifying and responding to the consequences of growing systemic risks to health. but the lessons of the covid-19 pandemic should not be limited to pandemic risk.16,17 instead, it is imperative that the suffering of frontline nations, including those in africa, be the core consideration at cop27: in an interconnected world, leaving countries to the mercy of environmental shocks creates instability that has severe consequences for all nations. the primary focus of climate summits remains to rapidly reduce emissions so that global temperature rises are kept to below 1.5 °c. this will limit the harm. but, for africa and other vulnerable regions, this harm is already severe. achieving the promised target of providing $100bn of climate finance a year is now globally critical if we are to forestall the systemic risks of leaving societies in crisis. this can be done by ensuring these resources focus on increasing resilience to the existing and inevitable future impacts of the climate crisis, as well as on supporting vulnerable nations to reduce their greenhouse gas emissions: a parity of esteem between adaptation and mitigation. these resources should come through grants not loans, and be urgently scaled up before the current review period of 2025. they must put health system resilience at the forefront, as the compounding crises caused by the climate crisis often manifest in acute health problems. financing adaptation will be more cost-effective than relying on disaster relief. some progress has been made on adaptation in africa and around the world, including early warning systems and infrastructure to defend against extremes. but frontline nations are not compensated for impacts from a crisis they did not cause. this is not only unfair, but also drives the spiral of global destabilisation, as nations pour money into responding to disasters, but can no longer afford to pay for greater resilience or to reduce the root problem through emissions reductions. a financing facility for loss and damage must now be introduced, providing additional resources beyond those given for mitigation and adaptation. this must go beyond the failures of cop26 where the suggestion of such a facility was downgraded to ‘a dialogue’.18 the climate crisis is a product of global inaction, and comes at great cost not only to disproportionately impacted african countries, but to the whole world. africa is united with other frontline regions in urging wealthy nations to finally step up, if for no other reason than that the crises in africa will sooner rather than later spread and engulf all corners of the globe, by which time it may be too late to effectively respond. if so far they have failed to be persuaded by moral arguments, then hopefully their self-interest will now prevail. acknowledgements this editorial is being published simultaneously in multiple journals. for the full list of journals see: https://www.bmj.com/content/full-list-authors-and-signatories-climate-emergency-editorial-october-2022. competing interests we have read and understood the bmj policy on declaration of interests and declare the following roles and relationships: j.k. is the ex-officio, president and secretary of the kenya orthopedic association, and the editor in chief of annals of african surgery. r.m. received a research grant on climate change and primary health care in africa with university ghent, funded by vlir (flemish interuniversity network). p.y. received payment for a cme lecture on antimicrobial use in the intensive care unit (icu) on behalf of novartis, for an educational event on antimicrobial stewardship on behalf of biomerieux, and for a cme lecture on adult vaccination updates on behalf of pfizer. p.y. also serves on the dsmb of the nhlbi-funded strengths trial focusing on hypertension and has served as a chair on a pfizer advisory board on pneumococcal vaccinations in adults. payment is received for both. c.z. received payment from the uk health alliance on climate change as senior advisor on the international journals project. j.m. is an unpaid board member on the international working group for health systems strengthening and employee of london school of hygiene & tropical medicine. doa has involvement with the inovio pharmaceuticals phase 1b vaccine trial and the glico healthcare ltd. the authors declare no further conflicts of interest beyond those inherent in the editorial roles listed above. authors’ contributions l.l-l. developed the idea of the editorial and led drafting along with c.z. j.m. contributed with l.a.’s guidance. all other authors contributed significantly to the editorial content. authors’ affiliated journals lukoye atwoli, editor-in-chief, east african medical journal; gregory e. erhabor, editor-in-chief, west african journal of medicine; aiah a. gbakima, editor-in-chief, sierra leone journal of biomedical research; abraham haileamlak, editor-in-chief, ethiopian jo urnal of health sciences; jean-marie kayembe ntumba, chief editor, annales africaines de medecine; james kigera, editor-in-chief, annals of african surgery; laurie laybourn-langton, university of exeter; bob mash, editor-in-chief, african journal of primar y health care & family medicine; joy muhia, london school of medicine and tropical hygiene; fhumulani mavis mulaudzi, editor-in-chief, curationis; david ofori-adjei, editor-in-chief, ghana medical journal; friday okonofua, editor-in-chief, african journal of reproductive health; arash rashidian, executive editor, and maha el-adawy, director of health promotion, eastern mediterranean health journal; siaka sidibé, director of publication, mali médical; abdelmadjid snouber, managing editor, journal de la facul té de médecine d’oran; james tumwine, editor-in-chief, african health sciences; mohammad sahar yassien, editor-in-chief, evidence-based nursing research; paul yonga, managing editor, east african medical journal; lilia zakhama, editor-in-chief, la tunisie médicale; chris zielinski, university of winchester. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. funding information respective authors were paid by their employers. chris zielinski’s time was funded by the uk health alliance on climate change. data availability data sharing is not applicable to this article, as no new data were created or analysed in this study. disclaimer the views and opinions in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references ipcc. climate change 2022: impacts, adaptation and vulnerability. working group ii contribution to the ipcc sixth assessment report. cambridge, uk and new york, ny: cambridge university press; 2022. un. the paris agreement [homepage on the internet]. united nations; 2022 [cited 2022 sep 12]. available from: https://www.un.org/en/climatechange/paris-agreement climate investment funds. climate change and health in sub-saharan africa: the case of uganda. climate investment funds; 2020. who. strengthening health resilience to climate change. geneva: world health organization; 2016. trisos ch, adelekan io, totin e, et al. africa [homepage on the internet]. in: climate change 2022: impacts, adaptation, and vulnerability. cambridge, uk and new york, ny: cambridge university press; 2022 [cited 2022 sep 26]. available from: https://www.ipcc.ch/report/ar6/wg2/ world bank. climate change adaptation and economic transformation in sub-saharan africa. washington, dc: world bank; 2021. opoku sk, leal filho w, hubert f, adejumo o. climate change and health preparedness in africa: analysing trends in six african countries. int j environ res public health. 2021;18(9):4672. https://doi.org/10.3390/ijerph18094672 evans m, munslow b. climate change, health, and conflict in africa’s arc of instability. perspectives in public health. 2021;141(6):338–341. https://doi.org/10.1177/17579139211058299 anugwom e.e. reflections on climate change and public health in africa in an era of global pandemic. in: stawicki sp, papadimos tj, galwankar sc, miller ac, firstenberg ms, editors. contemporary developments and perspectives in international health security volume 2 [homepage on the internet]. london: intechopen; 2021 [cited 2022 sep 12]. available from: https://www.intechopen.com/chapters/76312 african climate policy centre. climate change and health in africa: issues and options [homepage on the internet]. african climate policy centre; 2013 [cited 2022 sep 12]. available from: https://archive.uneca.org/sites/default/files/publicationfiles/policy_brief_12_climate_change_and_health_in_africa_issues_and_options.pdf un. climate change is an increasing threat to africa [homepage on the internet]. 2020 [cited 2022 sep 12]. available from: https://unfccc.int/news/climate-change-is-an-increasing-threat-to-africa atwoli l, muhia j, merali z. mental health and climate change in africa. bjpsych international. 2022;19(4):86–89. https://doi.org/10.1192/bji.2022.14 vulnerable twenty group. climate vulnerable economies loss report. switzerland: vulnerable twenty group; 2020. ritchie h. who has contributed most to global co2 emissions? our world in data [homepage on the internet]. 2019 [cited 2022 sep 12]. available from: https://ourworldindata.org/contributed-most-global-co2 bilotta n, botti f. paving the way for greener central banks. current trends and future developments around the globe. rome: edizioni nuova cultura for istituto affari internazionali (iai); 2022. who. cop26 special report on climate change and health: the health argument for climate action. geneva: world health organization; 2021. al-mandhari a, al-yousfi a, malkawi m, el-adawy m. ‘our planet, our health’: saving lives, promoting health and attaining well-being by protecting the planet – the eastern mediterranean perspectives. east mediterr health j. 2022;28(4):247–248. https://doi.org/10.26719/2022.28.4.247 evans s, gabbatiss j, mcsweeney r, et al. cop26: key outcomes agreed at the un climate talks in glasgow. carbon brief [homepage on the internet]. 2021 [cited 2022 sep 12]. available from: https://www.carbonbrief.org/cop26-key-outcomes-agreed-at-the-un-climate-talks-in-glasgow/ about the author(s) lebogang skosana national health laboratory services, tshwane academic division, pretoria, south africa department of medical microbiology, university of pretoria, pretoria, south africa farzana ismail department of medical microbiology, university of pretoria, pretoria, south africa centre for tuberculosis, national institute of communicable diseases, johannesburg, south africa nontombi mbelle † national health laboratory services, tshwane academic division, pretoria, south africa department of medical microbiology, university of pretoria, pretoria, south africa mohamed said national health laboratory services, tshwane academic division, pretoria, south africa department of medical microbiology, university of pretoria, pretoria, south africa citation skosana l, ismail f, mbelle n, said m. corrigendum: brucellosis – laboratory workers’ nightmare come true: a case study. afr j lab med. 2021;10(1), a1690. https://doi.org/10.4102/ajlm.v10i1.1690 note: doi of original article published: https://doi.org/10.4102/ajlm.v9i1.1114. †, 1956–2021. correction corrigendum: brucellosis – laboratory workers’ nightmare come true: a case study lebogang skosana, farzana ismail, nontombi mbelle, mohamed said published: 22 nov. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. in the version of this article initially published, skosana l, ismail f, mbelle n, said m. brucellosis – laboratory workers’ nightmare come true: a case study. afr j lab med. 2020;9(1), a1114. https://doi.org/10.4102/ajlm.v9i1.1114, the authors’ second affiliation was omitted in the ‘author’ and ‘affiliation’ sections. the authors’ affiliations are now corrected as: authors: lebogang skosana1,2 farzana ismail2,3 nontombi mbelle1,2† mohamed said1,2 affiliations: 1national health laboratory services, tshwane academic division, pretoria, south africa 2department of medical microbiology, university of pretoria, pretoria, south africa 3centre for tuberculosis, national institute of communicable diseases, johannesburg, south africa. this correction does not alter the study’s findings of significance or overall interpretation of the study’s results. the authors apologise for any inconvenience caused. article information authors: donald j. hamel1 jean-louis sankalé2 jay osi samuels3 abdoulaye d. sarr4 beth chaplin1 eke ofuche3 seema t. meloni1 prosper okonkwo3 phyllis j. kanki1 affiliations: 1department of immunology and infectious diseases, harvard school of public health, boston, ma, united states 2globomics, llc, decatur, ga, united states 3aids prevention initiative in nigeria ltd. gte., abuja, nigeria 4global aids program malawi, centers for disease control and prevention, nico house lilongwe 3, malawi correspondence to: phyllis kanki email: pkanki@hsph.harvard.edu postal address: 651 huntington avenue fxb405, boston ma 02115, united states dates: received: 06 may 2014 accepted: 03 nov. 2014 published: 14 may 2015 how to cite this article: hamel dj, sankalé jl, samuels jo, et al. building laboratory capacity to support hiv care in nigeria: harvard / apin pepfar, 2004–2012. afr j lab med. 2015;4(1), art. #190, 10 pages. http://dx.doi.org/10.4102/ajlm.v4i1.190 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. building laboratory capacity to support hiv care in nigeria: harvard/apin pepfar, 2004–2012 in this original article... open access • abstract • introduction • research methods and design    • clinic selection    • procurement of equipment    • laboratory modifications    • supply chain    • equipment maintenance    • data management    • laboratory trainings • results    • impact on health system strengthening    • training conferences    • electronic data management    • laboratory quality control and accreditation • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ introduction: from 2004–2012, the harvard/aids prevention initiative in nigeria, funded through the us president’s emergency plan for aids relief programme, scaled up hiv care and treatment services in nigeria. we describe the methodologies and collaborative processes developed to improve laboratory capacity significantly in a resource-limited setting. these methods were implemented at 35 clinic and laboratory locations. methods: systems were established and modified to optimise numerous laboratory processes. these included strategies for clinic selection and management, equipment and reagent procurement, supply chains, laboratory renovations, equipment maintenance, electronic data management, quality development programmes and trainings. results: over the eight-year programme, laboratories supported 160 000 patients receiving hiv care in nigeria, delivering over 2.5 million test results, including regular viral load quantitation. external quality assurance systems were established for cd4+ cell count enumeration, blood chemistries and viral load monitoring. laboratory equipment platforms were improved and standardised and use of point-of-care analysers was expanded. laboratory training workshops supported laboratories toward increasing staff skills and improving overall quality. participation in a world health organisation-led african laboratory quality improvement system resulted in significant gains in quality measures at five laboratories. conclusions: targeted implementation of laboratory development processes, during simultaneous scale-up of hiv treatment programmes in a resource-limited setting, can elicit meaningful gains in laboratory quality and capacity. systems to improve the physical laboratory environment, develop laboratory staff, create improvements to reduce costs and increase quality are available for future health and laboratory strengthening programmes. we hope that the strategies employed may inform and encourage the development of other laboratories in resource-limited settings. introduction top ↑ laboratories are fundamental and essential components of health systems, providing clinical staff and patients with test results that are the basis of disease diagnosis and treatment; yet, laboratories are often neglected by governments, development organisations and other stakeholders in plans to improve healthcare systems in developing countries. despite the scale-up of global health programmes in the last decade, sub-saharan africa continues to suffer the consequences of operating with some of the most poorly-equipped and under-resourced laboratories in the world.1 as such, by 2012, the us president’s emergency plan for aids relief (pepfar) blueprint, the world health organization (who) and the united nations millennium development goals (unmdg) each called for strengthened national laboratory systems as a critical component of scaling up hiv and tuberculosis (tb) prevention and treatment programmes.2,3 based on the nigerian national hiv sentinel surveillance surveys in 2005 and 2010, the national prevalence of hiv-1 has remained fairly stable at approximately 4%.4 the harvard school of public health received pepfar funds from 2004 to 2012 to support the development of prevention, care and treatment programmes in nigeria, botswana and tanzania. in nigeria, harvard partnered with the aids prevention initiative in nigeria (apin), an organisation developed through funding from the bill and melinda gates foundation from 2000–2006, to provide evidence-based hiv prevention in four states of the country. the harvard/apin pepfar programme was built upon this foundation of hiv prevention activities and initiated support of antiretroviral therapy (art) activities at six tertiary-level facilities in 2004; this expanded to 35 clinics and laboratories by 2009. to ensure sustainability, harvard helped to establish apin ltd./gte. as an independent, nigeria-based non-governmental organisation. beginning in 2009, and fully completed in february 2012, all harvard/apin pepfar programme activity was transitioned to apin management. from the beginning of the harvard/apin pepfar programme, it was determined that a fundamental component of the capacity-building efforts would be dedicated to laboratory infrastructure, with corresponding growth of logistics management for procuring supplies and laboratory staff training in order to ensure sustainability. in developing the programme frameworks and plans, we incorporated lessons learned from previously-developed art laboratories in both nigeria and senegal so as to elicit lasting gains in laboratory capacity and infrastructure.5 in this report, we describe the organisational framework that resulted in the establishment of and continuous quality improvements to laboratory capacity in nigeria over the eight years of the harvard/apin pepfar programme (2004–2012). we highlight the collaborative process, with details on specific strategies and methodologies, found to be essential for meaningful laboratory development in a resource-limited setting. research methods and design top ↑ our programme’s laboratories were organised with large tertiary facilities at the centre, providing support to secondary hospitals and associated primary health clinics using a hub-and-spoke model (figure 1). tertiary-level laboratories were associated with university teaching hospitals or research institutes with large hiv art programmes. secondary-level hospitals provided hiv serology, cd4+ cell count enumeration, haematology and clinical chemistry testing. they also had the capacity to store plasma samples for viral load (vl) testing, and dried blood spot (dbs) samples for early infant diagnosis, for up to two weeks before transport to an associated tertiary laboratory. primary health clinics were smaller health centres that provided basic care, performed hiv rapid testing, drew blood samples for testing to be done elsewhere and referred patients to the secondary or tertiary medical facilities. figure 1: illustration of hub-and-spoke laboratory organisation. clinic selection the selection of a clinic or hospital for development of laboratory capacity to support hiv care was a complex process and required accounting for a number of factors, including patient burden, existing infrastructure, prior collaborations, geographic proximity to other programme facilities and local politics. the harvard/apin pepfar programme both consulted and collaborated with in-country partners and funding organisations so as to identify candidate clinics. after a clinic was proposed, a detailed site visit was performed to survey the existing laboratory, clinical and personnel infrastructure. there were often substantial obstacles to laboratory development as a result of the poor existing infrastructure, such as the lack of running water or dependable electrical service. reliability of utilities was essential, as the programme’s protocol for laboratory testing was substantial, requiring electricity-driven instruments (table 1). table 1: antiretroviral therapy regimen testing schedule. procurement of equipment preliminary laboratory improvements began with a needs assessment and included ensuring reliable water and electricity supply, back-up generator, security and adequate air-conditioning capacity. whilst tertiary and secondary hospital laboratories had an existing patchwork of hiv diagnostics, clinical chemistry, haematology and cd4+ cell count analysers in place, the harvard/apin pepfar programme expanded and improved access to these critical technologies. in accordance with the who’s 2008 maputo declaration,6 we attempted to provide all laboratories with the same equipment manufacturer and models, supporting standardisation of platforms across sites. standardisation of laboratory equipment allowed for streamlined training and maintenance, eased acquisition of spare parts and reduced overall costs through higher-volume orders. the availability of in-country servicing along with predicted sustainability of manufacturers, vendors and platforms, were also important factors in selection criteria. for hiv testing, following the nigerian national rapid test algorithm guidelines at the time, the determine hiv rapid test (alere medical co., japan) was provided, followed by unigold (trinity biotech plc, ireland), with statpack (chembio diagnostic systems, medford, ny, united states) as the discordant result tiebreaker. if further hiv infection confirmation was required, western blot (immunetics, boston, ma, united states) was performed. immunologic monitoring of patients’ cd4+ cell counts was performed using the flow cytometry-based cyflow counter or cyflow ii (partec gmbh, munster, germany) platform. to monitor virologic treatment response, hiv vls were measured with the manual cobas amplicor hiv-1 monitor test, version 1.5 (roche diagnostics gmbh, mannheim, germany). eligibility for art and subsequent toxicity were evaluated using relevant blood chemistry assays (table 1) on the roche cobas c311, cobas c111 or equivalent. haematology monitoring was performed using the mindray bc-3200 (mindray medical ltd, shenzen, china) or equivalent. hiv-1 drug resistance was evaluated, when indicated, using the viroseq genotyping system version 2.0 (abbott molecular, des plaines, il, united states), with sequencing results being generated on the abi genetic analyser 3130xl (applied biosystems, foster city, ca, united states). laboratory modifications many laboratories required physical alterations to existing structures or reconfigurations to improve effective, logical sample processing. a laboratory’s ideal sample flow was established, beginning at the arrival bench, where samples were logged and separated as needed. sample aliquots were then sent to individual laboratory stations for routine testing, after which samples were moved to storage and to a final station where results were recorded and sent to data entry staff for entry to patients’ records. for more advanced testing, such as deoxyrobinucleic acid polymerase chain reaction (dna pcr), different steps of the assay protocol were performed in separate rooms, with access restricted to dedicated laboratory members in order to minimise risk of contamination. biosafety and fire preparedness procedures were reviewed and revised, and appropriate biohazard waste processing was ensured. security of laboratories was addressed through both physical and policy improvements, with signage, laboratory renovations and staff trainings that ensured the exclusion of non-essential staff from laboratory spaces. laboratory data were secured in locked locations with strict access controls and were maintained according to national standards. supply chain procurement processes were developed to maximise effective purchasing of equipment and consumables, from expanding use of non-cold chain reagents and regular meetings with in-country laboratory supply sales representatives to working with supply chain management systems (scms) and the clinton health access initiative (chai) so as to secure the necessary test kits. to store all materials for distribution to the sites, two warehouses were maintained – one in the south (lagos) and one in the centre (abuja) of the country. a programme logistics manager and head pharmacist were hired and trained, working together to organise and expedite distribution of supplies to the sites using programme vehicles with transport staff and/or by means of an express courier with a negotiated service contract. equipment maintenance maintenance of equipment is a critical aspect of ensuring strong laboratory infrastructure, particularly in a resource-limited setting. most laboratories had dedicated on-site engineers with varying levels of expertise. in addition, programme engineers were hired to travel to other sites for scheduled periodic preventive maintenance as well as specific repairs. retention of skilled engineers was a serious challenge and concern; accordingly, the programme made great efforts to build local capacity and allow flexible working hours. when soliciting quotes for large equipment purchases for the programme, every effort was made to include training for local engineers and application specialists. programme engineers also traveled to the united states, europe and elsewhere in africa for trainings for specific equipment maintenance on partec cyflow analysers (partec gmbh, munster, germany), nuaire laminar flow hoods (nuaire, inc., plymouth, mn, united states) and the roche cobas platform (roche gmbh, mannheim, germany). data management the harvard/apin pepfar data management team built an easy-to-use, electronic medical records system that allowed for consolidation of laboratory, clinical and pharmacy information using the filemaker pro platform (filemaker inc., santa clara, ca, united states). wherever possible, these key programme areas were linked by local computer networks within each site. database plug-ins or utility software tools were designed in order to import electronic laboratory results directly into databases when possible. every site also had dedicated data staff to maintain the electronic patient records. all databases were uploaded on a weekly basis to a secure server for compilation by the programme data team, for reporting and monitoring purposes. in addition, all laboratories were equipped with an internet-connected desktop computer for laboratory members to use for programme-related communication and a reference resource. laboratory trainings the larger tertiary laboratories carried much of the initial responsibility for training and mentoring their smaller secondary and primary satellite laboratories. programme satellite coordinators were the principal contact persons for all laboratory personnel and communicated problems needing attention to either local site management or up to programme management. laboratory quality conferences were held annually in-country, bringing members together from laboratories of every size. each conference typically had 70 to 90 attendees and were held in various locales within nigeria. this type of meeting was ideal for advancing overall laboratory quality, addressing changes to programme policy, developing consensus decisions and allowing smaller laboratory groups to interact closely with more experienced peers. results top ↑ in total, harvard/apin pepfar helped support and develop the infrastructure at 35 laboratories in nigeria. of the 18 major sites managed, 8 were tertiary and 10 were secondary laboratories. in addition, the nigerian federal ministry of health designated 7 as centres of excellence. all laboratories were housed in permanent buildings with electricity, back-up generators, running water and basic infrastructure; a number received substantial upgrades to ensure successful operation and future sustainability. notable examples of effective laboratory reorganisation were the infrastructure upgrades at the jos university teaching hospital (juth) tertiary laboratory (figure 2) and the logical workflow renovation of the molecular laboratory at the lagos university teaching hospital tertiary laboratory (figure 3). figure 2: infrastructure updates made to jos university teaching hospital in jos, plateau state. figure 3: diagram of the molecular biology laboratory renovations, produced in collaboration with us centers for disease control and prevention, for directional workflow at the lagos university teaching hospital, lagos state. all tertiary and secondary laboratory sites provided hiv serodiagnosis through rapid test technologies, automated haematology, clinical chemistry, laser-based cd4+ cell enumeration, vl quantitation and infant dna pcr diagnosis. the primary laboratories provided access to hiv rapid testing, haematology, clinical chemistry and cd4+ cell count enumeration. starting in late 2012, apin upgraded to automated vl equipment, using the cobas ampliprep/taqman hiv-1 test, version 2.0. all tertiary and secondary laboratories had the capacity for tb diagnosis with acid-fast bacilli (afb) staining and a subset of tertiary laboratories developed the infrastructure and capacity for the identification of multidrug-resistant tb (mdr-tb) through various assays, including genexpert (cepheid, sunnyvale, ca, united states) and genotype mtbdrplus (hain lifescience gmbh, nehren, germany). we introduced screening of select groups at two sites for mdr-tb using the mtbdrplus test and proposed using this test for expanded national surveillance to the national tb control programme.7 three tertiary laboratories had abi capillary sequencers (applied biosystems, foster city, ca, united states) for hiv drug resistance testing. in support of the national early infant diagnosis (eid) programme, with the support of chai and the us centers for disease control and prevention (cdc) office in nigeria, laboratories with pcr testing capacity were also able to provide eid testing of dbs samples. stock rooms were also improved with greater security mechanisms, such as bars on all doors and windows, sturdy shelving, stock cards and clear ordering and restocking procedures. by leveraging the high volume of regular laboratory tests required by the programme, contracts were secured for significantly reduced reagent costs from most vendors. by moving from a manual dynabeads (life technologies, grand island, ny, united states) method for cd4+ cell count enumeration, to automated partec cyflow platforms, test costs were reduced initially from us$22.00/test to us$5.00/test, to a cost in 2012 of under us$2.00/test. the cost of routine chemistry tests, such as alanine aminotransferase (alt) and creatinine, dropped with the implementation of automated platforms from over us$1.00/test to approximately us$0.29/test. vl test costs using manual roche amplicor kits were initially us$33.00/test, but by maintaining a high volume of tests over time and migrating to the automated roche cobas platform, costs were reduced to us$14.00/test by 2012. starting in 2010, harvard began shifting laboratory logistics responsibilities to apin. existing vendors began to bill apin directly, and supply chains were modified to increase local procurement of consumables and test kits. changing import regulations, supplier stock-outs and local strikes necessitated occasional aid from an established non-profit organisation that could provide additional mechanisms for import and customs clearance. the achievements of the eight-year laboratory scale-up in the harvard/apin pepfar programme have been significant. from 2004–2012, harvard/apin supported laboratories were able to provide haematology, chemistry, cd4+ cell count enumeration and vl results for over 2.5 million samples (table 2, figure 4). the collaboration for eid testing expanded rapidly in nigeria, with a greater than 10-fold increase in capacity from 2007 to 2008, when over 9000 hiv exposed infants were tested. table 2: number of tests performed annually by harvard/apin pepfar laboratories. figure 4: cumulative laboratory tests performed by harvard/apin pepfar laboratories. impact on health system strengthening beyond improvements of specific laboratory services to support the ongoing art programme, the training and mentorship activities expanded the goal of providing the highest quality of overall healthcare. the programme sought to extend beyond art delivery and to integrate quality processes and equipment benefits throughout the hospitals and clinics. for example, the purchase of portable tb-diagnostic x-ray equipment became available for use by other hospital departments. training of programme staff also benefited the overall institution’s laboratory capacity-building efforts, as most programme staff also had roles in the hospitals’ non-hiv laboratories. generally, only two to three persons from each laboratory were invited to attend the annual group trainings; subsequently, conference attendees provided step-down training in order to transfer new information to the entire local laboratory team. this also incentivised laboratory managers to maintain high levels of performance, helping to ensure leadership positions for their teams in future trainings. we found the utilisation of training-of-trainer and step-down training methods to be a very cost-effective system of providing training across programme laboratories and allowed for dissemination of training across local laboratory systems. training conferences centralised laboratory conferences provided programme management the opportunity to address the laboratory staff teams directly and to acknowledge and commend their hard work. these central workshops allowed programme staff continual assurance of standardisation across programme laboratories. over time, it was realised that workshop participation could be strengthened through administration of preand post-tests encompassing major topics. additionally, these meetings offered laboratory teams an opportunity to connect with others that were conducting similar work and allowed for generation of a network that could provide local troubleshooting and support. programme-wide laboratory trainings were attended by 211 laboratory staff over 59 training days; and programme management provided direct laboratory mentorship training over 526 days. in-country teams also developed trainings for more targeted topics such as equipment maintenance, new laboratory platform initiation and accreditation preparedness, training 159 laboratory staff over 84 training days. these numbers do not account for the step-down training days or for retraining sessions that took place upon return to individual laboratory sites. if programme management identified a laboratory with specific concerns, a site-specific training visit was organised. these localised trainings addressed a wide array of issues, from laboratory staff reorganisation, to launching a trial of a new diagnostic point-of-care platform, to troubleshooting an assay performing out of range. coordination was sought with clinical and pharmacy training teams so as to extend laboratory topics to their trainings and to apprise laboratory members of any updates to other programme areas that could have an impact on laboratory functions, such as the introduction of new drugs or drug regimens with a specific toxicity concern. internal and external trainings for laboratory engineers resulted in reduced service calls to factory technicians and less equipment downtime for sites.8 properly-functioning equipment ensured that test kits were consumed in a timely manner, prior to expiration, constituting another cost-saving goal. additionally, because of both interest and observed need, workshops were held to assist laboratory and research staff with grant writing and publication skill building. electronic data management significant gains were achieved in both electronic data capture and improving delivery of laboratory results to clinical staff and patients. electronic data capture decreased the opportunity of transcription errors and allowed laboratory staff more time for laboratory activities. laboratory result turnaround times were reduced by an average of two days through the use of electronic data, compared with the prior method that consisted of only handwritten logs and manual transcription. an advantage of creating a programme-specific database system was that it offered great flexibility, allowing data managers to revise clinical chemistry results to be reported in units that conformed to international standards, or the ability to introduce modifications rapidly as laboratory technologies evolved. for example, the databases were adapted to produce pop-up flags for critical values, such as low haemoglobin or elevated liver or renal enzymes. another unique electronic tool was the ‘viral load utility’, created to convert analyser data into standardised test results for direct database import. additionally, the database system was flexible enough to allow for transfer of data to national forms and provide aggregate reporting, when the federal ministry of health developed new registers and aggregate reporting forms. a major innovation by the harvard/apin pepfar laboratory and data teams was the design and implementation of an electronic database for both compiling patient laboratory information and then distributing reports over local networks to clinic and pharmacy locations. this ‘treatment response utility’ tool was developed primarily to provide medical staff with a comprehensive picture of a patient’s treatment profile over time and to transfer a greater number of laboratory results presented in a more useful format to the clinical decision-making team (figure 5). figure 5: example of a patient history in the treatment response utility. the utility provides a graphical layout that displays a timeline with vl, cd4+ cell count and drug pick-up data, as well as links to a more detailed clinical history for a particular patient. the visual snapshot aids the physicians in communicating and educating the patient on the positive health effects of arv drug adherence. the tool also assists clinicians in detecting failure of a drug regimen, assisting in a more rapid switch to a new drug regimen, or intervening for patients struggling with adherence. laboratory quality control and accreditation at the onset of the harvard/apin pepfar programme, very few laboratories had quality management systems in place. as laboratories became equipped and staff were trained, data quality processes and laboratory quality assurance systems were instituted. in-country external quality assurance (eqa) was scaled up, with distribution of standardised controls for vl testing. harvard/apin pepfar-supported laboratories subscribed to eqa programmes distributed by the college of american pathologists for blood chemistry and viral markers, as well as the united kingdom national external quality assessment service for cd4+ cell count monitoring. in addition, eqa for eid through dbs testing was conducted through the international laboratory branch of the cdc’s division of global hiv/aids. an eqa programme was not available for the viroseq drug resistance genotyping; however, genotyping and sequence analysis were verified at harvard. whilst being costly, the international eqa programmes were a requirement of laboratory accreditation programmes, and resulted in marked improvements in reported results for all laboratories by the second year of subscription. since 2009, addressing long-term sustainability, the harvard/apin pepfar laboratories have partnered with the nigerian national external quality assessment laboratory, managed by the medical laboratory science council of nigeria, to expand an accredited national eqa programme in lieu of international subscriptions. the slmta programme was launched in kigali, rwanda in 2009 by the who regional office for africa (who afro), the cdc, the chai and the american society for clinical pathology in an effort to promote accessible pathways to accreditation in sub-saharan africa.1 six laboratories from the harvard/apin programme were selected for inclusion in the initial slmta rollout in 2010. these initial laboratories were nominated by the government of nigeria and cdc’s office in nigeria and achieved marked improvements from 2010 to 2012 (table 3). table 3: slmta assessment update of harvard/apin pepfar-supported labs. six tertiary laboratories enrolled in the initial slmta rollout in 2010 and achieved exit scores of five stars (one laboratory) and four stars (five laboratories) on a five-star scale. the one five-star laboratory has also been iso 9001 certified and plans are in place to move additional secondary laboratory sites into future slmta quality assessment programmes.9 this programme has served as a springboard, not just for the initial laboratories enrolled, but also for all laboratories in the harvard/apin programme, to focus on the who afro assessment scheme and to make dramatic improvements to laboratory quality processes. discussion top ↑ quality laboratory services have become a foundation of the harvard/apin pepfar programmes in nigeria and capacity has grown to include automated clinical chemistries and haematology for monitoring art toxicity at 24 laboratories, pcr-based vl monitoring and eid at 10 laboratories and capillary-based genetic sequencing for hiv drug-resistance mutations at 3 laboratories. over the course of the programme, the number of patients supported with hiv care rose from 2439 in 2004 to 159 897 by 2012 and our programme laboratories provided over 2.5 million laboratory test results for these patients. in addition, many sites achieved documented improvements in quality services as they moved through the slmta programme toward accreditation. many of the outcomes detailed in this article originated from major international investments to expand global health programmes in the developing world (eg. pepfar). such scale-ups could represent an opportunity to apply proven methods and novel approaches to effect meaningful improvement in local laboratory capacities. sustainability of all laboratory improvement endeavours must be considered carefully, with attention being given to the realities of economic constraints, transitions to in-country support and management and integration with national strategic plans. it is critical to build a solid foundation of local laboratory leadership that can maintain improvements independently when international teams depart. the authors believe that without the described improvements to laboratory capacity and quality, the growth and achievements of the harvard / apin pepfar programme could not have been attained. various processes of the 12 quality system essentials that we used to scale up laboratory activities were effective. specifically, using multiple training methods worked well in ensuring sufficient numbers of trained laboratory staff at each site along with maintenance of high-quality, standardised services throughout the programme sites. the system of centralised procurement and supply distribution allowed for efficient monitoring of supply use and reduction in costs through bulk ordering. by implementing an electronic medical record system, we ensured increased use of data by clinical staff for improved patient care. furthermore, laboratory teams elevated the overall quality of care at the sites by providing data readily accessed by the electronic treatment response utility. the training efforts also resulted in personnel that were able to develop and maintain laboratories worthy of international accreditation. additionally, the laboratory scale-up and training efforts had many indirect effects. one major impact of the laboratory scale-up efforts was concomitant health system strengthening across the hospital settings in which our hiv programmes were located. other groups have documented similarly that the pepfar scale-up and integration within existing healthcare systems has improved the linkage of hiv and tb care10 and increased the number of in-hospital births in resource-limited settings.11 in addition, as a result of pepfar-associated training efforts, various programme-affiliated laboratory researchers from nigeria have been successful in gaining fogarty fellowships (fogarty international center, nih, bethesda, md, united states) to spend several months working on research projects in the harvard research laboratories in boston, ma, united states. the harvard/apin pepfar team has published research in peer-reviewed journals supporting the cost effectiveness and patient benefit from regular vl and drug resistance monitoring.12,13 finally, harvard worked with apin to expand testing capabilities by distribution of point-of-care equipment (including the partec cyflow minipoc) and are embarking on a new programme to test a point-of-care technology for measuring vls in an african resource limited setting.14 conclusion top ↑ in this article we have provided an overview of methods that may be useful in the development and support of a sustainable laboratory infrastructure, whilst simultaneously developing quality processes through a quality management system model and building upon the existing physical and human capital in a resource-limited setting such as nigeria. looking forward, as pepfar’s reorganisation of management by implementing partners occurs, apin endeavours to apply these strategies to strengthen laboratories at the hospitals it inherits and hopes that new partners at ceded locations continue to do the same. we hope that many of these lessons learned and strategies employed may assist and encourage the development of other laboratories in resource-limited settings. acknowledgements top ↑ the authors gratefully acknowledge the patients and the hard work and dedication of all the staff at collaborating clinics, hospitals, and laboratories. this work was funded, in part, by the us department of health and human services, health resources and services administration (u51ha02522). the contents are solely the responsibility of the authors and do not represent the official views of the funding institution. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions d.j.h. (harvard school of public health) conceived of the evaluation; conducted laboratory trainings and site visits; ordered equipment; and wrote the manuscript. j-l.s. (globomics) provided senior laboratory and programme development direction; contributed to the outline of the evaluation; edited the manuscript; and performed site visits. j.o.s (aids prevention initiative in nigeria ltd) assisted in the conception for the evaluation; served as apin laboratory programme director; consulted for new site additions; and conducted site visits. a.d.s. (cdc malawi) provided senior leadership; contributed to the outline of the evaluation; initiated and developed the laboratory quality programme, eqa management and who afro assessment processes; performed laboratory trainings and site visits; and advised on new equipment platforms. b.c. (harvard school of public health) contributed to the outline of the evaluation; edited the manuscript; developed molecular laboratory capacity in nigeria; and developed numerous laboratory databases, including the treatment response utility. e.o. (aids prevention initiative in nigeria ltd) contributed to the outline of the evaluation; performed site visits and laboratory trainings; and served as the primary liaison for the who afro laboratory accreditation processes. s.t.m. (harvard school of public health) contributed to the outline of the evaluation; reviewed and edited all versions of the manuscript; consulted on all site and laboratory activities; and oversaw collection and quality of all electronic laboratory data and reporting. p.o. (aids prevention initiative in nigeria ltd) provided medical leadership on all laboratory and programme activity in nigeria; contributed to the outline of the evaluation and reviewed and edited all versions of the manuscript; and contributed to laboratory trainings and performed site visits. p.j.k. (harvard school of public health) conceived of the evaluation; contributed to the outline of the evaluation; reviewed and edited all versions of the manuscript; provided senior leadership on all laboratory and programme activities; advised on laboratory expansion and development; and contributed to laboratory trainings. all authors read and approved the final manuscript. references top ↑ gershy-damet gm, rotz p, cross d, et al. the world health organization, african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm department of state. office of global aids coordinator. pepfar blueprint: creating an aids-free generation; 2012. http://www.pepfar.gov/documents/organization/201386.pdf. accessed december 2014. world health organization. guidance for development of national laboratory strategic plans. geneva: who; 2010. nigeria federal ministry of health. technical report: 2010 national hiv sero-prevalence sentinel survey. department of public health, abuja, nigeria; 2011. http://www.nigeria-aids.org/documents/2010_national%20hiv%20sero%20prevalence%20sentinel%20survey.pdf. accessed december 2014. kanki pj. the challenge and response in senegal. in: kanki p, marlink r (editors). a line drawn in the sand: responses to the aids treatment crisis in africa. cambridge, ma: harvard center for population and development studies, 2009; pp. 65–93. world health organization. the maputo declaration on strengthening of laboratory systems [document on the internet]. c2008. http://www.who.int/diagnostics_laboratory/maputo-declaration_2008.pdf. accessed december 2014. dinic l, akande p, idigbe eo, et al. genetic determinants of drug-resistant tuberculosis among hiv-infected patients in nigeria. j clin microbiol. 2012;50(9):2905–2909. http://dx.doi.org/10.1128/jcm.00982-12 fonjungo pn, kebede y, messele t, et al. laboratory equipment maintenance: a critical bottleneck for strengthening health systems in sub-saharan africa. j public health policy. 2012;33(1):34-45. http://dx.doi.org/10.1057/jphp.2011.57 guzel o, guner el. iso 15189 accreditation: reqirements for quality competence of medical laboratories, experience of a laboratory i. clin biochem. 2009;42(4–5); 274–278. http://dx.doi.org/10.1016/j.clinbiochem.2008.09.011 howard aa, gasana m, getahun m, et al. pepfar support for the scaling up of collaborative tb/hiv activities. j acquir immune defic syndr. 2012;60(suppl 3): s136–s144. http://dx.doi.org/10.1097/qai.0b013e31825cfe8e kruk me, jakubowski a, rabkin m, et al. pepfar programs linked to more deliveries in health facilities by african women who are not infected with hiv. health aff (millwood). 2012;31(7):1478–1488. http://dx.doi.org/10.1377/hlthaff.2012.0197 rawizza he, chaplin b, meloni st, et al. accumulation of protease mutations among patients failing second-line antiretroviral therapy and response to salvage therapy in nigeria. plos one. 2013;8(9):e73582. http://dx.doi.org/10.1371/journal.pone.0073582 rawizza he, chaplin b, meloni st, et al. immunologic criteria are poor predictors of virologic outcome: implications for hiv treatment monitoring in resource-limited settings. clin infect dis. 2011;53(12):1283–1290. http://dx.doi.org/10.1093/cid/cir729 lee hh, dineva ma, chua yl, et al. simple amplification-based assay: a nucleic acid-based point-of-care platform for hiv-1 testing. j infect dis. 2010;201(suppl 1): s65–s72. http://dx.doi.org/10.1086/650385 abstract background description of the intervention lessons learnt recommendations acknowledgements references about the author(s) denis omali infectious diseases institute, makerere university college of health sciences, kampala, uganda allan buzibye infectious diseases institute, makerere university college of health sciences, kampala, uganda richard kwizera infectious diseases institute, makerere university college of health sciences, kampala, uganda pauline byakika-kibwika infectious diseases institute, makerere university college of health sciences, kampala, uganda department of medicine, school of medicine, makerere university college of health sciences, kampala, uganda rhoda namakula infectious diseases institute, makerere university college of health sciences, kampala, uganda joshua matovu infectious diseases institute, makerere university college of health sciences, kampala, uganda olive mbabazi infectious diseases institute, makerere university college of health sciences, kampala, uganda emmanuel mande infectious diseases institute, makerere university college of health sciences, kampala, uganda christine sekaggya-wiltshire infectious diseases institute, makerere university college of health sciences, kampala, uganda damalie nakanjako infectious diseases institute, makerere university college of health sciences, kampala, uganda department of medicine, school of medicine, makerere university college of health sciences, kampala, uganda ursula gutteck department of clinical chemistry, university hospital zurich, university of zurich, zurich, switzerland keith mcadam department of clinical research, london school of hygiene and tropical medicine, london, united kingdom philippa easterbrook department of human immunodeficiency virus, world health organization, geneva, switzerland andrew kambugu infectious diseases institute, makerere university college of health sciences, kampala, uganda jan fehr department of clinical chemistry, university hospital zurich, university of zurich, zurich, switzerland barbara castelnuovo infectious diseases institute, makerere university college of health sciences, kampala, uganda yukari c. manabe infectious diseases institute, makerere university college of health sciences, kampala, uganda division of infectious diseases, johns hopkins school of medicine, baltimore, maryland, united states mohammed lamorde infectious diseases institute, makerere university college of health sciences, kampala, uganda daniel mueller department of clinical chemistry, university hospital zurich, university of zurich, zurich, switzerland concepta merry department of pharmacology and therapeutics, trinity college dublin, dublin, ireland citation omali d, buzibye a, kwizera r, et al. building clinical pharmacology laboratory capacity in lowand middle-income countries: experience from uganda. afr j lab med. 2023;12(1), a1956. https://doi.org/10.4102/ajlm.v12i1.1956 lessons from the field building clinical pharmacology laboratory capacity in lowand middle-income countries: experience from uganda denis omali, allan buzibye, richard kwizera, pauline byakika-kibwika, rhoda namakula, joshua matovu, olive mbabazi, emmanuel mande, christine sekaggya-wiltshire, damalie nakanjako, ursula gutteck, keith mcadam, philippa easterbrook, andrew kambugu, jan fehr, barbara castelnuovo, yukari c. manabe, mohammed lamorde, daniel mueller, concepta merry received: 17 may 2022; accepted: 30 nov. 2022; published: 07 feb. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: research and clinical use of clinical pharmacology laboratories are limited in lowand middle-income countries. we describe our experience in building and sustaining laboratory capacity for clinical pharmacology at the infectious diseases institute, kampala, uganda. intervention: existing laboratory infrastructure was repurposed, and new equipment was acquired. laboratory personnel were hired and trained to optimise, validate, and develop in-house methods for testing antiretroviral, anti-tuberculosis and other drugs, including 10 high-performance liquid chromatography methods and four mass spectrometry methods. we reviewed all research collaborations and projects for which samples were assayed in the laboratory from january 2006 to november 2020. we assessed laboratory staff mentorship from collaborative relationships and the contribution of research projects towards human resource development, assay development, and equipment and maintenance costs. we further assessed the quality of testing and use of the laboratory for research and clinical care. lessons learnt: fourteen years post inception, the clinical pharmacology laboratory had contributed significantly to the overall research output at the institute by supporting 26 pharmacokinetic studies. the laboratory has actively participated in an international external quality assurance programme for the last four years. for clinical care, a therapeutic drug monitoring service is accessible to patients living with hiv at the adult infectious diseases clinic in kampala, uganda. recommendations: driven primarily by research projects, clinical pharmacology laboratory capacity was successfully established in uganda, resulting in sustained research output and clinical support. strategies implemented in building capacity for this laboratory may guide similar processes in other lowand middle-income countries. keywords: therapeutic drug monitoring; building laboratory capacity; resource-limited setting; hiv; uganda. background clinical pharmacology is the study of drugs in humans.1 the central dogma of clinical pharmacology is ‘drug concentration determines drug actions’.2 therapeutic drug monitoring (tdm) is the laboratory measurement of the drug concentration in a sample matrix.3 one of the aims of measuring drug concentrations in tdm is to adjust drug dose to optimise clinical outcomes and minimise adverse events in hard-to-manage diseases like hiv infection and other infectious diseases.3,4,5,6,7 therapeutic drug monitoring is routinely utilised in developed countries but is used only infrequently in lowand middle-income countries (lmics).5,8,9 laboratories can measure drug concentrations in different sample matrices using immunoassay platforms10,11,12 and chromatographic methods like high-performance liquid chromatography (hplc) and liquid chromatography-mass spectrometry (lc-ms).3,9 because of its high specificity, hplc is preferred over immunoassays, and its lower cost and local availability make it the preferred choice over lc-ms in many lmic laboratories.13 however, both hplc and lc-ms platforms require high technical expertise that may not be available in lmics, and they have weak supply chains for equipment and spare parts.12 staff training and retention are also more challenging in lmics due to limited pre-service training opportunities and limited career options. as such, despite evidence of its relevance for specialised patient care in other settings,5 tdm is not included in the standard care package within the treatment guidelines outlined by the uganda ministry of health.14 the infectious diseases institute (idi), makerere university college of health sciences, is an hiv centre of excellence located in mulago national referral hospital complex in kampala, uganda.15 the institute, established through international partnerships, is an academic research centre that commenced operations in 2002. by 2019, through its clinic in mulago and partners, idi was supporting 329 335 hiv-positive patients actively receiving antiretrovirals. laboratory units in idi included a college of american pathologists-certified idi clinical core laboratory and a smaller translational research laboratory that was created in 2007 to develop laboratory research capacity for immunology, molecular biology, microbiology, and clinical pharmacology. generally, the need for a functional clinical pharmacology laboratory cannot be overlooked, especially in settings with a high disease burden like lmics. efforts to enhance clinical pharmacology laboratory capacity must consider multifaceted needs, including human resources, knowledge building, and infrastructure.16,17,18 across its programming, idi uses a systematic approach (capacity pyramid) to highlight gaps in interdependent types of capacity – both personal (e.g. skills) and institutional (e.g. systems) – and inform comprehensive interventions.19 furthermore, collaborative partnerships between institutions in developing and developed countries have been used as a key strategy to address challenges in strengthening laboratory capacity.9,16,18 this article describes idi’s experience with developing capacity by establishing and sustaining a clinical pharmacology laboratory in uganda. description of the intervention ethical considerations ethics committee approval was not required for this research. this research involved no human or animal subjects. repurposing of existing laboratory infrastructure in a clinical pharmacology laboratory for tdm, the process workflow is critical for assay accuracy and should be considered in the design of the laboratory facility.20 between january 2007 and december 2008, 292.53 square feet of space in the translational research laboratory was repurposed to host the clinical pharmacology laboratory. two fume extractors (to eliminate toxic chemical fumes for personnel health and safety) and one fume hood (for specific sample processing and storage) were already existent before the laboratory was repurposed. a lighting system akin to natural light was installed to ease visibility. strong workbenches made of non-porous material were already available in the acquired translational research laboratory, and these were arranged for smooth workflow and to support the instruments. air conditioning and air filtration systems were also already installed in the acquired laboratory space; these helped minimise dust exposure and ensure ambient temperature for the instruments and laboratory personnel. constant laboratory operation was supported by both the national electric power supply and a backup generator installed at the idi in 2004. laboratory equipment acquisition two hplc-ultraviolet (hplc-uv) machines with inbuilt detectors, an autosampler, and pumps (shimadzu lc-2010cht, shimadzu, kyoto, japan) controlled by class-vp software version 6.1 (shimadzu, kyoto, japan) were installed in the laboratory (figure 1). the machines were obtained as generous donations from trinity college dublin in 2007 and the university of zurich in 2013. with internally generated funds, the laboratory acquired other primary instruments like an analytical balance, a ph meter, and a magnetic stirrer. the laboratory also received a generous donation of a centrifuge from the united states national institutes of health. figure 1: high-performance liquid chromatography-ultraviolet detection machines installed in the clinical pharmacology laboratory at the infectious diseases institute, makerere university college of health sciences, kampala, uganda between 2007 and 2013. in 2018, leveraging its relationship with the university of zurich, idi received a donation of an lc-ms machine (thermo scientific lcq fleet ion trap lc/msn model, thermo fisher scientific, san jose, california, united states), the first of its kind in uganda for tdm and pharmacological research (figure 2). subsequently, in 2019, a nitrogen generator was purchased (figure 3) to support the operations of the lc-ms. a technical service engineer (originally from south africa, but subsequently from within uganda) authorised by the manufacturer services the hplc-uv instruments biannually. trained idi laboratory staff service the lc-ms with expert guidance from the university of zurich. also, idi engineers based at the site oversee the periodic servicing of other laboratory equipment, including the analytical balance, centrifuge, and ph meter. figure 2: a liquid chromatography-mass spectrometry instrument installed in the clinical pharmacology laboratory at the infectious diseases institute, makerere university college of health sciences, kampala, uganda in 2018. figure 3: a nitrogen generator installed to support continuous nitrogen gas supply to the mass spectrometer in the clinical pharmacology laboratory at the infectious diseases institute, makerere university college of health sciences, kampala, uganda in 2019. laboratory human resource development human resource establishment quality human resource, which is an important aspect of local capacity, is the centre of most laboratory capacity-building programmes.16,17,21,22,23 establishing quality human resource reduces both appraisal and failure costs, thereby reducing the overall cost of quality, which is a burden in lmics.24 building on the quality of the makerere university-johns hopkins university collaboration that led to the establishment of a college of american pathologists-certified laboratory, two laboratory technologists that had rotated through the idi clinical core laboratory were recruited. human resource knowledge building during the establishment phase, two laboratory technologists were trained in methods for measuring drug concentrations using the hplc instrument, one at the university of cape town and the other at both radboud university and the university of zurich. in 2018, through the strong idi-university of zurich research collaboration, one staff member was further trained in the use of the lc-ms platform, leading to the donation of the equipment to idi. the training activities were conducted both physically and via virtual media platforms to expand knowledge and skills in the areas of sample processing and measurement of drug concentrations in different human sample matrices, results analysis, equipment operation and maintenance, method development and validation, and the supply chain process. using cascade training (table 1), the formally trained staff member trained five other laboratory technologists to expand the laboratory’s human resource capacity. continuing medical education was encouraged for the laboratory staff throughout the capacity-building programme at the idi. currently, the laboratory has a strong human resource capacity composed of three laboratory technologists and continues to get technical and mentorship support from the university of zurich. table 1: human resource training programmes conducted between 2006 and 2019. lessons learnt current capacity for drug concentration measurements pharmacokinetic assays a clinical pharmacology laboratory was developed at idi and currently has the capacity to measure drug concentrations to guide tdm and clinical pharmacology studies using 10 analytical methods for hplc-uv and six methods for lc-ms either simultaneously or singly. these methods are used to measure the concentrations of anti-tuberculosis drugs such as ethambutol, rifampicin, isoniazid, pyrazinamide, rifapentine, rifabutin and moxifloxacin. antiretroviral drugs analysed in the laboratory include nevirapine, efavirenz, atazanavir, lopinavir, tenofovir, saquinavir, darunavir, etravirine, dolutegravir and raltegravir. antiepileptics (phenytoin and carbamazepine), antibiotics (vancomycin, gentamicin, kanamycin and amikacin), and antimalarial drugs (lumefantrine, artemether and its metabolites and halofantrine) are also analysed in the laboratory. currently, the lc-ms methods are used to determine the concentrations of tenofovir, dolutegravir, amikacin, and antimalarial drugs, while the concentrations of other drugs are measured using hplc-uv. the laboratory staff can also execute in-house innovations to develop, validate, and optimise methods for measuring the concentration of several drugs using the hplc-uv and lc-ms. drug concentration data from the laboratory has enabled researchers working on pharmacokinetic studies to detect drug interactions and sub-therapeutic concentrations and monitor patient antiretroviral therapy adherence (table 2). table 2: pharmacokinetic studies formally supported by the clinical pharmacology laboratory at the infectious diseases institute in kampala, uganda between 2009 and 2015. laboratory quality assurance programme in 2017, the laboratory commenced its participation in an external quality assurance (eqa) programme for nevirapine, efavirenz, atazanavir, and lopinavir through the stichting kwaliteitsbewaking klinische geneesmiddelanalyse en toxicologie (kkgt) (association for quality assessment in therapeutic drug monitoring and clinical toxicology, amstelveen, the netherlands).28 the eqa samples analysed in 2017 and 2019 were within the kkgt acceptance range in the four annual rounds. in 2018, eqa test results were within the kkgt acceptance range except for atazanavir in round one, lopinavir in rounds two and three, and efavirenz in round four. in 2019, anti-tuberculosis drugs, including ethambutol, rifampicin, isoniazid, pyrazinamide, rifapentine, and rifabutin, were included in two annual rounds of the eqa programme. the first attempt yielded results within the kkgt acceptance range in both rounds for all anti-tuberculosis drugs except isoniazid in round two. the second attempt in 2020 was not conducted for anti-tuberculosis drugs due to interruptions in the shipment of the eqa samples to the laboratory. antibiotics (only amikacin and vancomycin) and antiepileptics (only phenytoin and carbamazepine) were included in the 2017 eqa subscription, with four annual rounds. attempts to analyse the antiepileptics in all four rounds yielded results that were outside the kkgt acceptable range. after laboratory preparations, vancomycin was included in the eqa programme in 2020 and yielded results within the kkgt acceptance range in rounds one and four. however, the vancomycin eqa results for rounds two and three were out of the kkgt acceptance range. amikacin was not tested together with vancomycin in the eqa programme because the method to measure amikacin concentrations in the laboratory was not developed until 2020. with mutual interdependence with collaborators, the laboratory continues to develop capacity for other kkgt programmes, with continuous optimisation of existing assays for the measurement of drugs like the antiepileptics for which measurement was unsuccessful in the previous attempts. the lack of research studies requiring the measurement of carbamazepine and phenytoin concentrations may have contributed to the lesser focus on the antiepileptics programme. the eqa helped laboratory staff to identify pitfalls in routine laboratory analyses. interference of other analytes with the kkgt eqa samples was found to be the major cause of out-of-range low scores in the antiretroviral programme. this challenge was corrected by optimising the methods for simultaneous measurement of efavirenz, lopinavir, and atazanavir and using a different analytical method that had no interference between analytes. generally, participation in the eqa scheme improved staff confidence in supporting research studies and clinicians seeking tdm services. laboratory support for therapeutic drug monitoring and the need for a clinical pharmacology laboratory clinicians and clinical researchers at the idi clinic and external institutions have successfully used drug concentration results from the laboratory for tdm, specifically to assess non-adherence to regimens or to switch or discontinue patient therapy because of suspected drug resistance and toxicities. clinicians obtain patients’ blood samples and send the harvested plasma to the clinical pharmacology laboratory for testing with a corresponding request form attached. results from the laboratory are returned to clinicians to inform clinical management (counselling or dose adjustment, where appropriate). cumulatively, the laboratory tested 181 clinical care samples from the adult infectious diseases clinic at the idi between 2013 and 2019. with this support for clinical management, the need for a clinical pharmacology laboratory cannot be overemphasised, especially in lmics where there is a high disease burden. challenges and solutions encountered during laboratory capacity-building processes the instruments used for drug concentration measurements are costly and were acquired free of charge to idi through international collaborations (grants and donations). nevertheless, the costs of equipment preventive maintenance remained a challenge because of the high costs of service vendors and spare parts. the laboratory was able to incorporate these costs within research project budgets over the years, including from competitive grants. uganda has only a few authorised vendors that supply genuine high-purity reagents of hplc and lc-ms grade, and these are also costly to procure. before the acquisition of a nitrogen gas generator, obtaining high-purity gases like nitrogen and helium was difficult. initial training of laboratory technologists was limited to two staff members since this had to be conducted overseas. fortunately, efforts to cascade training to other laboratory staff were successful at minimal costs. notably, these challenges raise the cost of measuring the concentration of a drug in a single sample to approximately $40 united states dollars, which is outside the reach of many patients. from inception, the laboratory’s business case has thus focussed on funded research and clinic projects. for example, the tdm services at the idi clinic had to be paused in 2019, after analysing 181 samples from clinic patients, due to funding constraints. however, the clinical pharmacology laboratory remained operational since no such laboratory supporting clinical care was established in the country. recommendations the strategies employed in our laboratory yielded tangible results similar to those observed in related laboratory capacity-building programmes.16,17,18,22,23,29,30 clinical pharmacology laboratory capacity was developed to measure concentrations of antiretrovirals, anti-seizures, antibiotics, antimalarials, and anti-tuberculosis drugs for tdm. the capacity to develop, validate, and optimise analytical methods for other drugs was also developed. the success recorded for this laboratory capacity-building process reflects the gradual progress to strengthen capacity from inception to date with collaborative support. the focus of the institution’s research programme in the field of pharmacokinetics was sustained over the years, enabling continuous cash flow to the laboratory in the form of research costs covering sample analysis. however, with short-term project support as the main source of laboratory resources, factors beyond the laboratory’s direct control such as grant success or availability of collaborators with aligned goals could lead to shocks in the near term and impair long-term sustainability. we therefore recommend that laboratories in lmics (and their parent institutions) must be prepared to strengthen and sustain results from collaborative programmes when external support ends. laboratories in lmics intending to build and sustain long-term capacity for clinical pharmacology should build local capacity through training and infrastructure development.16,17,18,31 as with other capacity-building programmes, staff training in our setting was challenging,16,31 requiring international travel and time off work. we further recommend the introduction of practical coursework for clinical pharmacology sessions in pre-service laboratory education (bachelor’s degree-level laboratory technology training) to ease future on-the-job staff training. the african field epidemiology track programme developed and adopted this strategy, positively changing the laboratory profession in its programme member countries.17,32 using established centres like idi for postgraduate training in pharmacology or for placements to support new laboratories could reduce costs associated with expensive international training. further, pharmacology postgraduates can be included in laboratory mentorship programmes in lmics. investment in appropriate infrastructure at inception not only promotes staff safety but can also prolong the lifespan of the equipment. high-level air conditioning mitigates challenges of hot and humid climates that may affect laboratory instruments. the use of fume extractors to eliminate environmental dust and toxic fumes from laboratory reagents and processes is essential for staff health and safety. laboratory fume hoods should be used together with proper reagent segregation to minimise the chances of explosions resulting from flammable reagent fume reactions. the outcomes presented in this article represent success at a site where clinical research and laboratory infrastructure were already present and operating to international standards at baseline. our experience may thus not apply to all lmics due to variations in local guidelines and socio-economic factors. a clinical pharmacology laboratory was established, and laboratory capacity was developed and sustained at the idi through strong capacity-building collaborative relationships. the laboratory significantly contributed to research capacity development at idi and other external institutions by providing answers to questions from several pharmacology research studies. building clinical pharmacology laboratory capacity in lmics is feasible and necessary to support the global goal of managing hiv infection and other diseases. acknowledgements we acknowledge peter smith, university of cape town, lance heinle, pfizer global health fellows program, and david m. burger, nijmegen, radboud university, for organising staff training. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions d.o. and a.b. contributed equally. c.m. and dm. contributed equally. d.o. and a.b. conceived and designed the concept. d.o. wrote the initial manuscript. d.o., a.b., r.k., p.b.-k., r.n., j.m., o.m., e.m., d.n., p.e., b.c., y.c.m., m.l., d.m., and c.m. participated in critical revisions of the manuscript for intellectual content. c.s.-w., p.b.-k., u.g., m.l., d.m., and c.m. gave mentorship and technical support. o.m., j.f., d.n., b.c., y.c.m., p.e., k.m., and a.k. participated in administrative support. all named authors approved the final manuscript for publication. sources of support we are grateful to realta foundation, the irish department for foreign affairs, and the health research board of ireland (ghra06/02, ghra07/09) for initial support to establish the laboratory. we acknowledge the support given by the university of zurich in the provision of equipment and staff training. we acknowledge funding support for equipment, supplies, and maintenance from the european and developing countries clinical trials partnership and world health organization tropical diseases research (csa-ebola-2015-353). d.o. is supported by the fogarty international center of the national institutes of health under award number d43tw009771. a.b. is currently a phd scholar supported through the fogarty international centre, national institute of health grant #2d43tw009771-06 ‘hiv and co-infections in uganda’. the authors also acknowledge the deltas africa-funded makerere uganda virus research institute infection and immunity (muii) programme (grant #107743/z/15/z) that has collaborated with idi in building capacity of the translational research laboratory. we thank the united states national institutes of health for the generous donation of a centrifuge to the laboratory. r.k. is currently a phd scholar supported through the deltas africa initiative grant #del-15-011 to thrive-2, from wellcome trust grant #107742/z/15/z and the united kingdom government. e.m. is currently a msc fellow supported through the united states national institutes of health – fogarty international center, breca grant #1u2rtw010672. m.l. is supported by european and developing countries clinical trials partnership (ria2018ef-2083) which is part of the european and developing countries clinical trials partnership programme supported by the european union’s horizon 2020 research and innovation program. data availability all data underlying the findings are fully available without restriction in the manuscript from the corresponding author (d.o.). disclaimer the authors alone are responsible for the views expressed in this publication and they do not necessarily represent the views, decisions, or policies of the institutions with which they are affiliated. the views and opinions of authors expressed herein do not necessarily state or reflect those of european and developing countries clinical trials partnership. references huang s-m, lertora jj, markey sp, atkinson aj, jr. principles of clinical pharmacology. san diego, california: academic press; 2012. holford nh, atkinson aj, jr. time course of drug response. in: aj atkins, dr abernethy, ce daniels, rl dedrick, sp markey, editors. principles of clinical pharmacology. bethesda, maryland: elsevier, 2007; pp. 301–311. https://doi.org/10.1016/b978-012369417-1/50059-6 kang js, lee mh. overview of therapeutic drug monitoring. korean j intern med. 2009;24(1):1–10. https://doi.org/10.3904/kjim.2009.24.1.1 gutiérrez f, navarro a, padilla s, et al. prediction of neuropsychiatric adverse events associated with long-term efavirenz therapy, using plasma drug level monitoring. clin infect dis. 2005;41(11):1648–1653. https://doi.org/10.1086/497835 schoenenberger ja, aragones am, cano sm, et al. the advantages of therapeutic drug monitoring in patients receiving antiretroviral treatment and experiencing medication-related problems. ther drug monit. 2013;35(1):71–77. https://doi.org/10.1097/ftd.0b013e3182791f8c hallworth m, watson i. therapeutic drug monitoring: clinical guide. abbot park, illinois: abbott laboratories; 2010. ghiculescu r. therapeutic drug monitoring: which drugs, why, when and how to do it. aust prescr 2008;31:42–4. https://doi.org/10.18773/austprescr.2008.025 melker r, sackellares j, gold m. system and method for therapeutic drug monitoring. gainesville, us: university of florida research foundation inc.; 2005. nwobodo n. therapeutic drug monitoring in a developing nation: a clinical guide. jrsm open. 2014;5(8):2054270414531121. https://doi.org/10.1177/2054270414531121 hicks jm, brett emj. falsely increased digoxin concentrations in samples from neonates and infants. ther drug monit. 1984;6(4):461–464. https://doi.org/10.1097/00007691-198412000-00015 patel ja, clayton lt, lebel cp, mcclatchey kdj. abnormal theophylline levels in plasma by fluorescence polarization immunoassay in patients with renal disease. ther drug monit. 1984;6(4):458–460. https://doi.org/10.1097/00007691-198412000-00014 touw dj, neef c, thomson ah, vinks aaj. cost-effectiveness of therapeutic drug monitoring: a systematic review. ther drug monit. 2005;27(1):10–17. https://doi.org/10.1097/00007691-200502000-00004 lamorde m, fillekes q, sigaloff k, et al. therapeutic drug monitoring of nevirapine in saliva in uganda using high performance liquid chromatography and a low cost thin-layer chromatography technique. bmc infect dis. 2014;14:473. https://doi.org/10.1186/1471-2334-14-473 ministry of health. consolidated guidelines for prevention and treatment of hiv in uganda. kampala: ministry of health; 2016. buzibye a, musaazi j, von braun a, et al. antiretroviral concentration measurements as an additional tool to manage virologic failure in resource limited settings: a case control study. aids res ther. 2019;16(1):1–5. https://doi.org/10.1186/s12981-019-0255-x hamel dj, sankalé j-l, samuels jo, et al. building laboratory capacity to support hiv care in nigeria: harvard/apin pepfar, 2004–2012. afr j lab med. 2015;4(1):190. https://doi.org/10.4102/ajlm.v4i1.190 masanza mm, nqobile n, mukanga d, gitta snj. laboratory capacity building for the international health regulations (ihr [2005]) in resource-poor countries: the experience of the african field epidemiology network (afenet). bmc public health. 2010;10(1):1–7. https://doi.org/10.1186/1471-2458-10-s1-s8 paramasivan c, lee e, kao k, et al. experience establishing tuberculosis laboratory capacity in a developing country setting. int j tuberc lung dis. 2010;14(1):59–64. potter c, brough r. systemic capacity building: a hierarchy of needs. health policy plan. 2004;19(5):336–345. https://doi.org/10.1093/heapol/czh038 national research council. laboratory design, construction, and renovation: participants, process, and product. washington, dc: national academies press; 2000. fitzgibbon je, wallis cl. laboratory challenges conducting international clinical research in resource-limited settings. j acquir immune defic syndr. 2014;65(suppl. 1 [0 1]):s36–s39. https://doi.org/10.1097/qai.0000000000000038 maponga cc, monera-penduka tg, mtisi tj, et al. two decades (1998 to 2018) of collaborative human immunodeficiency virus clinical pharmacology capacity building in a resource constrained setting. cost eff resour alloc. 2021;19(1):1–8. https://doi.org/10.1186/s12962-021-00327-y mtisi tj, maponga c, monera-penduka tg, et al. strategic establishment of an international pharmacology specialty laboratory in a resource-limited setting. afr j lab med. 2018;7(1):1–6. https://doi.org/10.4102/ajlm.v7i1.659 elbireer a, gable ar, jackson jbj. cost of quality at a clinical laboratory in a resource-limited country. lab med. 2010;41(7):429–433. https://doi.org/10.1309/lmcz0zfr80qwibem lamorde m, byakika-kibwika p, okaba-kayom v, et al. nevirapine pharmacokinetics when initiated at 200 mg or 400 mg daily in hiv-1 and tuberculosis co-infected ugandan adults on rifampicin. j antimicrob chemother. 2011;66(1):180–183. https://doi.org/10.1093/jac/dkq411 von braun a, castelnuovo b, ledergerber b, et al. high efavirenz serum concentrations in tb/hiv-coinfected ugandan adults with a cyp2b6 516 tt genotype on anti-tb treatment. j antimicrob chemother. 2019;74(1):135–138. https://doi.org/10.1093/jac/dky379 sekaggya-wiltshire c, castelnuovo b, von braun a, et al. cohort profile of a study on outcomes related to tuberculosis and antiretroviral drug concentrations in uganda: design, methods and patient characteristics of the south study. bmj open. 2017;7(9):e014679. https://doi.org/10.1136/bmjopen-2016-014679 droste j, aarnoutse re, koopmans pp, hekster ya, burger dmj. evaluation of antiretroviral drug measurements by an interlaboratory quality control program. j acquir immune defic syndr. 2003;32(3):287–291. https://doi.org/10.1097/00126334-200303010-00007 niyonzima n, wannume h, kadhumbula s, et al. strengthening laboratory diagnostic capacity to support cancer care in uganda. am j clin pathol. 2021;156(2):205–213. https://doi.org/10.1093/ajcp/aqaa218 paglia mg, bevilacqua n, haji hs, et al. improvement of tuberculosis laboratory capacity on pemba island, zanzibar: a health cooperation project. plos one. 2012;7(8):e44109. https://doi.org/10.1371/journal.pone.0044109 petti ca, polage cr, quinn tc, ronald ar, sande ma. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377–382. https://doi.org/10.1086/499363 gitta sn, mukanga d, babirye r, dahlke m, tshimanga m, nsubuga p. the african field epidemiology network-networking for effective field epidemiology capacity building and service delivery. pan afr med j. 2011;10(suppl. 1):3. article information authors: rosemary a. audu1 catherine c. onubogu2 nkiru n. nwokoye2 eke ofuche3 shirematee baboolal4 odafen oke5 elizabeth t. luman6 emmanuel o. idigbe2 affiliations: 1human virology laboratory, nigerian institute of medical research, nigeria 2national tuberculosis reference laboratory, nigerian institute of medical research, nigeria 3 aids prevention initiative in nigeria, nigeria 4american society for microbiology, united states 5us centers for disease control and prevention, nigeria 6us centers for disease control and prevention, united states correspondence to: rosemary audu postal address: nigerian institute of medical research, 6 edmond crescent, yaba, nigeria dates: received: 21 may 2014 accepted: 08 july 2014 published: 08 sept. 2014 republished: 07 nov. 2014 how to cite this article: audu ra, onubogu cc, nwokoye nn, et al. improving quality in national reference laboratories: the role of slmta and mentorship. afr j lab med. 2014;3(2), art. #200, 7 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.200 note: article republished with updated references relating the special issue. copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. improving quality in national reference laboratories: the role of slmta and mentorship in this original research... open access • abstarct • introduction • research method and design    • implementation sites    • slmta implementation    • evaluation of laboratory performance    • quality improvement projects    • mentorship and additional support • results    • overall performance    • performance of quality system essentials    • quality improvement projects    • effect of changes to the audit checklist • discussion    • limitations    • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstarct top ↑ background: the nigerian institute of medical research houses two reference laboratories: the virology and tuberculosis laboratories. both were enrolled in the strengthening laboratory management toward accreditation (slmta) programme. objective: to describe the impact of slmta and discuss factors affecting the results, with an emphasis on mentorship. methods: the slmta programme was implemented from april 2010 through november 2012. participants attended three workshops and executed quality improvement projects; laboratory auditors evaluated performance using a standard checklist. the virology laboratory did not receive mentorship; however, the tuberculosis laboratory had an international mentor who visited the laboratory four times during the programme, spending two to four weeks embedded within the laboratory during each visit. results: there was an overall improvement in the performance of both laboratories, with the virology laboratory increasing 13% (from 80% at baseline to 93% at exit audit) and the tuberculosis laboratory increasing 29% (from 66% to 95%). these scores were maintained nine months later at the surveillance audit. conclusion: the slmta programme resulted in improved and sustained quality management performance for both laboratories. mentoring was a possible factor in the substantial improvement made by the tuberculosis laboratory and should be considered in order to augment the training received from the slmta workshops. introduction top ↑ the level of implementation of laboratory standards in the african region, verified through the process of accreditation, has historically been very low.1 until recently, most laboratories in africa have not emphasised quality management systems (qms) in the provision of healthcare services. in addition, lack of staff training and education, poor physical infrastructure, climate extremes and financial constraints2 have limited implementation of laboratory quality systems. the absence of national laboratory strategic plans to provide roadmaps for the implementation of quality laboratory services, as well as the lack of national laboratory quality standards to guide the provision of quality clinical laboratory services and accreditation in nigeria, are also obstacles for the implementation of quality laboratory systems. an earlier study has also reported that the culture of qms is uncommon in nigerian laboratories;3 as such, there is a need to build this culture. to strengthen the laboratory systems of african countries in a systematic approach, the us centers for disease control and prevention (cdc), in collaboration with the american society for clinical pathology, the clinton health access initiative and the world health organization’s regional office for africa (who afro), launched the strengthening laboratory management toward accreditation (slmta) training programme in 2009.4 this programme, which focuses on strengthening laboratory management to achieve immediate laboratory improvement and accelerate accreditation preparedness, has been implemented in 47 countries worldwide and is expanding rapidly.5 the programme includes workshops, improvement projects, site visits and, in some cases, mentoring.6 nigeria embraced the slmta programme in 2009 and, by 2010, seven laboratory experts were trained to roll out the slmta programme in 24 of the 344 nigerian laboratories supported by the us president’s emergency plan for aids relief (pepfar). the nigerian institute of medical research (nimr) has two reference laboratories, namely, the human virology laboratory (virology laboratory) and the national tuberculosis reference laboratory (tb laboratory), under its mandate ‘to conduct basic, applied and operational research for the prevention and control of communicable and non-communicable diseases of public health importance in the country in collaboration with the federal and state ministries of health and other stakeholders’.27 both laboratories were amongst the 24 enrolled in the slmta programme. the two laboratories are similar in their institutional management and were supported by the same pepfar implementation partner. however, the tb laboratory was assigned a mentor to assist with slmta implementation, whilst the virology laboratory was not. we describe the impact of the slmta programme and discuss potential factors affecting the results, with an emphasis on mentorship. research method and design top ↑ implementation sites the virology laboratory has provided laboratory services to the nigerian hiv treatment programme since 2002 and similar services for the pepfar hiv treatment project which commenced in 2004. the laboratory has the following sections: serology, immunology, chemistry, haematology and molecular diagnostics, which includes resistance testing for hiv. a total of 81 758 tests were performed by the virology laboratory in 2010. the laboratory staff comprised 14 degree-holding professionals, one diploma-holding professional, one certificate-holding professional, two data clerks, two phlebotomists, two cleaners, one driver and eight other administrative staff. this laboratory had some previous experience implementing qms and, in 2008, had received international organization for standardization (iso) 9001 accreditation,3 a general organisational certification of management processes. in preparing medical laboratories for international accreditation, the slmta programme employs iso 15189, which specifies standards for qms particular to medical laboratories. to help it meet these standards, which are more relevant to clinical laboratories, the virology laboratory enrolled in the slmta programme. the tb laboratory was established to meet the institute’s research mandate. from 2005, the scope of services increased with inclusion of a ‘directly observed treatment short-course’ (dots) diagnostic centre, which brought about the expansion and renovation of the laboratory in order to meet the tb diagnostic service needs of both the private and public sectors. with the dots centre, many more tb suspects were referred to the laboratory for diagnosis, treatment and follow-up, and the diagnostic workload increased dramatically. the laboratory was commissioned as a national tb reference laboratory in 2007 and offers the following services: smear microscopy for acid-fast bacilli; solid and liquid culture; identification of mycobacterium tuberculosis complex and mycobacteria other than tuberculosis (motts); and drug susceptibility testing using solid, liquid and molecular-based techniques. the laboratory is also involved in national tb drug resistance surveillance. a total of 80 799 tests were conducted in 2010. during the course of the slmta programme, the tb laboratory included 14 degree-holding staff, four diploma-holding staff, three microscopists, one administrative staff, one data clerk and one cleaner. each laboratory also had a director, laboratory manager, quality assurance officer and dedicated personnel who had consistent job responsibilities throughout the duration of the programme. slmta implementation the slmta programme was implemented in the nimr virology and tb laboratories over two years and seven months (figure 1). three workshops were conducted within this period, with an average eight-month interval between them. the laboratory managers and quality assurance officers from both laboratories attended the workshops, after which they trained the other laboratory staff. figure 1: slmta implementation timeline in nigerian reference laboratories. evaluation of laboratory performance a baseline audit was conducted in each laboratory in april 2010, six months before the first slmta workshop (figure 1). intermediate audits were conducted after each workshop in order to monitor progress and to help identify any remaining gaps. an exit audit was conducted in november 2012, four months after the third workshop and a surveillance audit was conducted in august 2013, nine months after the exit audit, in order to determine the ability of the laboratories to sustain the quality advances made. the baseline and first intermediate audits were conducted using the laboratory accreditation preparedness checklist developed in 2009 by who afro.8 the checklist had a total score of 250 points distributed into 12 sections corresponding to the 12 quality system essentials. in 2012, who afro revised the checklist by adding more details in the requirements for documents and records and management review, as well as modifying the sectional scores, with a new total of 258 points.9 this revised checklist was used in parallel with the older version for the second intermediate and surveillance audits to allow us to evaluate the effect of the revision; results presented are from the revised checklist. for the exit audit, only the revised checklist was used. based on the audit scores, laboratories were assigned a zeroto five-star rating, whereby < 55% = zero stars, 55% − 64% = one star, 65% − 74% = two stars, 75% − 84% = three stars, 85% − 94% = four stars and 95% − 100% = five stars. independent laboratory experts who had taken the slmta training-of-trainers course, which included one day of training on auditing, were engaged as auditors; the same team conducted the audits in both laboratories, although different auditors were scheduled for each round of audit. quality improvement projects quality improvement projects were selected by each laboratory after the first and second workshops based on laboratories’ specific needs within topics addressed at the workshops. the projects were implemented by all staff members and were monitored for effectiveness by their supervisors using the who afro checklist; reports were presented at the next workshop by the quality managers. the virology laboratory embarked on three quality improvement projects after the first workshop. the first was that staff were trained on the importance of monitoring the autoclave with emphasis being placed on effective sterilisation and proper waste segregation. they were then assessed daily by means of an in-house-developed checklist in order to improve competency regarding sterilisation and waste disposal. in addition, provision was made for stock level on inventory cards, expired reagents were disposed of and general improvements were made to the organisation of the store; and the storage media for documents were monitored monthly to ensure ease of retrieval of records, documents and policies. after the second workshop, another set of improvement projects was conducted: complaint types, root causes, corrective actions and effectiveness were monitored in order to improve customer satisfaction; specimens in and out of the laboratory were clocked for three months to measure and improve turnaround time; and the number and duration of items out of stock were monitored in order to reduce stock-outs of materials, kits and reagents. the officer responsible for storage monitored the stock-outs from the weekly requisition and issue records. for the tb laboratory, two quality improvement projects were implemented after the first workshop: monitoring inventory of reagents in order to improve turnaround time for acid-fast bacilli smear microscopy; and training staff on how to conduct internal audits. the laboratory conducted three improvement projects after the second workshop: monitoring media preparation and reviewing sputum-collection records in order to reduce the contamination rate of cultured samples; administering and analysing questionnaires from clients and effecting corrective actions in order to improve customer satisfaction; and improving documentation of inventory and establishing a requisition system for the stores. mentorship and additional support contrary to slmta’s implementation roadmap,5 time constraints on the laboratory experts who rolled out the slmta programme in nigeria prevented follow-up site visits at the virology laboratory between the workshops, which would have assisted in linking the training curriculum with on-site activities. for the tb laboratory, an experienced international mentor from the american society for microbiology was assigned to work with the laboratory throughout the slmta process. only tb laboratories were assigned mentors in this round of the slmta programme in nigeria. the mentor had a postgraduate degree in quality management systems and a doctoral degree in microbiology with a tb specialty, had previously managed a laboratory that successfully attained international accreditation and had attended a slmta training-of-trainers workshop. a facility-based approach was adopted as the mentor visited the laboratory on four occasions for an average duration of three weeks at a time, allowing an in-depth understanding of the laboratory. the mentor provided daily assistance to the staff in the implementation of qms, which included training in practical skills, assisting in improving quality of testing and giving assignments to be completed between visits. nonconformities reported from each audit were addressed by the mentor at each visit and management review meetings were established to identify opportunities for improvement and to formulate action plans. the mentor had administrative support from institutional management and the federal ministry of health, as well as technical and logistical support provided by cdc’s office in nigeria. within the time frame of the slmta programme, the aids prevention initiative in nigeria organised a five-day training on accreditation preparedness, with emphasis on quality management systems. staff from both of the laboratories participated alongside staff from other laboratories that the organisation supports. results top ↑ overall performance there was an overall improvement in the performance of both laboratories during the slmta programme (figure 2). the virology laboratory moved from an overall score of 80% at baseline, representing three stars, to 93% at the exit audit, representing four stars. the tb laboratory improved steadily from 66% at baseline audit, representing two stars, to 95% at exit audit, representing five stars. both laboratories maintained these gains at the nine-month surveillance audit. figure 2: comparison of performance of virology and tuberculosis laboratories over time using the who afro checklist. performance of quality system essentials examining the 12 quality system essentials closely revealed specific areas of strength, weakness and improvement (figure 3). the greatest improvements for the virology laboratory were in purchasing and inventory (from 67% to 90%) and in process control and internal and external quality assessment (from 74% to 94%) (figure 3a). the virology laboratory achieved 100% scores in five quality system essentials (documents and records; client management and customer service; internal audit; corrective action; occurrence and/or incident management and process improvement); however no progress was made in organisation and personnel, which remained at 80% for the exit audit. the tb laboratory generally started with lower scores than the virology laboratory, leaving more room for improvement. it made substantial improvements in management review (from 42% to 100%); internal audit (from 50% to 100%) and occurrence/incident management and process improvement (from 50% to 100%) (figure 3b). the tb laboratory also had very good performance at the exit audit in all the quality system essentials, obtaining 100% scores in six sections (documents and records; management review; client management and customer service; internal audit; corrective action; and occurrence/incident management and process improvement). figure 3: comparison of performance of the virology and tuberculosis laboratories in the 12 quality system essentials using the who afro checklist. quality improvement projects the impact of the quality improvement projects on the quality system essentials is shown in table 1. for the virology laboratory, after the first workshop the projects on documents and records and on purchasing and inventory impacted positively at the first intermediate audit and progress was sustained through the exit audit. the project on organisation and personnel did not have a positive impact on the score for the corresponding quality essential, as there was a drop in the next audit score, which did not improve beyond baseline at the exit audit. of the projects conducted after the second workshop, only the client management and customer service project corresponded to sustained performance, with audit scores remaining at 100%, as they were at the baseline audit. performance in information management was maintained at 93% after the second intermediate audit, but dropped to 83% at the exit audit, whilst purchasing and inventory improved from 93% before implementation to 97% at the second audit, but dropped to 90% at the exit audit. at the surveillance audit, improvements were generally sustained, except for purchasing and inventory, which reverted to the baseline score of 67%. for the tb laboratory, after the first workshop there was an improvement in the performance of the information management section, which continued through to the exit audit, where it reached 94% (table 1). the internal audit score dropped initially, but improved subsequently, attaining 100% at the exit audit. after the second workshop, there was a gradual improvement for process control and internal and external quality assessment to 91% at the exit audit; and maintenance of client management and customer service at 100%. purchasing and inventory scores decreased initially following project implementation (from 77% to 70%), then increased to 87% at the exit audit. the surveillance audit showed sustained or improved performance in all areas. table 1: impact of quality improvement projects on quality system essentials in the virology and tuberculosis laboratories of the nigerian institute of medical research. effect of changes to the audit checklist parallel audit scores using the revised (2012) checklist were slightly lower than those using the original (2009) checklist (table 2). differences ranged from 0% to 7% and were smaller at the surveillance audit than at the second intermediate audit, where they resulted in a change of star category for both the virology and tb laboratories. table 2: comparison of audit scores based on the original 2009 who afro checklist and the revised 2012 checklist. discussion top ↑ both the virology and tb laboratories successfully improved their quality scores, increasing by 13% and 29%, respectively. the virology laboratory started with more experience and higher scores at the baseline audit. however, improvement in the tb laboratory was steady and the exit score exceeded that of the virology laboratory. the two laboratories were from same institution and had the same management commitment and partner support, with similar test menu diversity, test volume and staff strength. the major difference between slmta implementation in the two laboratories was the presence of a facility-based laboratory mentor in the tb laboratory. other countries, such as kenya and botswana, have found similar results when implementing an accreditation-readiness programme, with mentored laboratories showing greater improvement than their non-mentored counterparts.10,11,12 whilst conclusive evidence is lacking, as none of these programmes were designed as case-control studies (i.e., with mentors randomly assigned to laboratories), the combined anecdotal evidence strongly supports the benefit of such mentorship. mentors who spend extended, well-structured periods in the laboratory working alongside the staff and helping participants to put quality improvement into practice through direct, daily coaching, can provide the needed support to fast-track laboratories toward quality improvement. the laboratories faced several challenges with regard to slmta implementation. firstly, whilst the laboratory checklist was used to help identify and correct problems, an understanding of some of the requirements was often a challenge, especially in the virology laboratory where an experienced mentor was not available to assist. similarly, the virology laboratory reported challenges in interpreting iso 15189 standard requirements and auditor recommendations. secondly, though many of the quality improvement projects were implemented successfully and increased performance of the quality system essentials, some of these advances were not sustained, especially in the virology laboratory. the purchasing and inventory section was affected worst as some records were not maintained. sustainability is a common concern for any improvement programme; once the intense focus of implementation ceases, special efforts and continued supervision are required so as to ensure that old habits do not return. it is possible that the root causes of the deficiencies were not properly identified and addressed. nevertheless, improvements in total scores were sustained, suggesting that quality improvement overall was maintained. successful achievement of the four to five star levels reached by the two nimr laboratories indicates a high level of laboratory functioning and gives credibility to the quality of the laboratory test results produced for improved healthcare services. the 22 other laboratories in nigeria’s first slmta round had similarly impressive results, moving from an average baseline of 60% to 87% at exit audit; 16 of the laboratories achieved four to five stars.13 these successes inspired nigeria to implement a second round of slmta in 2013 and to begin discussions regarding further national expansion of the programme. because of the potential benefit of on-site mentorship, national experts are being trained in nigeria to play this critical role. limitations our observations are subject to several limitations. the first of these is that mentorship was not assigned randomly. whilst factors that we examined, such as management support, laboratory size and testing volume, were similar for the two laboratories, there may have been other unobserved factors that could account for some of the differences. for example, the laboratories chose different quality improvement projects to implement between workshops. a report by maina et al. suggests that internal audits (which were implemented by the tb laboratory after the first workshop) may be a catalyst for improvements in other areas, as conducting self-review can identify areas that need improvement.14 whilst the virology laboratory was already conducting internal audits before slmta, the tb laboratory started with a 50% score in this area and increased to 100%, potentially helping to explain their improvement in other areas. the second limitation is that the checklists used for the baseline and exit audits were not exactly the same, potentially introducing bias in the results. comparison of the scores obtained by the two checklists used in parallel at the second intermediate and surveillance audits revealed that the revised checklist produced slightly lower results than the original checklist, suggesting that our overall improvement results are possibly conservative. the final limitation is that the auditors engaged in this study had only undertaken one day of training on auditing, which is not adequate to fully qualify them as auditors. whilst the use of a checklist helps to standardise the auditing process, some variability may have been introduced because of inexperience. conclusion the slmta programm was successful in improving the quality of the laboratory systems in these two laboratories, as evidenced by improved and sustained audit scores. the laboratory with expert on-site mentorship improved farther and steadier, achieving a score of five stars. our results suggest that laboratories should consider using on-site mentorship in order to augment the impact of slmta in implementing quality improvement. acknowledgements top ↑ the authors would like to thank the federal ministry of health and the management of nimr for their political commitment; and cdc’s nigeria office and the aids prevention initiative in nigeria for their support in conducting the slmta programme. we also appreciate the work of the auditors and facilitators who executed the project. the findings and conclusions in this article are those of the authors and do not necessarily represent the views of the cdc. this research has been supported by pepfar through the cdc. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions r.a.a. (human virology laboratory) analysed the data and initiated and wrote the manuscript. c.c.o. (national tuberculosis reference laboratory) provided the required information for the tb laboratory. n.n.n. (national tuberculosis reference laboratory) contributed to the write-up review. e.o. (aids prevention initiative in nigeria) coordinated the implementing partner support. s.b. (american society for microbiology) implemented the mentorship model. o.o. (cdc, nigeria) provided technical support. e.t.l. (cdc, united states) assisted with data analysis and interpretation and revised the manuscript extensively. e.o.i. (national tuberculosis reference laboratory) was responsible for the overall oversight with regard to the project implementation programme. references top ↑ 1.gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in african region. am j clin pathol. 2010;134(3):393–400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm 2.petti ca, polage cr, quinn tc, et al. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377–382. http://dx.doi.org/10.1086/499363 3.audu ra, sylvester-ikondu u, onwuamah ck, et al. experience of quality management system in a clinical laboratory in nigeria. afr j lab med. 2012;1(1), art. #18, 5 pages. http://dx.doi. org/10.4102/ajlm.v1i1.18 4.world health organization [who representative’s office for rwanda]. press release: kigali host the launch of a program to accelerate national laboratory service capacity building towards accreditation in the african region [document on the internet]. c2008 [cited 2014 jul 30]. available from: www.who.int/hiv/amds/diagnostics/amds_kigali_pr_lab.pdf 5.yao k, maruta t, luman et, nkengasong jn. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(1). in press. 6.yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3):401–409. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj 7.nigerian institute of medical research. development of a strategic plan (2011–2015) [document on the internet]. c2011 [cited 2014 jul 27]. available from: https://nimr.gov.ng/_data/nimr_strategic_plan.pdf 8.world health organization’s regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region (with checklist) [document on the internet]. c2011 [cited 2014 aug 10]. available from: http://www.afro.who.int/en/clusters-a-programmes/hss/bloodsafety-laboratories-a-health-technology/blt-highlights/3859-who-guide-for-thestepwise-laboratory-improvement-process-towards-accreditation-in-the-africanregion-with-checklist.html 9.world health organization’s regional office for africa. world health organization releases guide for the stepwise laboratory improvement process towards accreditation (slipta) in africa [page on the internet]. c2013 [cited 2014 jul 30]. available from: http://www.aslm.org/stay-informed/press-room/news-articles/world-healthorganization-releasesguide-for-the-stepwise-laboratory-improvementprocess-towards-accreditation-slipta-in-africa/ 10.gachuki t, sewe r, mwangi j, et al. attaining iso 15189 accreditation through slmta: a journey by kenya’s national hiv reference laboratory. afr j lab med. in press. 11.makokha e, mwalili s, basiye f, et al. using institutional mentorship to roll out slmta in kenya. afr j lab med. in press. 12.mokobela k, moatshe m, modukanele m. using slmta and mentorship to accelerate laboratory improvement in botswana. afr j lab med. in press. 13.yao k, luman e, slmta collaborating authors. evidence from 617 laboratories in 47 countries for slmta-driven improvement in quality management systems. afr j lab med. in press. 14.maina, rn, mengo dm, mohamud ad, et al. progressing beyond slmta: are internal audits the key drivers of accreditation? afr j lab med. in press. abstract introduction methods results discussion acknowledgements references about the author(s) abdul k. el karaaoui department of pathology and laboratory medicine, faculty of medicine, american university of beirut, beirut, lebanon nada assaf department of pathology and laboratory medicine, faculty of medicine, american university of beirut, beirut, lebanon citation el karaaoui ak, assaf n. using the slipta checklist to assess laboratory readiness for joint commission international accreditation. afr j lab med. 2023;12(1), a2044. https://doi.org/10.4102/ajlm.v12i1.2044 original research using the slipta checklist to assess laboratory readiness for joint commission international accreditation abdul k. el karaaoui, nada assaf received: 28 july 2022; accepted: 13 dec. 2022; published: 15 mar. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the stepwise laboratory improvement process towards accreditation (slipta) helps prepare laboratories in lowand middle-income countries to achieve international accreditation aligned with the iso 15189:2012 standards. accreditation by the joint commission international (jci) is among the highest sought by hospitals worldwide. while the readiness of laboratories with a five-star slipta score to undergo iso 15189:2012 accreditation was recently assessed, the compliance of the slipta checklist with jci is still unknown. objective: the study evaluated the slipta checklist’s utility in assessing laboratories to meet the jci standards. methods: we conducted a detailed gap analysis between slipta and jci laboratory standards from january 2021 to january 2022. we cross-matched the jci standard requirements to slipta clauses and categorised each standard into ‘met’, ‘partially met’, and ‘not met’. we highlighted similarities, discrepancies, and improvement areas. results: a total of 109 jci standards were included. the slipta checklist completely met 61 standards, partially met four, but did not meet 44. the unmet jci standards focused on the quality planning, control, and improvement sections. healthcare organisation management and quality control processes, including selecting an accredited reference laboratory, collecting quality management data, creating of post-analytical policies and procedures, and validating monitoring systems, constitute the basis of this preparation. conclusion: the slipta checklist covers major quality management system elements of the jci standards for laboratories. however, some components should be addressed to assure readiness for jci accreditation. what this study adds: this study identified additional areas not covered by the slipta checklist that are required for jci accreditation. keywords: standard culture; joint commission international; laboratory accreditation; lowand middle-income countries. introduction medical laboratories play a pivotal role in maintaining the healthcare chain in a nation. laboratory services are crucial for disease prevention, diagnosis, surveillance, and treatment.1,2 in developed countries, up to 70% of clinical decisions depend on laboratory test results.3,4 as such, maintaining quality laboratory services is essential for the efficient functioning of the healthcare system. accreditation is an external quality review that evaluates an organisation’s conformity with a predefined set of standards to receive formal recognition of compliance by an authorised external body.5 as a primary goal, laboratory accreditation aims to improve patient safety and the standard of care.6 in addition, accredited laboratories show superior test reliability, better operational efficiency, and higher competence.7 with increased outbreaks of diseases such as hiv, tuberculosis, and the recently emerging coronavirus disease 19 in lowand middle-income countries (lmics), there is increased attention on strengthening healthcare systems in limited-resource areas.8 in 2009, the world health organization regional office for africa, in collaboration with the african society for laboratory medicine, the united states center for disease control and prevention and african countries consorted on the creation of a stepwise laboratory improvement process towards accreditation (slipta) to implement quality management systems (qms) and organise quality improvement processes in lmic laboratories.9 the slipta programme consists of a checklist questionnaire linked to a five-star recognition depending on the level of compliance ranging from ≤ 55% (0 stars) to ≥ 95% (5 stars).8,9 it is an interim recognition, and laboratories that achieve a slipta five-star rating are encouraged to apply for international accreditations such as iso 15189:2012 and joint commission international (jci).8 founded in 1951, jci is today the largest international accreditation body for hospitals.11 the jci laboratory accreditation standards cover all laboratory needs for continual improvement within a quality management environment.11 it also addresses essential laboratory managerial and clinical functions while focusing on patient safety.11 because jci is a hospital accreditation scheme, laboratory jci clauses are clinically oriented. therefore, it is complementary to iso 15189 standards, which are process based. as an increasing number of hospitals worldwide are seeking jci accreditation, laboratories (including lmic laboratories) might undergo on-site evaluation by the jci during their hospital accreditation inspection. the suitability of the latest version of slipta to guide laboratories towards an iso 15189:2012 accreditation was recently assessed by datema et al.8 however, the readiness of slipta five-star laboratories to undergo jci accreditation is yet undetermined. the aim of this study was to evaluate the slipta checklist’s usefulness in assessing laboratories to meet jci standards. methods ethical considerations the local institutional review board (human research protection program at the american university of beirut) deemed the study exempt from review. this study did not involve human or non-human participants and did not require participant consent or data protections. study design this study was conducted at the college of american pathologists accredited department of pathology and laboratory medicine at the american university of beirut medical center, lebanon. the american university of beirut medical center is a jci-accredited tertiary healthcare facility. the assessors (the manuscript’s two authors) were selected based on their experience in the different accreditation processes and their certification as auditors for select certification bodies. the latest versions of slipta (version 2:2015) and jci standards for laboratories (3rd edition; january 2017) were retrieved from the world health organization regional office for africa and jci websites9,10,11. data analysis after thoroughly reviewing the jci and slipta standards, the authors performed a gap assessment separately between january 2021 and january 2022. first, each jci clause was compared to its counterpart in the slipta checklist and categorised as a ‘met’, ‘partially met’, or ‘not met’. ‘met’ jci clauses were addressed and totally covered by the slipta checklist, ‘partially met’ standards were addressed but not all elements were covered by the checklist, while ‘not met’ standards were not addressed. percentages were calculated using a spreadsheet, microsoft excel (microsoft, redmond, washington, united states). we then compared each author’s results, discussed areas of discrepancies, and issued a unified consensual analysis. the jci standards were divided into the three categories of the juran trilogy model (quality planning, quality control and quality improvement) as similarly done by juran.12 the results of our scoring system were used to determine the categories with the highest percentage of gaps. the slipta comprises 12 sections, each corresponding to a single qms element, which can be grouped into three categories: ‘quality planning’, ‘quality control’ and ‘quality improvement’, according to the juran trilogy model.12 the jci booklet comprises three general divisions, ‘accreditation participation requirements’, ‘patient-centred standards’, and ‘healthcare organisation management standards’, subdivided into seven sections. of the 152 jci standards, 109 were reviewed and compared to their counterparts in the slipta checklist. forty-three standards in sections 1 and 7 of jci were excluded from this review. section 1 targets accreditation participation requirements. this section is specific to jci and includes general principles such as the timely submission of accurate and complete data to the jci committee. section 1, including its 12 clauses, was excluded from the gap analysis due to lacking a counterpart. section 7 includes standards covering speciality quality control process (qcp) (qcp. 2 – qcp. 10.1 and qcp. 26 – qcp. 27.5: 31 clauses), which details requirements for single laboratory speciality sections. because slipta views hospital laboratories as a single unit, it lacks speciality-specific questions, and the corresponding standards (20%; 31/152) were excluded from the analysis. results the slipta v2:2015 addresses 60% of the 109 jci standards reviewed (56%; 61/109 wholly met and 4%; 4/109 partially met) but does not meet 40% (44/109) (table 1). among ‘partially met’ standards, 75% (3/4 standards) belong to the quality planning category and 25% (1/4) to quality improvement (table 2). among the ‘not met’ jci clauses, 11% (5/44) are quality planning clauses, 82% belong to quality control (36/44) and 7% (3/44) to quality improvement. table 1: gap analysis of joint commission international and stepwise laboratory improvement process towards accreditation, january 2021 – january 2022. table 2: distribution of non-conformities between joint commission international clauses and the stepwise laboratory quality improvement process towards accreditation checklist based on the categories of the juran trilogy, january 2021 – january 2022. gaps were noted in standards related to the organisation’s vision and mission statement, the accreditation of referral laboratories, ongoing communication with stakeholders, and quality monitoring. in terms of quality control, the validation of monitoring systems before implementation was also missing in the slipta checklist. slipta checklist coverage of joint commission international standards joint commission international section 2: patient safety goals this section focuses on patient safety goals and includes correct patient identification, effective communication of critical results, and hand hygiene standards. section 8 of the slipta (process control) addresses all the requirements for preanalytical quality checks, including patient identification, sample collection and reception. the first section of the slipta covers effective reporting of critical results, including adequate documentation and record keeping, while the facilities and biosafety section cover the hand hygiene standards. in addition, the slipta standards require proper staff training on hand hygiene practices (section 3.6 d). joint commission international section 3: healthcare organisation management the jci section 3 consists of healthcare organisation management standards, for which the initial step is the design of a leadership organisational chart and the determination of the laboratory’s mission and vision. the slipta checklist enquires about the presence of an organisational chart and a narrative description (section 3.2) of the different internal and external relationships within the laboratory. however, no points are assigned to developing a vision and mission statement. the following organisation management requirement is the appointment of a qualified laboratory manager, a clause fulfilled in the slipta checklist. despite the presence of a section in the slipta checklist addressing client management and customer service, there is no requirement for a qualified individual to oversee these procedures. in the jci, management is also responsible for service organisation and continuous communication with stakeholders; however, this is uncovered by the slipta checklist. resource planning is addressed in section 2.2 of the slipta checklist as part of the output review by leadership. the following section in jci regulates the contracts and services with reference laboratories, namely the presence of standard operating procedures to define the process for selecting and approving contracts and services by the leadership (governance, leadership and direction [gld] [gld.2.2]), the need for proof of accreditation or certification (gld.2.2.1), and performance monitoring (gld.2.2.2). while the slipta sets detailed requirements for the choice of referral laboratories and their continuous monitoring, along with the responsibility of the laboratory director in these tasks, it does not require checking the accreditation status of referral laboratories. the slipta checklist views the laboratory as a single unit rather than different laboratory speciality sections. thus, unlike the jci, it does not address standards for blood bank (gld.2.3) and point-of-care services (gld.2.4). nevertheless, the slipta standard 3.3, section 3, requires a laboratory director with adequate competency to supervise all laboratory workstations, which by extension includes blood bank and point-of-care services, as required by the jci gld.2.3 and gld2.4. in the jci, gld.3, 3.1 and 3.2 regulate effective interand intra-laboratory communication and efficient determination of clients’ needs; these are also detailed in slipta. the next jci section defines the different aspects of planning and coordination of a quality management programme, including the responsibilities of the leadership in developing the programme (gld.4) and implementing activities (gld.4.3), along with the required criteria for a well-functioning programme (gld.4.1) and its monitoring by a qualified individual (gld.4.2). the slipta checklist conforms to the jci requirements for quality management design. however, it does not completely cover quality management monitoring. for example, the jci quality management monitoring the leadership to determining processes to be measured (gld.4.5) and standards required to identify critical measures for clinical (gld.4.5.1) and managerial (gld4.5.2) structures, all of which slipta does not address. in addition, the analysis of measurement data requires skilled individuals (gld.5), an internal process to validate data (gld.6) and an appropriate investigation of off-target trends (gld.7). these criteria are met in the slipta checklist sections 1.5, 6.1 and 11.1 that determine the need for effective auditing, including personnel, procedures, and trend verification tools. the joint commission internationalgld.8 to 12.1 state principles for well-functioning safety and quality insurance programmes, including, but not limited to, the presence of an ongoing programme (gld.9), and the responsibility of the leaders to create, implement (gld.8), provide resources (gld.11) and monitor ongoing programmes (gld.10) while maintaining a safety culture throughout the laboratory (gld.12 and gld.12.1). laboratories with a five-star slipta accreditation are considered compliant with the safety and quality insurance programmes set by jci. joint commission international section 4: information management section 4 of the jci focuses on information management. it states the need for the presence of written policies and procedures addressing all testing phases (preanalytical: [management of information {moi}] moi.1, 2, 2.1, 2.2; analytical: moi.3; post-analytical: moi.4) and storage (moi.5), as well as their implementation (moi.1.1). it also requires the definition of a turnaround time (moi.4.1). the slipta checklist requires the presence of written policies and procedures (section 1.2) and their implementation (section 1.2). therefore, slipta meets all standards for all testing phases (preanalytical: sections 1.5, 8.4; analytical: sections 1.5, 8.7; post-analytical: section 1.5), including setting turnaround time and storage requirements for records and specimens. joint commission international section 5: staff qualifications and education section 5 standards are related to staff qualification and education. the first standard ([staff qualification and education {sqe}] sqe.1) mentions the role of laboratory leaders in defining the education and skills required for staff members, which is met by slipta section 3.3 on the role of laboratory directors. section 3 of slipta complies with the second standard in jci section 5, which discusses the training and experience required for supervisors and other leaders, licensure and registration of pathologists, and the need for defined responsibilities in the job descriptions. the next jci standard focuses on staff orientation, ongoing education, and yearly competency assessment needed to maintain and improve staff skills and knowledge (sqe.2, 2.1 and 3); the slipta sections 3.3, 3.6, and 3.7 meet these requirements. also, the slipta section 3.5. mentions the jci standard sqe.3.1 (maintenance of documented personnel information for every staff member) requirements. finally, the last standard in this section requires the laboratory to provide health services to all staff (sqe.4) and is met by the slipta checklist. joint commission international section 6: facility management laboratory facility management constitutes the focus of section 6. the first standard targets the responsibilities of laboratory leaders in providing the basic facilities and their facility’s compliance with laws and regulations. this requirement is met by slipta section 1.5 on accommodation and environmental conditions. the next standards state the requirement for sufficient storage space ([facility management and safety {fms}] fms.2.1) and proper record keeping (fms.2.2). resources for this goal should be provided by the laboratory leadership (fms.2). storage requirements, record keeping, and leadership roles are covered by the slipta sections 12.6, 1.5 (records control and sections) and 12.1. the jcistandards fms.3 to 4.1.1 mention the different aspects for efficient and effective operation of all utility systems and equipment. these aspects include inspection, testing of critical operating components, maintenance, and availability of emergency backup for critical utilities. interestingly, slipta section 1.5 on laboratory equipment and section 5 on equipment give a more detailed description of proper equipment protocol due to the question format checklist. the jci standard fms.4.2 on the requirement for a defined process for validating and maintaining computer software and laboratory information is met by slipta sections 9.9 and 9.10. the standards for periodic evaluation, labelling and documentation of reagents are met in slipta sections 7.4 (inventory control) and 8.8 (reagents acceptance testing). finally, section 12 in slipta (12.8–12.13) covers all safety aspects required by the last jci section 6 standard on safety (fms.5), including the management of hazardous materials, waste (fms.6 to 6.3), and fire (fms.7 and 7.1). joint commission international section 7: quality control the last jci section deals with quality control (qc) standards for all testing areas. it includes establishing qc processes for the different test methods, external and internal qc, verification of results accuracy, and methods comparison. it also targets new instrument validation and instrument monitoring systems implementation. the qc processes are addressed through several slipta sections with high compliance with jci clauses. however, the slipta checklist does not have questions on the validation of all monitoring systems before their implementation, as required by jci. discussion the level of compliance of slipta with jci requirements appears to be lower than its compliance with iso 15189, as determined by datema et al.8 this observation is because the slipta checklist is primarily based on the iso 15189 and the clinical and laboratory standards institute guideline qms01-a4 clauses in 2009.9 the slipta checklist and jci standards are structured differently. in addition to its checklist format, slipta is more detailed and extensive than jci, with specific instructions. the structure difference is because of their respective perspectives and objectives. the slipta checklist leans towards operations rather than management and targets laboratories with newly implemented qms. on the other hand, the jci accreditation standards are more outcome based than process based. they focus on the role of the laboratory leadership and management structure in achieving the desired quality outcomes, resource management, qc processes, the laboratory environment and development. the jci thus targets healthcare organisations with a particular quality background to achieve higher excellence. the jci booklet contains general standards addressing the laboratory as a single unit (similar to the slipta checklist). this approach is important to cover essential laboratory qms elements. in addition, requirements related to specific sections within the laboratory (blood bank, haematology, chemistry) are delineated. quality managers of laboratories undergoing jci evaluation might benefit from ensuring that section-specific requirements are met as they constitute up to 20% of jci standards. our analysis also reveals that the slipta quality planning and control sections contain the highest number of gaps when compared to jci (table 2). as an example, the definition of the organisation’s mission and vision, which slipta does not address, is crucial in the initial steps of the establishment of any qms. similarly, the role of leadership in service and resource planning and the implementation of culture safety should be emphasised in slipta. also, in the quality planning section, the safety programme should be improved in future versions, focusing on reducing infection risks and handling radioactive material. essential qc elements are missing in the slipta checklist. the relationship with external (referral) laboratories should be regulated. another important requirement for jci is the validation of qc systems before their implementation. the post-analytical testing phase should receive special focus for the improvement of the next versions, with the appropriate policies, procedures, and controls. in terms of quality improvement, our analysis reveals a need for the improvement of the managerial decisions of improvement activities and the associated data collection for quality measurement to comply with jci. however, the slipta checklist is skewed towards quality planning and qc sections, with quality improvement having the lowest number of allocated points.8 thus laboratories might focus on quality planning and qc clauses while neglecting quality improvement, which is a critical element in jci accreditation.8 limitations this study reviewed the slpita and jci checklists; thus, limitations are due to possible human errors during the review. each of the manuscript’s authors performed a full review independently to minimise errors. conclusion this article encourages all laboratories in lmics seeking a qms programme to start by implementing slipta, as per the world health organization regional office for africa recommendations. laboratories with high slipta scores seeking jci accreditation might benefit from addressing the following elements: selecting an accredited reference laboratory, collecting data for qm, creating post-analytical policies and procedures, and validating monitoring systems used in qc. because gap analysis is the initial step of any accreditation transition process, this analysis constitutes a useful guide for laboratory quality managers and directors in lmics planning to undergo the jci accreditation process as part of their healthcare organisations. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions n.a, and a.k.e.k. performed data collection and analysis, n.a. and a.k.e.k. wrote the manuscript. n.a. and a.k.e.k. reviewed drafts and approved the final version of the manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability data supporting the findings of this study are available from the corresponding author, n.a., on request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references datema ta, oskam l, van beers sm, klatser pr. critical review of the stepwise laboratory improvement process towards accreditation (slipta): suggestions for harmonisation, implementation and improvement. trop med int health. 2012;17(3):361–367. https://doi.org/10.1111/j.1365-3156.2011.02917.x nkengasong jn. strengthening laboratory services and systems in resource-poor countries. am j clin pathol. 2012;131(6):774. https://doi.org/10.1309/ajcp8gyx8ktkdatz forsman rw. why is the laboratory an afterthought for managed care organisations? clin chem. 1996;42(5):813–816. https://doi.org/10.1093/clinchem/42.5.813 centers for medicare & medicaid services. medicare and medicaid statistical supplement [homepage on the internet]. baltimore: centers for medicare & medicaid services [cited 2022 feb 03]. available from: https://www.cms.gov/research-statistics-data-and-systems/statistics-trends-and-reports/archives/mmss international organization for standardization. iso 15189:2012 medical laboratories – requirements for quality and competence [homepage on the internet]. geneva: international organization for standardization [cited 2022 feb 03]. available from: https://www.iso.org/standard/56115.html zima t. accreditation of medical laboratories – system, process, benefits for labs. j med biochem. 2017;36(3):231–237. https://doi.org/10.1515/jomb-2017-0025 peter tf, rotz pd, blair dh, khine aa, freeman rr, murtagh mm. impact of laboratory accreditation on patient care and the health system. am j clin pathol. 2010;134(4):550–555. https://doi.org/10.1309/ajcph1skq1hnwghf datema tam, oskam l, broerse jew, klatser pr. review of the stepwise laboratory quality improvement process towards accreditation (slipta) version 2:2015. afr j lab med. 2020;9(1):1068. https://doi.org/10.4102/ajlm.v9i1.1068 world health organisation, who regional office for africa. stepwise laboratory quality improvement process towards accreditation (slipta checklist version 2:2015 for clinical and public health laboratories) [homepage on the internet]. [cited 2022 feb 03]. available from: https://apps.who.int/iris/bitstream/handle/10665/204423/slipta-checkist0711.pdf?sequence=1&isallowed=y african society for laboratory medicine. slipta audited laboratories distribution map [homepage on the internet]. ethiopia:. african society for laboratory medicine. addis ababa. [cited 2022 feb 03]. available from: https://aslm.org/resource/slipta-map/ joint commission international. joint commission international accreditation standards for laboratories [homepage on the internet]. illinois: joint commission international. [cited 2022 feb 03]. available from: https://www.jointcommissioninternational.org/en/ juran jm. juran on leadership for quality – an executive handbook. new york, ny: collier macmillan. http://www.ajlmonline.org open access page 1 of 1 reviewer acknowledgement acknowledgement to reviewers in an effort to facilitate the selection of appropriate peer reviewers for the african journal of laboratory medicine, we ask that you take a moment to update your electronic portfolio on https://ajlmonline.org for our files, allowing us better access to your areas of interest and expertise, in order to match reviewers with submitted manuscripts. if you would like to become a reviewer, please visit the journal website and register as a reviewer. to access your details on the website, you will need to follow these steps: 1. log into the online journal at https:// ajlmonline.org 2. in your ‘user home’ [https://ajlmonline.org/ index.php/ajlm/user] select ‘edit my profile’ under the heading ‘my account’ 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fonte richard garfield lisa gerwing-adima malay haldar masoumeh hasani benjamin d. hedley marianne k. henderson lisa m. herzog laura hewitson carry hill hiroshi ichimura oni e.a. idigbe emmanuel o. irek mohamed a. ismail lise jamieson kithsiri b. jayasekara ali jebali feyisayo e. jegede derek jones lavania joseph aderemi kehinde stephen b. kennedy daniel kimani olayinka a. kotila taiwo kotila george boateng kyei direk limmathurotsakul han-qing liu lucio luzzato evelyn madoroba faithful makita-chingombe talkmore maruta marguerite massinga loembe itumeleng matle mohammed merza sandra miselem barend mitton reena d. mohanlal meade morgan gram (graham) mutandi jane mwangi jacob seroni mwebi jean ndjomou jeremy nel mae newton-foot apeksha niraula fatima b. nma jiya ifeanyi nsofor jacinta nwogu collins o. odhiambo abiodun r. ojewuyi olusegun s. ojo paul a. olaiya john a. olaniyi pascale ondoa libby onyeka chin-yih ou judith owen michael d. owens joão palma cihan papan rene paulussen maria paximadis m.l. pereira bueno lucy a. perrone sam peters deryn petty dimitri poddighe alexander m. popov joachim potgieter lance d. presser siriporn proungvitaya beatrice puije jennifer a. punt rahul rajbhar garshasb rigi theresa roussouw halima m. said jon salmanton-garcía moses samje christine e. schaner-tooley seema shetty caleb skipper julie smith anthony m. smith ajaykumar k. thirumala fabio timeus lara vojnov adolfo vubil shouwei wang azza a. zulfu http://www.ajlmonline.org https://ajlmonline.org https://ajlmonline.org https://ajlmonline.org https://ajlmonline.org/index.php/ajlm/user https://ajlmonline.org/index.php/ajlm/user mailto:publishing@aosis.co.za article information author: nicolas bouchet1 affiliation: 1quality assurance department, cnrfp (centre national de recherche et de formation sur le paludisme), burkina faso correspondence to: nicolas bouchet email: nikolas.bouchet@gmail.com postal address: 01 bp 2208, ouagadougou, burkina faso dates: received: 08 apr. 2014 accepted: 24 sep. 2014 published: 13 may 2015 how to cite this article: bouchet b. iso 15189: 2012: quels changements pour les laboratoires africains? afr j lab med. 2015;4(1), art. #181, 4 pages. http://dx.doi.org/10.4102/ajlm.v4i1.181 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. iso 15189: 2012 : quels changements pour les laboratoires africains? in this opinion paper... open access • introduction    • la norme iso 15189: 2012    • l'approche processus    • manuel qualité    • gestion des risques    • gestion des informations de laboratoire    • changements mineurs • conclusion • remerciements    • intérêts concurrents    • contributions des auteurs • références introduction top ↑ la norme iso 15189 est le standard international de référence en matière de biologie médicale,1 et l'accréditation est la reconnaissance d'un système qualité en pleine conformité avec cette norme. conscients des challenges actuels dans le domaine de la santé publique et de la recherche biomédicale, les laboratoires africains de biologie médicale pourraient être amenés à développer des systèmes de management de la qualité qui suivent les exigences de la norme iso 15189. sous l'impulsion de l'oms, les programmes slipta et slmta ont vu le jour ces dernières années en afrique, pour accompagner les laboratoires dans la mise en place de processus qualité. 2, 3, 4, 5 comme tout système qualité, les normes iso évoluent, et sont périodiquement révisées; c'est en décembre 2012 que l'iso, l'organisation internationale de normalisation, a publié la troisième édition de la norme iso 15189 6, qui remplace la version de 2007. 7 cet article a pour but de mettre en avant les changements majeurs entre les deux versions de cette norme, et les modifications que cela entraînera dans les systèmes qualité mis en place dans les laboratoires, et notamment les laboratoires africains. la norme iso 15189: 2012 la norme dans sa version 2012 est un document plus clair et mieux organisé que la version 2007. outre le titre qui s'est raccourci, la manière d'organiser les différentes rubriques a été changée, en suivant un ordre plus logique, ce qui permettra sans aucun doute aux utilisateurs de mieux appréhender les différentes exigences. lors de la présentation de la nouvelle version, l'iso a parlé de « révision technique »8, cela signifiant un document présentant chaque aspect avec plus de détails; mais la lecture de cette nouvelle version montre que les changements sont en réalité plus conséquents. l'approche processus un processus est « un ensemble d'activités corrélées ou interactives qui transforme des éléments d'entrée en éléments de sortie »9. l'approche processus décrit l'organisation du travail comme une suite d’étapes inter-reliées avec pour objectif la satisfaction optimale des clients et l'atteinte des objectifs fixés. le but est de rendre l'organisation plus fonctionnelle, et moins dépendante de la hiérarchie, avec l'introduction et le développement du management transversal, de la culture du résultat et du travail d’équipe au sein du laboratoire. la version 2007 abordait ce concept, mais laissait libre choix aux laboratoires de suivre ou non une approche processus dans leur système de management de la qualité (smq); cette approche existait déjà dans les smq basés sur la norme générale de la qualité, l'iso 9001, qui avait introduit ce concept dans sa version 20001, et l'avait maintenu dans sa version de 2008.1 la version 2012 de l'iso 15189 est beaucoup plus explicite, l'approche processus devenant une exigence (sous-chapitre 4.2.1); ceci pourrait s'avérer compliqué à mettre en œuvre pour les laboratoires africains. en effet, cette approche peut s'avérer difficile à mettre en place pour tous les laboratoires d'analyses de petite taille, avec un personnel peu nombreux, ces laboratoires représentant la majorité des laboratoires de biologie médicale en afrique sub-saharienne. l'approche processus implique en effet de cartographier les processus, c'est-à-dire de représenter l'ensemble des processus nécessaires pour le smq et leurs séquences en déterminant les interactions qui les lient, et en désignant des pilotes de processus avec une définition très claire des rôles de chacun. dans des structures de petite taille, une même personne peut avoir une vue d'ensemble et une vue détaillée des activités, et l'intérêt d'une cartographie précise des différents processus est souvent vu comme inutile. si par exemple, dans un laboratoire de 4 employés, chaque individu est impliqué dans tous les processus, la mise en place d'une cartographie peut paraître superflue. néanmoins, cette approche présente plusieurs avantages: tout d'abord, elle insiste sur le but final du smq du laboratoire, le résultat du patient, pour permettre par la suite de bien définir le rôle de chacun dans l'obtention du résultat du patient, et de le montrer clairement. elle permet enfin d'analyser et donc d'améliorer les performances du laboratoire. les laboratoires de biologie médicale ont un avantage pour mettre cette approche en œuvre, puisque leurs activités s'articulent autour de trois grands processus opérationnels, définis dans la norme: les processus pré-analytiques (chapitre 5.4), analytiques (5.5) et post-analytiques (5.7). nous pensons qu'en se concentrant sur ces trois processus majeurs, un laboratoire pourra être en mesure de rattacher les autres processus (comme les équipements et la gestion des stocks, les ressources humaines, la satisfaction des clients, l'amélioration continue, etc.) à une cartographie assez simple. manuel qualité alors que pour la rédaction du manuel qualité, la version 2007 (sous-chapitre 4.2.4) proposait un plan en 23 points, la version 2012 s'attache à décrire (sous-chapitre 4.2.2.2) les six grands points essentiels dans un manuel qualité de laboratoire: politique qualité, description du smq, organisation et structure du laboratoire, rôles et responsabilités de la direction du laboratoire, système documentaire et les politiques établies pour le smq avec référence aux activités sur lesquelles elles reposent. ce changement va donc laisser à chaque laboratoire le soin de définir le plan de son manuel en fonction de son propre système qualité, ce qui démontrera d'une part la maîtrise de la norme par le laboratoire, et d'autre part la capacité du laboratoire à adapter la norme aux réalités locales, ce qui s'avère être souvent le cas en afrique sub-saharienne. le tout en simplifiant l’écriture de ce document, pierre angulaire du smq. gestion des risques cet aspect est à nos yeux le changement majeur de la nouvelle version de la norme. il apparaît dans le sous-chapitre 4.1.1.4, relatif au directeur de laboratoire, où il est du rôle du directeur d’ « élaborer et appliquer un plan de fonctionnement dégradé » ; il est précisé que ces plans doivent être soumis à essai de manière périodique. le sous-chapitre 4.14.6 (gestion des risques) aborde également le sujet, en mettant l'accent sur les risques de défaillances éventuelles sur les résultats des analyses; tout doit être mis en œuvre pour réduire et/ou éliminer ces risques. le concept est également retrouvé dans le nouveau paragraphe consacré à la gestion des informations de laboratoire (5.10.3); il est précisé dans le point 5.3 que « le laboratoire doit disposer de plans de contingence […] en cas de défaillances ». ce concept de management des risques est une introduction importante de cette nouvelle norme; il implique un travail conséquent pour sa mise en œuvre, car toutes les situations d'urgence doivent être identifiées et évaluées (exemple: rupture en réactifs causée par un retard de livraison, panne d'un analyseur, coupure d’électricité, dysfonctionnement d'une imprimante, etc.). chaque situation doit être inscrite dans ce « plan de fonctionnement dégradé », avec pour chacune: la manière de résoudre ce problème (corrections = mesures palliatives); la manière de prévenir le patient et le médecin; la manière de communiquer le problème en interne; les actions correctives à mettre en œuvre pour éviter que le problème ne se reproduise; la manière de revenir à une situation normale. ces plans doivent ensuite être « testés périodiquement », et tout ce processus doit être documenté pour être justifié le cas échéant; ces tests doivent servir à améliorer le plan si nécessaire. ces plans doivent également être audités en cas d'audit interne du système qualité. si l'on prend des exemples de risques comme une coupure d’électricité ou le retard d'approvisionnement en réactifs, ce sont des évènements rares dans les pays industrialisés, mais ce sont des problèmes courants dans les pays d'afrique sub-saharienne. nombreuses sont les capitales africaines qui vivent encore au rythme des délestages durant certaines périodes de l'année, et les retards de livraison sont fréquents pour tout ce qui concerne les réactifs et consommables dans les laboratoires. ce qui sera un risque avec une probabilité d'apparition très faible dans un pays industrialisé sera un risque avec une fréquence d'apparition très élevée dans un pays de l'afrique sub-saharienne, ce qui va rendre la gestion des risques selon la norme iso plus difficile à mettre en œuvre pour les laboratoires africains. les plans de fonctionnement dégradé devront être mis en œuvre périodiquement pour certains aspects, cela faisant qu'une opération prévue pour être « exceptionnelle » deviendra une opération de routine. il est à noter que cette introduction de la notion de gestion des risques dans la nouvelle version de la norme iso 15189 s'inscrit dans une logique plus globale. ce concept, qui vient du monde industriel, a fait ces dernières années son entrée dans le monde réglementaire. dans le cadre du processus de révision de la norme iso 9001, qui est le référentiel de base en ce qui concerne les systèmes de management de la qualité (la nouvelle version est prévue pour 2015), le concept d'une approche de gestion des risques a été identifié par le groupe d'experts chargés de la révision de l'iso 9001 (26ème réunion de l'iso tc 176, tokyo, février 2010)8, et sera intégré à la nouvelle version. la maîtrise des risques est clairement l'un des objectifs de cette nouvelle version de la norme iso 9001, nécessitant de se projeter vers le futur pour anticiper tous les problèmes possibles qui pourraient empêcher la satisfaction client, objectif prioritaire de l'iso 9001. dans le domaine même d'application de l'iso 15189, dans les laboratoires de biologie médicale, ce concept a été introduit aux etats-unis par les nouvelles réglementations en matière de plan de contrôles de qualité internes, en suivant la ligne directrice du clsi (clinical and laboratory standards institute) ep23-a.12 cette ligne directrice a introduit le concept de gestion du risque dans le domaine des contrôles de qualité internes dans les laboratoires de biologie médicale13, 14, 15. gestion des informations de laboratoire la section 5.10 est une demi-nouveauté de cette version; il s'agit en effet de l'annexe b de la norme version 2007, qui passe donc du statut de ‘section informative’ à celui de ‘section normative’, avec obligation de mettre en œuvre ces exigences. toute cette section est centrée sur la protection des données et la gestion du système d'information; le laboratoire doit s'assurer que les outils informatiques impliqués dans la gestion des informations de laboratoire (collecte, traitement, enregistrement, compte-rendu, conservation des données) soient validés par les fournisseurs, soient régulièrement maintenus et soient sécurisés. les laboratoires devront tenir compte de ces nouvelles exigences et agir en conséquence, même si elles sont difficiles à mettre en œuvre, et tout aussi difficiles à documenter, encore plus dans notre contexte africain. changements mineurs le point 4.14, qui était dans l'ancienne version de la norme uniquement consacré aux audits internes, est dans cette version 2012 plus complet, puisqu'il y est question de tous les aspects des audits et évaluations: revue des prescriptions, procédures et exigences concernant les échantillons, les évaluations des retours d'informations de la part des clients, la prise en compte des suggestions du personnel, les audits internes et externes, la gestion des risques et le suivi des indicateurs qualité. tous ce processus d’évaluation / audit a pour objectif d'améliorer le système qualité du laboratoire, en s'assurant que tous les processus nécessaires à la qualité des résultats des analyses ont été réalisés pour répondre aux exigences des clients. un nouveau sous-paragraphe (4.1.1.3) présente des règles d’éthique au sujet des conflits d'intérêts potentiels, de l'intégrité du personnel, des considérations éthiques dans le cadre de la manipulation des échantillons d'origine humaine et de la confidentialité des patients. ce point est très important, car la culture de l’éthique des affaires / des entreprises est un concept peu développé en afrique sub-saharienne, contrairement au concept d’éthique médical, qui était traité dans la version 2007 de la norme en annexe c, ce qui a disparu dans la version 2012. conclusion top ↑ la norme iso 15189: 2012 reste le ‘gold standard’ en matière de qualité dans les laboratoires de biologie clinique. l'organisation plus claire de cette version permettra aux laboratoires de mieux s'imprégner du document pour mieux répondre aux exigences normatives. il est en effet impératif aux laboratoires d'appliquer ce modèle qualité pour obtenir l'accréditation pour s'assurer la confiance des patients et un respect national et international. le défi est de taille pour les laboratoires africains, qui doivent s'adapter à cette norme en prenant en compte les conditions particulières dans lesquelles ils évoluent. il est souhaitable que l'oms-afrique adapte sa liste slipta à cette nouvelle version de la norme, en adaptant de manière générale tous les programmes d'accompagnement pour inclure tous les changements de la version 2012. remerciements top ↑ j'adresse mes remerciements au dr issa nébié ouedraogo et au dr issiaka soulama (tous deux du cnrfp, burkina faso) pour leurs précieux commentaires et discussions autour du manuscrit, ainsi qu'au dr sodiomon b. sirima, administrateur délégué du cnrfp pour m'avoir permis de mener ce travail. intérêts concurrents aucun conflit d'intérêt: l'auteur déclare n'avoir aucun lien financier ou personnel l'ayant influencé de façon inappropriée pendant la rédaction de l'article. contributions des auteurs n.b. (centre de recherche et de formation sur le paludisme), rédaction de l'article. références top ↑ datema tam, oskam l, klatser pr. review and comparison of quality standards, guidelines and regulations for laboratories. afr j lab med. 2011;1(1), art.#3, 7 pages. gershy-damet g, rotz p, cross d. belabbes e, cham f, ndihokubwayo j, et al. the world health organization african region laboratory accreditation process. am j clin pathol. 2010; 134:393-400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm maruta t, motebang d, wanyoike j, peter t, rotz pj. impact of mentorship on who-afro strengthening laboratory quality improvement process towards accreditation (slipta). afr j lab med. 2012;1(1), art. #6, 8 pages. yao k, mckinney b, murphy a, rotz p, wafula w, sendagire h, okui s, et al. improving quality management systems of laboratories in developing countries. am j clin pathol. 2010;134:401-409. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj mothabeng d, maruta t, lebina m, lewis k, wanyoike j, mengstu y. strengthening laboratory management towards accreditation: the lesotho experience. afr j lab med. 2012;1(1), art. #9, 7 pages. organisation internationale de normalisation (iso). iso 15189: 2012. laboratoires de biologie médicale – exigences concernant la qualité et la compétence. troisième édition 2012-11-01, version corrigée 2013-03-01. organisation internationale de normalisation (iso). iso 15189: 2007. laboratoires d'analyses de biologie médicale – exigences particulières concernant la qualité et la compétence. deuxième édition 2007-04-15, version corrigée 2007-09-15. site internet de l'organisation internationale de normalisation (iso). disponible à cette adresse: www.iso.org organisation internationale de normalisation (iso). iso 9000: 2005 systèmes de management de la qualité – principes essentiels et vocabulaire. troisième édition 2005-09-15. organisation internationale de normalisation (iso). iso 9001: 2000 systèmes de management de la qualité – exigences. troisième édition 2000-12-15. organisation internationale de normalisation (iso). iso 9001: 2008 systèmes de management de la qualité – exigences. quatrième édition 2008-11-15, version corrigée 2009-07-15. clinical and laboratory standards institute. ep23-a: laboratory quality control based on risk management; approved guideline. 2011. person n. developing risk-based quality control plans: an overview of clsi ep23-a. clin lab med, 2013, 33, 15-26. http://dx.doi.org/10.1016/j.cll.2012.11.003 westgard j. perspectives on quality control, risk management, and analytical quality management. clin lab med, 2013, 33, 1–14. http://dx.doi.org/10.1016/j.cll.2012.10.003 nichols j. laboratory quality control based on risk management. ann saudi med. 2011 may-jun; 31(3): 223–228. http://dx.doi.org/10.4103/0256-4947.81526 abstract introduction methods results discussion acknowledgements references about the author(s) nomonde r. mvelase department of medical microbiology, kwazulu-natal academic complex, national health laboratory service, durban, south africa school of laboratory medicine and medical sciences, college of health sciences, university of kwazulu-natal, durban, south africa lindiwe p. cele department of public health, epidemiology and biostatistics unit, sefako makgatho health sciences university, pretoria, south africa ravesh singh department of medical microbiology, kwazulu-natal academic complex, national health laboratory service, durban, south africa school of laboratory medicine and medical sciences, college of health sciences, university of kwazulu-natal, durban, south africa yeshnee naidoo kwazulu-natal research innovation and sequencing platform, nelson r mandela school of medicine, university of kwazulu-natal, durban, south africa centre for epidemic response and innovation, school of data science and computational thinking, stellenbosch university, stellenbosch, south africa jennifer giandhari kwazulu-natal research innovation and sequencing platform, nelson r mandela school of medicine, university of kwazulu-natal, durban, south africa eduan wilkinson kwazulu-natal research innovation and sequencing platform, nelson r mandela school of medicine, university of kwazulu-natal, durban, south africa centre for epidemic response and innovation, school of data science and computational thinking, stellenbosch university, stellenbosch, south africa tulio de oliveira kwazulu-natal research innovation and sequencing platform, nelson r mandela school of medicine, university of kwazulu-natal, durban, south africa centre for epidemic response and innovation, school of data science and computational thinking, stellenbosch university, stellenbosch, south africa centre for the aids programme of research in south africa, university of kwazulu-natal, durban, south africa khine swe swe-han department of medical microbiology, kwazulu-natal academic complex, national health laboratory service, durban, south africa school of laboratory medicine and medical sciences, college of health sciences, university of kwazulu-natal, durban, south africa koleka p. mlisana department of medical microbiology, kwazulu-natal academic complex, national health laboratory service, durban, south africa school of laboratory medicine and medical sciences, college of health sciences, university of kwazulu-natal, durban, south africa centre for the aids programme of research in south africa, university of kwazulu-natal, durban, south africa citation mvelase nr, cele lp, singh r, et al. consequences of rpob mutations missed by the genotype mtbdrplus assay in a programmatic setting in south africa. afr j lab med. 2023;12(1), a1975. https://doi.org/10.4102/ajlm.v12i1.1975 original research consequences of rpob mutations missed by the genotype mtbdrplus assay in a programmatic setting in south africa nomonde r. mvelase, lindiwe p. cele, ravesh singh, yeshnee naidoo, jennifer giandhari, eduan wilkinson, tulio de oliveira, khine swe swe-han, koleka p. mlisana received: 06 june 2022; accepted: 24 oct. 2022; published: 06 feb. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: rifampicin resistance missed by commercial rapid molecular assays but detected by phenotypic assays may lead to discordant susceptibility results and affect patient management. objective: this study was conducted to evaluate the causes of rifampicin resistance missed by the genotype mtbdrplus and its impact on the programmatic management of tuberculosis in kwazulu-natal, south africa. methods: we analysed routine tuberculosis programme data from january 2014 to december 2014 on isolates showing rifampicin susceptibility on the genotype mtbdrplus assay but resistance on the phenotypic agar proportion method. whole-genome sequencing was performed on a subset of these isolates. results: out of 505 patients with isoniazid mono-resistant tuberculosis on the mtbdrplus, 145 (28.7%) isolates showed both isoniazid and rifampicin resistance on the phenotypic assay. the mean time from mtbdrplus results to initiation of drug-resistant tuberculosis therapy was 93.7 days. 65.7% of the patients had received previous tuberculosis treatment. the most common mutations detected in the 36 sequenced isolates were i491f (16; 44.4%) and l452p (12; 33.3%). among the 36 isolates, resistance to other anti-tuberculosis drugs was 69.4% for pyrazinamide, 83.3% for ethambutol, 69.4% for streptomycin, and 50% for ethionamide. conclusion: missed rifampicin resistance was mostly due to the i491f mutation located outside the mtbdrplus detection area and the l452p mutation, which was not included in the initial version 2 of the mtbdrplus. this led to substantial delays in the initiation of appropriate therapy. the previous tuberculosis treatment history and the high level of resistance to other anti-tuberculosis drugs suggest an accumulation of resistance. keywords: tuberculosis; rifampicin resistance; rpob mutations; mtbdrplus; discordance. introduction access to reliable, rapid and automated nucleic acid amplification tests remains one of the key factors in fulfilling the early tuberculosis diagnosis and universal access to drug susceptibility testing (dst) requirement of the world health organization (who) end tb strategy.1 the past decade has seen the development of many molecular tests, some of which have been endorsed by the who. the genotype mtbdrplus assay was the first to be endorsed by the who in 2008, followed by the xpert mtb/rif (xpert) in 2010.2,3 because of its ease of use, the xpert has been implemented in many settings for the initial diagnosis of tuberculosis and detection of rifampicin resistance. both the mtbdrplus and the xpert detect rifampicin resistance by identifying mutations in the 81-base pair region of the rpob gene, which spans codon 426–452 in the mycobacterium tuberculosis numbering system and codon 507–533 in the escherichia coli numbering system.4 this region is also called the rifampicin resistance-determining region (rrdr), as most of the rifampicin resistance-conferring mutations are found in this region.5 additionally, the mtbdrplus also detects resistance against isoniazid by identifying mutations in the katg gene and the promoter region of the inha gene. due to the limitations of the xpert and the mtbdrplus assays, phenotypic methods remain the gold standard for tuberculosis dst. both assays demonstrate variable performance in detecting heteroresistance and do not detect rpob gene mutations outside the rrdr.6 until recently, mutations outside the rrdr were believed to only account for less than 5% of overall rifampicin resistance.7,8 however, in a national drug resistance survey conducted in eswatini between 2009 and 2010, 30% of multidrug-resistant tuberculosis (mdr-tb: resistant to rifampicin and isoniazid) isolates carried the i491f rpob gene mutation located outside the rrdr.9 this caused concerns, especially in neighbouring countries like south africa, because while this mutation is rare globally, it might be more common in certain geographical settings. a subsequent study conducted in the northern provinces of south africa showed that 15% of isoniazid mono-resistant strains carried the i491 mutation, meaning they were mdr-tb strains.10 the same study also revealed that strains carrying this mutation may be driving outbreaks of mdr-tb in eswatini and south africa.10 rapid molecular tests that only detect mutations in the rrdr may fail to detect rifampicin resistance in patients with tuberculosis caused by strains carrying mutations outside the rrdr, and this may lead to inappropriate management, resulting in resistance selection, accumulation of resistance, treatment failure and increased transmission. in settings where molecular and phenotypic rifampicin dst are performed concurrently, discordant results often occur, especially with liquid culture-based assays.11,12 given the fact that rifampicin is the key determinant of the choice of a treatment regimen, the hesitancy caused by discordant results may also affect the decision to start appropriate treatment in the affected patients. often, an attempt is made to confirm a discordant result by either repeating the test or using another confirmatory assay (if available), thus causing further delay in initiating appropriate therapy. because the kwazulu-natal province accounts for almost 30% of south africa’s drug-resistant tuberculosis (dr-tb) cases, and since eswatini forms part of its northern border, we conducted this study in kwazulu-natal, south africa, to determine why phenotypically resistant isolates were reported as rifampicin susceptible on the mtbdrplus.13 considering the dearth of information on the clinical management of patients with rifampicin-discordant tuberculosis results globally, we also report on the programmatic management of these patients in our setting. methods ethical considerations ethics approval was obtained from the university of kwazulu-natal biomedical research ethics council (be267/18). individual patient consent was not required as only routine programmatic data was accessed; however, permission was obtained from the provincial department of health. for anonymity, patients’ names were only used for data collection and were not used during analysis. study design and setting the study was conducted in the kwazulu-natal province in south africa. the province has the second-highest population in the country with more than 11 million people. there are 11 districts in the province and one mdr-tb treatment facility per district. in health facilities in the kwazulu-natal province, the initial diagnosis of tuberculosis and rifampicin resistance is routinely done using the xpert (xpert mtb/rif, cepheid, sunnyvale, california, united states) in all patients suspected of tuberculosis disease. the xpert was previously used but was later replaced by its successor, the xpert mtb/rif ultra (xpert ultra, cepheid, sunnyvale, california, united states), in 2017. for patients with rifampicin-susceptible tuberculosis, no further dst is performed, and they are treated using first-line tuberculosis therapy. in patients with rifampicin-resistant tuberculosis on the xpert (ultra), a second sample is taken for culture and dst. other indications for tuberculosis culture include treatment failure and paucibacillary tuberculosis that shows a negative result on the xpert (ultra). during the study period between january 2014 and december 2014, the automated bactec mycobacteria growth indicator tube 960 system (becton dickinson, sparks, maryland, united states) was used for m. tuberculosis culture, and initial dst was done on all positive cultures using the mtbdrplus version 2 assay (hain lifescience, nehren, germany) to confirm rifampicin resistance and test for isoniazid resistance. the mtbdrplus assay uses dna strip technology where the strip contains both wild-type probes and mutation probes for the commonly occurring mutations (s450l, h455y, h455d, and d435v for rifampicin). the labelled polymerase chain reaction products from an amplified target are hybridised with specific probes immobilised on a strip (reverse hybridisation). resistance is reported when there is a lack of binding to the wild-type probe with or without binding to a mutation probe.14 isolates that were resistant to either rifampicin or isoniazid on the mtbdrplus assay were further tested for resistance to critical concentrations of isoniazid (0.2 µg/ml), rifampicin (1 µg/ml), ofloxacin (2 µg/ml), streptomycin (2 µg/ml), and kanamycin (5 µg/ml) using the 1% agar proportion method on middlebrook 7h10 agar (becton dickinson, sparks, maryland, united states).15 the simultaneous performance of molecular and phenotypic rifampicin dst allowed the detection of discordance between these two tests. laboratory analysis routine clinical isolates from specimens received at the inkosi albert luthuli central hospital laboratory of the kwazulu-natal province between january 2014 and december 2014 were used for this study. isolates were selected if they showed rifampicin susceptibility on the mtbdrplus but were rifampicin resistant on the 1% agar proportion method on middlebrook 7h10 agar at a critical rifampicin concentration of 1 µg/ml. the isolates from 2014 were chosen because simultaneous molecular and phenotypic rifampicin dst was performed during this time but was subsequently stopped. the selected isolates were then stored at –70 °c and later used for this study. of the isolates that had discordant rifampicin results, 36 were randomly selected for further evaluation using whole-genome sequencing. whole-genome sequencing stored isolates were grown on 7h11 middlebrook agar for over three weeks. genomic dna was extracted from isolates using the quick-dna™ miniprep kit (zymo research, irvine, california, united states). the concentration of dna was determined using the qubit dsdna hs assay kit (invitrogen, carlsbad, california united states). a minimum of 2 ng/µl dna was used for library preparation. libraries were prepared using the nextera dna library preparation kit and nextera cd index kit (illumina, san diego, california united states) according to the manufacturer’s protocol. each library was pooled and diluted to an equimolar concentration of 4 nm followed by denaturation and dilution to the final loading concentration. the library was spiked with 1% phix, which serves as an internal control to account for low-diversity libraries, and run on an illumina miseq platform (illumina, san diego, california, united states) using the miseq v2 500 cycle reagent kit (illumina, san diego, california, united states). drug resistance and strain-type profiles were determined using the tbprofiler pipeline (http:\\tbdr.lshtm.ac.uk\).16 mutations were called out at 100× depth of coverage. clinical data patients with discordant rifampicin susceptibility results were identified from the laboratory. further laboratory results (phenotypic dst, hiv status, cd4 count, and viral load results) were obtained from the laboratory information system. treatment data was obtained from the electronic drug-resistant tuberculosis treatment register of the kwazulu-natal provincial department of health. treatment outcomes were defined according to the who definitions.17 data analysis the data were captured into an excel file (microsoft corp., redmond, washington, united states) and cleaned and coded before being imported into stata version 13 (statacorp, college station, texas, united states) for statistical analysis. patient names and ages were used to remove duplicate entries. descriptive analysis was conducted on data for all patients with rifampicin-discordant tuberculosis results, as well as those selected for whole-genome sequencing. categorical variables such as sex, hiv status, previous tuberculosis treatment, as well as the xpert, phenotypic dst and mtbdrplus results, were presented as proportions and percentages. continuous variables such as age, cd4 count, and the time taken to treatment initiation were presented as means with standard deviation. a bivariate analysis was conducted using the two-sample t-test to compare the mean time taken to treatment initiation between the xpert-susceptible and xpert-resistant results, and a p-value of < 0.05 was considered indicative of statistical significance. results in 2014, out of 12 279 m. tuberculosis complex cases detected using the mtbdrplus assay, 505 (4.1%) were isoniazid mono-resistant. from the 505 isoniazid mono-resistant cases, 145 (28.7%) were mdr-tb based on the phenotypic 1% agar proportion method (i.e., had discordant rifampicin dst results). the median age of the patients with discordant rifampicin dst results was 33.8 years, and 52.4% were male (table 1). table 1: patient characteristics of isolates with rpob gene mutations missed by the genotype mtbdrplus assay at inkosi albert luthuli central hospital laboratory in kwazulu-natal, south africa between january 2014 and december 2014. microbiology results out of the 145 isolates with discordant rifampicin dst results, phenotypic dst showed that 79 (54.5%) were mdr-tb plus streptomycin resistant, 43 (29.7%) were mdr-tb, 10 (6.9%) were mdr-tb plus resistance to a fluoroquinolone, seven (4.8%) were mdr-tb plus resistance to a fluoroquinolone and any second-line injectable agent, five (3.5%) were mdr-tb plus resistance to a second-line injectable agent, and one (0.7%) was rifampicin mono-resistant. xpert results were available for 97 (66.9%) of the 145 patients. of these, 37 (38.1%) were rifampicin resistant, and 60 (61.9%) were susceptible. treatment details patient records were found for 108 (74.5%) of the 145 isolates on the dr-tb treatment register. of these, 71 (65.7%) patients had a previous tuberculosis treatment history. sixty-seven (62.0%) patients had favourable treatment outcomes (cured and treatment completed) and the average treatment duration was 18.4 months. seventy-six (70.4%) patients on the dr-tb treatment register had an xpert result; 34 of these were rifampicin resistant and 42 were rifampicin susceptible (table 2). the mean time to dr-tb treatment initiation was 32.7 days for patients with rifampicin-resistant xpert results, and 186.6 days for patients with rifampicin-susceptible xpert results. the difference in the time to treatment between the two groups was statistically significant (p < 0.001). table 2: time from availability of results to initiation of drug-resistant tuberculosis treatment in patients with m. tuberculosis isolates with discordant rifampicin susceptibility results in kwazulu-natal, south africa between january 2014 and december 2014. when calculating the mean time to results in reference to the mtbdrplus assay and phenotypic dst results, the 34 xpert rifampicin resistant cases were removed. additionally, in two other patients initiated on treatment, the date of initiation was not recorded. of the remaining 72 patients, 11 patients started treatment before mtbdrplus results became available, while 61 started treatment after a mean of 93.7 days from the availability of results. similarly, for phenotypic dst, 49 of the 72 patients started treatment prior to the availability of results while 23 started treatment after (mean 19.8 days) results were available. whole-genome sequencing results out of the 36 isolates whose whole genomes were sequenced, 19 (52.8%) had single rpob mutations outside the rrdr, namely i491f (16 isolates; 44.4%), v170f (2; 5.6%) and p483l (1; 2.8%) (figure 1). there were 14 (38.9%) isolates with single rpob mutations located within the rrdr, namely 12 isolates (33.3%) with l452p mutation and two isolates (5.6%) with s450l mutation. the three remaining isolates (8.3%) had double rpob mutations, including one isolate with a combination of the s450l mutation (located within the rrdr) and t400a mutation (located outside the rrdr), and two isolates with double mutations located within the rrdr – one with d435g and l452p mutations and another one with d435y and l452p mutations. figure 1: whole-genome sequencing results of 36 isolates with rpob gene mutations missed by the genotype mtbdrplus assay at inkosi albert luthuli central hospital laboratory in kwazulu-natal, south africa, between january 2014 and december 2014. the most common isoniazid resistance-conferring mutation was the s315t mutation, (29 isolates; 80.6%). this was followed by the inha promoter region mutation t-8a (15 isolates; 41.7%), which was found together with the katg mutation in all isolates. besides resistance to rifampicin and isoniazid, most isolates were also resistant to other first-line drugs, including pyrazinamide (25 isolates; 69.4%), ethambutol (30; 83.3%), and streptomycin (25; 69.4%). some isolates were also resistant to second-line drugs, including the second-line injectable agents (three isolates; 8.3%), fluoroquinolones (six; 16.7%), and ethionamide (18; 50.0%). one isolate had an insertion in the mmpr5 gene, which is associated with bedaquiline and clofazimine resistance. among the 16 isolates with an i491f rpob mutation, one belonged to sub-lineage 2.2.1, while the remaining 15 isolates belonged to three distinct sub-lineages of lineage 4. of these 15 isolates, three isolates each had unique mutation patterns, while 12 clustered into two groups based on mutation patterns. one group of six isolates belonging to the 4.4.1.1 sub-lineage carried katg s315t, pnca h51d, embb m306i, and rpsl k43r, which confer resistance to isoniazid, pyrazinamide, ethambutol, and streptomycin. another group of six isolates belonging to sub-lineage 4.3.3 had mutations conferring isoniazid (inha [fabg1 8t > a] plus katg s315t), pyrazinamide (pnca g132a), ethambutol (embb m306v), streptomycin (gidb 130bp deletion), and ethionamide (etha 11a > g) resistance. the 12 isolates carrying a single l452p mutation also belonged to lineage 4. among the 12 isolates, there were two clusters with common resistance-conferring mutations. the first group consisted of eight isolates belonging to sub-lineage 4.3.3 and had the inha fabg1c.-8t > a plus katg s315t isoniazid resistance-conferring mutations, as well as the pnca 456_457 c insertion (pyrazinamide resistance), embb m306v (ethambutol resistance), etha_11a > g (ethionamide resistance), and the gidb 130bp deletion (streptomycin resistance) mutations. the second group consisted of three isolates belonging to sub-lineage 4.4.1.1.1 and carrying katg s315t and embb m306i mutations for isoniazid and ethambutol resistance. discussion in this study, almost 29% of the isoniazid mono-resistant tuberculosis cases detected using the mtbdrplus assay were mdr-tb cases. this led to significant delays in the initiation of dr-tb treatment. the main cause of rifampicin resistance missed by the mtbdrplus assay was the presence of mutations outside the rrdr (mainly i491f), as well as the l452p rpob mutation. mutations outside the rrdr are not detected by the currently used who-endorsed rapid molecular assays, while the l452p mutations were missed by the previous version of the mtbdrplus assay. importantly, isolates carrying these mutations were also resistant to other first-line anti-tuberculosis drugs whose resistance is not routinely tested in tuberculosis patients globally, and the isolates also clustered into distinct groups with unique mutation profiles. the rpob l452p mutation was left out of the earlier version (version 2, released in 2011) of the mtbdrplus assay as it was thought to be clinically insignificant.18 this was later corrected in an updated version of the assay launched in 2014.18,19 at the time of this study, the older version 2 was still in use, hence the discordant rifampicin results between the mtbdrplus assay and the phenotypic assay in isolates harbouring this mutation. the mtbdrplus assay may also miss heteroresistance. one belgian study from 2019 found that the limit of detection of rifampicin heteroresistance was 5% – 10%.6 this may explain the other rrdr mutations missed by the mtbdrplus assay in this study. notably, among isolates that had an xpert result and had the l452p mutation as detected by whole-genome sequencing, the xpert assay detected rifampicin resistance. mutations outside the rrdr were found in just over half (19/36) of isolates with discordant results that were tested using whole-genome sequencing. if we assume that this proportion is representative of the whole 145 samples with discordant results (i.e. 52.7% of all discordant results are due to mutations outside the rrdr), this will equate to 76 out of 145 discordant isolates. this means that about 15% (76/505) of isoniazid mono-resistant cases had mutations outside the rrdr. this is the same prevalence found by makhado et al. from pretoria, south africa, when they screened isoniazid mono-resistant cases for the i491f mutation in clinical samples collected between 2013 and 2016.10 unlike makhado et al., who used molecular methods to screen for the i491f mutation, we used the 1% agar proportion method to test for rifampicin resistance missed by the mtbdrplus assay. phenotypic methods, especially liquid-based methods, can fail to detect rifampicin resistance caused by the i491f mutation. in a 2019 study conducted in belgium by torrea et al., the agar proportion method detected rifampicin resistance in 75% of isolates with i491f that was missed by the mycobacteria growth indicator tube dst.12 it is therefore likely that the occurrence of these mutations is more frequent than what we found in this study. while the overall prevalence of i491f mutation among tuberculosis patients is reportedly low, in patients with isoniazid resistance, the prevalence is high.9,10,20 the who defines universal access to dst as performing rapid dst for at least rifampicin in all patients with bacteriologically confirmed tuberculosis plus additional dst for at least fluoroquinolones and second-line injectable agents in patients with rifampicin resistance.1 the use of xpert as an entry point to tuberculosis care without investigating isoniazid resistance would prove disastrous for patients infected with m. tuberculosis strains that have mutations outside the area of detection and are resistant to all other first-line drugs. recent studies conducted between 2015 and 2017 have shown that isoniazid resistance generally develops before rifampicin resistance.21,22 notwithstanding the importance of testing for rifampicin resistance, the neglect of isoniazid testing leads to inappropriate therapy, treatment failure and accumulation of resistance in patients with initial isoniazid resistance.23 we therefore propose an algorithm to optimise dr-tb detection (figure 2). we submit that the initial dst should include both isoniazid and rifampicin. importantly, if resistance is found to any of these two drugs, it should trigger further dst of other first-line and second-line drugs that will be used for treatment. moreover, an attempt should be made to look for the i491f mutation in isolates from patients with isoniazid mono-resistant tuberculosis as this mutation may be missed by both phenotypic and genotypic dst methods that are routinely used for the detection of rifampicin resistance. figure 2: proposed algorithm for the diagnosis of drug-resistant tuberculosis. the largest global cluster of extensively drug-resistant tuberculosis that was ever reported was from tugela ferry in kwazulu-natal in 2005 and it was caused by a strain named f15/lam4/kzn.24 a study conducted by pillay et al. using m. tuberculosis isolates collected between 1994 and 2002 showed how this extensively drug-resistant strain accumulated resistance over time under a tuberculosis programme that lacked appropriate dst.25 patients received inappropriate therapy, thus allowing the selection and spread of resistant strains and leading to treatment failure with dire consequences, especially among patients who were also hiv-positive.24 with the current tuberculosis diagnostic algorithm that only tests for rifampicin resistance, we find ourselves in a similar circumstance that calls for swift action if we are to avoid the same unfortunate outcome. targeted next-generation sequencing can overcome some of the challenges of rapid molecular assays and phenotypic dst by allowing the rapid detection of rpob mutations outside the rrdr and additional mutations conferring resistance to other anti-tuberculosis drugs, including those that are difficult to test by phenotypic methods (e.g. pyrazinamide). however, the cost, skill levels and expertise required to perform next-generation sequencing and interpret its results remain the prohibiting factors limiting the implementation of this technology, especially in high-burdened, low-resource countries where it is needed the most.26 therefore, in many countries, including south africa, next-generation sequencing remains confined to the reference and research laboratories. although molecular tests have decreased the time to tuberculosis dst results from weeks, using the previous phenotypic tests, to hours and days, discordant results may reverse this benefit. given the fact that mtbdrplus results in this study showed rifampicin-susceptible m. tuberculosis, appropriate treatment (dr-tb treatment) could not be initiated until phenotypic dst results showing rifampicin resistance became available. even so, due to the inferiority of second-line tuberculosis treatment compared to the standard first-line treatment, clinicians may be reluctant to change patient treatment based on a discordant result. the patients in this study often had multiple results, showing that the clinicians sought more evidence before committing patients to dr-tb treatment regimens. as shown in this study, there were significant delays in the initiation of dr-tb treatment, which devalues the benefits of rapid molecular tests. it was alarming to find such high levels of resistance to other first-line drugs (pyrazinamide and ethambutol), as well as ethionamide and streptomycin. phenotypic dst for pyrazinamide and ethambutol is not routinely performed in many settings because of poor reproducibility and reliability.27,28,29 in the south african setting where xpert (ultra) is used for the initial diagnosis of tuberculosis and no further dst is performed for rifampicin-susceptible cases, these patients would be treated with first-line therapy. in fact, given the high number of patients with a previous tuberculosis treatment history and current indications for performing m. tuberculosis culture, phenotypic dst was probably performed for these patients because they had already failed tuberculosis therapy. the presence of resistance to streptomycin suggests that these patients may have failed a few rounds of tuberculosis therapy because streptomycin was previously used as part of a standard re-treatment regimen in patients who had failed first-line therapy. in south africa, this regimen was stopped after the rollout of xpert, which allowed universal testing of all tuberculosis patients. the rollout of xpert was completed towards the end of 2013. isolates in this study belonged predominantly to lineage 4, which is known to predominate among dr-tb cases in the kwazulu-natal province.30 most of the isolates clustered based on the i491f and l452p rpob mutations, with each cluster carrying a unique set of mutations conferring resistance against isoniazid, pyrazinamide, ethambutol, streptomycin, or ethionamide. this suggests that these highly resistant strains may have been spreading undetected in the community. furthermore, lineage 4.4.1.1 strains with rpob i491f, katg s315t, pnca h51d, embb m306i, and rpsl k43r mutations have been linked to an outbreak that originated in eswatini and later spread to south africa.10 limitations this study reports old data on m. tuberculosis isolates from 2014. however, we examined this period because this was when both phenotypic and genotypic rifampicin dst were performed simultaneously in our setting. moreover, the tuberculosis diagnostic algorithm has not changed since then, although phenotypic rifampicin dst was subsequently stopped. another limitation of this study was our use of phenotypic dst to select isolates with possible mutations outside the rrdr instead of using molecular screening. this may have underestimated the prevalence of isolates with these mutations as some of them remain susceptible on the phenotypic assay. due to limited resources, we sequenced only a subset of the isolates with discordant results. the data from the susceptible tuberculosis treatment register was not available to compare with that on the dr-tb register to determine if patients not listed on the dr-tb register were treated with first-line tuberculosis therapy. finally, the study was performed in one province of south africa so the findings may not apply to other regions. nonetheless, this province has the highest prevalence of dr-tb cases in the country and similar findings have been reported in the northern provinces. conclusion the presence of highly drug-resistant m. tuberculosis strains with mutations missed by the routine rapid molecular assays highlights the need for the revision of the who definition of universal access to dst so that tuberculosis diagnostic algorithms include testing for both isoniazid and rifampicin in all patients with bacteriologically confirmed tuberculosis. the recent endorsement of the xpert mtb/xdr by the who for detection of isoniazid, fluoroquinolone and second-line injectable agent resistance in xpert (ultra)-confirmed tuberculosis cases provides an opportunity to close the gap in isoniazid testing.1 the i491f mutation remains the most commonly detected mutation outside the rrdr and its frequent occurrence in isoniazid-resistant cases calls for its inclusion in assays that detect rifampicin resistance. this codon is not too far away from the rrdr, so current assays can be upgraded to include it to avoid the use of inappropriate therapy, prevent the accumulation of resistance, and reduce community spread. acknowledgements the authors would like to thank the national health laboratory service tuberculosis laboratory staff at the inkosi albert luthuli central hospital who performed the routine clinical work for the province of kwazulu-natal. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions n.r.m. and k.p.m. were involved in the original conceptualisation of the study as well as obtaining the study funding. n.r.m. also carried out laboratory work, data acquisition and analysis, project administration and writing of the manuscript. l.p.c. performed the data analysis and writing of the manuscript. r.s. was involved in performing laboratory work. y.n. and j.g. performed whole-genome sequencing. e.w. did the whole-genome sequencing analysis. t.d.o. supervised the overall whole-genome sequencing work. k.s.s.-h. assisted with the writing and reviewing of the manuscript and k.p.m. was the overall supervisor of the project including the writing of the manuscript. sources of support this work was supported by the university capacity development programme of the university of kwazulu-natal and the national health laboratory service research trust. data availability all data generated from this study are available on request from the corresponding author, n.r.m. disclaimer the views expressed in this manuscript are those of the authors and not an official position of the institutions involved or any funders. references who. who operational handbook on tuberculosis. module 3: diagnosis – rapid diagnostics for tuberculosis detention, 2021 update. geneva: world health organization; 2021. who. molecular line probe assays for rapid screening of patients at risk of multidrug-resistant tuberculosis (mdr-tb). geneva: world health organization; 2008. who. automated real-time nucleic acid amplification technology for rapid and simultaneous detection of tuberculosis and rifampicin resistance: xpert mtb/rif system. policy statement. geneva: world health organization; 2011. andre e, goeminne l, cabibbe a, et al. consensus numbering system for the rifampicin resistance-associated rpob gene mutations in pathogenic mycobacteria. clin microbiol infect. 2017;23(3):167–172. https://doi.org/10.1016/j.cmi.2016.09.006 telenti a, imboden p. detection of rifampicin-resistance mutations in mycobacterium tuberculosis. lancet. 1993;341(8846):647–650. https://doi.org/10.1016/0140-6736(93)90417-f ng kcs, supply p, cobelens fgj, et al. how well do routine molecular diagnostics detect rifampin heteroresistance in mycobacterium tuberculosis? j clin microbiol. 2019;57(11):e00717–e00719. https://doi.org/10.1128/jcm.00717-19 siu gkh, zhang y, lau tck, et al. mutations outside the rifampicin resistance-determining region associated with rifampicin resistance in mycobacterium tuberculosis. j antimicrob chemother. 2011;66(4):730–733. https://doi.org/10.1093/jac/dkq519 heep m, brandstätter b, rieger u, et al. frequency of rpob mutations inside and outside the cluster i region in rifampin-resistant clinical mycobacterium tuberculosis isolates. j clin microbiol. 2001;39(1):107–110. https://doi.org/10.1128/jcm.39.1.107-110.2001 sanchez-padilla e, merker m, beckert p, et al. detection of drug-resistant tuberculosis by xpert mtb/rif in swaziland. n engl j med. 2015;372:1181–1182. https://doi.org/10.1056/nejmc1413930 makhado na, matabane e, faccin m, et al. outbreak of multidrug-resistant tuberculosis in south africa undetected by who-endorsed commercial tests: an observational study. lancet infect dis. 2018;18(12):1350–1359. https://doi.org/10.1016/s1473-3099(18)30496-1 van deun a, barrera l, bastian i, et al. mycobacterium tuberculosis strains with highly discordant rifampin susceptibility test results. j clin microbiol. 2009;47(11):3501–3506. https://doi.org/10.1128/jcm.01209-09 torrea g, ng kcs, van deun a, et al. variable ability of rapid tests to detect mycobacterium tuberculosis rpob mutations conferring phenotypically occult rifampicin resistance. sci rep. 2019;9:11826. https://doi.org/10.1038/s41598-019-48401-z nicd. microbiologically confirmed tuberculosis 2004–15 south africa. johannesburg: national institute for communicable diseases; 2017. lifescience h. genotype mtbdrplus ver 2.0: molecular genetic assay for identification of the m. tuberculosis complex and its resistance to rifampicin and isoniazid from clinical specimens and cultivated samples. 2015; nehren, germany: hain lifescience. woods gl, brown-elliott ba, conville ps, et al. clsi standards: guidelines for health care excellence. susceptibility testing of mycobacteria, nocardiae, and other aerobic actinomycetes. wayne, pa: clinical and laboratory standards institute; 2011. schleusener v, köser cu, beckert p, niemann s, feuerriegel s. mycobacterium tuberculosis resistance prediction and lineage classification from genome sequencing: comparison of automated analysis tools. sci rep. 2017;7:46327. https://doi.org/10.1038/srep46327 who. meeting report of the who expert consultation on drug-resistant tuberculosis treatment outcome definitions, 17–19 november 2020. geneva: world health organization; 2021. huang wl, chen hy, kuo ym, jou r. performance assessment of the genotype mtbdrplus test and dna sequencing in detection of multidrug-resistant mycobacterium tuberculosis. j clin microbiol. 2009;47(8):2520–2524. https://doi.org/10.1128/jcm.02499-08 köser cu, georghiou sb, schön t, salfinger m. on the consequences of poorly defined breakpoints for rifampin susceptibility testing of mycobacterium tuberculosis complex. j clin microbiol. 2021;59(4):e02328-20. https://doi.org/10.1128/jcm.02328-20 shea j, halse ta, kohlerschmidt d, et al. low-level rifampin resistance and rpob mutations in mycobacterium tuberculosis: an analysis of whole-genome sequencing and drug susceptibility test data in new york. j clin microbiol. 2021;59:e01885-20. https://doi.org/10.1128/jcm.01885-20 manson al, cohen ka, abeel t, desjardins ca, armstrong dt, barry ce 3rd, et al. genomic analysis of globally diverse mycobacterium tuberculosis strains provides insights into the emergence and spread of multidrug resistance. nat genet. 2017;49:395–402. https://doi.org/10.1038/ng.3767 cohen ka, abeel t, manson mcguire a, et al. evolution of extensively drug-resistant tuberculosis over four decades: whole genome sequencing and dating analysis of mycobacterium tuberculosis isolates from kwazulu-natal. plos med. 2015;12:e1001880. https://doi.org/10.1371/journal.pmed.1001880 gegia m, winters n, benedetti a, van soolingen d, menzies d. treatment of isoniazid-resistant tuberculosis with first-line drugs: a systematic review and meta-analysis. lancet infect dis. 2017;17(2):223–234. https://doi.org/10.1016/s1473-3099(16)30407-8 gandhi nr, moll a, sturm aw, et al. extensively drug-resistant tuberculosis as a cause of death in patients co-infected with tuberculosis and hiv in a rural area of south africa. lancet. 2006;368(9547):1575–1580. https://doi.org/10.1016/s0140-6736(06)69573-1 pillay m, sturm aw. evolution of the extensively drug-resistant f15/lam4/kzn strain of mycobacterium tuberculosis in kwazulu-natal, south africa. clin infect dis. 2007;45:1409–1414. https://doi.org/10.1086/522987 who. the use of next-generation sequencing technologies for the detection of mutations associated with drug resistance in mycobacterium tuberculosis complex: technical guide. geneva: world health organization; 2018. schön t, juréen p, giske cg, et al. evaluation of wild-type mic distributions as a tool for determination of clinical breakpoints for mycobacterium tuberculosis. j antimicrob chemother. 2009;64(4):786–793. https://doi.org/10.1093/jac/dkp262 zhang y, permar s, sun z. conditions that may affect the results of susceptibility testing of mycobacterium tuberculosis to pyrazinamide. j med microbiol. 2002;51(1):42–49. https://doi.org/10.1099/0022-1317-51-1-42 werngren j, sturegård e, juréen p, ängeby k, hoffner s, schön t. reevaluation of the critical concentration for drug susceptibility testing of mycobacterium tuberculosis against pyrazinamide using wild-type mic distributions and pnca gene sequencing. antimicrob agents chemother. 2012;56(3):1253–1257. https://doi.org/10.1128/aac.05894-11 gandhi nr, brust jcm, moodley p, et al. minimal diversity of drug-resistant mycobacterium tuberculosis strains, south africa. emerg infect dis. 2014;20(3):426–433. https://doi.org/10.3201/eid2003.131083 article information authors: keoratile ntshambiwa1 winnie ntabe-jagwer1 chandapiwa kefilwe1 fredrick samuel1 sikhulile moyo2 affiliations: 1sekgoma memorial hospital, ministry of health, botswana2botswana-harvard aids institute partnership and botswana-harvard hiv reference laboratory, princess marina hospital, botswana correspondence to: keoratile ntshambiwa postal address: po box 11299, palapye, botswana dates: received: 27 june 2014 accepted: 15 sept. 2014 published: 03 nov. 2014 how to cite this article: ntshambiwa k, ntabe-jagwer w, kefilwe c, samuel f, moyo s. translating a national laboratory strategic plan into action through slmta in a district hospital laboratory in botswana. afr j lab med. 2014;3(2), art. #209, 5 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.209 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. translating a national laboratory strategic plan into action through slmta in a district hospital laboratory in botswana in this original research... open access • abstract • introduction • research methods and design    • improvement project 1: reducing sample turnaround time    • improvement project 2: increasing patient and clinician satisfaction    • improvement project 3: ‘6s’    • improvement project 4: improving specimen management and document control • results • discussion    • limitations of the study    • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: the ministry of health (moh) of botswana adopted strengthening laboratory management toward accreditation (slmta), a structured quality improvement programme, as a key tool for the implementation of quality management systems in its public health laboratories. coupled with focused mentorship, this programme aimed to help moh achieve the goals of the national laboratory strategic plan to provide quality and timely clinical diagnoses.objectives: this article describes the impact of implementing slmta in sekgoma memorial hospital laboratory (smhl) in serowe, botswana. methods: slmta implementation in smhl included trainings, improvement projects, site visits and focused mentorship. to measure progress, audits using the stepwise laboratory quality improvement process towards accreditation (slipta) checklist were conducted at baseline and exit of the programme, with scores corresponding to a zeroto five-star scale. turnaround times, customer satisfaction, and several other health service indicators were tracked. results: the laboratory scored 53% (zero stars) at the baseline audit and 80% (three stars) at exit. nearly three years later, the laboratory scored 85% (four stars) in an official audit conducted by the african society for laboratory medicine. turnaround times became shorter after slmta implementation, with reductions ranging 19% to 52%; overall patient satisfaction increased from 56% to 73%; and clinician satisfaction increased from 41% to 72%. improvements in inventory management led to decreases in discarded reagents, reducing losses from us $18 000 in 2011 to $40 in 2013. conclusion: the slmta programme contributed to enhanced performance of the laboratory, which in turn yielded potential positive impacts for patient care at the hospital. introduction top ↑ laboratory medicine plays a critical role in healthcare and public health,1,2,3 and expanding laboratory capacity has been a focus of funding partners such as the us president’s emergency plan for aids relief (pepfar).4 implementation of laboratory quality management systems (qms) and accreditation to international standards contribute to increased accuracy of diagnoses and improvement in patient management.3 although considerable resources have been invested in recent years for the improvement of laboratory systems in resource-limited settings, lack of access to reliable laboratory testing still remains a barrier to effective healthcare and treatment for diseases, including tuberculosis, hiv and malaria.5,6,7 amongst the numerous challenges identified are the lack of national standards and policies specifically addressing qms.2,3 in 2008, the world health organization’s (who) maputo declaration8 called for the adoption of national laboratory strategic plans; nkengasong et al. concurred, arguing that such plans are one of the essential requirements for laboratory capacity building.9the ministry of health (moh) in botswana recognises the need for a collaborative and coordinated effort to raise the standards of its national laboratories. however, over the years there has been slow progress in implementing qms. previous training of healthcare workers has focused on general management topics rather than identifying tangible tasks to bring about change, making the training difficult to apply in the laboratory.10 in 2009, the moh developed a health sector laboratory strategic plan for 2009–2014, in which laboratory accreditation was a stated goal: to have four district-level laboratories accredited by 2013 and two national-level laboratories accredited by 2014. it became apparent that a radical shift in strategy would be required in order to realise these ambitious goals. to spearhead the moh’s national laboratory strategic plan, strengthening laboratory management toward accreditation (slmta), a structured quality improvement programme, was selected to assist in the development of qms within healthcare facilities.10,11 since its launch in 2009, slmta has demonstrated success in many countries12because of its task-oriented measureable design. sekgoma memorial hospital laboratory (smhl), a district laboratory of the sekgoma memorial hospital, was selected by the moh as one of eight laboratories to participate in the first slmta training cohort. sekgoma memorial hospital is a 350-bed facility located in serowe, a trade and commerce centre located in central district in eastern botswana. smhl has eight units: chemistry, haematology, microbiology, cd4, viral load, blood bank, serology and reception. patient samples for laboratory testing originate from the hospital wards, from 18 clinics within the hospital’s catchment area, and from outpatient services. on average, approximately 100 samples are received daily for chemistry, cd4 and haematology testing; 150 samples for viral load testing; and 30 samples for microbiology testing. this article describes the implementation and results of the slmta training and mentorship programme at smhl. research methods and design top ↑ the botswana slmta training programme was conducted in accordance with the standard slmta implementation model.111 training consisted of a series of three workshops, each lasting four days, held in august 2010, november 2010 and february 2011. attendees from smhl included the laboratory manager, the quality officer and two section supervisors. at each workshop, attendees selected improvement projects to implement in the laboratory based on the slmta modules presented. two supervisory visits of one day each were conducted by the three slmta trainers following each workshop.programme performance was measured by audits conducted before (baseline, july 2010) and after (exit, november 2011) slmta implementation using the who’s regional office for africa (afro) stepwise laboratory quality improvement process towards accreditation (slipta) checklist.113 slipta is an accreditation preparation framework, which measures and rewards the incremental progress of implementing qms requirements using a comprehensive audit checklist addressing the 12 quality system essentials of the laboratory.114 the slipta checklist and scoring system rate laboratory quality on a scale of zero to five stars; a rating of five stars signifies that the laboratory is ready to seek international accreditation. in-country auditors trained by a global healthcare public foundation, an independent external contractor based in kenya, conducted the baseline and exit audits. in august 2014, smhl received an official who afro slipta audit by the african society for laboratory medicine. from april to june 2011, smhl received focused mentorship from the botswana bureau of standards (bobs) to augment the slmta training. the bobs mentorship programme consisted of monthly visits, each lasting one week, during the three-month mentoring period. the visits included training on the international organization for standardization (iso) 15189 standard and qms documentation, with a focus on the management requirements of iso 15189. all laboratory staff members participated in the training sessions, whilst key staff members received additional mentorship on essential documentation of the qms. the laboratory developed and implemented four overarching improvement projects to address deficiencies identified during the baseline audit. a plan-do-check-act (pdca) cycle was implemented in order to facilitate continuous quality improvements, with past results driving future activities. progress of improvement projects was monitored using audit items selected from the slipta checklist, which were tracked and recorded. below we describe the improvement projects implemented: reducing sample turnaround time; increasing customer satisfaction; the ‘6s’ project; and improving specimen management and documentation. improvement project 1: reducing sample turnaround time in clinical laboratories, turnaround time is the total elapsed time from when a test is ordered to when the results are verified and released. a target turnaround time of two hours was established for haematology, chemistry and cerebrospinal fluid (csf) tests; a target of one hour was established for pregnancy tests. to assess this improvement project, data were obtained from the laboratory computer system, the integrated patient management system, on a monthly basis. average turnaround time for tests was analysed for two 6-month periods: april to september 2011 and october 2011 to march 2012. turnaround time data from before slmta implementation were not available. improvement project 2: increasing patient and clinician satisfaction customer satisfaction surveys are mandated by moh guidelines. separate questionnaires were administered for both patients and clinicians once a year in 2011, 2012 and 2013. the questionnaires utilised a rating system of ‘poor’, ‘fair’, ‘good’ and ‘very good’, with the latter two responses interpreted as ‘satisfied’ for purposes of analysis. the patient questionnaire asked patients to evaluate the laboratory reception, the staff members’ ability to answer questions, staff behaviours and attitudes, staff availability, the waiting time for test results, and overall satisfaction. patient questionnaires were distributed to patients every morning for a period of one month at the end of each year in 2011, 2012 and 2013. the clinician questionnaire asked staff to rate hospital services, turnaround times, and overall satisfaction; evaluate the attitudes of laboratory staff; and suggest ways to respond to enquiries. twenty doctors and nurses in each of the 10 hospital wards completed the clinician questionnaires for each year of evaluation. improvement project 3: ‘6s’ the slmta team implemented ‘6s’ (sorting, straightening, shining, standardising, sustaining and safety) in order to improve storage space and reduce expired products. an action plan was developed for each issue identified. wastage generated from discarded products as a result of expiry was tracked and their value calculated for fiscal years (fy) 2011 and 2013. improvement project 4: improving specimen management and document control to improve specimen management, missing and deficient essential documents were identified based on the slipta checklist and iso 15189 requirements. standard operating procedures (sops) were created and revised, including system procedures, safety procedures and technical procedures; staff were then trained on the relevant sops. signs with directions were placed in all testing areas so as to facilitate movement of staff and clients throughout the facility. selected hospital drivers attended a half-day workshop of presentations and simulation exercises, where they were trained on safe specimen handling, proper transportation and timely result distribution. results top ↑ the laboratory audit score improved from zero stars (53%) at baseline in july 2010 to three stars (80%) at the exit audit in november 2011. at the official who afro slipta audit in august 2014, the laboratory received four stars (85%).turnaround times decreased for all tests monitored: 19% reduction for haematology tests, 44% for chemistry tests, 30% for csf analyses and 52% for pregnancy tests (figure 1). pre-set targets of one hour for pregnancy tests and two hours for the remaining tests were all met. figure 1: overall patient satisfaction increased from 56% in 2011 to 85% in 2012, then decreased to 73% in 2013 (figure 2a). this pattern was consistent in each of the individual questions. clinician satisfaction increased steadily from 41% in 2011 to 64% in 2012 and 72% in 2013 (figure 2b), with steady improvements in all areas. figure 2a: figure 2b: the ‘6s’ improvement project led to the reorganisation of workstations and removal of unused items, which resulted in the creation of space and the alleviation of staff overcrowding. storage shelves were labelled with contents and the storeroom was cleaned and reorganised; all expired items were removed. systematic tracking of reagent expiration dates and improved inventory management led to a decrease in discarded reagents, with a subsequent drop in laboratory losses from p128 000 (approximately $18 000) in fy2011 to p280 (approximately $40) in fy2013. improved sample management and documentation revealed problems with specimen collection, especially a high sample rejection rate caused by poor phlebotomy. as a result, a specimen collection manual was developed and distributed to all clinics and wards in the hospital and guidance was provided. a total of 154 sops were developed and implemented, including management and technical documents for specimen receipt and results dispatch. long turnaround times during lunch hours were reduced by implementing an improved mid-day staff coverage system. a sign-in book for sample delivery was initiated in order to reduce delays in the transport of samples from the laboratory reception to the benches. all urgent samples were tagged with a white sticker on the cap to make their priority visible. discussion top ↑ implementation of slmta and targeted mentorship enabled smhl to develop an effective qms, including mechanisms to monitor critical aspects of laboratory service in pre-analytical, analytical and post-analytical processes. this resulted in rapid and measurable changes as evidenced by the jump from zero stars at the baseline audit to three stars at the exit audit. these improvements were sustained and further increased, with the laboratory reaching four stars at the official who slipta audit nearly three years later.reduction of turnaround time and improved customer satisfaction after completion of the slmta programme also indicated positive and sustained impact on patient care. the decrease in turnaround time for haematology occurred despite an increase in testing volume; this was because of a combination of reduced equipment downtime as a result of effective preventive maintenance of analysers and reduced stock-outs from the introduction of an effective inventory management system. slmta training enabled the laboratory staff to take on the daily challenges of increased volume that could otherwise strain the laboratory system. one important lesson learned was that staff motivation and management buy-in are critical for the success of the slmta programme. involving management in the early stages of slmta planning and periodic structured updates facilitated their understanding of qms and built consensus on the collaborative role of each department in achieving the goals of the national laboratory strategic plan. productive and frequent discussions between clinicians and laboratory staff paved the road to success and became a vehicle for translating strategic plans to action. the slmta programme brought about a cultural shift at smhl by providing the staff with a step-by-step process to break down tasks into actionable items with the common goal of providing high-quality laboratory services. the focus on continuous quality improvement ensures that the gains are maintained in a practical way, with active involvement of management and clinicians in identifying and addressing remaining challenges. staff motivation was also noted as a key driver to implementing qms. for instance, immediate changes in the organisation and utilisation of space as a result of the ‘6s’ project improved staff morale, largely because staff members were involved directly in the results-oriented process. this immediate transformation encouraged and motivated the staff to find other solutions using existing resources. improvement projects that were identified and planned during the slmta training stimulated creativity, ownership and action amongst laboratory and hospital staff; and were a great asset to the quality improvement programmes at smhl. it was noted by management that the more the staff were trained on slmta, the more enthusiastic and confident they became, thus enhancing the working relationship between staff and clients. in addition to slipta scores and stars, monitoring quality indicators such as turnaround time and customer satisfaction was valuable for assessing the laboratory’s success and identifying areas needing improvement. for example, customer satisfaction surveys alerted laboratory staff to declining patient satisfaction with waiting times from 2012 to 2013. after brainstorming sessions, the team concluded that this decline may have been caused by a failure to communicate to patients about the newly-introduced practice of prioritising services to patients needing urgent assistance such as pregnant women, the elderly and the disabled. armed with results from the survey, laboratory staff were able to address the problem head-on, by improving communication with patients to ensure that they understood the purpose of the new triage system. limitations of the study we acknowledge some limitations associated with this work. primarily, data for turnaround time and customer satisfaction were not collected before slmta implementation, preventing a pre-/post-slmta comparison. thus, our results are likely an underestimate of the true effect of slmta implementation. conclusion the botswana moh has made great strides in its drive toward continuous quality improvement in public-sector laboratories by adopting the slmta programme and focused mentorship as tools to help translate the national laboratory strategic plan into action. whilst tuberculosis, hiv, malaria and other diseases remain a challenge in botswana, the achievement of quality, timely and accurate laboratory results will play an important role in reducing the incidence of infections and improving treatment outcomes. at smhl, the slmta programme has been essential for the strengthening of laboratory management systems and has helped lay a firm foundation for further advancements in patient care. acknowledgements top ↑ we would like to thank all laboratory staff members of smhl, the slmta master trainers and management of serowe district health management team, all of whom contributed to the success story of smhl. we acknowledge and appreciate the moh clinical services for guidance and assistance, as well as for selecting smhl for participation in the first cohort. we would also like to acknowledge support from the chief medical laboratory scientist, mr david matema. in addition, we would like to thank the cdc botswana office for their unwavering support and contribution to smhl. we also acknowledge the bobs and moh for their guidance and assistance. we acknowledge the slmta master trainers and in-country slipta auditors for their enthusiasm and objectivity, and for raising the standards of implementation of qms in botswana. we would also like to acknowledge drs katy yao and elizabeth luman, along with global scientific solutions for health, for their contribution and support during the preparation of this manuscript. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions k.n., w.n-j., c.k. and f.s. (all sekgoma memorial hospital) implemented the slmta programme and wrote the manuscript. s.m. (princess marina hospital) provided technical writing support and analysis and contributed to writing of the manuscript. references top ↑ 1. hay burgess dc, wasserman j, dahl ca. global health diagnostics. nature. 2006;444(suppl 1):1–2. http://dx.doi.org/10.1038/nature054402. nkengasong jn. strengthening laboratory services and systems in resource-poor countries. am j clin pathol. 2009;131:774. http://dx.doi.org/10.1309/ajcp8gyx8ktkdatz 3. peter tf, rotz pd, blair dh, et al. impact of laboratory accreditation on patient care and the health system. am j clin pathol. 2010;134(4):550–555. http://dx.doi.org/10.1309/ajcph1skq1hnwghf 4. the president’s emergency plan for aids relief. fy 2014 country operational plan (cop) guidance. version 2 [document on the internet]. c2013 [cited 2013 dec 15]. available from: http://www.pepfar.gov/documents/organization/217765.pdf 5. petti ca, polage cr, quinn tc, et al. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377–382. http://dx.doi.org/10.1086/499363 6. abimiku ag. building laboratory infrastructure to support scale-up of hiv/aids treatment, care, and prevention: in-country experience. am j clin pathol. 2009;131(6):875–886. http://dx.doi.org/10.1309/ajcpelmg6gx6rqsm 7. birx d, de souza m, nkengasong jn. laboratory challenges in the scaling up of hiv, tb, and malaria programs: the interaction of health and laboratory systems, clinical research, and service delivery. am j clin pathol. 2009;131(6):849–851. http://dx.doi.org/10.1309/ajcpgh89qdswfons 8. world health organization. the maputo declaration on strengthening of laboratory systems [document on the internet]. c2008 [cited 2014 sep 08]. available from: http://www.who.int/diagnostics_laboratory/maputo-declaration_2008.pdf 9. nkengasong jn, mesele t, orloff s, et al. critical role of developing national strategic plans as a guide to strengthen laboratory health systems in resource-poor settings. am j clin pathol. 2009;131(6):852–857. http://dx.doi.org/10.1309/ajcpc51blobbpakc 10. yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3):401–409. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj 11. yao k, maruta t, luman et, nkengasong jn. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(2), art. #194, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i2.194 12. yao k, luman et, slmta collaborating authors. evidence from 617 laboratories in 47 countries for slmta-driven improvement in quality management systems. afr j lab med. 2014;3(2), art. #262, 11 pages. http://dx.doi.org/10.4102/ajlm.v3i2.262 13. world health organization’s regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region (with checklist) [document on the internet]. c2013 [cited 2014 may 27]. available from: http://www.afro.who.int/en/clusters-a-programmes/hss/blood-safety-laboratories-a-health-technology/blt-highlights/3859-who-guide-for-the-stepwise-laboratory-improvement-process-towards-accreditation-in-the-african-region-with-checklist.html 14. clinical and laboratory standards institute. application of a quality management system model for laboratory services; approved guidelines – third edition. clsi document gp26-a3 [isbn 1-56238-553-4]. wayne, pa: clinical and laboratory standards institute; 2004. article information authors: linda r. andiric1 charles g. massambu2 affiliations: 1globalhealth, american society for clinical pathology (ascp), chicago, united states 2ministry of health and social welfare, tanzania correspondence to: linda andiric postal address: globalhealth, american society for clinical pathology (ascp), 33 west monroe street, suite 1600, chicago, united states dates: received: 22 may 2014 accepted: 08 aug. 2014 published: 03 nov. 2014 how to cite this article: andiric lr, massambu cg. one laboratory’s progress toward accreditation in tanzania. afr j lab med. 2014;3(2), art. #202, 4 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.202 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. one laboratory’s progress toward accreditation in tanzania in this lessons from the field... open access • abstract • introduction • programme implementation • results • discussion • acknowledgements    • competing interests    • authors’ contributions    • disclosure statement • references abstract top ↑ introduction: the amana regional hospital laboratory in tanzania was selected, along with 11 other regional and district laboratories, to participate in a pilot programme for laboratory quality improvement using the strengthening laboratory management toward accreditation (slmta) training programme. programme implementation: the slmta programme entailed hands-on learning, improvement projects between and after a three-workshop series, supervisory visits from an oversight team and an expert laboratory mentor to facilitate and coach the process. audits were conducted at baseline, exit (approximately one year after baseline) and follow-up (seven months after exit) using the stepwise laboratory quality improvement process towards accreditation (slipta) checklist. quality stars (zero to five) were awarded based on audit scores. results: with a dedicated staff and strong leadership from laboratory management, amana laboratory implemented processes, policies and procedures recommended as elements of best laboratory practices. the laboratory improved from zero stars (36%) at baseline to successfully achieving three stars (81%) at exit. this was the highest score achieved by the 12 laboratories in the programme (the median exit score amongst the other laboratories was 58%). seven months after completion of the programme, the laboratory regressed to one star (62%). discussion: as the slmta improvement programme progressed, amana laboratory’s positive attitude and hard work prevailed. with the assistance of a mentor and the support of the facility’s management a strong foundation of good practices was established. although not all improvements were maintained after the conclusion of the programme and the laboratory dropped to a one-star rating, the laboratory remained at a higher level than most laboratories in the programme. introduction top ↑ until recently, laboratories in africa were largely ignored, distrusted and negated so that, for example, fewer than 50% of patients treated for malaria actually had laboratory confirmation of the disease.1 given the fact that in the united states as many as 94% of all patients’ treatment and diagnoses are based on laboratory data for confirmation,2 laboratory utilisation in africa was, by comparison, undervalued, underused and limited in its capacity. clinicians often rationalised that laboratory tests were an unnecessary additional cost because diagnoses and treatment protocols were based solely on the physician’s clinical judgement. if or when laboratory data were available, they were perceived as unreliable. if test results were received and were contradictory to clinical impressions, those test results were frequently ignored and only the clinical indicators were considered.3 financial resources from funding organisations and programmes such as the us president’s emergency plan for aids relief (pepfar) focused on the prevention and care of infectious diseases such as hiv, malaria and tuberculosis.4 because control of these infectious diseases is dependent upon accurate laboratory data, laboratory improvement became a priority. in july 2009 in kigali, rwanda, the world health organization’s regional office for africa (who afro), in partnership with the us centers for disease control and prevention (cdc), the clinton health access initiative and the american society for clinical pathology (ascp), launched the strengthening laboratory management toward accreditation (slmta) training programme,5 along with a stepwise accreditation preparation scheme that provides a five-step assessment method rather than a pass-fail one.6 in july 2010, the ministry of health and social welfare (mohsw) of tanzania began a pilot programme for the improvement of the country’s medical laboratory services using the slmta programme. twelve hospitals, comprising six regional and six district laboratories, were enrolled in slmta as tanzania’s first cohort. the minimum criteria for selection into the programme, as set by the country’s laboratory task force, were sufficient and qualified staff, a recently remodelled infrastructure to include sufficient space and utilities to carry out quality testing, and a basic knowledge of quality management by prior attendance in a quality management systems (qms) workshop. participation in an external quality assessment (eqa) programme was also preferred. this article describes amana regional hospital laboratory’s success in the improvement of its practices using the slmta programme and examines possible factors contributing to this success. programme implementation top ↑ amana regional hospital is located in dar es salaam city centre, tanzania. in 2010, the newly-remodelled hospital laboratory was both spacious and well-endowed with modern laboratory equipment. there were 23 staff members, including phlebotomists and cleaners. the laboratory was maintained neatly and was well organised. when the tanzanian mohsw notified amana laboratory that it had been selected to participate in the slmta programme, the laboratory manager was aware of international organization for standardization (iso) 15189, but he and his staff had no previous knowledge of slmta or the who afro accreditation preparation scheme. the improvement programme for amana laboratory and the other 11 selected facilities began in july 2010 with a baseline audit by three teams of auditors. two team members were trained south african national accreditation system (sanas) auditors, two were ascp slmta facilitators (one of whom was also a college of american pathologists [cap] inspector) and three others were slmta facilitators from the cdc office in tanzania. the audits were conducted using the who afro stepwise laboratory quality improvement process towards accreditation (slipta) checklist, which is based on iso 15189. with this checklist, recognition is given for progressive improvement; the lowest level of recognition (one star) requires a minimum of 55% of the total score possible on the checklist, two stars require 65% – 74%, three stars require 75% – 84%, four stars require 85% – 94% and five stars require ≥ 95% compliance. at the highest level (five stars), a laboratory is considered ready to seek accreditation to iso 15189 standard by any international accreditation agency.6 following the baseline audits, the slmta programme consisted of three five-day workshops where participants (laboratory managers and quality officers of the selected facilities) were taught the skills they needed in order to improve the quality of their laboratory services. each participant received a laboratory management framework document5 that defined the tasks laboratory managers must perform in order to meet the standards for best laboratory practices as set forth by the iso 15189 standard. a copy of the slipta checklist and a slmta toolkit with modules defining best laboratory practices were also given. the three workshops were held with three-month intervals between them so that skills taught in the workshop could be applied in the participants’ home laboratories. at the conclusion of each workshop, improvement projects were assigned that had either been taught during the previous workshop or were selected to address the common nonconformities found at the baseline audits. after completion of the programme in august 2011, exit audits were conducted in the 12 laboratories by the same team that conducted the baseline audits. in february 2012, seven months later, an official who afro slipta audit was conducted in amana laboratory by mohsw on behalf of the african society for laboratory medicine. results top ↑ the median baseline score for the 12 laboratories was 31% (figure 1). amana laboratory scored 36% at the baseline audit, well below the 55% needed for a one-star rating. during the audit debrief, it was clear that this low score was both a surprise and a disappointment to the amana laboratory staff. the laboratory manager, quality officer and mentor, an experienced tanzanian laboratory professional with good laboratory practices assigned to assist amana laboratory in its implementation of the improvement projects, responded positively, however, with determination to resolve the underlying quality problems in the laboratory. by the exit audit, improvements were seen in all of the participating laboratories, with a median exit score of 62% (figure 1). amana laboratory earned a score of 81% at the exit audit, corresponding to a three-star recognition level, which was the highest score in the cohort. much had been accomplished. for example, a quality manual for the laboratory had been developed by the quality officer in collaboration with the laboratory manager, which documented policies on how the laboratory would ensure quality in every aspect of service delivery. each policy was either described in detail in the quality manual or was referenced to a full standard operating procedure (sop) denoted by both the document title and control number. however, despite this exemplary improvement, seven months later in the official who afro slipta audit, the laboratory had regressed to one star (62%). figure 1: baseline and exit audit scores for the 12 laboratories in the first slmta cohort in tanzania, including slipta audit score for laboratory 12, the amana laboratory. discussion top ↑ at the exit audit, all 12 laboratories in tanzania’s first slmta cohort showed improvements in slipta scores, with five sites achieving a two-star recognition level, one achieving one-star recognition and five achieving no stars but showing improvement. amana laboratory achieved three stars and the highest score in the cohort, standing out as an example of the remarkable progress that can be achieved within a single year. when asked about the important factors that contributed to the successful outcome, the laboratory manager responded that the programme, in its entirety, contained all the essentials that were important for success. firstly, the tools and job aids taught and practised in the slmta programme, including the laboratory management framework and audit checklist, provided the crucial ‘how-to’ guidance for accomplishing best laboratory practices. secondly, there were supportive visits by the tanzanian slmta supervisory team that provided guidance for the implementation of improvement projects and clarified some translation misunderstandings that resulted from the workshops being taught in english rather than the native kiswahili. thirdly, encouragement from the facility medical officer and the regional medical officer was important with regard to supporting the efforts of the laboratory as they implemented improvements. fourthly, the assigned laboratory mentor assisted in all aspects of implementing the improvements and reinforced the skills learned from the slmta curriculum. finally, the laboratory staff’s team spirit, involvement and commitment to achieveing quality were crucial in meeting the goals of the programme. whilst all 12 laboratories in the cohort underwent the same training and supportive supervisory visits, it is possible that varied levels of support from facility medical officers contributed to differences in outcomes. in addition, amana laboratory’s relatively greater improvement could be attributed to laboratory leadership; amana’s laboratory manager made an effort to inspire a team spirit within his staff which, in turn, may have motivated them to strive for achievement of the improvement goals. also important was maintaining an open mind and positive attitude, despite the low baseline audit score, as amana’s laboratory management team approached the slmta improvement projects. they took the slmta curriculum seriously and were diligent in its implementation. they believed in the overall goal and the value of this initiative for the improvement of laboratory service and, by example to their staff, dedicated themselves to the extra hours and hard work required. the establishment of these essential elements ensured that the slmta curriculum was utilised to its fullest potential; the supervisory team was welcomed; and the mentor was well-received, enabling him to assist and to carry out his assignment. despite amana laboratory staff’s remarkable success during the programme, they were not able to maintain the improvements after the programme ended. seven months later, the score had dropped to the one-star level. as is often the case, once the focus on the programme ended, the laboratory found it difficult to maintain and update records and reviews. amana laboratory has learned that quality improvement is a continuous process and that, whilst progress may not always be steady, they are committed to continue to both improve and implement their quality systems. acknowledgements top ↑ the authors wish to acknowledge the support of the ascp, the cdc, the mohsw of tanzania and the slmta workshop facilitators. the authors are also grateful for the excellent assistance provided by the cdc editorial staff and the excellent peer review that was received to make this article better. competing interests the authors declare that they have no financial or personal relationship(s), which may have inappropriately influenced them in writing this article. authors’ contributions l.r.a. (globalhealth, ascp) is a technical consultant for ascp, a master trainer for slmta in tanzania and co-authored this article. c.g.m. (mohsw, tanzania) provided data on laboratory audit outcomes and co-authored this article. disclosure statement this report was made possible through support provided by the cdc, under the terms of cooperative agreement number 5u2gps001285-03. the opinions herein are the opinions of the author and do not necessarily reflect the views of the cdc. references top ↑ 1.reyburn h, mbata r, drakeley c, et al. overdiagnosis of malaria in patient with severe febrile illness in tanzania: a prospective study. bmj. 2004:329:1212–1217. http://dx.doi.org/10.1136/bmj.38251.658229.55 2.forsman rw. the value of the laboratory professional in the continuum of care. clin leadersh manag rev. 2002;16(6):370–373. 3.petti ca, polage cr, quinn tc, et al. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377–382. http://dx.doi.org/10.1086/499363 4.masanza mm, nqobili n, mukanga d, et al. laboratory capacity building for the international health regulations (ihr[2005]) in resource-poor countries: the experience of the african field epidemiology network (afenet). bmc public health. 2010;10(suppl 1):s8. http://dx.doi.org/10.1186/1471-2458-10-s1-s8 5.yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3):401–409. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj 6.gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. http://dx.doi.org/10.1309/ ajcptuuc2v1wjqbm article information authors: john n. nkengasong1 deborah birx1 affiliations: 1division of global hiv/aids, center for global health, us centers for disease control and prevention, atlanta, united states correspondence to: john nkengasong postal address: 1600 clifton road, atlanta, ga 30333, united sates dates: received: 03 sept. 2014 accepted: 11 sept. 2014 published: 03 nov. 2014 how to cite this article: nkengasong jn, birx d. quality matters in strengthening global laboratory medicine. afr j lab med. 2014;3(2), art. #239, 4 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.239 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. quality matters in strengthening global laboratory medicine in this commentary... open access • introduction    • squaring the circle    • time to stop doing more of the same    • slipta and lqms-sip, the companions of slmta • evident evidence and the up-shot    • slmta, nearing the tipping point?    • conclusion • acknowledgements    • competing interests    • authors’ contributions    • disclaimer • references introduction top ↑ around 300 bc, during the time of the ancient greek physician, hippocrates, the first documented examination of human bodily fluids was conducted. this gave birth to laboratory medicine, which is the use of laboratory tests to guide clinical investigations.1 ever since, as a result of its multi-faceted nature, ensuring the quality of testing in laboratory medicine has remained a challenge, but is an evolving practice in many countries. in the developed world, laboratory medicine has been transformative and, in most cases, is the science behind clinical care and disease surveillance. central to the practice of laboratory medicine in the developed world is the recognition of the importance of quality assurance (qa). as such, there is regular review of existing policies in order to ensure the continuous improvement of quality systems. for instance, in the united states, reports by the institute of medicine, to err is human: building a safer health system (1999), 2 and the committee on quality of health care in america, crossing the quality chasm: a new health system for the 21st century (2001),3 helped refocus attention on the need to minimise medical errors and improve quality. in developing countries, qa in laboratory medicine has been severely neglected and has become a serious impediment to effective healthcare delivery and disease surveillance.4,5,6 in fact, a vicious cycle has been established whereby most physicians in developing countries rely on history-taking and physical examination for patient management, since they have little confidence in laboratory test results, even if laboratory facilities exist.5 as such, inadequate resources are allocated to laboratory services, which in turn results in less-than-optimal quality-assured results, further leading to the neglect of laboratory systems (figure 1). nonetheless, many countries are currently making great strides in implementing quality management systems (qms), which is leading to laboratory accreditation to international standards.7,8 the importance of quality in laboratory medicine cannot be overstated: it adds significant value to patient outcomes and management;9 reduces wastage, minimises sample rejection and enhances client satisfaction;10 prevents unneeded diagnostic testing; improves turnaround times for accurate diagnosis; and reduces the use of inappropriate treatment. because laboratory errors occur at a rate of 32% – 75% in the pre-analytic phase, 13% – 32% in the analytic phase and 9% – 31% in the post-analytic phase,11,12 it is vital that assuring the quality of laboratory medicine be considered a continuum of a total testing process of all analytical phases. errors that occur in the pre-analytical phase in the spectrum of laboratory testing can have a direct effect on patient outcomes in the post-analytical stage. figure 1: quality matters: a catalyst to a tipping point for strengthening laboratory medicine? squaring the circle because of the acute challenges of implementing qa in laboratory medicine, healthcare providers in developing countries have unwisely neglected the important role of laboratory diagnosis in patient care. as a result, achieving the international organization for standardization (iso) 15189 requirements for clinical laboratory has become a lofty aspiration. understandably, but unfortunately, these countries seem to find themselves at an impasse of practising laboratory medicine in the 21st century by squaring the circle: relying on inadequate quality-assured test results or empiricism for patient management, resulting in disproportionate administration of antibiotics and high cost to patients.5 in the last decade, there has been a massive focus on global health, with funding reaching an unprecedented us $28.2 billion in 2010.13 the surge in funding has resulted in the recognition that strengthening health systems, including laboratory services, is critical to healthcare delivery. thus, an unparalleled opportunity has presented itself to strengthen quality-assured laboratory medicine, using innovative approaches to addressing old, neglected challenges. time to stop doing more of the same increased funding for global health has placed a sense of urgency on stakeholders to act now and collectively, but in a different manner.4 previous approaches that attempted to strengthen qms did not use a holistic approach, focusing rather on individual activities: qa, proficiency testing and theoretical concepts. this approach has shown severe limitations with regard to advancing quality-assured laboratory medicine in developing countries. to continue with such strategies would be analogous to pounding square pegs into round holes. rather, novel holistic approaches that place emphasis on task-based and results-driven quality improvement projects are needed urgently. to achieve these goals, laboratory medicine in developing countries must innovate and create performance-enablers that would both incentivise and energise the field, thereby facilitating adoption. successful performance-enablers must focus on four chief aspects: implementation, measurement, reward and improvement. in order to ensure a sustainable culture of the practice of quality-assured laboratory medicine, countries need to embrace innovative qms programmes, which require commitment to the process of continuous quality improvement of laboratory medicine. some countries have made remarkable progress by using innovative approaches to implementing laboratory accreditation. for example, between 1961 and 1998, south korea endorsed the laboratory accreditation program (lap) of the college of american pathologists (cap); however, during that time only 11 laboratories were accredited.15 because of the challenges in implementation of lap, in 1999, south korea modified the cap-lap into a step-wise laboratory accreditation process known as the korea laboratory accreditation process (klap). in klap, laboratories with a score of > 90% received a two-year certificate; laboratories with scores between 60% and 89% received a one-year certificate; and those with a score of < 60% failed the certification. as a result of klap, 227 laboratories were certified between 1999 and 2006 and many laboratories were enrolled in the programme across the country. in 2001, thailand established a step-wise national accreditation programme as a local alternative for improvement of laboratory quality. the accreditation programme was established with standards based on iso 15189 and, from 2003 to 2009, 724 (50.6%) of 1432 laboratories in thailand were assessed. of these, 197 (27.2%) were accredited, primarily in the government sector.16 the programme has thus far been affordable, feasible, scalable, sustainable and effective.16 over the past five years, through global partnership, innovative performance-enablers have been developed to guide the implementation of a sustainable qms leading to accreditation in developing countries: (1) the stepwise laboratory quality improvement process towards accreditation (slipta);17 (2) the caribbean laboratory quality management system – stepwise improvement process (lqms-sip) towards accreditation;18 and (3) the task-based strengthening laboratory management toward accreditation (slmta) programme.19 slipta and lqms-sip, the companions of slmta both slipta and lqms-sip are innovative performance-enabling tools and companions of slmta. these tools were designed to motivate the implementation of qms with measurable delivery through slmta. slipta is a framework endorsed by the world health organization regional office for africa (who afro) and jointly implemented by the african society for laboratory medicine (aslm) for improving the quality of public health laboratories in african countries to achieve accreditation to the iso 15189 standard. the who afro slipta checklist is based on iso 15189/17025, with 111 items and a possible 258 points, which are further divided into five star levels: one to five.17 lqms-sip has been endorsed in the caribbean region and consists of a three-tiered system: the first tier represents the minimum requirements that correspond to mandatory criteria required for the granting of a licence based on legislation enacted by the ministries of health. the next two tiers are quality-improvement levels representing achievements in meeting specific requirements of a qms. the caribbean regional organization for standards and quality hosts the lqms-sip secretariat and works directly with countries and other laboratory stakeholders to coordinate the rollout and implementation of the regulatory activities and the recognition process. the caribbean public health agency (carpha) helps with the coordination of the caribbean public health laboratory network and participates in the development of quality standards. evident evidence and the up-shot top ↑ slmta is grounded in its ability to identify deficiencies in a laboratory, improve them and measure the outcomes. after completing a full slmta round, which typically lasts for up to 18 months, changes are evident and outcomes are visible. laboratories experience a net progression from one to five stars on the slipta scoring checklist upon completion of the slmta programme. more significant is the enduring impact on personnel who have undergone slmta training, achieving positive changes in attitude toward the culture of quality, as well as recognition of quality-assured laboratory medicine. in this special issue of the african journal of laboratory medicine, several countries have shared remarkable evidence regarding how slmta has been transformative in their laboratories7,8 and is beginning to stimulate changes in hospital management.20 since 2009, when slmta was launched in kigali, rwanda, it has expanded exponentially. as of the end of 2013 it has been implemented in 47 countries in africa, the caribbean, latin america and southeast asia. with the introduction of slmta, the prospects of implementing sustainable quality-assured laboratory medicine seem to be a reality in developing countries. in total, the 302 laboratories that have completed the slmta programme conduct approximately 43.5 million diagnostic tests annually. based on baseline audit scores, laboratories that had at least one quality star prior to slmta participation conducted only one out of every six tests. this number quadrupled to two out of three after slmta training. these gains have also proven to be sustainable; of the 92 laboratories that have conducted surveillance audits at five to 28 months after slmta completion, 62% showed a further increase in their score from the exit audit, with more than half increasing their score by more than 10 additional percentage points.8 at present, countries that have implemented slmta are caught between a state of cautious optimism and open-minded concern about the rollout and sustainability of the programme.d open-minded concern about the rollout and sustainability of the programme. slmta, nearing the tipping point? in order to ensure sustainability, it is urgent to identify system drivers that will enable the country to reach a tipping point; these include expanded coverage and demonstration of the impact on patient care. such a tipping point, defined in this context as the number of laboratories that will constitute a critical mass for the demand of the programme to become a nation-wide requirement, once attained, will begin to increase confidence in quality-assured laboratory medicine for evidence-based patient management. this may lead to an increased uptake and use of laboratory test results, encouraging greater investment of resources in laboratory services and, ultimately, breaking the vicious cycle of the neglected laboratory systems in developing countries (figure 1). potential drivers that could facilitate a tipping point for slmta include incorporating slmta into a pre-service curriculum for schools of medical laboratory sciences; strengthening the clinical-laboratory interface; developing country-specific national strategic plans for rolling out slmta and other qms tools; accelerating the process by engaging aslm or similar organisations to audit and reward laboratories that have undergone the slmta process; incorporating basic laboratory information systems as part of qms; designing slmta-like training tools for hospital and clinic certification and accreditations; and, lastly, encouraging donors and funders to prioritise qa and continuous quality improvement as a core component of laboratory health system strengthening. conclusion slmta eliminates redundant procedures by reorganising the laboratory set-up so that personnel spend less time handling and processing specimens. slmta is not a destination; rather, it is a journey that relies on ongoing cooperation at all levels, including senior management, laboratory staff and end users. in the 21st century, as the economies of developing countries continue to grow, many individuals will begin to seek affordable quality-assured healthcare; as such, there is bound to be increasingly consumer-oriented healthcare that holds physicians and laboratory workers more accountable for errors. because of improved quality-assured laboratory medicine, healthcare providers will broaden their practice from using laboratory tests to confirm clinical diagnoses to using tests to detect clinically unapparent diseases, as well as to support outbreak surveillance responses, such as the recent outbreak of ebola virus in west africa. in order to meet this demand, both slmta and slipta will need to be rolled out in developing countries so as to stimulate healthcare service providers to focus on a systematic work flow for quality services rendered to patients, resulting in increased efficiency and quality whilst lowering waste and cost and improving safety. critically, a patient-centered continuous quality improvement approach will become indispensable. hospital accreditation, as in thailand, will most likely drive the need for laboratory quality, as active involvement of managers will create collective organisational commitment of quality improvement and a focus on patient-oriented thinking. acknowledgements top ↑ competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions j.n.n. (cdc, atlanta) contributed to the conception and development of the manuscript. d.b. (cdc, atlanta) contributed to the writing and review of the manuscript. disclaimer the comments and conclusions in this report are those of the authors and do not necessarily represent the official position of the united states department of health and human services, public health service, or centers for disease control and prevention. references top ↑ 1.berger d. a brief history of medical diagnosis and the birth of the clinical laboratory. mlo med lab obs. 1999;31(7):28–30, 32, 34–40. 2.institute of medicine. to err is human: building a safer health system [document on the internet]. c1999 [cited 2014 sep 22]. available from: http://www.iom.edu/~/media/files/report%20files/1999/to-err-is-human/to%20err%20is%20human%201999%20%20report%20brief.pdf 3.committee on quality of health care in america. crossing the quality chasm: a new health system for the 21st century [document on the internet]. c2001 [cited 2014 sep 20]. available as ebook from: http://www.nap.edu/openbook.php?record_id=10027 4.petti ca, polage cr, quinn tc, et al. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377–382. http://dx.doi.org/10.1086/499363 5.polage cr, bedu-addo g, owusu-ofori a, et al. laboratory use in ghana: physician perception and practice. am j trop med hyg. 2006;75(3):526–531. 6.nkengasong jn, nsubuga p, nwanyanwu o, et al. laboratory systems and services are critical in global health: time to end the neglect? am j clin pathol. 2010;134(3):368–373. http://dx.doi.org/10.1309/ajcpmpsinq9brmu6 7.luman et, yao k, nkengasong jn. a comprehensive review of the slmta literature part 2: measuring success. afr j lab med. 2014;3(2), art. #276, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i2.276 8.yao k, luman et, slmta collaborating authors. evidence from 617 laboratories in 47 countries for slmta-driven improvement in quality management systems. afr j lab med. 2014;3(2), art. #262, 11 pages. http://dx.doi.org/10.4102/ajlm.v3i2.262 9.carter jy, lema oe, wangai mw, et al. laboratory testing improves diagnosis and treatment outcomes in primary health care facilities. afr j lab med. 2012;1(1), art. #8, 6 pages. 10.zeh ce, inzaule sc, magero vo, et al. field experience in implementing iso 15189 in kisumu, kenya. am j clin pathol. 2010;134(3):410–418. http://dx.doi.org/10.1309/ajcpzirkdus5lk2d 11.leatherman s, ferris tg, berwick d, et al. the role of quality improvement in strengthening health systems in developing countries. int j qual health care. 2010;22(4):237–243. http://dx.doi.org/10.1093/intqhc/mzq028 12.bonini p, plebani m, ceriotti f, et al. errors in laboratory medicine. clin chem. 2002;48(5):691–698. 13.institute for health metrics and evaluation. financing global health 2012: the end of the golden age? [report on the internet]. c2012 [cited 2013 feb 05]. available from: http://www.healthmetricsandevaluation.org/publications/policy-report/financing-global-health-2012-end-golden-age 14.nkengasong jn. a shifting paradigm in strengthening laboratory health systems for global health: acting now, acting collectively, but acting differently. am j clin pathol. 2010;134(3):359–360. http://dx.doi.org/10.1309/ajcpy5asuejyq5rk 15.shin bm, chae sl, min wk, et al. the implementation and effects of a clinical laboratory accreditation program in korea from 1999 to 2006. korean j lab med. 2009;29(2):163–170. http://dx.doi.org/10.3343/kjlm.2009.29.2.163 16.wattanasri n, manoroma w, viriyayudhagorn s. laboratory accreditation in thailand: a systemic approach. am j clin pathol. 2010;134(4):534–540. http://dx.doi.org/10.1309/ajcpzyy19wmkmazt 17.world health organization regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region (with checklist) [document on the internet]. c2011 [cited 2013 aug 05]. available from: http://www.afro.who.int/index.php?option=com_docman&task=doc_download&gid=8642&itemid=2593 18.pan american health organization. regional office of the world health organization. caribbean guidance on the stepwise improvement process for strengthening laboratory quality management systems towards accreditation [document on the internet]. c2012 [cited 2014 sep 10]. available from: http://www.cmedlabsfoundation.net/images/standards/guidance%20stepwise%20final%20-%2004%2010%202012.pdf 19.yao k, maruta t, luman et, nkengasong jn. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(2), art. #194, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i2.194 20.eno lt, asong t, ngale e, et al. driving hospital transformation with slmta in a regional hospital in cameroon. afr j lab med. 2014;3(2), art. #221, 5 pages. http://dx.doi.org/10.4102/ajlm.v3i2.221 about the author(s) passoret vounba economic community of central african states (eccas) commission/fourth phase of the regional disease surveillance systems enhancement project (redisse iv), libreville, gabon severin loul economic community of central african states (eccas) commission/fourth phase of the regional disease surveillance systems enhancement project (redisse iv), libreville, gabon ludovic f. tamadea economic community of central african states (eccas) commission/fourth phase of the regional disease surveillance systems enhancement project (redisse iv), libreville, gabon joël f.d. siawaya department of laboratory services, chu mère-enfant fondation jeanne ebori, libreville, gabon regional integrated surveillance and laboratory network (rislnet) for central africa, libreville, gabon citation vounba p, loul s, tamadea lf, siawaya jfd. corrigendum: microbiology laboratories involved in disease and antimicrobial resistance surveillance: strengths and challenges of the central african states. afr j lab med. 2023;12(1), a1913. https://doi.org/10.4102/ajlm.v12i1.1913 note: doi of original article published: https://doi.org/10.4102/ajlm.v11i1.1570 correction corrigendum: microbiology laboratories involved in disease and antimicrobial resistance surveillance: strengths and challenges of the central african states passoret vounba, severin loul, ludovic f. tamadea, joël f.d. siawaya published: 09 may 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. in the published article, vounba p, loul s, tamadea lf, siawaya jfd. microbiology laboratories involved in disease and antimicrobial resistance surveillance: strengths and challenges of the central african states. afr j lab med. 2022;11(1), a1570. https://doi.org/10.4102/ajlm.v11i1.1570, there is an omission in the acknowledgements statement. the corrected acknowledgements statement appears below. the original incorrect wording: we sincerely thank dr skander hathroubi (from humboldt university of berlin, germany) for his contribution by reading and correcting the first draft of the manuscript. the revised and updated wording: we sincerely thank dr skander hathroubi (from humboldt university of berlin, germany) for his contribution by reading and correcting the first draft of the manuscript. this study was commissioned and supported by the regional disease surveillance systems enhancement (redisse) project in central africa, which is financially supported by the world bank and managed by the economic community of central african states (eccas). we would like to thank eccas and the world bank for supporting our efforts through redisse. the authors apologise for this error. the correction does not change the study’s findings of significance or overall interpretation of the study’s results or the scientific conclusions of the article in any way. abstract introduction methods results discussion acknowledgements references about the author(s) abass abdul-karim tamale zonal public laboratory, ghana health service, tamale, ghana david opare national public health laboratory, ghana health service, accra, ghana ulysses balis department of pathology, university of michigan school of medicine, ann arbor, michigan, united states lee f. schroeder department of pathology, university of michigan school of medicine, ann arbor, michigan, united states citation abdul-karim a, opare d, balis u, schroeder lf. providing specimen transport through an online marketplace in the northern region of ghana. afr j lab med. 2023;12(1), a2062. https://doi.org/10.4102/ajlm.v12i1.2062 note: additional supporting information may be found in the online version of this article as online supplementary document 1. original research providing specimen transport through an online marketplace in the northern region of ghana abass abdul-karim, david opare, ulysses balis, lee f. schroeder received: 12 nov. 2022; accepted: 22 may 2023; published: 20 july 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: integrated diagnostic networks, which are themselves dependent on robust specimen transport solutions, are fundamental to effective healthcare systems. objective: this study aimed to pilot an online marketplace for the transport of specimens throughout a laboratory network in ghana. methods: independent drivers were matched with health facilities that required specimen transport using a suite of mobile applications and web portals developed for this study. this marketplace was piloted with seven drivers, two laboratories, and five health facilities in ghana’s northern region from march 2019 to october 2019. results: during the pilot, 182 deliveries were completed for 691 patients, including 4118 laboratory tests for antenatal care, disease surveillance, and clinical testing. testing included 34 tests for communicable and non-communicable diseases. all but two specimens (laboratory cancellations) were successfully delivered and tested. the median time from request to encrypted emailing of results was 19.7 h, while that for a drop-off request was 0.9 h. in the midwife registry, the median time from patient visit to result recording was 1 day, compared to 4 days in the same months in 2018, and the number of mothers without documented testing decreased from 41 to 3. similarly, the proportion of tuberculosis specimen deliveries from buipe polyclinic to tamale zonal laboratory taking over 1 day fell from 62% at baseline to 3% during the pilot. conclusion: an online marketplace successfully orchestrated the delivery of laboratory specimens under a variety of clinical circumstances, reducing overall turn-around time without diminution of the overall specimen delivery process. what this study adds: this study established the efficacy of an online marketplace to orchestrate timely and high-quality delivery of specimens within a laboratory network. keywords: laboratory network; specimen transport; specimen referral; public health laboratory; essential diagnostics; diagnostic network. introduction fundamental to any effective national healthcare system is a functional laboratory network with hierarchical tiers.1,2 through a laboratory network, peripheral health centres without full testing capacity can access a larger portfolio of diagnostics by sending patient specimens to higher-tier laboratories. a robust courier solution that ensures specimens are shipped efficiently and reliably is essential to the laboratory network but is often non-existent in many lowand middle-income countries, except for some select diseases of public health importance.3,4 in ghana, specimen transport is limited but critical for clinical testing and disease surveillance and is a particular concern in the northernmost regions of the country.5,6 the maputo declaration of 2008 called for the strengthening of laboratory networks throughout sub-saharan africa.1 this was in response to the escalating efforts to curb endemic conditions like hiv, tuberculosis, and malaria. since then, other major global efforts for the surveillance of antimicrobial resistance and epidemic-prone diseases2 like coronavirus disease 2019 have identified the need for tiered laboratory networks.7,8,9 tiered laboratory networks permit knowledge sharing between tiers, strengthened reagent supply chains, shared resources for instrument servicing, and testing access for remote health facilities through specimen transport.10 recent efforts to strengthen specimen transportation revealed the feasibility of a wide variety of strategies, with perhaps the greatest barrier being a lack of engagement.3,11,12,13,14,15,16,17 in ghana, various ad-hoc approaches to specimen transport are utilised and the results are not always satisfactory. for instance, the time to deliver specimens for meningitis testing, a disease requiring rapid results, varied between 3 and 7 days in the yendi and talensi districts in ghana.5,18 the rate of laboratory-confirmed meningitis is thus low in ghana and throughout the entire meningitis belt, where fewer than 10% of cases are confirmed through testing.19 online marketplaces like uber™ and airbnb™ have had major impacts in multiple industries. uber technologies incorporated (san francisco, california, united states) revolutionised the transportation industry by tapping into the sharing economy. this study aimed to create and pilot an online marketplace in ghana to match drivers with health facilities that need specimens sent to higher-tier laboratories. our goal was to harness the existing transportation resources in ghana to efficiently move specimens throughout the laboratory network. this approach also allowed for the most effective use of diagnostic resources in a health system where instruments are rarely used to full capacity due to a lack of locally available specimens. methods ethical considerations this study was conducted according to the ethical approval provided by ghana health service ethical review committee (ghs-erc 002/05/18). written consent was obtained for participation from the prospective survey participants, as well as from patients submitting samples to be used in the study. all data were anonymised and maintained on health insurance portability and accountability act-compliant servers and password-protected laptops. development of the mobile and web-based application a mobile and web-based application suite formed the technological foundation for our online marketplace (figure 1). the custom suite was created for this study by app emporio (rajkot, india) on a technological stack including nodejs (https://nodejs.org/en), angular2 (https://angular.io/), and mongodb (https://www.mongodb.com/). the suite included different persona-based applications for health facilities, drivers, and laboratories. there was also a web portal for the participating laboratories, where test menus, turn-around times (tats), and prices could be added. similarly, an administrative portal provided real-time visualisation of operations, including global positioning system locations of drivers and active deliveries. figure 1: screenshots of the health facility mobile phone application developed for the specimen transport network piloted at health facilities in the northern region of ghana between march 2019 and october 2019. the application includes (a) the laboratory test menus from which healthcare staff select tests, (b) the tracking features of the application, and (c) visualisation of real-time driver location during the delivery. workflow of the specimen transport network patients were identified through routine clinical care and brought to the health facility’s laboratory for phlebotomy and possible participation in the pilot. there, specimens were collected from the patients and packaged in triple containers with ice packs and paper requisitions to facilitate proper identification of samples and confirmation of tests to be performed.20 the packages were then sealed to prevent exposure of drivers to potential biohazards. drivers were thus not required to use personal protective equipment. a trained health facility liaison then used the application to request a driver from a list of active drivers, sorted by distance. if the desired driver did not accept, the application automatically queried other drivers in order of distance from the pick-up point, based upon real-time geospatial telemetry from the driver-persona-based application. once a driver accepted the task, they travelled to the health facility, picked up the package, and delivered the same to the selected laboratory. during this time, the driver was trackable in real time through the application or web interface. upon delivery, the driver confirmed completion through the application, and the laboratory confirmed receipt of the adequate samples for testing, as well as the availability of the required testing reagents. laboratory test results were delivered to the health facilities electronically through proton ag (geneva, switzerland), a secure end-to-end encrypted email service. quality assurance staff from the project team monitored the requested deliveries and ensured that samples arrived safely and that the laboratories performed the tests and communicated the results. the application also included a rating system for health facilities and drivers. a pilot study of the online marketplace was conducted from march 2019 to october 2019 at five public health facilities in the northern region of ghana (online supplementary document – supplementary methods), and a medical records review collected data on test utilisation and tats (online supplementary document – supplementary methods). additionally, prospective surveys of patients (online supplementary document – supplementary survey 1) and providers (online supplementary document – supplementary survey 2) collected data on challenges and preferences for laboratory testing. data analysis all data on specimen delivery were stored on our servers and made available through our web administration dashboard. no private health information was stored on this server. data from the abstraction of medical records and laboratory registers were entered into microsoft excel spreadsheets (microsoft corporation, redmond, washington, united states) and checked for consistency. data were imported to the r statistical environment (v3.6.1)21 for statistical analysis. for dichotomous outcomes, statistical evaluations were conducted using the mcnemar test (mcnemar.test function within the r stats package) with continuity correction, and tats were compared using the two-sample kolmogorov-smirnov test (ks.test function within the r stats package), with approximated p-values where exact computation was not possible due to ties (p < 0.05 were considered significant). due to technical difficulties, deliveries were often cancelled and had to be re-requested by facilities after the driver had already started the collection or delivery. in these instances, timestamps would underestimate actual delivery times. therefore, in the tat calculations, any deliveries with collection or delivery durations of less than 10 min were excluded, as the shortest driving time between the health facilities and laboratories was about 10 min. results in the medical record review of 15 health facilities (pre-pilot), 23 laboratories were identified that performed external testing, with a median of four laboratories per health facility (online supplementary document – supplementary figure 1). outside tamale, test results for 99% of in-house tests were delivered by the laboratory within 1 day of request, compared to 82% of tests sent to external laboratories (p < 0.001). in tamale, the proportions of test results resulted by the laboratories within 1 day of test request were similar for in-house tests (100%) and tests conducted at other laboratories (98%). for health facilities outside tamale, test results for 90% of in-house tests were recorded by providers within the same day of the test request compared to 74% of tests performed outside the facility (p < 0.001). within tamale, test results for 51% of in-house tests were recorded on the same day of the test request compared to 40% of tests performed outside the facility (p = 0.50). within tamale, the median tat from test request to recording of results was 3.1 h for in-house testing compared to 1.7 h for send-out testing (p < 0.001). likewise, the median tat from request to recording by the provider was higher for in-house testing (12.7 h) compared to send-out testing (4.8 h; p = 0.08). the types of tests conducted in-house were similar to those conducted elsewhere, with a total of 23 different tests identified. the most common of these were malaria, complete blood count, haemoglobin, typhoid, urinalysis with microscopy, sickle solubility, urine pregnancy, and hepatitis b surface antigen tests, which collectively accounted for 95% of testing. other less-commonly ordered tests included stool microscopy, hepatitis c test, glucose-6-phosphate dehydrogenase test, tuberculosis sputum microscopy, liver function tests, renal function tests, glucose tests, and hiv tests. interviews were conducted with 73 patients, including representatives from each of our pilot sites. when asked about the frequency with which they experienced different barriers to testing, 58% of patients described test availability as ‘often’ or ‘always’ a barrier, 15% reported test expense as ‘often’ or ‘always’ a barrier, and 1% reported the physician not ordering tests as ‘often’ or ‘always’ a barrier (figure 2). there were no responses of ‘other’ reasons. nearly equal numbers of patients responded that they would either transport the samples to a laboratory themselves (44%) or that a specimen would be sent by some other means (46%). others (8%) reported that they had experienced both, while 1% reported that they had experienced neither. due to the structure of the question, it was difficult to interpret how patient samples were transported to outside laboratories (e.g., by a system organised by the health facility or by the patient’s family or friends). nearly all patients (96%) said they would be interested in the specimen transport service being piloted, mostly to avoid travel or reduce transport costs; two patients were, however, concerned about the possibility of mislabelled specimens or loss of confidentiality. figure 2: results of a patient survey conducted at health facilities in the northern region of ghana between march 2019 and october 2019. patients reported that test availability was the most common cause preventing laboratory testing. seven providers were interviewed, with representation from each health facility (figure 3). twenty-nine percent responded that tests are ‘often’ or ‘always’ returned promptly. when testing was not available in their health facility, 57% of providers responded that samples were ‘often’ or ‘always’ sent to outside laboratories, 43% stated that patients were transferred to higher-tier facilities, and 14% stated that patients travelled themselves to outside laboratories. the most commonly experienced barriers to accessing diagnostic testing were tat (80% responded ‘often’ or ‘always’) and cost (50% responded ‘often’ or ‘always’). all seven providers said that the specimen transport services would be useful if made available at their facilities. figure 3: results of a survey conducted among health providers at health facilities in the northern region of ghana between march 2019 and october 2019. related questions are grouped between dashed lines. over the 8 months of the pilot, 182 deliveries were conducted for 691 patients, including 4118 laboratory tests (table 1). other than laboratory cancellations for two specimens due to spillage (samples were re-collected), all deliveries were completed, and results were posted. of the 182 deliveries, 120 were excluded from tat calculations as their collection or delivery durations were less than 10 min, indicating a technical difficulty with the application that required the resubmission of delivery requests. at the individual testing level, the median time from health facility request to sending of results by encrypted email was 19.7 h, with an interquartile range (iqr) of 18.6 h – 20.7 h. the median time from request to collection of the specimen by the driver was 21 min (iqr: 11 min – 38 min), and the median time from specimen pick-up to drop-off at the laboratory was 28.8 min (iqr: 19 min – 65 min). the median time from drop-off to reporting of results was 18.5 h (iqr: 16.2 h – 19.4 h), accounting for 94% of the total tat (iqr: 89% – 96%). turn-around times observed during the pilot could not be compared with those observed during the retrospective analysis because most medical records only contained dates, not times (hours and minutes). driver fees varied from $1.75 united states dollars (usd) to $8.77 usd per delivery, depending on the requesting facility. multiple patients were pooled for each delivery. some surveillance testing for tuberculosis required variably higher fees for delivery due to longer travel times (> 2 h; up to $17.54 usd). the direct payments to drivers on average were $0.77 usd per patient and $0.11 usd per test, excluding the longer travel to the buipe district for surveillance testing (18 deliveries). table 1: characteristics and performance of the specimen transport network piloted at health facilities in the northern region of ghana between march 2019 and october 2019. one hundred and ten deliveries (60% of all deliveries) included samples for clinical testing (figure 4). deliveries were conducted mostly for haemoglobin (n = 146 tests), malaria (n = 142), and urinalysis (n = 103) testing. surveillance testing initiated by disease control officers was included in 18% of deliveries, and these included tests for tuberculosis (n = 63), meningitis (n = 5), measles (n = 7), and yellow fever (n = 4). figure 4: laboratory tests ordered during the pilot of a specimen transport network at health facilities in the northern region of ghana between march 2019 and october 2019. the figure shows tests performed for antenatal care, general clinical care, and public health surveillance. antenatal care guidelines recommend a specific set of tests, which explains the consistent ordering patterns for that patient population. however, much general clinical testing included the same tests as for antenatal care. surveillance testing was largely for tuberculosis but also included testing for bacterial meningitis and other priority public health targets. at the antenatal care (anc) facility reviewed, there were 248 mothers at baseline and 250 in the pilot phase. more mothers were without any documented testing in the anc facility during the baseline months in 2018 (41 not tested; 207 tested) compared to the corresponding months during the pilot (3 not tested; 247 tested; mcnemar test: p < 0.001). in the baseline months, 136 mothers had dates recorded in the midwife register for when their laboratory test results were entered. during the pilot, laboratory test result dates were entered in the register for 115 mothers. the median time from the initial visit to result recording in the anc register (which may be later than the actual return of results) was significantly shorter during the pilot compared to the baseline months (1 day vs 4 days; two-sample kolmogorov-smirnov test: p < 0.001) (figure 5). figure 5: antenatal care testing times and screening rates as extracted from a midwife registry in the northern region of ghana between may 2018 – august 2018 (baseline) and may 2019 – august 2019 (on-demand delivery). the histograms represent the number of patients (y-axis) with documented test results within a given time frame from the initial patient visit (x-axis). the bars are overlaid on top of each other. in the review of zonal laboratory records for tuberculosis testing, there were 77 specimens during the baseline months and 32 specimens during the pilot. during the pilot, the median delivery time for specimens was 0 days (same day) compared to a median of 2 days at baseline. this translated to 97% of specimens being delivered in less than 1 day (time from specimen collection to receipt in laboratory) during the pilot compared to 38% of specimens being delivered in less than 1 day at baseline (two-sample kolmogorov-smirnov test: p < 0.001) (figure 6). figure 6: specimen transport times for tuberculosis testing as documented in the public health laboratory register from the buipe polyclinic in the northern region of ghana between 2009 and 2014 (baseline) and march 2019 – october 2019 (on-demand delivery). the histogram shows the number of specimens (y-axis) and the time intervals within which they were delivered for testing after specimen pick-up (x-axis). the bars are overlaid on top of each other. discussion this pilot of an online marketplace for the transport of laboratory specimens revealed that specimens could be successfully delivered under a variety of clinical circumstances, including for general clinical testing at health facilities and hospitals, anc testing with daily deliveries of bundled patient specimens, and surveillance testing as requested by disease control officers. for tuberculosis testing, tats improved from a baseline period, and for anc testing, both tat and the number of mothers screened improved. a 2021 study found that most health facilities in the northern region of ghana did not offer the majority of tests recommended in the world health organization essential diagnostics list.6 our review of medical records revealed that the testing that does occur is performed by a combination of on-site and external laboratories. outside of testing for hiv early infant diagnosis and some limited diseases for public health surveillance, none of the facilities reviewed operated formalised specimen transport systems for clinical testing (a.a.-k., 2021, personal communication, march 31). presumably, therefore, samples tested at external laboratories were mostly transported through ad-hoc means (e.g., patient presentation to the laboratory, delivery of the specimen by family or friends, etc.). more on-site tests were completed within 1 day of request compared to external testing, and this difference was greater outside tamale. this suggests that specimen transport should be prioritised in rural areas, where there are longer distances to external laboratories. nevertheless, patients in both urban and rural settings would likely benefit from such services. the sustainable development goals demand quality essential healthcare services with universal health coverage, where timeliness is a key component of quality.22,23,24,25 in line with this, the benefits of integrated laboratory networks to healthcare delivery include increased availability, accuracy, and tat of laboratory testing.26,27 during the pilot, the time from the initial visit to recording of test results in the midwife register at the anc decreased from 4 days to 1 day, and tuberculosis testing times decreased from 2 to 0 days (same day). as specimen transport systems are a key component of laboratory networks, multiple models have been implemented in different settings, and although the published models are not directly comparable (different metrics and different geographic and disease characteristics), they provide some context to the findings in this study. in a 2013 study in uganda, a motorcycle hub-and-spoke delivery system for centralised hiv early infant diagnosis led to a reduction in tat from 1–2 months to 5–10 days.11 the same service led to a tat reduction for tuberculosis testing from 21 to 3 days.3 in ethiopia, a private–public partnership involving the ethiopian postal service enterprise led to hiv early infant diagnosis tats decreasing from 7 to 2 days in addis ababa and from 10 to 5 days in the amhara region.3,11,12 the current pilot performed comparably, if not better than these. in addition to tat, laboratory networks can also improve the rates at which patients receive testing and downstream interventions. for example, in a 2015 study in haiti, the number of patients enrolled on antiretroviral therapy for new sites joining the specimen referral network increased by 182% within 6 months.28 antenatal care screening rates for mothers increased in this study compared to the prior year (3 out of 250 mothers not tested compared to 41 out of 248 mothers not tested at baseline). antenatal care screening is important for the prevention of mother-to-child transmission of infectious diseases, monitoring for disorders such as pre-eclampsia, and ensuring that expectant mothers with anaemia deliver at facilities with transfusion capabilities. while this study did not follow such patient outcomes, it is reasonable to expect that there would be a commensurate positive impact with improved screening.29,30 regarding tuberculosis, at least six previously undiagnosed patients were diagnosed during the pilot. one of these patients reported prior symptoms for 1 year, and another described her son’s recent death from a condition with similar symptoms. the early detection of tuberculosis, enabled through laboratory networks, can have an important impact on transmission (and thus prevalence), patient outcomes, and the economic burden on affected families.31,32 this pilot was designed to assess the feasibility and effectiveness of the transport model, not the costs. nonetheless, when a health system is determining whether to provide testing on site or via specimen transport, specimen transport cost is a factor that must be considered. in some settings, overall testing costs may be lower with external testing. in the 2013 study in uganda, the implementation of the specimen transport system for centralised testing led to a 62% reduction in operational costs.3 in a 2023 study in uganda, researchers found similar per-test costs in the decentralised versus centralised model for tuberculosis testing.33 although we documented driver fees, which varied based on distance travelled, we did not perform a comprehensive cost analysis. these deliveries often included batches of patient specimens, thus reducing the per-specimen transportation cost considerably. in this study, excluding the longer deliveries for surveillance in buipe district, average driver fees were $0.73 usd per patient and $0.11 usd per test. in other settings, this amount would vary based on the use of taxis versus motorcycles (with motorcycles likely costing less), the number of patients per delivery, and the number of tests per delivery. another cost was for smartphones, as most of the drivers had phones, but not smartphones. the study required providing them with smartphones and data. however, this augmented provisioning to drivers may not be necessary if smartphone adoption increases over time.34 a full analysis would need to include the costs of application development and maintenance, platform operation, as well as the economies of scale that depend on the number of deliveries per period, among other costs. ghana was the first sub-saharan african country to introduce a tax-funded national health insurance scheme (nhis 2003). the nhis provides payments to accredited public and private laboratories for testing. however, nhis reimbursement to laboratories is often less than what laboratories would charge when patients pay ‘out of pocket’. a 2021 study in ghana found that the greater the difference between the nhis reimbursement and the market rate, the less available the test was in health facilities and laboratories.6 nonetheless, the nhis represents a foundation upon which payments for diagnostic testing can be provided. the nhis provides a single payment per test and does not pay for specimen transport, the cost of which would thus be borne by the health facility, the patient (who might otherwise have to pay for transport themselves), or the laboratories, as they desire more specimens. because such a transport system would increase testing demand, laboratories may be willing to pay for some, or possibly all, of the transport costs. this would be particularly true if specimens are transported in batches such that per-test delivery costs are low compared to testing costs. regarding sustainability, future work would be needed to further explore the scenarios (e.g., frequency, distance, and urgency of pick-ups) in which this service would be optimal for both the ‘buyer’ and ‘seller’. this includes understanding the ‘willingness to pay’ of the potential buyer. it is also important to understand the ‘willingness to sell’ of the potential seller of this service under each scenario (i.e., drivers). payment could be improved through telebanking, and different payment contracts could be explored (e.g., simple payment per delivery or flat rates plus per-delivery fee). limitations there were challenges experienced during this pilot. first, facilities often had to resend requests and that may have impacted tat estimates, although this was mitigated by excluding unreasonably short collection or delivery durations of less than 10 min. these difficulties could have been due to unstable cellular networks leading to the cancellation of active deliveries. in the next phase, cellular message event buffering with a closed-loop confirmation messaging protocol could mitigate this shortcoming. second, while the surveillance testing pilot was successful, disease control officers sometimes made requests from very distant locations (> 2-h drive), thus leading to unsustainably high driver fees. recruitment of bus drivers in a daisy-chained fashion to allow deliveries to and from bus stations and subsequent bus delivery between cities is a possible solution. third, requests from district hospitals were fewer than anticipated. this may have represented a reluctance to engage in a change of procedure. it is also possible that providers preferred to refer patients to nearby private laboratories where the providers had existing relationships. further investigation would be necessary to determine the root causes of low district hospital order rates. conclusion one path towards improving diagnostic testing is additional in-house testing, but a complementary approach using specimen transport could build on the laboratory network approach. instead of investing in fleets of drivers for this purpose, an online marketplace taps into existing transportation resources that are often under-utilised. through such a specimen transport service as piloted in this study, existing laboratory capacity could be better harnessed for patient care and disease surveillance. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.a.-k. contributed to study conceptualisation, methodology, investigation, project administration, writing, review and editing. d.o. contributed to the supervision of the project as well as writing, review, and editing of the article. u.b. contributed to conceptualisation, methodology, writing, review and editing. l.f.s. contributed to conceptualisation, methodology, formal analysis, writing of the original draft, review and editing, software, resources, and supervision of the project. sources of support funding for the study was received from the bill and melinda gates foundation (opp1182131). data availability the datasets generated and analysed during the current study are available from the corresponding author, l.f.s., on reasonable request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references world health organization. the maputo declaration on strengthening of laboratory systems. annex e list of laboratory tests by level. geneva: who; 2008. global health security agenda. driving outcomes toward the global health security agenda action package commitments [homepage on the internet]. [cited 2018 jul 18]. available from: https://www.ghsagenda.org/packages fonjungo pn, alemnji ga, kebede y, et al. combatting global infectious diseases: a network effect of specimen referral systems. clin infect dis. 2017;64(6):796–803. https://doi.org/10.1093/cid/ciw817 dowdy dw. minding the gap: specimen referral systems for diagnosis of infectious diseases. clin infect dis. 2016;64:ciw820. https://doi.org/10.1093/cid/ciw820 kaburi bb, kubio c, kenu e, et al. evaluation of the enhanced meningitis surveillance system, yendi municipality, northern ghana, 2010–2015. bmc infect dis. 2017;17:306. https://doi.org/10.1186/s12879-017-2410-0 ward cw, schroeder lf. availability and prices of who essential diagnostics in laboratories in west africa: a landscape survey of diagnostic testing in northern ghana. j appl lab med. 2021;6(1):51–62. https://doi.org/10.1093/jalm/jfaa190 o’neill j. tackling drug-resistant infections globally: final report and recommendations. the review on antimicrobial resistance. 2016. united kingdom. wellcome trust. fleming fund launched to tackle global problem of drug-resistant infection [homepage on the internet]. [cited 2018 jun 04]. available from: https://wellcome.ac.uk/press-release/fleming-fund-launched-tackle-global-problem-drug-resistant-infection world health organization (who). global antimicrobial resistance surveillance system (glass) infection [homepage on the internet]. [cited 2018 jun 04]. available from: http://www.who.int/glass/en/ nkengasong jn, nsubuga p, nwanyanwu o, et al. laboratory systems and services are critical in global health: time to end the neglect? am j clin pathol. 2010;134(3):368–373. https://doi.org/10.1309/ajcpmpsinq9brmu6 kiyaga c, sendagire h, joseph e, et al. uganda’s new national laboratory sample transport system: a successful model for improving access to diagnostic services for early infant hiv diagnosis and other programs. plos one. 2013;8:1–7. https://doi.org/10.1371/journal.pone.0078609 borchert jn, tappero jw, downing r, et al. rapidly building global health security capacity – uganda demonstration project, 2013. mmwr morb mortal wkly rep. 2014;63:69–72. joloba m, mwangi c, alexander h, et al. strengthening the tuberculosis specimen referral network in uganda: the role of public-private partnerships. j infect dis. 2016;213(suppl_2):s41–s46. https://doi.org/10.1093/infdis/jiw035 kebede y, fonjungo pn, tibesso g, et al. improved specimen-referral system and increased access to quality laboratory services in ethiopia: the role of the public-private partnership. j infect dis. 2016;213(suppl_2):s59–s64. https://doi.org/10.1093/infdis/jiv576 calcagno c, lobatto me, robson pm, millon a. specimen referral network to rapidly scale-up cd4 testing: the hub and spoke model for haiti. j aids clin res. 2016;28:1304–1314. https://doi.org/10.1002/nbm.3369 nguyen g. vietnam: better tb specimen referral system improves mdr-tb diagnostic accuracy and safety [homepage on the internet]. 2013 [cited 2018 jun 04]. available from: https://www.msh.org/news-events/stories/vietnam-better-tb-specimen-referral-system-improves-mdr-tb-diagnostic-accuracy nguyen g. improved tb specimen referral system enhances mdr-tb diagnostic accuracy and specimen safety in vietnam [homepage on the internet]. 2014 [cited 2018 jun 04]. available from: http://www.stoptb.org/news/frompartners/2014/fp14_041.asp apanga pa, awoonor-williams jk. an evaluation of meningitis surveillance in northern ghana. int j trop dis health. 2016;12(2):1–10. https://doi.org/10.9734/ijtdh/2016/22489 world health organization. meningitis outbreak response in sub-saharan africa who guideline. geneva: who; 2014. dangerous goods regulations (packing instruction 650) 62nd edition. montreal: iata (international air transport association); 2021. r core team. r: a language and environment for statistical computing [homepage on the internet]. vienna: r foundation for statistical computing; 2017. available from: https://www.r-project.org/ sdg target 3.8 | achieve universal health coverage, including financial risk protection, access to quality essential health-care services and access to safe, effective, quality and affordable essential medicines and vaccines for all [homepage on the internet]. [cited 2023 jan 23]. available from: https://www.who.int/data/gho/data/themes/topics/indicator-groups/indicator-group-details/gho/sdg-target-3.8-achieve-universal-health-coverage-(uhc)-including-financial-risk-protection kruk me, gage ad, arsenault c, et al. high-quality health systems in the sustainable development goals era: time for a revolution. lancet global health. 2018;6(11):e1196–e1252. https://doi.org/10.1016/s2214-109x(18)30386-3 the phcpi conceptual framework. in: phcpi [homepage on the internet]. 2018 [cited 2023 jan 23]. available from: https://improvingphc.org/phcpi-conceptual-framework committee on improving the quality of health care globally, board on global health, board on health care services, health and medicine division, national academies of sciences, engineering, and medicine. crossing the global quality chasm: improving health care worldwide. washington, dc: national academies press, 2018; p. 25152. https://doi.org/10.17226/25152 ondoa p. preparing national tiered laboratory systems and networks to advance diagnostics in africa and meet the continent’s health agenda: insights into priority areas for improvement. african j lab med. 2020;9(2):1–10. fleming ka, horton s, wilson ml, et al. the lancet commission on diagnostics: advancing equitable access to diagnostics. lancet. 2021;393(10185):2018–2020. https://doi.org/10.1016/s0140-6736(19)31052-9 louis fj, osborne aj, elias vj, et al. specimen referral network to rapidly scale-up cd4 testing: the hub and spoke model for haiti. j aids clin res. 2015;6(8):1000488. https://doi.org/10.4172/2155-6113.1000488 world health organization. who recommendations on antenatal care for a positive pregnancy experience. geneva: who; 2016. campbell omr, graham wj. strategies for reducing maternal mortality: getting on with what works. lancet. 2006;368(9543):1284–1299. early detection of tuberculosis: an overview of approaches, guidelines and tools [homepage on the internet]. [cited 2023 jan 23]. available from: https://www.who.int/publications-detail-redirect/who-htm-stb-psi-2011.21 zwerling a, shrestha s, dowdy dw. mathematical modelling and tuberculosis: advances in diagnostics and novel therapies. adv med. 2015;2015:907267. https://doi.org/10.1155/2015/907267 thompson rr, nalugwa t, oyuku d, et al. multicomponent strategy with decentralised molecular testing for tuberculosis in uganda: a cost and cost-effectiveness analysis. lancet global health. 2023;11(2):e278–e286. https://doi.org/10.1016/s2214-109x(22)00509-5 broadband commission for sustainable development. working group report on smartphone access: strategies towards universal smartphone access. geneva: itu; 2022. article information authors: bernard nkrumah1 beatrice van der puije2 veronica bekoe3 rowland adukpo4 nii a. kotey2 katy yao5 peter n. fonjungo5 elizabeth t. luman5 samuel duh2 patrick a. njukeng6 nii a. addo3 fazle n. khan7 celia j.i. woodfill1 affiliations: 1us centers for disease control and prevention, us embassy, ghana2global health systems solutions, c75/20 amanfro street, abelenkpe, ghana 3national aids control program, ghana health service, ghana 4national public health reference laboratory, ghana health service, ghana 5us centers for disease control and prevention, atlanta, united states 6global health systems solutions, cameroon 7us centers for disease control and prevention, cote d’ivoire correspondence to: bernard nkrumah postal address: us centers for disease control and prevention, us embassy, number 4 forth circular road, cantonments, ghana dates: received: 15 july 2014 accepted: 06 aug. 2014 published: 03 nov. 2014 how to cite this article: nkrumah b, van der puije b, bekoe v, et al. building local human resources to implement slmta with limited donor funding: the ghana experience. afr j lab med. 2014;3(2), art. #214, 7 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.214 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. building local human resources to implement slmta with limited donor funding: the ghana experience in this original research... open access • abstract • introduction • research method and design    • programme implementation approach    • baseline audits and site selection    • development of a local mentorship programme    • slmta programme implementation    • programme monitoring and evaluation    • preparation for slmta expansion    • data entry and analysis • results    • measuring the impact of slmta    • local capacity building and cost of slmta implementation • discussion    • limitations of the study    • recommendations    • conclusion • acknowledgements    • competing interests    • disclaimer    • authors’ contributions • references abstract top ↑ background: in 2009, ghana adopted the strengthening laboratory management toward accreditation (slmta) programme in order to improve laboratory quality. the programme was implemented successfully with limited donor funding and local human resources.objectives: to demonstrate how ghana, which received very limited pepfar funding, was able to achieve marked quality improvement using local human resources. method: local partners led the slmta implementation and local mentors were embedded in each laboratory. an in-country training-of-trainers workshop was conducted in order to increase the pool of local slmta implementers. three laboratory cohorts were enrolled in slmta in 2011, 2012 and 2013. participants from each cohort attended in a series of three workshops interspersed with improvement projects and mentorship. supplemental training on internal audit was provided. baseline, exit and follow-up audits were conducted using the stepwise laboratory quality improvement process towards accreditation (slipta) checklist. in november 2013, four laboratories underwent official slipta audits by the african society for laboratory medicine (aslm). results: the local slmta team successfully implemented three cohorts of slmta in 15 laboratories. seven out of the nine laboratories that underwent follow-up audits have reached at least one star. three out of the four laboratories that underwent official aslm audits were awarded four stars. patient satisfaction increased from 25% to 70% and sample rejection rates decreased from 32% to 10%. on average, $40 000 was spent per laboratory to cover mentors’ salaries, slmta training and improvement project support. conclusion: building in-country capacity through local partners is a sustainable model for improving service quality in resource-constrained countries such as ghana. such models promote country ownership, capacity building and the use of local human resources for the expansion of slmta. introduction top ↑ the recent drive by the world health organization’s regional office for africa (who afro) and the us president’s emergency plan for aids relief (pepfar) toward strengthening laboratory systems in africa is a historic step in the improvement of health systems. however, this effort is hampered by the lack of locally-qualified laboratory personnel.1,2 the need to strengthen weak laboratory networks, systems and services in developing countries was highlighted in 2008 in a series of advocacy meetings: the maputo declaration (january 2008) for strengthening laboratory health systems,1 the lyon statement (april 2008) on the need for developing countries to establish practical quality management systems,3 and the yaoundé resolution (september 2008) issued by who afro in recognition of the dilapidated state of the laboratory health systems and the need to strengthen all laboratory tiers in order to fight multiple diseases.4 in the following year in kigali (july 2009), who afro launched a stepwise laboratory accreditation preparation scheme, which recognises and encourages incremental progress toward fulfilment of the requirements of the international organization for standardisation (iso) 15189 standard; and, at the 59th session of the who afro regional committee (september 2009), member states adopted resolutions afr/rc59/r2 and afr/rc59/wp/3 aimed at strengthening public health laboratories and other centres of excellence in order to improve disease prevention and control.4 also launched in kigali was the strengthening laboratory management toward accreditation (slmta) programme, developed by the us centers for disease control and prevention (cdc) and partners so as to guide countries toward achieving the called-for improvements. as pepfar’s flagship programme for strengthening laboratory systems, slmta has been implemented in 47 countries, demonstrating measurable and positive impact.5ghana has more than 420 public sector laboratories organised into a four-tier laboratory system: national/central, regional/zonal, district and subdistrict levels. nationaland regional-level laboratories provide technical assistance and supportive supervision for the district and subdistrict level laboratories. in 2012, the ministry of health (moh) and the ghana health service (ghs) drafted a five-year national laboratory strategic plan. this strategic plan clearly addressed local capacity building and highlighted the government’s commitment to fulfil their mandate of providing quality healthcare through the adoption of slmta as the systematic approach for implementation of laboratory quality management systems (qms) in ghana. in conjunction with the strategic plan, ghana drafted a national laboratory accreditation policy. accreditation ensures the quality, precision, dependability and timeliness of laboratory testing results.6,7,8 two private laboratories in ghana are accredited, but none of the public sector laboratories, which perform the bulk of patient testing, are accredited to any national or international standards. called the largest health initiative ever implemented by a single country to address a disease, since 2003 pepfar has provided financial and technical support to developing countries to fight hiv, saving millions of lives.9 laboratory strengthening is a critical component of the pepfar strategy. in 2013, the institute of medicine report on pepfar noted that ‘its substantial support for laboratory strengthening has had fundamentally positive effects for the response to hiv and has been leveraged to improve the functioning of entire health systems’.9 ghana is one of pepfar’s targeted assistance countries, which receive limited financial support for key populations or priority technical areas, capacity building and/or technical assistance.10 ghana’s annual laboratory budget from pepfar is about $1.1m (8% of the total ghana pepfar funding). this notwithstanding, pepfar is a major source of support for ghana’s laboratory system strengthening programmes. given the limited funding, ghana sought a strategy to implement the slmta programme for laboratory system strengthening in an economical and sustainable manner. this paper describes how a country like ghana, which receives very limited funding, was able to achieve marked improvement in laboratory quality management by empowering local partners to implement the slmta programme. research method and design top ↑ programme implementation approach a top-down programme implementation approach was adopted. under this model, the country sought to first build capacity and strengthen the quality of laboratory services within the nationaland regionallevel laboratories. in turn, these higher-level laboratories would be equipped to support capacity building and strengthen quality laboratory services at the lower tier levels. two local implementing partners, the governmental agency ghs and a non-government not-for-profit organisation, global health systems solutions (ghss), were engaged by the cdc’s ghana office to implement slmta through cooperative agreements. baseline audits and site selection eighteen public sector laboratories (three national, 12 regional and three district level) were considered initially for enrolment into the slmta programme. ghs conducted a baseline audit of all 18 laboratories using the stepwise laboratory quality improvement process towards accreditation (slipta) checklist, which provides a quantitative measure of adherence to iso 15189 requirements. the scored checklist quantifies a laboratory’s quality status using a zeroto five-star rating: 0–141 points (< 55%) = zero stars, 142–166 points (55% – 64%) = one star, 167–192 points (65% – 74%) = two stars, 193–218 points (75% – 84%) = three stars, 219–243 points (85% – 94%) = four stars and 244–258 points (95% – 100%) = five stars.11fifteen of the 18 audited laboratories were selected to enrol in the slmta programme based on several factors: their baseline scores; infrastructural availability and suitability; geographical distribution; staffing; and management and staff willingness to participate. the 15 laboratories comprised two national-level laboratories, 12 regional-level laboratories and one district-level laboratory (table 1). geographically, these laboratories cover all 10 regions in ghana (figure 1). these laboratories were grouped further into three cohorts: cohort 1 comprised four laboratories; cohort 2, five laboratories; and cohort 3, six laboratories. table 1: level and function of laboratories in the slmta programme. figure 1: geographic location of laboratories involved in slmta implementation in ghana, 2011–2013. development of a local mentorship programme in october 2011, ghss, working with ghs and other stakeholders, initiated a local mentorship programme, training and placing full-time local mentors at each of the selected laboratories. potential mentors were recruited through newspaper advertisements in an open competitive process; minimum qualifications included a degree in medical laboratory technology or equivalent and previous experience in working with ghs. their training included classroom-based lectures on topics such as qms and the 12 quality system essentials (qses); iso 15189 requirements; conducting audits using the slipta checklist; and project management. trainee mentors then spent one to three weeks with ghss senior mentors during a field practicum before they were sent to their post. mentors were closely supervised by ghss, ghs and cdc by means of a weekly and monthly reporting systems. slmta programme implementation the first cohort of laboratories implemented slmta from april 2011 to april 2012, the second from may 2012 to may 2013 and the third from february 2013 to november 2013. four staff members from each enrolled laboratory, namely, the laboratory manager, quality manager and two other staff members, participated in the three slmta workshops which were coordinated by ghss and ghs. laboratories selected their own participants, with assistance from their upper management, by following the above-mentioned selection criteria. slmta implementation in the three cohorts followed the prescribed slmta process,12 with the three workshops taught by qualified slmta facilitators from ghs and ghss, implementation of improvement projects after each workshop and quarterly site visits to monitor progress, track quality indicators and provide technical assistance. participants also attended complementary training on iso 15189 and internal audit conducted by the ghana standards authority (gsa). site visits were conducted using the slmta site visit monitoring tool by either ghss alone or by a combined technical team from ghs, cdc’s ghana office and ghss. written reports detailing observations, technical assistance provided and recommendations were delivered to the laboratory, the regional directors of health and hospital directors in order to ensure that management within each region were well informed regarding the programme and the progress made by their laboratories. programme monitoring and evaluation intermediate audits were conducted semi-annually in order to measure the progress made by the laboratories whilst helping partners to review their work plan, implementation approach and the mentorship programme. the audits were conducted by trained in-country auditors using the slipta checklist and results were communicated to the participating laboratories so as to guide corrective actions. mentors who were cross-trained as auditors did not conduct audits in laboratories that they mentored. an exit audit was conducted for each laboratory in the three cohorts at the end of the slmta training. follow-up audits were conducted six months after the exit audits in order to monitor the performance of the laboratories and to ensure that recommendations from the exit audits were addressed. one laboratory from cohort 3 (l12) did not receive an intermediate audit because of delayed communication to the site. another laboratory in cohort 3 (l15) did not receive an exit audit because of its high scores at baseline and intermediate audits; in november 2013, this laboratory and the 3 highest-performing laboratories from cohort 1 underwent official slipta audits by the african society for laboratory medicine (aslm).additional indicators, such as specimen rejection rates and patient satisfaction, were also tracked in cohort 1 laboratories so as to assess the progress and impact of implementation. specimen rejection rates were calculated as a percentage of samples rejected as a result of non-conformity to specimen acceptance criteria. because of a lack of data on the specimen rejection rate in ghanaian laboratories prior to the implementation of slmta, specimen rejection rates were only monitored after baseline, during slmta implementation and thereafter for three years. patient satisfaction was measured using questionnaires given to patients and comments received from patients through the suggestion box. the cost in us dollars for the implementation of slmta was reported by the implementing partners. estimated costs included mentor salaries, slmta training and improvement project support. salaries of mentors were determined by the implementing partners, in accordance with ghana’s labour laws. slmta training costs were calculated based on four personnel from each laboratory and two trainers participating in the three five-day workshops. costs included per diem, local transportation to the training venue, training materials for all participants and the workshop venue package. workshop trainers were staff members from the implementing partner; their salaries were considered to be an in-kind contribution and were not included in the estimates. similarly, salary and time missed from work for participants were not included. expenditures sustained by the partner in order to support laboratory improvement projects, such as colour-coded bin liners, emergency eye-wash kits and iso training for internal auditors, were also estimated. preparation for slmta expansion in april 2013, a laboratory auditors training course was organised by the clinical and laboratory standards institute (clsi) and aslm for selected professionals with experience in qms, iso 15189 and slmta. the training programme equipped the participants to plan, prepare and conduct independent laboratory quality audits based on the slipta checklist and iso 15189 requirements. the training format consisted of classroom didactic presentations and a field practicum through mock audits.in september 2013, a slmta training-of-trainers (tot) workshop was conducted in order to increase the pool of local trainers and implementers for nationwide slmta scale-up. this workshop was led by two slmta master trainers from ghana and one from nigeria, all previously trained to lead tot workshops.13 local laboratory professionals who had implemented slmta were selected for the training. data entry and analysis the results of the audits were entered into a microsoft® excel spreadsheet (microsoft® 2010). statistical analysis was done using stata se 12.1 (stanford university it services 2012) after the data had been cleaned and exported. continuous variables such as slipta scores were summarised and presented as medians. percentage scores were determined by dividing the respective scores by the maximum possible points and expressing the results as a percentage. results top ↑ measuring the impact of slmta laboratories in all three cohorts demonstrated a steady improvement in the implementation of qms, as was illustrated by the median slipta scores at each audit (figure 2). median improvements from baseline to exit were 23 percentage points for cohort 1, 29 percentage points for cohort 2 and 20 percentage points for cohort 3. the most improved laboratory (l5) demonstrated an increase from 2% at baseline to 50% at exit, which increased further to 59% at the follow-up audit six months later. laboratory 2, on the other hand, scored 38% at baseline and 46% at exit, but decreased to 36% at the follow-up audit (figure 3). at baseline, only one of the 15 laboratories was at the one-star level. by the exit audit, three laboratories had reached at least one star; of the eight laboratories that conducted follow-up audits, seven had reached at least one star. figure 2: median slipta scores for the three ghana slmta cohorts. each of the 12 qses improved from baseline to exit. the most improved areas were process control and internal and external quality assessment (54%); customer service (50%); organisation and personnel (48%); and documents and records (44%) (figure 4). information management showed the least improvement (9%), followed by purchasing and inventory (18%) and internal audit (20%). the areas with the lowest median exit audit scores were internal audit (20%), occurrence management (25%), corrective action (33%) and management reviews (35%). figure 4: median performance of all 15 ghana slmta laboratories across the 12 quality systems essentials, as measured by the slipta checklist at the baseline and exit audits. the three highest-performing laboratories at exit audit (l1, l3 and l4, all from cohort 1), plus l15 from cohort 3, which did not receive an exit audit but earned three stars at both the baseline and intermediate audits, underwent official slipta audits by aslm. official slipta audit results overall were slightly higher than exit audit results (figure 3). three of these laboratories (l1, l3 and l15) earned four official slipta stars and one (l4) earned one star. figure 3: performance of 15 ghana slmta laboratories at baseline, intermediate, exit, follow-up and official slipta audit. average specimen rejection rates across the four slmta laboratories in cohort 1 decreased from 32% (range 23% – 44%) in 2011 to 25% (range 12% – 35%) in 2012 and 10% (range 3%–12%) in 2013. patient satisfaction increased from 25% to 70% over the same time period (figure 5). figure 5: specimen rejection and customer satisfaction trends for slmta cohort 1 laboratories. local capacity building and cost of slmta implementation the ghana slmta team presently comprises 18 slmta trainers, 15 mentors, 11 clsi/aslm trained auditors and two master trainers. this team has been responsible for the successful implementation of three cohorts of slmta with a total of 15 laboratories, training 60 laboratory professionals in qms. to date, two senior mentors and six trainers have completed phase one of the slipta auditor training conducted by aslm and clsi.the cost for slmta implementation for each laboratory was incurred in the areas of project management, slmta trainings and embedded mentorship. taken together, the implementing partners reported a total expenditure of $600 000 to implement slmta in the 15 laboratories. on average, $40 000 total was spent per laboratory to cover mentors’ salaries ($24 000), slmta training ($6000) and improvement project support ($10 000). discussion top ↑ the ghana national laboratory strategic plan prioritises the development of local capacity as a sustainable way to support the delivery of quality results for improved patient care and treatment. in 2009, ghana adopted slmta using limited funding and only local human resources to drive the programme forward. results have been remarkable in the 15 laboratories enrolled in slmta – audit scores doubled from baseline to exit and more than half of those laboratories reached one or more stars, including three laboratories that have achieved four stars. the success of the ghana slmta programme can be attributed to three strong factors: management engagement and commitment, the use of local partners and implementation of a local mentorship programme.management engagement and commitment have been shown to be critical elements in promoting quality laboratory services.14 in ghana, management was engaged at three levels: central, regional and facility. in june 2012, shortly after completion of the first slmta cohort, ghs organised a one-day meeting, chaired by the director general of ghs, to sensitise regional health directors, hospital medical directors and laboratory managers from all 10 regions of the country on the slmta programme and how it could benefit their facilities. a medical director whose facility had recently completed slmta gave a presentation on how the programme had transformed his facility. this medical director became the ‘slmta ambassador’ amongst his peers. the meeting served as a catalyst in the acceleration of slmta implementation in ghana; afterward, regional directors of health engaged regularly with participating laboratories by means of site visits, courtesy calls and review of audit reports. some medical directors at the facility level even joined in-house slmta trainings, site visits and debriefing activities. others personally took on responsibilities to oversee some improvement projects. this high-level engagement created tremendous enthusiasm within the facilities and contributed to staff morale. with a limited budget, it was important for ghana to incorporate cost-effective and results-oriented approaches into its programme implementation. to achieve the country’s priority of developing sustainable human capacity, local partners were engaged from the beginning, which proved to be advantageous in several respects: (1) they were familiar with the local administration, culture and language; (2) they were well accepted into the facilities as peers; and (3) their service was less expensive than international partners. local partners contributed directly to workforce development through the training and hiring of local human resources. care was taken to ensure that hiring was done through a merit-based process and was well specified in the organisations’ policies and procedures. key components included training, capacity building, salary structure and working conditions for technical, administrative and financial staff. to ensure that programme objectives and targets were met, partners were assisted in adhering to reporting requirements in a timely and consistent manner. mentorship is an important vehicle to establish and solidify qms and to help laboratories achieve their quality improvement goals.15 guidance regarding the implementation of a structured laboratory mentorship programme has been documented.15 similar to the lesotho mentorship approaches,16,17 ghana adopted a full-time, resident mentorship approach. in this approach, each mentor was assigned to a laboratory and resided within the locality where the laboratory was situated. effective mentoring requires full understanding of a laboratory’s culture, processes, procedures and people; we found that this embedded mentorship approach was further enhanced with the placement of indigenous professionals who already understood the in-country laboratory culture. it has been suggested that mentorship visits of four to eight weeks may be more effective than shorter periods.17 in our embedded mentorship approach, mentors stayed at the facility full time for the duration of the programme (18–24 months) and worked with laboratory staff to help raise the laboratory’s level of performance. one positive result of slmta implementation in ghana was improved patient satisfaction in the quality of service delivery and a reduction in the rejection of specimens. these results are consistent with previously-published findings.7 patient satisfaction was improved by the introduction of: customer service managers; suggestion boxes; client satisfaction surveys; and posters which displayed in bold text the cost of tests, expected turnaround time for tests and other vital information to patients. specimen rejection was reduced through three interventions: development of quality manuals that defined clearly the policies, processes and procedures for all the laboratories; development of a clinicians’ handbook that defined in detail to all clinicians, nurses and midwives the specimen acceptance and rejection criteria for the laboratory; and consistent training for both laboratory and non-laboratory staff. despite the improvements, challenges remain in the slmta laboratories. some qses, such as internal audit, occurrence management, corrective actions and management reviews, scored a median of ≤ 35% at the exit audit, indicating that these critical areas are not functioning adequately. these qses are common low-scoring areas, as reported in lesotho16 and other countries.18 in ghana, these deficiencies were largely a result of a lack of internal audit skills, inadequate staffing, limited experience of mentors, inefficient communication channels and institutional bottlenecks, such as administrative and procurement7 processes. as a result of these findings, training on internal audit has been conducted for all laboratory and quality managers, in collaboration with gsa. inadequate staffing levels and lack of motivation amongst some staff members, coupled with increased workloads, may have contributed to delays in the implementation of improvement projects. these issues are not easy to address, although advocacy continues for the prioritisation of laboratory human resource needs and identification of incentive options for overworked staff. many of the mentors used in these three cohorts had been trained recently and this was their first mentoring experience; whilst some settled in quickly, others needed more time to adjust to their new environment and tasks assigned. with time, these mentors have gained tremendous experience and are more effective in assisting laboratories to implement quality systems. once slmta roll-out is complete, these mentors will have skills that will make them valuable assets as quality managers. finally, inefficient communication and institutional bottlenecks remain a challenge; however at some facilities, especially in those where management was engaged in the slmta process, efforts are being made to simplify administrative processes and streamline communication channels. most countries implementing slmta have relied heavily on pepfar funding for implementation support. limited pepfar funding in ghana meant building local capacity that could be sustained and replicated as the slmta program grows. because slmta activities such as training, mentoring, monitoring and auditing are inter-related, we adopted a cross-training approach such that slmta trainers were also trained as mentors and certified as auditors, in an effort to maximise their potential. limitations of the study one limitation of this study was the absence of control laboratories that were followed over the same period of time. as a result we could not compare the improvements with laboratories that were not enrolled in slmta. whilst it is possible that some improvements observed in this programme were a result of secular influences or random factors, the magnitude of the observed impact strongly suggests a positive impact of the slmta programme. recommendations local partners may be considered for in-country programme implementation. however, capacity building of partner staff in administrative, technical and financial areas must be an integral part of the programme to ensure utmost compliance with reporting requirements. conclusion the slmta programme in ghana has shown substantial laboratory improvements as evidenced by progress in 15 laboratories, including four that have been audited officially by aslm. this experience demonstrates that local partners, when supported and managed adequately, can achieve great results at a reasonable cost. our programme also demonstrates the feasibility of indigenous capacity building and sustainability in an era of reduced pepfar funding, as countries are encouraged to do more with less. acknowledgements top ↑ we are grateful to the leadership of the moh, ghs and the national aids/sti control program. we appreciate the assistance of our implementing partner ghss and all participating laboratories. this activity has been supported by pepfar through cdc under the terms of cooperative agreement numbers ps002987 and ps002762. the findings and conclusions in this paper are those of the authors and do not necessarily represent the views of the cdc. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. disclaimer the findings and conclusions in this article are those of the authors and do not necessarily represent the official position of the cdc. authors’ contributions b.n. (cdc, ghana) was the activity manager and headed the writing of the manuscript; b.v.d.p. (ghss, ghana), v.b. (national aids control program), r.a. (national public health reference laboratory) and n.a.k. (ghss, ghana) conducted slmta trainings, mentoring and contributed to the writing of the manuscript; k.y., p.n.f. and e.t.l. (all cdc, atlanta) provided technical assistance and contributed to the writing of the manuscript; s.d. (ghss, ghana), p.a.n. (ghss, cameroon), n.a.a. (national aids control program), f.n.k. (cdc, cote d’ivoire) and c.j.i.w. (cdc, ghana) managed the programme and contributed to the writing of the manuscript. all authors have read and agreed to this manuscript. references top ↑ 1. world health organization’s regional office for africa. the maputo declaration on strengthening of laboratory systems [document on the internet]. c2008 [cited 2013 jan 02]. available from: http://www.who.int/diagnostics_laboratory/maputo-declaration_2008.pdf2. fonjungo pn, kebede y, arneson w, et al. preservice laboratory education strengthening enhances sustainable laboratory workforce in ethiopia. hum resour health. 2013;11:56. http://dx.doi.org/10.1186/1478-4491-11-56 3. world health organization. joint who–cdc conference on health laboratory quality systems. who/hse/ihr/lyo/2008.3 [document on the internet]. c2008 [cited 2013 jan 03]. available from: http://www.who.int/ihr/lyon/report20080409.pdf 4. gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm 5. yao k, luman et, slmta collaborating authors. evidence from 617 laboratories in 47 countries for slmta-driven improvement in quality management systems. afr j lab med. 2014;3(2), art. #262, 11 pages. http://dx.doi.org/10.4102/ajlm.v3i2.262 6. cobbina e, agbezudor jy, amuzu ps, et al. the current status and future of medical laboratory quality regulation and accreditation in ghana. accred qual assur. 2012;17:613–619. http://dx.doi.org/10.1007/s00769-012-0927-x 7. zeh ce, inzaule sc, magero vo, et al. field experience in implementing iso 15189 in kisumu, kenya. am j clin pathol. 2010;134(3):410–418. http://dx.doi.org/10.1309/ajcpzirkdus5lk2d 8. peter tf, rotz pd, blair dh, et al. impact of laboratory accreditation on patient care and the health system. am j clin pathol. 2010;134(4):550–555. http://dx.doi.org/10.1309/ajcph1skq1hnwghf 9. institute of medicine. evaluation of pepfar. washington, dc: the national academies press; 2013. 10. the president’s emergency plan for aids relief. fy 2014 country operational plan guidance. version 2 [document on the internet]. c2013 [cited 2013 dec 15]. available from: http://www.pepfar.gov/documents/organization/217765.pdf 11. world health organization’s regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region (with checklist) [document on the internet]. c2011 [cited 2013 jan]. available from: http://www.afro.who.int/index.php?option=com_docman&task=doc_download&gid=8642&itemid=2593 12. yao k, maruta t, luman et, nkengasong jn. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(2), art. #194, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i2.194 13. maruta t, yao k, ndlovu n, moyo s. training-of-trainers: a strategy to build country capacity for slmta expansion and sustainability. afr j lab med. 2014;3(2), art. #196, 7 pages. http://dx.doi.org/10.4102/ajlm.v3i2.196 14. opio a, wafula w, amone j, et al. country leadership and policy are critical factors for implementing laboratory accreditation in developing countries: a study on uganda. am j clin pathol. 2010;134(3):381–387. http://dx.doi.org/10.1309/ajcp6kmotclisgj3 15. maruta t, rotz p, trevor p. setting up a structured laboratory mentoring programme. afr j lab med. 2013;2(1), art. #77, 7 pages. 16. mothabeng d, maruta t, lebina m, et al. strengthening laboratory management towards accreditation: the lesotho experience. afr j lab med. 2012;1(1), art. #9, 7 pages. 17. maruta t, motebang d, wanyoike j, et al. impact of mentorship on who-afro strengthening laboratory quality improvement process towards accreditation (slipta). afr j lab med. 2012;1(1), art. #6, 8 pages. 18. luman et, yao k, nkengasong jn. a comprehensive review of the slmta literature part 2: measuring success. afr j lab med. 2014;3(2), art. #276, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i2.276 article information authors: thomas gachuki1 risper sewe1 jane mwangi2 david turgeon3 mary garcia4 elizabeth t. luman3 mamo umuro1 affiliations: 1kenya ministry of health, national hiv reference laboratory, kenya2division of global hiv/aids, us centers for disease control and prevention, nairobi, kenya 3division of global hiv/aids, us centers for disease control and prevention, atlanta, georgia, united states 4clinical pathology laboratories, austin, texas, united states correspondence to: thomas gachuki postal address: kenyatta national hospital grounds off ngong road, po box 20750 00202, nairobi, kenya dates: received: 24 july 2014 accepted: 10 sept. 2014 published: 03 nov. 2014 how to cite this article: gachuki t, sewe r, mwangi j, et al. attaining iso 15189 accreditation through slmta: a journey by kenya’s national hiv reference laboratory. afr j lab med. 2014;3(2), art. #216, 9 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.216 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. attaining iso 15189 accreditation through slmta: a journey by kenya’s national hiv reference laboratory in this lessons from the field... open access • abstract • introduction • research method and design    • study site    • slmta process and evaluation    • team formation    • improvement projects    • training    • mentorship • results    • audit scores and accreditation    • improvement projects    • quality indicators and costs • discussion    • conclusion • acknowledgements    • competing interests    • authors’ contributions    • attribution of support    • disclaimer • references abstract top ↑ background: the national hiv reference laboratory (nhrl) serves as kenya’s referral hiv laboratory, offering specialised testing and external quality assessment, as well as operating the national hiv serology proficiency scheme. in 2010, the kenya ministry of health established a goal for nhrl to achieve international accreditation.objectives: this study chronicles the journey that nhrl took in pursuit of accreditation, along with the challenges and lessons learned. methods: nhrl participated in the strengthening laboratory management toward accreditation (slmta) programme from 2010–2011. improvement projects were undertaken to address gaps in the 12 quality system essentials through development of work plans, team formation, training and mentorship of personnel. audits were conducted and the scores used to track progress along a five-star grading scale. standard quality indicators (turn-around time, specimen rejection rates and service interruptions) were measured. costs of improvement projects and accreditation were estimated based on expenditures. results: nhrl scored 45% (zero stars) at baseline in march 2010 and 95% (five stars) after programme completion in october 2011; in 2013 it became the first public health laboratory in kenya to attain iso 15189 accreditation. from 2010–2013, turn-around times decreased by 50% – 95%, specimen rejections decreased by 93% and service interruptions dropped from 15 to zero days. laboratory expenditures associated with achieving accreditation were approximately us $36 500. conclusion: international accreditation is achievable through slmta, even for a laboratory with limited initial quality management systems. key success factors were dedication to a shared goal, leadership commitment, team formation and effective mentorship. countries wishing to achieve accreditation must ensure adequate funding and support. introduction top ↑ the burden of hiv in kenya is high, with 1.6 million people living with the infection as of december 2011, including 621 813 patients who had been placed on antiretroviral therapy (art) by 2010. in order to support diagnostic testing and laboratory monitoring of hiv patients, there is a high demand for quality laboratory services, as 5.7 million hiv tests were performed in 2012 alone.1gershy-damet et al. pointed out that high-quality laboratory testing is critical for patient care, disease prevention and surveillance.2 laboratory test results play a crucial role in medical decision making; and accurate and reliable diagnostic testing and monitoring are critical to the successful management of hiv. in order to ensure the reliability and accuracy of testing, a quality system that addresses all aspects of testing is essential. however, establishing and maintaining high-quality testing standards presents major challenges in resource-poor settings.3 key amongst these challenges is lack of adherence to international standards as a result of inadequate quality management systems (qms)4 that focus on achieving quality testing services. in addition, because most hiv diagnostic testing is done by non-laboratory staff, reference laboratories play a critical role in monitoring field testing.2,5 to ensure quality results at every level, the world health organization (who) recommends that national reference laboratories seek accreditation to international standards.6 in 2003, the kenya ministry of health established the national hiv reference laboratory (nhrl), a public health facility designed to monitor the quality of hiv testing by providing a serology proficiency scheme, conducting external quality assessment (eqa) testing and acting as the centre of excellence in the laboratory monitoring of hiv patients. initially, the nhrl did not have qms in place and was not benchmarking itself against international standards. the quality of analytical testing and services was not validated, limiting its ability and authority to act as a centre of excellence. in 2010, the nhrl adopted the ministry of health’s goal to accredit all national and regional level public laboratories in kenya to the international organization for standardization (iso) 15189 standard, which is specifically designed to encourage medical laboratories to develop a highly disciplined approach to improving the quality of services.7 iso 15189 assesses the competence of the qms within the laboratory,8 provides a framework for increased analytical quality9 and verifies that laboratories are not deviating from quality and competency standards.10 the accreditation journey at the nhrl began in 2009 when laboratory management invited a consultant from a global healthcare public foundation (aghpf) to review the current laboratory qms and provide advice on needed improvements. the findings of this review stirred the management to seek assistance in the development and implementation of a more robust qms. in 2010, nhrl adopted the strengthening laboratory management toward accreditation (slmta) programme and enrolled in kenya’s first cohort along with 12 other laboratories, with the goal of attaining iso 15189 accreditation. this paper chronicles the journey that the nhrl took in the pursuit of international accreditation, along with the challenges and lessons learnt. we show how management commitment, team formation, culture change and mentorship were instrumental in the successful completion of this journey. research method and design top ↑ study site the nhrl is located in the capital city of nairobi and consists of three main sections: serology, molecular, and art monitoring. in addition, there are two cross-cutting sections: logistics, and monitoring and evaluation. each section is managed by a team lead.in its role as an hiv referral laboratory and centre of excellence, the nhrl is responsible for strengthening laboratory systems for hiv diagnosis, care, treatment and surveillance. it provides leadership and support to the national hiv laboratory programme by formulating policy and guidelines on hiv laboratory-related issues and coordinating activities and partners. the nhrl offers reference services in hiv testing and laboratory art monitoring, including hiv viral load testing, early infant diagnosis, cd4 lymphocyte enumeration and the evaluation and monitoring of the quality of hiv testing reagents and equipment. it also provides and coordinates eqa services in hiv testing by running the national hiv serology quality assurance program for over 7000 laboratory and non-laboratory testing personnel. additionally, the nhrl is responsible for eqa programmes in cd4 lymphocyte enumeration, haematology and chemistry. the nhrl also provides support and mentoring to hiv testing and art monitoring personnel, as well as building in-country capacity to design, implement and evaluate hiv-related surveillance systems and surveys. slmta process and evaluation the slmta programme uses the stepwise laboratory quality improvement process towards accreditation (slipta) checklist in order to identify strengths and weaknesses and to measure progress. the slipta checklist provides an evaluation score based on laboratory quality in the 12 quality system essentials (qses). laboratories are assigned a ‘star’ level based on their scores: zero stars (0% – 54%), one star (55% – 64%), two stars (65% – 74%), three stars (75% – 84%), four stars (85% – 94%), and five stars (≥ 95%). laboratories that score five stars are encouraged to pursue iso 15189 accreditation.11a baseline audit was conducted in march 2010 by slmta in-country trainers using the slipta checklist. this was followed by the first slmta workshop in april 2010, then the second workshop in september of the same year and the third workshop in january 2011. an exit audit was conducted by auditors from the kenya accreditation service (kenas) in october 2011. in february 2012, a consultant from the south africa national accreditation service (sanas) performed a pre-accreditation assessment utilising the sanas 15189 checklist in order to determine readiness for accreditation. several quality indicators were monitored weekly, monthly or annually so as to assess the impact of the slmta programme on laboratory service quality and patient care. specimen turnaround times for viral load, enzyme-linked immunosorbent assay (elisa) and cd4 tests were calculated using data from the laboratory information management system (lis). information on service interruptions because of equipment downtime and stockouts was obtained from the lis monthly and averaged over a calendar year. customer satisfaction was estimated from patient feedback forms that were availed either in laboratory reception areas or by mailing to customers. specimen rejections were tallied from the lis. corrective actions and occurrence management were evaluated based on completed corrective action forms and quarterly reports. these were divided into three phases: pre-analytic, analytic and post-analytic. routine results from eqa panel tests for all analytes were collated and performance evaluated using microsoft® excel 2007, by aggregating the score achieved in every eqa challenge and obtaining a percentage score. a score of 100% was desirable, whilst any score below this would call for corrective action. costs of programme implementation were estimated in us dollars based on expenditures made by the laboratory on quality improvement. these costs include fees paid to the accrediting body, kenas, and the cost of various improvements such as access control, safety equipment, equipment service contracts, iso training, eqa enrolment, storage area renovation and electronic temperature-monitoring system. for this analysis, in-kind contributions such as mentorship provided by the us centers for disease control and prevention (cdc) and costs borne by the ministry of health, such as slmta training, were not included. the opportunity cost of staff time to participate in training, complete the improvement projects and prepare for accreditation was also not included. team formation to implement the qms, a strategic, tiered, accreditation team structure with a clear reporting mechanism was formed. the structure included a management team, a quality assurance (qa) team and section teams.the management team was composed of the laboratory manager, deputy laboratory manager/qa manager, section team leads, the safety officer and the logistician. this core group guided the accreditation process and held regular review meetings in order to track progress and monitor the quality indicators adopted by the laboratory. they also reviewed gaps identified in both internal and external audits and formulated plans for continuous quality improvement. the qa team, reporting to the management team, was chaired by the qa manager/deputy laboratory manager and two qa officers (one also serving as the safety officer). this team was responsible for monitoring the accreditation process, offering leadership and coordinating the implementation of various improvement projects. each member of this team was assigned to a section and mentored by the qa manager. section team leads were given authority to make decisions and were ultimately responsible for improvement projects within their section. the section teams held weekly meetings to discuss problems and possible solutions and to track the progress of improvement projects within their section. section team leads reported to the qa team on all critical issues pertaining to qms implementation. annual staff retreats were held at the beginning of each year, during which work plans were developed with clear timelines and action points that incorporated all 12 qses and were based on iso 15189 requirements. team building also took place during the annual staff retreats. these plans were posted on bulletin boards where they were visible to all staff. regular monthly staff meetings were held in order to review work plans and monitor progress of the quality improvement initiative. after every internal and external audit, work plans were modified so as to reflect progress made and to redirect efforts where needed. individual staff members set annual accreditation goals and targets against which they were appraised for their annual staff performance contracts. an employee recognition scheme was put in place and incentives were provided. laboratory management led the way by prioritising accreditation and making sure that all personnel were keenly aware of the accreditation goal; accreditation was the main agenda item in all meetings and took priority in budget considerations, ensuring that resources required for the process were secured. improvement projects improvement projects were undertaken for all 12 qses in order to address the gaps identified in the audits. each member of the nhrl staff was responsible for at least one project with clear timelines. the findings of routine audits were used to make continual improvements within the qms. the laboratory undertook more improvement projects (table 1) than required by kenya’s slmta team, including changes to the design of the laboratory and development of workflow diagrams. the plan–do–check–act cycle was adopted in implementation of the quality improvement projects.12 most importantly, method validation was performed in order to assess the methods and equipment utilised in the laboratory.13 table 1: gaps identified and corresponding improvement projects conducted for slmta implementation at kenya’s national hiv reference laboratory, 2010–2013. all staff members were actively involved in the quality improvement projects. work plans were developed at the beginning of each year and after every audit. the work plans involved establishing a strategic goal and objective, with responsibility and project timeline assigned. work plans were reviewed regularly in staff meetings and were located centrally in the laboratory for easy reference. the work plans served as valuable tools for setting realistic targets, measuring progress and enforcing individual responsibility, leading to a focused implementation of improvement projects. flow diagrams were developed to assist in identifying weak areas and making necessary improvements. training all laboratory personnel were trained on the iso 15189 standard by management sciences for health and on good laboratory practice by the kenya aids vaccine initiative. staff also underwent 14 days of mentorship training in three of kenya’s internationally-accredited research laboratories: the kenya medical research institute (kemri)/cdc hiv research laboratory in kisumu, the us army walter reed laboratory in kericho and the academic model for treatment laboratory in eldoret. the qa manager also attended internal audit training conducted by sanas. mentorship two mentors from cdc’s international laboratory branch, division of global hiv/aids in atlanta spent a total of eight weeks in the nhrl during the slmta process. an initial three-week visit was made in january 2011 following the second slmta workshop. to make effective use of the mentors’ time on site, a brief report was prepared by the nhrl in advance of the first visit and shared with the mentors, including information on test methods and equipment used in the nhrl. at the beginning of the visit an internal audit was performed and a work plan developed based on the findings, in collaboration with the qa team and individual members of the various laboratory sections. at the end of the visit another audit was performed and the entire team participated in development of another work plan for outstanding issues.long-distance support then followed via email for a six-month period. an additional two-week visit was made by one of the mentors, who is also a member, inspector and team lead for the college of american pathologists. a final three-week visit was made by both mentors in january 2012, three months after the exit audit, in order to prepare the laboratory for the iso accreditation pre-assessment. results top ↑ audit scores and accreditation at the baseline audit in march 2010 before slmta implementation, nhrl scored 45%, corresponding to zero stars. at the october 2011 exit audit, the laboratory more than doubled their score to 95%, earning five stars. in march 2013, three years after initiation of slmta, the nhrl achieved accreditation to iso 15189. improvement projects gaps were identified in all 12 qses after the baseline audit. improvement projects were undertaken to address these problems (table 1). some projects were one-time activities, such as development of policies and procurement; for example a policy on environmental control was developed and room thermometers were procured. other projects implemented more comprehensive on-going changes to laboratory procedures, such as quarterly analysis of occurrence management and keeping minutes at staff meetings. all the improvement projects that were undertaken were completed by the time the laboratory attained accreditation. quality indicators and costs average turn-around time for viral load testing decreased from 20 days in 2010 to six days in 2013 (70%). similarly, elisa turn-around time decreased from 191 days to 10 days (95%). cd4 turn-around time decreased from 24 hours to 12 hours (50%). the number of rejected specimens decreased from 133 in 2010 to nine in 2013 (93%) and the number of service interruption days decreased from 15 to zero (100%) (table 2). table 2: trends in quality indicators, kenya’s national hiv reference laboratory, 2010–2013. the cost to the laboratory to conduct slmta improvement projects and to continue through to iso 15189 accreditation was us$36 500 (table 3). table 3: expenditures by kenya’s national hiv reference laboratory to achieve iso 15189 accreditation discussion top ↑ the nhrl was successful in achieving accreditation to iso 15189 in march 2013, three years after beginning the quality improvement process. high-quality laboratory testing is critical for patient care, disease prevention and disease surveillance.5 although the majority of laboratory testing is done by public laboratories, no laboratory in the public sector had been accredited previously in kenya, as all eight accredited laboratories were private or research laboratories. in fact, in all of sub-saharan africa except south africa, only two public laboratories had been accredited previously to international standards: one in namibia and one in botswana.14the success of nhrl was a result of several factors. firstly, the team was built with a shared vision, all striving to meet iso 15189 requirements. collective involvement has been shown elsewhere to be important in implementing change.15,16 the slmta trainees shared their projects with all staff, who then took up responsibility; this helped to prevent the mentality that quality improvement was ‘someone else’s job’ and ensured shared ownership of the process. in the weekly section meetings, brainstorming led to development of local solutions and sharing of best practices, ensuring there was no slackening of momentum. these meetings also enhanced the cohesiveness of the entire nhrl staff team. secondly, the old adage is true: what gets measured, gets done. slipta scores and star levels provided a framework for identifying strengths and weaknesses and quantifying progress. the baseline audit offered an objective analysis of processes in the laboratory, revealed critical gaps in the system and guided the team in initiating a gradual process of preparedness for accreditation. the exit audit documented how far the laboratory had come, giving leadership and staff the motivation to continue improving and the confidence to seek international accreditation. thirdly, the slmta programme provided nhrl staff the training needed to make qms improvements quickly and to prepare for accreditation. the laboratory used slmta improvement projects as a springboard to implement additional projects with a wider scope in order to cover all aspects of the qms. changes to the design of the laboratory and workflow diagrams allowed efficient and logical flow of work processes. improvement in testing turn-around time was achieved by preventing service disruptions, ensuring uninterrupted reagent supply, establishing equipment service contracts and creating a back-up programme. fourthly, mentorship was key in helping the laboratory customise solutions. effective mentorship has been shown to be a success factor in the implementation of slmta in various settings.16,17 the two cdc mentors not only spent periods of time in the facility but also offered guidance and assistance remotely. the intense preparation conducted by laboratory staff before the visits enhanced focus and sustainability when the mentors left. the mentors did not perform tasks, but instead guided laboratory staff to do them, fostering ownership and building capacity. contact was maintained with mentors after they left, ensuring continuity. the mentees identified high-priority areas in which they required assistance, saving time onsite. a positive staff attitude facilitated the productive relationships with mentors; no time was wasted in finding common ground because all shared the same goal of nhrl accreditation. finally, continued focus on accreditation after slmta allowed the laboratory to reach even higher levels. the pre-accreditation assessment conducted by the sanas assessor offered an objective in-depth analysis using a different checklist and gave laboratory staff an idea of what to expect in the accreditation visit. findings from this assessment were used to address remaining gaps prior to the official inspection. nhrl faced many critical challenges in implementing qms, as summarised in table 4. one serious problem that remains unsolved is staff attrition. because the government handles staff deployment, trained staff members are often transferred to other laboratories. nhrl is working with the ministry of health to prioritise continuity of staff and training for new staff members in order to sustain quality levels. the nhrl spent approximately us$36 500 in pursuit of iso 15189 accreditation, in addition to that spent by the ministry of health on slmta training and by partners for mentorship and additional training. one of the largest expenses was the placement of equipment on service contracts. to reduce costs, the laboratory adopted the equipment placement model, whereby an equipment manufacturer places equipment in a laboratory at no cost, recovering their expenses by selling reagents to the laboratory. other substantial expenses included the renovation of a storage room to overcome space shortages and installation of a temperature-monitoring system in order to improve the archiving of specimens. the largest single expense was the purchase of a back-up generator; this purchase also benefited other users within the national public health laboratories complex. many key components of the programme were paid for by various partners and were thus not included in the cost estimate. for example, iso training was sponsored by management sciences for health and included staff from other laboratories. personnel were immunised by the division of vaccination in the ministry of health. finally, the aghpf consultant and cdc mentors, critical for readying the laboratory for accreditation, were sponsored by their respective organisations. cost considerations must be weighed against the benefits of quality improvement. some improvements will result in large cost savings over time. human resource management has been made easier as staff competencies are assessed annually; personnel are now more efficiently assigned to specific responsibilities based on their core competencies. the process of hiv results confirmation, which used to take more than one month, now takes less than 10 days, ensuring rapid resolution nationwide for clients with discrepant hiv results. hiv viral load results are now received in less than 10 days; this information is critical with regard to alerting clinicians to the need to change treatment regimens for patients with treatment failure, thus reducing their likelihood of developing drug resistance. services are no longer interrupted because of reagent shortages or equipment downtime and adherence to sample handling guidelines has greatly reduced rejected samples, decreasing both costs and wastage.18 accreditation also provides immeasurable benefits in enabling the nhrl to fulfil its mission as the country’s reference laboratory for hiv testing. it has accorded the nhrl international recognition and elevated customer confidence with respect to the reliability of services as they fulfil their mandate. pursuit of accreditation has led to significant improvement in the quality of both analytical test results and customer service. because of the central role the laboratory plays in kenya, these benefits have a direct impact on the quality of hiv testing and monitoring throughout the country. conclusion the experience of kenya’s nhrl shows that it is feasible to attain international accreditation through the implementation of the slmta programme, even in settings with poor resources and laboratories without initial systems. acknowledgements top ↑ the authors wish to thank the staff and management of the nhrl, kenya for their efforts leading to the first accreditation of a public laboratory in kenya. we would also like to thank the cdc-kenya and cdc-atlanta offices for their support and technical assistance. we would also like acknowledge the assistance provided by the division of vaccine (ministry of health, kenya) and the management sciences for health. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions t.g. (nhrl) led the slmta programme and accreditation process at nhrl and wrote the manuscript. r.s. (nhrl) provided assistance with the accreditation process and reviewed the manuscript. j.m. (international laboratory branch, cdc, kenya) supported slmta implementation and reviewed the manuscript. d.t. (international laboratory branch, cdc, atlanta) supported implementation of the qms and reviewed the manuscript. m.g. (clinical pathology laboratories) supported implementation of the qms and reviewed the manuscript. e.t.l. (international laboratory branch, cdc, atlanta) assisted with analysis and interpretation of the data and substantially revised the manuscript. m.u. (nhrl) led implementation of the qms and reviewed the manuscript. attribution of support this publication was made possible by support from the u.s. president's emergency plan for aids relief (pepfar) through cooperative agreement 5u19 gh000069-02 from cdc, division of global hiv/aids. disclaimer the findings and conclusions in this article are those of the author(s) and do not necessarily represent the official position of the cdc or the government of kenya. references top ↑ 1.national aids control council. the kenya aids epidemic: update 2012 [document on the internet]. c2012 [cited 2014 sep 15]. available from: http://nascop.or.ke/library/3d/final%20kenya%20update%202012,%2030%20may.pdf2.gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm 3.petti ca, polage cr, quinn tc, et al. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377–382. http://dx.doi.org/10.1086/499363 4.zeh ce, inzaule sc, magero vo, et al. field experience in implementing iso 15189 in kisumu, kenya. am j clin pathol. 2010;134(3):410–418. http://dx.doi.org/10.1309/ajcpzirkdus5lk2d 5.bates i, maitland k. are laboratory services coming of age in sub-saharan africa? [editorial] clin infect dis. 2006;42(3):383–384. http://dx.doi.org/10.1086/499368 6.world health organization. joint who – cdc conference on health laboratory quality systems, lyon, france, 9 – 11 april 2008. who/hse/ihr/lyo/2008.3 [document on the internet]. c2008 [cited 2014 sep 15]. available from: http://www.who.int/csr/ihr/lyon/report20080409.pdf?ua=1 7.international organization for standardization. iso 15189:2007: medical laboratories – particular requirements for quality and competence. 2nd ed. geneva, switzerland: international organization for standardization, 2007. 8.international laboratory accrediation cooperation. guidance for the implementation of a medical laboratory accreditation system. ilac-g26:07/2012 [document on the internet]. c2012 [cited 2014 sep 15]. available from: http://www.ats.rs/sites/default/files/download/ilac_g26_07_2012.pdf 9.peter tf, rotz pd, blair dh, et al. impact of laboratory accreditation on patient care and the health system. am j clin pathol. 2010;134(4):550–555. http://dx.doi.org/10.1309/ajcph1skq1hnwghf 10.rabinovitch a. the college of american pathologists laboratory accreditation program. accred qual assur. 2002;7:473–476. http://dx.doi.org/10.1007/s00769-002-0537-0 11.world health organization regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region (with checklist) [document on the internet]. c2011 [cited 2013 may 31]. available from: http://www.afro.who.int/index.php?option=com_docman&task=doc_download&gid=8642&itemid=2593 12.woodcock s, fine g, mcclure k, et al. the role of standards and training in preparing for accreditation. am j clin pathol. 2010;134(3):388–392. http://dx.doi.org/10.1309/ajcp03tfpbkeyynt 13.chan cc. principles and practices of analytical method validation: validation of analytical methods is time‐consuming but essential. qual assur j. 2011;14(3–4):61–64. http://dx.doi.org/10.1002/qaj.477 14.schroeder lf, amukele t. medical laboratories in sub-saharan africa that meet international quality standards. am j clin pathol. 2014;141(6):791–795. http://dx.doi.org/10.1309/ajcpq5ktkagsscfn 15.mcalearney as, terris d, hardacre j, et al. organizational coherence in health care organizations: conceptual guidance to facilitate quality improvement and organizational change. qual manag health care. 2013;22(2):86–99. http://dx.doi.org/10.1097/qmh.0b013e31828bc37d 16.audu ra, onubogu cc, nwokoye nn, et. al. improving quality in national reference laboratories: the role of slmta and mentorship. afr j lab med 2014. in press. afr j lab med. 2014;3(2), art. #200, 7 pages. http://dx.doi.org/10.4102/ajlm.v3i2.200 17.makokha ep, mwalili s, basiye f, et al. using institutional mentorship to roll out slmta in kenya. afr j lab med. 2014. in press. 2014;3(2), art, #220, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i2.220 18.carlson ro, amirahmadi f, hernandez js. a primer on the cost of quality for improvement of laboratory and pathology specimen processes. am j clin pathol. 2012;138(3):347–354. http://dx.doi.org/10.1309/ajcpsmqyaf6x1hut article information authors: edwin shumba1 phoebe nzombe1 absolom mbinda1 raiva simbi2 douglas mangwanya2 peter h. kilmarx3 elizabeth t. luman4 sibongile n. zimuto1 affiliations: 1zimbabwe national quality assurance programme (zinqap) trust, zimbabwe 2ministry of health and child welfare (mohcw), zimbabwe 3us centers for disease control and prevention (cdc), zimbabwe 4us centers for disease control and prevention (cdc), united states correspondence to: edwin shumba postal address: po box a1955, avondale, harare, zimbabwe dates: received: 09 sept. 2014 accepted: 13 sept. 2014 published: 03 oct. 2014 how to cite this article: shumba e, nzombe p, mbinda a, et al. weighing the costs: implementing the slmta programme in zimbabwe using internal versus external facilitators. afr j lab med. 2014;3(2), art. #248, 6 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.248 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. weighing the costs: implementing the slmta programme in zimbabwe using internal versus external facilitators in this original research... open access • abstract • introduction • research methods and design    • slmta programme    • expenditure analysis    • theoretical estimate of expenditures    • projected financial costs of national expansion • results • discussion    • limitations    • conclusion • acknowledgements    • competing interests    • authors’ contributions    • cdc disclaimer • references abstract top ↑ background: in 2010, the zimbabwe ministry of health and child welfare (mohcw) adopted the strengthening laboratory management toward accreditation (slmta) programme as a tool for laboratory quality systems strengthening. objectives: to evaluate the financial costs of slmta implementation using two models (external facilitators; and internal local or mohcw facilitators) from the perspective of the implementing partner and to estimate resources needed to scale up the programme nationally in all 10 provinces. methods: the average expenditure per laboratory was calculated based on accounting records; calculations included implementing partner expenses but excluded in-kind contributions and salaries of local facilitators and trainees. we also estimated theoretical financial costs, keeping all contextual variables constant across the two models. resource needs for future national expansion were estimated based on a two-phase implementation plan, in which 12 laboratories in each of five provinces would implement slmta per phase; for the internal facilitator model, 20 facilitators would be trained at the beginning of each phase. results: the average expenditure to implement slmta in 11 laboratories using external facilitators was approximately us$5800 per laboratory; expenditure in 19 laboratories using internal facilitators was approximately $6000 per laboratory. the theoretical financial cost of implementing a 12-laboratory slmta cohort keeping all contextual variables constant would be approximately $58 000 using external facilitators; or $15 000 using internal facilitators, plus $86 000 to train 20 facilitators. the financial cost for subsequent slmta cohorts using the previously-trained internal facilitators would be approximately $15 000, yielding a break-even point of 2 cohorts, at $116 000 for either model. estimated resources required for national implementation in 120 laboratories would therefore be $580 000 using external facilitators ($58 000 per province) and $322 000 using internal facilitators ($86 000 for facilitator training in each of two phases plus $15 000 for slmta implementation in each province). conclusion: investing in training of internal facilitators will result in substantial savings over the scale-up of the programme. our study provides information to assist policy makers to develop strategic plans for investing in laboratory strengthening. introduction top ↑ public health laboratories play a central role in disease detection, prevention and control. high-quality diagnostics and monitoring are critical to ensure better patient outcomes. in response, developing countries have started paying more attention to efforts to expand laboratory capacity, strengthen laboratory systems and improve laboratory quality.1 the strengthening laboratory management toward accreditation (slmta) programme was launched in 20092and has since been implemented in 47 countries in africa, southeast asia, the caribbean and latin america.3 the programme has demonstrated measurable improvement in laboratories as a result of an approach that incorporates improvement projects and structured supervisory visits, allowing extension of learning beyond the classroom.4 zimbabwe is a resource-limited country with an annual per capita health expenditure of us$16,5 well below the world health organization’s recommended minimum of $34.6 inadequate numbers of well-trained laboratory staff and lack of training on laboratory quality management systems are major challenges facing the laboratory system in zimbabwe.1,7 to address these gaps, the us president’s emergency plan for aids relief (pepfar), through the us centers for disease control and prevention (cdc), in 2010 made available a five-year grant to the zimbabwe ministry of health and child welfare (mohcw). this funding was earmarked for strengthening laboratory systems through a cooperative agreement with the zimbabwe national quality assurance program trust (zinqap), a local not-for-profit organisation which focuses on laboratory quality assurance issues in zimbabwe. in 2010, the mohcw’s department of laboratory services embarked on a five-year strategic plan, one objective of which was to implement quality management systems.8 the laboratory services directorate adopted slmta as a tool for laboratory quality systems strengthening and engaged zinqap as the programme provider. evaluation of financial costs, or expenditures, of implementing the slmta programme is essential in order to guide policy makers and prioritise scarce resources.9 cost analysis is a critical tool in understanding the value of programmes and projecting budgets for scale up.10 the aim of this study is to provide a partial financial expenditure analysis of the slmta programme in zimbabwe from the programme provider’s perspective, with a focus on expenditure comparison between using external (international) versus internal (country-based mohcw) facilitators. these two models have substantial upfront and ongoing cost implications and zimbabwe has used both in its slmta implementation and expansion. we evaluate the financial costs of slmta implementation using both external and internal facilitators and project the financial costs of scaling up the programme nationally. research methods and design top ↑ slmta programme details of slmta programme implementation have been described elsewhere.2 to date, the slmta programme in zimbabwe has been implemented in three cohorts; only cohorts i (implemented in 2010–2011) and iii (implemented in 2012–2013) are included in this analysis. cohort ii was conducted using a different model and detailed cost data were not available. the slmta programme in zimbabwe included four components: (1) baseline audits conducted by zinqap staff; (2) three slmta workshops of four days each; (3) supervision of the implementation of improvement projects following each workshop; and (4) exit audits. mentorship, which is often incorporated into slmta implementation, was not conducted for these two cohorts during the training because of financial constraints and availability of staff.two implementation models were used – one with external workshop facilitators and auditors; and the other with internal workshop facilitators and auditors following extensive training. for cohort i, 11 laboratories were trained using an external facilitator based in uganda, along with three local facilitators. the exit audit was conducted by three external auditors from uganda, lesotho and botswana. cohort iii used four internal facilitators who were trained in a 13-day training-of-trainers course followed by a six-day auditor training. the exit audit was also conducted by these internal facilitators. this cohort included 19 laboratories (13 in zimbabwe and six in namibia). expenditure analysis a partial expenditure analysis was conducted of the implementation of the slmta programme, using both external and internal facilitators. we evaluated the expenditures from a programme perspective, including only direct costs borne by zinqap, the programme provider, whilst excluding in-kind contributions and salaries of local facilitators and trainees. expenditures were collected using a detailed inventory (payment vouchers and general ledger) of all resources associated with the slmta programme and the training of local facilitators. expenditures were tallied for each of the four programme components and then categorised into training equipment, training (facilities and materials), trainers and supervisors (transport; accommodation and per diem; and fees) and participants (transport; accommodation and per diem). for cohort iii, expenditures for supervision and auditing of the six laboratories from namibia were not available. we therefore estimated costs as though they had been in-country, using an inflation factor based on average expenditures for these activities for the zimbabwean laboratories. we also calculated the cost of training local facilitators in training-of-trainers and auditing courses. these expenditures were entered into costit software version 4.5 (world health organization 2007) and analysed in the financial analysis mode. costit is software designed to record and analyse economic and financial data. all expenditures are expressed in us dollars, which has been the official national currency of zimbabwe since 2009. we used a ‘top-down’ cost accounting approach, which starts with total expenditures and then divides by the number of trained individuals and the number of laboratories to yield the average cost per trained individual and average cost per trained laboratory, respectively. theoretical estimate of expenditures expenditures of the two cohorts had considerable inherent variability resulting from contextual factors. for example, the rental cost of the training facility varied from workshop to workshop because of seasonality and fluctuations in the economy. also, for cohort i, zinqap paid per diems to participants, whilst for cohort iii, no participant per diem was paid because of budget constraints. to better compare the two models, we estimated slmta implementation costs based on a theoretical scenario, keeping all contextual variables constant across the two models. contextual variables included facility rental fees, training materials, number of laboratories and participants. expenditure assumptions for this model were based on the expert estimation of zinqap programme implementers, using lessons learned from conducting the two previous cohorts and a normative budgeting approach. projected financial costs of national expansion outputs for the two models based on theoretical financial costs when holding constant contextual variables were used to project the cost of scaling up the slmta programme nationally. we based these estimates on a two-phase implementation plan in which half of the 10 provinces in zimbabwe would implement slmta in each phase. slmta would be rolled out in a series of five cohorts (one per province) of 12 laboratories each, for a total of 60 laboratories per phase. for the internal facilitator model, one facilitator training would be held per phase, training four people per province to conduct the slmta programme locally. time required to implement a phase will depend on resources available; we therefore did not specify a timeframe. results top ↑ the total expenditures for implementation of the slmta programme in 11 laboratories using external facilitators were approximately $64 000 (table 1). the total expenditures in 19 laboratories using internal facilitators were approximately $28 000, plus $84 000 for facilitator training. this yields an average cost per laboratory of approximately $5800 using external facilitators and $5900 using internal facilitators ($3200 and $4700 per person trained, respectively). table 1: partial expenditure estimates for the slmta program in zimbabwe. when keeping all contextual variables constant across the two models, the theoretical financial cost of implementing slmta in 12 laboratories using external facilitators would be approximately $58 000, whilst the theoretical cost of implementing slmta in the same 12 laboratories using internal facilitators would be approximately $15 000, plus $86 000 to train 20 facilitators (table 2). the estimated financial cost to implement subsequent slmta cohorts would remain $58 000 per cohort using external facilitators and would be approximately $15 000 per cohort using the previously-trained internal facilitators, yielding a break-even point of 2 cohorts, at $116 000 for either model. table 2: theoretical cost estimates for the slmta program in zimbabwe. in the external facilitator model, the majority of the financial costs would be spent on slmta workshops (59%) and exit audits (37%) (figure 1a). for the internal facilitator model, the majority of the cost for the first slmta cohort would be spent on conducting the facilitator training (85%), with approximately 12% being spent on conducting workshops. for subsequent cohorts with internal facilitators, the majority of the financial cost would go toward the workshops (83%), with approximately 9% for supervision. figure 1: projected distribution of costs by (a) slmta component, and (b) expenditure category. examining expenditures by expenditure category, the majority of the cost of the external facilitator model would be fees for trainers and supervisors (78%), whilst the remainder would be spent on training (22%) (figure 1b). for the first cohort using the internal facilitator model, financial costs would be divided fairly evenly between participant expenses (44%), training (30%) and trainers and supervisors (22%). in subsequent cohorts using internal facilitators, training costs would account for the majority of expenditures (83%), with trainers and supervisor expenses accounting for the remaining 17%. the projected financial cost of scaling up the slmta programme nationally is shown in figure 2. implementation in 120 laboratories (12 in each of the 10 provinces) would cost approximately $580 000 using the external facilitator model and $322 000 using the internal facilitator model. the initial investment of training 20 internal facilitators ($86 000) will pay for itself by the second slmta cohort, after which the programme will benefit from a cost saving of $43 000 (74%) per cohort for the remainder of the cycle. figure 2: projected cost of scaling up strengthening laboratory management toward accreditation to 10 provinces using external and internal facilitators. discussion top ↑ this is the first detailed expenditure analysis of slmta implementation to be published. we found that programme expenditures to implement slmta were less than $6000 per laboratory. yao et al. have shown that slmta implementation results in substantial improvements in laboratory quality.3 collectively, zimbabwe’s cohort i laboratories conduct more than 1.5 million tests each year and their median audit scores more than doubled from preto post-slmta.3 given the dramatic improvements and relatively low financial cost of slmta implementation found in the current study, it can be argued that slmta is well worth the investment in time and resources.whilst the estimated financial cost to implement a single slmta cohort is lower using external facilitators, the upfront investment of training local facilitators pays for itself by the second cohort, resulting in a 44% cost savings over national scale up of the programme. in addition, training local laboratorians to be slmta facilitators builds long-term indigenous capacity of the laboratory programme, enabling these individuals to conduct in-country laboratory audits, assist laboratory staff in other activities and improve the overall quality of laboratory management throughout the country. in this case, it is critical to minimise staff turnover between the training and slmta implementation so as to ensure a return on investment. some strategies may include an agreement between the mohcw and supervisors not to reassign the trainers for a period of 2 years; financial incentives to the participants upon completion of the slmta programme; and binding contracts in which the participant agrees to remain on the job for a specified period or until the conclusion of slmta implementation in their province. bringing in external facilitators requires high transport costs as well as facilitators’ fees. because we assumed that the internal facilitators would be employed by mohcw and work within their own provinces, these costs would be minimised. we found that the financial costs of slmta implementation are sensitive to participant and trainer travel and per diems. in cohort i, when participants were provided accommodation and per diem, this cost was nearly 40% of the entire financial cost of slmta implementation. hence, decentralising the slmta programme to each province, minimising necessary travel for participants, will reduce costs substantially. a study in cameroon also found that decentralised slmta training enables more laboratory staff to be trained, increasing local capacity and sustainability.11 use of internal facilitators will make decentralisation more feasible, as facilitators can be selected strategically from a wide geographic distribution. slmta is often coupled with on-site mentorship in a variety of models so as to boost improvements even further.12 our analysis did not include mentorship costs, because mentorship was not incorporated in the slmta programme for these two cohorts. bringing in external mentors would increase the cost of the programme substantially; however, given that internal facilitators can receive mentorship training, they could also be used in this capacity to reduce costs and have a positive impact on programme implementation. limitations this study is subject to some limitations. firstly, several expenses associated with slmta implementation, such as overhead costs, vehicles and salaries, were provided in kind and thus not included in this analysis. our results may therefore underestimate the financial cost of implementing slmta in other settings. also not included was the cost of conducting improvement projects within the laboratories, as this was financed by the individual laboratories; however, slmta is designed to focus on management behaviours and participants are encouraged to identify solutions to problems within their existing resources. expenditures were reported as incurred at the time of implementation, not adjusted for inflation or annuitised over years of useful life. because inflation has varied widely over the past decade in zimbabwe,13 we did not attempt to adjust for it, but based estimates on approximate costs in 2013–2014. secondly, there is a level of uncertainty inherent in the assumptions of our theoretical models. however, the resulting financial cost estimates were fairly close to the actual financial costs experienced by the programme, suggesting that they may indeed be realistic. finally, this analysis is not meant to be an exhaustive assessment of all potential models, but rather a comparison of two models that have been used in zimbabwe. additional studies are needed to identify the most cost-effective models for various situations and to conduct an overall economic evaluation of the slmta programme. countries considering the implementation of slmta will need to examine the expected costs of each of the various components of programme implementation, taking into account local parameters and potential support from partners. conclusion our study suggests that national scale up of the slmta programme in zimbabwe would cost approximately $322 000. countries considering slmta implementation should weigh the pros and cons of investing in training of internal staff to act as programme facilitators. in zimbabwe, investing in training of local facilitators will result in a nearly 50% decrease in the costs of national expansion, as well as develop in-country capacity of laboratory managers and mentors, supporting programme sustainability. acknowledgements top ↑ we would like to acknowledge the zinqap finance office staff for their support and sharing data. this research has been supported by pepfar through the cdc. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions e.s. (zinqap) designed the study, collected and analysed data and wrote the manuscript. p.n. (zinqap), r.s.1 (mohcw), d.m. (mohcw), p.h.k. (cdc, zimbabwe) and s.n.z.1 (zinqap) implemented the programme and revised the manuscript. a.m. (zinqap) reviewed the manuscript and assisted in the data analysis. e.t.l. (cdc, united states) designed the study and substantially revised the manuscript. cdc disclaimer the findings and conclusions in this paper are those of the authors and do not necessarily represent the official position of the cdc. references top ↑ 1. nkengasong jn, nsubuga p, nwanyanwu o, et al. laboratory systems and services are critical in global health: time to end the neglect? am j clin pathol. 2010;134(3):368–373. http://dx.doi.org/10.1309/ajcpmpsinq9brmu62. yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3):401–409. 3. yao k, luman et, slmta collaborating authors. evidence from 617 laboratories in 47 countries for slmta-driven improvement in quality management systems. afr j lab med. 2014;3(2), art. #262, 11 pages. http://dx.doi.org/10.4102/ajlm.v3i2.262 4. yao k, maruta t, luman et, nkengasong jn. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(2), art. #194, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i2.194 5. community working group on health in zimbabwe. post health budget analysis 2013. harare; 2013 (unpublished). 6.commission on macroeconomics and health. macroeconomics and health: investing in health for economic development. report of the commission on macroeconomics and health (chaired by jeffrey d. sachs) [document on the internet]. c2001. [cited 2014 sep 26]. available from: http://whqlibdoc.who.int/publications/2001/924154550x.pdf 7. birx d, de souza m, nkengasong jn. laboratory challenges in the scaling up of hiv, tb, and malaria programs: the interaction of health and laboratory systems, clinical research, and service delivery. am j clin pathol. 2009;131(6):849–851. http://dx.doi.org/10.1309/ajcpgh89qdswfons 8. zimbabwe ministry of health and child welfare. laboratory service national strategic plan 2010–2014. harare; 2010 (unpublished). 9. de allegri m, marschall p, flessa s, et al. comparative cost analysis of insecticide-treated net delivery strategies: sales supported by social marketing and free distribution through antenatal care. health policy plan. 2010;25(1):28–38. http://dx.doi.org/10.1093/heapol/czp031 10. guinness l, levine r, weaver m. 10 best resources in … cost analysis for hiv/aids programmes in low and middle income countries. health policy plan. 2004;19(4):242–245. http://dx.doi.org/10.1093/heapol/czh029 11. ndasi j, dimite l, mbome v, et al. decentralised facility-based training as an alternative model for slmta implementation: the cameroon experience. afr j lab med. 2014;3(2), art. #231, 6 pages. http://dx.doi.org/10.4102/ajlm.v3i2.231 12. nzombe p, luman et, shumba e, et al. maximising mentorship: variations in laboratory mentorship models implemented in zimbabwe. afr j lab med. 2014;3(2), art. #241, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i2.241 13. central intelligence agency. the world factbook [page on the internet]. c2013 [cited 2014 aug 31]. available from: https://www.cia.gov/library/publications/the-world-factbook/index.html abstract introduction antiretroviral therapy monitoring low-level viraemia conclusion acknowledgements references about the author(s) nicholus nanyeenya department of epidemiology and biostatistics, school of public health, makerere university college of health sciences, kampala, uganda ministry of health central public health laboratories, kampala, uganda noah kiwanuka department of epidemiology and biostatistics, school of public health, makerere university college of health sciences, kampala, uganda damalie nakanjako department of medicine, school of medicine, makerere university college of health sciences, kampala, uganda gertrude nakigozi rakai health sciences project, masaka, uganda simon p.s. kibira department of community health and behavioral sciences, school of public health, makerere university college of health sciences, kampala, uganda susan nabadda ministry of health central public health laboratories, kampala, uganda charles kiyaga ministry of health central public health laboratories, kampala, uganda isaac sewanyana ministry of health central public health laboratories, kampala, uganda esther nasuuna infectious diseases institute, makerere university college of health sciences, kampala, uganda fredrick makumbi department of epidemiology and biostatistics, school of public health, makerere university college of health sciences, kampala, uganda citation nanyeenya n, kiwanuka n, nakanjako d, et al. low-level viraemia: an emerging concern among people living with hiv in uganda and across sub-saharan africa. afr j lab med. 2022;11(1), a1899. https://doi.org/10.4102/ajlm.v11i1.1899 review article low-level viraemia: an emerging concern among people living with hiv in uganda and across sub-saharan africa nicholus nanyeenya, noah kiwanuka, damalie nakanjako, gertrude nakigozi, simon p.s. kibira, susan nabadda, charles kiyaga, isaac sewanyana, esther nasuuna, fredrick makumbi received: 23 mar. 2022; accepted: 29 june 2022; published: 20 oct. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract attaining viral load (vl) suppression for over 95% of the people living with hiv on antiretroviral therapy is a fundamental step in enabling uganda and other sub-saharan african countries to achieve global sustainable development goal targets to end the hiv/aids epidemic by 2030. in line with the 2013 world health organization recommendations, several sub-saharan african countries, including uganda, use a threshold of 1000 hiv viral rna copies/ml to determine hiv viral non-suppression. the united states centers for disease control and prevention and the international association of providers of aids care deem this threshold very high, and hence recommend using 200 copies/ml to determine viral non-suppression. using 1000 copies/ml as a threshold ignores people living with hiv who have low-level viraemia (llv; hiv vl of at least 50 copies/ml but less than 1000 copies/ml). despite the 2021 world health organization recommendations of using intensive adherence counselling for people living with hiv with llv, several sub-saharan african countries have no interventions to address llv. however, recent studies have associated llv with increased risks of hiv drug resistance, virologic failure and transmission. the purpose of this narrative review is to provide insights on the emerging concern of llv among people living with hiv receiving antiretroviral therapy in sub-saharan africa. the review also provides guidance for uganda and other sub-saharan african countries to implement immediate appropriate interventions like intensive adherence counselling, reducing vl thresholds for non-suppression and conducting more research to manage llv which threatens progress towards ending hiv by 2030. keywords: hiv/aids; low-level viraemia; viral load testing; non-suppression; virologic failure. introduction by the end of 2020, an estimated 37.7 million people were living with hiv/aids; two-thirds (67%) were living in sub-saharan africa, and 27.5 million were accessing antiretroviral therapy (art).1 the increased access to art has necessited the monitoring of its efficacy among people living with hiv, hence the increased scale-up of hiv viral load (vl) monitoring. as the world strives to control the hiv epidemic, attaining vl suppression for over 95% of people living with hiv on art is a fundamental step in enabling uganda, and other sub-saharan african countries, to achieve the global targets of ending the hiv/aids epidemic by 2030, as stipulated in target 3.3 of the sustainable development goal 3.1,2,3 hiv viral non-suppression is defined as hiv rna viral copies equal to or greater than 1000 copies/ml as recommended by the world health organization (who).4 this is also an indication of hiv virologic treatment failure in uganda, and several other sub-saharan african countries like kenya, zambia and sierra leone, among others.5 low-level viraemia (llv) defined as having an hiv vl of at least 50 copies/ml but less than 1000 copies/ml (≥ 50 copies/ml to < 1000 copies/ml) is considered as being virally suppressed. despite the recent 2021 who recommendation to offer intensive adherence counselling (iac) to people living with hiv on art with llv,6 no special intervention is given to people living with hiv with llv in uganda and several other sub-saharan african countries; yet, llv has been associated with various clinically poor outcomes like hiv drug resistance and virologic failure.7,8 this narrative review was completed in january 2022, and the reviewed articles were published from march 2000 to june 2021. several databases including google scholar, pubmed, and science direct were searched using search terms like ‘low-level viraemia’, ‘viral load testing’, ‘non-suppression threshold’ and ‘virologic failure’. this review aims at guiding uganda and other sub-saharan african countries to take immediate appropriate interventions to manage llv. antiretroviral therapy monitoring highly active art has been the springboard in the management and control of hiv/aids over the years; the goal of highly active art is to lead to hiv viral suppression with an increase in the body immunity function.4 globally, access to art has increased, thereby improving the hiv treatment outcomes,1 and this has been a great milestone in achieving epidemic control. in uganda, an estimated 1.2 million (out of 1.4 million) people living with hiv were on art by 2019.9 as the efforts to scale-up people living with hiv on art continue, there is an inevitable need to monitor the efficacy of art among people living with hiv10 and several methods of art monitoring are recommended by who. these include patient monitoring involving clinical events and adherence monitoring, immunological monitoring involving cd4 cell count, and virologic monitoring comprising hiv vl testing.11, 12 virologic monitoring virologic monitoring which comprises hiv vl testing is the preferred way of monitoring treatment outcomes in people living with hiv on art, and who recommends use of a threshold of 1000 copies/ml to determine vl non-suppression, indicative of either poor drug adherence or virologic failure.6 however, the united states centers for disease control and prevention and international association of providers of aids care recommend a threshold of 200 copies/ml for vl non-suppression.13, 14 a number of sub-saharan african countries initiated the scale-up of vl testing from 2014 to 2015, following the who 2013 recommendation.15, 16 uganda began scaling up hiv vl testing for all eligible people living with hiv on art in 2014.16 upon initiation of art, the first vl test is done for people living with hiv at six months, and then at 12 months, after which it is repeated annually for suppressed adults, and every six months for suppressed children and adolescents. people living with hiv with a vl result below 1000 copies/ml have a suppressed vl and are routinely given adherence counselling in which they are encouraged to continue with art, and no other intervention is given.5 a decreased vl is associated with better clinical outcomes and slowed disease progression,17 and is also associated with reduced hiv incidence at community level.17, 18 however, there is limited data about the cut-off of viral copies at which the slowed disease progression and reduced hiv incidence at community level happens. people living with hiv/aids who have been on art for at least six months with a vl of 1000 copies/ml or more are non-suppressed. these people are offered monthly iac sessions for three months. after the iac sessions, a vl test is repeated in the fourth month to determine whether they have achieved vl suppression.5, 6, 12 if the repeat vl result after iac is still non-suppressed, this is considered as virologic failure provided non-adherence to art is ruled out. a switch committee is then convened to discuss switching the patient to another art line.5 several predisposing factors like poor art adherence, unawareness about the art benefits and other existing chronic illness, among others, have been associated with vl non-suppression.19, 20, 21 people living with hiv on art with non-suppressed vl are at increased risk of faster progression to aids, which is also associated with poor clinical outcomes.21, 22 tremendous progress has been achieved by several sub-saharan african countries in establishing comprehensive vl testing programmes23; for instance, in uganda, the number of annual vl tests has steadily increased annually from 16 411 vl tests (2% of people living with hiv on art) in 2014 to 1 332 335 vl tests (95% of people living with hiv on art) in 2020.24 challenges affecting the scale-up of vl testing, like suboptimal sample transportation and results delivery, limited human resource in health facilities, unawareness about vl testing among hiv healthcare providers and people living with hiv, protracted procurement processes, and poor adherence to national and who vl testing guidelines, have limited full coverage of vl testing in the country.23, 25 the other key important issue to note with the introduction of vl testing is llv. low-level viraemia definition and risk factors the who defines llv as a low but detectable vl that is, viraemia (≥ 50 copies/ml to < 1000 copies/ml).26 people living with hiv/aids on art with viraemia ≥ 50 copies/ml to < 1000 copies/ml are virally suppressed, but have llv.5, 27 previous studies have associated llv with baseline cd4 + t cell counts below 200 cells/mm3, art duration longer than 60 months, art regimens comprising of zidovudine, lamivudine and nevirapine (aor: 2.26, p < 0.001) or didanosine-based regimen, and existing subtype b′ infection.27, 28 justification for using a threshold of 1000 copies/ml for viral load non-suppression the use of 1000 copies/ml as a threshold for determining vl non-suppression in sub-saharan african countries including uganda, has generated enormous debate over the years, since it can lead to the accumulation of people living with hiv on art categorised as suppressed vl, but having llv.29 the recommendation by who to use this threshold of 1000 copies/ml30 was based on a public health perspective where dried blood spot samples were used instead of plasma as a specimen type for hiv-1 vl testing, facilitate the decentralisation of specimen collection and can increase access to vl testing in resource-limited settings like uganda due to their cost-effectiveness, as compared to plasma.31 unfortunately, the performance of dried blood spot samples for vl testing is lower, when compared to the gold standard sample type plasma, with a low sensitivity and specificity to detect treatment failure, and favours using a threshold of 1000 copies/ml.29, 32 slowed disease progression has been demonstrated in people living with hiv on art with a vl of less than 1000 copies/ml,17, 33 though the exact threshold at which llv predicts disease progression varies, and remains debatable.34 furthermore, a reduced risk of hiv transmission for people living with hiv on art with vl results less than; 1000 copies/ml,35 1500 copies/ml,36 and less than 1700 copies/ml,37 has been shown. no hiv transmission risks have been demonstrated below 400 copies/ml,38 but the range of viraemia between 400 copies/ml and 1000 copies/ml at which hiv transmission occurs, still remains unclear.39 prevalence and effects sustained vl suppression has been shown to reduce the occurrence of hiv drug resistance among people living with hiv on art and also leads to improved treatment outcomes.40, 41, 42 however new emerging resistance mutations have been demonstrated in people living with hiv on art with llv,7,8 where persistent llv has been associated with increased risk of virologic failure43; which is also associated with increased risk of adverse treatment outcomes.44 for the case of the sub-sahara african region, 19.3% of the participants had llv while 7.8% of the participants had persistent llv in a study, which aimed to evaluate virologic failure and its predictors in four african countries including uganda, kenya, tanzania and nigeria.45 furthermore, 57.5% of participants with persistent llv (plasma hiv rna > 50 copies/ml at two consecutive visits) in this study would later have confirmed virologic failure. however this study did not examine hiv drug resistance, which could be a key driver of virologic failure (failure of people living with hiv with a non-suppressed vl to suppress after three sessions of iac, and poor drug adherence has been ruled out), and also assessment of drug adherence was mainly through self reports which could have been affected by social desirability and recall bias.45 furthermore, in south africa, llv occurred in 23% of people living with hiv on art with an incidence of 11.5 per 100 person-years of follow-up (95% confidence interval: 11.4–11.7), during first-line art. in this study, llv was associated with increased hazards ratios of virological failure and the subsequent switch to second-line art, as compared with a non-dectectable vl of less than 50 copies/ml; and the risk of virological failure was increased more with increased ranges of llv.7 this study did not look at hiv drug resistance testing results in llv, because they were not available. in another south african study looking at hiv viraemia and mother-to-child transmission risk after art initiation in pregnancy in cape town, the risk of early hiv vertical transmission was greatly associated with vl at delivery, with noted risks of 0.25%, 2.0% and 8.5% among women with vl < 50 copies/ml, 50 copies/ml – 1000 copies/ml and > 1000 copies/ml at delivery, respectively (p < 0.001). this implies that women with llv at delivery had eight times the risk of hiv vertical transmission, as compared to women with a non-detectable vl < 50.46 in malawi, a study was conducted that comprised 1274 mothers and described vl suppression among hiv-positive mothers at 4–26 weeks postpartum, the factors associated with vl suppression, and the impact of vl suppression levels on mother-to-child transmission. this study indicated that 8.7% of these hiv-positive mothers had llv and were more likely to be adolescents, who had been on art for less than six months, with suboptimal adherence. llv was associated with 7.0% risk of mother-to-child transmission, as compared to 0.9% risk for mothers with a non-detectable vl.47 elsewhere, hiv drug resistance was detected in about 30.0% of people living with hiv with a vl test having the first episode of llv in british columbia; and these patients had an increased risk of developing virologic failure, compared to those without hiv drug resistance.48 another study indicated that there were 44.0% accumulated resistance mutations in 47 people living with hiv on art with two or more episodes of llv, and surprisingly the median viraemia was 267 copies/ml; and virologic failure followed in 16.0% of these people living with hiv.49 in the united kingdom, 30.0% of people living with hiv with llv acquired at least one drug resistant mutation50; and major resistant mutations were detected in 12.7% of people living with hiv with llv.51 proposed interventions reduced drug adherence has been associated with residual llv,52 and this implies that interventions to enhance treatment adherence could be offered to people living with hiv on art with llv to achieve a non-detectable vl, which is the recommended target of art in various international guidelines.53, 54 in the recent 2021 who consolidated guidelines, iac has been recommended to be offered to people living with hiv with llv,6 though most sub-sahara african countries have not yet started offering this intervention. furthermore, there is no data in uganda and other sub-sahara african countries to determine whether iac can be effective in achieving a non-detectable vl among people living with hiv on art with llv. few sub-sahara african countries like south africa7, 46 and malawi47 have undertaken research to understand llv and its implications, which has created a knowledge gap about the subject, thereby affecting key policy decisions in the region. conclusion there is an increasing frequency of llv among people living with hiv on art in uganda and across other sub-sahara african countries. this llv is associated with increased risks of hiv drug resistance and virologic failure which can affect the efficacy of art, and lead to accelerated hiv disease progression. with the advancing global targets to end the hiv epidemic, reduce transmission rates, and improve the clinical outcomes of the people living with hiv on art, there is an inevitable need to re-assess the use of the threshold of 1000 copies/ml to determine vl non-suppression, and to establish strategies to address the rising proportions of people living with hiv on art with unmanaged llv in the region. more research about the association between llv and drug resistance and virologic failure in sub-sahara africa also needs to be done. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions n.n., n.k., d.n., g.n. and f.m. designed the concept of the article. n.n., e.n. and i.s. drafted the article with guidance and inputs from n.k., d.n., g.n., f.m., s.p.s.k., s.n. and c.k. all of the authors read through the final article and approved it. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors references joint united nations programme on hiv/aids. global hiv & aids statistics – fact sheet [homepage on the internet]. [cited 2021 jun 21]. available from: https://www.unaids.org/en/resources/fact-sheet joint united nations programme on hiv/aids. fast-track ending the aids epidemic by 2030. [homepage on the internet]. [cited 2021 jun 17]. available from: https://www.unaids.org/sites/default/files/media_asset/jc2686_wad2014report_en.pdf united nations sustainable development goals. goal 3: ensure healthy lives and promote well-being for all at all ages [homepage on the internet]. [cited 2021 jun 17]. available from: https://www.un.org/sustainabledevelopment/health/ world health organization. consolidated guidelines on the use of antiretroviral drugs for treating and preventing hiv infection: recommendations for a public health approach. 2nd edition. [homepage on the internet]. world health organization; 2016 [cited 2021 jun 20]. available from: https://apps.who.int/iris/handle/10665/208825 consolidated guidelines for prevention and treatment of hiv in uganda.pdf [homepage on the internet]. [cited 2021 jun 20]. available from: http://library.health.go.ug/publications/hivaids/consolidated-guidelines-prevention-and-treatment-hiv-uganda world health organization. consolidated guidelines on hiv prevention, testing, treatment, service delivery and monitoring: recommendations for a public health approach [homepage on the internet]. [cited 2022 jan 05]. available from: https://www.who.int/publications-detail-redirect/9789240031593 hermans le, moorhouse m, carmona s, et al. effect of hiv-1 low-level viraemia during antiretroviral therapy on treatment outcomes in who-guided south african treatment programmes: a multicentre cohort study. lancet infect dis. 2018;18(2):188–197. https://doi.org/10.1016/s1473-3099(17)30681-3 taiwo b, gallien s, aga e, et al. antiretroviral drug resistance in hiv-1-infected patients experiencing persistent low-level viremia during first-line therapy. j infect dis. 2011;204(4):515–520. https://doi.org/10.1093/infdis/jir353 unaids. uganda. country fact sheet. [homepage on the internet]. [cited 2021 jun 21]. available from: https://www.unaids.org/en/regionscountries/countries/uganda world health organization. antiretroviral therapy for hiv infection in infants and children: towards universal access: recommendations for a public health approach. geneva: world health organization; 2010. paintsil e. monitoring antiretroviral therapy in hiv-infected children in resource-limited countries: a tale of two epidemics. aids res treat. 2010;2011:e280901. https://doi.org/10.1155/2011/280901 antiretroviral therapy for hiv infection in infants and children: towards universal access: recommendations for a public health approach: 2010 revision 7 clinical and laboratory monitoring. geneva: world health organization; 2010. available from: https://www.ncbi.nlm.nih.gov/books/nbk138582/ centers for disease control and prevention. understanding the hiv care continuum. 2019;4. available from: https://www.cdc.gov/hiv/policies/continuum.html international advisory panel on hiv care continuum optimization. iapac guidelines for optimizing the hiv care continuum for adults and adolescents. j int assoc provid aids care. 2015;14(1_suppl):s3–s34. https://doi.org/10.1177/2325957415613442 lecher s, ellenberger d, kim aa, et al. scale-up of hiv viral load monitoring – seven sub-saharan african countries. morb mortal wkly rep. 2015;64(46):1287–1290. https://doi.org/10.15585/mmwr.mm6446a3 uganda aids commission. uga_2017_countryreport.pdf [homepage on the internet]. [cited 2021 jun 20]. available from: https://www.unaids.org/sites/default/files/country/documents/uga_2017_countryreport.pdf thiébaut r, morlat p, jacqmin-gadda h, et al. clinical progression of hiv-1 infection according to the viral response during the first year of antiretroviral treatment. groupe d’epidémiologie du sida en aquitaine (gecsa). aids. 2000;14(8):971–978. https://doi.org/10.1097/00002030-200005260-00008 das m, chu pl, santos gm, et al. decreases in community viral load are accompanied by reductions in new hiv infections in san francisco. plos one. 2010;5(6):e11068. https://doi.org/10.1371/journal.pone.0011068 alave j, paz j, gonzález e, et al. [risk factors associated with virologic failure in hivinfected patients receiving antiretroviral therapy at a public hospital in peru]. rev chilena infectol. 2013;30(1):42–48. https://doi.org/10.4067/s0716-10182013000100006 joseph davey d, abrahams z, feinberg m, et al. factors associated with recent unsuppressed viral load in hiv-1-infected patients in care on first-line antiretroviral therapy in south africa. int j std aids. 2018;29(6):603–610. https://doi.org/10.1177/0956462417748859 nabukeera s, kagaayi j, makumbi fe, mugerwa h, matovu jkb. factors associated with virological non-suppression among hiv-positive children receiving antiretroviral therapy at the joint clinical research centre in lubowa, kampala uganda. plos one. 2021;16(1):e0246140. https://doi.org/10.1177/0956462417748859 world health organization. consolidated guidelines on the use of antiretrovir.pdf [homepage on the internet]. 2016 [cited 2021 jun 22]. available from: https://apps.who.int/iris/bitstream/handle/10665/208825/9789241549684_eng.pdf%20?sequence=1 lecher s, williams j, fonjungo pn, et al. progress with scale-up of hiv viral load monitoring – seven sub-saharan african countries, january 2015 – june 2016. mmwr morb mortal wkly rep. 2016;65(47):1332–1335. https://doi.org/10.15585/mmwr.mm6547a2 uganda viral load. dashboard [homepage on the internet]. [cited 2021 jun 22]. available from: https://vldash.cphluganda.org/ roberts t, cohn j, bonner k, hargreaves s. scale-up of routine viral load testing in resource-poor settings: current and future implementation challenges. clin infect dis. 2016;62(8):1043–1048. https://doi.org/10.1093/cid/ciw001 world health organization. updated recommendations on hiv prevention, infant diagnosis, antiretroviral initiation and monitoring. 2021. available at: https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=&cad=rja&uact=8&ved=2ahukewjn7oluwjb5ahuhecakhcrxbhwqfnoecb0qaq&url=https%3a%2f%2fapps.who.int%2firis%2frest%2fbitstreams%2f1336192%2fretrieve&usg=aovvaw3erchcjcauatpudczjrouf cohen c. low-level viremia in hiv-1 infection: consequences and implications for switching to a new regimen. hiv clin trials. 2009;10(2):116–124. https://doi.org/10.1310/hct1002-116 zhang t, ding h, an m, et al. factors associated with high-risk low-level viremia leading to virologic failure: 16-year retrospective study of a chinese antiretroviral therapy cohort. bmc infect dis. 2020;20(1):147. https://doi.org/10.1186/s12879-020-4837-y ellman tm, alemayehu b, abrams ej, arpadi s, howard aa, el-sadr wm. selecting a viral load threshold for routine monitoring in resource-limited settings: optimizing individual health and population impact. j int aids soc. 2017;20(suppl 7):e25007. https://doi.org/10.1002/jia2.25007 world health organization. who recommendations 2013. available at: https://www.who.int/publications parkin nt. measurement of hiv-1 viral load for drug resistance surveillance using dried blood spots: literature review and modeling of contribution of dna and rna. aids rev. 2014;16(3):160–171. taieb f, hong tt, ho ht, et al. first field evaluation of the optimized ce marked abbott protocol for hiv rna testing on dried blood spot in a routine clinical setting in vietnam. plos one. 2018;13(2):e0191920. https://doi.org/10.1371/journal.pone.0191920 misgina kh, weldu mg, gebremariam th, et al. predictors of mortality among adult people living with hiv/aids on antiretroviral therapy at suhul hospital, tigrai, northern ethiopia: a retrospective follow-up study. j health popul nutr. 2019;38(1):37. https://doi.org/10.1186/s41043-019-0194-0 opportunistic infections project team of the collaboration of observational hiv epidemiological research in europe (cohere), mocroft a, reiss p, et al. is it safe to discontinue primary pneumocystis jiroveci pneumonia prophylaxis in patients with virologically suppressed hiv infection and a cd4 cell count <200 cells/microl? clin infect dis. 2010;51(5):611–619. https://doi.org/10.1086/655761 ioannidis jp, abrams ej, ammann a, et al. perinatal transmission of human immunodeficiency virus type 1 by pregnant women with rna virus loads <1000 copies/ml. j infect dis. 2001;183(4):539–545. https://doi.org/10.1086/318530 quinn tc, wawer mj, sewankambo n, et al. viral load and heterosexual transmission of human immunodeficiency virus type 1. rakai project study group. n engl j med. 2000 mar 30;342(13):921–929. https://doi.org/10.1056/nejm200003303421303 gray rh, wawer mj, brookmeyer r, et al. probability of hiv-1 transmission per coital act in monogamous, heterosexual, hiv-1-discordant couples in rakai, uganda. lancet. 2001;357(9263):1149–1153. https://doi.org/10.1016/s0140-6736(00)04331-2 attia s, egger m, müller m, zwahlen m, low n. sexual transmission of hiv according to viral load and antiretroviral therapy: systematic review and meta-analysis. aids. 2009;23(11):1397–1404. https://doi.org/10.1097/qad.0b013e32832b7dca dieffenbach cw. preventing hiv transmission through antiretroviral treatment-mediated virologic suppression: aspects of an emerging scientific agenda. curr opin hiv aids. 2012;7(2):106–110. https://doi.org/10.1097/coh.0b013e32834f3f13 deeks sg. antiretroviral treatment of hiv infected adults. bmj. 2006;332(7556):1489. https://doi.org/10.1136/bmj.332.7556.1489 egger m, may m, chêne g, et al. prognosis of hiv-1-infected patients starting highly active antiretroviral therapy: a collaborative analysis of prospective studies. lancet. 2002;360(9327):119–129. https://doi.org/10.1016/s0140-6736(02)09411-4 grinsztejn b, hosseinipour mc, ribaudo hj, et al. effects of early versus delayed initiation of antiretroviral treatment on clinical outcomes of hiv-1 infection: results from the phase 3 hptn 052 randomised controlled trial. lancet infect dis. 2014;14(4):281–290. https://doi.org/10.1016/s1473-3099(13)70692-3 gupta-wright a, fielding k, van oosterhout jj, et al. virological failure, hiv-1 drug resistance, and early mortality in adults admitted to hospital in malawi: an observational cohort study. lancet hiv. 2020;7(9):e620–e628. https://doi.org/10.1016/s2352-3018(20)30172-7 ryscavage p, kelly s, li jz, harrigan pr, taiwo b. significance and clinical management of persistent low-level viremia and very-low-level viremia in hiv-1-infected patients. antimicrob agents chemother. 2014;58(7):3585–3598. https://doi.org/10.1128/aac.00076-14 kiweewa f, esber a, musingye e, et al. hiv virologic failure and its predictors among hiv-infected adults on antiretroviral therapy in the african cohort study. plos one. 2019;14(2):e0211344. https://doi.org/10.1371/journal.pone.0211344 myer l, phillips tk, mcintyre ja, et al. hiv viraemia and mother-to-child transmission risk after antiretroviral therapy initiation in pregnancy in cape town, south africa. hiv med. 2017 feb;18(2):80–88. https://doi.org/10.1111/hiv.12397 landes m, van lettow m, nkoma e, et al. low detectable postpartum viral load is associated with hiv transmission in malawi’s prevention of mother-to-child transmission programme [homepage on the internet]. j int aids soc. 2019 [cited 2021 dec 14];22(6):e25290. available from: https://pubmed.ncbi.nlm.nih.gov/31180186/ swenson lc, min je, woods ck, et al. hiv drug resistance detected during low-level viremia is associated with subsequent virologic failure. aids. 2014;28(8):1125–1134. https://doi.org/10.1097/qad.0000000000000203 li jz, gallien s, do td, et al. prevalence and significance of hiv-1 drug resistance mutations among patients on antiretroviral therapy with detectable low-level viremia. antimicrob agents chemother. 2012;56(11):5998–6000. https://doi.org/10.1128/aac.01217-12 delaugerre c, gallien s, flandre p, et al. impact of low-level-viremia on hiv-1 drug-resistance evolution among antiretroviral treated-patients. plos one. 2012;7(5):e36673. https://doi.org/10.1371/journal.pone.0036673 mackie ne, phillips an, kaye s, booth c, geretti am. antiretroviral drug resistance in hiv-1-infected patients with low-level viremia. j infect dis. 2010;201(9):1303–1307. https://doi.org/10.1086/651618 maggiolo f, di filippo e, comi l, et al. reduced adherence to antiretroviral therapy is associated with residual low-level viremia. pragmat obs res. 2017;8:91–97. https://doi.org/10.2147/por.s127974 guidelines for the use of antiretroviral agents in hiv-1-infected adults and adolescents [homepage on the internet]. aids education and training centers national coordinating resource center (aetc ncrc). [cited 2021 jun 25]. available from: https://aidsetc.org/resource/guidelines-use-antiretroviral-agents-hiv-1-infected-adults-and-adolescents european aids clinical society. eacs guidelines [homepage on the internet]. [cited 2021 jun 25]. available from: https://www.eacsociety.org/guidelines/eacs-guidelines/ article information authors: mary n. mataranyika1 landine k. beukes1 affiliations: 1ministry of health and social services, namibia correspondence to: mary mataranyika postal address: private bag 70046, khomasdal, windhoek, namibia dates: received: 14 may 2014 accepted: 15 sept. 2014 published: 03 nov. 2014 how to cite this article: mataranyika mn, beukes lk. view from the top: involvement of namibia’s health ministry in laboratory quality improvement. afr j lab med. 2014;3(2), art. #195, 2 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.195 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. view from the top: involvement of namibia’s health ministry in laboratory quality improvement in this commentary... open access • introduction • implementing laboratory quality management in namibia    • lessons learned    • sustainability • acknowledgements    • competing interests    • authors’ contributions • references introduction top ↑ laboratories form the backbone of health systems, providing critical test results that aid diagnoses and identify the causes of disease outbreaks. accurate laboratory results improve medical interventions, minimise surgical and treatment errors, decrease the length of hospital stays and reduce the cost of hospitalisation.1 weak reporting systems and delayed or inaccurate processing of test results contribute to increased morbidity and mortality from major diseases of public health concern, such as tuberculosis, malaria, cholera and hiv.2in 2012, namibia’s ministry of health and social services (mohss), working with stakeholders from both the private and public sectors, developed namibia’s first national public health laboratory policy3 and strategic plan4 (approved by the cabinet and launched in 2013) to guide the delivery of quality laboratory improvements and services. according to the laboratory policy, the mohss is mandated to offer high-quality laboratory services; support clinical care providers in disease treatment and prevention; provide disease surveillance and response; and develop policies that adhere to the national health principles of service equity, accessibility, affordability and sustainability.3,4 in order to address issues of poor reporting systems and inaccurate diagnostic results, the mohss provides oversight over the namibia institute of pathology, the blood transfusion service of namibia and all private laboratories. the government of namibia endorses the world health organization (who) resolutions and programs aimed at laboratory improvements, including the 2008 maputo declaration to strengthen laboratory systems,5 the who regional office for africa’s (afro) stepwise laboratory quality improvement process towards accreditation (slipta) framework6 and the strengthening laboratory management toward accreditation (slmta) programme.7 implementing laboratory quality management in namibia top ↑ in 2010, the mohss, with support from partners, began to implement the slmta programme and slipta framework. the national public health laboratory policy encourages all laboratories to embrace slmta and slipta, and a concerted effort is now being planned to assist the country's 64 laboratories to implement quality improvement. in june 2012, the mohss supported eight people from six namibian laboratories to participate in a series of slmta workshops held at the zimbabwe national quality assurance programme in harare, zimbabwe. slmta in namibia follows the standard three-workshop model, with improvement project implementation periods of approximately two to four months following each workshop.7 progress of the enrolled laboratories is monitored through audits using the slipta checklist. in addition to overall score and star ratings on a zeroto five-star scale, the checklist evaluates 12 quality areas. the african society for laboratory medicine (aslm) and the clinical and laboratory standards institute (clsi) conducted official slipta audits for four of the laboratories post-slmta; the remaining two laboratories did not receive audits because of lack of permission. one laboratory achieved three stars, two laboratories achieved two stars and one laboratory received zero stars. the highest scoring quality areas were client management, facilities and safety and purchasing and inventory. the lowest scores were observed in internal audit, management reviews and corrective action. internal audits were either not being carried out or were conducted inconsistently. management reviews did not address all appropriate topics and did not include action plans, responsibilities, timelines or status information. most corrective actions and action plans were conducted by untrained staff. lessons learned to impact health outcomes and improve laboratory services, all stakeholders – including government agencies, funding bodies and staff – must be involved in and committed to achieving shared laboratory improvement goals. it is suggested that ministries of health adopt quality practices and standards as an integral part of their programmes and understand that external reviews and the accreditation of medical laboratories involve more than the mere assessment of conformance to standards for organisational processes. in namibia, there is a need for the mohss and the audited laboratories, with the assistance of aslm, to jointly develop and follow up on a plan of action to address the gaps identified in the audits. amongst the gaps in staff knowledge identified during these audits were how to manage non-conforming events; how to conduct internal audits and root cause analysis; and how to carry out preventive and corrective actions and monitor their effectiveness. in addition, staff lacked necessary skills in management of quality control and monitoring for trends, biases and shifts, as well as mulitirule (also called ‘westgard rules’) violations and client/customer survey feedback. nonconformities should be shared routinely with staff so that they can implement corrective actions. furthermore, on-site quality assurance responsibilities for the chief medical laboratory technologists/technologists in charge were overloaded. additional leaders should be trained in international organization for standardization (iso) 15189:2012 standard requirements and management responsibilities. it would be advisable for the mohss to develop a system of assessing all laboratories and addressing gaps identified, and then to use those assessments as a means of ensuring that quality becomes a condition for re-registration or licensing to operate a laboratory. sustainability an essential component in implementing a new programme is to plan for sustainability. the mohss is taking ownership of the quality improvement programmes by budgeting for future implementation of slmta and slipta activities throughout the country. the mohss plans to build sustainability into its laboratory quality initiatives by increasing the number of in-country auditors, mentors and trainers, as well as by hosting training-of-trainers workshops so as to remove dependency on costly, external implementers. it is anticipated that this strategy will build capacity and promote self-sufficiency in namibian medical laboratories. the mohss plans to build capacity for writing grant applications to support slmta and slipta, trainings, operational research, dissemination of information and integration of all laboratory services with the aim of attaining accreditation to iso standards in select laboratories. it would be advisable for the mohss to set up a national structure to audit and recognise laboratories; encourage development of innovative procedures and methods; allocate funding for equipment maintenance; and collaborate with accreditation bodies and other institutions in order to implement laboratory quality improvements. as quality improvements become institutionalised in hospital laboratories, it is becoming evident that entire hospital systems are in dire need of similar quality improvement programmes. the namibia mohss calls on international agencies to develop and adapt programmes such as slipta and slmta for use throughout hospital systems so as to ensure continuous quality patient care. acknowledgements top ↑ we would like to acknowledge the mohss permanent secretary, mr. andrew ndishishi; the mohss director, ms. p.k. nghipandulwa; and the us centers for disease control and prevention (cdc) office in namibia, for their continuous encouragement toward this cause. we would also like to thank mr. harold kaura of the namibia institute of pathology, mr. theron slinger of maxi medical laboratory, dr. van finckenstein of the national blood transfusion service of namibia, caprivi pathology centre laboratory management; and the permanent secretary of the ministry of defence for allowing their staff to participate in this programme. much appreciation is also given to talkmore maruta (aslm) and patrick mateta (clsi) for constructive criticism of the mohss during the audits. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions m.n.m. (mohss) was the lead author and researcher for this article. l.k.b. (mohss) was co-author and reviewed and revised the manuscript. references top ↑ 1. nkengasong jn, nsubuga p, nwanyanwu o, et al. laboratory systems and service are critical in global health: time to end the neglect? am j clin pathol. 2010;134(3):368–373. http://dx.doi.org/10.1309/ajcpmpsinq9brmu62. peter tf, rotz pd, blair dh, et al. impact of laboratory accreditation on patient care in the health system. am j clin pathol. 2010;134(4):550–555. http://dx.doi.org/10.1309/ajcph1skq1hnwghf 3. namibia ministry of health and social services, national public health laboratory policy (establishing a strong public health laboratory system) [document on the internet]. c2012 [cited 2014 oct 14]. available from: http://www.mhss.gov.na/files/downloads/dcc_3182_nphl_policy_final_new%20copy.pdf 4. namibia ministry of health and social services, national public health laboratory strategic plan (establishing a strong public health laboratory system) [document on the internet]. c2012 [cited 2014 oct 14]. available from: http://www.mhss.gov.na/files/downloads/687_3184_nphl_strategic%20plan_final_new%20copy.pdf 5. world health organization regional office for africa. the maputo declaration on strengthening of laboratory systems: [document on the internet]. c2008 [cited 2013 jan 02]. available from: http://www.who.int/diagnostics_laboratory/maputo-declaration_2008.pdf 6. world health organization’s regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region (with checklist) [document on the internet]. c2012 [cited 2014 oct 20]. available from: http://www.afro.who.int/en/clusters-a-programmes/hss/blood-safety-laboratories-a-health-technology/blt-highlights/3859-who-guide-for-the-stepwise-laboratory-improvement-process-towards-accreditation-in-the-african-region-with-checklist.html 7. yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin path. 2010;134(3):401–409. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj abstract introduction methods results discussion acknowledgements references about the author(s) joseph anejo-okopi department of microbiology, university of jos, jos, nigeria aids prevention initiative in nigeria, jos university teaching hospital, jos, nigeria edith okeke department of internal medicine, university of jos, jos university teaching hospital, jos, nigeria pantong m. davwar department of internal medicine, university of jos, jos university teaching hospital, jos, nigeria chika onwuamah center for human virology and genomics nigeria institute of medical research, lagos, nigeria harris onywera institute of infectious disease and molecular medicine, university of cape town, cape town, south africa division of medical virology, department of pathology, university of cape town, cape town, south africa research, innovations, and academics unit, tunacare services health providers limited, nairobi, kenya patience omaiye department of internal medicine, university of jos, jos university teaching hospital, jos, nigeria mary duguru department of internal medicine, university of jos, jos university teaching hospital, jos, nigeria ocheme j. okojokwu department of microbiology, university of jos, jos, nigeria otobo i. ujah department of community and family health, college of public health, university of south florida, tampa, florida, united states bulus jonathan department of family medicine, plateau state specialist hospital, jos, nigeria chima a. george department of family medicine, bingham university teaching hospital, jos, nigeria ramyil s. crown department of medical microbiology and parasitology, bingham university teaching hospital, jos, nigeria fiyaktu b. yakubu department of chemical pathology, jos university teaching hospital, jos, nigeria judith o. sokei center for human virology and genomics nigeria institute of medical research, lagos, nigeria leona c. okoli center for human virology and genomics nigeria institute of medical research, lagos, nigeria onyemocho audu department of epidemiology and community health, benue state university, makurdi, nigeria seth c. inzaule department of hiv and global hepatitis program, world health organization, geneva, switzerland isaac o. abah department of pharmacology, university of jos, jos university teaching hospital, jos, nigeria patricia agaba aids prevention initiative in nigeria, jos university teaching hospital, jos, nigeria department of family medicine, university of jos, jos university teaching hospital, jos, nigeria oche o. agbaji department of internal medicine, university of jos, jos university teaching hospital, jos, nigeria atiene s. sagay department of obstetrics and gynaecology, university of jos, jos university teaching hospital, jos, nigeria claudia hawkins department of medicine, feinberg school of medicine, northwestern university, chicago, illinois, united states citation anejo-okopi j, okeke e, davwar pm, et al. molecular detection of hepatitis b virus genotype e with immune escape mutations in chronic hepatitis b patients on long-term antiviral therapy in jos, nigeria. afr j lab med. 2022;11(1), a1677. https://doi.org/10.4102/ajlm.v11i1.1677 original research molecular detection of hepatitis b virus genotype e with immune escape mutations in chronic hepatitis b patients on long-term antiviral therapy in jos, nigeria joseph anejo-okopi, edith okeke, pantong m. davwar, chika onwuamah, harris onywera, patience omaiye, mary duguru, ocheme j. okojokwu, otobo i. ujah, bulus jonathan, chima a. george, ramyil s. crown, fiyaktu b. yakubu, judith o. sokei, leona c. okoli, onyemocho audu, seth c. inzaule, isaac o. abah, patricia agaba, oche o. agbaji, atiene s. sagay, claudia hawkins received: 19 july 2021; accepted: 28 apr. 2022; published: 18 oct. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: previous studies in nigeria have reported the presence of hepatitis b virus (hbv) genotype e and the availability of immune escape mutants. there is a paucity of data on chronic patients on long-term antiviral therapy for hbv infection. objective: this study assessed hbv genotypes and drug resistance variants among patients with chronic hbv infection receiving tenofovir in jos, nigeria. methods: this cross-sectional study consecutively enrolled 101 patients (51 with hiv/hbv co-infection and 50 with hbv infection only) on antiviral therapy from february 2018 to may 2019 at four hospitals in jos, nigeria. dna quantification of hbv was performed on all samples; 30 samples with detectable viral load were selected for genotyping using sanger sequencing by targeting the full-length sequences of reverse transcriptase gene of the hbv genome. phylogenetic analysis was performed with reference sequences from genbank. escape mutant and drug resistance analysis were performed using hbv drug resistance interpretation and geno2pheno. results: only 30 (29.7%) of the 101 study participants had detectable hbv dna. of these, six (20.0%) isolates were successfully amplified and sequenced. the identified genotype was e, including escape mutations l127r (16.7%) and g145a (16.7%). conclusion: this study revealed exclusive dominance of genotype e in nigeria. the s gene mutations g145a and l271r are known to be associated with modified antigenicity and impaired serologic assays, which may cause false negatives in the detection of anti-hbv surface antigen. the presence of mutants that are associated with vaccine immune escape may also have diagnostic and vaccine immune response implications. keywords: hepatitis b virus (hbv); genotyping; antiviral drug resistance; chronic hepatitis b. introduction hepatitis b virus (hbv) infection is a public health problem worldwide, with an estimated global infection of 2 billion, 257 million of whom are chronically infected.1,2 hepatitis b virus infection causes about 880 000 deaths yearly due to cirrhosis and hepatocellular carcinoma (hcc).3 the prevalence of hcc is rapidly increasing with most cases being attributable to chronic hepatitis b. the disease is endemic in sub-saharan africa, affecting more than 8.0% of the population, most of whom are infected in early childhood.3 individual infections may resolve, become sub-clinical, chronic or result in liver damage. sub-saharan africa has one of the highest hbv-related liver cancer rates in the world.4,5 hepatitis b virus infection increases the risk of developing liver cirrhosis and hcc by 25.0% to 40.0%.6 hepatitis b virus prevalence in nigeria varies among different populations (ranging between 9.2% and 18.0%), including hbv/hiv co-infected population.7,8,9 there is mounting evidence for heterogeneity of hbv treatment outcomes and vaccination efficacy attributed to the variations in the distribution of hbv genotypes.10 hepatitis b virus has been characterised into 10 genotypes: a–i4,11 and j which was recently identified in a single case in japan.12 genotypes a, d and e circulate in africa, with the latter genotype prevailing in western africa,4,13 including nigeria.14,15,16,17,18 looking at nigeria’s huge population and cross-border movement, it is important that the molecular characterisation of hbv genotypes and prevalence of drug resistance mutations is determined for better management and epidemiological purposes. the investigation of hbv genotype is becoming more important due to its role in the pathogenesis and treatment response in both hbv-mono and hbv/hiv co-infected patients and the wide availability of antiviral therapy.19 equally, drug resistance is a major clinical concern impacting clinical outcomes. studies have reported the impact of some mutations that showed significant role of viral persistence on liver disease, immune escape and resistance to antiviral therapy.20,21 the reverse transcriptase encoded by the pol gene lacks effective proofreading ability, which has implications in hbv therapy.22 however, the use of low genetic barrier drugs such as lamivudine has contributed to the development of a drug-associated vaccine-escape mutant which is becoming a growing health concern. this mutant alters envelope antigen that permits the virus to escape the neutralisation antibody to hepatitis b surface antigen.21 this called for the need of drugs with higher genetic barriers to resistance than lamivudine or telbivudine.19 however, there is a paucity of knowledge regarding the hbv genotypes and resistant strains in chronic adult patients on long-term antiviral therapy. we therefore characterised hbv in chronic patients on tenofovir to identify circulating genotypes and drug resistance variants in nigeria. methods ethical considerations the study protocol was approved by the four study sites research ethics committees, namely: jos university teaching hospital, faith alive foundation, bingham university teaching hospital and plateau state specialist hospital (study approval numbers: juth/adm/11/0517, fa/0112f, buth/ec/01-25-18b and plashrec/12018). after explaining the study objectives and procedures to the participants, a written informed consent was obtained from each patient before data and sample collection. the patients’ data were coded and reported without divulging personal health information. all other aspects of the study were conducted in accordance with the ethical standards of the helsinki declaration. study design and population we conducted a cross-sectional study between february 2018 and may 2019 at jos university teaching hospital, plateau state specialist hospital, faith alive foundation and bingham university teaching hospital, all in jos metropolis, nigeria. the 101 consecutively enrolled adult patients, including 51 patients co-infected with hiv, were confirmed to have chronic hbv infection after testing positive for hepatitus b surface antigen antibodies. for hbv sero-diagnosis, 5 ml of blood sample was collected, centrifuged at 2500 revolutions per minute for 1 min, and 1 ml of plasma was used for testing hbsag by third-generation enzyme-linked immunosorbent assay (monolisa, bio-rad, paris, france) according to manufacturer’s instruction; a positive hbsag result indicates current infection. all participants were on antiviral therapy (hbv/hiv: lamivudine/tenofovir combination, hbv-mono: tenofovir alone) ≥ 12 months. sample preparation the 5 ml of blood samples collected were centrifuged, and the sera separated, aliquoted, and stored at –80 °c until use. the hbv dna detection and quantification were performed using automated cobas® ampliprep taqman hbv test version 2.0 (roche diagnostics international ag, rotkreuz, switzerland). a total of 30 samples with detectable viral load were selected for dna sequencing reactions. hepatitis b virus dna extraction dna extraction, amplification and direct sequencing of the full-length hbsag and the reverse transcriptase genes were performed at center for human virology and genomics, nigeria institute of medical research, lagos, nigeria. viral dna was extracted from 200 µl of plasma samples using the zr viral dna kittm (zymo research, irvine, california, united states), according to the manufacturer’s protocol. primers and nested polymerase chain reaction universal primers from previous study were used for detection of hbv genotypes,23 which were synthesised by inqaba biotech (pretoria, south africa). two outer primer sets were used. primer set 251f (5ʹ-gga tgt gtc tgc ggc gtt t-3ʹ) and 1797r (5ʹ-gac cca caa ttc ktt gac ata ctt tcc-3ʹ) were used for the initial amplification, and 251f (5ʹ-cga acc act gaa caa atg gc-3ʹ) and 1190r (5ʹ-tca cca tat tct tgg gaa caa ga-3ʹ) used for the second amplification, as previously described.23 these primer sets were designed to generate overlapping fragments, and polymerase chain reaction (pcr) was performed using platinum™ superfi™ pcr master mix (thermofisher, waltham, massachusetts, united states). each 25.0 µl reaction mixture contained 12.5 µl of 2× premix, 1.25 µl of each 10.0 µm primer, 5.0 µl of pcr water and 5.0 µl dna template for first pcr while for the second pcr, 3.0 µl of first pcr product was used. both pcr were carried out in applied biosystems miniamp plus thermal cycler (thermofisher, waltham, massachusetts, united states) with the following cycling conditions: 95 °c for 3 min followed by 35 cycles of 97 °c for 30 s, 54 °c for 60 s and 72 °c for 60 s for the first round of pcr while 31 cycles were done for the second round of pcr. each round of pcr was followed by a final extension of 72 °c for 10 min. amplified products were verified using 1% agarose gel stained with gelred® (biotium, fremont, california, united states) on a cyfox gel documentation system (sysmex-partec, münster, germany). the amplified products were purified using exosap-it (thermofisher, waltham, massachusetts, united states) and quantified using dsdna high-sensitivity (thermofisher, waltham, massachusetts, united states) reagent on the qubit-4tm (thermofisher, waltham, massachusetts, united states). samples with dna concentration ≥ 5 ng/µl and gel bands of appropriate size were selected for further processing. purification and sanger sequencing verified nested pcr products were purified using exosap-ittm pcr product cleanup reagent (thermofisher, waltham, massachusetts, united states) at 37 °c for 15 min (to digest excess primers and deoxynucleoside triphosphates), and 80 °c for 15 min (to inactivate the enzymes) according to manufacturer’s protocol. the sequencing reaction mixture contained 1.0 µl of bigdye® terminator version 3.1 ready reaction mix (applied biosystems, foster city, california, united states), 4.0 µl of 5× sequencing buffer, 2.0 µl of template, 11.0 µl of double distilled water, and 2.0 µl of 10.0 µm 251f and 1190r primers. cycle sequencing was performed using 251f and 1190r primers in a miniamp plus thermal cycler with the following program: 25 cycles of 96 °c for 10 s, 50 °c for 5 s and 60 °c for 4 min. the cycle sequencing product was purified using the bigdye xterminator® solution (applied biosystems, waltham, massachusetts, united states) followed by the capillary electrophoresis on the 3130xl genetic analyzer using a 50 cm capillary array, and pop-7 polymer (applied biosystems, waltham, massachusetts, united states). all the generated sequences were analysed and assembled using clc genomic workbench version 8.0.3 (clc bio, aarhus, denmark) and then subjected to ncbi nucleotide blast (https://blast.ncbi.nlm.nih.gov/blast.cgi) for quality check. in this study, a chromatogram was considered of good quality if (1) at least 80% of total amplicon length was sequenced and (2) the noise-to-signal ratio was estimated to be < 5%. phylogenetic analysis of hepatitis b virus sequences to infer the evolutionary relationship between the six hbv nucleotide sequences in our study and hbv sequences from different parts of the world, we used molecular evolutionary genetics analysis (mega) x version 10.1.7 (mega software, dortmund, germany).24 first, a total 102 sequences (including a sequence from a chimpanzee) representing the hbv genotypes from different parts of the world were mined from the ncbi genbank nucleotide database (https://www.ncbi.nlm.nih.gov/nucleotide/). the sequence from the chimpanzee was used as an out-group. the generated nucleotide sequence data were deposited in the genbank database under the accession numbers on236588–on236593. these sequences, together with the six from our cohort, were aligned using multiple sequence comparison by log-expectation (muscle) software,25 which is a sequence alignment option in mega. the multiple sequence alignment was trimmed to 846-base pairs, followed by determination of the dna model that best describes the nucleotide substitution pattern. this was performed using the maximum likelihood (ml) statistical method. the general time reversible (gtr) model with discrete gamma distribution (+g = 0.51, with 5 rate categories) and some invariant evolutionary sites (+i = 35.51% sites) was chosen as the best model, based on its low bayesian information criterion score (18 980). for the phylogeny reconstruction the ml statistical method was used with a bootstrap of 1000 replicates and the sub tree-pruning-regrafting (extensive; spr level 5) as the ml heuristic method. moreover, we used the option of ‘moderate branch length filter’ to enable a semi-stringent exhaustive optimisation of the branch lengths of the phylogenetic trees. initial trees for the heuristic search were generated using neighbor-joining and bionj algorithms and the phylogenetic tree with superior log likelihood value selected. phylogenetic trees were produced in newick format employing interactive tree of life interface (https://itol.embl.de/tree/). to determine the genetic diversity of the six hbv sequences, we performed an alignment analysis using mega.24 the overall mean (average) and maximum distance were used as proxies for genetic diversity. assessment of hepatitis b virus drug resistance mutations the obtained sequences of overlapping surface (s) and pol gene were translated to the protein sequences and aligned with the references in bioedit version 7.1.3.0.26 escape mutant analysis and drug resistance analysis were performed using hbv drug resistance interpretation and geno2pheno (hbv) version 2.0 (https://hbv.geno2pheno.org/index.php). it is a program that searches for homology between the input sequence and other dna sequences in the existing stored database for drug resistance and surface gene mutations. genotype assignments were confirmed using the genbank blast and hbvseq tools from the hiv drug resistance database.26,27 statistical analysis the obtained data were analysed using statistical package for social science software (version 19.0; ibm corp., armonk, new york, united states) and expressed as medians (with interquartile ranges [iqr]) for continuous variables, and as counts and percentages for discrete variables. chi-squared test was used for discrete data. results thirty (29.7%) of the 101 had detectable hbv dna. of the 30 patients, 11 (36.7%) were co-infected with hiv while 19 (63.3%) had hbv-mono infection. seventeen (56.7%) were female; the mean age was 41 years. the proportion of patients with hbv dna copies/ml of < 20 copies/ml was 22/30 (73.3%); 6/19 (31.6%) of hbv-mono and 2/11 (18.2%) of hiv/hbv had 20 copies/ml – 20 000 copies/ml. median aspartate aminotransferase u/l was 28 (23.8–36.0) for hiv/hbv co-infected patients and 27 (iqr: 19–32) for those with hbv-mono infection. median alanine aminotransferase was 26.1 (iqr: 19.8–35.5) for those with hiv/hbv dual infection and 27 (iqr: 20–41) for those with hbv-mono infection while platelet level was 259 (iqr: 198.3–298.8) for those with hiv/hbv dual infection and 195 (168–257) for those with hbv-mono infection. phylogeny and genetic diversity of the hepatitis b virus sequences all the generated sequences were from the hbv-mono infected patients. the genotypes of the six hbv nucleotide sequences were inferred from the phylogenetic tree (figure 1), which showed that all the sequences were hbv genotype e. all the six sequences clustered with hbv genotype e, most of which were from the west african region. figure 1: phylogenetic maximum likelihood circular consensus tree with branch node statistics as viewed using interactive tree of life (https://itol.embl.de/tree/). hepatitis b virus drug resistance report according to the geno2pheno (hbv) 2.0 report, all the six hbv genotype e sequences were susceptible to lamivudine, adefovir, entecavir, tenofovir disoproxil fumarate, and telbivudine. two (33.3%) of the six genotypes, all from hbv-mono infected individuals, had escape mutations, the sh2 domain-containing adapter protein b protein of the surface-gene. one sample had l127r escape mutation while the other had g145a at a position first described as vaccine escape mutation. these mutations are amino acid substitutions within the major hydrophilic region called the ‘a’ determinant (124–147). discussion our findings showed a predominance of genotype e consistent with previous studies from nigeria and other west african regions.4,14,16,17,18,28,29,30 we however found that the genotypes had a lower diversity compared to a previous report,14 although other studies have equally observed a low genetic diversity of hbv genotype e in west africa.18,31 a possible explanation may be due to natural selection, drug pressure and hyperendemicity of hbv genotype e infection in other west african countries.28 the ease of transmission and epidemiological pattern of hbv have been linked to genotype variability, as well as clinical virological parameters. the pathogenicity of hbv has been shown to vary with genotype and certain mutations have been associated with genotype diversity that influences disease outcome, hcc development, diagnostic testing and treatment outcomes.28 studies have shown a higher severity of liver disease progression and hcc in patients infected with c and d genotypes than those infected with a or b genotypes.32,33 although the clinical implications of genotype e, the predominant genotype in our study, have not been well reported, it is speculated that patients with genotype e have better liver disease prognosis than other genotypes.29 larger studies are needed to provide further evidence on good clinical prognosis for patients infected with genotype e. nonetheless, these findings suggest the need for adoption of individualised genotypic testing, which may play a key role in guiding future treatment strategies to combat the hbv disease. in the interim, early treatment is warranted to ensure good hbv prognosis. as reported in other studies, we did not find any tenofovir or underlying lamivudine-associated mutations including the rta194t that was earlier reported to be resistant to tenofovir.34,35 this suggests that tenofovir-containing and monotherapy regimen is still effective against the virus in our setting. however, we observed vaccine escape mutations (l127r and sg145a) in two (33.3%) of the six genotypes. the finding of sg145a is consistent with findings from nigeria,16 ghana36 and gabon.37 these hbsag vaccine escape mutants may have arisen as a result of specific selection such as the host immune system due to vaccination or antiviral selective pressure that truncates the production of hbsag resulting in low plasma hbv dna levels.38 although we did not take the history of patients’ vaccination, but all the patients were on antiviral therapy (≥ 12 months), including mono-lamivudine or lamivudine-containing regimen before switching to tenofovir, and this may suggest a possible reason for emergence of drug-associated potential vaccine-escape mutants (sg145a) in the study patients. similarly, mutations arising in the s orf region because of inherent or selective pressure by antiviral drugs can end up in the emergence of virus escape mutants in the adjacent s region with subsequent lack of response to hbv vaccine. the presence of this mutant over time could lead to partial replicative capacity of resistant hbv variant, and lack of response to hbv vaccine.39 this mutation may be due to poor adherence to antiviral therapy and the use of drugs with a low genetic barrier to resistance such as lamivudine, which was the major drug used in nigeria before switching to tenofovir among hbv-mono infected patients. this mutation sg145 confers immune escape competence on the virus,40,41 which has negative impact on humoral immune response to hbv vaccine. however, a recent study has shown that the risk of transmission of hbv infection is low in a vaccinated individual.42 similarly, the presence of this mutation in immune-compromised patients could be responsible for hbv reactivation among persons previous immune to hepatitus b surface antigen antibodies, suggesting immune escape mutant.43 also, this mutation among others can be responsible for virus detection failure in most routine screening tests.44 this is because an assay used for hbv screening may give false-negative results if the test kit was not designed to detect mutants in the ‘a’ determinant. this is common in the clinical setting where the patients have hbsag negative result, yet will have classical symptoms and, if tested for hbv dna, will have positive high hbv dna result. the detection of some of these emerging mutants has become a major challenge to commercially available immunoassays. the l127r escape mutation in the ‘a’ determinant region observed in our study has been associated with hbv antigenicity, diagnostics and immunogenicity.45 overall, l127r mutation was mostly associated with patients who had experienced lamivudine treatment in other african countries.36,37 the long-term impact of hbsag vaccine escape mutations on the natural history of chronic hbv remains unclear. however, it has been hypothesised that the presence of stop codons may favour the production of a truncated hbsag (despite low serum hbv dna), which accumulates in the endoplasmic reticulum and induces oxidative stress to enhance cell proliferation.38 this may also explain the transmission of hbv genotype e infection even in vaccinated individuals. studies on the hbv genotypes, immune escape and antiviral drug resistance mutations in nigeria are scarce. this is the first published study, to the best of our knowledge, that characterised hbv genotypes and drug resistance in chronic hepatitis b patients (hbv/hiv co-infected and hbv-mono infected patients) on long-term antiviral therapy (tenofovir) in nigeria. however, one recent study assessed the effect of hbv mutations with liver severity in hiv/hbv co-infected antiretroviral therapy-naïve patients in nigeria.27 in this study, hbv mutations were independently associated with liver disease severity, but the effect declined after antiretroviral therapy initiation. our study however, highlighted the need to assess hbv mutations due to the potential link with disease severity. limitations there are limitations to this study that need to be highlighted. it is a cross-sectional study with a small sample size, and low amplification and sequencing rate, yielding only 20.0% of the intended samples. many factors could be responsible for this. for example, several freeze-thawing cycles and poor storage due to frequent power outage could have affected the sample integrity, resulting in decreased hbv dna quantity. other causes include intrinsic reagent-related issues and impact of hbv/e on the sensitivity of hbsag assays due to surface antigen gene mutations located outside the sequenced region.46 also, there is a growing concern that the accumulation of escape mutations may lead to detection failure of hbsag using the available commercial diagnostic assays. this in turn may lead to a false-negative hbv result. moreover, this has an impact on risk of transmission, including rendering the vaccination ineffective due to undiagnosed mutant carriers. conclusion our study confirmed the dominance of genotype e, and it has great public health significance, given that the protective vaccine currently in use by the national vaccination programme is from the hbv-a2 strain. additionally, no patients had hbv drug resistant mutations, suggesting that the use of tenofovir is an effective treatment in the management of hbv infection in both chronic hbv/hiv co-infected and hbv-mono infected patients. the study also showed the presence of mutation associated with immune escape mutant in chronic hbv-infected patients on long-term antiviral therapy. therefore, further larger study is required to understand the dynamics of immune escape mutant on hbv diagnosis, and impact of genotypes on treatment outcomes. acknowledgements we thank the patients who participated in this study. we also thank the staff and management of jos university teaching hospital, faith alive foundation hospital, bingham university teaching hospital, and plateau state specialist hospital for their support during patients’ enrolment and sample collection. finally, we thank the of the management and staff of center for human virology and genomics, and nigeria institute of medical research, lagos, nigeria, for providing the laboratory space and equipment during the analysis. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions j.a.-o., e.o. and c.h. were involved in conceptualisation, methodology, funding acquisition, project administration, investigation, writing original draft preparation and supervision. a.s.s. and o.a. assisted with funding acquisition, supervision, project administration, resources, writing, reviewing and editing. h.o. and c.o. performed data curation, assisted with software and contributed to the formal analysis, validation, preparation of the original draft and validation. p.m.d., p.o. and m.d. were responsible for investigation, project administration, resources, writing, reviewing and editing. all authors discussed the results, contributed to and approved the final manuscript. sources of support research reported in this publication was partly supported by the fogarty international center and national institute of mental health, of the united states national institutes of health under award number d43 tw010543. data availability the data sets generated with unique identifiers and analysed during the study are available on request from the corresponding author, j.a.-o., but are not publicly available. disclaimer the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health. references stanaway jd, flaxman ad, naghavi m, et al. the global burden of viral hepatitis from 1990 to 2013: findings from the global burden of disease study 2013. lancet. 2016;388(10049):1081–1088. https://doi.org/10.1016/s0140-6736(16)30579-7 who. hepatitis b fact sheet. geneva: world health organization (editor); 2017. hou j, liu z, gu f. epidemiology and prevention of hepatitis b virus infection. int j med sci. 2005;2(1):50–57. https://doi.org/10.7150/ijms.2.50 kramvis a, kew mc. epidemiology of hepatitis b virus in africa, its genotypes and clinical associations of genotypes. hepatol res. 2007;37(s1):s9–s19. https://doi.org/10.1111/j.1872-034x.2007.00098.x schweitzer a, horn j, mikolajczyk rt, krause g, ott jj. estimations of worldwide prevalence of chronic hepatitis b virus infection: a systematic review of data published between 1965 and 2013. lancet. 2015;386(10003):1546–1555. https://doi.org/10.1016/s0140-6736(15)61412-x kim bk, han kh, ahn sh. prevention of hepatocellular carcinoma in patients with chronic hepatitis b virus infection. oncology. 2011;81(suppl. 1):41–49. https://doi.org/10.1159/000333258 olayinka ta, oyemakinde a, balogun sm, et al. seroprevalence of hepatitis b infection in nigeria: a national survey. am j trop med hyg. 2016;95(4):902–907. https://doi.org/10.4269/ajtmh.15-0874 akindigh mt, abba oj, robert oc, okojokwu jo, okechalu nj, anejo-okopi aj. seroprevalence of hepatitis b virus co-infection among hiv-1-positive patients in north-central nigeria: the urgent need for surveillance. afr j lab med. 2019;8(1):622. https://doi.org/10.4102/ajlm.v8i1.622 musa b, bussell s, borodo mm, samaila aa, femi ol. prevalence of hepatitis b virus infection in nigeria, 2000-2013: a systematic review and meta-analysis. niger j clin pract. 2015;18(2):163–172. https://doi.org/10.4103/1119-3077.151035 croagh cm, desmond pv, bell sj. genotypes and viral variants in chronic hepatitis b: a review of epidemiology and clinical relevance. world j hepatol. 2015;7(3):289–303. https://doi.org/10.4254/wjh.v7.i3.289 yu h, yuan q, ge sx, et al. molecular and phylogenetic analyses suggest an additional hepatitis b virus genotype ‘i’. plos one. 2010;5(2):e9297. https://doi.org/10.1371/journal.pone.0009297 tatematsu k, tanaka y, kurbanov f, et al. a genetic variant of hepatitis b virus divergent from known human and ape genotypes isolated from a japanese patient and provisionally assigned to new genotype j. j virol. 2009;83(20):10538–10547. https://doi.org/10.1128/jvi.00462-09 yousif m, mudawi h, bakhiet s, glebe d, kramvis a. molecular characterization of hepatitis b virus in liver disease patients and asymptomatic carriers of the virus in sudan. bmc infect dis. 2013;13:328. https://doi.org/10.1186/1471-2334-13-328 forbi jc, ben-ayed y, xia gl, et al. disparate distribution of hepatitis b virus genotypes in four sub-saharan african countries. j clin virol. 2013;58(1):59–66. https://doi.org/10.1016/j.jcv.2013.06.028 ayodele mbo, ogugbue cj, frank-peterside n, tatfeng ym. prevalent hepatitis b virus genotypes circulating among hiv co-infected patients in an urban city, south-south nigeria. j aids clin res. 2019;10:5. faleye cot, adewumi om, ifeorah mi, et al. detection of hepatitis b virus isolates with mutations associated with immune escape mutants among pregnant women in ibadan, southwestern nigeria. springerplus. 2015;4:43. https://doi.org/10.1186/s40064-015-0813-1 ahmad ae, bakari ag, olayinka at. pattern of prevalent hepatitis b virus genotypes in zaria, nigeria. niger postgrad med. 2019;26(2):80–86. https://doi.org/10.4103/npmj.npmj_59_19 assih m, ouattara ka, diarra b, et al. genetic diversity of hepatitis viruses in west-african countries from 1996 to 2018. world j hepatol. 2018;10(11):807–821. https://doi.org/10.4254/wjh.v10.i11.807 de clercq e, ferir g, kaptein s, neyts j. antiviral treatment of chronic hepatitis b virus (hbv) infections. viruses. 2010;2(6):1279–1305. https://doi.org/10.3390/v2061279 mokaya j, mcnaughton al, hadley mj, et al. a systematic review of hepatitis b virus (hbv) drug and vaccine escape mutations in africa: a call for urgent action. plos negl trop dis. 2018;12(8):e0006629. https://doi.org/10.1371/journal.pntd.0006629 locarnini s, mason ws. cellular and virological mechanisms of hbv drug resistance. j hepatol. 2006;44(2):422–431. https://doi.org/10.1016/j.jhep.2005.11.036 sayan m, akhan sc, meric m. naturally occurring amino-acid substitutions to nucleos(t)ide analogues in treatment naïve turkish patients with chronic hepatitis b. j viral hepat. 2010;17(1):23–27. https://doi.org/10.1111/j.1365-2893.2009.01149.x chook jb, teo lw, ngeow fy, et al. universal primers for detection and sequencing of hepatitis b virus genomes across genotypes a to g. j clin microbiol. 2015;53(6):1831–1835. https://doi.org/10.1128/jcm.03449-14 kumar s, stecher g, li m, knyaz c, tamura k. mega x: molecular evolutionary genetics analysis across computing platforms. mol biol evol. 2018;35(6):1547–1549. https://doi.org/10.1093/molbev/msy096 edgar rc. muscle: a multiple sequence alignment method with reduced time and space complexity. bmc bioinformatics. 2004;5:113. hall ta. bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt. nucleic acids symp. 1999;41:95–98. neumann-fraune m, beggel b, kaiser r, obermeier m. hepatitis b virus drug resistance tools: one sequence, two predictions. intervirology. 2014;57(3–4):232–236. https://doi.org/10.1159/000361076 grant j, agbaji o, hawkins c, et al. hepatitis b virus sequencing and liver fibrosis evaluation in hiv/hbv co-infected nigerians. trop med int health. 2017;22(6):744–754. https://doi.org/10.1111/tmi.12873 mulders mn, venard v, njayou me, et al. low genetic diversity despite hyperendemicity of hepatitis b virus genotype e throughout west africa. j infect dis. 2004;190(2):400–408. https://doi.org/10.1086/421502 fujiwara k, tanaka y, orito e, et al. distribution of hbv genotypes among hbv carriers in benin: phylogenetic analysis and virological characteristics of hbv genotype e. world j gastroenterol. 2005;11(41):6410–6415. https://doi.org/10.3748/wjg.v11.i41.6410 dongdem za, dzodzomenyo m, adjei aa, et al. hepatitis b virus genotypes among chronic hepatitis b patients reporting at korle-bu teaching hospital, accra, ghana. pan afr med j. 2016;25(suppl 1):5. https://doi.org/10.11604/pamj.supp.2016.25.1.6170 shi y-h. correlation between hepatitis b virus genotypes and clinical outcomes. jpn j infect dis. 2012;65(6):476–478. https://doi.org/10.7883/yoken.65.476 kramvis a. clinical implications of hepatitis b virus genotypes and hbeag in pediatrics. rev med virol. 2016;26(4):285–303. https://doi.org/10.1002/rmv.1885 sunbul m. hepatitis b virus genotypes: global distribution and clinical importance. world j gastroenterol. 2014;20(18):5427–5434. https://doi.org/10.3748/wjg.v20.i18.5427 amini-bavil-olyaee s, herbers u, sheldon j, luedde t, trautwein c, tacke f. the rta194t polymerase mutation impacts viral replication and susceptibility to tenofovir in hepatitis b e antigen-positive and hepatitis b e antigen-negative hepatitis b virus strains. hepatology. 2009;49(4):1158–1165. https://doi.org/10.1002/hep.22790 mahmood m, anwar ma. analysis of resistant mutations in reverse transcriptase domain of hepatitis b virus from patients from islamabad, pakistan. j unexplored med data. 2017;2:60–64. https://doi.org/10.20517/2572-8180.2017.13 liu y, wang cm, cheng j, et al. hepatitis b virus in tenofovir-naive chinese patients with chronic hepatitis b contains no mutation of rta194t conferring a reduced tenofovir susceptibility. chin med j (engl). 2009;122:1585–1586. malagnino v, salpini r, maffongelli g, et al. high rates of chronic hbv genotype e infection in a group of migrants in italy from west africa: virological characteristics associated with poor immune clearance. plos one. 2018;13(3):e0195045. https://doi.org/10.1371/journal.pone.0195045 bivigou-mboumba b, francois-souquiere s, deleplancque l, et al. broad range of hepatitis b virus (hbv) patterns, dual circulation of quasi-subgenotype a3 and hbv/e and heterogeneous hbv mutations in hiv-positive patients in gabon. plos one. 2016;11(1):e0143869. https://doi.org/10.1371/journal.pone.0143869 perazzo p, eguibar n, gonzã¡lez rh, nusblat ad, cuestas ml. hepatitis b virus (hbv) and s-escape perazzo p mutants: from the beginning until now. j hum virol retrovirol. 2015;2(3):00046. https://doi.org/10.15406/jhvrv.2015.02.00046 caligiuri p, cerruti r, icardi g, bruzzone b. overview of hepatitis b virus mutations and their implications in the management of infection. world j gastroenterol. 2016;22(1):145–154. https://doi.org/10.3748/wjg.v22.i1.145 sheldon j, ramos b, garcia-samaniego j, et al. selection of hepatitis b virus (hbv) vaccine escape mutants in hbv-infected and hbv/hiv-coinfected patients failing antiretroviral drugs with anti-hbv activity. j acquir immune defic syndr. 2007;46(3):279–282. https://doi.org/10.1097/qai.0b013e318154bd89 salpini r, colagrossi l, bellocchi mc, et al. hepatitis b surface antigen genetic elements critical for immune escape correlate with hepatitis b virus reactivation upon immunosuppression. hepatology. 2015;61(3):823–833. https://doi.org/10.1002/hep.27604 cao gw. clinical relevance and public health significance of hepatitis b virus genomic variations. world j gastroenterol. 2009;15(46):5761–5769. https://doi.org/10.3748/wjg.15.5761 zeid wma, ramadan di, shemis ma. prevalence of mutations within major hydrophilic region of hepatitis b virus and their correlation with genotypes among chronically infected patients in egypt. arab j gastroenterol. 2016;17(1):34–40. https://doi.org/10.1016/j.ajg.2016.03.001 launay o, masurel j, rosenberg ra, et al. high levels of serum hepatitis b virus dna in patients with ‘anti-hbc alone’: role of hbsag mutants. j viral hepat. 2011;8(10):721–729. https://doi.org/10.1111/j.1365-2893.2011.01482.x article information authors: julia driessen1 henry limula2 oliver j. gadabu3 gervase gamadzi2 edwin chitandale2 anne ben-smith4 noor alide2 gerald p. douglas5 affiliations: 1department of health policy and management, university of pittsburgh, pittsburgh, united states 2kamuzu central hospital, ministry of health, lilongwe, malawi 3baobab health trust, lilongwe, malawi 4department of biomedical informatics, university of pittsburgh, united states 5center for health informatics for the underserved, university of pittsburgh, united states correspondence to: julia driessen email: driessen@pitt.edu postal address: 130 de soto street, a614 crabtree hall, pittsburgh, united states dates: received: 10 mar. 2014 accepted: 13 apr. 2015 published: 11 june 2015 how to cite this article: driessen j, limula h, gadabu oj. informatics solutions for bridging the gap between clinical and laboratory services in a low-resource setting. afr j lab med. 2015;4(1), art. #176, 7 pages. http://dx.doi.org/10.4102/ajlm.v4i1.176 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. informatics solutions for bridging the gap between clinical and laboratory services in a low-resource setting in this original research... open access • abstract • introduction    • problem statement       • key focus       • contribution to field • research method and design    • setting    • procedure    • ethical considerations • results • discussion    • limitations of the study    • recommendations    • conclusion • trustworthiness    • reliability and validity • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: there has been little formal analysis of laboratory systems in resource-limited settings, despite widespread consensus around the importance of a strong laboratory infrastructure. objectives: this study details the informational challenges faced by the laboratory at kamuzu central hospital, a tertiary health facility in malawi; and proposes ways in which informatics can bolster the efficiency and role of low-resource laboratory systems. methods: we evaluated previously-collected data on three different aspects of laboratory use. a four-week quality audit of laboratory test orders quantified challenges associated with collecting viable specimens for testing. data on tests run by the laboratory over a one-year period described the magnitude of the demand for laboratory services. descriptive information about the laboratory workflow identified informational process breakdowns in the pre-analytical and post-analytical phases and was paired with a 24-hour sample of laboratory data on results reporting. results: the laboratory conducted 242 242 tests over a 12-month period. the four-week quality audit identified 54% of samples as untestable. prohibitive paperwork errors were identified in 16% of samples. laboratory service workflows indicated a potential process breakdown in sample transport and results reporting resulting from the lack of assignment of these tasks to any specific employee cadre. the study of result reporting time showed a mean of almost six hours, with significant variation. conclusions: this analysis identified challenges in each phase of laboratory testing. informatics could improve the management of this information by streamlining test ordering and the communication of test orders to the laboratory and results back to the ordering physician. introduction top ↑ problem statement the healthcare challenges faced by resource-limited countries require an efficient and accessible laboratory infrastructure. laboratory testing plays an irreplaceable role in the diagnosis and treatment of diseases such as hiv, tuberculosis, and malaria.1 monitoring the progression of hiv through laboratory measures such as viral load and cd4 counts is important for managing patients on antiretroviral therapy (art) and identifying treatment failure. the serious public health threat of drug-resistant tuberculosis also requires laboratory testing for drug sensitivity.2 whilst rapid diagnostic tests are available for malaria and are in use in many health facilities, many countries still consider microscopy performed in the laboratory as the gold standard because it detects a wider array of species.3 if laboratory tests are not available or used, there is a substantial risk of inappropriate treatment, which harms the patient, wastes already limited resources and contributes to increased drug resistance.4,5 despite the need for laboratory testing in addressing the infectious disease burden, many laboratories are ill-equipped to play a central role in the diagnostic and care delivery process in resource-constrained countries.6 as a result of poor laboratory services and staffing, there is a diagnostic culture amongst physicians of circumventing laboratory testing and using other, less reliable signals to diagnose diseases such as malaria, or treating for malaria despite a negative laboratory result.7,8,9,10 in many low-income countries, there is reluctance to order tests such as a sputum microscopy for tuberculosis, since the patient often dies before the results are received.11 in malawi, for example, 40% of hospitals have only one trained laboratory technician.12,13 malawi's national art programme has relied primarily on clinical criteria for art treatment initiation.14 whilst compelling, these anecdotal insights and macro-level measures offer an incomplete picture of laboratory testing in low-resource settings and do not indicate how these shortages impact healthcare delivery and health outcomes. for example, investing in additional microscopes may have a limited effect if the necessary reagents are unavailable or physicians rarely order tests that require microscopes. a better understanding of the micro-level dynamics of laboratory testing, as well as the role of the laboratory within a low-resource health system, can identify high-value steps to laboratory strengthening. key focus laboratory testing is typically described as a three-stage process: pre-analytical, analytical and post-analytical.15,16 most studies of laboratory testing, both in lowand high-resource settings, focus on the analytical phase, or the analysis step within the laboratory; it has also been noted that the preand post-analytical phases are common sources of delays and errors.17 understanding the dynamics of a low-resource laboratory will also highlight the potential role of informatics in bolstering the efficiency of the laboratory system. laboratory testing is both materially and informationally intensive. whereas most coverage of laboratory systems in low-resource settings focuses on the material needs, such as sufficient quantities of reagents and working centrifuges, the information burden is just as demanding, but far less well-understood. informatics streamlines the management and transfer of data and thus may be appropriate for addressing these information barriers. however, better evidence is needed around the informational demands and barriers experienced in laboratories in low-resource settings. one problem where informatics solutions have been employed in low-resource settings is in improving clinic access to centralised laboratory testing results.18,19 other systems have been implemented in facilities with on-site laboratories in order to improve management of laboratory histories. in most cases, these systems involve data entry of paper forms, with the goal of maintaining accurate patient histories.19 contribution to field in this article we present detailed data on the role and workflow of clinical laboratory testing at kamuzu central hospital (kch), an 800+ bed tertiary health facility in lilongwe, malawi's capital city, with the goal of understanding other ways that informatics can be used to address laboratory challenges. kch has been an incubator for developing informatics interventions in the clinical setting since 2001. the hospital has an extensive local area network connecting more than 60 computers across the hospital campus. this is, in turn, connected to a dedicated power backup system and linked to a wireless metropolitan area network spanning greater lilongwe. aspects of this work have been described elsewhere.20,21,22 laboratory systems strengthening and the promotion of good laboratory practices are supported by a number of technical partners and the laboratory is currently in the process of preparing for an audit under the framework of strengthening laboratory management toward accreditation (slmta). four different aspects of the laboratory are assessed: (1) the demand for laboratory testing in the hospital; (2) the burden of untestable samples; (3) the process of laboratory testing in a typical patient visit; and (4) the time required for reporting of results. research method and design top ↑ setting kch is a tertiary care facility in the capital city of malawi. it serves an area of over four million people, with approximately 50 000 admissions and 245 000 outpatient visits per year.23 the laboratory at kch houses nine different departments: haematology, parasitology, microbiology, molecular biology, serology, flow cytometry, biochemistry, histology and blood bank. the laboratory operates continuously with 27 professional staff, although staffing levels are reduced at nights and on weekends. procedure we analysed previously-collected data from laboratory records and the results of studies done as part of a quality improvement effort by the kch laboratory. in 2009, a quality audit was performed on the samples sent for testing to the kch laboratory over a four-week period.24 a total of 3549 samples were evaluated for completeness of the test orders and viability of the samples. if a test order or sample was deemed untestable, the reason for this classification was noted. issues with test orders included incorrect or incomplete forms and unlabeled samples. samples were classified as non-viable if the sample quantity was insufficient, or if the samples were clotted, haemolysed, too old, or in the wrong container. results were stratified by department. an additional step as part of this quality improvement effort was to analyse the time required for results reporting. this consisted of tracking the amount of time that results were waiting in the laboratory for pick-up over a 24-hour period. a total of 25 patients had test orders sent to the laboratory during this observation period. no patient details were captured for these samples. information about test volume was obtained from the kch laboratory for the time period july 01, 2010 to june 30, 2011. this information is routinely collected by the laboratory and included, for each assay conducted, the total number of tests performed per month. overall test volume indicates a conservative estimate of the demand for testing in this setting, since some tests could not be performed because of equipment malfunctions and/or reagent shortages. the demand for certain assays is evidence of the types of pressures faced by the laboratory, since different assays have different time sensitivities and testing demands. we describe the critical steps of the laboratory testing workflow that involve information transfer. the three phases of testing are commonly broken down into nine discrete steps (order, collection, identification, transportation, preparation, analysis, reporting, interpretation, action); and we define the stages within kch, specifying for each step the location and staff members involved, as well as other pertinent details.15 the goal of this exercise is to identify the informational demands and potential process breakdowns in clinical laboratory testing at kch to better understand the potential role of informatics. ethical considerations this article describes a rationale for introducing informatics interventions to improve the quality of laboratory services in low-resource settings. the motivation is supported by results from previously-conducted quality improvement audits. no primary research was conducted, thus the work described here does not meet the criteria for requiring institutional review board approval. results top ↑ of the 3549 samples evaluated as part of the quality audit, 54% (n = 1923) were not testable (table 1). there was variation in this rate across the departments within the laboratory, ranging from 5% of samples for microbiology to 70% of samples sent to the blood bank department. an insufficient sample volume was the most common reason for a sample being deemed untestable (n = 1606). this characterised over 80% of untestable samples (n = 1923) and 45% of all audited samples (n = 3549). of the high number of samples that were considered untestable because of insufficient blood volume, the vast majority came from the paediatric department, where it is challenging to get sufficient blood from a sick and frequently dehydrated infant. test order forms were filled out either incorrectly or incompletely in just over 16% (n = 591) of audited samples. the least frequent problems identified were samples that were mixed up, too old, or in the wrong container. table 1: results of 2009 quality audit. between july 01, 2010 and june 30, 2011 the kch laboratory conducted 242 242 tests (table 2). the number of tests carried out for the parasitology and blood bank departments accounted for over half of the laboratory's total test workload during this time. the most common tests were: malaria parasites; full blood count; blood grouping and cross matches; and cd4 count. there was considerable monthly variation in the number of tests conducted, reflecting the seasonality of diseases such as malaria. the average monthly test load was 20 187 tests, with a standard deviation of 3263 tests. there were several classes of tests, including blood lipids and hormones, which were not conducted at all during this time period because of inoperative equipment and/or lack of reagents. table 2: kamuzu central hospital laboratory test workload, july 01, 2010 – june 30, 2011. figure 1 defines the total testing process workflow at kch according to the commonly-specified nine stages of laboratory testing, and indicates the staff responsible for the task.15,25 the process begins and ends on the patient ward with the physician, who makes the initial request for a laboratory test and also determines a course of action based on the interpretation of the test results. the pre-analytical phase is initiated with the ordering of a test and also includes the sample collection and identification or matching of the sample with the patient. these two tasks are both completed by a clinician or nurse and are associated with some of the issues identified in the quality audit, such as incomplete or incorrect labeling and sample mix-ups. incomplete or illegible labeling result from a number of factors, including insufficient label space and lack of necessary information at time of ordering. the pre-analytical phase then extends beyond the patient ward to include transport of the sample to the laboratory and preparation of the sample for testing by a laboratory technician. the transportation phase represents the transfer of the specimen and associated information from one department to another within the hospital; this task is not assigned to a specific job title in the hospital. it is most likely to be carried out by a nurse, a patient attendant, or a janitor, but there is no established routine for sample transport. samples waiting for transport to the laboratory are typically stored in a treatment room or at a nursing station, and are taken with varying frequency to the laboratory. it is thus clear that one challenge is a lack of awareness of the time required for sample transport, so issues such as delays or misplaced samples are not proactively addressed. in addition, time is spent during this stage transcribing various information from the test order form, so information is being duplicated. this phase concludes with the preparation phase, in which the laboratory technician uses the test order information to prepare the sample for analysis. informational gaps may cause delays at this point in the process because, just as the sample transport does not have a systematic workflow, the reporting of sample and test order errors back to the wards is similarly unstructured. figure 1: total testing process workflow at kch.1 description of staff involved in total test process at kch. steps shaded in grey indicate that information transfer is occurring. the analytical phase includes a single step, namely, the analysis of the laboratory sample. information is generated as part of this process and is combined with information supplied during the pre-analytical phase (patient age, gender, etc.) to generate a result. the manual nature of matching the test results with the corresponding patient based on patient name is another informational step that can cause delays and potentially lead to reporting errors. the process of reporting the result back to the physician is the start of the post-analytical phase and, again, is not a formalised process. it could be performed by a variety of personnel at unspecified frequencies. results are left in the laboratory entryway in cubby-style pigeon holes for pick-up. often, when someone is sent from a ward to drop off laboratory samples, they will also pick up and deliver any results that are available for that ward. this means that critical results may not be reported to the wards in an expedited manner. table 3 presents the duration of the reporting stage for laboratory results from a 24-hour observation of the laboratory. results were processed for all 25 patients who had test orders sent to the laboratory during the observation period. test orders were reduced as the haematology instrument was not operational at that time and polymerase chain reaction results for outpatients were delivered through a different mechanism. additionally, the hospital census was unusually low that day. of the 25 results, 18 were collected during the 24-hour observation window, whilst seven remained in the pigeon holes. the average duration of the reporting stage was just under six hours, with significant variation. two of the results were collected immediately because they were related to a critical patient and the laboratory called the ward when the results were available. on the other hand, over one-fifth of results spent more than 16 hours in the laboratory before being collected. table 3: result reporting turnaround time from a 24-hour quality audit (n = 18).† discussion top ↑ the results present a multi-faceted depiction of laboratory testing in a hospital in a low-resource setting, focusing on both the demand for laboratory testing and the informational challenges in meeting that demand. with more than 240 000 tests conducted at the kch laboratory during a one-year period, it is clear that laboratory services are very much in demand within the hospital, matching the rhetoric around the importance of accessible laboratory services in low-resource settings. however, the diagnostic process is information-intensive; and the quality audit and workflow analysis suggest that the capture, management, and transfer of this information are a significant barrier to maximising the laboratory's role at kch. the quality audit identified informational barriers in the collection and identification of samples. almost one-sixth of samples were untestable because of incomplete or incorrect paperwork. the quality audit was conducted as a quality improvement effort and, as motivation for the study, laboratory employees articulated several challenges associated with incorrect or incomplete test orders. firstly, patient details, such as age and gender, affect the interpretation of the results; and test orders that omit these data increase the likelihood of an interpretation error. secondly, the time and date of the sample is particularly important for tests sent to either the microbiology or the biochemistry departments, so the absence of this information compromises the accuracy of the test. finally, the current system for results reporting relies on ward information from the test order, so when this information is not included it is common for results to never reach the patient. these pre-analytical errors and delays are similar to those found in other low-resource laboratory environments.26 post-analytical delays were also evident from the analysis of results reporting, although the sample size was small and further examination is warranted. these inaccuracies have implications for both the hospital and the patients. untestable samples equate to wasted resources, including the physical supplies for the sample, such as the syringe and collection vessels, as well as employee time involved in collecting the sample and communicating the error. for patients, these errors amount to delays in care; surgical patients may face delays in scheduled surgeries if laboratory results are not ready, whilst for others it may mean an extra night in the hospital. efficient laboratory testing is particularly important for patients in critical condition, namely, those who face delays in life-saving care and/or empirical treatments prescribed in the absence of confirmatory laboratory results. the findings of the quality audit also informed the interpretation of the data around the frequency of laboratory testing. the information about test volume reflected tests conducted by the kch laboratory and therefore serves as a conservative measure of the demand for laboratory services at kch. there are at least two reasons that demand for laboratory services may be greater than that shown in table 1. first, samples deemed untestable may not always be corrected and re-sent to the laboratory, as the process for informing clinicians of untestable samples is unstructured and thus possibly lengthy; and clinicians may choose to pursue diagnosis and treatment without confirmatory laboratory results. second, material shortages, such as reagents and properly functioning equipment, may prohibit the performance of certain kinds of tests. the workflow analysis mapped the laboratory testing process at kch to the standard stages of testing and identified the informational components of each stage. this exercise identified a lack of formalised workflow around the transport of samples from the wards to the laboratory and the reporting of results from the laboratory to the wards. the reporting delays were confirmed in the study of reporting times, although a limitation of this component of the results is the small sample size. these gaps suggest that one unique aspect of the informational challenges faced by laboratory systems is the geographic scale of the testing process. unlike the paperwork issues identified in the quality audit, the workflow issue involves interactions amongst multiple agents across different departments. the standard approach to laboratory testing does not necessarily reconcile these multiple players and environments. information systems tend to have a departmental focus. for example, electronic medical records are patient-centric systems focusing primarily on managing clinical patient data. whilst this often includes laboratory test results, electronic medical records functionality does not extend into the laboratory. laboratory information systems, on the other hand, are typically specimen-centric systems, focusing on processes and workflows within the laboratory. in this scenario the generation of test orders and reporting of test results often falls within the gap between the electronic medical records and the laboratory information system. we propose a system for supporting specimen management, increasing visibility into the status of all orders for both clinical as well as laboratory staff and following a model embraced by courier companies (dhl/federal express/ups) to track and manage packages. this simple model has three main components: (1) the generation of a test order and associated paperwork; (2) the ability to monitor the status of the order, complete with exception alerts when applicable; and (3) electronic results reporting back to the ward. the approach of real-time monitoring of specimens as they move through each stage of the process is novel and is likely to have a higher impact in a low-resource setting, where challenges are arguably greater. in table 4, we summarise problems identified in the kch workflow and present proposed informatics interventions for each. table 4: problems identified and proposed informatics solutions. the proposed informatics interventions address problems identified at kch that would not generally be considered as being within the scope of a traditional laboratory information system implementation.27 this application of informatics to the pre-analytical and post-analytical phases is novel and these stages are natural targets for informatics interventions because they are information-intensive and often where errors arise.17 such an approach, which recognises that much of the total testing process takes place outside the laboratory, may also improve the perception of laboratory testing amongst clinicians. whilst clinicians in many of these settings have a tendency to circumvent laboratory testing in favour of more superficial, less accurate diagnostic signals, they may be more likely to opt for laboratory testing if they perceive it to be quick and accurate.7,8 we recognise that informatics interventions cannot solve all problems. whilst our proposed informatics intervention does not prevent insufficient samples from being sent to the laboratory, it provides a formal mechanism for reporting and correcting those errors, potentially saving time in the laboratory testing process and improving the timely delivery of care. a variation of the proposed intervention could include decision support tools that remind the nurse preparing the order of the sample requirements (sample amount, container type, etc.). the need for investment in laboratory infrastructure for disease prevention and control is recognised in the literature.28 resource shortages, such as laboratory technicians, microscopes and access to electricity, are commonly cited as limiting factors in improving laboratory services.29 the informational challenges of laboratory testing in low-resource settings, whilst more challenging to identify than the resource limitations, will limit the impact of additional resources if unaddressed. this article synthesised an array of data about laboratory operations in a low-resource hospital setting, calling attention to these informational challenges. next steps will include a larger-scale effort to document the pre-analytical and post-analytical phases at kch and other low-resource hospital laboratories. in addition, whilst the proposed interventions are hypothetical at this point, we are in the early stages of modeling such systems. limitations of the study this study focuses on the informational challenges associated with laboratory testing at kch and does not consider other challenges that may serve as limiting factors, such as the availability of reagents and other physical resources required for testing. resolving informational challenges in the laboratory workflow may have a limited impact on overall laboratory performance if resource-based constraints are present. furthermore, the findings from the results reporting study must be interpreted with caution, as the sample size was small. recommendations future research will attempt to further quantify the workflows and challenges within the laboratory at kch as well as those in other low-resource settings. in addition, evaluation of informatics solutions targeting the laboratory will speak to the extent that informational challenges are limiting the stature and role of the laboratory at kch. conclusion in this article, we present a multi-faceted depiction of the laboratory testing process and informational challenges in a low-resource setting. one criticism of laboratory process analyses, even in more advanced settings, is the focus on the analytical phase.27 we presented evidence that encompassed all three stages of testing and identified two specific informational challenges: (1) complete testing paperwork; and (2) efficient, timely communication between the wards and laboratory. indeed, these issues were identified because of the wider focus on the preand post-analytical phases, which capture the multiple players and locations involved in the complete laboratory testing process. informational barriers and inefficiencies arose at the transition points, the transfer of responsibility from one role or location to another during the testing process. whilst information such as test orders and results should support workflow and decision making, in this case it appears that challenges in information management are undermining these processes. the plight of laboratory services in low-resource settings is at once loudly decried and woefully under-investigated. here we presented examples of informational barriers in the preand post-analytical phases of the total testing process in a hospital in a low-resource setting – challenges which, by their nature, are predisposed to be mitigated or potentially even eliminated by informatics interventions. future work will attempt to design, implement, and evaluate informatics solutions to these and other barriers to more efficient and integral laboratory systems in low-resource settings. trustworthiness top ↑ the results presented represent the actual findings of the analyses described in the research method and design section, without alteration. reliability and validity the quality audit and turnaround time study reflect standard methods of measuring laboratory performance; and test volume is a standard measure of laboratory workload. acknowledgements top ↑ the authors would like to acknowledge the contributions of kch staff who conducted the data collection. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions j.d. (university of pittsburgh) led the analysis and manuscript writing; n.a., h.l., g.g. and e.c. (kamuzu central hospital) assisted with study concept and data acquisition; o.j.g. (baobab health) contributed to study design; a.b.-s. (university of pittsburgh) assisted with study concept, data analysis, and manuscript drafting; and g.p.d. (university of pittsburgh) assisted with study concept, data acquisition, interpretation of data and manuscript writing. all authors critically revised the manuscript. references top ↑ sokhna c, mediannikov o, fenollar f, et al. point-of-care laboratory of pathogen diagnosis in rural senegal. plos negl trop dis. 2013;7(1):e1999. wilson d, howell v, toppozini c, et al. against all odds: diagnosing tuberculosis in south africa. j infect dis. 2011;204(suppl 4):s1102–s1109. http://dx.doi.org/10.1093/infdis/jir453 wilson ml. laboratory diagnosis of malaria: conventional and rapid diagnostic methods. arch pathol lab med. 2013;137(6):805–811. http://dx.doi.org/10.5858/arpa.2011-0602-ra el-amin eo, elbashir mih, elamin oe, et al. the underlying aetiologies of coma in febrile sudanese children. trans r soc trop med hyg. 2013;107(5):307–312. http://dx.doi.org/10.1093/trstmh/trt013 moon am, biggs hm, rubach mp, et al. evaluation of in-hospital management for febrile illness in northern tanzania before and after 2010 world health organization guidelines for the treatment of malaria. plos one. 2014;9(2):e89814. http://dx.doi.org/10.1371/journal.pone.0089814 abreha t, alemayehu b, tadesse y, et al. malaria diagnostic capacity in health facilities in ethiopia. malar j. 2014;13:292. http://dx.doi.org/10.1186/1475-2875-13-292 polage cr, bedu-addo g, owusu-ofori a, et al. laboratory use in ghana: physician perception and practice. am j trop med hyg. 2006;75(3):526–531. nankabirwa j, zurovac d, njogu jn, et al. malaria misdiagnosis in uganda – implications for policy change. malar j. 2009;8:66. http://dx.doi.org/10.1186/1475-2875-8-66 rambau pf. pathology practice in a resource-poor setting: mwanza, tanzania. arch pathol lab med. 2011;135(2):191–193. choge jk, magak ng, akhwale w, et al. symptomatic malaria diagnosis overestimate malaria prevalence, but underestimate anaemia burdens in children: results of a follow up study in kenya. bmc public health. 2014;14:332. http://dx.doi.org/10.1186/1471-2458-14-332 nkengasong jn, nsubuga p, nwanyanwu o, et al. laboratory systems and services are critical in global health: time to end the neglect? am j clin pathol. 2010;134(3):368–373. http://dx.doi.org/10.1309/ajcpmpsinq9brmu6 mundy c, ngwira m, kadewele g, et al. evaluation of microscope condition in malawi. trans r soc trop med hyg. 2000;94(5):583–584. http://dx.doi.org/10.1016/s0035-9203(00)90098-1 harries a, zachariah r, bergström k, et al. human resources for control of tuberculosis and hiv-associated tuberculosis [unresolved issues]. int j tuberc lung dis. 2005;9(2):128–137. ministry of health, malawi. clinical management of hiv in children and adults. lilongwe, malawi; 2011. hawkins rc. laboratory turnaround time. clin biochem rev. 2007;28(4):179–194. lundberg gd. acting on significant laboratory results. jama. 1981;245(17):1762–1763. http://dx.doi.org/10.1001/jama.1981.03310420052033 manor pg. turnaround times in the laboratory: a review of the literature. clin lab sci. 1999;12(2):85–89. fraser hs, allen c, bailey c, et al. information systems for patient follow-up and chronic management of hiv and tuberculosis: a life-saving technology in resource-poor areas. j med internet res. 2007;9(4):e29. http://dx.doi.org/10.2196/jmir.9.4.e29 fraser hsf, biondich p, moodley d, et al. implementing electronic medical record systems in developing countries. inform prim care. 2005;13(2):83–95. driessen j, cioffi m, alide n, et al. modeling return on investment for an electronic medical record system in lilongwe, malawi. j am med inform assoc. 2013;20(4):743–748. http://dx.doi.org/10.1136/amiajnl-2012-001242 allain tj, van oosterhout jj, douglas gp, et al. applying lessons learnt from the ‘dots’tuberculosis model to monitoring and evaluating persons with diabetes mellitus in blantyre, malawi. trop med int health. 2011;16(9):1077–1084. http://dx.doi.org/10.1111/j.1365-3156.2011.02808.x douglas gp, gadabu oj, joukes s, et al. using touchscreen electronic medical record systems to support and monitor national scale-up of antiretroviral therapy in malawi. plos med. 2010;7(8):e1000319. http://dx.doi.org/10.1371/journal.pmed.1000319 hoffman m, mofolo i, salima c, et al. utilization of family members to provide hospital care in malawi: the role of hospital guardians. malawi med j. 2013;24(4):74–78. gamadzi g, chitandale e, yohane i. kch lab quality assurance programme update (internal report). lilongwe, malawi: kamuzu central hospital; 2009. plebani m, lippi g. closing the brain-to-brain loop in laboratory testing. clin chem lab med. 2011;49(7):1131–1133. http://dx.doi.org/10.1515/cclm.2011.617 mbah ha. phlebotomy and quality in the african laboratory. afr j lab med. 2014;3(1), art. #132, 4 pages. http://dx.doi.org/10.4102/ajlm.v3i1.132 lober wb, revere d, hills r. a lab-emr interoperability profile as an ehealth architecture component for resource-constrained settings. stud health technol inform. 2010;160(pt 1):257–261. nkengasong jn. a shifting paradigm in strengthening laboratory health systems for global health: acting now, acting collectively, but acting differently. am j clin pathol. 2010;134(3):359–360. http://dx.doi.org/10.1309/ajcpy5asuejyq5rk petti ca, polage cr, quinn tc, et al. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377–382. http://dx.doi.org/10.1086/499363 abstract background description of the intervention results lessons learnt recommendations acknowledgements references about the author(s) emma e. kploanyi school of public health, university of ghana, legon, accra, ghana joseph kenu school of public health, university of ghana, legon, accra, ghana benedicta k. atsu school of public health, university of ghana, legon, accra, ghana david a. opare national public health and reference laboratory, ghana health service, accra, ghana franklin asiedu-bekoe public health division, ghana health service, accra, ghana lee f. schroeder department of pathology and clinical laboratories, university of michigan, ann arbor, michigan, united states david w. dowdy department of epidemiology, johns hopkins bloomberg school of public health, baltimore, maryland, united states alfred e. yawson department of community health, university of ghana medical school, accra, ghana ernest kenu school of public health, university of ghana, legon, accra, ghana citation kploanyi ee, kenu j, atsu bk, et al. an assessment of the laboratory network in ghana: a national-level atlas survey (2019–2020). afr j lab med. 2023;12(1), a1844. https://doi.org/10.4102/ajlm.v12i1.1844 note: additional supporting information may be found in the online version of this article as online supplementary document 1. lessons from the field an assessment of the laboratory network in ghana: a national-level atlas survey (2019–2020) emma e. kploanyi, joseph kenu, benedicta k. atsu, david a. opare, franklin asiedu-bekoe, lee f. schroeder, david w. dowdy, alfred e. yawson, ernest kenu received: 11 feb. 2022; accepted: 29 sept. 2022; published: 08 feb. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: integrated health systems with strong laboratory networks are critical in improving public health. the current study assessed the laboratory network in ghana and its functionality using the assessment tool for laboratory services (atlas). intervention: a national-level laboratory network survey was conducted among stakeholders of the ghanaian laboratory network in accra. face-to-face interviews were conducted from december 2019 to january 2020, with follow-up phone interviews between june and july 2020. also, we reviewed supporting documents provided by stakeholders for supplementary information and transcribed these to identify themes. where possible, we completed the laboratory network scorecard using data obtained from the atlas. lessons learnt: the laboratory network (labnet) scorecard assessment was a valuable addition to the atlas survey as it quantified the functionality of the laboratory network and its overall advancement toward achieving international health regulations (2005) and global health security agenda targets. two significant challenges indicated by respondents were laboratory financing and delayed implementation of the ghana national health laboratory policy. recommendations: stakeholders recommended a review of the country’s funding landscape, such as funding laboratory services from the country’s internally generated funds. also, they recommended laboratory policy implementation to ensure adequate laboratory workforce and standards. keywords: laboratory systems; laboratory network; atlas; epidemic-prone diseases; labnet scorecard; laboratory capacity; laboratory readiness; laboratory strengthening. background integrated health systems with effective and efficient laboratory networks1,2 are gaining wide recognition for their critical role in managing high-burden endemic infections, particularly hiv, tuberculosis and malaria,3,4 and accelerating disease outbreak response. the importance of laboratory outbreak response has been highlighted by the severe acute respiratory syndrome (sars, 2003), h1n1 (2009), meningitis (2009), cholera (2010), middle east respiratory syndrome (2012), ebola virus disease (2014), zika virus disease (2015), and yellow fever (2016–2017) outbreaks, as well as the current coronavirus disease 2019 (covid-19) pandemic.5,6,7,8,9 there are considerable delays in outbreak detection and communication for most infectious disease outbreaks originating from africa,10,11 as exemplified in the delayed response to the west african ebola outbreak, which was reported four months after the index infection and, more recently, the delayed diagnosis of suspected covid-19 cases in africa.6,12,13 also, the covid-19 pandemic has underscored the diagnostic capacity challenges facing african countries. as of january 2022, ghanaian sars coronavirus 2 (sars-cov-2) daily testing rates was around 70 per 1000 people, in contrast to over 2300 per 1000 people in the united states.14 improving laboratory networks is a critical step toward controlling health emergencies.15 the global health security agenda (ghsa) requires countries to establish tiered national laboratory systems and ‘determine an appropriate level of diagnostic capability at each level of the public health hierarchy from national to the district’.16 the laboratory network in ghana was assessed in 2006, focusing on hiv diagnostic resources, using the assessment tool for laboratory services (atlas).17 the assessment identified the need for guidelines in biosafety, inventory control and logistics management information systems, and the application of policies and procedures.18 however, there have been no publicly available assessments of the laboratory network in ghana since then. in this study, we conducted the atlas survey with a wider scope, including a laboratory network (labnet) scorecard evaluation.19 the atlas survey was conducted using the central-level questionnaire to describe the national laboratory network in terms of its organisational structure, management of laboratory services and logistics, as well as quality regulation. a portion of the labnet evaluation was added to provide a quantitative measure of functionality to corroborate the qualitative findings from the atlas survey on core capabilities. the assessment was conducted to identify strengths and weaknesses of the laboratory network and provide recommendations for its improvement. description of the intervention ethical considerations the study obtained ethical approval from the ghana health service ethical review committee (ghs-erc 005/05/19) and noguchi memorial institute for medical research institutional review board (fwa 00001824). in addition, the director-general of the ghana health service (ghs) granted permission before the study was conducted. written consent was obtained from every participant before conducting an interview. study design and setting this was a qualitative study conducted among key stakeholders in the laboratory network in accra, ghana. it is part of a more extensive, ongoing study to map and model the laboratory network in ghana for three epidemic-prone diseases (epds) – bacterial meningitis, measles and yellow fever – and three diseases of public health importance (dphi): hiv, tuberculosis and hepatitis c virus (hcv). these diseases guided our choice of stakeholder interviewed. data collection we had a consultation with stakeholders from the national public health & reference laboratory (nphrl) and disease surveillance department of ghs in november 2019 to adapt the national-level united states agency for international development atlas20 to suit the ghanaian context, as recommended in the atlas guidelines. we identified stakeholders to be interviewed with the atlas during the consultation session. the atlas comprises nine sections: organisation, policy, forecasting and procurement, financing, storage and distribution, inventory control system, laboratory information management system, supervision, and general questions. face-to-face interviews were conducted from december 2019 to january 2020, with follow-up phone interviews conducted between june 2020 and july 2020. also, supporting documents provided by stakeholders were reviewed for supplementary information, and responses were transcribed to identify themes. where possible, data were used to complete the labnet scorecard.19 the labnet scorecard has been validated for assessing a country’s laboratory network functionality in implementing the international health regulations (2005) and attaining the ghsa targets.19 our labnet scorecard assessment was based on five of the nine core capabilities closely related to aspects of the laboratory network assessed by the atlas: political, legal, regulatory and financial framework; structure and organisation of the laboratory networks; laboratory information (management system); infrastructure, equipment and supplies; and quality of laboratory services. the scores were converted to percentages reflecting overall advancement toward standards or targets, and interpretations were drawn on each component’s functionality stage based on the weakest score (supplementary table 1). survey implementation a total of 12 respondents were interviewed: two representatives from clinical laboratories unit (clu) under the institutional care division, ghs; deputy directors at supplies, stores and drugs management (ssdm) and the finance division, ghs; the head and quality manager for nphrl; and a representative each from the health facilities regulatory agency and allied health professionals council. relevant stakeholders from disease control programmes including the expanded programme of immunisation (epi) with focus on epds, national tuberculosis control programme (ntp), national aids and sexually transmitted infections control programme (nacp) and national viral hepatitis control programme (nvhcp) were also interviewed. results organisational structure of clinical and public health laboratories the ghs tiers into the national, regional, district and sub-district levels with the clu coordinating vertical laboratory activities (figure 1). respondents reported over 800 public clinical laboratories, including 10 regional, 112 district and about 692 sub-district health facility laboratories. based on data from the national database district health information management system ii (dhims2) (ghana health service with technical assistance from the university of oslo health informatics department, accra, ghana), the ghs laboratories conduct biochemistry (n = 342), haematology (n = 397) and microbiology tests (n = 365). the public health division of the ghs is responsible for the public health laboratories (phls) that surveil measles, rubella, yellow fever, tuberculosis, hiv and other diseases. the nphrl in accra has oversight responsibility for three other phls situated in tamale (northern zone), kumasi (middle zone), and sekondi (western zone). figure 1: organisational structure of clinical and public health laboratories in ghana as of january 2020. the atlas assessment provided the number of laboratory facilities in each category in january, 2020. laboratories in the ghs organisational structure are tiered into three levels: tertiary (nphrl and teaching hospital laboratories), secondary (regional hospital laboratories and zonal phls) and primary laboratories (laboratories in district, sub-district facilities and health centres). the relationship between tiers is based on referrals, flowing from lower-tier to higher-tier laboratories, with higher-tier facilities assigned some oversight responsibilities on lower-tier laboratories (e.g., outreach training and supportive supervision [otss]). the four teaching hospitals, although directly under ministry of health (moh) but not ghs, only play a key role in the referral system. the regional laboratory scientists also serve as laboratory coordinators in various regions. however, the districts lack such coordinators due to inadequate staff capacity. the epi, nacp, ntp and nvhcp collaborate with clinical laboratories and phls to conduct laboratory testing. the epi has outsourced all laboratory services and logistics management for epds to nphrl. while the nacp has art sites at 488 facilities, including laboratories, the ntp works with about 337 laboratories in the health system. the nphrl coordinates national-level activities of nvhcp, whereas the health directorates coordinate activities at the regional and district levels. there are no specific laboratories designated for hcv testing under the current programme. policies and other guidelines the moh had no dedicated laboratory policy development unit at the time of assessment. thus, the clu represents the ghs on clinical laboratory policy issues. the laboratory technical committee comprising members from all agencies of moh and ghs developed the ghana national health laboratory policy21 that was approved in 2013; however, the ghana national health laboratory policy is still not operational. the policy describes the laboratory organisation, services and test menu by level, staffing norms, logistics management, quality management and laboratory information system. the primary level test menu covers basic parasitology and bacteriology, cytology, histopathology, serology, clinical chemistry and haematology tests. in contrast, the secondary level test menu covers more sophisticated testing such as immunohistochemistry and nucleic acid testing. finally, the tertiary level test menu covers more specialised tests and the phls focus on epd-dphis in support of public health surveillance (figure 2). figure 2: test menu for primary, secondary and tertiary tiers in the ghanaian laboratory network, 2020. the policy document on infection prevention and control22 is fully operational. it contains guidelines that cover infection prevention and biosafety. the phls perform tests using standard operating procedures (sops). also, the clinical laboratories conform to international and funding programme standards. however, harmonised general clinical testing is lacking, particularly the use of different automated equipment at the facility level for haematology, immunology and chemistry. financing funding for laboratory services in the country is fragmented for infrastructure, supplies and equipment. there are different funding sources for target programmes and phls; general clinical testing relies on internally generated funds and national health insurance system (nhis) reimbursement on diagnostic services and tests listed on the nhis benefits package.23 the government of ghana only provides funding for infrastructure, some equipment and workers’ salaries. the primary funding donors were the global fund (figure 3), which provided the most funding for hiv and tuberculosis, and the world health organization (who), which funds epds. currently, no donor funding is available for hcv laboratory services, nhis covers only hcv screening and remaining costs are borne by patients. stakeholders estimated a 20% – 50% gap in funding for the six epd-dphis. figure 3: financing of laboratory services for priority disease conditions in ghana, 2020. different units and divisions coordinate programme-specific testing for epd-dphis. most programmes procure laboratory supplies at the central level rather than allocate funds to laboratories. the nphrl receives most supplies and funds from donors, allocating these to the zonal phls through their divisional heads. outbreaks also determine the allocation of financial resources. target programmes and the nphrl have separate budgetary line items for laboratory services, supplies and equipment. however, the finance unit of the ghs works with a highly aggregated budget, the medium term expenditure framework plan for moh24; hence, there is no specific budget line for laboratory services. since ghana attained lower-middle-income status, a transition plan was implemented in 2015, requiring the government to increase funding each year with a full transition scheduled in 2022. forecasting and procurement the target programmes, nphrl and health facilities consistently forecast and procure needed laboratory supplies; however, there is no standardised forecasting method. the epi does not prepare forecasts for epds because they have outsourced this role to nphrl. the situation is quite peculiar for nvhcp as they also prepare forecasts but do not receive funding for laboratory supplies. unless otherwise requested by funders, the public procurement act 663 guides procurement as amended in 2016. for example, at ntp, a procurement request to ssdm initiates an open tender with the selection of vendors by the central tender committee. a diverse stakeholder team monitors the procurement process. the average lead time is programme-specific, being six months for nacp and inconsistent for ntp due to time lags between contract award and item supply. although the programmes operate independently, there are instances when they carry out joint activities, especially between the ntp and nacp. their procurement units lead procurement and monitoring for general clinical testing of their target disease conditions at the facilities. all programmes indicated adequate laboratory supplies, except the nvhcp, which has no funding for laboratories. the nacp supplies reagents and haematology and chemistry analysers to the art sites, whereas the ntp supplies reagents to all tuberculosis testing laboratories. however, there was little information on the adequacy of supplies at the phls and clinical laboratories, as this assessment was conducted only at the national level. although multiple systems manage laboratory supplies, respondents reported that duplicating efforts had been minimised. storage and distribution the integrated scheduled delivery system implements the last mile distribution strategy of laboratory supplies and equipment to all levels other than phls. currently, there are four storage facilities for laboratory supplies and cold chain reagents at the central level, and although these storage facilities have adequate cold chain capacity, the regional stores do not. thus, a central medical store is being built for future storage needs. a third-party logistics firm is contracted for distribution as ghs delivery vehicles are insufficient. the nphrl handles supplies at the national level for onward distribution to the zonal phls. the programmes intervene when there is a need for redistribution or emergency distribution. inventory control the international organization for standardization (iso) 15189 requires establishing an inventory system with preset minimum and maximum stock levels. hence, sops have been developed by the ssdm indicating the minimum, reorder points and maximum stock levels for facilities. the minimum and maximum stock levels are two and three months for the central level and three and six months for the regional medical stores. the laboratory facilities determine order quantities for general clinical testing supplies, higher-level authorities at the central level for programmes, and the head of nphrl for all phls. the laboratory scientists at the facilities collaborate with their procurement units to conduct stock-taking twice annually, using various mechanisms, such as whatsapp platforms (meta platforms, menlo park, california, united states), microsoft excel spreadsheets (microsoft, redmond, washington, united states), and gx-alerts (cepheid, sunnyvale, california, united states), aside from the monthly stocks reports. the who monitors stock for measles, rubella and yellow fever through kit management reports regularly submitted by the nphrl, while the nphrl monitors activities, including stock balances, at the zonal phls. specimen transport system based on the current testing algorithm, sputum samples from all new suspected cases are tested using genexpert® machines (cepheid, sunnyvale, california, united states). thus, the available 127 genexperts® are strategically placed within a 2-h drive to most facilities, easing specimen referral. the ghana postal courier service is contracted to transport samples on specific pickup days, ensuring collection within two days. tuberculosis drug resistance testing is performed at five laboratories, with specimen referral arranged by the requesting facility through public transport or courier service. hiv viral load testing and early infant diagnosis are performed at 10 regional and four teaching hospitals. as with tuberculosis diagnosis, the courier service transports hiv specimens on facility-specific pickup days. the requesting facility arranges specimen transport for all other specimen referrals. laboratory information system management the health information management system used in all health facilities is the dhims2. this database is an upgrade from the dhims, which incorporates tracking by target programmes and improves cause-of-death statistics. like most clinical databases, the dhims2 captures laboratory service data at an aggregate level down to facility, but not patient level. the basic laboratory information system also reports into dhims2 periodically. similarly, e-tracker data for hiv viral load are captured in the dhims2. two other databases do not directly report data into dhims2: gx-alert for genexpert, and epiinfo (epi info™, centers for disease control and prevention, atlanta, georgia, united states) or data on epds. gx-alert data are aggregated at the facility level and entered manually into dhims2 whereas epiinfo captures data on the who disease network that are reported directly to the who. no entries are made into dhims2 (figure 4). figure 4: reporting system for laboratory services management information in ghanaas of 2020. the phls prepare weekly yellow fever reports and communicate positive results via phone. other reports are submitted either electronically or as hard copies or by both methods. standard national forms used to collect and report data into dhims2 include case-based forms and other national forms developed by the clu for haematology, chemistry and microbiology testing. they also provide demographic data crucial for planning immunisation activities. there are also logbooks at the laboratories that provide information on the laboratory tests requested and conducted. however, only aggregated data are available at the national level. as described, reports from these databases are useful for quantifying commodities during forecasting, procurement, inventory control, targeted screening, planning public health interventions, notifying international organisations on disease burden and outbreaks, and seeking donor support. however, not all facilities submit data as and when due. the nphrl monitors reporting rates and supports supervision exercises organised by phls. supervision the clu does not supervise private laboratories. instead, it carries out supportive supervision for general clinical testing at the regional level. another regional-level team supervises the district and sub-district levels. in addition, the clu performs supervisory visits every six months using the otss checklist for malaria diagnosis and the integrated monitoring and supervision checklist for general supervision. for the phls, a national-level supervisory team monitors reagent availability, storage and shelf life, and staff competencies and credentials using checklists. supervision is quarterly at the nphrl but less frequent at the zonal phls due to insufficient logistics; however, the laboratory managers monitor daily activities. target programmes, mainly nacp and ntp, organise supervisory visits to laboratories every quarter or twice annually, depending on resources. these programmes are building local capacity so that representatives can supervise activities at the district level. the epi only provides supervision and support for the national and regional cold rooms. quality regulation and accreditation at the time of assessment, no laboratory had been iso accredited. however, the clu has assessed some laboratories preparing for accreditation using the stepwise laboratory improvement process towards accreditation checklist.25 the clu also carries out external quality assurance through otss (supplementary figure 1), supporting the implementation of iso 15189. the nphrl and zonal phls have implemented quality management systems based on this standard with self-regulation. the health facilities regulatory agency under the moh regulates laboratory quality independently of the clu. they issue laboratory operation licenses based on an intensive checklist inspection, with tiered accreditation: 50% (6-month license) or 90% (3-year renewal) of items. unannounced laboratory monitoring is carried out twice annually and could lead to a reassignment of the tier, influencing the laboratory’s nhis reimbursement. the allied health professionals council regulates laboratory professionals through licensing examinations and sanctions. although the council is enjoined by act 857 to supervise practitioners, this is lacking due to fund constraints. instead, their interventions are responses to malpractice brought to their attention. the assessment of laboratory network functionality using the laboratory network scorecard the self-report of stakeholders on five of the nine core capabilities of the laboratory network using the labnet scorecard indicated that the laboratory information management system advanced the most (74%) toward achieving the international health regulations (ihr) and ghsa targets (figure 5; supplementary table 1). three capabilities were similarly rated: structure and organisation of the laboratory networks (58%), infrastructure, equipment and supplies (62%), and quality of the laboratory services (63%). however, the political, legal, regulatory and financial framework rated the lowest (33%), with the finance component at the least functional stage as it lacked vital attributes. figure 5: country results for assessment of the laboratory network in ghana using the labnet scorecard, 2020. (a) overall advancement of core capabilities towards the standards taking all scores into consideration; (b) stages of functionality highlighting weak scores. lessons learnt experiences although atlas guidelines recommend group discussions for administering the tools, we adopted key informant interviews due to stakeholders’ conflicting and limited availability for a group discussion. multiple stakeholders with in-depth knowledge and experience in one or more tool sections were interviewed to obtain a holistic picture of the current laboratory network. this mode of administering the tool offered the added advantage of receiving more detailed responses than obtained in a time-limited group discussion. moreover, the key informant interviews served as a basis to receive further information from respondents to consolidate findings. some respondents recommended other stakeholders that could better respond to some sections of the tool; hence, the earlier list of stakeholders generated from the consultative meeting was updated a few times. the labnet scorecard assessment was a valuable addition to the atlas survey as it provided quantitative responses that served as a reflection of the overall advancement toward standards and targets set by ihr (2005) and ghsa. based on the experiences described, we drew lessons, and identified some strengths and challenges: lessons the atlas was a useful baseline assessment of the entire laboratory network because multiple stakeholders with in-depth knowledge and experience in one or more tool sections were interviewed. the labnet scorecard assessment was a useful addition to the atlas survey as it assessed the functionality of the laboratory network through quantitative responses that served as a reflection of the overall advancement toward targets set by ihr (2005) and ghsa. the lowest labnet score was obtained in the ‘legal and regulatory framework’ assessment; the ghana national health laboratory policy although approved has not yet been implemented, driving the lack of harmonised test menu, reagents and equipment. thus, the laboratory policy needs to be implemented to ensure an adequate workforce and standardisation of laboratories. a weak legal and regulatory framework impacts all other labnet core components and must be addressed first. vertical programmes rely heavily on external funding, whereas health facilities solely rely on internally generated funds for laboratory supply procurement and service delivery. this presents the opportunity for a better collaboration with vertical programmes to access external funding or adapt the funding mechanisms of health facilities through targeted policy and administrative interventions. the supervision and quality regulation organised by the administrative units and vertical programmes align with iso standards and could be leveraged for the accreditation of laboratories. strengths the assessment found a collaboration between disease control programmes, clinical and phls in the forecasting, procurement and distribution of laboratory supplies, testing for priority target disease conditions, and supportive supervision for laboratory testing. the assessment also found nhis reimbursement on diagnostic services and tests listed on the benefits package as an opportunity to increase local financing of laboratory services. some gaps identified by the first atlas assessment18 have been addressed over the years through the development and implementation of the policy for infection prevention and control, and sops guiding inventory control and laboratory testing. it is commendable that the country has a national database into which data from clinical and phls are reported and easily accessed for decision-making. the labnet assessment indicated that the laboratory information management system advanced the most toward achieving ihr and ghsa targets. the specimen transport arrangements between some target programmes (ntp and nacp) and ghana postal courier service serve as a good foundation to developing a specimen referral and transport system for all priority conditions. challenges laboratory funding was a major challenge indicated by most respondents. target programmes rely heavily on external funding; the clu reported that the internally generated funds and the often-delayed nhis reimbursements were insufficient to maintain testing capacity (figure 3). funding from the government of ghana was insufficient for laboratory services, and there was no specific budget line for these services at the central level. inadequate funds at the central level affect holistic and integrated supervision. currently, the clu takes advantage of the otss for malaria diagnosis to conduct general supervision. three out of five stakeholders from clu, nphrl and epi indicated that some laboratories run out of reagents and other supplies due to insufficient funds. the nvhcp has especially suffered from inadequate funding for its activities. programmes that receive external funding do not have the liberty of discretionary expending as funders dictate spending. another challenge is the delayed implementation of the laboratory policy approved in 2013. as a result, test menu harmonisation and reagents and equipment standardisation are a challenge, affecting specimen and patient referral to higher tiers. in addition, there is no organised system at the national level for sample transportation except for hiv and tuberculosis diagnoses. study limitations this assessment was carried out to characterise the laboratory network in ghana using the atlas, a qualitative tool. in addition, some aspects of the labnet scorecard were used to measure functionality quantitatively in support of findings on five out of the nine core capabilities that were related to some aspects of the atlas administered. ideally, the entire qualitative and quantitative labnet evaluation should be applied as part of a multisectoral workshop led by the moh, with results based on consensus. thus, future assessments should consider administering the entire labnet tool and convene workshops to validate findings. conclusion laboratories under the ghana health service are tiered into three levels with the clinical laboratories vertically coordinated by the clu whereas the public health unit coordinates the phls. the laboratories collaborated with target programmes in the forecasting, procurement and distribution of laboratory supplies, supportive supervision and testing for priority conditions. no laboratory had yet received iso accreditation at the time of assessment although some had been assessed using the stepwise laboratory improvement process towards accreditation checklist. in terms of functionality, the laboratory information management system advanced the most toward achieving ihr and ghsa targets whereas the political, legal, regulatory and financial framework lagged behind with the finance component at the lowest stage of functionality. major gaps were identified in laboratory financing and the implementation of the national health laboratory policy. these should represent the focus of future initiatives to strengthen the laboratory network in ghana. recommendations stakeholders recommended that the funding landscape for the country be reviewed. considerations should be made to fund laboratory services from the country’s internally generated funds and expand the scope of nhis. also, the moh should create a laboratory unit to collaborate with administrative units for laboratories in all their agencies to update and implement the national health laboratory policy to guide improvement of the laboratory network towards achieving the ghsa targets. moreover, plans for a national sample referral and transportation system that accommodate a wide range of clinical testing needs should be operationalised. the collaboration between administrative units and target programmes could be improved through joint supervision to optimise the resources available. also, the quality regulation in laboratories align with iso standards and could be leveraged for the accreditation of laboratories. a follow up labnet assessment could be conducted to help document the laboratory network’s progress towards attaining the ghsa targets on all nine core capabilities. acknowledgements the authors are grateful to all stakeholders in the ghanaian laboratory network who provided their inputs during data collection and validation. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions e.k., l.f.s., d.w.d. and a.e.y. designed the study; all authors contributed to the implementation of the research. e.e.k. analysed the results and wrote the manuscript with support from j.k., b.k.a., l.f.s., d.a.o. and f.a.-b. all authors provided critical feedback and contributed to the final manuscript. sources of support the study was funded by the united states national institutes of health (1 r01 ai136977-01a1) under the terms of the agreement awarded to the regents of the university of michigan. data availability the data that support the findings of this study are available on request from the corresponding author, e.k. disclaimer the views expressed in this article are the authors’ own, not an official position of the collaborating institutions or the funding agency responsible and no official endorsement should be inferred. references olmsted ss, moore m, meili rc, et al. strengthening laboratory systems in resource-limited settings. am j clin pathol. 2010;134(3):374–380. https://doi.org/10.1309/ajcpdqosb7qr5glr world health organization. the maputo declaration on strengthening of laboratory systems. in: consensus meeting on clinical laboratory testing harmonization and standardization [homepage on the internet]. maputo: world health organization – regional office for africa; 2008 [cited 2021 nov 10]. available from: https://cdn.who.int/media/docs/default-source/inaugural-who-partners-forum/lab-system-strengthening-maputo-declaration-200850aaec2a-1644-4209-801a-cb477d270e2e.pdf?sfvrsn=9d2694f0_1&download=true kassebaum nj, arora m, barber rm, et al. global, regional, and national disability-adjusted life-years (dalys) for 315 diseases and injuries and healthy life expectancy (hale), 1990–2015: a systematic analysis for the global burden of disease study 2015. lancet. 2016;388(10053):1603–1658. https://doi.org/10.1016/s0140-6736(16)31460-x wang h, naghavi m, allen c, et al. global, regional, and national life expectancy, all-cause mortality, and cause-specific mortality for 249 causes of death, 1980–2015: a systematic analysis for the global burden of disease study 2015. lancet. 2016;388(10053):1459–1544. acheampong g, owusu m, nkrumah b, et al. laboratory capacity in covid-19 diagnosis and the need to enhance molecular testing in ghana. glob j health sci. 2021;6(1):10–17. https://doi.org/10.1080/23779497.2021.1908157 oladipo ek, ajayi af, odeyemi an, et al. laboratory diagnosis of covid-19 in africa: availability, challenges and implications. drug discov ther. 2020;14(4):153–160. https://doi.org/10.5582/ddt.2020.03067 ondoa p, kebede y, loembe mm, et al. covid-19 testing in africa: lessons learnt. lancet microbe. 2020;1(3):e103. https://doi.org/10.1016/s2666-5247(20)30068-9 sands p, mundaca-shah c, dzau vj. the neglected dimension of global security – a framework for countering infectious-disease crises. n engl j med. 2016;374(13):1281–1287. https://doi.org/10.1056/nejmsr1600236 world health organization. meningitis outbreak response in sub-saharan africa who guideline. geneva: world health organization; 2014. kaburi bb, kubio c, kenu e, et al. evaluation of the enhanced meningitis surveillance system, yendi municipality, northern ghana, 2010–2015. bmc infect dis. 2017;17(1):306. https://doi.org/10.1186/s12879-017-2410-0 kluberg sa, mekaru sr, mciver dj, et al. global capacity for emerging infectious disease detection, 1996–2014. emerg infect dis. 2016;22(10):e1–e6. https://doi.org/10.3201/eid2210.151956 who ebola response team, agua-agum j, allegranzi b, et al. after ebola in west africa – unpredictable risks, preventable epidemics. n engl j med. 2016;375(6):587–596. https://doi.org/10.1056/nejmsr1513109 meltzer mi, atkins cy, santibanez s, et al. estimating the future number of cases in the ebola epidemic--liberia and sierra leone, 2014–2015. mmwr surveill summ [serial online]. 2014 [cited 2021 may 12];63 suppl 3(3):1–14. available from: http://www.cdc.gov/vhf/ebola/outbreaks/guinea/index.html our world in data. total covid-19 tests per 1,000 people [homepage on the internet]. 2022 [cited 2022 jan 27]. available from: https://ourworldindata.org/grapher/full-list-cumulative-total-tests-per-thousand?tab=table&country=ind~idn~ita~zaf~kor~usa~dnk~nzl~can~gha world health organization. technical guidelines for integrated disease surveillance and response in the african region [homepage on the internet]. 3rd ed. brazzaville: who regional office for africa; 2019 [cited 2021 may 12]. available from: https://www.afro.who.int/publications/technical-guidelines-integrated-disease-surveillance-and-response-african-region-third global health security agenda. global health security agenda: action packages [homepage on the internet]. 2014 [cited 2021 may 12]. available from: https://ghsagenda.org/laboratory-systems/ diallo a, teclemariam l, felling b, et al. assessment tool for laboratory services (atlas) 2006 [homepage on the internet]. arlington, tx: deliver, for the u.s. agency for international development; 2006 [cited 2021 may 26]; p. 1–82. available from: https://pdf.usaid.gov/pdf_docs/pnado529.pdf addo na, adukpo r, bekoe v, et al. assessment of the ghana laboratory logistics system and services. arlington, tx: deliver, for the u.s. agency for international development; 2006. ondoa p, datema t, keita-sow m-s, et al. a new matrix for scoring the functionality of national laboratory networks in africa: introducing the labnet scorecard. afr j lab med. 2016;5(3):a498. https://doi.org/10.4102/ajlm.v5i3.498 usaid project deliver. assessment tool for laboratory services (atlas). washington, dc: usaid; 2017. ministry of health. ghana national health laboratory policy. accra: moh; 2013. ministry of health. national policy and guidelines for infection prevention and control in health care settings [homepage on the internet]. 2015 [cited 2021 may 12]. available from: https://ghs.gov.gh/ooboxyky/2022/10/ipc-policy-and-guidelines-final-15517-1.pdf national health insurance scheme. tariffs for diagnostic centre: g-drg revised tariffs 2019. accra: moh; 2019. ministry of health. medium term expenditure framework (mtef) for 2018–2021: programme based budget estimates for 2018 [homepage on the internet]. 2018 [cited 2021 may 26]. available from: https://www.mofep.gov.gh/sites/default/files/pbb-estimates/2018/2018-pbb-moh.pdf world health organization regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region (with checklist). brazzaville: who afro; 2012. abstract introduction methods results discussion acknowledgements references about the author(s) nada a. abdelrahim department of medical microbiology, faculty of medical laboratory sciences, nile university, khartoum, sudan nahla mohamed department of virology, faculty of clinical microbiology, umeå university, umeå, sweden magnus evander department of virology, faculty of clinical microbiology, umeå university, umeå, sweden clas ahlm department of infection and immunology, faculty of clinical microbiology, umeå university, umeå, sweden imad m. fadl-elmula department of pathology and clinical genetics, faculty of medicine, al-neelain university, khartoum, sudan assafa academy, kartoum, sudan citation abdelrahim na, mohamed n, evander m, ahlm c, fadl-elmula im. human herpes virus type-6 is associated with central nervous system infections in children in sudan. afr j lab med. 2022;11(1), a1718. https://doi.org/10.4102/ajlm.v11i1.1718 original research human herpes virus type-6 is associated with central nervous system infections in children in sudan nada a. abdelrahim, nahla mohamed, magnus evander, clas ahlm, imad m. fadl-elmula received: 29 aug. 2021; accepted: 25 may 2022; published: 22 sept. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: human herpes virus type-6 (hhv-6) is increasingly recognised as a febrile agent in children. however, less is known in sub-saharan african countries, including sudan. objective: we investigated the involvement of hhv-6 in paediatric central nervous system (cns) infections in khartoum, sudan. methods: febrile patients aged up to 15 years with suspected cns infections at omdurman hospital for children from 01 december 2009 to 01 august 2010 were included. viral dna was extracted from leftover cerebrospinal fluid (csf) specimens and quantitatively amplified by real-time polymerase chain reaction (pcr) at umeå university in sweden. results: of 503 csf specimens, 13 (2.6%) were positive for hhv-6 (33.0% [13/40 of cases with proven infectious meningitis]). the median thermal cycle threshold for all hhv-6-positive specimens was 38 (range: 31.9–40.8). the median number of virus copies was 281.3/pcr run (1 × 105 copies/ml csf; range: 30–44 × 103 copies/pcr run [12 × 103 – 18 × 106 copies/ml csf]). all positive patients presented with fever and vomiting; 86.0% had seizures. the male-to-female ratio was 1:1; 50.0% were toddlers, 42.0% infants and 8.0% teenagers. most (83.0%) were admitted in the dry season and 17.0% in the rainy season. cerebrospinal fluid leukocytosis was seen in 33.0%, csf glucose levels were normal in 86.0% and low in 14.0%, and csf protein levels were low in 14.0% and high in 43.0%. conclusion: among children in sudan with cns infections, hhv-6 is common. studies on the existence and spread of hhv-6 chromosomal integration in this population are needed. keywords: hhv-6; viral neuroinfections; viral meningitis; aseptic meningitis; real-time pcr. introduction human herpes virus type-6 (hhv-6) is a major cause of acute febrile illnesses in young children1 where most are infected by the age of three.2 the virus remains latent in white blood cells (i.e. monocytes and macrophages) following a primary infection and persistence in salivary glands.3 reactivation of hhv-6 can occur in the case of an immunosuppression which may result in complications affecting various systems including the central nervous system (cns).3 young immunocompetent children who suffer from fever and seizures can develop cns disease in primary hhv-6 infections.4,5 in this matter, several authors have described the major role that could be played by the virus as a cause of paediatric neuroinfections.6,7,8,9,10,11 detecting hhv-6 nucleic acids in the cerebrospinal fluid (csf) by molecular assays indicates active virus replication, hence cns infection. this interpretation is complicated by the phenomenon of hhv-6 chromosomal integration.12,13 the only human herpes virus that is found integrated in the human genome and can be passed on vertically from parent to child is hhv-6.14 this occurs occasionally, and is claimed to be detected by simply identifying persistently high concentrations of hhv-6 nucleic acids in blood because of chromosomal integration in white blood cells.12,13 in contrast, integrated hhv-6 dna is highly unexpected in normal cell-free body compartments, including the csf.11,13,15 ward16 stated that viral load should be high to identify a condition as chromosomal integration and low virus copies would indicate an infection. normal csf glucose with normal or elevated proteins is the usual finding in viral infections of the cns.17 increased numbers of white blood cells (˃ 5 cell/mm3) in csf indicates inflammation; however, normal csf cellular counts in patients with proven cns infections have been frequently reported.17,18,19,20 it is therefore recommended not to limit hhv-6 testing to patients with increased csf cellular counts.21 most patients recover fully from an hhv-6 infection, but rare complications leading to neurological impairments or death can also occur.22,23 little is known on hhv-6 infections in saharan and sub-saharan africa, and the infection has never been investigated in sudan. we, therefore, intended to identify the involvement of hhv-6 in cns infections in a large group of children in khartoum, sudan. this report is a part of a larger study on the microbial aetiologies of cns infections in this population. methods ethical considerations the ethical clearance for conducting this study was obtained from the ethical review board of the national center for neurological sciences in september 2009. patient consent was determined to be unnecessary and was waived. patients were not contacted directly; data were obtained from hospital files and were kept anonymous at all stages of the study. excess specimens were obtained from the hospitals main laboratory after all officially requested tests were applied. permission to collect data and specimens was granted from hospital authorities verbally (from hospital general director; authors’ attestation on file) and in writing (from laboratories administration, federal ministry of health). study materials a total of 503 csf specimens were obtained from febrile (˃ 37 °c) meningitis-suspected attendees of the omdurman hospital for children in khartoum, sudan. patients aged 0 to 15 years who were admitted between 01 december 2009 and 01 august 2010 were included. clinical and demographic data were obtained from hospital records retrospectively. routine csf analyses were performed at the microbiology laboratory of omdurman hospital. an aliquot of csf was frozen at −80 °c for further analysis for hhv-6 dna at the department of clinical microbiology, umeå university, umeå, sweden. human herpes virus type-6 dna was extracted then quantitatively amplified by real-time polymerase chain reaction (pcr) using taqman® (eurogentec®, seraing, liège, belgium) method. viral dna extraction the qiaamp ultrasens virus technology kit (qiagen®, hilden, germany) was used for viral dna extraction in csf. one ml specimen was used to concentrate viral dna: buffer ac (qiagen®, hilden, germany) was added, shortly incubated, then sediment by low g-force (i.e. 1000–1200 × g) centrifugation (eppindorf centrifuge 5415d®, hamburg, germany) to pellet nucleic acid complexes. the supernatant was discarded; the pellet was re-suspended in buffer ar (qiagen®, hilden, germany) and proteinase k and incubated for 10 min at 40°c. binding conditions were adjusted by adding buffer ab (qiagen®, hilden, germany); the lysate was applied to the silica gel membrane spin column. during brief centrifugation (i.e. 3000–5000 × g), dna selectively binds to the membrane. remaining contaminants and enzyme inhibitors were removed by centrifugation (i.e. 3000–5000 × g) in two wash steps using buffer aw1 then buffer aw2. pure viral nucleic acids were eluted in 30 µl low-salt buffer ave (qiagen®, hilden, germany) twice. each elute (60 µl) was divided into two aliquots (30 µl each) and preserved at −80 °c. taqman® real-time polymerase chain reaction viral gdna amplification and detection was performed by real-time analysis using the applied biosystems® 7900ht fast real-time pcr system (foster city, california, united states) and the taqman® (eurogentec®, seraing, liège, belgium) probe (reporter dye famtm on 5´ end and quencher dye tamratm on 3´ end). the lower assay detection limit is one virus copy per ml specimen. forward and reverse primers (figure 1) and probes (dna technology®, aarhus, denmark) were diluted to reach final working concentration of 25 µm (standardised concentration by umeå university hospital). commercially provided oligonucleotide products were diluted to the suitable working solution and the recommendation of quantitect® (qiagen®, hilden, germany) q pcr protocol was followed. a pcr master mix solution was prepared for harry (hhv-6) primers (figure 1) as follow: volumes of 12.5 µl of 2 × quantitecttm probe pcr master mix (qiagen®, hilden, germany), 1.0 µl forward primer (final concentration 25 µm), 1.0 µl reverse primer (final concentration 25 µm), 0.8 µl probe (final concentration 20 µm) and 7.2 µl rnase-free water (ambion®, waltham, massachusetts, united states) were added into 2.5 µl template gdna to complete a total reaction volume of 25.0 µl per single pcr reaction. the mixture was pulse-vortexed (vortex-genie pulse®, bohemia, new york, united states) and centrifuged briefly (i.e. 3000 × g). then, 22.5 µl single reaction mix was transferred into a well of a microamptm optical 96-well reaction plate (applied biosystems®, foster city, california, united states) to which 2.5 µl template gdna was added to reach total volume of 25.0 µl per one reaction. figure 1: specifications of human herpes virus type 6 primers and probe in khartoum, sudan, december 2009 – august 2010. the pcr plates were covered by microamptm optical adhesive film (applied biosystems®), concentrated at the bottom of the plate wells by spinning at low speed in a cold centrifuge (i.e. 1200 × g) using allegratm x-12r centrifuge (beckman coulter®, brea, california, united states). each pcr reaction plate was designed for carrying eight standard dilutions, one negative reverse transcriptase preparation, one non-template control containing ddh2o and duplicates or triplicates of each experimental dna template. the standard used for pcr had 5 × 106 (5e6) virus copies (obtained from the virology department, umeå university hospital). the first standard preparation was used without dilution, having a concentration of 5e6 copies, followed by 1/10 serial dilutions for the subsequent seven preparations to reach final concentrations of 5e5, 5e4, 5e3, 5e2, 5e1, 5 and 1 virus copy. real-time cycler thermal condition was as follows: heating at 50 °c for 2 min, initial activation of hotstartaq® dna polymerase (qiagen®, hilden, germany) at 95 °c for 10 min, denaturation at 94 °c for 15 s and combined annealing and extension at 60 °c for 60 s. the cycle was repeated 45 times taking an approximate duration of 90 min including generation of amplification curve. data were analysed using the software abi 7900ht sds plate utility version 2.4 (applied biosystems®). statistical analysis statistical package program statistical package for social sciences version 21 (ibm corp., chicago, illinois, united states) was used. categorical variables were expressed in frequencies and percentages and cross-tabulated and pearson chi-square tested for statistically significant differences under the 0.05 level. numerical variables were described using measures of central tendency and of dispersion; pearson’s correlation and its 95% confidence intervals were calculated. results clinical, demographic and conventional laboratory findings all of the following describe patients with positive csf hhv-6 dna: all patients with clinical data (100%; 7/7; six cases out of a total of 13 had missing clinical data) presented with fever (˃ 37 °c) and vomiting and six (86%) with seizures (figure 2). for the 12 patients with available demographic data, the male-to-female ratio was 1:1 (figure 2). figure 2: demographic, clinical and conventional laboratory data for human herpes virus type-6 positive patients, khartoum, sudan, december 2009 – august 2010. half of the patients were toddlers, followed by 42% (5/12; 1 missing case) infants and 8% (1/12) a 15-year-old teenager. most patients (83%; 10/12) were admitted to the hospital in the dry season (december to june) and the remaining 17% (2/12) admitted in the rainy season (july to august). cerebrospinal fluid specimens from 42% (5/12) were traumatic; cytological and chemical analyses were not performed. the remaining 58% (7/12) were clear; 33% (1/7) showed increased csf white blood cells (50 cells/mm3) with 60% lymphocytosis and the remaining 67% (6/7) showed normal csf white blood cell count (< 5 cells/mm3). cerebrospinal fluid glucose levels for most specimens (86%; 6/7) were normal (45 mg/dl – 100 mg/dl) and one specimen (14%) was low (30 mg/dl). cerebrospinal fluid proteins levels were high (> 45 mg/dl) in 43% (3/7), normal (14 mg/dl – 45 mg/dl ) in 43% (3/7) and low (11 mg/dl) in 14% (1/7) (figure 2). all 13 hhv-6 positive cases did not show evidence of rapid-growing-bacteria coexisting in csf on gram’s stain and in vitro bacterial culture. however, 23% (3/13) were positive for other non-cultivable microbes. all patients (100%; 7/7) recovered and were discharged. real-time polymerase chain reaction findings polymerase chain reaction testing for hhv-6 dna in csf revealed 13 (2.6%) positive specimens out of a total of 503. median cycle threshold (ct) for all hhv-6 positive specimens was 38 with a range of 31.9 to 40.8. median virus copy was 281.3 per pcr run (1 × 105 virus copies/ml csf) with a range of 30 to 44 × 103 copies per pcr run (12 × 103 and 18 × 106 virus copies/ml csf). individual pcr data for all 13 positive specimens are shown in table 1. standard dilutions with viral copies of 5e6 to 5e2 produced amplification curves between ct 24 and ct 36. the generated standard curve plot showed perfect negative association between ct and viral quantity. this was repeatable in all pcr runs. table 1: thermal cycle and virus load for human herpes virus type-6 positive individual cerebrospinal fluid specimens using quantitative taqman real-time polymerase chain reaction, khartoum, sudan, december 2009 – august 2010. the final classification of cases based on clinical and molecular findings is shown in table 2. table 2: classification of cases based on clinical and molecular findings, khartoum, sudan, december 2009 – august 2010. discussion human herpes virus type-6 is becoming increasingly recognised as an emerging cns pathogen; nevertheless, it has never been investigated in sudan and very little is known about it in sub-saharan african countries or those of the meningitis belt. in the present study, hhv-6 dna was identified in the csf of 2.6% of paediatric patients with suspected meningitis. this result is close to the findings of hosseininasab24 who identified csf hhv-6 dna in 1.5% (1/65) of children with aseptic meningitis in southern iran. tavakoli21 reported similar findings of 1.8% (27/1482) in new york, united states. another study in new york by yao11 revealed a significantly higher prevalence of 40.0% (14/35) in patients with cns infections who tested negative for other cns pathogens. among well-defined groups in our study, the prevalence was also high, accounting for 33.0% out of 40 cases with proven infectious meningitis (table 2). in the present study, unlike yao’s approach, cases with other detectable cns pathogens were not excluded because of possible co-infections, as frequently reported.21,25,26,27 it has been reported that primary infection with hhv-6 always occurs in young children (age 0 to 2 years).5,28,29 human herpes virus type-6 infections, especially to the cns, can also occur in healthy older children and adults, but it is thought to be due to virus reactivation.22 among 24 hhv-6 positive patients in tavakoli’s study,21 42% were infants ≤ 3 years and 12.5% were teenagers 11–17 years old. out of our 13 hhv-6 positive cases, 92% were infants ≤ 2.3 years and one (8%) was a 15-year-old teenager. the ratio of boys to girls in this study was 1:1 which agrees with tavakoli’s findings of 1.3:1.1. clinical signs and symptoms in hhv-6 meningitis are not specific.30 out of the 24 examined patients, tavakoli reported fever in 71.0%, altered mental status in 67.0%, headache in 29.0% and seizures in 33.0%. other reported symptoms were muscle weakness, muscle pain and stiff neck, which are general symptoms for meningitis or encephalitis.21 in the present study, all hhv-6 positive cases presented with fever and vomiting, 86.0% presented with seizures, 14.3% with chills and 14.3% with a stiff neck. none of our patients developed skin rash, which is the only specific – but rare – symptom in hhv-6 meningitis.30 findings of both tavakoli and hosseininasab concur. normal csf glucose with normal or elevated proteins is the usual finding in viral infections of the cns,17 as found in this study, where all specimens showed normal csf glucose concentration, 33% normal proteins and 67% high proteins. unfortunately, chemical analysis of the csf is ineffective in case of viral infections; however, its significance is in distinguishing bacterial from aseptic aetiologies which is a crucial preliminary step in deciding adequate treatment. cerebrospinal fluid leukocytosis was seen in 33%. increased csf white blood cell numbers indicate inflammation; however, normal csf cellular counts do not rule out viral aetiologies. in fact, normal csf cellular counts in patients with proven cns infections were frequently reported.17,18,19,20 normal csf profile was reported in 25% of hhv-6 positive cases in tavakoli’s study21; accordingly, hhv-6 testing should not be limited to patients with abnormal csf profiles. thermal ct is defined as the number of cycles required for the fluorescent signal to cross the threshold (i.e. exceed background level). median ct for our positive cases was 38 with a range of 31.9 to 40.8. in tavakoli’s study, ct values ranged from 25.03 to 39.92. in quantitative real-time pcr, ct values inversely correlate with viral loads; therefore, a low ct value indicates a high viral load and vice versa. our ct values and viral loads were found to be significantly (p = 0.029) inversely correlated (r = −0.6, 95% confidence interval: −0.1 to −0.9) indicating significant variation of viral loads among our patients. substantial variation among viral loads in patients was also reported by tavakoli.21 the phenomenon of hhv-6 chromosomal integration is in debate; while some11,12,13 consider it an easily identifiable condition based on the presence or absence of nucleus containing blood cells in different body compartments, ward16 believes the few leukocytes that are usually present in normal csf can reveal positive viral dna in the case of hhv-6 integration. in the present study, the csf was clear with no observed cells in most cases (86%), while a single case (14%) showed an increased csf cell count. ward16 elaborated that in order to identify a condition as chromosomal integration, viral load should be high while low virus copies would indicate an infection. they16 reported a significantly lower (i.e. 2.4 log10 copies/ml) csf hhv-6 dna concentration in 9 children with primary infection in comparison with 21 patients with viral chromosomal integration (i.e. 4.0 log10 copies/ml). in the present study, the median csf virus concentration was 1 × 105 copies per ml with a minimum virus concentration of 12 × 103 copies per ml and a maximum of 18 × 106 copies per ml. while ward16 recommended identifying low virus copies (≤ 103) as an acute hhv-6 infection and high virus copies (≥ 104) as chromosomal integration, collot31 identified viral integration in approximate concentrations of 103 to 106 copies per ml. in the present study, we identified viral concentrations as low as 104 and as high as 107. several other studies reported high csf viral loads in patients with hhv-6 cns infections.11,21 in addition, other authors12,13 insist that chromosomal integration is a rare condition. accordingly, we assume the detected hhv-6 dna in our mostly cell-free csf specimens is more likely to be from free replicating virus than from chromosomally integrated virus. in infants, primary hhv-6 infection is an important cause of febrile seizures with an incidence of 13% in the united states.4 knowing that febrile seizures and vomiting were dominant symptoms among our population and the most frequent age group was children up to the age of 2.3 years, therefore, further supported our assumption. despite this, and for the sake of scientific relevance, we are not ruling out the possibility of integration among our identified cases. for this reason, studies to identify the prevalence of hhv-6 integration among healthy sudanese population are warranted. alongside hhv-6, mixed microbial infections were identified in three cases in this study. several authors also reported mixed viral infections to the cns.21,25,26,27 despite moderate neurological sequel, and less likely death, patients usually recover fully from an hhv-6 infection22,23, as fortunately observed in this population. limitations a major limitation in this study, however, is that we were unable to further genotype our identified hhv-6 viral dna because of a limited csf volume (i.e. all available csf was consumed in testing and identifying multiple microbes – reported in other publications). conclusion human herpes virus type-6 cns infection is frequent in this population (i.e. identified in one-third of cases with proven infectious meningitis). we recommend studying the existence and spread of hhv-6 chromosomal integration in the healthy sudanese population. acknowledgements we acknowledge all laboratory technologists in the bacteriology departments of omdurman hospital for children, the national health laboratory and the central public laboratories at the ministry of health for providing clinical data and specimens. we also thank the administrations of sudan medical and scientific research institute (sumasri) of the university of medical sciences and technology (umst) and that of the faculty of medical laboratory sciences of al-neelain university in khartoum, sudan, for providing working space and allowing use of laboratory ware. we are indebted for the kind gesture of a complete bench fee waiver that was provided by the department of clinical microbiology, umeå university in umeå, sweden, to perform all molecular investigations. we thank professor hassan mohammed ali for his great efforts in revising the manuscript. competing interests we, the authors, declare that we have no competing interests with respect to the research, authorship or publication of this article. authors’ contributions n.a.a. developed research questions and design, collected and managed all data, performed all laboratory work, statistical analysis and interpretation, wrote and edited the text. n.m. advised on the approach and methodology and guided and supervised the molecular laboratory work. m.e. and c.a. contributed to the approach and methodology, supervised the molecular laboratory work, edited and proofread the manuscript and were major contributors in the overall study. i.m.f.-e. supervised the research process throughout, contributed in the development of research questions, design and methodology, and managed all logistics and clinic-based activities. all authors have read and approved the final manuscript. sources of support molecular laboratory investigations were supported by the department of clinical microbiology at umeå university in umeå, sweden. all remaining project expenses were self-financed. data availability the data sets used and analysed during the current study will be available from the corresponding author, n.a., on reasonable request once all related articles are published. disclaimer views expressed in this article are the authors’ own; they are not an official position of the institution or funder. references pruksananonda p, hall cb, insel ra, et al. primary human herpesvirus 6 infection in young children. n engl j med. 1992;326(22):1445–1450. https://doi.org/10.1056/nejm199205283262201 briggs m, fox j, tedder rs. age prevalence of antibody to human herpesvirus 6. lancet. 1988;1(8593):1058–1059. https://doi.org/10.1016/s0140-6736(88)91883-1 lusso p, gallo rc. human herpesvirus 6 in aids. immunol today. 1995;16(2):67–71. https://doi.org/10.1016/0167-5699(95)80090-5 kondo k, nagafuji h, hata a, tomomori c, yamanishi k. association of human herpesvirus 6 infection of the central nervous system with recurrence of febrile convulsions. j infect dis. 1993;167(5):1197–1200. https://doi.org/10.1093/infdis/167.5.1197 hall cb, long ce, schnabel kc, et al. human herpesvirus-6 infection in children; a prospective study of complications and reactivation. n engl j med. 1994;331(7):432–438. https://doi.org/10.1056/nejm199408183310703 koskiniemi m, rautonen j, lehtokoski-lehtiniemi e, vaheri a. epidemiology of encephalitis in children: a 20-year survey. ann neurol. 1991;29(5):492–499. https://doi.org/10.1002/ana.410290508 kolski h, ford-jones el, richardson s, et al. etiology of acute childhood encephalitis at the hospital for sick children, toronto, 1994–1995. clin infect dis. 1998;26(2):398–409. https://doi.org/10.1086/516301 yoshikawa t, asano y. central nervous system complications in human herpesvirus-6 infection. brain dev. 2000;22(5):307–314. https://doi.org/10.1016/s0387-7604(00)00113-3 whitley rj, gnann jw. viral encephalitis: familiar infections and emerging pathogens. lancet. 2002;359(9305):507–513. https://doi.org/10.1016/s0140-6736(02)07681-x theodore wh, epstein l, gaillard wd, shinnar s, wainwright ms, jacobson s. human herpes virus 6b: a possible role in epilepsy? epilepsia. 2008;49(11):1828–1837. https://doi.org/10.1111/j.1528-1167.2008.01699.x yao k, honarmand s, espinosa a, akhyani n, glaser c, jacobson s. detection of human herpesvirus-6 in cerebrospinal fluid of patients with encephalitis. ann neurol. 2009;65(3):257–267. https://doi.org/10.1002/ana.21611 clark da, nacheva ep, leong hn, et al. transmission of integrated human herpesvirus 6 through stem cell transplantation: implications for laboratory diagnosis. j infect dis. 2006;193(7):912–916. https://doi.org/10.1086/500838 ward kn, leong hn, nacheva ep, et al. human herpesvirus 6 chromosomal integration in immunocompetent patients results in high levels of viral dna in blood, sera, and hair follicles. j clin microbiol. 2006;44(4):1571–1574. https://doi.org/10.1128/jcm.44.4.1571-1574.2006 tanaka-taya k, sashihara j, kurahashi h, et al. human herpesvirus 6 (hhv-6) is transmitted from parent to child in an integrated form and characterization of cases with chromosomally integrated hhv-6 dna. j med virol. 2004;73(3):465–473. https://doi.org/10.1002/jmv.20113 leong hn, tuke pw, tedder rs, et al. the prevalence of chromosomally integrated human herpesvirus 6 genomes in the blood of uk blood donors. j med virol. 2007;79(1):45–6. https://doi.org/10.1002/jmv.20760 ward kn, leong hn, thiruchelvam ad, atkinson ce, clark da. human herpesvirus 6 dna levels in cerebrospinal fluid due to primary infection differ from those due to chromosomal viral integration and have implications for diagnosis of encephalitis. j clin microbiol. 2007;45(4):1298–1304. https://doi.org/10.1128/jcm.02115-06 logan sa, macmahon e. viral meningitis. bmj. 2008;336(7634):36–40. https://doi.org/10.1136/bmj.39409.673657.ae big c, reineck la, aronoff dm. viral infections of the central nervous system: a case-based review. clin med res. 2009;7(4):142–146. https://doi.org/10.3121/cmr.2009.864 lee be, davies hd. aseptic meningitis. curr opin infect dis. 2007;20(3):272–277. https://doi.org/10.1097/qco.0b013e3280ad4672 abdelrahim na, fadl-elmula im, ali hm. bacterial meningitis in sudanese children; critical evaluation of the clinical decision using clinical prediction rules. bmc pediatr. 2019;19(1):319. https://doi.org/10.1186/s12887-019-1684-3 tavakoli np, nattanmai s, hull r, et al. detection and typing of human herpesvirus 6 by molecular methods in specimens from patients diagnosed with encephalitis or meningitis. j clin microbiol. 2007;45(12):3972–3978. https://doi.org/10.1128/jcm.01692-07 isaacson e, glaser ca, forghani b, et al. evidence of human herpesvirus 6 infection in 4 immunocompetent patients with encephalitis. clin infect dis. 2005;40(6):890–893. https://doi.org/10.1086/427944 takanashi j, barkovich aj, tada h, takada n, fujii k, kohno y. cortical liquefaction in severe human herpesvirus 6 encephalopathy. neurology. 2006;66(3):452–453. https://doi.org/10.1212/01.wnl.0000196486.42635.f8 hosseininasab a, alborzi a, ziyaeyan m, et al. viral etiology of aseptic meningitis among children in southern iran. j med virol. 2011;83(5):884–888. https://doi.org/10.1002/jmv.22056 ibrahim ai, obeid mt, jouma mj, roemer k, mueller-lantzsch n, gärtner bc. prevalence of herpes simplex virus (types 1 and 2), varicella-zoster virus, cytomegalovirus, and human herpesvirus 6 and 7 dna in cerebrospinal fluid of middle eastern patients with encephalitis. j clin microbiol. 2005;43(8):4172–4174. https://doi.org/10.1128/jcm.43.8.4172-4174.2005 minjolle s, michelet c, jusselin i, joannes m, cartier f, colimon r. amplification of the six major human herpesviruses from cerebrospinal fluid by a single pcr. j clin microbiol. 1999;37(4):950–953. https://doi.org/10.1128/jcm.37.4.950-953.1999 tang yw, espy mj, persing dh, smith tf. molecular evidence and clinical significance of herpesvirus coinfection in the central nervous system. j clin microbiol. 1997;35(11):2869–2872. https://doi.org/10.1128/jcm.35.11.2869-2872.1997 yoshikawa t, suga s, asano y, yazaki t, kodama h, ozaki t. distribution of antibodies to a causative agent of exanthem subitum (human herpesvirus-6) in healthy individuals. pediatrics. 1989;84(4):675–677. https://doi.org/10.1542/peds.84.4.675 ward kn, gray jj, fotheringham mw, sheldon mj. igg antibodies to human herpesvirus-6 in young children: changes in avidity of antibody correlate with time after infection. j med virol. 1993;39(2):131–138. https://doi.org/10.1002/jmv.1890390209 schvoerer e, fréchin v, fritsch s, et al. atypical symptoms in patients with herpesvirus dna detected by pcr in cerebrospinal fluid. j clin virol. 2006;35(4):458–462. https://doi.org/10.1016/j.jcv.2005.11.005 collot s, petit b, bordessoule d, et al. real-time pcr for quantification of human herpesvirus 6 dna from lymph nodes and saliva. j clin microbiol. 2002;40(7):2445–2451. https://doi.org/10.1128/jcm.40.7.2445-2451.2002 article information authors: rosemary a. audu1 rosemary n. okoye2 chika k. onwuamah1 fehintola a. ige1 adesola z. musa3 nkiruka n. odunukwe4 daniel i. onwujekwe4 oliver c. ezechi4 emmanuel o. idigbe1 phyllis j. kanki5 affiliations: 1human virology laboratory, nigerian institute of medical research, lagos, nigeria 2clinical diagnostic laboratory, nigerian institute of medical research, lagos, nigeria 3monitoring and evaluation unit, nigerian institute of medical research, lagos, nigeria 4clinical sciences division, nigerian institute of medical research, lagos, nigeria 5harvard school of public health, boston, massachusetts, united states correspondence to: rosemary audu email: rosemaryaudu@yahoo.com postal address: human virology laboratory, nigerian institute of medical research, 06 edmond crescent, pmb 2013 yaba, lagos, nigeria dates: received: 17 mar. 2014 accepted: 11 aug. 2015 published: 30 sept. 2015 how to cite this article: audu ra, okoye rn, onwuamah ck, et al. potential for false-positive hiv test results using rapid hiv testing algorithms. afr j lab med. 2015;4(1), art. #178, 5 pages. http://dx.doi.org/10.4102/ajlm.v4i1.178 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. potential for false-positive hiv test results using rapid hiv testing algorithms in this original research... open access • abstract • introduction • methods    • study setting    • national testing algorithm    • study design    • data abstraction and inclusion criteria    • laboratory methods    • statistical analyses    • ethical considerations • results • discussion    • limitations    • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: in order to scale up access to hiv counselling and testing in nigeria, an hiv diagnostic algorithm based on rapid testing was adopted. however, there was the need to further evaluate the testing strategy in order to better assess its performance, because of the potential for false positivity. objectives: the objective of this study was to compare positive hiv test results obtained from the approved rapid testing algorithm with results from western blot tests performed on samples from the same patient. methodology: a retrospective review was conducted of hiv screening and confirmatory results for patients seen between 2007 and 2008. rapid test and western blot results were extracted and compared for concordance. discordant results were further reviewed using a combination of hiv-1 rna viral load and cd4+ cell count test results and clinical presentation from medical records. results: analysis of 2228 western blot results showed that 98.3% (n = 2191) were positive for hiv-1, 0.4% (n = 8) were positive for hiv-2 and 0.3% (n = 7) were dual infections (positive for both hiv-1 and hiv-2); 0.6% (n = 13) were indeterminate and 0.4% (n = 9) were negative. further investigation of the 13 indeterminate results showed nine to be hiv-1 positive and four to be hiv-negative, for a total of 13 negative results. the positive predictive value of the hiv counselling and testing algorithm was 99.4%. conclusion: using the rapid testing algorithm alone, false positives were detected. therefore, effective measures such as training and retraining of staff should be prioritised in order to minimise false-positive diagnoses and the associated potential for long-term psychological and financial impact on the patients. introduction top ↑ the diagnosis of hiv infection is most often based on the detection of serum antibodies to hiv. these serological tests could be classified either as screening tests, such as rapid tests or enzyme linked immuno-sorbent assay (elisa), or confirmatory tests, such as western blot (wb). prior to the availability of rapid testing, same-day results were not obtainable and an estimated one third of individuals tested did not return to learn their hiv status.1 compared with elisa and wb, rapid test assays are cheaper, easier to perform and the results are readily available on the same day. in 1999, the world health organization and the joint united nations programme on hiv/aids recommended the use of a combination of rapid test assays or elisas to confirm positive results, employing a highly sensitive test as the first screening test and a second, highly specific confirmatory test to verify the detection of antibodies specific to hiv. this was considered to be as reliable for confirmation as wb but at a much lower cost.2 in 2007, the government of nigeria adopted a serial testing algorithm with three combinations of the following rapid test assays: determine® (abbott, tokyo, japan), stat-pak® (chembio diagnostic systems, medford, new york, united states) or uni-gold™ (trinity biotech, jamestown, new york, united states). samples with discordant results were further tested with bundi rt (bundi international diagnostics ltd, aba, nigeria) as the tiebreaker.3 the adopted algorithm could be used in any of the combinations shown in table 1. because of quality issues with the bundi test kits, the use of the kit was suspended; the third combination was retained and is currently in use in the country. table 1: algorithm adopted in 2007 for hiv rapid testing in nigeria. as with all screening assays, hiv rapid test screening has a certain degree of false-positive results, irrespective of the algorithm in use.4 for this reason, a confirmatory test is necessary for a definitive diagnosis. evaluations of hiv rapid testing strategies have been reported in some african countries where the results have been used to formulate alternative hiv testing strategies.5,6 regardless of which rapid test algorithm is used, false-positive results may still occur;7,8 1% – 2% of reactive rapid test results have been found to be negative with an hiv nucleic acid test.7 since the introduction of the 2007 hiv rapid test strategy in nigeria, its performance has not been evaluated. therefore, the assessment of the algorithm’s performance remains an important priority to better inform national policy and ensure accurate hiv diagnosis and surveillance. the aim of this study was to compare positive hiv test results obtained from the approved rapid testing algorithm with results from wb tests performed on samples from the same patient. methods top ↑ study setting the study was conducted at the nigerian institute of medical research (nimr), lagos, a parastatal of the federal ministry of health. nimr has an hiv treatment centre that currently provides comprehensive hiv care, antiretroviral treatment (art) and support for over 18 000 patients. the centre is supported by the government of nigeria and the us president’s emergency plan for aids relief-funded programme for art; as a result, all services are provided free to patients. patients who screen positive for hiv at the hiv counselling and testing (hct) centre or any government of nigeria-approved hiv counselling and testing centre are referred to the treatment programme for further management. these patients are clinically assessed and sent to the nimr virology laboratory for baseline laboratory assessment. the nimr virology laboratory is an iso 9001:2008 certified laboratory and employs well trained and competent personnel to perform the laboratory assays. quality assurance measures undertaken by the laboratory include: participation in external quality assessment for all assays; equipment maintenance; and several measures to verify pre-analytical, analytical and post-analytical processes. national testing algorithm the approved national serial testing algorithm used for this study was: determine, unigold and stat-pak as tie breaker. this algorithm is currently in use in nigeria; thus, the results of this study are relevant to the current situation in the country. study design this was a retrospective study, which analysed data from the programme’s electronic database. cases with discordant results were further reviewed using a combination of plasma viral load, cd4+ cell counts and clinical data to determine a presumptive hiv diagnosis or progression of infection. hiv test results generated between january 2007 and august 2008 were analysed. discordant results were reviewed until december 2011. data abstraction and inclusion criteria data were abstracted from records of patients aged 18 years or older who had tested positive for hiv using the 2007 nigerian rapid test algorithm at the nimr hct, satellite sites or private clinics. data for patients sent to the nimr virology laboratory for confirmation of hiv diagnosis by wb were abstracted for this study. data from all patients who had given informed consent to participate in research studies were included. wb test result data included all positive, negative and indeterminate results. indeterminate results were defined as reactivity profiles that were not compatible with either a positive or a negative interpretation. laboratory methods the nimr hct site conducted hiv testing using the national testing algorithm; this required the use of non-cold chain dependent rapid test kits supplied by the government of nigeria through the central medical stores. the satellite sites and private clinics who referred hiv-positive patients for care and treatment also indicated the rapid test kits used in obtaining the positive hiv results. at the nimr virology laboratory, the baseline assessment included: hiv confirmation using the wb technique (immunetics inc., boston, massachusetts, united states) to detect specific viral proteins for both hiv-1 and hiv-2; cd4+ cell count (cells/µl) using the flow cytometry technique employed by the partec cyflow counter ii (gorlitz, germany) to determine eligibility for the initiation of art; and quantitation of hiv-1 rna viral load in plasma (roche amplicor v1.5, mannheim, germany) with a lower limit of detection of 400 copies/ml. the viral load assay was further used to monitor the effectiveness of therapy and for timely identification of poor adherence and possible virologic failure. statistical analyses the positive predictive value was calculated based on the results of the rapid tests and wb using standard formulae. exact 95% confidence intervals for these proportions were calculated. analyses were conducted using statistical package for social sciences version 17 for windows (ibm corporation, chicago, illinois, united states). ethical considerations ethical approval for this study was obtained from the nimr and harvard school of public health institutional review boards. written informed consent (dated 14 november 2006) was obtained prior to enrolment of patients for both services and research. results top ↑ results from 2228 different patients meeting the inclusion criteria were extracted from the programme’s electronic database. there was a male: female ratio of 1.9:1 and the mean age was 38.8 ± 9.0 years. the nimr hct facility accounted for 967 (43.4%) of the results abstracted, whereas 1261 (56.6%) were from satellite sites and private clinics. the analyses of the wb results showed that 98.3% were positive for hiv-1, 0.4% were positive for hiv-2 and 0.3% had hiv-1 and hiv-2 dual infections. there were also 13 (0.6%) indeterminate and nine (0.4%) negative results (table 2). the 13 indeterminate results were further investigated using plasma viral load levels and nine (69.2%) were found to be infected with hiv-1 based on very high hiv-1 viral load levels (median = 310 377 rna copies/ml; range = 2150–991 706 rna copies/ml), whereas four (30.8%) were considered to be hiv-negative based on undetectable viral load results (<400 rna copies/ml). thus, a total of 13 results that had been positive according to the rapid test algorithm were actually negative. this yielded a false-positive rate of 0.6%. table 2: hiv-positive rapid test results obtained by western blot technique, nimr, january 2007 − august 2008. clinical records of cases with negative wb results were reviewed. after a minimum of 12 months with no clinical evidence of infection or laboratory evidence of hiv disease, these cases were discontinued from the clinic. the four cases with indeterminate wb results and undetectable viral load results were monitored for an additional two years. in the absence of any clinical manifestation of hiv infection and repeat undetectable viral load results, they were discharged from the clinic. taking both the true positives and false positives into consideration, the positive predictive value was calculated to be 99.4% (99.0% – 99.6% ci). the satellite sites and private clinics were responsible for 46.2% (6/13) of the false-positive results. upon further investigation, we found that all patients at these facilities (100%) had been screened using determine and stat-pak test kits, including those with false-positive results. since both kits produced concordant results, a third kit was not required as a tie breaker for any of these cases. the immunological and virological data of these false positives were analysed and it was found that their median cd4+ cell count was 668 cells/µl (range = 50–1500 cells/µl) and all had an undetectable viral load (< 400 rna copies/ml). discussion top ↑ rapid hiv antibody tests help to improve access to testing in both clinical and nonclinical settings, as well as increasing the proportion of people who receive their results once tested. as good as the rapid hiv screening assays may be, a confirmatory test is still required for a definitive diagnosis because of the possibility of false-positive results. false positives may result from numerous causes, including the inherent characteristics of the assay, or may be because of the genetic diversity of the virus.4 the percentage of false positivity reported in this study was 0.6%, which is similar to that reported in other studies,3,9 but is lower than the 2% reported in cameroon.10 as part of nigeria’s global aids response progress report in 2012, 11.7% of the population of 162 265 000 had received an hiv test within the last 12 months and knew their hiv status.11 this corresponds to 18 985 005 tests performed in nigeria within the year. given the false-positive rate of 0.6% found in this study, approximately 113 910 people per year who are not hiv-positive receive a false hiv-positive diagnosis. the us president’s emergency plan for aids relief reported the yearly cost of management of an hiv patient to be us $338.12 these 113 910 false-positive diagnoses could result in us $38.5m per year in inappropriate but significant costs for hiv management. these resources could be better used for the estimated 1 512 720 hiv-infected individuals still requiring art. possible ways to identify patients with false-positive results include: lack of risk factors for hiv, normal cd4+ cell count and undetectable viral load.13 in nigeria, the cd4+ cell count reference value has been reported to be 365–1571 cells/µl.14 causes of false-positive results include: fictitious hiv exposure/infection15 and hiv vaccination,16 the latter being unlikely because hiv vaccine trials have not been conducted in nigeria. other likely reasons for false-positive results could be either clerical or technical. the possibility of these errors in the nimr virology reference laboratory is low because of the laboratory’s stringent quality management system. none of the laboratories that carried out the hct for the results included in this study had implemented a quality management system. this is not unusual, considering the dearth of knowledge about quality management systems in most laboratories in africa, including nigeria.17 the inconsistent or incorrect use of the rapid test algorithm has also been reported as a possible cause of false-positive results.18 in addition, false-positive results could also be a result of poor algorithm planning or implementation.19 the incorrect interpretation of weak-positive test lines and non-usage of tie-breaker algorithms, which occurs in our setting, could explain the latter. the use of confirmatory wb allowed for the diagnosis of hiv-1 and hiv-2, between which these particular rapid test assays cannot discriminate. as seen in previous studies,20 hiv-1 was the major hiv type amongst patients accessing hiv care in our clinic. the very low rate of hiv-2 and hiv-1/2 dual infections observed in this study could be attributed to the decline in its prevalence, as reported in other west african countries,21 resulting in part from the inefficiency of transmission of hiv-2.22 in this study, men had a higher uptake of testing than women. this is similar to observations made between 2003 and 2007 (about the same timeframe as this study), but by 2011, this finding had been reversed.11 this is most likely attributable to the increased availability of prevention of mother-to-child transmission treatment, as well as treatment including paediatric therapy. the delivery of a false-positive hiv result to a patient can be devastating, as it often results in psychological stress and trauma, particularly with subsequent stigmatisation. in a study from three african countries, namely, the democratic republic of congo, burundi and ethiopia, some patients who were falsely diagnosed as hiv-positive had been abandoned by their partners after they started on art or prophylaxis.23 there are also substantial financial costs related to misdiagnosis, where patients might lose time and compensation from work in order to attend clinic visits. in addition, the possible side effects and toxicities of unnecessary art need to be considered. in our study, patients with false-positive results were found to have high median cd4+ cell counts and undetectable viral loads. neither of these conditions can rule out the possibility of hiv infection, particularly if patients are natural elite controllers24 or receiving art. it has also been reported that there are apparently healthy nigerians who have cd4+ cell counts below 350 cells/µl.17,25 it is important to note that the cost of side effects, psychological trauma and lifelong treatment is unquantifiable. there have been reports of suicides resulting from a diagnosis of hiv,26 hence the gravity of false-positive results should be well-recognised. it is therefore essential that great care and caution is exercised in hiv diagnosis. in spite of the challenge of false-positive results, the use of rapid test kits doubled the proportion of individuals who had been tested and received their test results between 2003 and 2007.11 the calculated positive predictive value of 99.4% in this study is similar to the established national positive predictive value of 99.5% (96.8% – 99.9% ci) based on the national algorithm’s sensitivity of 100% and specificity of 99.7%.3 this implies that the use of this algorithm is still very essential in the expansion of hiv testing in a highly-populated country such as nigeria, if the spread of this virus is to be controlled. limitations this study is, however, not without limitations. the sensitivity, specificity and negative predictive values could not be estimated, because only hiv-positive patients were referred to the treatment centre for further follow up, including the wb re-testing. in like manner, not all cases referred from the hct centres eventually reported to the treatment centre. it is also important to note that although wb is a confirmatory test, it does have a window period of six weeks; thus, its usefulness for the detection of acute infections is limited,27,28 as there is a possibility of missing early infections. although rapid test kits also have a window period, the higher sensitivity assay is used initially, then followed by a very specific test. the long follow up of patients with discordant test results and repeat testing should have reduced the effect of the six-week window period. the wb assay also generates some indeterminate results. however, an indeterminate result is still acceptable in comparison to a false-positive result, as it allows for further evaluation.29 conclusion in this study, we found a 0.6% rate of hiv false positives with the implementation of the 2007 nigerian hiv rapid testing algorithm. it is therefore recommended that clinicians should refer suspicious false-positive patients for wb confirmation. all possible measures should be taken to prevent false-positive diagnoses associated with rapid testing alone, as the long-term costs of treatment, side effects and psychological trauma to patients may be considerable. acknowledgements top ↑ we are grateful to the harvard/aids prevention initiative in nigeria (apin) us president’s emergency plan for aids relief (pepfar) programme, which provided the financial support for this project. this work was supported, in part, by the us department of health and human services, health resources and services administration (u51ha02522). the contents are solely the responsibility of the authors and do not represent the official views of the funding institutions. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions r.a.a (nigerian institute of medical research) was the project leader. r.n.o., c.k.o. and f.a.i. (nigerian institute of medical research) performed most of the experiments and retrieval of records. a.z.m. (nigerian institute of medical research) was the data manager and responsible for data analysis. n.n.o., d.i.o. and o.c.e. (nigerian institute of medical research) were responsible for clinical management and manuscript preparation. e.o.i. (nigerian institute of medical research) made conceptual contributions, whilst p.j.k. (harvard school of public health) was responsible for project design and manuscript preparation. references top ↑ us centers for disease control and prevention, advancing hiv prevention: new strategies for a changing epidemic – united states, 2003. mmwr. 2003;52(15):329–332. unaids/world health organization. operational characteristics of commercially available assays to determine antibodies to hiv-1 and/or hiv-2 in human sera. report 11. geneva: world health organization; 1999. federal ministry of health, nigeria. laboratory based hiv rapid test validation in nigeria, phase 1, april 2007. nigeria: the nigeria hiv rapid test evaluation working group; 2007. cordes rj, ryan me. pitfalls in hiv testing. application and limitations of current tests. postgrad med. 1995;98(5):177–180, 185–186, 189. lyamuya ef, aboud s, urassa wk, et al. evaluation of simple rapid hiv assays and development of national rapid hiv test algorithms in dar es salaam, tanzania. bmc infect dis. 2009;9:19. http://dx.doi.org/10.1186/1471-2334-9-19 andersson s, da silva z, norrgren h, et al. field evaluation of alternative testing strategies for diagnosis and differentiation of hiv-1 and hiv-2 infections in an hiv-1 and hiv-2-prevalent area. aids. 1997;11(15):1815–1822. http://dx.doi.org/10.1097/00002030-199715000-00005 wesolowski lg, delaney kp, meyer wa, et al. use of rapid hiv assays as supplemental tests in specimens with repeatedly reactive screening immunoassay results not confirmed by hiv-1 western blot. j clin virol. 2013;58(1):240-244. http://dx.doi.org/10.1016/j.jcv.2013.06.019 baveewo s, kamya mr, mayanja-kizza h, et al. potential for false positive hiv test results with the serial rapid hiv testing algorithm. bmc res notes. 2012;5:154. http://dx.doi.org/10.1186/1756-0500-5-154 anzala o, sanders ej, kamali a, et al. sensitivity and specificity of hiv rapid tests used for research and voluntary counselling and testing. east afr med j. 2008; 85(10):500–504. aghokeng af, mpoudi-ngole e, dimodi h, et al. inaccurate diagnosis of hiv-1 group m and o is a key challenge for ongoing universal access to antiretroviral treatment and hiv prevention in cameroon. plos one. 2009;4(11):e7702. http://dx.doi.org/10.1371/journal.pone.0007702 national agency for the control of aids, federal republic of nigeria global aids response, country progress report [document on the internet]. c2012 [cited 2014 sep 12]. available from: http://www.unaids.org/en/dataanalysis/knowyourresponse/countryprogressreports/2012countries/nigeria2012garprreportrevised.pdf [updated link viewed 2015 sep 07, from: http://www.unaids.org/sites/default/files/en/dataanalysis/knowyourresponse/countryprogressreports/2012countries/nigeria%202012%20garpr%20report%20revised.pdf] holmes cb, atun r, avila c, et al. expanding the generation and use of economic and financial data to improve hiv program planning and efficiency: a global perspective. j acquir immune defic syndr. 2011;57(suppl 2):s104–s108. http://dx.doi.org/10.1097/qai.0b013e31821fa12d mylonakis e, paliou m, greenbough tc, et al. report of a false-positive hiv test result and the potential use of additional tests in establishing hiv serostatus. arch intern med. 2000;160 (15):2386–2388. http://dx.doi.org/10.1001/archinte.160.15.2386 oladepo dk, idigbe eo, audu ra, et al. establishment of reference values of cd4 and cd8 lymphocyte subsets in healthy nigerian adults. clin vaccine immunol. 2009;16(9):1374–1377. http://dx.doi.org/10.1128/cvi.00378-08 craven de, steger ka, la chapelle r, et al. factitious hiv infection: the importance of documenting infection. ann intern med. 1994;121(10):763–766. http://dx.doi.org/10.7326/0003-4819-121-10-199411150-00006 belshe rb, clements ml, keefer mc, et al. interpreting hiv serodiagnostic test results in the 1990s: social risks of hiv vaccine studies in uninfected volunteers. niaid aids vaccine clinical trials group. ann intern med. 1994;121(8):584–589. http://dx.doi.org/10.7326/0003-4819-121-8-199410150-00005 gershy-damet g, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm aniedobe mn, onwuamah ck, onubogu cc, et al. proficiency of private laboratories in diagnosis of hiv in nigeria. oral presentation at the 18th union conference, africa region, 3rd – 5th march 2011. available from: http://www.afro.who.int/en/nigeria/press-materials/item/2884-the-18th-conference-of-the-international-union-against-tuberculosis-and-lung-disease-iuatld-african-region-held-from-3-5th-march-2011-abuja-nigeria.html klarkowski d, o’brien dp, shanks l, et al. causes of false-positive hiv rapid diagnostic test results. expert rev anti infect ther. 2014;12(1):49–62. http://dx.doi.org/10.1586/14787210.2014.866516 kashyap b, gautam h, chadha s, et al. delayed progression and inefficient transmission of hiv-2. southeast asian j trop med publ health. 2010;41(3): 570–573. madec y, boufassa f, porter k, et al. spontaneous control of viral load and cd4 cell count progression among hiv-1 seroconverters. aids. 2005;19(17):2001–2007. http://dx.doi.org/10.1097/01.aids.0000194134.28135.cd us centers for disease control and prevention. interpretation and use of the western blot assay for serodiagnosis of human immunodeficiency virus type 1 infections. mmwr. 1989;38(suppl 7):1–7. owen sm, yang c, spira t, et al. alternative algorithms for human immunodeficiency virus infection diagnosis using tests that are licensed in the united states. j clin microbiol. 2008;46(5):1588–1595. http://dx.doi.org/10.1128/jcm.02196-07 shanks l, klarkowski d, o’brien dp. false positive hiv diagnoses in resource limited settings: operational lessons learned for hiv programmes. plos one. 2013;8(3):e59906. http://dx.doi.org/10.1371/journal.pone.0059906 audu ra, idigbe eo, akanmu as, et al. values of cd4+ t lymphocyte in apparently healthy individuals in lagos, nigeria. ejsr. 2007;16(2):168–173. keiser o, spoerri a, brinkhof mw, et al.; swiss hiv cohort study; swiss national cohort. suicide in hiv-infected individuals and the general population in switzerland, 1988–2008. am j psychiatry. 2010;167(2):143–150. http://dx.doi.org/10.1176/appi.ajp.2009.09050651 olaleye od, bernstein l, ekweozor cc, et al. prevalence of human immunodeficiency virus types 1 and 2 infections in nigeria. j infect dis. 1993;167(3):710–714. http://dx.doi.org/10.1093/infdis/167.3.710 bock p, markovitz d. infection with hiv-2. aids. 2001;15(suppl 5):s35–s45. http://dx.doi.org/10.1097/00002030-200100005-00006 ngan ccl, thoe sys, chan kp, et al. alternative strategies for confirmation of human immunodeficiency virus infection require judicious use. j. clin. microbiol. 2002;40(1):314–315. http://dx.doi.org/10.1128/jcm.40.1.314-315.2002 article information authors: oladipo a. aboderin1,2 olufemi adefehinti3 babatunde w. odetoyin1 amadin a. olotu2 iruka n. okeke4 olugbenga o. adeodu3,5 affiliations: 1department of medical microbiology and parasitology, obafemi awolowo university, ile-ife, nigeria2department of medical microbiology and parasitology, obafemi awolowo university teaching hospitals complex, ile-ife, nigeria 3department of paediatrics, obafemi awolowo university teaching hospitals complex, ile-ife, nigeria 4department of biology, haverford college, haverford, united states 5department of paediatrics and child health, obafemi awolowo university, ile-ife, nigeria correspondence to: oladipo abo postal address: department of medical microbiology and parasitology, college of health sciences, obafemi awolowo university, 220005, ile-ife, nigeria dates: received: 25 june 2011 accepted: 19 jan. 2012 published: 04 june 2012 how to cite this article: aboderin oa, adefehinti o, odetoyin bw, olotu aa, okeke in, adeodu oo. prolonged febrile illness due to ctx-m-15 extended-spectrum β-lactamase-producing klebsiella pneumoniae infection in nigeria. afr j lab med. 2012;1(1), art. #16, 4 pages. http://dx.doi.org/10.4102/ ajlm.v1i1.16 note: this report was presented as a poster (abstract c-119) at the 111th general meeting, american society for microbiology, may 21–24, new orleans, louisiana, united states. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. prolonged febrile illness due to ctx-m-15 extended-spectrum β-lactamase-producing klebsiella pneumoniae infection in nigeria in this case studies... open access • abstract • introduction • case report • discussion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ we report on an 8-year-old patient with septicaemia unresponsive to therapy for five weeks. undetected, extended-spectrum β-lactamase (esbl) production by the infecting klebsiella strain was regarded as responsible for treatment failure. intravenously administered imipenem during the sixth week led to sustained resolution of fever. resource-limited hospitals can incur prohibitive costs from esbl-producer infections because of diagnostic limitations and consequent treatment failure involving prolonged supportive therapy. introduction top ↑ the first report of plasmid-encoded β-lactamase capable of hydrolysing the extended-spectrum cephalosporins was published in 1983.1 since then, extended-spectrum β-lactamases (esbls) have become an increasingly important resistance mechanism among enterobacteriaceae worldwide. a 2006 report of the infectious diseases society of america listed esbl-producing klebsiella pneumoniae and escherichia coli among drug-resistant microbes for which new therapies are urgently needed.2 reports show that the esbl problem is rapidly evolving and increasing in severity and scope with the discovery of new esbls, particularly the ctx-m types, which have become the most prevalent.3 the β-lactamases are the greatest threat to the usefulness of β-lactam antibiotics such as the penicillins and cephalosporins. of all the different types of β-lactamases, esbls currently have the greatest clinical impact in terms of diversity and distribution as well as the ability to hydrolyse expanded-spectrum third generation cephalosporins. the earliest variants of esbls originated as a result of point mutations in the genes for broad-spectrum β-lactamases whilst newer ones, including the most successful such as ctx-m, arose by acquisition from the environmental metagenome through horizontal gene transfer.4 cephalosporins as bactericidal, cell wall-active β-lactam agents were introduced in the 1980s and as a result of effectiveness against broad-spectrum β-lactamases became standard for treatment of severe conditions such as bloodstream infections, pneumonia and intra-abdominal infections, until esbls started compromising usefulness in response to overuse and selective pressure. organisms that produce esbls are an important reason for therapy failure with cephalosporins and have serious consequences for infection control. furthermore, ctx-m esbl enzymes have been associated with coresistance to other agents including trimethoprim-sulphamethoxazole, tetracycline, gentamicin, tobramycin and ciprofloxacin.5 it is essential that clinical microbiology laboratories rapidly and reliably detect and report esbl-producing organisms. case report top ↑ an eight-year-old girl presented with a two-week history of fever, abdominal pain, passage of watery stool and recurrent vomiting. there was also a history of frequent micturition with occasional dysuria but neither haematuria nor passage of dark-coloured urine. prior to presentation in the teaching hospital, she had been admitted to a distant private hospital for five days, where she was treated with amoxicillin, ciprofloxacin and artesunate (doses and duration of treatment are unknown). she was discharged from that hospital because her state of health was not improving significantly and also because of the need for better family support. there was no other history of hospital admission or blood transfusion and no history suggestive of haemoglobinopathy. immunisation and nutritional history were essentially normal.general physical examination revealed a conscious but ill-looking, somewhat pale, febrile (temperature 38.5 °c) girl. she was moderately dehydrated and jaundiced. her weight was 22 kg (86% of expected weight for age). there was no facial or pedal oedema. the respiratory rate was 32 cycles per min and breathing was regular; pulse regular at 120 beats per min and with good volume. her blood pressure was 90/50 mmhg. the significant systemic findings on examination at admission were severe suprapubic tenderness, moderate hepatosplenomegaly (firm, not tender) and negative renal angle tenderness. all other systems were normal. conventional blood cultures were done at five different times after admission. there was a growth of klebsiella sp. on three occassions. once, the isolate was sensitive to gentamicin and ceftriaxone but resistant to all available antibiotics tested on the other two occasions. urine and stool cultures did not yield growth of any pathogens. screening for human immunodeficiency virus (hiv), hepatitis c virus (hcv) and hepatitis b surface antigen (hbsag) was negative. full blood counts showed a haematocrit ranged between 14% and 30%, white cell counts of 8 x 109/l – 9.2 x 109/l and an essentially normal platelet count (260 x 109/l). the erythrocyte sedimentation rate was 80 mm/hr (westergreen method) and the haemoglobin phenotype (by electrophoresis) was as. serum biochemistry parameters were all normal except for conjugated hyperbilirubinaemia. repeated abdominal ultrasonography showed findings that are consistent with hepatosplenomegaly in a septicaemic patient. whilst in hospital and when fever was uncontrolled and persistent, the patient was given fresh whole blood transfusions thrice and exchange blood transfusions twice, amongst other forms of treatment. fever remained persistent for five weeks following admission, despite different courses of antibiotics involving ciprofloxacin, gentamicin, ceftazidime, ceftriaxone and amoxicillin-clavulanic acid (table 1). it was only at this point that the possibility of infection with an esbl-producing organism was considered. esbl-producers are not routinely sought in the diagnostic laboratory. during the sixth week, the klebsiella sp. isolate from the patient was tested and confirmed to be producing an esbl. immediately following this test result, treatment was commenced with imipenem (not routinely available in the hospital) and there was dramatic resolution of fever. the patient remained free of fever for one week after receiving imipenem and was subsequently discharged. two weeks later, when she reported for follow-up, she was still fever-free and healthy. table 1: treatment interventions. the klebsiella sp. isolate was identified as klebsiella pneumoniae subspecies pneumoniae using the api 20e identification strips for enterobacteriaceae (biomérieux, marcy-l’étoile, france). presumptive esbl phenotypic testing and confirmation in the organism was performed by disc diffusion tests on mueller hinton agar by employing ceftazidime (30 µg) and cefpodoxime (10 µg) alone and in combination with clavulanic acid as ceftazidime-clavulanic acid (30/10 µg) and cefpodoxime-clavulanic acid (10/1 µg) respectively. results were interpreted using the clinical and laboratory standards institute (clsi) criteria for disc diffusion.6 antimicrobial susceptibility testing for the organism was carried out by the disc diffusion technique according to the guidelines and recommendations of clsi.6 the isolate was resistant to streptomycin, gentamicin, chloramphenicol, tetracycline, nalidixic acid, ciprofloxacin, ampicillin, trimethoprim, sulphamethoxazole, ceftriaxone, cefepime and amoxicillin-clavulanic acid, but susceptible to imipenem.genomic dna was extracted from the isolate using the wizard genomic extraction kit (promega) according to the manufacturer’s directions and used as template for pcr reactions targeting resistance elements and genes. platinum pcr supermix (invitrogen) was used for all reactions, and pcr cycle conditions were as recorded in the original articles describing the primers (table 2). we employed oligonucleotides that prime the conserved ends of the cassette regions of class 1 and 2 integrons respectively to screen for these elements (table 2).7,8 as shown in figure 1, we were able to determine that the strain harboured a class 1, but not a class 2 integron. sequencing of the 1.6 kb amplified class 1 cassette region revealed that it was identical to the cassette region of plasmid pip1206 (genbank accession number nc_010558), containing two integrated cassettes: a dfra17 cassette encoding resistance to trimethoprim, and an aada4 aminoglycoside resistance cassette.9 since the integron did not contain an esbl cassette, we screened the isolate for blactx-m type genes, employing primers that amplify an internal fragment from multiple blactx-m alleles (table 2)10. the resulting 550 bp product shown in figure 2 was sequenced and found to be identical to the corresponding region of blactx-m-15. table 2: oligonucleotides for pcr reactions. figure 1: pcr amplification of the variable regions of class 1 and class 2 integrons. (a) class 1 integron-variable regions amplified using lev5’cs and lev3’cs primers. lane 1: no template; lane 2: e. coli strain 042 bearing an aada cassette within a class 1 integron; lane 3: e. coli strain 17-2 bearing the dfra1-sat-aada cassette sequence within a class 2 integron; lane 4: k. pneumoniae subsp. pneumoniae isolate k01 from this study; lane 5: 1 kb ladder plus (invitrogen). marker size fragments are indicated to the right of the gel in kilobase pairs. (b) class 2 integron-variable regions amplified using hep51 and hep74 primers with samples shown in (a) loaded on to the gel. (c) cassette content and orientation of the k01 integron amplified in (a), as predicted from the dna sequence. figure 2: pcr amplification of a ctx allele from k. pneumoniae subsp. pneumoniae isolate k01 from this study. lane 1: 1 kb ladder plus (invitrogen). lane 2: k01. lane 3: esbl-negative e. coli strain 042. lane 4: no template. marker size fragments are indicated to the left of the gel in kilobase pairs. in the absence of a positive control, the identity of the band amplified from strain k01 was determined by sequencing. the cost of repeated investigations (table 3) was n14 000.00 ($90.92), which is more than a tenfold increase on projected diagnostic expenses, had a diagnosis estimate been made immediately on admission. antibiotics and fresh whole blood transfusions (thrice) as well as exchange blood transfusions (twice) cost n89 900.00 ($583.77). these and other treatment interventions effectively doubled the cost of treatment as compared to that for what would normally have been appropriate therapy after admission. finally, prolonged hospital accommodation, feeding and nursing care over 48 days amounted to n20 400.00 ($132.46) in contrast to n2975.00 ($19.32) for admission for one week, an almost tenfold increase. table 3: investigations. discussion top ↑ the occurrence and spread of infections resulting from esbl-producing organisms have been well documented in countries of europe, asia and north america.11 in contrast, data on the epidemiology of esbl enzymes is very limited in nigeria. molecular analysis of eight nigerian esbl-producing enterobacter species in 2001 detected only tem and shv-like esbls and no ctx-m types.12 in a study of klebsiella pneumoniae isolates associated with community-acquired urinary tract infections between 2002 and 2003 in ibadan, nigeria, ctx-m group 1, -like enzymes were found in 17 (57%), but ctx-m-15 was identified in only two isolates.13 olowe et al. investigated the occurrence of ctx-m esbl-producing e. coli and found nine of 79 ampicillin-resistant hospital isolates to be esbl producers.14 more recently, a case of necrotising fasciitis was reported in a nigerian patient in the uk.15 morganella morganii and citrobacter freundii carrying the ctx-m-15 esbl gene were isolated from the patient, highlighting the presence of ctx-m genes in africa even though there is a scarcity of reports in the literature. here, we describe a case of prolonged, uncontrolled fever found to be due to esbl-producing k. pneumoniae. to the best of our knowledge, this is the first documented clinical course and outcome of esbl-producing bacterial infection in nigeria. failure to recognise and initially diagnose the presence of an esbl-producing organism resulted in considerable expense in the management of the infection. this includes the cost of different courses of ineffective antibiotics for five weeks, exchange blood transfusions and whole blood transfusions, as well as hospital charges resulting from prolonged stay in hospital. the estimated avoidable cost of supportive therapy and investigation related to possible alternative diagnoses was almost $600 in a country where the average annual per capita income is $2300 and health care resources are severely limited. the clinical diagnostic microbiology laboratory plays a crucial part in the detection and reporting of esbl-producing bacteria, and it is important that laboratories be fully aware of the significance of esbl-producing organisms and the best methods for detecting them, as in our case. resource-limited hospitals can incur prohibitive costs associated with esbl-producer infections because of prolonged supportive therapy and treatment failure following the use of readily available antibiotics. diagnostic improvements to allow routine detection and reporting of esbl production in enterobacteriaceae will help greatly in avoiding these costs. acknowledgements top ↑ this work was supported by a branco weiss fellowship (awarded to ino) from the society-in-science, swiss federal institute of technology (eth), zurich, switzerland. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions o.a.a. (obafemi awolowo university) coordinated the research. o.a. (obafemi awolowo university teaching hospitals complex) a.a.o. (obafemi awolowo university teaching hospitals complex), o.a.a. and o.o.a. (obafemi awolowo university teaching hospitals complex) managed the patient (case) clinically. a.a.o., o.a.a. and b.w.o. (obafemi awolowo university) performed microbiological testing whilst i.n.o. carried out molecular experiments. a.o.a. and i.n.o. (haverford college) drafted the paper, to which o.a., b.w.o., o.o.a. and a.a.o. contributed. o.a.a. and i.n.o. undertook revision of the manuscript. all authors approved the final version. references top ↑ 1. knothe h, shah p, krcmery v, antal m, mitsuhashi s. transferable resistance to cefotaxime, cefoxitin, cefamandole and cefuroxime in clinical isolates of klebsiella pneumoniae and serratia marcescens. infection. 1983;11:315–317. http://dx.doi.org/10.1007/bf01641355, pmid:6321357 2. talbot gh, bradley j, edwards je, gilbert d, scheld m, bartlett jg. bad bugs need drugs: an update on the development pipeline from the antimicrobial availability task force of the infectious diseases society of america. clin infect dis. 2006;42:657–668. http://dx.doi.org/10.1086/499819, pmid:16447111 3. cantón r, coque tm. the ctx-m β-lactamase pandemic. curr opin microbiol. 2006;9:466–475. http://dx.doi.org/10.1016/j.mib.2006.08.011, pmid:16942899 4. poirel l, kāmpfer p, nordmann p. chromosome-encoded ambler class a β-lactamase of kluyvera georgiana, a probable progenitor of a subgroup of ctx-m extended-spectrum β-lactamases. antimicrob agents chemother. 2002;46:4038–4040. http://dx.doi.org/10.1128/aac.46.12.4038-4040.2002, pmid:12435721, pmcid:132763 5. pitout jdd. multiresistant enterobacteriaceae: new threat of an old problem. expert rev anti-infect ther. 2008;6:657–669. http://dx.doi.org/10.1586/14787210.6.5.657, pmid:18847404 6. clinical and laboratory standards institute. performance standards for antimicrobial susceptibility testing: twentieth informational supplement. clsi document m100-s20. wayne, pa: clinical and laboratory standards institute; 2010. 7. lévesque c, piché l, larose c, roy ph. pcr mapping of integrons reveals several novel combinations of resistance genes. antimicrob agents chemother. 1995;39:185–191. http://dx.doi.org/10.1128/aac.39.1.185, pmid:7695304, pmcid:162507 8. white pa, mciver cj, rawlinson wd. integrons and gene cassettes in the enterobacteriaceae. antimicrob agents chemother. 2001;45:2658–2661. http://dx.doi.org/10.1128/aac.45.9.2658-2661.2001, pmid:11502548, pmcid:90711 9. périchon b, bogaerts p, lambert t, frangeul l, courvalin p, galimand m. sequence of conjugative plasmid pip1206 mediating resistance to aminoglycosides by 16s rrna methylation and to hydrophilic fluoroquinolones by efflux. antimicrob agents chemother. 2008;52:2581–2592. http://dx.doi.org/10.1128/aac.01540-07, pmid:18458128, pmcid:2443923 10. bonnet r, recule c, baraduc r, et al. effect of d240g substitution in a novel esbl ctx-m-27. j antimicrob chemother. 2003;52:29–35. http://dx.doi.org/10.1093/jac/dkg256, pmid:12775683 11. pitout jdd, laupland kb. extended-spectrum β-lactamase-producing enterobacteriaceae: an emerging public-health concern. lancet infect dis. 2008;8:159–166. http://dx.doi.org/10.1016/s1473-3099(08)70041-0, pmid:18291338 12. aibinu ie, ohaegbulam vc, adenipekun ea, ogunsola ft, odugbemi to, mee bj. extended-spectrum β-lactamase enzymes in clinical isolates of enterobacter species from lagos, nigeria. j clin microbiol. 2003;41:2197–2200. http://dx.doi.org/10.1128/jcm.41.5.2197-2200.2003, pmid:12734278, pmcid:154721 13. soge oo, queenan am, ojo kk, adeniyi ba, roberts mc. ctx-m-15 extended-spectrum β-lactamase from nigerian klebsiella pneumoniae. j antimicrob chemother. 2006;57:24–30. http://dx.doi.org/10.1093/jac/dki429, pmid:16319181 14. olowe oa, grobbel m, büchter b, lübke-becker a, fruth a, wieler lh. detection of blactx-m-15 extended-spectrum β-lactamase genes in escherichia coli from hospital patients in nigeria. int j antimicrob agents. 2010;35:206–207. http://dx.doi.org/10.1016/j.ijantimicag.2009.10.004, pmid:20022221 15. soleimanian s, gordon nc, wareham dw. polymicrobial necrotizing fasciitis involving enterobacteria producing ctx-m-15 extended-spectrum β-lactamases. j med microbiol. 2011;60:135–137. http://dx.doi.org/10.1099/jmm.0.021998-0, pmid:20813849 abstract introduction methods results discussion acknowledgements references about the author(s) noutin f. michodigni department of molecular biology and biotechnology, pan african university institute for basic sciences technology and innovation (pausti), nairobi, kenya department of reproductive health and biology, institute of primate research (ipr), nairobi, kenya atunga nyachieo department of reproductive health and biology, institute of primate research (ipr), nairobi, kenya juliah k. akhwale department of zoology, school of biological sciences, jomo kenyatta university of agriculture and technology (jkuat), nairobi, kenya gabriel magoma department of molecular biology and biotechnology, pan african university institute for basic sciences technology and innovation (pausti), nairobi, kenya department of biochemistry, college of health sciences, jomo kenyatta university of agriculture and technology (jkuat), nairobi, kenya abdoul-salam ouédraogo department of medical microbiology laboratories, souro-sanou teaching hospital, bobo-dioulasso, burkina faso andrew n. kimang’a department of medical microbiology, college of health sciences, jomo kenyatta university of agriculture and technology (jkuat), nairobi, kenya citation michodigni nf, nyachieo a, akhwale jk, magoma g, ouédraogo a-s, kimang’a an. formulation of phage cocktails and evaluation of their interaction with antibiotics in inhibiting carbapenemase-producing klebsiella pneumoniae in vitro in kenya. afr j lab med. 2022;11(1), a1803. https://doi.org/10.4102/ajlm.v11i1.1803 note: additional supporting information may be found in the online version of this article as online supplementary document 1. original research formulation of phage cocktails and evaluation of their interaction with antibiotics in inhibiting carbapenemase-producing klebsiella pneumoniae in vitro in kenya noutin f. michodigni, atunga nyachieo, juliah k. akhwale, gabriel magoma, abdoul-salam ouédraogo, andrew n. kimang’a received: 28 nov. 2021; accepted: 12 may 2022; published: 18 july 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the development of alternative control measures, such as phage therapy or adjunctive therapy, is urgently needed to manage the dissemination of carbapenemase-producing klebsiella pneumoniae. objective: this study aimed to evaluate the therapeutic potential of formulated phage cocktails and their interaction with select antibiotics in inhibiting the growth of carbapenemase-producing k. pneumoniae clinical isolate in vitro in kenya. methods: the study was conducted from february 2021 to october 2021 at the institute of primate research, nairobi, kenya. phage cocktails were formulated based on the morphology and biological properties of precipitated klebsiella phages. the efficacy of individual bacteriophages and phage cocktails as well as their combination with antibiotics were determined for their inhibitory activity on carbapenemase-producing k. pneumoniae (kp20). results: the precipitated bacteriophages were members of myoviridae, siphoviridae and podoviridae. regarding the evaluation of the phage cocktails, the absorbances at 600 nm of the bacterial culture treated with the two-phage cocktail (2φ ma) ranged from 0.173 to 0.246 at 16 h and 20 h whereas it peaked from 2.116 to 2.190 for the positive control. moreover, the results of the adjunctive therapy showed that the optical density at 600 nm of the bacterial culture treated with 2φ ma was 0.186 at 24 h post-incubation time while it was 0.099 with the bacterial culture treated with imipenem in combination with 2φ ma. conclusion: this study demonstrated that the two-phage cocktail in combination with imipenem was able to synergistically delay the increase in carbapenemase-producing k. pneumoniae growth in vitro. keywords: phage cocktail; antibiotics; klebsiella pneumoniae; carbapenemase; absorbances. introduction the development of alternative control measures is urgently needed to manage the dissemination of carbapenemase-producing (cp) klebsiella pneumoniae because of the decrease in the effectiveness of antibiotic drugs.1,2 bacteriophage therapy has, therefore, been proposed as an alternative strategy in controlling multidrug-resistant (mdr) bacterial infections, including mdr k. pneumoniae clinical isolates.3,4 some of the beneficial effects of bacteriophages over antibiotics include their abundance, host specificity and exponential replication.5 however, the narrowness of phage host range and the ever emergence of novel pathogen variants will at minimum represent some limitations for phage therapy.6,7 this challenge can be managed by formulating phage cocktails that contain different phages infecting one species or by combining phages with antibiotics, which may result in a broad spectrum of activity.8 the characterisation of therapeutic phages in terms of their biology and bactericidal activity is mandatory before any preclinical trials because the application of non-characterised lytic bacteriophages may cause undesirable virulence as an adaptive response to bacteriophage infection.5,9 in other words, the development of effective strategies, such as phage growth parameters, host range spectrum, the presence of lytic proteins, formulation of phage cocktail and phage synergistic interaction with antibiotic to enhance phage efficacy, is a key prerequisite for optimal treatment of mdr infections caused by k. pneumoniae. several studies have reported the advantages and drawbacks of phage cocktails in treating mdr bacteria.10,11 unfortunately, bacteriophages employed as cocktail or adjunctive therapy have not been well investigated in the african continent to effectively control mdr bacterial infections.12 this study aimed to evaluate the therapeutic potential of formulated phage cocktails and their interaction with select antibiotics in inhibiting the growth of cp k. pneumonia clinical isolate in vitro in kenya. methods ethical considerations this study was not human or animal research. however, clearance was issued by the nairobi city water & sewerage company limited (ncwsc/hr/trg.14/vol.8/14/mmm/ak) for collection of waste water samples. bacterial isolate collection, phage isolation and purification the study was conducted from february 2021 to october 2021 in the phage biology research laboratory at the institute of primate research, nairobi, kenya. the clinical isolate of cp k. pneumoniae (kp20) was obtained from the stock of the recently published data in kenya.13 bacteriophages were isolated from waste water samples of dandora estate sewage treatment works (ruai) and rongai effluent, nairobi, kenya. they were purified from waste water samples through spot assay and double-layer plaque method as described, previously.14,15 determination of minimum inhibitory concentrations for kp20 the minimum inhibitory concentrations of a panel of antibiotics including carbapenems for kp20 were determined using vitek 2 systems version 9.2 and ast-xn05 (bar code 1481424403205844) card (biomerieux, inc., hazelwood, missouri, united states), according to the manufacturer’s instructions. the detailed procedure is described in online supplementary method 1. bacteriophage propagation and precipitation phage lysate (≥ 108 pfu/ml or 109 pfu/ml) was propagated in large volume, concentrated and cleaned up in the presence of sodium chloride and polyethylene glycol as described elsewhere.14,16 morphological characterisation of precipitated bacteriophages the morphological characterisation of the precipitated klebsiella phages was conducted at the university of leicester, united kingdom. negative staining with 1% (weight/volume) uranyl acetate on 3 mm carbon-coated copper grids of the precipitated klebsiella phages was conducted at the university of leicester and the bacteriophages were visualised with a transmission electron microscope (jeol uk ltd., welwyn garden city, united kingdom).17 bacteriophages were then classified according to their morphology.18 bacteriophage host range determination a spot assay was conducted to determine the host range of precipitated bacteriophages and phage cocktails as described by kutter.19 bacterial strains tested in our study included reference strains and mdr bacterial strains reported by michodigni et al.13 and described in online supplementary methods 2. one-step growth experiment a one-step growth experiment for determination of latent period, and burst size of precipitated bacteriophages, was performed as described elsewhere, with some modifications.20 exponential-growth-phase culture of cp k. pneumoniae (≈ 2.98 × 108 cfu/ml) and phage precipitate (≈ 109–1010 pfu/ml) were mixed with bacterial suspension to obtain a multiplicity of infection (moi) of 0.1.14 the suspension was incubated at 37 °c in a shaking incubator for 10 min at 120 rotations per minute following with centrifugation. the pellet was then suspended in 7 ml of fresh tryptic soy broth and incubated at 37 °c in a shaking incubator at 125 rotations per minute. at 3-min intervals, from 0 min to 36 min, 500 µl of the suspension was taken and phage titres were estimated.20,21 formulation of phage cocktails and evaluation of their in vitro activity on carbapenemase-producing klebsiella pneumoniae three klebsiella phages, designated as cprsa, cprsb and esbla, were used for the formulation of two-phage cocktail (2φ ma) and three-phage cocktail (3φ mb). the two-phage cocktail comprised cprsa and cprsb while the three-phage cocktail consisted of cprsa, cprsb and esbla. bacteriophage cocktails consisting of two and three tested phage lysates were formulated by combining in equal ratios of 1:1 and 1:1:1. the evaluation of the phage cocktails’ effectiveness was carried out based on the method described by merabishvili with some modifications.22 two-phage (2φ ma) and three-phage (3φ mb) cocktails were composed and their ability to inhibit the growth of kp20 at its exponential-growth-phase was evaluated. subsequently, the efficacy of two individual bacteriophages, designated as klebsiella phage cprsa and cprsb at moi 1.0, 0.1 and 0.001, was also investigated. only broth media (tryptic soy broth) and individual bacteriophages represented the negative controls. bacterial culture with a final resulting concentration of ≈ 2.98 × 106 cfu/ml served as positive controls. the od600 of the host bacterium was measured every 4 h for 24 h, and finally at 48 h, to assess the inhibitory activity of bacterial growth by both individual phages and phage cocktails. the different absorbances obtained at 600 nm were compared to the ones of the positive control sample. the different concentrations of individual bacteriophages and phage cocktails are summarised in online supplementary table 1. the experiment was performed in duplicate. table 1: minimum inhibitory concentrations of a panel of antibiotic and carbapenemase produced to kp20, nairobi, kenya, february 2021 – october 2021. evaluation of inhibitory activity of phage cocktails in combination with antibiotics on carbapenemase-producing klebsiella pneumoniae in vitro the inhibitory activity of the phage cocktail alone, and in combination with imipenem or tigecycline was assessed on kp20 at its optimal exponential growth (od600 ≈ 0.6). the two antibiotics meropenem and tigecycline (glentham life sciences ltd. wiltshire, united kingdom) were tested in combination with 2φ ma and 3φ mb. the phage cocktails 2φ ma and 3φ mb with optimal concentrations (moi 0.1 and 0.001) were used in this experiment in combination with the single and double of the minimum inhibitory concentrations of imipenem and tigecycline. sterile broth media (tryptic soy broth), bacterial culture treated with antibiotics, and bacteriophage cocktails represented the negative controls. bacterial culture with a final resulting concentration of ≈ 2.98 × 108 cfu/ml served as a positive control sample. the growth of the host bacterium was monitored at 3-h intervals, between 0 h and 24 h, and at 48 h of incubation by measuring od600. the optical densities obtained at 600 nm were compared to the ones of positive and negative control samples. the different concentrations of phage cocktails and antibiotics are indicated in online supplementary table 2. the experiment was performed in duplicate. table 2: spectrum of activity of individual klebsiella phages isolated from ruai and rongai in february 2021 (cprsa, cprsb and esbla) and phage cocktail (2φ ma and 3φ mb) on reference strains and different bacterial species. data analysis a two-tailed t-test was performed to determine the significance levels of phage cocktails in combination with select antibiotics using graphpad prism version 9.2.0. (graphpad software, san diego, california, united states). a p < 0.05 was considered as significantly different. results minimum inhibitory concentrations of antibiotics and detected carbapenemase genes the minimum inhibitory concentrations of imipenem and tigecycline to kp20 were 4 µg/ml and 1 µg/ml (table 1). we previously reported the presence of extended-spectrum beta-lactamase (esbl) genes (blatem and blaoxa) and carbapenemase genes (blandm-1, blaimp).13 the esbl and carbapenemase genes produced by kp20 are indicated in table 1. morphological characteristics of precipitated klebsiella phages a transmission electron microscope showed that the bacteriophages included in this cocktail were all tailed phages (figure 1). among the three imaged bacteriophages, two were myoviruses (cprsa and cprsb) and one was a podovirus (esbla). figure 1: transmission electron microscope images of the precipitated klebsiella bacteriophages isolated from ruai and rongai in february 2021. (a) and (b) bacteriophages belonging to the family of myoviridae with contractile tails consisting of a sheath and a central tube (cprsa and cprsb); (c) and family of podoviridae short tail (esbla). precipitated klebsiella phages growth characteristics the burst sizes of the precipitated phages in the cocktail were 610 (1.812 × 1011 ÷ 2.98 × 108), 1 (2.294 × 108 ÷ 2.98 × 108) and 10 (3.123 × 109 ÷ 2.98 × 108) for klebsiella phage cprsa, cprsb, esbla. regarding their latent period, klebsiella phages cprsa and cprsb had the same latent period (9 min) and the one of esbla was 18 min as shown in online supplementary figure 1. host range of klebsiella phage lysates klebsiella phages cprsa, cprsb and esbla were used for the formulation of the two-phage cocktail (2φ ma) and the three-phage cocktail (3φ mb). the two-phage cocktail comprised cprsa and cprsb while the three-phage cocktail consisted of cprsa, cprsb and esbla. the two-phage cocktail was the combination of two bacteriophages belonging to the family myoviridae while the three-phage cocktail was the combination of 2φ ma and one belonging to the family podoviridae. the results of the host range analysis showed that both the individual phages and phage cocktails (2φ ma and 3φ mb) were active on bacterial strains belonging to klebsiella species. the three carbapenem-resistant k. pneumoniae were susceptible to only one individual phage, the klebsiella phage cprsa. the two-phage preparations were also active on the three cp k. pneumoniae strains, in addition to k2, which was described as an extended-spectrum beta-lactamase producer in our previous study. inhibitory activity of individual klebsiella phages and phage cocktails on carbapenemase-producing klebsiella pneumoniae culture the ability of the mixture of two phages (ma) and three phages (3φ mb) to inhibit the growth of kp20 culture at an absorbance of ≈ 0.3 was determined at three different multiplicities of infection (moi 1.0, 0.1 and 0.001). it was observed in our study that there was an increase in cp k. pneumoniae growth to the individual phage cprsb at its three different doses and to the phage cprsa at mois 0.1 and 0.001 after 8 h and 16 h of incubation (figure 2a). the two-phage combination (2φ ma) was able to control the rapid growth of cp k. pneumoniae after 16 h of incubation and up to 24 h (figure 2b). this bactericidal activity of the phage cocktail was observed at mois 0.1 and 0.001 whereas the three-phage cocktail (3φ mb) could slightly control the increase in cp k. pneumoniae growth after 16 h and at mois 1.0 and 0.001. at mois 0.1 and 0.001, the dose response of the two-phage cocktail was not significantly different in the ability to inhibit the bacterial growth at this incubation time (p = 0.105). at the most effective phage dose (moi: 0.001), the absorbances at 600 nm of the bacterial culture treated with the individual phage cprsa, and the two-phage cocktail (2φ ma), increased from 0.019 to 0.515, and 0.173 to 0.246 at 16 h and 20 h, while it rose from 2.116 to 2.190 for the positive control during the same interval of time (figure 2b). for the most active individual phage (cprsa) and phage cocktail (2φ ma), the dose responses were not significant (p = 0.12) at moi 0.1 after 16 h of treatment time (table 3). significant difference (p = 0.01) was observed between the two treatments at moi 0.001. figure 2: efficacy of phage cocktails in inhibiting the growth of carbapenem-resistant k. pneumoniae (kp20), nairobi, kenya, february 2021 – october 2021. optical density of kp20 cultures (≈ 106 cfu/ml) infected with (a) individual phages cprsa and cprsb at moi 1.0, 0.1 and 0.001 (b) the 2-phage cocktail (2φ ma at moi 1.0, 0.1 and 0.001) or 3-phage cocktail (3φ mb at moi 1.0, 0.1 and 0.001). the digits 1, 2 and 3 coming next after the id of the phages represents moi 1.0, 0.1 and 0.001. the most effective phages were the individual phage cprsa and phage cocktail 2φ ma at moi 0.1 (cprsa2 & 2φ ma2) and 0.001 (cprsa3 & 2φ ma3). table 3: statistical significance between the concentrations of individual phages (cprsa and cprsb) and phage cocktails (2φ ma and 3φ mb), nairobi, kenya, february 2021 – october 2021. interactive properties between the phage cocktails and antibiotics combinations in inhibiting the growth of carbapenemase-producing klebsiella pneumoniae in vitro the in vitro studies showed that the same phage cocktails (2φ ma and 3φ mb) maintained their bactericidal activity after 24 h of incubation at the lowest phage concentration (moi 0.001) (figure 3a). at different timepoints of 18 h and 24 h, the optical density at 600 nm of the bacterial culture treated with 2φ ma decreased from 0.217 to 0.186 whereas it increased from 0.226 to 0.269 for bacterial culture treated with 3φ mb. the results of the adjunctive treatment (phage and antibiotic combination therapy) experiments revealed that the absorbance of the bacterial culture treated with the combination of imipenem and two-phage cocktail (imp2 + 2φ ma3) decreased from 0.256 (21 h) to 0.099 (24 h) at the lowest phage concentration (figure 3b). when the phage cocktails were combined with tigecycline, the od600 decreased from 0.294 (21 h) to 0.189 (24 h) for bacterial culture treated with tigecycline and the two-phage preparation at the lowest phage concentration, moi 0001 (tg1 + 2φ ma3) while it increased slightly from 0.207 (21 h) to 0.331 (24 h) in the case of bacterial culture treated with tigecycline and the three-phage preparation (tg1 + 3φ mb3). the absorbances of the bacterial culture treated with tigecycline was 0.249 (21 h) and 0.314 (24 h) while it ranged from 1.212 (21 h) to 1.245 (24 h) for the bacterial culture treated with imipenem. the positive control sample exhibited an optical density of 1.166 (21 h) and 1.270 (24 h). the level of significance between the bacterial culture treated with 2φ ma and the bacterial culture treated with imp2 + 2φ ma3 after 21 h of incubation are shown in table 4. the results of bacterial cultures treated with phage cocktail alone, and in combination with imipenem, were statistically significant compared with the result of bacterial culture treated with only imipenem (p = 0.02). no statistical difference was observed between the results of the bacterial culture treated with a combination of phage and antibiotic and those of bacterial culture treated with tigecycline alone. a significant difference (p = 0.04) was observed between the bacterial culture treated with 2φ ma at moi 0.001 and the adjunctive therapy (bacterial culture treated with imp2 + 2φ ma3). table 4: level of significance between bacterial culture treated with two-phage cocktail and the bacterial culture treated with two-phage cocktail and imipenem combination, nairobi, kenya, february 2021 – october 2021. figure 3: bactericidal activity of klebsiella phage cocktail alone or in combination with antibiotics, nairobi, kenya, february 2021 – october 2021. (a) carbapenemase-producing k. pneumoniae culture (od600≈0.6) treated with phage cocktail 2φ ma and 3φ mb at moi 0.1 or 0.001, imipenem (imp, mic = 4 µg/ml), and tigecycline (tg, mic = 1µg/ml), separately. imp2 and tg2 were considered as double the volume of each antibiotic; (b) carbapenemase-producing k. pneumoniae culture treated with phage cocktail + imipenem or phage cocktail + tigecycline. imp2 + 2φ ma3 was the treatment of kp20 culture with the double volume of imipenem combined with two-phage cocktail at moi 0.001, and tg1 + 3φ mb2 was the treatment of kp20 culture with the single volume of tigecycline combined with the three-phage cocktail at moi 0.1. discussion this study aimed to evaluate the therapeutic potential of formulated phage cocktails and their interaction with select antibiotics in inhibiting the growth of the kp20 clinical isolate. on one hand, the phenotypic results revealed that the precipitated phages obtained in this study had lytic activity with high titers and they were related to tailed phages. the latter are generally divided into three families including myoviridae with contractile tails consisting of a sheath and a central tube (25% of tailed phages), siphoviridae with long and noncontractile tails (61%), and podoviridae having short tails (14%).23 klebsiella tailed phages identified in our study belonged to the family of myoviridae and podoviridae.24 previous studies reported similar observations regarding the morphological characters of klebsiella phages.25 on the other hand, this study showed that one of the precipitated phages (cprsa) had the shortest latency period (9 min) and high burst size (610). our findings were supported by horváth et al. in 2020. the authors reported that their isolated klebsiella phage had a relatively short latency period of 18 min and its burst size was ~220 phage particles per infected bacteria.20 indeed, a high burst size is key to achieve a productive adsorption and replication of bacteriophages and to reach the benefits that phages could have in comparison with antibiotics while a short latency period is recommended from a phage therapy perspective.26 both the individual bacteriophages and phage cocktails were not susceptible to the reference bacterial strains tested in this study and, hence, displayed high specificity for their host bacteria, which were kp20 clinical isolates. previous studies on host ranges of klebsiella phages reported similar results.27,28,29 the major consequence of phage host specificity is that it demands an appropriate diagnosis of the bacteria involved in the infection before initiation of phage treatment.7,30 nevertheless, this narrowness of phage host range to the strains of the same bacterial species could limit its lytic activity on microbiota.7,30 the phage host range is indeed affected by a number of factors including the absence of required accessory proteins for phage adsorption, restriction-modification and clustered regularly interspaced short palindromic repeats (crispr) systems.31,32 despite their narrow host range, bacteriophages with lytic activity may still be useful in phage therapy and the use of bacteriophages as cocktails for adjunctive treatment can represent a highly attractive strategy.33 besides the choice of bacteriophages with different bacterial cell wall receptor recognition sites in formulating a phage cocktail, the most important criterion for successful phage cocktail preparation also includes the compatibility of bacteriophages in mixtures.22 therefore, our study demonstrated that the mixture of two phages (2φ ma) was able to significantly delay the resurgence of bacterial cells in culture, as compared to the application of individual phage or the mixture of three phages (3φ mb) (p = 0.02). our result was in line with the previous data published on phage cocktail efficacy.11 this synergistic activity of two-phage cocktail over the mixture of three phages in our study suggested that the individual phages might have employed different receptors to adsorb to the bacterial cells and, hence, effectively inhibited the bacterial growth. some authors have reported that one phage in their two-phage cocktail used an outer membrane protein (ompc) as a receptor, and another one employed a lipopolysaccharide component as its receptor to effectively control bacterial resistance.34,35 moreover, the mixture of many phages may be less effective in the absence of identification of specific bacteriophage receptors because the same phages might share the same receptors and interfere with one another.36,37 the adjunctive treatment (imp2 + 2φ ma3) significantly inhibited the growth of kp20 in vitro compared to the two-phage treatment alone (p = 0.04). at moi 0.001, the two-phage cocktail combined with imipenem had significantly lysed the bacterial cell compared to the two-phage cocktail. this statistical difference might be related to the beneficial effect of bacteriophage treatment in adjunctive therapy. our finding contradicted the study of pacios et al., who indicated the inability of phage-imipenem combination to kill imipenem-resistant isolate harbouring oxa-245 b-lactamase.38 this divergence might be related to the use of a single lytic phage in adjunctive treatment instead of phage cocktail. furthermore, an antibacterial effect was also observed between the phage cocktails in combination with the most effective antibiotic (tigecycline) without significant difference (p = 0.99). a number of studies have reported the positive effect of bacteriophages in combination with antibiotics.39 this synergistic activity between phage cocktail in combination with imipenem might have been due to the sensitivity of the bacterial strains to the select antibiotic after bacteriophage action. indeed, it was reported in a previous study that phage-resistant bacterial strains are more susceptible to antibiotics and the rate of their growth is slower in comparison with wild bacterial strains.40 our study also demonstrated the efficacy of phage cocktail in the absence of phage receptor analysis in formulating phage cocktails. interestingly, this study pointed out the repurposing of imipenem using phage cocktail therapy, and further encourages the repurposing of the efficacy of food and drug administration-approved antibiotics for acceptance of phage therapy worldwide, especially in africa. limitations the limitation of this study was the absence of genomics, which could enable us to further conclude on the taxonomic classification of the bacteriophages. the conclusions were made based on a single carbapenem-resistant strain, which was also a limiting factor in our study due to the high host specificity of phages, and the diverse k. pneumoniae strain types and resistance mechanisms which may affect the utility of the adjunctive therapy. the potential impact of phages on the normal microbiota was not considered in the current study, especially given that the phage cocktails might have activity against k. pneumoniae strains other than those that are carbapenem-resistant, and e. coli strains. conclusion this current study revealed the presence of lytic tailed klebsiella phages belonging to the family myoviridae, siphoviridae and podoviridae in nairobi sewage systems with relatively short latent periods and optimal burst sizes, indicating their therapeutic potential in composing phage cocktails and synergistic interaction in combination with non-sensitive antibiotic (imipenem) against carbapenem-resistant k. pneumoniae clinical isolate in vitro. acknowledgements we are grateful to the pan african university under the african union for financial support. we are thankful to ms ivy mutai, angela juma, mercy munini and mr dennis kotty for their collaboration on phage isolation. our gratitude to dr jessica sacher of the phage directory for having facilitated the transmission electron microscope analysis. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions n.f.m., a.n., j.k.a., g.m. and a.n.k. conceived and designed the study. n.f.m. conducted the laboratory experiments, data analysis and drafted the original manuscript. n.f.m., a.n., j.k.a., g.m. and a.-s.o. reviewed and approved the final manuscript for submission. sources of support this research project was supported by the pan african university. data availability all data are available from the corresponding author, n.f.m., upon request. disclaimer the views and opinions expressed in this article are only those of the authors. references tacconelli e, carrara e, savoldi a, et al. discovery, research, and development of new antibiotics: the who priority list of antibiotic-resistant bacteria and tuberculosis. lancet infect dis. 2018;18(3):318–327. https://doi.org/10.1016/s1473-3099(17)30753-3 ssekatawa k, byarugaba dk, wampande e, ejobi f. a systematic review: the current status of carbapenem resistance in east africa. bmc res notes. 2018;11(1):1–9. https://doi.org/10.1186/s13104-018-3738-2 inal jm. phage therapy: a reappraisal of bacteriophages as antibiotics. arch immunol ther exp ed. 2003;51(4):237–244. li m, xiao y, li p, et al. characterization and genome analysis of klebsiella phage p509, with lytic activity against clinical carbapenem – resistant klebsiella pneumoniae of the kl64 capsular type. arch virol. 2020;165:2799–2806. https://doi.org/10.1007/s00705-020-04822-0 cao f, wang x, wang l, et al. evaluation of the efficacy of a bacteriophage in the treatment of pneumonia induced by multidrug resistance klebsiella pneumoniae in mice. biomed res int. 2015;2015:752930. https://doi.org/10.1155/2015/752930 drulis-kawa z, majkowska-skrobek g, maciejewska b, delattre a, lavigne r. learning from bacteriophages – advantages and limitations of phage and phage-encoded protein applications. curr protein pept sci. 2012;13(8):699–722. https://doi.org/10.2174/138920312804871193 loc-carrillo c, abedon st. pros and cons of phage therapy. bacteriophage. 2011;1(2):111–114. https://doi.org/10.4161/bact.1.2.14590 knezevic p, hoyle ns, matsuzaki s, gorski a. editorial: advances in phage therapy: present challenges and future perspectives. front cell infect microbiol. 2021;12:1390. https://doi.org/10.3389/fmicb.2021.701898 iszatt jj, larcombe an, chan h, stick sm, garratt lw, kicic a. phage therapy for multi-drug resistant respiratory tract infections. viruses. 2021;13(9):1809. https://doi.org/10.3390/v13091809 abd-allah im, el-housseiny gs, yahia is. rekindling of a masterful precedent; bacteriophage: reappraisal and future pursuits. front cell infect microbiol. 2021;11:477. https://doi.org/10.3389/fcimb.2021.635597 chan bk, abedon st, loc-carrillo c. phage cocktails and the future of phage therapy. futur microbiol. 2013;8(6):769–783. https://doi.org/10.2217/fmb.13.47 makumi a, mhone al, odaba j, guantai l, svitek n. phages for africa: the potential benefit and challenges of phage therapy for the livestock sector in sub-saharan africa. antibiotics. 2021;10(9):1085. https://doi.org/10.3390/antibiotics10091085 michodigni nf, nyachieo a, akhwale jk, magoma g. molecular identification of co-existence of carbapenemase and extended-spectrum β-lactamase genes in klebsiella pneumoniae clinical isolates, and their phylogenetic patterns in kenya. adv microbiol. 2021;11(8):399–415. https://doi.org/10.4236/aim.2021.118030 gutiérrez d, fernández l, rodríguezz a, garcía p. practical method for isolation of phage deletion mutants. methods protoc. 2018;1(1):6. https://doi.org/10.3390/mps1010006 oduor jmo, onkoba n, maloba f, nyachieo a. experimental phage therapy against haematogenous multi-drug resistant staphylococcus aureus pneumonia in mice. afr j lab med. 2016;5(1):1–7. https://doi.org/10.4102/ajlm.v5i1.435 pajunen m, kiljunen s, skurnik m, molineux i, molineux i. bacteriophage φ yeo3-12, specific for yersinia enterocolitica serotype o: 3, is related to coliphages t3 and t7 this study. am soc microbiol. 2000;182(18):5114–5120. https://doi.org/10.1128/jb.182.18.5114-5120.2000 haines mek, hodges fe, nale jy, et al. analysis of selection methods to develop novel phage therapy cocktails against antimicrobial resistant clinical isolates of bacteria. front med. 2021;12:613529. https://doi.org/10.3389/fmicb.2021.613529 bradley de. ultrastructure of bacteriophages and bacteriocins. am soc microbiol. 1967;31:230–314. https://doi.org/10.1128/br.31.4.230-314.1967 kutter e. isolation, characterization, and interactions. in: martha rj, clokie amk, editors. bacteriophages: methods and protocols. volume 1. rijeka: 2009 humana press, a part of springer science+business media; 2014. p. 141–148. horváth m, kovács t, koderivalappil s, ábrahám h. identification of a newly isolated lytic bacteriophage against k24 capsular type, carbapenem resistant klebsiella pneumoniae isolates. sci rep. 2020;10:1–11. https://doi.org/10.1038/s41598-020-62691-8 baer a, kehn-hall k. viral concentration determination through plaque assays: using traditional and novel overlay systems. j vis exp. 2014;(93):e52065. https://doi.org/10.3791/52065 merabishvili m, pirnay j, de vos d. guidelines to compose an ideal bacteriophage cocktail. in: sillankorva jas, editor. bacteriophage therapy. rijeka: springer science and business media llc; 2018. p. 99–109. ackermann h. bacteriophage observations and evolution. res microbiol. 2003;154(4):245–251. https://doi.org/10.1016/s0923-2508(03)00067-6 white he, orlova ev. biology and applications. in: savva rs, editor. bacteriophages. london: intechopen; 2019. p. 5–14. gao m, wang c, qiang x, et al. isolation and characterization of a novel bacteriophage infecting carbapenem – resistant klebsiella pneumoniae. curr microbiol. 2020;77:722–729. https://doi.org/10.1007/s00284-019-01849-8 mirzaei mk, nilsson as. isolation of phages for phage therapy: a comparison of spot tests and efficiency of plating analyses for determination of host range and efficacy. plos one. 2015;10(5):e0118557. https://doi.org/10.1371/journal.pone.0127606 anand t, virmani n, kumar s, et al. phage therapy for treatment of virulent klebsiella pneumoniae infection in a mouse model. j glob antimicrob resist. 2020;21:34–41. https://doi.org/10.1016/j.jgar.2019.09.018 bhetwal a, maharjan a, shakya s, et al. isolation of potential phages against multidrug-resistant bacterial isolates: promising agents in the rivers of kathmandu, nepal. biomed res internantional. 2017;2017:3723254. https://doi.org/10.1155/2017/3723254 domingo-calap p, beamud b, vienne j, gonzález-candelas f, sanjuán r. isolation of four lytic phages infecting klebsiella pneumoniae k22 clinical isolates from spain. int j mol sci. 2020;21:425. https://doi.org/10.3390/ijms21020425 skurnik m, pajunen æm, kiljunen æs. biotechnological challenges of phage therapy. biotechnol lett. 2007;29(7):995–1003. https://doi.org/10.1007/s10529-007-9346-1 xie y, wahab lgj. development and validation of a microtiter plate-based assay for determination of bacteriophage host range and virulence. viruses. 2018;10(4):189. https://doi.org/10.3390/v10040189 labrie sj, samson je, moineau s. bacteriophage resistance mechanisms. nat publ gr. 2010;8:317–327. https://doi.org/10.1038/nrmicro2315 microbiol c, chegini z, khoshbayan a, et al. bacteriophage therapy for inhibition of multi drug – resistant uropathogenic bacteria: a narrative review. ann clin microbiol antimicrob. 2021;20:30. https://doi.org/10.1186/s12941-021-00433-y paulo l, carla a, angela c, romalde l, nunes ml. bacteriophages with potential to inactivate salmonella typhimurium: use of. virus res. 2016;220:179–192. https://doi.org/10.1016/j.virusres.2016.04.020 tanji y, shimada t, yoichi m, miyanaga k, hori k, unno h. toward rational control of escherichia coli o157: h7 by a phage cocktail. appl microbiol biotechnol. 2004;64(2):270–274. https://doi.org/10.1007/s00253-003-1438-9 goodridge ld. designing phage therapeutics. curr pharm biotechnol. 2010;11(1):15–27. https://doi.org/10.2174/138920110790725348 hall ar, de vos d, friman v, pirnay j, buckling a. effects of sequential and simultaneous applications of bacteriophages on populations of pseudomonas aeruginosa in vitro and in wax moth. appl environ microbiol. 2012;78(16):5646–5652. https://doi.org/10.1128/aem.00757-12 pacios o, fernández-garcía l, bleriot i, blasco l, gonzález-bardanca m, lópez m. enhanced antibacterial activity of repurposed mitomycin c and imipenem in combination with the lytic phage vb _ kpnmvac13 against clinical isolates of klebsiella pneumoniae. antimicrob agents chemother. 2021;65(9):e0090021. https://doi.org/10.1128/aac.00900-21 li x, he y, wang z, et al. a combination therapy of phages and antibiotics: two is better than one. int j biol sci. 2021;17(13):3573–3582. https://doi.org/10.7150/ijbs.60551 oechslin f. resistance development to bacteriophages occurring during bacteriophage therapy. viruses. 2018;10(7):351. https://doi.org/10.3390/v10070351 abstract introduction research method and design results discussion acknowledgements references about the author(s) joseph h.k. bonney noguchi memorial institute for medical research, university of ghana, legon, ghana edward o. nyarko 37 military hospital, public health division, accra, ghana sally-ann ohene world health organization ghana country office, accra, ghana joseph amankwa disease surveillance department, ghana health service, accra, ghana ralph k. ametepi 37 military hospital, public health division, accra, ghana shirley c. nimo-paintsil noguchi memorial institute for medical research, university of ghana, legon, ghana badu sarkodie disease surveillance department, ghana health service, accra, ghana prince agbenohevi 37 military hospital, public health division, accra, ghana michael adjabeng disease surveillance department, ghana health service, accra, ghana nicholas n.a. kyei 37 military hospital, public health division, accra, ghana samuel bel-nono 37 military hospital, public health division, accra, ghana william k. ampofo noguchi memorial institute for medical research, university of ghana, legon, ghana citation bonney jhk, nyarko eo, ohene s-a, et al. molecular confirmation of lassa fever imported into ghana. afr j lab med. 2016;5(1), a288. http://dx.doi.org/10.4102/ajlm.v5i1.288 original research molecular confirmation of lassa fever imported into ghana joseph h.k. bonney, edward o. nyarko, sally-ann ohene, joseph amankwa, ralph k. ametepi, shirley c. nimo-paintsil, badu sarkodie, prince agbenohevi, michael adjabeng, nicholas n.a. kyei, samuel bel-nono, william k. ampofo received: 10 dec. 2014; accepted: 02 feb. 2016; published: 25 apr. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: recent reports have shown an expansion of lassa virus from the area where it was first isolated in nigeria to other areas of west africa. two ghanaian soldiers on a united nations peacekeeping mission in liberia were taken ill with viral haemorrhagic fever syndrome following the death of a sick colleague and were referred to a military hospital in accra, ghana, in may 2013. blood samples from the soldiers and five asymptomatic close contacts were subjected to laboratory investigations. objective: we report the results of these investigations to highlight the importance of molecular diagnostic applications and the need for heightened awareness about lassa fever in west africa. methods: we used molecular assays on sera from the two patients to identify the causative organism. upon detection of positive signals for lassa virus ribonucleic material by two different polymerase chain reaction assays, sequencing and phylogenetic analyses were performed. results: the presence of lassa virus in the soldiers’ blood samples was shown by l-gene segment homology to be the macenta and las803792 strains previously isolated in liberia, with close relationships then confirmed by phylogenetic tree construction. the five asymptomatic close contacts were negative for lassa virus. conclusions: the lassa virus strains identified in the two ghanaian soldiers had molecular epidemiological links to strains from liberia. lassa virus was probably responsible for the outbreak of viral haemorrhagic fever in the military camp. these data confirm lassa fever endemicity in west africa. introduction lassa virus is a single-stranded rna virus and a member of the arenaviridae family and the genus arenavirus. the virus is the aetiologic agent of lassa fever, which is an acute and often fatal illness, endemic in portions of west africa.1 there are an estimated 300 000 to 500 000 cases of lassa fever each year,2 with a reported mortality rate of 15% – 20% for hospitalised patients. rodents of the genus mastomys are the reservoir for the virus, which is transmitted through direct contact with materials contaminated with urine and/or droppings of infected rodents. human-to-human transmission of the virus may give rise to nosocomial or community-based outbreaks. subsequent to its first isolation in lassa, nigeria and its restricted endemicity to two geographical regions in west africa, recent reports have shown expanded areas of spread.3,4 the upturn in global travel activities and international commitments in conflict and disaster situations have made the import of lassa fever and other viral haemorrhagic fevers (vhfs) into non-endemic countries a more likely event than in the past, which has been well documented.5,6,7,8 presently, an epidemic of another vhf, ebola virus disease, has spread throughout guinea and beyond its borders. as of december 6, 2014, a total of 17 895 ebola virus cases, including 6394 deaths, had been reported in six west african countries (guinea, liberia, sierra leone, nigeria, senegal and mali) and the democratic republic of the congo.9 the use of molecular diagnostic tools has resulted in highly sensitive and specific tests for infectious organisms and genetic diseases. these tools, which include polymerase chain reaction (pcr) and molecular sequencing, have come in handy for the rapid identification of lassa virus and other vhf agents from suspected cases. in may 2013, an outbreak of acute febrile illness affecting three military personnel occurred in the ghanaian united nations mission in liberia military contingent in zorzor, liberia. of the three cases, two were evacuated to a military hospital in ghana, where molecular diagnostic methods were used to determine the causative agent. two of the three cases were fatal. research method and design ethical considerations verbal consent was sought from the close relatives of the patients. the officers were also spoken to personally by the medical team at the 37 military hospital. setting and patients the index case was a male soldier with severe symptoms of malaria who died on may 11, after admission to a level ii hospital for about a week. two male colleagues, aged 27 years (patient 1) and 33 years (patient 2), who were living in the same camp, subsequently developed severe fever and myalgia on may 15 (patient 1) and 21 (patient 2). their medical conditions deteriorated, necessitating their medical evacuation to the 37 military hospital, a level iv facility, in accra, ghana, on may 26, 2013 after five days of illness for patient 1 and 11 days for patient 2. patient 1 died two days after admission. at the military hospital in accra, the medical staff ruled out malaria through microscopic examination of thick and thin blood smears and focused on the clinical manifestations, which were classified as vhf. a presumptive clinical diagnosis of lassa fever was made on the strength of previous outbreaks of lassa fever in liberia.10 to investigate these clinical suspicions, blood samples from the patients and five asymptomatic close contacts were analysed. laboratory investigations five millilitres of blood were collected from each of the two patients (patient 1 sample number: 60fsd_28052013; patient 2 sample number: 59_pn_28052013) and five asymptomatic contacts (medical staff who had initially attended the two patients without barrier nursing). the samples were transported to the virology department of the noguchi memorial institute for medical research at the university of ghana in legon, ghana. the blood samples were processed into serum by low-speed centrifugation at 4 °c and the resultant 3 ml aliquots of sera were cryopreserved at -80 °c. real-time reverse transcription-pcr pan filoviridae assay for marburg and ebola viral rna was extracted from 140 µl of the blood samples using the qiaamp viral rna mini kit (qiagen, hilden, germany) according to the manufacturer’s instructions. a diagnostic assay developed by panning et al.,11 for filovirus species and carried out with a onestep rt-pcr reaction kit (qiagen, hilden, germany) was used in a 25 µl total reaction volume, including 3 µl of the extracted rna. the real-time diagnostic assay used five optimised large (l)-gene primers and three probes, as well as an internal control with a separate detection probe. reactions were supplemented with 40 ng/ml bovine serum albumin and 400 mmol/l each dntp. the primers and probes used were designed and published by panning et al.11 (table 1). all probes were synthesized by tib-molbiol (berlin, germany). amplification in a 96-well abi 7300 real time pcr system (life technology holdings, singapore) involved the following steps: 50 °c for 30 minutes; 95 °c for 15 minutes; and 45 cycles of denaturation at 95 °c for 15 seconds, annealing at 52 °c for 25 seconds and extension at 72 °c for 20 seconds. fluorescence was measured at the end of each 52 °c annealing step. table 1: reverse transcription-pcr test assays used in the laboratory investigations, ghana, 2013. gel-based pan flaviviridae assay for yellow fever and dengue fever an endpoint reverse transcription (rt)-pcr protocol for the detection and identification of flaviviruses, developed by pierre, drouet and deubel in 199412 with a set of universal oligonucleotide primers, was used for laboratory investigation of flaviviruses present in the serum samples. these primers correspond to the 3’ non-coding region of the ns5 gene, which is highly conserved amongst the mosquito-borne flaviviruses. the onestep rt-pcr kit (qiagen, hilden, germany) was used with the following cycling conditions: reverse transcription at 50 °c for 30 minutes; initial activation step at 95 °c for 15 minutes; 45 cycles in a 3-step cycling of 95 °c for 30 seconds, 52 °c for 30 seconds and 72 °c for 30 seconds, with a cooling step of 30 seconds at 30 °c. gel-based lassa fever s-gene segment assay a gel-based conventional rt-pcr assay was performed using abi 2720 thermal cycler (life technology holdings, singapore) for the detection of lassa virus with primers specific for regions of the s rna segment. the rt-pcr (45 cycles) contained onestep rt-pcr kit reagents (qiagen, hilden, germany) with the sense primer 36e2 and the antisense primer lvs-339-rev, as described by ölschläger et al.13 (table 1). cycling conditions for the rt-pcr involved the following steps: 50 °c for 30 minutes; 95 °c for 15 minutes; and 45 cycles of 95 °c for 15 seconds, 52 °c for 30 seconds and 72 °c for 30 seconds. the amplification products (expected size: 320 bp) were electrophoresed on a 2% agarose gel (peqlab biotechnologie, erlangen, germany), stained with ethidium bromide and viewed under a kodak gel logic 100 imaging system (cole-parmer int., chicago, illinois, united states). lassa virus l-gene segment amplification by rt-pcr an rt-pcr assay specific to the l-gene segment of the lassa arenavirus on the rna extracted from the processed blood samples. the 45 cycle rt-pcr used a agpath-id one-step rt-pcr kit (ambion, life technologies, thermo fisher scientific, new york, new york, united states) with the primers as described by vieth et al.14 (table 1). the reaction solution consisted of: 2.5 μl nuclease-free h2o, 12 μl 2x rt-pcr buffer, 2 μl of the upstream and downstream primers (10 μm each), 1.5 μl 2x rt-pcr buffer enzyme mix and 5 µl dna. nucleic acid amplification started with a 30 minute rt step at 50 °c, then an initial denaturation step of 2 minutes at 95 °c and a 45-cycle amplification of 20 seconds at 98 °c, 30 seconds at 55 °c and 60 seconds at 72 °c. a gel was used to separate the products of the nucleic acid amplification. images of the dna bands were captured using a gel logic 100 imaging system (eastman kodak company, rochester, new york, united states). the dna bands of nearly 400 bp in size were analysed and purified for sequencing. real-time rt-pcr assay for lassa fever a real-time rt-pcr assay was performed using the abi 7300/7500 real time pcr system (life technology holdings, singapore) for the detection of lassa virus with primers specific for regions of the small (s) rna gene segment. the reagent used in the preparation of the master mix for the 45-cycle rt-pcr was the power sybr® green rna-to-ct™ 1-step kit (life technologies, carlsbad, california, united states) with the sense primer 36e2 and the antisense primer lvs-339-rev, as described in detail by ölschläger et al.13 (table 1). the amplification protocol was as follows: 48 °c for 30 minutes; 95 °c for 10 minutes; and 45 cycles of 95 °c for 15 seconds, 60 °c for 1 minute. at the end of each cycle, fluorescence was measured at 60 °c. sequencing and phylogenetic analysis the pcr products generated in the conventional rt-pcrs from the l-gene segment fragments were purified in accordance with the manufacturer’s instructions using a commercial kit (big dye xterminator purification kit, applied biosystems, carlsbad, california, united states). the purified products were sequenced on both strands using the l-gene pcr primers and an abi prism 3130 genetic analyser (hitachi high technology, singapore). the sequences were assembled with lasergene software (dnastar, thermo fisher scientific, new york, new york, united states) and automated base calling was proofread by visual inspection of the electropherograms. other published lassa virus sequences were obtained from the national center for biotechnology information’s15,16 genbank (table 2) to align with the generated sequences for phylogenetic analysis. with the use of the national library of medicine’s basic local alignment search tool17 (blast) program, we compared the patients’ lassa virus sequences with other reference lassa virus sequences to locate regions that were similar. all sequences obtained were copied into bioedit software (north carolina state university, raleigh, north carolina, united states) and put together into an ideal sequence alignment file with the use of a multiple sequence alignment tool, clustalw18 (version 2; embl-ebi, cambridge, united kingdom). we then used molecular evolutionary genetics analysis (mega) software (version 5.05) in a kimura two-parameter model19 to infer a phylogenetic tree from the aligned nucleotide sequences following a neighbour-joining method. to establish reliability and to infer the strength of similarity between the patient’s sequence and the referenced sequences from the phylogenetic tree, a bootstrap analysis of 1000 replicates was used. table 2: lassa fever viral strains used for homology and phylogenetic analysis of ghana case strains, 2013. results detection and characterisation of nucleic acid in clinical specimens the gel-based rt-pcr flavivirus tests for yellow fever and dengue fever (types 1–4), as well as the real-time rt-pcr filovirus test for marburg and ebola, were negative (table 3). however, the rt-pcr amplification of the s-gene segment of arenaviruses detected a 320 bp dna band in the well with the patients’ clinical specimens. the length and position of these bands were on a par with the positive lassa virus control (an inactivated culture supernatant of cells infected with lassa virus strain csf); no band was observed in the negative control (pcr-grade water) well. the real-time rt-pcr amplification of the s-gene segment of the arenavirus test produced a clear peak with a sigmoid-shaped curve for the patient’s samples, whereas no peak was observed for the negative control, which in the figure is covered by the threshold line, or baseline (figure 1). this two-result signal indicated the presence of lassa virus in the patients’ sera. the samples from the five asymptomatic close contacts were tested in the same assay run and no indication of the presence of lassa virus was observed (data not shown). figure 1: real-time pcr amplification of patient sera and controls. table 3: summary of suspected patient information and their test results, ghana, 2013. lassa virus l-gene segment sequencing and homology analysis the gel-based rt-pcr amplification assay for the large l-gene segment of the arenavirus showed ~400 bp products that matched the expected size (figure 2). the comparison of the nucleotide sequences from the amplified products with the genbank database showed that the patients’ sequences had high similarities to known lassa virus strains. genetically, the sequences were close to strains that had originated in guinea and been reported in liberia. the highest similarity (85% maximum identity in nucleotides) was to lassa virus strain macenta20 and lassa virus strain las803792.14 figure 2: ethidium bromide-stained 2% agarose gel image of the pcr products generated from the two patients samples run in duplicate. phylogenetic tree analysis the phylogenetic analysis with genbank data revealed that the nucleotide sequences from the patients had close phylogenetic relationships with the reported lassa virus strains macenta20 (genbank accession number: ay628200) and las80379214 (genbank accession number: ay693638) (figure 3). the sequence data from the patient who died (patient 1, sample no. 60fsd_28052013) was sent to genbank and assigned accession number kf425246. figure 3: phylogenetic relationship between the nucleotide sequences of reported lassa virus strains and that of the two patients. discussion we identified the aetiological agent responsible for suspected cases of vhf imported into ghana. the patients were soldiers with the ghanaian united nations mission in liberia military contingent in zorzor, liberia, who had had close contact earlier with another soldier suspected of dying from vhf. these patients were medically evacuated, with deteriorating health conditions, to a level iv military hospital in ghana, where viral nucleic acid was detected and characterised from the patients’ blood specimens. the characteristic arenavirus signal indicated on both the gel-based and real time rt-pcrs was confirmed as lassa virus by capillary dna sequencing. these results confirmed lassa virus as the aetiological agent that caused the outbreak in liberia, where lassa fever is known to be endemic.21 it has been documented that lassa fever seems to have two geographically-separate endemic areas: the mano river region in the west (guinea, sierra leone and liberia) and nigeria in the east.22 moreover, literature indicates that, since the initial discovery of lassa fever in nigeria in 1969, nosocomial outbreaks have occurred repeatedly in three localities, specifically zorzor, phebe and ganta in liberia.23 molecular analyses of patients’ samples from the outbreak we report are indicative of clues regarding the source of the aetiological agent. the nucleotide sequences from the samples aligned closely with the lassa fever strain isolated in guinea in 200424 and also reported subsequently in liberia.21 the 85% proportion of alignment between genbank-reported strains and sequences from the patients’ sera suggests a link between the source of the patients’ lassa virus infection and the district of zorzor in liberia from which they were evacuated. in addition, this finding supports the assertion that persons participating in humanitarian missions or peacekeeping activities in the regions comprising sierra leone and liberia are at risk for lassa fever.23,24,25 phylogenetic analysis of our patients’ sequences with genbank-reported lassa virus strains yielded a single parsimonious tree rooted with the prototype lp strain of lassa virus. our results indicate that our patients’ samples had close genetic relationships to the macenta20 and las80379214 strains of lassa virus, both of which were reported in liberia but isolated in guinea in 2004. the sequence data from the patient who died (patient 1; sample no. 60fsd_28052013) had close homology with reported lassa strain las803792, which was isolated from a fatal case in 2004.14 this observation is consistent with a study that suggested that lassa virus strains differ in virulence potential.1 limitations our report was limited by our inability to conduct a battery of tests, including serological assays, for either an ideal suspected case(s) of vhf or for the five asymptomatic close contacts of the evacuated patients. that notwithstanding, this report underscores the importance of preventive measures for all visitors and workers including peacekeeping forces to endemic regions in west africa. this is because there is currently no effective lassa fever vaccine is available. recommendations it is recommended that medical support plans for peacekeeping operations should be built purposefully and in consideration of existing endemicity and history of endemicity in the host nations. such support plans should be duly informed by frequently-updated research on endemic agents and other health concerns in the visiting country. sensitisation of officers and other travellers to infectious agents in the host country and the importance of speedy reporting to health facilities when unwell should be given sufficient emphasis. this report of laboratory investigations of imported cases of lassa fever and other documented medical fatalities on past peacekeeping operations supports the need for essential medical organisational changes in future operations. this would involve a good balance of proximity to medical care and transportation time for medical emergencies. conclusion in conclusion, it is envisaged that the importation of vhfs into non-endemic countries will increase in likelihood as a result of increased travel and international commitments in conflict and disaster situations to vhf-endemic countries in west africa. thus, healthcare providers should: (1) have a high index (low threshold) of suspicion for vhf amongst travellers returning from endemic areas; (2) promptly implement appropriate infection prevention and control measures; and (3) rapidly report suspected cases to avert undue nosocomial transmission. acknowledgements we are most grateful to the medical and para-medical staff of the public health department of the 37 military hospital and the staff of virology department of the noguchi memorial institute for medical research, college of health sciences, university of ghana, legon, particularly acknowledging the technical support by juliana naa dedei aryeequaye. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. sources of support this work was supported by the noguchi memorial institute for medical research, university of ghana, legon. authors’ contributions j.h.k.b. performed the laboratory testing, analysed and interpreted the results and wrote the manuscript. e.o.n. coordinated sample collection and surveillance and edited the manuscript. s.-a.o. coordinated surveillance and edited the manuscript. j.a. supervised surveillance and edited the manuscript. r.k.a. was involved in surveillance and sample collection. s.c.n.-p. participated in sample testing and analysis and edited the manuscript. b.s. took part in the surveillance and edited the manuscript. p.a. participated in sample collection and surveillance. m.a. edited the manuscript and was involved in surveillance. n.n.a.k. was involved in surveillance and sample collection. s.b.-n. was involved in surveillance and edited the manuscript. w.k.a. supervised the design and implementation of the work and edited the manuscript. references günther s, lenz o. lassa virus. crit rev clin lab sci. 2004; 41(4):339–390. http://dx.doi.org/10.1080/10408360490497456 mccormick jb. epidemiology and control of lassa fever. curr top microbiol immunol. 1987;134:69–78. http://dx.doi.org/10.1007/978-3-642-71726-0_3 sogoba n, feldmann h, safronetz d. lassa fever in west africa: evidence for an expanded region of endemicity. zoonoses public health. 2012;59(suppl 2):43–47. http://dx.doi.org/10.1111/j.1863-2378.2012.01469.x dzotsi ek, ohene sa, asiedu-bekoe f, et al. the first cases of lassa fever infection in ghana. ghana med j. 2012;46(3):166–170. unit for surveillance and communication, unit for preparedness and response, editorial team. case of lassa fever imported into germany from sierra leone. euro surveill. 24 july 2006;11(30):pii=3008. günther s, emmerich p, laue t, et al. imported lassa fever in germany: molecular characterization of a new lassa virus strain. emerg infect dis. 2000;6(5):466–476. http://dx.doi.org/10.3201/eid0605.000504 günther s, weisner b, roth a, et al. lassa fever encephalopathy: lassa virus in cerebrospinal fluid but not in serum. j infect dis. 2001;184(3):345–349. http://dx.doi.org/10.1086/322033 schmitz h, köhler b, laue t, et al. monitoring of clinical and laboratory data in two cases of imported lassa fever. microbes infect. 2002;4(1):43–50. http://dx.doi.org/10.1016/s1286-4579(01)01508-8 world health organization. ebola virus disease, west africa – update [page on the internet]. c2014 [cited 2014 dec 06]. available from: http://who.int/csr/don/2014_05_15_ebola/en/ frame jd, jahrling pb, yalley-ogunro je, et al. endemic lassa fever in liberia. ii. serological and virological findings in hospital patients. trans r soc trop med hyg. 1984;78(5):656–660. http://dx.doi.org/10.1016/0035-9203(84)90232-3 panning m, laue t, ölschlager s, et al. diagnostic reverse-transcription polymerase chain reaction kit for filoviruses based on the strain collections of all european biosafety level 4 laboratories. j infect dis. 2007;196(suppl 2):s199–204. http://dx.doi.org/10.1086/520600 pierre v, drouet mt, deubel v. identification of mosquito-borne flavivirus sequences using universal primers and reverse transcription/polymerase chain reaction. res virol. 1994;145(2):93–104. http://dx.doi.org/10.1016/s0923-2516(07)80011-2 ölschläger s, lelke m, emmerich p, et al. improved detection of lassa virus by reverse transcription-pcr targeting the 5’ region of s rna. j clin microbiol. 2010;48(6):2009–2013. http://dx.doi.org/10.1128/jcm.02351-09 vieth s, drosten c, lenz o, et al. rt-pcr assay for detection of lassa virus and related old world arenaviruses targeting the l gene. trans r soc trop med hyg. 2007; 101(12):1253–1264. http://dx.doi.org/10.1016/j.trstmh.2005.03.018 sayers ew, barrett t, benson da, et al. database resources of the national center for biotechnology information. nucleic acids res. 2009;40(database issue):d13–d25. benson da, karsch-mizrachi i, clark k, et al. genbank. nucleic acids res. 2009;40(database issue):d48–d53. http://dx.doi.org/10.1093/nar/gkn723 altschul sf, gish w, miller w, et al. basic local alignment search tool. j mol biol. 1990;215(3):403–410. http://dx.doi.org/10.1016/s0022-2836(05)80360-2 larkin ma, blackshields g, brown np, et al. clustalw and clustalx version 2. bioinformatics. 2007;23(21):2947–2948. http://dx.doi.org/10.1093/bioinformatics/btm404 tamura k, peterson d, peterson n, et al. mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. mol biol evol. 2011;28(10):2731–2739. http://dx.doi.org/10.1093/molbev/msr121 hajjaj a, chain psg, do lh, et al. lassa virus strain macenta segment l, complete sequence. accession number: ay628200; 2004. jahrling pb, frame jd, smith sb, et al. endemic lassa fever in liberia. iii. characterization of lassa virus isolates. trans r soc trop med hyg. 1985;79(3):374–379. http://dx.doi.org/10.1016/0035-9203(85)90386-4 fichet-calvet e, rogers dj. risk maps of lassa fever in west africa. plos negl trop dis. 2009;3(3):e388. http://dx.doi.org/10.1371/journal.pntd.0000388 ter meulen j, koulemou k, wittekindt t, et al. detection of lassa virus antinucleoprotein immunoglobulin g (igg) and igm antibodies by a simple recombinant immunoblot assay for field use. j clin microbiol. 1998;36(11):3143–3148. fair j, jentes e, inapogui a, et al. lassa virus-infected rodents in refugee camps in guinea: a looming threat to public health in a politically unstable region. vector borne zoonotic dis. 2007;7(2):167–171. http://dx.doi.org/10.1089/vbz.2006.0581 khan sh, goba a, chu m, et al. new opportunities for field research on the pathogenesis and treatment of lassa fever. antiviral res. 2008;78(1):103–115. http://dx.doi.org/10.1016/j.antiviral.2007.11.003 article information authors: phidelis m. maruti1 ekesa a. mulianga1 lorna n. wambani1 melda n. wafula1 fidelis a. mambo2 shadrack m. mutisya3 eric n. wakaria4 erick m. mbati5 angela a. amayo4 jonathan m. majani1 bryan nyary6 kilian a. songwe3 affiliation: 1ministry of health-kenya, kenya2department of health sciences, masinde muliro university, kenya 3a global healthcare public foundation, kenya 4management sciences for health-kenya, kenya 5aids, population and health integrated assistance plus (aphia plus) western, kenya 6international healthcare and development, nigeria correspondence to: phidelis maruti postal address: bungoma district hospital, 14-50200 bungoma, kenya dates: received: 22 may 2014 accepted: 02 july 2014 published: 16 sept. 2014 republished: 07 nov. 2014 how to cite this article: maruti pm, mulianga ea, wambani ln, et al. creating a sustainable culture of quality through the slmta programme in a district hospital laboratory in kenya. afr j lab med. 2014;3(2), art. #201, x pages. http://dx.doi.org/10.4102/ ajlm.v3i2.201 note: article republished with updated references relating the special issue. copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. creating a sustainable culture of quality through the slmta programme in a district hospital laboratory in kenya in this original research... open access • abstract • introduction • research method and design • results and discussion    • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: bungoma district hospital laboratory (bdhl), which supports a 200-bed referral facility, began its strengthening laboratory management toward accreditation (slmta) journey in 2011 together with eight other laboratories in the second round of slmta rollout in kenya.objectives: to describe how the slmta programme and enhanced quality interventions changed the culture and management style at bdhl and instilled a quality system designed to sustain progress for years to come. methods: slmta implementation followed the standard three-workshop series, mentorship site visits and audits. in order to build sustainability of progress, bdhl integrated quality improvement processes into its daily operations. the lab undertook a process of changing its internal culture to align all hospital stakeholders – including upper management, clinicians, laboratory staff and maintenance staff – to the mission of sustainable quality practices at bdhl. results: after 16 months in the slmta programme, bdhl improved from zero stars (38%) to four stars (89%). over a period of two to three years, external quality assessment results improved from 47% to 87%; staff punctuality increased from 49% to 82%; clinician complaints decreased from 83% to 16; rejection rates decreased from 12% to 3%; and annual equipment repairs decreased from 40 to 15. twelve months later the laboratory scored three stars (81%) in an external surveillance audit conducted by kenya accreditation service (kenas). conclusion: management buy-in, staff participation, use of progress-monitoring tools and feedback systems, as well as incorporation of improvement processes into routine daily activities, were vital in developing and sustaining a culture of quality improvement. introduction top ↑ laboratory systems are one of the core capacities that countries must develop in order to comply with world health organization (who) international health regulations, since they play a major role in the key processes of detection, assessment, response, notification and monitoring of events.1 as laboratory results influence up to 70% of medical diagnoses,2 reliable laboratory services are essential for the provision of safe and effective treatment to patients. the quality of laboratory services is a major factor that directly affects the quality of healthcare in a country.2the strengthening laboratory management toward accreditation (slmta) programme promotes rapid, measurable improvement in laboratories of developing countries. slmta is implemented through multiple workshops with intervening site visits to support improvement projects. kenya began the slmta implementation process with 12 laboratories in april 2010. bungoma district hospital laboratory (bdhl) was enrolled in the second slmta round in february 2011, along with eight other laboratories. after the first three months of slmta implementation, bdhl management noted, from both the internal audit report and general observations, that little progress had been made. as a result, radical changes were phased in to encourage all laboratory staff to participate in improvement activities, adopt more disciplined and stringent work duties and schedules, engage in laboratory planning and include hospital management and other stakeholders in the process. this article describes how the slmta programme and enhanced interventions changed the culture and management style at bdhl, instilling a system designed to sustain progress for years to come. research method and design top ↑ bungoma district hospital, a primary care facility with very limited resources, started operating in 1952 as a chief’s native health centre. located in bungoma town, it now serves as a referral hospital for the north-western region of kenya. with 200 in-patient beds, it offers both out-patient and in-patient services and provides laboratory services for haematology, serology, clinical chemistry, immunology, microbiology, parasitology and blood banking.consistent with the slmta protocol,3 a baseline audit was conducted in february 2011, followed by three workshops, two one-week mentorship site visits after each workshop, a mid-term audit and an exit audit in march 2012. to determine the impact of changes made at bdhl and the sustainability of the new systems, an external surveillance audit was conducted by the kenya accreditation service (kenas) in february 2013, 12 months after slmta concluded. the non-profit charity a global healthcare public foundation played an important role in conducting the three workshops, on-site mentorship and conference call follow up. in addition, the foundation provided funding for laboratory facility and equipment upgrades. efforts were made to engage all hospital management in the process, as well as other stakeholders, including the hospital maintenance unit, the procurement and/or supplies unit and clinicians. slmta was integrated by laboratory staff into routine work processes and a succession plan was developed for laboratory management to ensure the sustainability of the quality improvements. this included appointing a deputy for each key function, performing on-the-job mentorship of staff on slmta, as well as regular internal reviews of progress. further engaging stakeholders and ensuring continued progress, the laboratory staff conducted weekly customer surveys (for patients/clients), which informed improvement projects. slmta uses the who’s regional office for africa’s (who afro) stepwise laboratory quality improvement process towards accreditation (slipta) framework to both guide improvement activities and evaluate programme effectiveness.4unlike traditional pass/fail accreditation schemes, slipta uses a zeroto five-star scale to recognise the evolving fulfilment of international organization for standardization (iso) 15189 requirements. laboratories that fail to achieve at least 55% on their audit score receive a zero-star rating; 55% − 64% yields one star, 65% − 74% yields two stars, 75% − 84% yields three stars, 85% − 94% yields four stars and laboratories that achieve 95% or more receive five stars.4 this stepwise approach acknowledges laboratories where they stand, supports them with a series of evaluations to demonstrate improvement and both recognises and rewards their progress. the slipta process is not intended to replace established accreditation schemes, but rather to provide an interim pathway to the realisation of international laboratory standards.5 several indicators were measured to assess the impact of slmta implementation. firstly, results from routine external quality assessment (eqa) panels from human quality assessment services (huqas), conducted three times per year for 22 analytes, were compared; the average annual percentage of correct responses from 2010 to 2013 are presented. secondly, staff punctuality in 2011–2013 was assessed based on data from an employee time clock, defined as the average overall percentage of person-days that staff arrived on time for their shift. thirdly, clinician and customer satisfaction were assessed by means of a ‘how do you rate us’ form that was made available to all patients in 2012–2013 and clinicians in 2011–2013; the proportion of forms submitted with complaints was calculated. fourthly, annual average sample rejection rates for all laboratory tests were calculated for 2011–2013; and finally, equipment repairs and the proportion carried out by external engineers versus internal staff from the hospital’s biomedical engineering department were assessed for 2011–2013. results and discussion top ↑ bdhl’s baseline audit score was 38% (0 stars). after slmta implementation, the laboratory scored 89% (4 stars) at the exit audit. the surveillance audit carried out 12 months afterward yielded a score of 81% (3 stars) (figure 1). figure 1: results for baseline, mid-term, exit and surveillance audits, bungoma district hospital laboratory, kenya. without hospital management support, sustainable changes are difficult to achieve. poor eqa data for chemistry and haematology, as well as an increased mortality rate in medical wards from 3% in 2009 to 9% in 2011, were presented to management to demonstrate that laboratory failures could be contributing to deaths, especially amongst hiv patients for whom treatment depends heavily on chemistry results. hospital management approved the purchase of a new fully-automated analyser to replace the old semi-automated analyser and control materials, and convinced partners to donate air conditioners for the laboratory. as a result, erratic temperatures no longer interfered with the quality of results or turnaround time and overall eqa results improved from 47% in 2010 to 87% in 2013 – above the set target of 80% (table 1). because staff buy-in is also crucial, further efforts were made to encourage participation throughout the hospital. an annual award scheme for the entire hospital was established in order to motivate staff to improve patient care. in addition, a ‘wall of fame’ and a ‘wall of shame’ were instituted in order to further inculcate a culture of friendly competition amongst staff and to ensure conformity to the set standards. these activities led to the prompt release of test results, thereby improving turnaround time and the efficiency and quality of patient care throughout the hospital. however, the laboratory experienced substantial staff turn-over in 2013, with four laboratory personnel being transferred, including the medical superintendent and laboratory manager. this left the laboratory with nine staff members, thus causing acute staff shortages and gaps in laboratory management, with the result being that customer complaints increased from 3% in 2012 to 22% in 2013. a new policy for clocking-in and -out for laboratory staff and the introduction of a leave request form resulted in an increase in staff punctuality from 49% in 2011 to 82% in 2013 (table 2), as well as an increase in staff availability to perform assigned tasks. this temporarily addressed staff shortages. quarterly meetings for one-on-one mentorship with each laboratory staff member were introduced. during these meetings, laboratory management reminded staff members of strategic objectives, thanked them for their hard work, provided feedback on their performance and suggested areas of improvement. in response to positive feedback and this collaborative approach, laboratory staff members reported feeling appreciated, more engaged and willing to be part of a team to improve healthcare quality in the hospital. the laboratory also began holding quarterly meetings with clinicians to discuss sample rejection rates, clinicians’ perception of the laboratory and suggestions for improvement. the proportion of clinicians who reported complaints on the feedback form decreased from 83% in 2011 to 16% in 2013, whilst the total number of form submissions increased from 76 to 252. sample rejection rates declined from 12% in 2011 to 3% in 2013 and clinicians reported in meetings that their confidence in laboratory results had improved. the laboratory also met regularly with stakeholders from the maintenance and procurement departments in order to advocate for prompt routine preventive equipment maintenance. consequently, the number of equipment repairs decreased from 40 in 2011 to 15 in 2013 and the proportion of repairs conducted by an external engineer versus the hospital biomedical engineering department decreased from 80% to 20% (table 1). table 1: summary of quality indicators before and after slmta implementation. in order to sustain the gains achieved, slmta was integrated into daily routines, building a foundation for continuous improvement. discussions of improvement projects are now included in regular laboratory staff meetings; the laboratory conducts weekly hands-on continuous medical education sessions; and all staff members are now involved in budget and planning discussions. these changes were designed in order to improve the laboratory staff’s customs, beliefs and attitudes in the workplace, leading to widespread and lasting staff support of laboratory quality improvement activities. sustainability is further enhanced by quarterly internal audits, conducted by the laboratory quality officer. to monitor processes, staff members identify causes of problems and suggest possible solutions. a root cause analysis is conducted and corrective action is identified, following a two-step procedure. step 1 involves the development of a cause-and-effect diagram (figure 2) in order to categorise probable causes of non-conformity under ‘the 6 ms’ of machinery, methods, measurement, manpower, materials and milieu (environment).6 using objective evidence, the quality officer then examines each probable cause and, based on the evaluation, the staff then works by process of elimination to identify those items which were most likely associated with the non-conformity. figure 2: cause-and-effect diagram. step 2 is the root cause investigation. using the problems identified in step 1, an investigation into their root cause is performed. for example, the quality officer may recognise that laboratory personnel do not have proper competency records. to find the source of this problem, the officer may ask ‘why?’ and then receive a variety of answers, including: • staff are not aware of the need for competency assessment. • staff were not trained on the procedure for competency assessment. • the quality officer thought the procedure was covered during training. • no records of training are kept. • the training procedure does not mention the need to keep records. after this questioning, the root cause of the problem may become clear: for example, perhaps the training procedure does not fully address the need for record keeping. the quality officer may then recommend that, in order to improve the system, the training procedure must be revised. this process of identifying problems and selecting improvement activities allows for a clear understanding of what is hindering efficient and reliable work in the laboratory and provides appropriate solutions for improvement (table 2). table 2: summary of improvement activities. conclusion bdhl successfully used slmta to progress from zero to four stars within a 16-month period and to maintain a three-star rating for 12 months thereafter. this quality improvement required substantial effort and a collaborative approach. fundamental steps were necessary in order to create and maintain a culture that supports continuous quality improvement. firstly, universal rules were established and enforced, such as adopting written protocols and practices that prescribe clear policies, procedures, values and behaviours. secondly, the principles and techniques of quality improvement and their associated behaviours were taught so that staff members could learn both the concepts and how to apply them. finally, it was critical to reinforce these principles and behaviours on a continual basis. bdhl employees were recognised and rewarded when they demonstrated adherence and consequences were made clear for noncompliance. leaders and managers did not allow laboratory staff to become complacent, simply meeting basic or minimum requirements. everyone was pressed continually for professional excellence, growth and improvement.for quality management systems to be implemented effectively and sustained, the hospital’s management and staff must be involved and willing to participate. bdhl can attest that sustainable improvement is achieved by engaging all hospital stakeholders, leading to increased confidence in the laboratory on the part of clinicians, nurses and patients. long-term sustainability will rely on continued vigilance, training of new staff and participation of all stakeholders. acknowledgements top ↑ the authors wish to acknowledge the commitment of the bungoma district hospital management for nursing the implementation to reality, including the entire bdhl staff, procurement unit staff and hospital maintenance unit staff who all worked tirelessly to achieve the implementation. gratitude goes to a global public health foundation for its mentorship and support; to the us centers for disease control and prevention (cdc) kenya office for its support and coordination; to the us president’s emergency plan for aids relief (pepfar) for providing funds to implement slmta; and to global scientific solutions for health for their manuscript support. finally, we wish to acknowledge talkmore maruta for his training and encouragement. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions p.m.m. (ministry of health-kenya) was responsible for experimental and project design. e.a.m., l.n.w. and m.n.w. (all ministry of health-kenya) performed most of the experiments. f.a.m. (masinde muliro university), s.m.m. (a global healthcare public foundation), e.m.m. (aphia plus), a.a.a. (management sciences for health-kenya), j.m.m. (ministry of health-kenya) and b.n. (international healthcare and development) all made conceptual contributions. e.n.w. (management sciences for health-kenya) was responsible for the calculations; and k.a.s. (a global healthcare public foundation) was the project leader. references top ↑ 1.masanza mm, ngobile n, mukanga d, et al. laboratory capacity building for the international health regulations (ihr[2005]) in resource-poor countries: the experience of the african epidemiology network (afenet). bmc public health. 2010;10(suppl 1):s8. http://dx.doi.org/10.1186/1471-2458-10-s1-s82.guzel o, guner ei. iso 15189 accreditation: requirements for quality and competence of medical laboratories, experience of a laboratory i. clin biochem. 2009;42(4–5):274–278. http://dx.doi.org/10.1016/j.clinbiochem.2008.09.011 3.yao k, maruta t, luman e, et al. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. in press. 4.who afro. who guide for the stepwise laboratory improvement process towards accreditation in the african region (with checklist) [document on the internet]. c2012 [cited 2014 jul 20]. available from: http://www.afro.who.int/en/clusters-a-programmes/hss/blood-safety-laboratories-a-health-technology/blt-highlights/ 3859-who-guide-for-the-stepwise-laboratory-improvement-process-towards-accreditation-in-the-african-region-with-checklist.html 5.gershy-damet gm, rotz p, cross d, et al. world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm 6.wikipedia. ishikawa diagram [page on the internet]. n.d. [cited 20 jul 2014]. available from: http/en.wikipedia.org/wiki/ishikawa_diagram abstract introduction signs and symptoms of inherited metabolic disorders basic methods for the laboratory investigation of inborn errors of metabolism purine metabolism disorder conclusion acknowledgements references about the author(s) john i. anetor department of chemical pathology, faculty of basic medical sciences, university of ibadan, ibadan, nigeria bose e. orimadegun department of chemical pathology, faculty of basic medical sciences, university of ibadan, ibadan, nigeria gloria o. anetor department of public health science, faculty of health sciences, national open university of nigeria, abuja, nigeria citation anetor ji, orimadegun be, anetor go. a pragmatic approach to the diagnosis of inborn errors of metabolism in developing countries. afr j lab med. 2023;12(1), a1946. https://doi.org/10.4102/ajlm.v12i1.1946 review article a pragmatic approach to the diagnosis of inborn errors of metabolism in developing countries john i. anetor, bose e. orimadegun, gloria o. anetor received: 30 apr. 2022; accepted: 05 apr. 2023; published: 30 may 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract inborn errors of metabolism (iem) are a group of genetically derived diseases that are individually rare but collectively common and can be very severe. while high-income countries usually employ modern scientific technologies like tandem mass spectrometry for iem investigation, these disorders are, in contrast, only rarely screened for in developing countries due to misconceptions that the required facilities are beyond the reach of these countries. this paper attempts to educate scientists and clinicians in developing countries on low-technology iem screening methods that only require moderate facilities. although a definitive diagnosis of iem may require specialised laboratory investigations and attendant interpretation, in most cases, the basic facilities available in the average clinical chemistry laboratory in developing countries can allow the early detection of iem. this early detection would facilitate critical early decision making, thus leading to better management, optimised treatment, and reduced morbidity and or mortality of iem in these resource-limited countries. with this approach, a few referral centres for confirmatory investigation, comparable to those existing in developed countries, could be established. this can be integrated into creative health education for healthcare professionals and families who have individuals with iem. what this study adds: iems are important enough that every country, developed or developing, should have screening plans and basic laboratory facilities that are adequate for initial iem diagnosis. no country should therefore give up on testing for iems on the excuse of a paucity of advanced facilities. keywords: basic laboratory investigations; developing countries; inborn errors of metabolism; health education; optimisation of treatment. introduction inborn errors of metabolism (iem), also referred to as inherited metabolic disorders, are mutational disorders that impair the function of proteins in every way (mostly enzymatic functions). archibald garrod, who was the first to explain the relationship between genotype and phenotype, helped bring attention to iem through his pioneering work on one of the first types of iem discovered – alkaptonuria.1,2 his work helped to elucidate iem, thus paving the way for the realisation that every aspect of human anatomy and physiology is determined by biochemical reactions catalysed by specific enzymes, and that enzymes are determined by genetic constitution. since then, several additional types of iem have been described, and many more are being investigated (table 1).3 although iems are quite common collectively, individual iem disorders are rare. the most frequently encountered iems are monogenic forms.4,5 in most cases, iems are autosomal recessive. table 1: types of inborn errors of metabolism commonly investigated using laboratory procedures. according to the 2003 institute of medicine report ‘reducing birth defects: meeting the challenge in the developing world’,6 birth defects are the leading cause of infant mortality in many regions of developing countries.7 in these countries, malnutrition, poverty, disease, and lack of access to healthcare elevate the prenatal risk of birth defects and impose enormous personal and societal consequences. experts in developed countries often have similar concerns because birth defects are also the highest single cause of infant death in many regions.8 there is a link between birth defects and genetic abnormalities such as iems,7 and environmental and genetic factors are inextricably linked. as these gene-environment interactions are important in iem, people in developing countries are at a greater risk of developing an iem.9 this highlights the need for scientists and doctors to prioritise iem detection, management, and prevention through research aimed at identifying environmental red flags. a holistic and integrated approach is also required for the effective investigation and treatment of iem. to achieve this, collaboration across the distinct disciplines of genetics, evolutionary and developmental biology, environmental science, and sociology is necessary.7 this article is, in part, an extended response one of the authors gave at a laboratory medicine congress in north africa in late 2019, where a questioner inferred that iems are not screened for or diagnosed in developing nations because the needed specialist laboratory facilities are not available. although a recent report from colombia (a developing country) by echeverri et al.10 outlines the activities in their reference centre that support the questioner’s position, their diagnostic approach is not pragmatic or applicable to most developing countries but may apply to referral centres in developing countries. nevertheless, they did make a notable point about the importance of health education for both health and non-health professionals, who are critical and underappreciated in the field of iem. a recent report by civallero, de kremer and giugliani11 proposed the use of a few simple and affordable instruments such as spectrophotometer and gas chromatography-mass spectrometry for iem investigations, as these can give an enormous amount of information that can be adapted to the needs of health facilities in developing countries. this approach contrasts that of echeverri et al.10 and may be more relevant to health facilities in developing countries. the mayo clinic has also outlined another diagnostic approach that can be modified for the qualitative and quantitative determination of diagnostic markers of iem (table 2).12 table 2: general methods for the qualitative and quantitative determination of diagnostic markers of inborn errors of metabolism. clinically, iem can be grouped into three categories, namely, disorders that cause intoxication, disorders that affect energy metabolism, and disorders that affect complex molecules.13,14 in disorders of intoxication, metabolites accumulate at toxic concentrations and the accumulating metabolites are usually proximal to the metabolic block. consequently, identifying these metabolites provides a basis for diagnosis using basic laboratory facilities, which are readily affordable in resource-limited settings. therefore, this article aims to raise awareness of basic investigations that can detect iem early and do not require expensive or sophisticated equipment commonly used in developed countries. a quasi-systematic review was conducted where the science citation index and pubmed databases were searched, independent of date of publication, for articles on iem. we prioritised articles that were published in developing countries and articles published by leading authorities and institutions. we also included the early lectures of the ‘father of inborn errors of metabolism’, archibald garrod.1,2 we conducted a limited synthesis of all retrieved articles and found evidence of the paucity of investigations of iem in developing countries due to the lack of facilities, which we have tried to address in this article. a major limitation of our approach is that it only provides a cursory overview of the topic. signs and symptoms of inherited metabolic disorders there are some common signs and symptoms that raise the index of suspicion of iem.15 these include acute life-threatening crisis, lethargy or coma with or without a symptom-free period, seizures, persistent vomiting, respiratory distress syndrome (apnoea), poor feeding/failure to thrive, hypotonicity/hypertonicity (ataxia, posturing), hepatosplenomegaly/jaundice, dysmorphic features (facial coarsening), macroglossia, ocular features (cataract, corneal clouding, abnormal eye movements), coarse hair with abnormal texture, abnormal odour of urine/body, loss of developmental milestones, unusual responses to fasting and intercurrent infection (von gierke’s disease, type 1 glycogenosis), and fluctuating neurological status. basic methods for the laboratory investigation of inborn errors of metabolism alkaptonuria alkaptonuria is one of the earliest iems described by garrod.2 this is a benign disorder that poses no danger to life except for ochronosis (darkening) of the cartilage, and patients with this condition are prone to arthritis from the fourth decade of life. alkaptonuria is characterised by the darkening of standing urine, and a simple benedict’s reaction on the urine sample will give a positive reaction. all that is required for this test is copper sulphate in an alkaline medium, which is inexpensive. phenylketonuria some basic investigations can also aid in the early detection of phenylketonuria, which is one of the most well-known and potentially fatal iems that is associated with profound intelligence quotient loss and severe mental retardation (mr). phenylketonuria can easily be diagnosed using the fecl3 test (on which the phenistix test is based) that requires only a 10% fecl3 solution, the guthrie test, amino acid analysis using high-performance liquid chromatography or tandem mass spectrometry. the latter two methods may be expensive but could be made available in a referral centre. the guthrie screening test for increased phenylalanine in serum is one alternative to specialised high-performance liquid chromatography for amino acid analysis that is within the reach of many laboratories in developing countries. it is a microbiological test that relies on phenylalanine’s ability to counteract the effects of a metabolic antagonist on the growth of a bacillus subtilis strain that requires phenylalanine as a growth factor. the test procedure involves the suspension of b. subtilis spores in an agar medium containing a minimum amount of growth nutrients plus a fixed amount of the metabolic antagonist, β-2-thienylalanine, which is a non-protein amino acid similar in structure to phenylalanine. when the infant is 6 days old (or as soon as possible if the baby is being treated with antibiotics that interfere with the test), blood is drawn by heel prick onto filter paper, dried, and sent to the laboratory. these blood spots are cut into uniform discs and then placed on agar plates inoculated with the b. subtilis isolate, with the only source of phenylalanine being the blood spot. the blood spot discs are compared to discs containing known amounts of phenylalanine. if the serum contains a high level of phenylalanine, the phenylalanine will diffuse from the sample disc into the bacterial medium to counteract the inhibitory effect of thienylalanine, resulting in a ring of bacterial growth around the disc. if the phenylalanine level is less than 2 mg/dl, the growth inhibition will not be overcome, and no bacterial growth will be observed. as false positives can occur, positive results should be confirmed using a chemical method or chromatography. to avoid false positive results due to liver immaturity, tests should not be performed on premature infants or full-term babies immediately after birth. the infant should also be on a phenylalanine-containing diet for at least 24 h before the sample is collected.3 maple syrup urine disease in maple syrup urine disease, there is a deficiency of the branched chain α-ketoacid dehydrogenase enzyme that decarboxylates the branched chain amino acids (leucine, isoleucine, and valine). this results in the accumulation of these amino acids and their metabolites (a-ketoacids) in the blood. maple syrup urine disease is a severe iem that begins in the first year of life and progresses to severe mr, acidosis, coma, and death within 5 years if left untreated. as an alternative to tandem mass spectrometry and high-performance liquid chromatography analysis of organic acids, maple syrup urine disease can easily be detected in the laboratory using the rothera’s test. this requires a simple sodium nitroprusside reagent (a common reagent in most clinical chemistry laboratories) and ammonium hydroxide. when mixed and pulverised, the test retains its positive reaction, unlike in cases of metabolic derangement such as type 1 diabetes mellitus (diabetes ketoacidosis), where the ketone bodies (acetoacetate, acetone, and 3-hydroxy butyrate) are responsible for the positive reaction. the clinical observation of the characteristic offensive odour of the urine (isovaleric aciduria), which may be the first suggestive sign of abnormal metabolite excretion, may be an indication for this investigation. alpha-1-antitrypsin deficiency another iem that can readily be investigated with the moderate facilities available in most developing countries, is alpha-1-antitrypsin deficiency. alpha-1-antitrypsin is an antiprotease synthesised in the liver, and its deficiency arises from point mutations in the serpina1 gene (single amino acid substitution), with over 70 alleles described.16 the normal genotype is pimm, and pimz is the heterozygous genotype for the z gene. alpha-1-antitrypsin deficiency frequently arises from pizz (the homozygous genotype for the z gene).17 this genetic defect causes the protein to form aggregates in the liver that cannot be excreted, thus causing liver damage. the absence of its antiprotease activity also causes emphysema and cirrhosis in children. although alpha-1-antitrypsin deficiency is commonly investigated using polymerase chain reaction in advanced laboratories, the combination of traditional liver function tests and cellulose acetate serum protein electrophoresis, when the α-band is either significantly reduced or completely absent, is highly suggestive. wilson’s disease wilson’s disease (hepatolenticular degeneration) is another iem that can easily be investigated in a basic clinical laboratory. the disease is due to a mutation in the atp7b gene, which encodes the copper-transporting protein, ceruloplasmin. wilson’s disease is easily identified by kayser fleischer’s rings (almond-coloured rings around the cornea). in the laboratory, wilson’s disease can be diagnosed by determining serum copper levels using simple flame atomic absorption spectrophotometry and ceruloplasmin by standard spectrophotometric methods. the result will show low ceruloplasmin concentrations and serum copper levels accompanied by massive copper excretion in the urine. if necessary, a liver biopsy can be done to detect excessive amounts of copper in the liver. cystic fibrosis cystic fibrosis is a common iem that affects one out of every 2500 live births in the united kingdom.18 cystic fibrosis is inherited in a recessive mendelian pattern, and individuals with this disease experience a generalised exocrine secretion disorder that is characterised by abnormally viscous secretions. the functional defect is caused by the cystic fibrosis transmembrane conductance regulator protein, which controls transmembrane chloride transport.19 surprisingly, cystic fibrosis patients who do not have functional copies of the protein have a slew of lung and digestive problems.20 symptoms include recurrent respiratory infections, irreversible lung disease, and pancreatic insufficiency leading to malabsorption. in neonates, intestinal obstruction can occur due to the increased viscosity of faecal material, which is useful in laboratories for disease screening. cystic fibrosis can be identified clinically through slit-lamp examination of affected patients’ eyes, which may reveal pathognomonic crystals of cystine in the cornea. this corneal defect can cause photophobia, beginning around the age of two. cystic fibrosis can be screened for using immunoreactive trypsin in blood. also, faecal pancreatic elastase-1 is the most widely used test to diagnose pancreatic insufficiency in people with cystic fibrosis. sweat electrolyte analysis is used to make the final diagnosis; when sweat chloride level is less than 30 mmol/l, cystic fibrosis is not likely, but when it is greater than or equal to 60 mmol/l, it is diagnostic.21 pilocarpine iontophoresis (using an electric device to stimulate sweating) is also a standard test used in the diagnosis of cystic fibrosis. galactosaemia galactosaemia is caused by a lack of galactose-1-phosphate uridyltransferase, a rate-limiting enzyme that causes hypoglycaemia. this leads to increased levels of galactose-1-phosphate due to blockage of the typical metabolic pathway, and this may lead to alternative metabolism and direct tissue damage. some key biochemical features of galactosaemia include impaired bilirubin uptake and conjugation, increased unconjugated hyperbilirubinemia, hepatomegaly, jaundice, severe mr, accumulation of free galactose, and deposition of galactose-1-phosphate in renal tubules with attendant renal damage and associated generalised aminoaciduria. simple and practical tests that can be used to investigate galactosaemia include benedict’s test to detect the presence of reducing substances, thin-layer chromatography to determine the presence of galactose in urine, and galactose-1-phosphate uridyltransferase enzyme assay to detect elevated blood galactose. amniocentesis may be used for prenatal diagnosis, and the galactose tolerance test may be performed if the expected rise in the blood glucose level after galactose administration is not observed. in advanced laboratories, molecular analysis is available.22 the diagnosis of galactosaemia is well within the capabilities of most basic medical facilities and the treatment is simple, only requiring the elimination of galactose from the diet. von gierke’s disease in von gierke’s disease, there is deficiency of glucose-6-phosphatase, an enzyme that splits glucose from glucose-6-phosphate (glucose-6-po4 + g-6-phosphatase → glucose + po4). owing to this metabolic block, glucose-6-phosphate accumulates in the liver. the presence of fasting hypoglycaemia and hepatomegaly are strongly suggestive of von gierke’s disease and clearly distinguishes it from other glycogenoses such as pompe’s, mcardle’s, and many others. urea cycle disorders type 1 hyperammonaemia type 1 hyperammonaemia is one of the common urea cycle disorders (ucds). it is an autosomal recessive disorder that is caused by a deficiency of the carbamoyl phosphate synthetase 1 enzyme. the main biochemical characteristics of ucds are extremely high blood ammonia levels, mr, and a low urea level. because of the mr that comes with this metabolic disorder, early detection is critical, and the diagnosis is within the capabilities of a typical laboratory. to screen for type 1 hyperammonaemia, advanced methods such as amino acid analysis using high-performance liquid chromatography or tandem mass spectrometry may be required. in a resource-limited medical facility, blood gas and electrolyte abnormalities are important for detecting ucds. the presence of reduced blood urea levels, increased ammonia levels, elevated transaminase (alanine transaminase and aspartate transaminase) activity, and coagulopathy (increased prothrombin time and partial thromboplastin time) should be strongly suggestive of a urea cycle defect and risk of encephalopathy and should be sufficient basis for referring patients for specialist investigation and initiating early patient management. hyperammonaemia type ii the biochemical lesion of hyperammonaemia type ii is a deficiency of the ornithine transcarbamoyl transferase enzyme, and the inheritance pattern is x-linked. the key features of this disorder include elevated ammonia levels in the blood, cerebrospinal fluid and urine, as well as orotic aciduria caused by carbamoyl phosphate channelling into pyrimidine synthesis. hyperammonaemia is caused by a lack of any of the urea cycle enzymes. if the metabolic block occurs in one of the earlier steps of the urea cycle, ammonia, which is neurotoxic, accumulates and causes a more severe condition. conversely, the absence of enzymes catalysing reactions in the later stages of the urea cycle results in the accumulation of less toxic intermediates and less severe symptoms. hence, the exact point of the metabolic block needs to be identified as it determines the prognosis. because the brain is sensitive to ammonia, hyperammonaemia, as well as elevated ammonia levels in other body fluids, causes toxic symptoms and neurological damage. understanding the situation is critical because it guides treatment, which primarily consists of a low-protein diet and frequent small feeds. hippuric acid formation (conjugation product between glycine and benzoyl is a detoxification step) or phenylacetyl glycine can also eliminate amino nitrogen. citrullinaemia citrullinaemia is another example of ucds. citrullinaemia is caused by arginosuccinate synthetase deficiency, which results in elevated ammonia and citrulline levels in the blood. breast milk should be avoided in this condition because it contains high amounts of citrulline. hyperornithinaemia another urea cycle enzyme deficiency is hyperornithinaemia, which is an ornithine transport defect. it is an autosomal recessive condition in which ammonia and ornithine levels in the blood are elevated. hyperargininaemia hyperargininaemia, which results from arginase deficiency and causes arginine accumulation in the blood and cerebrospinal fluid, is another metabolic abnormality seen in ucds. it is worth noting that in this condition, cysteine and lysine are excreted in the urine instead of arginine. mass spectrometry can be used to screen for hyperargininaemia. purine metabolism disorder purine metabolism disorder is best illustrated by lesch-nyhan’s (l-n) syndrome, an x-linked inherited purine metabolism disorder. the biochemical lesion is caused by a hypoxanthine-guanine phosphoribosyltransferase deficiency, which impairs the purine salvage pathway. this results in the accumulation of phosphoribosyl pyrophosphate, an intermediate in the purine synthetic pathway, and other purine synthetic cycle intermediates in the biosynthetic pathway. hypoxanthine-guanine phosphoribosyltransferase activity is normal in the brain and at low levels in the liver and spleen.23 some of the neurological manifestations of l-n syndrome are thought to be due to the toxicity of the purine degradation products to the developing central nervous system or the absence of hypoxanthine-guanine phosphoribosyltransferase, which results in an imbalance in adenine and guanine nucleotides at a critical developmental phase for the infant.24 self-mutilation, mr, raised uric acid levels (described as petit gout), nephrolithiasis (renal stone), and gout later in life are the main characteristics of l-n syndrome. the elevated uric acid level in relation to the patient’s age and other clinical manifestations, such as mr, are sufficient indications for l-n syndrome diagnosis in resource-limited countries. molecular investigation for confirmation may be requested from referral centres. tangier’s disease tangier’s disease is one of the common iems of lipid metabolism. tangier’s disease is an autosomal dominant metabolic disorder that derives from the deficiency of adenosine triphosphate-binding cassette transporter-1. the key biochemical and clinical characteristics of tangier’s disease include defective efflux of cholesterol from cells, reduction in high-density lipoprotein levels in the blood, absence of the α-band in serum protein electrophoresis, accumulation of cholesterol esters in tissues, presence of large orange-yellow tonsils, and muscle atrophy associated with recurrent neuropathies and atherosclerosis. the aforementioned features of the disease are sufficient indicators for the early detection of tangier’s disease before the onset of the later features, such as neuropathy and atherosclerosis. porphyrias the porphyrias are a group of iems associated with the absence of genes encoding enzymes that catalyse reactions in the haem biosynthetic pathway (porphyria means purple). the porphyrias are characterised by increased production and excretion of porphyrin precursors (delta aminolaevulinic acid and porphobilinogen). most are inherited as autosomal dominant traits and are classified into three broad groups: hepatic porphyrias, erythropoietic porphyrias, and combined erythropoietic and hepatic abnormalities. the classification is based on the major site where the enzyme deficiency is manifested. the clinical manifestations of porphyrias vary and are not usually associated with anaemia. one of the most common clinical manifestations is acute intermittent porphyria, which is an inherited genetic disorder.25 the biochemical lesion in acute intermittent porphyria is a lack of porphobilinogen deaminase (uroporphyrinogen-1-synthetase), which causes a secondary increase in aminolaevulinic acid synthase activity (negative feedback mechanism abolished). delta aminolaevulinic acid and porphobilinogen, two key intermediates in the haem biosynthetic pathway, are elevated in blood and urine of patients with porphyrias. the porphobilinogen assay should be conducted using fresh urine samples transported in dark bottles. the key reagent required for porphobilinogen is p-dimethylaminobenzaldehyde (ehrlich’s reaction), which is commonly employed in testing for urobilinogen, a basic test conducted in all laboratories. elevated urinary porphobilinogen excretion confirms the presence of hepatic porphyria. if porphobilinogen excretion exceeds aminolaevulinic acid excretion, lead poisoning can be ruled out.26,27 acute abdominal pain and neurological manifestations, such as sensory and motor disturbances, including confusion and agitation, are among the clinical symptoms that may be intermittent and vague (dubbed ‘little imitators’). a simple ultraviolet fluorescence test with a wood’s lamp to demonstrate porphyrin is also useful and easily accessible. congenital hypothyroidism all newborns in every hospital should be screened for congenital hypothyroidism before they are discharged. congenital hypothyroidism is a clinically significant disorder, especially due to its adverse neurological outcome. this adverse neurological outcome can however be easily reversed if detected early. most infants are asymptomatic at birth because thyroxin from the maternal circulation diffuses across the placenta. congenital hypothyroidism may be suggested by clinical features such as lethargy, umbilical hernia, slow movement, hoarse cry, macroglossia, hypothermia, and hypotonia.28 a simple panel of thyroid function tests, especially thyroid-stimulating hormone, are useful investigations to confirm these clinical findings where they manifest. the thyroid-stimulating hormone value is particularly important as it is very sensitive.29 congenital adrenal hyperplasia congenital adrenal hyperplasia is an inherited endocrinopathy caused by the genetic absence of certain enzymes involved in steroidogenesis, particularly the cortisol pathway. due to the lack of a negative feedback mechanism, this biochemical lesion results in the accumulation of intermediates, some of which are potent androgens such as 17-hydroxyprogesterone, which accounts for virilism and genital ambiguity in affected patients. the most common of these enzyme deficiencies is a 21-hydroxylase deficiency. the enzyme responsible for producing the mineralocorticoid aldosterone may also be altered, which could explain the salt-loss presentation (hyponatraemia and hyperkalaemia). some of the key clinical and biochemical features include precocious growth, increased testosterone, increased 17-hydroxyprogesterone, suboptimal cortisol level with basal and adrenocorticotropic hormone stimulation tests, small testes, and shock. though genetic tests like karyotyping may be used to detect congenital adrenal hyperplasia, basic tests such as sodium assay (hyponatraemia), potassium assay (hyperkalaemia), urinary sodium test (increased sodium level), and plasma renin activity (increased plasma renin activity level) may be within the reach of laboratories in resource-limited countries. owing to the severity of congenital adrenal hyperplasia, we think it should be a candidate for newborn screening programs in these countries. inherited hyperbilirubinaemias congenital hyperbilirubinaemias arise from the absence of enzymes involved in bilirubin metabolism and transportation, especially uridine diphosphate glycosyltransferase. some of these hyperbilirubinaemias include crigler najjar, gilbert, dubin-johnson, and rotors. these can be easily diagnosed by basic bilirubin determination (total and conjugated) and related to clinical presentation. a combination of blood and urine findings helps in the differential diagnosis. newborn screening programmes newborn screening programmes are useful for the early detection of conditions with a presumptive period during which treatment can dramatically improve the outcome. these programmes are especially useful in resource-poor countries where facilities for managing ensuing complications are unavailable in delayed cases. while screening programmes are available in many developed countries, they are not available in many developing countries, and those that were previously available have degenerated. the assumption that the observed degeneration is due to a lack of advanced scientific equipment required for investigation is false as many iems can be detected using basic clinical chemistry laboratory facilities; this has been demonstrated in this brief article and supported by brown and lo14 and, more recently, civallero, de kremer and giugliani.11 even in the absence of tandem mass spectrophotometry in these countries, basic laboratory investigations can accomplish a great deal. inherited metabolic disorders such as congenital hypothyroidism (cretinism), phenylketonuria, and cystic fibrosis are commonly screened for in high-income countries like the united kingdom. in developing countries, these disorders, as well as others such as maple syrup urine disease, galactosaemia, alpha-1-antitrypsin deficiency, ucds, and l-n syndrome, can also routinely be investigated using basic laboratory facilities. for early diagnosis and to avoid the serious and irreversible consequences of iem, close collaboration between clinics and metabolic laboratories, or just clinical laboratories, is critical. a structured approach should be in place and may include targeted laboratory investigation and a comprehensive patient examination to assess drug use, feeding history, transfusion history, and family medical history. at the minimum, an iem team consisting of clinicians (paediatricians), a clinical chemist or laboratory scientist, and a dietician must be available. interpretation of findings should take a multidisciplinary approach as this is one area where clinicians require a great deal of guidance from laboratory specialists. many physicians who request laboratory tests are unfamiliar with the metabolic derangements associated with various iems. brown and lo14 propose that metabolic investigation reports should include age-related reference intervals for quantitative results. with a differential diagnosis derived from relevant abnormal and/or normal data, recommendations for further specialist investigations, and sufficient contact information for such institutions, there should be enough collaboration for clinicians to feel free to contact the laboratory if questions arise. laboratory reports are best interpreted when clinical and relevant laboratory information are provided along with the test requisition, as is well-known in pathology and laboratory medicine. results pointing to multiple iems should be correlated with the patient’s clinical and laboratory data to narrow the differential diagnosis before further investigation.14,30 it is recommended to conduct basic investigations that are useful in the diagnosis of iems as these are beneficial to the community (table 3).31 table 3: basic investigations useful in the diagnosis of inborn errors of metabolism. conclusion although the individual types of iems are rare, this category of diseases is common and often severe. because severe morbidity and/or mortality can be avoided in some forms of iem, it is critical to make accurate diagnoses early, especially in developing countries with limited complication management facilities. though the definitive diagnosis of iems may require specialised laboratory investigations and interpretation, basic investigations that can be conducted in the average clinical chemistry laboratory in developing countries can provide valuable information for critical and early decision making in most cases. referrals to specialised centres for further investigation can then be done. appropriate interpretation of investigation findings requires interdisciplinary collaboration between clinical laboratory scientists and clinicians and is critical for the early diagnosis of patients with iem and improved management of complex cases. a good team of paediatricians, clinical chemists (or laboratory scientists), and a dietician can be extremely useful. it is critical to emphasise that education and training for both health and non-health professionals are both essential and beneficial. health facilities in developing countries need support from international organisations like the international federation of clinical chemistry to improve current diagnostic capacity. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions j.i.a., b.e.o. and g.o.a. contributed equally to the conceptualisation, review and final approval of the manuscript. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability data sharing does not apply to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this manuscript are those of the authors and do not reflect the official policy or position of any affiliated agency of the authors. references garrod ae. inborn errors of metabolism. the croonian lectures. london: oxford university press; 1909. garrod ae. the incidence of alkaptonuria: a study in chemical individuality. med chir trans. 1902;85:69–78. hortin gl. amino acids, peptides and proteins. in: burtis ca, bruns de, editors. tietz fundamentals of clinical chemistry and molecular diagnostics. st. louis, mo: saunders, elsevier inc.; 2015. pp. 286–317. leonard jv, morris aam. inborn errors of metabolism around the time of birth. lancet. 2000;356:583–587. https://doi.org/10.1016/s0140-6736(00)02591-5 marshall wj, lapsley m, day a, ayling r. clinical biochemistry: metabolic and clinical aspects. 3rd ed. edinburgh: churchill livingstone, 2014; pp. 461–483. institute of medicine committee on improving birth o. in: bale jr, stoll bj, lucas ao, editors. reducing birth defects: meeting the challenge in the developing world. washington (dc): national academy of sciences press, usa; 2003. weinhold b. environmental factors in birth defects: what we need to know. environ health perspect. 2009;117(10):a440–a447. https://doi.org/10.1289/ehp.117-a440 arnold gl. inborn errors of metabolism in the 21(st) century: past to present. annals of translational medicine. 2018;6(24):467. https://doi.org/10.21037/atm.2018.11.36 edwards tm, myers jp. environmental exposures and gene regulation in disease etiology. environ health perspect. 2007;115(9):1264–70. https://doi.org/10.1289/ehp.9951 echeverri ox, guevara jm, espejo j, et al. research, diagnosis, and education in inborn errors of metabolism in colombia: 20 years’ experience from a reference center. orphanet j rare dis. 2018;13(1):141. https://doi.org/10.1186/s13023-018-0879-2 civallero g, de kremer r, giugliani r. high-risk screening and diagnosis of inborn errors of metabolism: a practical guide for laboratories. j inborn errors metab screen. 2018;6. https://doi.org/10.1177/2326409818792065 mayo clinic medical laboratories. biochemical genetics – inborn errors of metabolism. rochester, mn: mayo foundation for medical education and research; 2011. fernandes j, saudubray jm, van den berghe g. inborn metabolic diseases: diagnosis and treatment. 4th ed. berlin: springer verlag, 2000; p. 561. brown sm, lo sf. inborn errors of metabolism. in: clark w, editor. contemporary practice in clinical chemistry. 3rd ed. london: aacc press, 2016; p. 651. bakerman s. bakerman’s abc of interpretive laboratory data. chicago, il: yearbook medical publishers; 2002. stoller jk, hupertz v, aboussouan ls. alpha-1 antitrypsin deficiency. in: adam mp, everman db, mirzaa gm, et al., editors. genereviews®. seattle, wa: university of washington, 2006; pp. 1993–2022. demir n, diken eo, karabulut gh, karnak d, kayacan o. alpha-1 antitrypsin levels and polymorphisms in interstitial lung diseases. turk j med sci. 2017;47(2):476–482. https://doi.org/10.3906/sag-1508-76 nickle t, barrette-n i. mutation and variation usa [homepage on the internet]. libretexts; c2021 [cited 2021 feb 01]. available from: https://bio.libretexts.org/bookshelves/genetics/book%3a_online_open_genetics_(nickle_and_barrette-ng)/04%3a_mutation_and_variation ooi cy, durie pr. cystic fibrosis transmembrane conductance regulator (cftr) gene mutations in pancreatitis. j cyst fibrosis. 2012;11(5):35–562. https://doi.org/10.1016/j.jcf.2012.05.001 gibson-corley kn, meyerholz dk, engelhardt jf. pancreatic pathophysiology in cystic fibrosis. j pathol. 2016;238(2):311–320. https://doi.org/10.1002/path.4634 schmidt h, sharma g. sweat testing. in: statpearls [homepage on the internet]. treasure island, fl: statpearls publishing; 2022 [updated 2022 may 08]. available from: https://www.ncbi.nlm.nih.gov/books/nbk547728/ timson dj. the molecular basis of galactosemia – past present and future. gene. 2015;589(2):133–141. https://doi.org/10.1016/j.gene.2015.06.077 green a, morgan i, gray j. neonatology and laboratory medicine. london: acb venture publication; 2003. sciver cr, beaudet al, sly wl, et al., editors. the metabolic and molecular basis of inherited disease. 8th ed. new york, ny: mcgraw-hill; 2001. peters tj, sarkany r. porphyria for the general physician. clin med. 2005;5(3):275–281. https://doi.org/10.7861/clinmedicine.5-3-275 anderson ka, sassa s, bishop d, desnick r. disorders of heme biosynthesis: x-linked sideroblastic anemia and the porphyrias. in: sciver c, beaudet a, sly w, valle d, childs b, editors. metabolic and molecular bases of inherited disease [homepage on the internet]. new york, ny: mcgraw-hill, 2001; pp. 2991–3062. shedlofsky si. young woman with recurrent abdominal pain. in: scott mg, grownowski am, eby cs, editors. tietz’s applied laboratory medicine. hoboken, nj: john wiley, 2007; pp. 513–522. ain. the fatigued attorney. in: scott mg, grownowski am, eby cs, editors. tietz’s applied laboratory medicine. hoboken, nj: john wiley, 2007; pp. 513–522. demers lm, spencer ca. laboratory medicine practice guidelines: laboratory support for the diagnosis and monitoring of thyroid disease. clin endocrinol (oxf). 2003;58(2):138–140. https://doi.org/10.1046/j.1365-2265.2003.01681.x anetor ji, durojaiye oo, iyanda a, adeniyi a, agbedana eo. a basic investigation for inherited metabolic disease in two institutions for the handicapped in ibadan: indication for genomics. j biomed invest. 2006;4(1):23–27. https://doi.org/10.4314/jbi.v4i1.30411 kruszka p, regier d. inborn errors of metabolism: from preconception to adulthood. am fam physician. 2019;99(1):25–32. abstract introduction methods results discussion acknowledgements references about the author(s) alshymaa a. ahmed department of clinical pathology, faculty of medicine, zagazig university, zagazig city, egypt alia a. el shahawy department of medical microbiology and immunology, faculty of medicine, zagazig university, zagazig city, egypt heba m. kadry department of medical microbiology and immunology, faculty of medicine, zagazig university, zagazig city, egypt nora m. said department of clinical pathology, faculty of medicine, zagazig university, zagazig city, egypt citation ahmed aa, el shahawy aa, kadry hm, said, nm. performance of two multiplex flow cytometric assays for antibody detection in egyptian patients. afr j lab med. 2023;12(1), a2099. https://doi.org/10.4102/ajlm.v12i1.2099 note: additional supporting information may be found in the online version of this article as online supplementary document 1 original research performance of two multiplex flow cytometric assays for antibody detection in egyptian patients alshymaa a. ahmed, alia a. el shahawy, heba m. kadry, nora m. said received: 14 oct. 2022; accepted: 22 mar. 2023; published: 18 may 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: autoantibodies are vital biomarkers for the diagnosis, assessment and prognostic determination of various autoimmune disorders. objective: this study aimed to evaluate the performance of the two athena multi-lyte® systems for the detection of various autoantibodies. methods: a total of 105 systemic lupus erythematosus patients, 35 patients with other autoimmune diseases (diseased controls), and 30 healthy volunteers (healthy controls) at zagazig university hospitals, zagazig city, al sharqia governorate were tested for anti-double-stranded dna (anti-dsdna) antibodies using indirect immunofluorescence (iif) and the athena multi-lyte® anti-nuclear antibodies-ii system between may 2020 and april 2022. seventy-five patients with clinically suspected autoimmune vasculitis (aiv) and 25 healthy volunteers were also tested for anti-myeloperoxidase and anti-proteinase 3 antibodies using iif, the athena multi-lyte® aiv system, and enzyme-linked immunosorbent assay (elisa). results: the athena anti-dsdna test (98.5%) was more specific than iif (96.9%) for diagnosing systemic lupus erythematosus, but both tests had the same sensitivity (38.1%). combining both methods increased sensitivity to 47.6%, while increasing the cut-off of the athena anti-dsdna test to 134 international units/ml increased specificity to 100%. the athena multi-lyte aiv system exhibited substantial agreement with iif regarding anti-myeloperoxidase testing (κ = 0.65) and almost perfect agreement with elisa (κ = 0.85). the athena multi-lyte® aiv system exhibited perfect agreement with iif (κ = 1) and substantial agreement with elisa for anti-proteinase 3 testing (κ = 0.63). conclusion: athena multi-lyte® systems appear to be reliable for anti-dsdna, anti-myeloperoxidase, and anti-proteinase 3 screening and may be an optimal choice for monitoring anti-dsdna levels. what this study adds: it is necessary to evaluate various autoantibodies detection assays to increase both sensitivity and specificity of autoimmune diseases diagnostic approaches. athena multi-lyte® systems appear to be reliable for anti-dsdna, anti-myeloperoxidase, and anti-proteinase 3 screening and may be an optimal choice for monitoring anti-dsdna levels. keywords: autoantibodies; anti-double-stranded dna; anti-neutrophil cytoplasmic antibodies; multiplex; performance. introduction systemic lupus erythematosus (sle) and systemic vasculitis are multisystem autoimmune diseases characterised by the generation of circulating autoantibodies specific to different target antigens. the incidence and prevalence of both sle and anti-neutrophil cytoplasmic antibody (anca)-associated vasculitis (aav) show wide geographical variations.1 the overall incidence of sle ranges from 0.3 cases per 100 000 population per year in ukraine to 31.5 per 100 000 per year among afro-caribbean people living in the united kingdom.2 in egypt, the estimated prevalence of adult sle is 6.1 cases per 100 000 persons, with a higher prevalence (11.3 per 100 000) reported among women.3 anti-neutrophil cytoplasmic antibody-associated vasculitis inflicts considerable global effects, including elevated mortality rates, poor quality of life, and socio-economic burdens,4 and studies have demonstrated an aav prevalence of between 48 and 184 cases per million individuals.1 in egypt, aav constitutes about 3.6% of vasculitides.5,6 however, there is inadequate data to identify the prevalence of aav in africa and south asia.7 autoantibodies are crucial biomarkers that guide the diagnosis, treatment, and follow-up of autoimmune disorders.8 new automated high-throughput assays are continuously being developed to replace conventional assays for detecting these autoantibodies. however, an accurate evaluation of these new techniques is mandatory to ensure that they are clinically valuable and can improve various medical decisions.9 according to the guidelines provided by the european league against rheumatism and the american college of rheumatology, patients are classified as positive for sle if they have serum anti-nuclear antibody titres ≥ 1:80.10 the presence of antibodies against double-stranded dna (anti-dsdna) is the serological hallmark for a diagnosis of sle. an increased anti-dsdna titre in association with low levels of the complement components c1q, c3, and c4 is indicative of an acute sle exacerbation.11,12 diverse methods have been developed for the detection of anti-dsdna. the crithidia luciliae immunofluorescence test is remarkably specific and yields a high positive predictive value for sle. however, the sensitivity of c. luciliae immunofluorescence test for sle diagnosis is low (ranging from 20% to 55%, depending on the kit supplier), and being a semi-quantitative test, it is not an optimal choice for clinical follow-ups and disease flare predictions.13,14,15 in contrast, the enzyme-linked immunosorbent assay (elisa) is more sensitive (> 60%) than c. luciliae immunofluorescence test but has a lower specificity for sle.15,16 multiplex bead-based assays provide the advantage of assessing multiple antibody specificities simultaneously in small volumes of serum. one such technique is the addressable laser bead immunoassay, as exemplified by the commercially available luminextm platforms. the sensitivity of addressable laser bead immunoassay is similar to that of elisa for the detection of anti-dsdna, while the specificity is comparable to that of c. luciliae immunofluorescence test.17 existing guidelines for the diagnosis and follow-up of sle such as ‘the 2019 european league against rheumatism and the american college of rheumatology classification criteria for systemic lupus erythematosus’18 do not specify a certain assay for anti-dsdna detection. however, the guidelines advise that positive results should be confirmed by indirect immunofluorescence (iif) or a farr assay.15,19 the detection of ancas is vital for the diagnosis of the unique group of small-vessel vasculitic disorders named aav. this group includes three main diseases: granulomatosis with polyangiitis, microscopic polyangiitis, and eosinophilic granulomatosis with polyangiitis. patients with granulomatosis with polyangiitis are mainly proteinase 3-anca positive, while those with microscopic polyangiitis and eosinophilic granulomatosis with polyangiitis are mainly myeloperoxidase-anca positive.20,21 indirect immunofluorescence is used as a screening test for anca in accordance with the 1999 international recommendations for anca detection.22 this guideline advises the use of an antigen-specific assay to confirm a positive iif test. however, the revised 2017 international consensus of anca testing23 stated that the combined use of both iif and antigen-specific immunoassays is not necessary. specifically, a high-quality antigen-specific immunoassay is sufficient and does not need iif confirmation.24,25 international guidelines and recommendations have emphasised the roles of anti-dsdna and anca in the diagnosis and follow-up of autoimmune rheumatic diseases and vasculitic disorders.15,23 with this study, therefore, we aimed to assess the performance of the athena multi-lyte® test systems for the detection of these antibodies. methods ethical considerations ethical approval for this study was obtained from the zagazig university-institutional research board (zu-irb) (approval number: 5926). written informed consent was obtained from participants and parents or guardians of patients younger than 18 years. all procedures were performed according to the principles of the declaration of helsinki.26 patients’ files and samples were de-identified and coded, and all laboratory members were blinded to the participants’ data. study population this study was conducted in the clinical pathology department of the faculty of medicine at zagazig university, zagazig, al sharqia, egypt, from may 2020 to april 2022. all participants were of the same ethnicity. the required sample sizes were calculated using the openepi program version 3 (https://www.openepi.com/samplesize/sscc.htm) at 95% confidence, 90% power, and a 10% dropout rate.27 we assumed the anti-dsdna sensitivity in sle patients to be 33% and that up to 3% of healthy controls and 6% of diseased controls can be anti-dsdna positive.15 we also assumed an anca sensitivity of 46% in vasculitis patients and that up to 6% of healthy controls may be anca positive.28 sera for anti-dsdna testing were collected from 170 subjects, including 105 patients who met the revised criteria (european league against rheumatism and the american college of rheumatology) for sle classification18 and 65 control subjects matched for age and gender. the control subjects included 35 patients diagnosed with autoimmune diseases other than sle (diseased controls) and 30 healthy participants (healthy controls). sera for anca testing were collected from 75 patients with clinically suspected autoimmune vasculitis (aiv) and 25 healthy volunteers matched for age and gender (control group). individuals who were pregnant, had a severe infection or systemic disease,29 or refused to sign the informed consent were excluded from the study. personal data (age, gender, ethnicity) and medical data were collected from patients’ files retrieved from the rheumatology department after obtaining the required permissions. for the diseased control group, the disease diagnosis was confirmed clinically and by the presence of specific autoantibodies. serum rheumatoid factor and anti-ccp measured using the cobas 6000 autoanalyzer (roche diagnostics, forrenstrasse, rotkreuz, switzerland) were used to confirm rheumatoid arthritis. using the anti-nuclear antibodies profile 3 immunoglobulin g (igg) euroline immunoblot assay (euroimmun, seekamp, lübeck, germany), we measured anti-sjögren’s-syndrome-related antigen a and anti-sjögren’s-syndrome-related antigen b for primary sjögrens’ syndrome diagnosis, anti-scl70 for systemic sclerosis diagnosis, and anti-jo1 and anti-pm/scl100 for polymyositis diagnosis. sample collection two millilitres of whole blood were collected from each patient at the time of diagnosis. the blood samples were left to clot at room temperature for 15 min – 30 min and then centrifuged at 1000−2000 × g for 10 min. sera were separated and stored at −80 °c prior to use. multiplex microbead-based immunoassay the athena multi-lyte ana-ii plus test system (zeus scientific, evans way, branchburg, new jersey, united states) was used for the quantitative detection of igg-class anti-dsdna, the qualitative detection of igg-class anti-nuclear antibodies, and the semi-quantitative detection of igg-class antibodies specific for eight different nuclear antigens (sjögren’s-syndrome-related antigen a, sjögren’s-syndrome-related antigen b, sm, rnp, scl-70, jo-1, centromere b, and histone) in the sera of enrolled subjects. the athena multi-lyte aiv plus test system (zeus scientific, evans way, branchburg, new jersey, united states) was used for the semi-quantitative and qualitative detection of igg-class antibodies specific for three analytes (myeloperoxidase, proteinase 3, and glomerular basement membrane) in the sera of enrolled subjects. results > 120 international units/ml were considered positive. the microsphere suspension was prepared according to the manufacturer’s instructions and analysed using the luminex200 platform (a diasorin company, technology boulevard, austin, texas, united states). all samples were run in duplicate. indirect immunofluorescence assay anti-dsdna was detected via iif using nova lite® dsdna c. luciliae kits (inova diagnostics, inc., san diego, california, united states). the screening was performed at a serum dilution of 1/10, after which positive samples were diluted in the range of 1/20 to 1/640 for anti-dsdna titre determination. anti-neutrophil cytoplasmic antibody was detected via iif using nova lite® anca anti-neutrophil cytoplasmic autoantibody kits (inova diagnostics, inc., san diego, california, united states). the screening was performed at a serum dilution of 1/20. both tests were performed according to the manufacturer’s instructions. positive anti-myeloperoxidase appears as perinuclear staining (p-anca) while positive anti-proteinase 3 appears as cytoplasmic staining (c-anca).30 enzyme-linked immunosorbent assay for anti-myeloperoxidase and anti-proteinase 3 the orgentec anti-myeloperoxidase (p-anca) and anti-proteinase 3 (c-anca) elisa kits (orgentec diagnostika gmbh, mainz, germany) were used for the quantitative measurement of igg-class autoantibodies specific for myeloperoxidase and proteinase 3. results ≥ 5 iu/ml were considered positive. all samples were run in duplicate. we performed the assays and calculated the results according to the manufacturer’s instructions. data analysis the spss® 20.0 software package (ibm corporation, armonk, new york, united states) was used for data processing and analysis. the overall detection results are expressed as percentages of the total number of samples. agreement between different assays was assessed using cohen’s kappa (κ) coefficient, with the following levels of agreement: 0.01–0.2 = slight agreement, 0.21–0.4 = fair agreement, 0.41–0.61 = moderate agreement, 0.61–0.8 = substantial agreement, and 0.81–1 = almost perfect or perfect agreement. receiver operating characteristic curves were constructed, and the areas under the curves were calculated at 95% confidence intervals. the t-test and one-way analysis of variance with post hoc tests were used to compare ages, and the chi-square test was used to compare frequencies. the spearman correlation coefficient test (r) was performed to examine the correlation between anti-dsdna concentrations measured by athena multi-lyte® ana-ii (continuous variable) and anti-dsdna titres estimated by iif (ordinal variable).31 results were considered significant at a p-value < 0.05. sensitivity, specificity, accuracy, and positive and negative predictive values were calculated using medcalc (https://www.medcalc.org/) (medcalc software ltd, acacialaan, ostend, belgium).32 in addition to the clinical and analytical performance evaluations conducted, we compared the general characteristics of the assays used, including time taken for the run, estimated cost per test, and the technical experience required. results general characteristics of patients anti-dsdna antibodies were studied in 105 sle patients and participants in control groups (30 healthy participants and 35 patients with autoimmune diseases other than sle). among patients with other autoimmune diseases, 18 had systemic sclerosis, 15 had rheumatoid arthritis, one had polymyositis, and one had primary sjögren syndrome. within the anca group, we examined 75 patients with aiv and 25 healthy participants matched for age and gender. systemic lupus erythematosus patients’ ages ranged from 5 to 60 years (mean ± standard deviation: 32.4 ± 12.8 years), and 80 (76.1%) of them were women (table 1). the ages of the aiv patients ranged from 14 to 77 years (mean ± standard deviation: 38.4 ± 15.7 years), and 50 (66.7%) of them were women. table 1: demographic characteristics of sle patients and control subjects at the zagazig university hospitals, zagazig, al sharqia, egypt, may 2020 – april 2022. the most common symptom observed in the sle group was skin manifestations (86.7%; 91/105) in the form of photosensitivity, malar, or discoid rash (table 2). articular manifestations in the form of arthritis and arthralgia were observed in 82.8% (87/105) of patients, while 75.2% (79/105) of patients had nephritis. autoimmune vasculitis patients presented mainly with constitutional symptoms (80.0%; 60/75) and skin manifestations (57.3%; 43/75) in the form of skin ulcerations and palpable purpura. the lungs (66.7%; 50/75) and cardiovascular system (56.0%; 42/75) were the most frequently affected organs in these patients. anti-nuclear antibodies were detected in all sle patients (100%; 105/105) and 77.1% (27/35) of the diseased control group. other autoantibodies were detected in both groups with variable frequencies. table 2: clinical manifestations and serological findings of sle patients, aiv patients, and diseased controls, zagazig university hospitals, zagazig, al sharqia, egypt, may 2020 – april 2022. anti-double-stranded dna testing by iif and athena multi-lyte ana-ii test overall, 38.1% (40/105) of sle patients were positive for anti-dsdna by iif or athena multi-lyte® ana-ii. additionally, athena multi-lyte® ana-ii yielded equivocal results for 9.5% (10/105) of samples (anti-dsdna 100 iu/ml – 120 iu/ml). all healthy controls were negative for anti-dsdna using both iif and athena multi-lyte® ana-ii. one patient with rheumatoid arthritis had positive anti-dsdna results with iif, and another one had positive results with both methods. there was a moderate correlation between the anti-dsdna concentrations measured using athena multi-lyte ana-ii and the anti-dsdna titres estimated using iif (r = 0.55, p < 0.001). the athena multi-lyte® ana-ii and iif results exhibited substantial agreement with respect to anti-dsdna testing (κ = 0.66, 95% confidence intervals: 0.53–0.79, percent agreement: 87.6%, p < 0.001). at the manufacturer’s cut-off (120 iu/ml), the sensitivity of athena multi-lyte ana-ii for sle diagnosis was 38.1%, and the specificity was 98.5% (online supplementary table 1, figure 1). the best youden’s index was obtained when the cut-off was lowered to 109 iu/ml, at which the sensitivity increased to 46.7% and the specificity decreased to 96.9%. full (100%) specificity was reached by increasing the cut-off to 134 iu/ml, but this caused the sensitivity to decrease to 37.1%. for iif, the sensitivity was 38.1% and the specificity was 96.9%. however, the combination of both methods at the manufacturer’s cut-off (120 iu/ml) increased the sensitivity to 47.6% and the specificity to 96.9%. figure 1: receiver operating characteristic curves showing the clinical performance of iif and athena multi-lyte ana-ii when conducted separately and in combination for the diagnosis of sle among patients attending zagazig university hospitals, zagazig, al sharqia, egypt, between may 2020 and april 2022. the european league against rheumatism and the american college of rheumatology criteria was used as the gold standard. to determine its analytical performance, we compared the results of athena multi-lyte® ana-ii to those of iif, the gold-standard method. athena multi-lyte® ana-ii had a sensitivity of 73.8% (95% confidence intervals: 57.96% – 86.14%), a specificity of 92.19% (86.10% – 96.19%), a positive predictive value of 75.61% (62.47% – 85.24%), a negative predictive value of 91.47% (86.56% – 94.70%), an accuracy of 87.65% (81.74% – 92.19%), and an area under the curves of 0.830 (0.746–0.914) (figure 2). figure 2: receiver operating characteristic curve showing the analytical performance of athena multi-lyte ana-ii in the detection of anti-double-stranded dna among patients with sle attending zagazig university hospitals, zagazig, al sharqia, egypt, between may 2020 and april 2022. indirect immunofluorescence was used as the gold standard. anti-myeloperoxidase and anti-proteinase 3 analyses using iif, athena multi-lyte® iif, and elisa overall, 62.7% (47/75) and 14.7% (11/75) of patients tested positive for p-anca and c-anca by iif, while only 40.0% (30/75) and 14.7% (11/75) tested positive for anti-myeloperoxidase and anti-proteinase 3 by athena multi-lyte aiv. using the elisa method, 32.0% (24/75) of patients were positive for ani-myeloperoxidase, and 28.0% (21/75) were positive for anti-proteinase 3. all healthy volunteers were negative for anti-myeloperoxidase and anti-proteinase 3 using all three methods. all subjects were negative for anti-glomerular basement membrane. regarding the results of anti-myeloperoxidase testing, we observed a substantial agreement between iif and athena multi-lyte aiv (κ = 0.653), a moderate agreement between iif and elisa (κ = 0.525), and an almost perfect agreement between athena multi-lyte aiv and elisa (κ = 0.853) (table 3). for anti-proteinase 3 testing, there was a perfect agreement between athena multi-lyte aiv and iif (κ = 1), and a substantial agreement between elisa and both iif and athena multi-lyte aiv (κ = 0.635). moreover, the elisa results were highly correlated with the athena multi-lyte aiv results for the analyses of both anti-myeloperoxidase (r = 0.606, p < 0.001) and anti-proteinase 3 (r = 0.363, p = 0.006). table 3: agreement between the studied methods (athena multi-lyte aiv, iif, and elisa) for anca detection among aiv patients attending zagazig university hospitals, zagazig, al sharqia, egypt, between may 2020 and april 2022. the clinical performances of the anti-myeloperoxidase and anti-proteinase 3 detection methods could not be assessed because a definitive diagnosis of aiv relies on histopathology, which was not performed routinely for all patients. however, we assessed the analytical performances of athena multi-lyte aiv and elisa versus iif, the gold-standard method. for anti-myeloperoxidase, athena multi-lyte aiv had a higher sensitivity than elisa (63.8% vs 51.6%), while both tests had a specificity of 100% (online supplementary table 2). for anti-proteinase 3, both methods had a sensitivity of 100%, while athena multi-lyte aiv had a higher specificity than elisa (100% vs 88.8%). a comparison of the general characteristics of the methods used showed that iif had the highest estimated cost (240.00 egyptian pounds [egp]) for anti-dsdna testing as further dilutions were required to obtain the antibody titre using iif (table 4). however, for anca testing, elisa had the highest cost (200.00 egp) and time consumption (180 min) due to the need for two separate kits to obtain antigen-specific identification of anti-myeloperoxidase and anti-proteinase 3. in addition to being a quantitative rather than semi-quantitative assay, the athena multi-lyte also required the least amount of technical expertise (a bachelor’s degree in clinical laboratory science), was the least time-consuming (90 min), and had intermediate costs (120.00 egp). table 4: comparison of the general characteristics of athena multi-lyte, iif, and elisa used for anti-dsdna and anca detection among sle and aiv patients attending zagazig university hospitals, zagazig, al sharqia, egypt, between may 2020 and april 2022. discussion this work was designed to evaluate the performance of two commercially available multiplex bead-based flow cytometric assays, the athena multi-lyte ana-ii and athena multi-lyte aiv systems, for the detection of anti-dsdna and ancas. the multiplex assays had reasonable performance compared with traditional methods used for the detection of autoantibodies. for the detection of anti-dsdna, our data demonstrated a substantial agreement between the athena multi-lyte test system and iif. however, we observed a highly significant correlation between anti-dsdna concentration by athena multi-lyte test system and iif antibody titre. our findings are in concordance with the results of previous studies conducted in canada in 201033 and 2013,34 and in italy in 2018.15 these studies used a device from a different manufacturer (bioplex 2200, bio-rad laboratories, hercules, california, united states) and reported the same level of agreement and correlation with disease activity as achieved with iif. according to our data, the athena multi-lyte test system had the same sensitivity as iif for anti-dsdna detection, as well as improved specificity. moreover, the combination of both methods increased the sensitivity to 47.6%. two studies performed by infantino and colleagues in italy in 201514 and 201815 reported that the multiplex bead-based assay was more sensitive than iif but was slightly less specific.15 based on our observations, the athena multi-lyte test system for anti-dsdna detection provides multiple advantages, as it is a simple and rapid method with a reasonable cost and has acceptable clinical and analytical performance. although the combination of athena multi-lyte ana-ii and iif provided better sensitivity, economic factors might hinder the routine use of this combination for screening. our findings favour the use of the athena multi-lyte test for patient follow-ups because this one-step quantitative analysis is simpler than the multiple dilutions required for the semi-quantitative iif determination of antibody titres. the athena multi-lyte test systems exhibited almost perfect agreement with elisa and moderate agreement with iif with respect to anti-myeloperoxidase detection. additionally, the athena multi-lyte assay exhibited an almost perfect agreement with elisa and a perfect agreement with iif for anti-proteinase 3 detection. these results are consistent with the results of previous studies, including one performed in the netherlands in 2007, and another multicentre study performed in 2017 that recruited patients from germany, denmark, the netherlands, and belgium.35,36 only one study, conducted in the united states in 2018,37 reported a moderate agreement between a multiplex bead-based assay and elisa for both anti-myeloperoxidase (κ = 0.35) and anti-proteinase 3 (κ = 0.53) detection. this difference might be attributable to the use of kits produced by a different manufacturer (bio-plex® 2200 testing platform, bio-rad laboratories, hercules, california, united states). the analytical performance of athena multi-lyte aiv was superior to that of elisa for both anti-myeloperoxidase and anti-proteinase 3 detection. for anti-myeloperoxidase testing, athena multi-lyte aiv was more sensitive than elisa, while both methods were equally specific. for anti-proteinase 3 testing, both methods were equally sensitive, whereas athena multi-lyte aiv was more specific. regarding anca detection, histopathology is the definitive diagnosis for aiv.10 however, these data were not available for the patients included in this study. therefore, we were unable to assess the clinical performance of athena multi-lyte aiv and anca iif assays. still, the athena multi-lyte test systems provided good analytical performances and significant agreement with the results of conventional assays, warranting their use for anti-myeloperoxidase and anti-proteinase 3 screening. limitations this was a single-centre study with a relatively small sample size. the study was also limited by a lack of histopathology data for patients with suspected aiv. conclusion the athena multi-lyte test systems are reliable and robust for the simultaneous detection of autoantibodies. these tests aid in the diagnosis and follow-up of systemic rheumatic disorders and the diagnosis of vasculitic disorders. multiplex autoantibody testing is less time-consuming than traditional single-antibody assessment and requires only small sample volumes. future studies that include the definitive diagnosis of aiv according to international standards are required to yield a better understanding of the clinical performances of anti-myeloperoxidase and anti-proteinase 3 tests. we recommend the conduct of a multicentre study with a larger sample size to confirm our findings. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.a.a., a.a.e.s., h.m.k. and n.m.s contributed to the study conceptualisation and design. n.m.s. and a.a.a. contributed to sample processing, data analysis and manuscript writing. a.a.e.s. and h.m.k. contributed to patient’s selection, data collection and manuscript revision. finally, the manuscript has been read and approved by all the authors. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability data supporting the findings of this study are available from the corresponding author, a.a.a., on request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references kitching ar, anders h-j, basu n, et al. anca-associated vasculitis. nat rev dis primers. 2020;6(1):71. https://doi.org/10.1038/s41572-020-0204-y carter ee, barr sg, clarke ae. the global burden of sle: prevalence, health disparities and socioeconomic impact. nat rev rheumatol. 2016;12(10):605–620. https://doi.org/10.1038/nrrheum.2016.137 gheita ta, noor ra, abualfadl e, et al. adult systemic lupus erythematosus in egypt: the nation-wide spectrum of 3661 patients and world-wide standpoint. lupus. 2021;30(9):1526–1535. https://doi.org/10.1177/09612033211014253 phillip r, luqmani r. mortality in systemic vasculitis: a systematic review. clin exp rheumatol. 2008;26(5 suppl 51):s94–s104. attia dhs, abdel noor ra, salah s. shedding light on vasculitis in egypt: a multicenter retrospective cohort study of characteristics, management, and outcome. clin rheumatol. 2019;38(6):1675–1684. https://doi.org/10.1007/s10067-019-04441-4 shahin aa, zayed hs, elrefai rm, et al. the distribution and outcome of vasculitic syndromes among egyptians: a multi-centre study including 630 patients. egypt rheumatol. 2018;40(4):243–248. https://doi.org/10.1016/j.ejr.2018.01.001 watts ra, mahr a, mohammad aj, gatenby p, basu n, flores-suárez lf. classification, epidemiology and clinical subgrouping of antineutrophil cytoplasmic antibody (anca)-associated vasculitis. nephrol dial transplant. 2015;30 suppl 1:i14–i22. https://doi.org/10.1093/ndt/gfv022 bruner bf, guthridge jm, lu r, et al. comparison of autoantibody specificities between traditional and bead-based assays in a large, diverse collection of patients with systemic lupus erythematosus and family members. arthritis rheum. 2012;64(11):3677–3686. https://doi.org/10.1002/art.34651 albon s, bunn c, swana g, karim y. performance of a multiplex assay compared to enzyme and precipitation methods for anti-ena testing in systemic lupus and systemic sclerosis. j immunol methods. 2011;365(1–2):126–131. https://doi.org/10.1016/j.jim.2010.12.010 yates m, watts ra, bajema i, et al. eular/era-edta recommendations for the management of anca-associated vasculitis. ann rheum dis. 2016;75(9):1583–1594. https://doi.org/10.1136/annrheumdis-2016-209133 zhao j, wang k, wang x, et al. the performance of different anti-dsdna autoantibodies assays in chinese systemic lupus erythematosus patients. clin rheumatol. 2018;37(1):139–144. https://doi.org/10.1007/s10067-017-3771-x abuaf n, desgruelles c, moumaris m, et al. detection by flow cytometry of anti-dna autoantibodies and circulating dna immune complexes in lupus erythematosus. j immunol res. 2019;2019:6047085. https://doi.org/10.1155/2019/6047085 slater ng, cameron js, lessof mh. the crithidia luciliae kinetoplast immunofluorescence test in systemic lupus erythematosus. clin exp immunol. 1976;25(3):480–486. infantino m, meacci f, bentow c, et al. clinical comparison of quanta flash dsdna chemiluminescent immunoassay with four current assays for the detection of anti-dsdna autoantibodies. j immunol res. 2015;2015:902821. https://doi.org/10.1155/2015/902821 infantino m, manfredi m, merone m, et al. analytical variability in the determination of anti-double-stranded dna antibodies: the strong need of a better definition of the old and new tests. immunol res. 2018;66(3):340–347. https://doi.org/10.1007/s12026-018-8992-9 pisetsky ds. anti-dna antibodies – quintessential biomarkers of sle. nat rev rheumatol. 2016;12(2):102–110. https://doi.org/10.1038/nrrheum.2015.151 enocsson h, sjowall c, wirestam l, et al. four anti-dsdna antibody assays in relation to systemic lupus erythematosus disease specificity and activity. j rheumatol. 2015;42(5):817–825. https://doi.org/10.3899/jrheum.140677 aringer m. eular/acr classification criteria for sle. semin arthritis rheum. 2019;49(3s):s14–s17. https://doi.org/10.1016/j.semarthrit.2019.09.009 agmon-levin n, damoiseaux j, kallenberg c, et al. international recommendations for the assessment of autoantibodies to cellular antigens referred to as anti-nuclear antibodies. ann rheum dis. 2014;73(1):17–23. https://doi.org/10.1136/annrheumdis-2013-203863 csernok e, damoiseaux j, rasmussen n, et al. evaluation of automated multi-parametric indirect immunofluorescence assays to detect anti-neutrophil cytoplasmic antibodies (anca) in granulomatosis with polyangiitis (gpa) and microscopic polyangiitis (mpa). autoimmun rev. 2016;15(7):736–741. https://doi.org/10.1016/j.autrev.2016.03.010 csernok e, mahrhold j, hellmich b. anti-neutrophil cytoplasm antibodies (anca): recent methodological advances – lead to new consensus recommendations for anca detection. j immunol methods. 2018;456:1–6. https://doi.org/10.1016/j.jim.2018.01.007 savige j, gillis d, benson e, et al. international consensus statement on testing and reporting of antineutrophil cytoplasmic antibodies (anca). am j clin pathol. 1999;111(4):507–513. https://doi.org/10.1093/ajcp/111.4.507 damoiseaux j, csernok e, rasmussen n, et al. detection of antineutrophil cytoplasmic antibodies (ancas): a multicentre european vasculitis study group (euvas) evaluation of the value of indirect immunofluorescence (iif) versus antigen-specific immunoassays. ann rheum dis. 2017;76(4):647–653. https://doi.org/10.1136/annrheumdis-2016-209507 conrad k, roggenbuck d, reinhold d, sack u. autoantibody diagnostics in clinical practice. autoimmun rev. 2012;11(3):207–211. https://doi.org/10.1016/j.autrev.2011.05.014 sowa m, grossmann k, knutter i, et al. simultaneous automated screening and confirmatory testing for vasculitis-specific anca. plos one. 2014;9(9):e107743. https://doi.org/10.1371/journal.pone.0107743 shrestha b, dunn l. the declaration of helsinki on medical research involving human subjects: a review of seventh revision. j nepal health res counc. 2020;17(4):548–552. https://doi.org/10.33314/jnhrc.v17i4.1042 sullivan km, dean a, soe mm. openepi: a web-based epidemiologic and statistical calculator for public health. public health rep. 2009;124(3):471–474. https://doi.org/10.1177/003335490912400320 guchelaar nad, waling mm, adhin aa, van daele pla, schreurs mwj, rombach sm. the value of anti-neutrophil cytoplasmic antibodies (anca) testing for the diagnosis of anca-associated vasculitis, a systematic review and meta-analysis. autoimmun rev. 2021;20(1):102716. https://doi.org/10.1016/j.autrev.2020.102716 wei q, jiang y, xiao m, et al. comparison of chemiluminescence microparticle immunoassay, indirect immunofluorescence assay, linear immunoassay and multiple microbead immunoassay detecting autoantibodies in systemic lupus erythematosus. scand j immunol. 2020;91(3):e12849. https://doi.org/10.1111/sji.12849 mclaren js, stimson rh, mcrorie er, coia je, luqmani ra. the diagnostic value of anti-neutrophil cytoplasmic antibody testing in a routine clinical setting. qjm. 2001;94(11):615–621. https://doi.org/10.1093/qjmed/94.11.615 schober p, boer c, schwarte la. correlation coefficients: appropriate use and interpretation. anesth analg. 2018;126(5):1763–1768. https://doi.org/10.1213/ane.0000000000002864 saboor soomro r, shah ia, saboor a, bhutto aub, memon s. sensitivity and specificity of hepatitis b virus screening via rapid immunoassay chromatographic test. cureus. 2021;13(1):e12909. https://doi.org/10.7759/cureus.12909 hanly jg, thompson k, mccurdy g, fougere l, theriault c, wilton k. measurement of autoantibodies using multiplex methodology in patients with systemic lupus erythematosus. j immunol methods. 2010;352(1–2):147–152. https://doi.org/10.1016/j.jim.2009.10.003 venner aa, ibañez d, gladman dd, et al. comparison of three anti-dsdna assays: performance and correlation with systemic lupus erythematosus disease activity. clin biochem. 2013;46(4–5):317–320. https://doi.org/10.1016/j.clinbiochem.2012.12.004 damoiseaux j, vaessen m, knapen y, et al. evaluation of the fidis vasculitis multiplex immunoassay for diagnosis and follow-up of anca-associated vasculitis and goodpasture’s disease. ann n y acad sci. 2007;1109(1):454–463. https://doi.org/10.1196/annals.1398.051 bossuyt x, rasmussen n, van paassen p, et al. a multicentre study to improve clinical interpretation of proteinase-3 and myeloperoxidase anti-neutrophil cytoplasmic antibodies. rheumatology. 2017;56(9):1533–1541. https://doi.org/10.1093/rheumatology/kex170 sun q, calderon b, zhao z. discrepancies between two immunoassays for the determination of mpo and pr3 autoantibodies. clin chim acta. 2017;470:93–96. https://doi.org/10.1016/j.cca.2017.05.008 references about the author(s) rajiv t. erasmus department of chemical pathology, faculty of medicine and health sciences, stellenbosch university, cape town, south africa citation erasmus rt. point-of-care testing: connecting communities in africa and ensuring equity in access to health and diagnostics. afr j lab med. 2022;11(1), a2072. https://doi.org/10.4102/ajlm.v11i1.2072 editorial point-of-care testing: connecting communities in africa and ensuring equity in access to health and diagnostics rajiv t. erasmus copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. according to the african society for laboratory medicine, ‘[m]edical laboratories play a pivotal role in global disease diagnosis, surveillance, outbreak investigation, initiation and monitoring of therapy, as well as research and development’. however medical laboratories in africa cannot meet the testing demands of a rapidly growing health delivery service,1 due to a lack of resources and inadequate diagnostic services, which compromise patient care. this leads to misdiagnosis, resulting in underor over-treatment of disease, giving rise to numerous societal health challenges.1 a recent lancet commission review2 revealed a lack of access to diagnostics at the lowest tier of the healthcare system – the community – where the highest population in need of services exists, but only 47% globally have access. in its mapping exercise undertaken between november 2019 and april 2020, the european centre for disease prevention and control3 found that point-of-care (poc) testing could present a possible solution as it plays: a critical role in rapidly diagnosing both infectious and noninfectious diseases and is not only for appropriate and timely treatment but also for the detection of outbreaks and controlling the rapid spread of infectious diseases. (p. 1) in 2013, almost a decade ago, jani and peter4 had stressed ‘how point-of-care testing could drive innovation in global health’. indeed, if one fast-forwards to 2022, poc testing has made great strides in diagnosing and managing the disease burden in africa. according to world health organization,5 the diseases afflicting the african population are responsible for a substantial loss in health, estimated at 704 765 879 disability adjusted life years in 2015 alone. in the world health organization african region, total losses amounted to 629 603 271 disability adjusted life years; 59.1% were from communicable, maternal, perinatal and nutritional conditions with diabetes, anaemia, malaria and syphilis contributing to the greatest burden of disease.5 the international organization for standardization defines poc testing and near-patient testing for any disease (not just infectious diseases) as ‘testing that is performed near or at the site of a patient with the result leading to possible change in the care of the patient’.6 the small footprint of the instrument means that they can be easily transported. according to the united states’ national institutes of health, ‘empowering clinicians to make decisions at the “point-of-care” has the potential to significantly impact healthcare delivery and to address the challenges of health disparities’ and this could result in ‘the success of a potential shift from curative medicine, to predictive, personalised, and pre-emptive medicine’.7 although poc testing is progressively being used for the identification and management of various disease states in africa, controlling various poc instruments at numerous sites can be difficult, particularly when such instruments are run by non-technical staff. however, these challenges can be overcome with recent advances in connectivity and digitisation, as well as standardisation of software and middleware, which allow the quality of testing to be monitored in real time. connectivity allows for poc instruments to hook up with a laboratory or hospital information system, as well as with electronic medical records.8 for this purpose an open-access data management system is critical, permitting connections between instruments from any manufacturer. this process automatically validates and transfers patient results, including those from electronic medical records, as well as monitoring and managing data, multiple testing devices, and operators.8 as these are intimately linked to operator training and competency, harm to the patient is minimised by reducing analytical errors. faster decision-making can also occur due to significantly lower turnaround times.8 with the advent of 5g and artificial intelligence, greater focus on personalised care is likely to occur resulting from big data analysis and development of algorithms; communities in africa stand to benefit from this. wireless technology, the internet of things and big data will not only allow patients in africa to take charge of their health needs, but hugely improve the way health-related data is transmitted and interpreted, making it possible for healthcare delivery to be more efficient, precise and, ultimately, more affordable. in addition, accessibility would mean increased equity in healthcare and diagnostics to communities in africa. smartphone-based operation, paper-based sensing assays, and lab-on-a-chip are being turned into mobile laboratories, which are furthering this trend in remote areas.9 already there are well-established programmes that have changed the health landscape in africa. the most recent cochrane review10 reported that poc viral load testing showed both high sensitivity and specificity for the diagnosis of hiv viral load, at or above the clinical threshold of 1000 copies/ml, among people living with hiv who attended healthcare facilities for their hiv tests. furthermore, poc viral load testing has been reported to enhance viral suppression and retention in hiv care.10,11 rapid antigen lateral flow tests were particularly useful during the coronavirus disease 2019 (covid-19) pandemic and were used for rapid screening and tracing of cases in communities.12 the introduction of poc lateral flow urine lipoarabinomannan assays has improved tuberculosis case detection and lowered diagnostic holdups in people living with hiv and is particularly useful in african communities where traditional methods have a long turnaround time. although education, training, data governance and quality management will remain key to ensure the reliability and accuracy of test results,13,14 these results demonstrate that poc testing can simplify treatment and improve outcomes for hiv-positive adults receiving antiretroviral therapy in resource-limited settings and that poc testing presents a great advantage for disease diagnosis, monitoring and its management for patients in africa. the african union11 has put forward a digital transformation strategy for africa 2020–2030. if it is successfully implemented, it will no doubt further propel poc testing, ensuring optimal governance and quality. thus, africa is in a unique position to use poc and digital technologies, including machine learning and artificial intelligence, into a highly effective strategy for delivering speedy, high-quality, data-driven care to communities in africa. africa must take advantage of the convergence of poc testing and digital technologies, as this will surely enhance diagnostics and healthcare on the continent. references messele t. about aslm [homepage on the internet]. aslm; 2014 [cited n.d.]. available from: https://na.eventscloud.com/ehome/65245/aboutaslm fleming ka, horton s, wilson ml, et al. the lancet commission on diagnostics: transforming access to diagnostics. lancet. 2021;398(10315):1997–2050. https://doi.org/10.1016/s0140-6736(21)00673-5 european centre for disease prevention and control. assessment of point-of-care testing devices for infectious disease surveillance, prevention and control – a mapping exercise. stockholm: european centre for disease prevention and control; 2022. jani iv, peter tf. how point-of-care testing could drive innovation in global health. n engl j med. 2013;368(24):2319–2324. https://doi.org/10.1056/nejmsb1214197 world health organization. a heavy burden: the productivity cost of illness in africa. brazzaville: who regional office for africa; 2019. international organization for standardization. iso 22870: 2016 point-of-care testing (poct) – requirements for quality and competence. [cited 2016 nov]. available from: https://www.iso.org/standard/71119.html omnia health. point-of-care diagnostic testing: empowering the clinicians. omnia health, informa markets; 2020 [cited 2020 jan 16]. available from: https://insights.omnia-health.com/laboratory/point-care-diagnostic-testing-empowering-clinicians erasmus rt, sumedha s, el-sharkawy r. connectivity strategies in managing a poct service. ejifcc. 2021;2:190–194. kumar s, nehra m, khurana s, et al. aspects of point-of-care diagnostics for personalized health wellness. int j nanomed. 2021;16:383–402. https://doi.org/10.2147/ijn.s267212 ochodo ea, olwanda ee, deeks jj, mallett s. point-of-care viral load tests to detect high hiv viral load in people living with hiv/aids attending health facilities. cochrane database syst rev. 2022;3(3):cd013208. https://doi.org/10.1002/14651858.cd013208.pub2 drain pk, dorward j, bender a, et al. point-of-care hiv viral load testing: an essential tool for a sustainable global hiv/aids response. clin microbiol rev. 2019;32(3):e00097-18. https://doi.org/10.1128/cmr.00097-18 crozier a, rajan s, buchan i, mckee m. put to the test: use of rapid testing technologies for covid-19. bmj. 2021;372:n208. https://doi.org/10.1136/bmj.n208 jalavu tp, rensburg m, erasmus r. clinical staff knowledge and awareness of point-of-care-testing best practices at tygerberg hospital, south africa. afr j lab med. 2020;9(1):a853. https://doi.org/10.4102/ajlm.v9i1.853 gous n, boeras di, cheng b, takle j, cunningham b, peeling rw. the impact of digital technologies on point-of-care diagnostics in resource-limited settings. expert rev mol diagn. 2018;18(4):385–397. https://doi.org/10.1080/14737159.2018.1460205 article information authors: orji bassey1 kyle bond1 adebayo adedeji2 odafen oke1 ado abubakar1 kachiro yakubu2 tapdiyel jelpe1 ezekiel akintunde3 patrick ikani4 adeniyi ogundiran5 ali onoja6 issa kawu2 gabriel ikwulono2 idris saliu7 okey nwanyawu1 varough deyde1 affiliations: 1us centers for disease control and prevention (cdc), nigeria 2federal ministry of health (fmoh), nigeria 3us department of defense (dod), nigeria 4global hiv aids initiative in nigeria (ghain), nigeria 5world health organization (who), nigeria 6african health project (ahp), nigeria 7safe blood for africa foundation (sbfaf), nigeria correspondence to: varough deyde email: che5@cdc.gov postal address: 1140 prospect street, 2nd floor, bldg 3, pretoria, south africa dates: received: 20 aug. 2014 accepted: 14 feb. 2015 published: 29 may 2015 how to cite this article: bassey o, bond k, adedeji a, et al. evaluation of nine hiv rapid test kits to develop a national hiv testing algorithm in nigeria. afr j lab med. 2015;4(1), art. #224, 17 pages. http://dx.doi.org/10.4102/ajlm.v4i1.224 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. evaluation of nine hiv rapid test kits to develop a national hiv testing algorithm in nigeria in this original research... open access • abstract • introduction • research method and design    • strategy, sampling and testing    • test kit selection and characteristics    • source and size of specimens    • testing procedure    • reference testing/gold standard    • quality control reference laboratory testing    • data collection, management and analysis    • cost estimations    • ethical considerations • results    • sensitivity and specificity of the evaluated individual test kits    • kits dropped from consideration    • accuracy of testing algorithms    • cost estimates for the algorithms    • proposed test algorithms • discussion    • limitations of the study    • conclusion • trustworthiness    • reliability    • validity • acknowledgements    • competing interests    • authors’ contributions • references • appendix 1 • appendix 2 • appendix 3 • appendix 4 abstract top ↑ background: non-cold chain-dependent hiv rapid testing has been adopted in many resource-constrained nations as a strategy for reaching out to populations. hiv rapid test kits (rtks) have the advantage of ease of use, low operational cost and short turnaround times. before 2005, different rtks had been used in nigeria without formal evaluation. between 2005 and 2007, a study was conducted to formally evaluate a number of rtks and construct hiv testing algorithms. objectives: the objectives of this study were to assess and select hiv rtks and develop national testing algorithms. method: nine rtks were evaluated using 528 well-characterised plasma samples. these comprised 198 hiv-positive specimens (37.5%) and 330 hiv-negative specimens (62.5%), collected nationally. sensitivity and specificity were calculated with 95% confidence intervals for all nine rtks singly and for serial and parallel combinations of six rtks; and relative costs were estimated. results: six of the nine rtks met the selection criteria, including minimum sensitivity and specificity (both ≥ 99.0%) requirements. there were no significant differences in sensitivities or specificities of rtks in the serial and parallel algorithms, but the cost of rtks in parallel algorithms was twice that in serial algorithms. consequently, three serial algorithms, comprising four test kits (bunditm, determinetm, stat-pak® and uni-goldtm) with 100.0% sensitivity and 99.1% – 100.0% specificity, were recommended and adopted as national interim testing algorithms in 2007. conclusion: this evaluation provides the first evidence for reliable combinations of rtks for hiv testing in nigeria. however, these rtks need further evaluation in the field (phase ii) to re-validate their performance. introduction top ↑ nigeria is the tenth most populous country in the world and the most populous country in africa, with an estimated population of 162.3 million.1 the first hiv case in nigeria was reported in 1986.2 this stimulated interest in the screening of various populations in nigeria for hiv. the national hiv sero-prevalence sentinel survey amongst populations of pregnant women attending antenatal clinics (anc) commenced in nigeria in 1991 and has since become a biennial activity. the trend of hiv infection amongst this anc population since the commencement showed a steady increase − 1.8% (1991), 3.8% (1993), 4.5% (1995, 1996), 5.4% (1999), to a high of 5.8% in 2001 – before declining to 5.0% in 2003 and then stabilising subsequently at 4.4% in 2005, 4.6% in 2008 and 4.1% in 2010.3 nigeria has a generalised hiv epidemic – each of the 36 states and the federal capital territory has over 1.0% hiv prevalence4 – and an estimated 3.5 million people are infected with the virus in the country. there are about 0.4 million estimated new infections per year, 1.5 million persons requiring antiretroviral therapy and an estimated 2.2 million total aids orphans currently living in the country.5,6 in 2005, the nigeria national action committee on aids (naca) strategic framework set out to provide antiretrovirals (arvs) to 80.0% of adults and children with advanced hiv infection and to 80.0% of hiv-positive pregnant women, all by 2010. the implications of these efforts entail screening several million people for hiv infection. a 2005 survey of types of rapid test kits (rtks) used in facilities participating in anc in two of the six geopolitical zones of nigeria revealed 19 different brands ranging from cold chain-dependent to non-cold chain-dependent (adedeji aa, personal communication, march 2005). the lack of coordinated use of hiv rtks also resulted in some discrepancies observed in results from the same sample within a health facility or at different health facilities, thereby making it difficult to provide centralised quality assurance or a post-marketing validation programme in-country (adedeji aa, personal communication, march 2005). as a result of these problems, the nigerian government saw the need to adopt the use of non-cold chain-dependent hiv rtks for hiv testing. hiv rapid testing remains a key entry point to hiv prevention, treatment, care and support in resource-limited settings.7 its main advantages include the relative ease of use, low cost and faster turn-around time over enzyme immunoassays (eias) and western blot (wb) assays. with an hiv rapid testing strategy, increased awareness of hiv status amongst many groups who would otherwise have been unaware of their status has been achieved.8,9,10 providing quality-assured and accurate rapid hiv serological testing is critical in the early diagnosis and timely counselling of hiv-infected people for referral to care and treatment as well as prevention of mother-to-child transmission and monitoring of hiv prevalence in the population.7,9 hiv rapid testing also readily provides access to and enhances hiv counselling and testing in hard-to-reach rural populations,11,12 as well as in hard-to-reach, high-risk target populations, such as men who have sex with men.10 high-risk groups with acute hiv infection in nigeria have previously been characterised by use of a combination of rapid hiv testing in mobile units and laboratory-based specimens pooling for nucleic acid amplification testing.13 to date, several african countries have conducted evaluation studies and implemented rapid hiv testing as a tool for fighting the hiv epidemic. these studies have demonstrated that the use of rapid testing can be an important part of the overall hiv testing strategies in resource-limited settings, where cold storage capacity, reliable power, efficient transportation and sufficient numbers of skilled laboratorians may not be readily available.14,15,16,17,18,19,20 a number of sub-saharan african countries follow the world health organization (who) 2009 guidelines21,22 on the use of hiv antibody detection tests, where the recommended test algorithm includes a sensitive enzyme-linked immunosorbent assay (elisa) as a screening test, followed by a confirmatory test done on all positive samples using wb.23,24,25,26 recent studies have shown that diagnostic algorithms based on two or more serological tests are dependable and significantly lower the rate of recurrence of false positivity, thereby minimising misdiagnosis.26,27 recently, the use of rapid testing combined with elisa has increased significantly in africa and asia and tends to replace the use of wb assays.26,28,29,30,31,32 accurate hiv diagnosis in resource-limited settings, as is the case in most regions of nigeria, can be affected by emergence of new hiv subtypes and recombinant forms, hence the importance of occasionally assessing and selecting the best-performing serological assays before their wide-scale usage within the country.26,33,34,35 the goal of this evaluation was to assess and select non-cold chain-dependent hiv rtks for the development of evidence based national testing algorithms based on key criteria such as performance, ease of use and cost, amongst others. it also sought to develop a list of highly-sensitive and specific hiv rtks with documented good performance to serve as alternative algorithms in times of stock-outs of the rtks included in the primary algorithms. the present evaluation allowed the identification and recommendation of three national interim algorithms for hiv rapid testing in the country. to our knowledge, these recommendations are still implemented by the federal ministry of health (fmoh) and a second, field evaluation, phase has been conducted, although the results are not yet available. the methodology applied by the present evaluation could be used by other countries planning to develop hiv testing algorithms. research method and design top ↑ strategy, sampling and testing in august 2005, a multi-agency working group was set up by the government of nigeria for the evaluation of hiv rtks. the working group included participants from the fmoh and other organisations, specifically, the national aids and stis control program (nascp), naca, the national agency for food, drug administration and control (nafdac), the national institute for pharmaceutical research and development (niprd), the who, the centers for disease control and prevention – global aids program (cdc-gap) and other partners implementing the us president's emergency plan for aids relief (pepfar) programme in nigeria, who had international experience in rtk evaluations. test kit selection and characteristics the hiv rtks used in this evaluation were chosen based on the following who 2001 and 2009 recommended criteria: (1) stability within the climate in the country and not cold chain-dependent; (2) ability to test whole blood; (3) easy to use and interpret; (4) low test price (≤ us$3.20); (5) ability of manufacturers to produce and provide adequate numbers of testing kits to meet the needs of testing programmes in the country; (6) prior experience and validation – documented performance in the country and other african countries; (7) ability to detect hiv-1, hiv-2 and hiv type o subtypes; (8) ability to detect both igg and igm antibodies in order to reduce the window period; (9) do not require additional equipment to run tests or read results; (10) packaging of test kits not excessively bulky; (11) long shelf life (at least one year) and robust; and (12) test results provided in 30 minutes or less.21,22 in addition to the criteria above, test kits were selected based on their sensitivity and specificity when used singly and in combination using the minimum sensitivity and specificity (both ≥ 99.0%) criteria.17,36 the criteria were ranked in order of importance and relevance to the nigerian context. a total of nine test kits were selected for the evaluation (table 1). all the tests studied in this evaluation are qualitative tests for the detection of antibodies to hiv-1 and hiv-2 and use immunochromatographic technology. table 1: characteristics of hiv rapid tests included in this evaluation. source and size of specimens specimens were collected from sites in five geopolitical zones of nigeria between 2005 and 2006. ten health facilities were originally planned to contribute specimens for this evaluation; however, because of logistical challenges, specimens from only five facilities were used for the study. these sites still provide a good representation of the population. patient identification information was removed from all specimens and only hiv sero-status was reported. all specimens included in this study were unlinked and anonymised before inclusion and no blood specimen was drawn solely for the purpose of this validation. the specimen panel used for this evaluation was prepared from two sources. the first was existing sample archives (leftover plasma or serum collected routinely for diagnostic purposes) in hiv testing laboratories at federally administrated teaching hospitals. the second was the remaining samples from a joint cdc/university of maryland hiv sero-conversion project. the following specimen acceptance or rejection criteria were put in place to ensure that specimens of high quality were used in this evaluation: (1) properly collected, no haemolysis; (2) properly processed, no obvious signs of fungal or bacterial contamination/growth; (3) properly stored, freshly collected, at 20 °c, not stored for longer than two months at the collection sites; (4) clear hiv eia sero-status, positive or negative. hiv-positive specimens had to contain high titres of hiv-specific antibody and an eia signal-to-cutoff ratio of 3.0 or higher. hiv-negative specimens had to have eia results comparable to that of the kit negative control; and (5) adequate specimen volume (at least 3.0 ml). all specimens were treated and prepared based on the cdc/who guidelines.15,16 specimens were then given new identification numbers, logged into a database and divided into about three aliquots (volume permitting) to avoid repeated freeze-thaw cycles that may affect antibody titres. the aliquots were stored at -20 °c for a maximum of two months until being characterised and used in the evaluation. to avoid several freeze-thaw cycles, aliquots were kept in a refrigerator whilst in use during the validation period. approximately 200 hiv-positive and 200 hiv-negative specimens are needed to provide 95% confidence intervals of less than ± 2.0% for both the estimated sensitivity and specificity. thus, to meet the minimum acceptable test characteristics of the hiv rapid test as stated above, the final panel sample contained 528 specimens, of which 198 (37.5%) were hiv-1 positive and 330 (62.5%) were hiv negative. testing procedure all specimens were assigned new identification numbers between 1 and 528, then ordered by their reactivity (positive or negative) and randomised to allow for blinded testing. ten skilled and experienced laboratorians working on the serology bench at the sites that contributed the specimens for the evaluation were recruited and were then provided with background information on the evaluation, refresher training on good laboratory practice and an orientation to the data entry forms. job aids were provided for each rtk and each test was demonstrated. under the supervision of cdc and nascp laboratory staff, the laboratorians practised on control specimens prior to evaluating the panel. testing of each assay was implemented using the specimens according to the manufacturer's instructions for each individual test kit. laboratorians worked in pairs; each pair evaluated approximately 100 specimens over a half-day period per test product. specimen sets were rotated between the testers. each test result was read independently by two individuals. all the laboratorians then completed a questionnaire concerning various aspects of the rtk they had just evaluated (see appendices). the laboratorians appraised each rtk based on the following criteria: ease of running and reading test results, including ease of reading the reaction line; ease of interpreting the test results; ease of learning the test procedure; overall ease of running the assay; packaging size; and waste generation. this was done in an effort to capture information, in addition to accuracy, which is also critical in identifying tests for an algorithm. reference testing/gold standard all specimens were fully characterised using standardised reference testing (gold standard): two third-generation eias, plus wb for all eia-reactive specimens. all specimens with discordant eia and wb results were excluded from the panel. specimens with indeterminate wb results were also excluded. the two eias selected for this validation, namely, vironostika® hiv uniform ii plus o (biomerieux, france) and genscreen® 1/2 version 2 (bio-rad, usa), were both third-generation eias targeting both igg and igm of hiv-1 and -2, plus type o antibodies using recombinant antigens covering all group m, hiv-1 subtypes a-h. both assays have been widely used throughout africa12,18,19,25 and have consistently produced reliable data and detected hiv-specific antibodies. an antibody-only test is the most appropriate for comparison with hiv antibody-detecting rapid tests. the wb kit selected was new lav-blot i (bio-rad). all reference testing was conducted as per the manufacturer's instructions. quality control reference laboratory testing all laboratory work associated with this evaluation was carried out at the asokoro training laboratory, located at the asokoro general hospital in abuja. this work included specimen characterisation, storage and the evaluation exercise. this institute of human virology, nigeria (ihvn)-supported site was selected for the following reasons: current status as a national hiv laboratory training facility; central location within federal capital territory; constant electrical power; ongoing external quality assurance/laboratory monitoring programme; appropriate infrastructure for reference testing (eia equipment); and adequate specimen storage space. data collection, management and analysis all test results were collected on paper forms and entered into a spreadsheet database (microsoft® excel™) for analysis. access to the project databases was limited to only key project staff through password-protected computers and all paper forms were kept in locked filing cabinets. during the data analysis, the sensitivity and specificity of each rtk were calculated by comparing the rtk results with reference results derived from eia/wb testing. cost estimations the supply chain management system (scms) was established in nigeria in 2007 following the who hiv test kit bulk procurement scheme established in 1989, which is aimed at facilitating access to high-quality test kits at a low cost through an easy purchasing procedure. the scms coordinates pooled procurement systems for hiv arvs and rtks and provided pricing information for the analysis as negotiated with the manufacturers and/or companies or their agents.37,38 each of the rtks under consideration was evaluated in both parallel and serial testing algorithms and anticipated costs for each algorithm were determined in us dollars based on the negotiated scms price. for the parallel testing algorithms, the price of each screening rtk was added to that of the confirmatory (i.e., second) rtk. the cost of the tie-breaker rtk was not included, since the frequency of use of a tie breaker is low (at most, 1.8% of the time). for the serial algorithms, the full price of the screening rtk was added to the price of the confirmatory rtk at 10.0% hiv prevalence (since the second test would only be used to confirm positive test results), plus the price of the tie breaker when needed at an hiv prevalence of 10.0%. ethical considerations the protocol for this evaluation was developed following the who's regional office for africa (who afro) guidelines21 and received ethical approval from the niprd, nigeria and institutional review board (irb) as well as the cdc irb (approval dated 03.21.2006). results top ↑ sensitivity and specificity of the evaluated individual test kits the sensitivity and specificity results were calculated for each individual test (table 2). all nine tests performed well in this evaluation, as indicated by high sensitivity and specificity values. the sensitivity value for seven of the nine tests was 100.0%, indicating that none of these tests produced false-negative results. two tests, first response® and instantchektm, had lower sensitivities (98.9% and 96.9%, respectively). specificity varied slightly between the tests, ranging from 96.0% to 100.0%; oraquick® and stat-pak® were each 100.0% specific. table 2: sensitivity and specificity calculations for the nine selected rapid test kits. kits dropped from consideration figure 1 shows the kit selection process and results. after the initial performance of each individual kit was tested, three of the nine kits (instantchektm, first response® and oraquick®) were removed from further consideration. both instantchektm and first response® ere excluded because of their performance (sensitivity and specificity) and the complexity of the result interpretation. oraquick® was dropped because of its cost and short shelf-life. figure 1: process for selecting hiv rapid test kits for development of interim national hiv testing algorithms. commercial kits available in nigeria were selected for evaluation singly based on who guidelines (step 1). of the nine kits, six were retained for inclusion in the algorithm testing exercise and three were dropped (step 2). serial and parallel testing algorithms were assessed for performance (sensitivity and specificity), cost and the country context (step 3). serial algorithms using four kits were selected and recommended as interim national guidelines in 2007 (step 4). figure 2 presents a summary of findings from the questionnaire on rtk characteristics administered to the laboratorians who performed the testing. figure 2: results from questionnaires administered to laboratorians conducting the evaluation. respondents were asked to rate all nine kits based on the following criteria: ease of reading the reaction line (panel a); ease of interpreting the test results (panel b); ease of learning how to perform the test (panel c); and overall ease of using and running the kit (panel d). scores ranged from very easy (1) to very difficult (5) for this set of four questions; panels a–d represent average scores. respondents were also asked about the size of the packaging (panel e), with scores ranging from 1 (very bulky) to 5 (very compact); and about quantity of waste generated (panel f), with scores ranging from 1 (a lot of waste) to 5 (minimal waste). panels e-f represent average scores. accuracy of testing algorithms in diagnostic settings, rtks are used in testing algorithms, not as individual tests. one major advantage of evaluating rtks using a single, well-characterised specimen panel is that sensitivity and specificity can be calculated for all possible combinations of tests. this was completed for both parallel and serial algorithms (box 1, appendix 3 and 4). box 1: cost (us $), sensitivity and specificity of parallel and serial testing algorithms†. all possible parallel algorithms had a sensitivity of 100.0%, which indicates that none of the specimens in this panel had a false-negative result with more than one test product (appendix 3). specificity was also high, ranging from 99.1% to 100.0%. this represents from zero to three false-positive results for each algorithm. over one-third (n = 24/60) of the possible test combinations had the highest possible (100.0%) sensitivity and specificity. for all 120 possible serial algorithms, sensitivity was 100.0% (appendix 4). specificity ranged from 99.1% to 100.0%. over half (n = 67) of the proposed algorithms had 100.0% sensitivity and specificity. this included five of the eight algorithms utilising the two rtks currently in wide use in nigeria (determinetm and stat-pak®). the number of specimens (out of 528) requiring a tie-breaker test because of discordant results between the first two tests is also reported for serial and parallel algorithms (appendix 3 and 4). eight of 30 serial algorithms (two test combinations) did not require the use of a tie-breaker test. other combinations, for both algorithm types, ranged from one to 10, representing at most 2.0% of specimens. cost estimates for the algorithms the cost for the serial testing algorithms was found to range from us$0.70 to us$1.90, whilst parallel algorithms ranged from us$1.40 to us$3.30 (table 3, appendix 3 and 4). in general, the cost for the serial testing algorithms was about half the cost of the parallel testing algorithms, since the latter require two tests to be run on all clients, even the 90.0% of clients who are hiv negative. table 3: national interim serial hiv rapid testing algorithm implemented in 2007. proposed test algorithms determinetm and stat-pak®, both of which have been evaluated and used widely internationally, have also been used widely in the nigerian hiv programme since the 2001 and 2006 anc surveys, respectively. tremendous investment has been made in training large numbers of laboratory staff, including the adaptation of the training package for both test kits for use in nigeria. determinetm has also been used widely throughout africa. in this evaluation, both tests had high sensitivity and specificity individually and in the serial algorithms. determinetm, with its high sensitivity (100.0%), is strongest as a screening test and was recommended as the first test in any proposed algorithm. determinetm was not recommended as a confirmatory test because of its lower specificity (97.8%). use as a tie-breaker was only recommended in the event that stat-pak® is not available. uni-goldtm has also been used widely internationally and performed well in this evaluation. in light of the fact that larger numbers of tests will soon be available in nigeria to support hiv diagnostic testing programmes, it was also included in the interim national hiv rapid testing algorithm. based on its performance and the need for ongoing quality assurance, adequacy of supply of kits and the development of a track record, it was recommended that bunditm be included as a tie-breaker test. this would allow for continued monitoring of this new product. based on the above findings, the construction of the three interim serial testing algorithms was based on four of the six rapid hiv test kits, namely determinetm, stat-pak®, uni-goldtm and bunditm (table 3). discussion top ↑ the expansion of hiv prevention and care services in resource-constrained settings comes with great challenges regarding how to maintain quality-assured and accurate hiv testing as the number of hiv testing facilities increases.21 in addition, there are challenges associated with the quest for alternative, less expensive and efficient rapid hiv testing strategies, devoid of the supplemental confirmatory testing using the expensive wb assay and capable of retaining a high level of sensitivity in the face of the divergent hiv-1 subtypes dominating most sub-saharan african countries.14,15,16,17,32,33 this is even further complicated by the move to decentralise hiv testing by involving fewer skilled and experienced laboratory/non-laboratory personnel.17,32 this study evaluated nine hiv rtks using double eias as the reference test and wb as a supplemental confirmatory test for eia-concordant reactive specimens. this serves as a gold-standard testing method for this evaluation and is comparable to the methods adopted in similar studies.29,32,39,40 all of these studies were in line with the cdc/who afro guidelines17 for hiv testing technologies in africa. of the nine rtks selected for the evaluation, three (oraquick®, instantchektm and first response®) were dropped because of short shelf-life, poor performance, cost or complexity following the who phase 1 hiv rtk evaluation criteria. the remaining six rtks (bunditm, determinetm, double-check goldtm, stat-pak®, surecheck® and uni-goldtm) were then subjected to parallel and serial testing algorithms in several possible combinations, resulting in combinations with high levels of sensitivity and specificity, as well as a high accuracy for diagnosing hiv infection. sixty possible parallel algorithms had costs ≤ us$3.20, had a sensitivity of 100.0% – indicating that no-false negative results were obtained with the panel of specimens – and had specificities ranging from 99.1% – 100.0%. this represents zero to three false-positive results for each of the algorithms. it was also observed that over one-third (n = 24/60) of the possible test combinations had the highest possible (100.0%) sensitivity and specificity. of note, the remaining 60 possible parallel combinations of the rtks were not presented in this report because of their higher cost (> $3.20). similarly, of the 120 possible serial algorithms, sensitivity was 100%, whilst specificity ranged from 99.1% – 100.0%. additional analysis revealed that over half (n = 67/120) of the possible serial algorithms had 100.0% sensitivity and specificity, indicating that none of the panel specimens showed a false-negative or false-positive result with more than one test product. a comparative analysis of the performance characteristics between the parallel and serial testing algorithms revealed no differences in accuracy regarding individual performance in diagnosing hiv infection. similar comparative analyses of performance of combinations of elisas and rtks in parallel or serial testing algorithms have shown that these combinations can also produce accurate results for hiv infections diagnosis.17,27 however, a comparative cost analysis between the two testing strategies showed a substantial difference, as the cost of carrying out a parallel testing algorithm is twice as expensive as the cost of a serial testing algorithm. these findings are comparable to those previously reported.14,17 besides sensitivity, specificity and the cost of the testing algorithms, other important factors were also considered before making a choice of assay for the national testing algorithm. over the years, the fmoh, through its hiv/aids division in its efforts to implement national programmes has also significantly invested in terms of training the laboratorians and non-laboratorians involved in hiv testing using the stat-pak® and determinetm hiv rtks. this shows that both test kits are both commerciallyand readily available and had wide-scale use in nigeria. furthermore, based on laboratorians’ evaluation and rating of the nine rapid test kits using questionnaires and the test selection criteria recommended by the who, determinetm was identified as the most compact test kit, allowing for less expensive transport and generating the least waste, thereby alleviating concerns about biohazard waste disposal at testing sites, whilst stat-pak® ranked high in terms of readability of the reaction line and result interpretation. cost-wise, a serial testing algorithm comprising determinetm and stat-pak® was found to be inexpensive, as it costs less than one us dollar. the cost of the serial testing algorithm is vital, considering the expected testing targets and the large size of the voluntary counselling and testing programme in nigeria. also rated highly was uni-goldtm, which is known to be used widely internationally. bunditm, on the other hand, was included in the serial algorithm as a tie-breaker, because it is assembled locally and readily available, in addition to its high performance in the evaluation. the ease and convenience of performing the assay were also considered as in previous, similar studies.17 based on the above findings and criteria, nigeria adopted the serial hiv testing algorithm as an interim national testing algorithm (table 3). similar considerations and decision strategies were also adopted in a comparable study conducted in 11 african countries.17 the six non-cold chain-dependent test kits (bunditm, determinetm, double-check goldtm, stat-pak®, surecheck® and uni-goldtm) performed well in this laboratory-based evaluation, both as individual tests and in serial testing algorithms. data from this initial evaluation suggest that any combination of these six rtks would perform well in a three-test, serial algorithm and that the tests with the highest sensitivity, such as determinetm and uni-goldtm, should be used as the screening test, whereas those with highest specificity, such as stat-pak®, should be used for confirmation. limitations of the study the present evaluation is not without limitations. first of all, this evaluation was limited to stored frozen plasma specimens and oral fluid was not collected for evaluation. as a result, the comparative advantage of using test kits with oral fluid or fresh specimens could not be evaluated. another limitation was the variability in performance of some of the test kits in the hands of different testing personnel; this phenomenon has also been observed previously.14,15,16,17 in addition, the sensitivity of these test kits/testing algorithms is not well established and may differ based on hiv-1 subtypes, given the great genetic diversity of hiv-1 in africa.17 as a result, the who developed guidelines to help country-based evaluation and implementation of rapid hiv testing.17 considering these limitations, a formal hiv test kit performance evaluation should be an ongoing process that starts before testing implementation and continues after testing processes have been implemented in the field. as a result, since this evaluation provided data on laboratory-based validation, the selected rtks should be field tested (phase ii) in varying combinations before a final national testing algorithm is selected. furthermore, it is critical to ensure that the hiv test algorithms currently in place and future ones be monitored continuously through a quality-assurance programme (phase iii) developed within nigeria. this quality-assurance programme should have the capacity to rapidly identify and correct testing problems related to the selected test kits and use of those kits in algorithms. finally, it is important to note that, at the time of preparing the present manuscript, field monitoring had revealed a performance issue with bunditm and the kit was removed from the algorithm in 2008. only three kits thus remain in use (determinetm, uni-goldtm and stat-pak®). the second phase (field-based evaluation) was conducted in 2012, however, the results are not yet available (adedeji aa, personal communication, june 2012). conclusion three hiv testing algorithms with high sensitivity, specificity and accuracy to diagnose hiv infections were identified and recommended for use as interim national algorithms. these hiv testing algorithms provide a cheaper and more efficient alternative to wb supplemental confirmatory testing. the results of this analysis showed further that serial testing algorithms are not only sensitive and specific, but also less expensive. finally, the present evaluation provides the first evidence-based and reliable combination of hiv test kits in nigeria. it is important that a field, ‘point-of-care testing’ evaluation is conducted and the findings used to inform future decisions on what test kits to use in the country for accurate hiv testing. trustworthiness top ↑ the current report reflects the findings observed by the technical working group, those who performed the testing, as well as the team that analysed the data. reliability the results of the experiments presented in this report were obtained using specimens collected in nigeria and these results have been confirmed using who-recommended gold standard testing procedures for evaluating hiv rapid test kits. however, the methods of the evaluation can be applied in other countries. validity the development and recommendation of an interim hiv rapid testing algorithm in nigeria demonstrated the study's success in achieving its goal. not only were the test kits evaluated based on gold standard methods and procedures, but also the outcome of the study were scientific evidence-based recommendations that allowed the government of nigeria to make informed decisions on what kits to use in their hiv testing programmes. acknowledgements top ↑ this publication was made possible by support from pepfar through the department of health and human services, cdc, division of global hiv/aids. the findings and conclusions in this manuscript are those of the authors and do not necessarily represent the official position of the cdc. the authors are deeply grateful to other members of phase i laboratory evaluation working group for their immense involvement and supportive supervision during the rapid testing phase and contributions to the initial report writing phase. members of this group include: hiv/aids division, fmoh (a. akinsete, a. lawanson, o. salawu, t.o. adonye, a. uwah, mrs g.m. bassey, f. simon); naca (a. ikpeazu); nafdac (c.u. anyakora); who (a.h. fagbami); institute of human virology, nigeria (a. abubakar, b. okelade, j. jugu, p.j. nwadike); ghain (i.g. audu); national blood transfusion service (a. abayomi, s.m. aminu); national institute for pharmaceutical research and development (o. dauda); cbge university of jos (m. njoku); nigeria ministry of defense – emergency plan implementation committee (epic) (l. ukachukwu); university of abuja teaching hospital, gwagwalada (m. rubainu); university college hospital, ibadan (d. olaleye); university of port harcourt (s. esiet); usman danfodio univserity teaching hospital (d.b. idowu); aminu kano teaching hospital (h. takalmawa); university of benin teaching hospital (f. agbontaen); university of maiduguiri teaching hospital (m. anietie); asokoro general hospital (e. kayode); plateau state specialist hospital virology research center (p. amangam); nnamdi azikiwe university teaching hospital (k. stephen); university of nigeria, teaching hospital (a. abah). we want to especially acknowledge the following subject matter experts from the cdc in atlanta for reviewing and editing the manuscript: dr bharat parekh, de anindya, and taiwo abimbola. we also appreciate terrell peggy for coordinating these efforts. in addition, we thank the cdc nigeria staff: dr ahmed mukhtar, dr obinna ogbanufe, dr solomon odafe, dr mustapha bello, dr bertrand odume, dr ibrahim jahun and mr raphael akpan for their review and discussions of the manuscript. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions o.b. (cdc-nigeria) performed some of the experiments and wrote the manuscript. a.a., k.y., i.k. (all fmoh-nigeria) and a.o. (who-nigeria) co-led the project and experimental design. t.j., a.a., o.o. (all cdc-nigeria), p.i. (ghain), g.i. (fmoh), i.s. (sbfaf), a.o. (ahp) and e.a. (dod) made conceptual contributions and performed the experiments. k.b., v.d. and o.n. (all cdc-nigeria) contributed to the experimental design and wrote the manuscript. references top ↑ federal republic of nigeria. global aids response progress reporting 2012; 2012. nasidi a, harry to. the epidemiology of hiv/aids in nigeria. in: o adeyi, p kanki, o odutola, et al, editors. aids in nigeria: a nation on the threshold. harvard, ma: harvard center for population and development studies, 2006; p. 17–36. federal ministry of health, nigeria. technical report: 2010 national hiv sero-prevalence sentinel survey among pregnant women attending ante-natal clinics in nigeria. department of public health, national aids/sti control programme; 2010. irin africa. authorities predict 250,000 people on arvs by mid-2006 [page on the internet]. c2013 [cited 2014 aug 1]. available from: http://www.irinnews.org/report/38272/nigeria-authorities-predict-250-000-people-on-arvs-by-mid-2006 idoko j, taiwo b, murphy r. treatment and care of hiv disease. in: o adeyi, p kanki, o odutola, et al, editors. aids in nigeria: a nation on the threshold. harvard, ma: harvard center for population and development studies, 2006; p. 385–436. national agency for the control of aids, nigeria. national hiv/aids strategic plan 2010–2015. abuja, nigeria; 2010. franco-paredes c, tellez i, del rio c. rapid hiv testing: a review of the literature and implications for the clinician. curr hiv/aids rep. 2006;3(4):169–175. http://dx.doi.org/10.1007/s11904-006-0012-3 centers for disease control and prevention. rapid hiv testing in outreach and other community settings – united states, 2004–2006. mmwr. 2007;56(47):1233–1237. shah s, haag a, purohit a, et al. utility of rapid hiv testing in rural settings. the 14th international aids conference, barcelona, spain, july 7–12; 2002. yu y. community-based hiv testing among msm: anonymous hiv counseling and testing in wuhan. the 19th international aids conference, washington, dc, july 22–29; 2012. mckenna sl, muyinda gk, roth d, et al. rapid hiv testing and counseling for voluntary testing centers in africa. aids. 1997;11(suppl 1):s103–s110. respess ra, rayfield ma, dondero tj. laboratory testing and rapid hiv assays: applications for hiv surveillance in hard-to-reach populations. aids. 2001;15(suppl 3):s49–s59. http://dx.doi.org/10.1097/00002030-200104003-00007 charurat m, nasidi a, delaney k, et al. characterization of acute hiv-1 infection in high-risk nigerian populations. j infect dis. 2012;205(8):1239–1247 http://dx.doi.org/10.1093/infdis/jis103 world health organization. the importance of simple/rapid tests in hiv diagnostics. weekly epidemiological record. 1998;73:321–328. alemnji g, nkengasong j, parekh bs. hiv testing in developing countries: what is required? indian j med res. 2011;134:779–786. http://dx.doi.org/10.4103/0971-5916.92625 world health organization. guidelines for assuring the accuracy and reliability of hiv rapid testing: applying a quality system approach. geneva, switzerland; 2005. plate dk. evaluation and implementation of rapid hiv tests: the experience in 11 african countries. aids res hum retroviruses. 2007;23(12):1491–1498. http://dx.doi.org/10.1089/aid.2007.0020 owen sm, yang c, spira t, et al. alternative algorithms for human immunodeficiency virus infection diagnosis using tests that are licensed in the united states. j clin microbiol. 2008;46(5):1588–1595. http://dx.doi.org/10.1128/jcm.02196-07 foglia g, royster gd 4th, wasunna km, et al. use of rapid and conventional testing technologies for human immunodeficiency virus type 1 serologic screening in a rural kenyan reference laboratory. j clin microbiol. 2004;42(8):3850–3852. http://dx.doi.org/10.1128/jcm.42.8.3850-3852.2004 bebell lm, pilcher cd, dorsey g, et al. acute hiv-1 infection is highly prevalent in ugandan adults with suspected malaria. aids. 2010;24(12):1945–1952. http://dx.doi.org/10.1097/qad.0b013e32833bb732 world health organization. guidelines for appropriate evaluations of hiv testing technologies in africa. harare, zimbabwe; 2002. world health organization. guidelines for using hiv testing technologies in surveillance: selection, evaluation and implementation. geneva, switzerland; 2009. world health organization. revised recommendations for the selection and use of hiv antibody tests. weekly epidemiological record. 1997;72(12):81–88. sommerfelt ma, ohlsson i, flolid i, et al. a simple semi-rapid hiv-1& 2 confirmatory immunoassay using magnetic particles. j virol methods. 2004;115(1):191–198. http://dx.doi.org/10.1016/j.jviromet.2003.09.031 beelaert g, vercauteren g, fransen k, et al. comparative evaluation of eight commercial enzyme linked immunosorbent assays and 14 simple assays for detection of antibodies to hiv. j virol methods. 2002;105(2):197–206. http://dx.doi.org/10.1016/s0166-0934(02)00102-7 andersson s, da silva z, norrgren h, et al. field evaluation of alternative testing strategies for diagnosis and differentiation of hiv-1 and hiv-2 infections in an hiv-1 and hiv-2-prevalent area. aids. 1997;11(15):1815–1822. http://dx.doi.org/10.1097/00002030-199715000-00005 nkengasong jn, maurice c, koblavi s, et al. evaluation of hiv serial and parallel serologic testing algorithms in abidjan, côte d’ivoire. aids. 1999;13(1):109–117. http://dx.doi.org/10.1097/00002030-199901140-00015 lyamuya ef, aboud s, urassa wk, et al. evaluation of simple rapid hiv assays and development of national rapid hiv test algorithms in dar es salaam, tanzania. bmc infect dis. 2009;9:19. http://dx.doi.org/10.1186/1471-2334-9-19 nunn aj, biryahwaho b, downing rg, et al. algorithms for detecting antibodies to hiv-1: results from a rural ugandan cohort. aids. 1993;7(8):1057–1061. http://dx.doi.org/10.1097/00002030-199308000-00005 phili r, vardas e. evaluation of a rapid human immunodeficiency virus test at two community clinics in kwazulu-natal. s afr med j. 2002;92(10):818–821. urassa wk, bredberg-rådén u, mbena e, et al. alternative confirmatory strategies in hiv-1 antibody testing. j acquir immune defic syndr. 1992;5(2):170–176. zeh c, oyaro b, vandenhoudt h, et al. performance of six commercial enzyme immunoassays and two alternative hiv-testing algorithms for the diagnosis of hiv-1 infection in kisumu, western kenya. j virol methods. 2011;176(1–2):24–31. http://dx.doi.org/10.1016/j.jviromet.2011.05.021 yang c, li m, cowart f, et al. characterization of human immunodeficiency virus type-1 from hiv-1 seropositive cases with undetectable viremia. j clin virol 2004;30(3):224–228. http://dx.doi.org/10.1016/j.jcv.2003.11.007 kline rl, dada a, blattner w, et al. diagnosis and differentiation of hiv-1 and hiv-2 infection by two rapid assays in nigeria. j acquir immune defic syndr. 1994;7(6):623–626. constantine nt, zekeng l, sangare ak, et al. diagnostic challenges for rapid human immunodeficiency virus assays. performance using hiv-1 group o, hiv-1 group m, and hiv-2 samples. j hum virol. 1997;1(1):45–51. stetler hc, granade tc, nunez ca, et al. field evaluation of rapid hiv serologic tests for screening and confirming hiv-1 infection in honduras. aids. 1997;11(3):369–375. http://dx.doi.org/10.1097/00002030-199703110-00015 united states intenational agency for development. acquisition and assistance policy directive (aapd 05-01). procurement of hiv-aids test kits from code 935 countries; 2005. world health organization. who hiv test kit – bulk procurement scheme. geneva: who; 2002. wilkinson d, wilkinson n, lombard c, et al. on-site hiv testing in resource-poor settings: is one rapid test enough? aids. 1997;11(3):377–381. http://dx.doi.org/10.1097/00002030-199703110-00016 delaney kp, branson bm, uniyal a, et al. evaluation of the performance characteristics of 6 rapid hiv antibody tests. clin infect dis. 2011;52(2):257–263. http://dx.doi.org/10.1093/cid/ciq068 appendix 1 top ↑   data collection sheet appendix 2 top ↑   testers’ ratings of rapid test kits (rtks) during laboratory evaluation appendix 3 top ↑ table 1-a3: parallel algorithms. appendix 4 top ↑ table 1-a4: serial algorithms. article information authors: giselle guevara1 floris gordon2 yvette irving2 ismae whyms3 keith parris1 songee beckles4 talkmore maruta6 nqobile ndlovu5 rachel albalak1 george alemnji1 affiliations: 1us centers for disease control and prevention, caribbean regional office, barbados 2african field epidemiology network, caribbean office 3princess margaret hospital, bahamas 4ladymeade reference unit, barbados 5african field epidemiology network, uganda 6clinton health access initiative, botswana correspondence to: giselle guevara postal address: us centers for disease control and prevention, caribbean regional office, us embassy, bridgetown, wildey business park, wildey, st. michael bb14006, barbados dates: received: 19 may 2014 accepted: 18 june 2014 published: 03 nov. 2014 how to cite this article: guevara g, gordon f, irving y, et al. the impact of slmta in improving laboratory quality systems in the caribbean region. afr j lab med. 2014;3(2), art. #199, 9 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.199 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the impact of slmta in improving laboratory quality systems in the caribbean region in this original research... open access • abstract • introduction • research method and design    • advocacy strategy with governments    • laboratory audits       • slmta workshops       • mentorship for the laboratories • results    • case studies       • case study 1 – inventory management       • case study 2 – improving documents and records management in the microbiology laboratory • discussion    • early engagement of key stakeholders    • an implementation roadmap    • structured improvement approach    • mentorship    • key challenges and recommendations    • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: past efforts to improve laboratory quality systems and to achieve accreditation for better patient care in the caribbean region have been slow. objective: to describe the impact of the strengthening of laboratory management toward accreditation (slmta) training programme and mentorship amongst five clinical laboratories in the caribbean after 18 months. method: five national reference laboratories from four countries participated in the slmta programme that incorporated classroom teaching and implementation of improvement projects. mentors were assigned to the laboratories to guide trainees on their improvement projects and to assist in the development of quality management systems (qms). audits were conducted at baseline, six months, exit (at 12 months) and post-slmta (at 18 months) using the stepwise laboratory quality improvement process towards accreditation (slipta) checklist to measure changes in implementation of the qms during the period. at the end of each audit, a comprehensive implementation plan was developed in order to address gaps. results: baseline audit scores ranged from 19% to 52%, corresponding to 0 stars on the slipta five-star scale. after 18 months, one laboratory reached four stars, two reached three stars and two reached two stars. there was a corresponding decrease in nonconformities and development of over 100 management and technical standard operating procedures in each of the five laboratories. conclusion: the tremendous improvement in these five caribbean laboratories shows that slmta coupled with mentorship is an effective, user-friendly, flexible and customisable approach to the implementation of laboratory qms. it is recommended that other laboratories in the region consider using the slmta training programme as they engage in quality systems improvement and preparation for accreditation. introduction top ↑ improving laboratory quality systems and attaining accreditation are important benchmarks in national health laboratory practice, as accreditation is a process that gives formal recognition of the technical competence of a laboratory to perform specific tests.1 in many cases, the added value of accreditation far outweighs the necessary investment in human resources, finances and time, since it is an independent method of determining and monitoring laboratory performance, whilst assuring the validity of the results to the users.2,3implementation of laboratory quality management systems (qms) and achievement of accreditation amongst laboratories in the caribbean region has been limited. available data report only three accredited government-owned or public clinical laboratories in the caribbean as of 2011.4 over the years, many caribbean laboratory staff have been provided with information on qms and accreditation in various forms, including training, conferences, meetings and printed material. however, using this knowledge collectively and developing a comprehensive plan in order to address quality gaps and begin the journey toward accreditation have been challenging. during a preliminary laboratory needs assessment survey conducted in 2009, laboratory managers and other stakeholders discussed the problems of an undertrained laboratory workforce, the lack of motivation and, most importantly, the perception that the quality improvement process was cumbersome.4 the need to put strategies in place to eliminate these hindrances as soon as possible was emphasised. the recommendation was that a more user-friendly, stepwise approach to quality systems implementation, in combination with task-based training tools to improve staff knowledge, could lead to more substantial improvement in quality systems. the strengthening laboratory management toward accreditation (slmta) programme was launched in 2009 and has been implemented in 47 countries worldwide.5 it is a management training programme that utilises a series of workshops interspersed with on-site projects designed to improve laboratory quality. evidence from other settings has shown that the slmta training programme yields observable and measurable laboratory improvements.6 furthermore, the training empowers laboratory staff and enhances management’s ability to improve their own laboratories by making use of existing resources.7 a laboratory quality improvement mentorship intervention programme in lesotho that incorporated the slmta training and a stepwise approach to accreditation preparedness has resulted in significant measurable improvements in the quality of enrolled laboratories over a period of 12 months.8 the reauthorisation of the us president’s emergency plan for aids relief (pepfar ii) in 2008 resulted in the establishment of the pepfar caribbean regional program and the development of the pepfar partnership framework with 12 caribbean countries (barbados; trinidad and tobago; belize; suriname; jamaica; the bahamas; st. lucia; st. vincent and the grenadines; grenada; antigua and barbuda; st. kitts and nevis; and dominica). since then, the pepfar laboratory-strengthening working group has worked closely with the ministries of health (mohs) in these countries to improve the quality and reliability of laboratory results and to offer basic testing services for persons living with hiv. the need to engage laboratories in these countries in quality improvement and accreditation was identified very early during this collaboration when it became apparent that laboratory services, systems and infrastructure in the region were weak, with various populations lacking access to timely, low-cost and high-quality laboratory services.4 with the aim of improving laboratory quality in the region, the us centers for disease control and prevention (cdc) caribbean regional office laboratory team, the international laboratory branch of the division of global hiv/aids at cdc atlanta and the african field epidemiology network (the laboratory implementing partner) collaborated to research options for effective laboratory quality improvement. the decision was made to use the slmta training programme, coupled with the world health organization regional office for africa’s (who afro) stepwise laboratory quality improvement process towards accreditation (slipta) checklist, along with mentorship, in order to improve the quality systems of five laboratories in four of the caribbean partnership framework countries. this article discusses improvements in the laboratory quality systems during the 18-month implementation of the slmta training programme and mentorship in these laboratories. research method and design top ↑ advocacy strategy with governments at the initiation of the regional laboratory strengthening activities, following the signing of the pepfar caribbean regional partnership framework in 2010, key sensitisation meetings were held with policymakers and other stakeholders in each of the four countries to highlight the need, importance and advantages of improved laboratory quality systems and accreditation. these meetings included chief medical officers, permanent secretaries, laboratory directors and other regional partners. in addition to discussing an overall strategy for collaboration and strengthening of the entire laboratory health system, a presentation was made highlighting the stepwise approach toward accreditation, the slmta training programme and the use of mentors as innovative approaches to implementing quality systems and eventually achieving accreditation. the proposed strategy for laboratory strengthening began by engaging the national reference laboratories in each of the four selected countries. although each laboratory was unique in its operation, size and workload, it was agreed that the challenges faced were similar and they would, therefore, all benefit from the proposed interventions. to ensure buy-in and to highlight the need for providing additional resources to address the deficiencies previously identified during the laboratory needs assessment survey in 2009 and the subsequent baseline audits in 2011, key senior officials from the human resources, procurement and maintenance departments of the mohs and hospitals were invited to attend the audit debrief meetings in their respective countries. laboratory audits periodic audits spanning three to four days were conducted in each of the five national reference laboratories by experienced auditors using the slipta checklist. the slipta programme uses a stepwise accreditation preparedness scheme that recognises laboratories according to their level of compliance with the the international standard iso 15189 – medical laboratories – particular requirements for quality and competence. the results of the laboratory audits were reported for each of the 12 sections of the checklist covering the 12 quality systems essentials (clsi gp 26-a3 [2004]), including 111 main items for a total of 258 possible points (table 1). the score obtained by each laboratory indicates the level of performance, which determines the star rating from 0 to five stars. table 1: slipta scoring system for laboratories. the audits were conducted in each of the five participating laboratories at baseline, after six months (mid-term audit), after 12 months (exit audit) and after 18 months (follow-up audit) to ensure continuous monitoring of the laboratories and their performance (figure 1). each laboratory audit began with an introductory meeting convening the laboratory director and departmental heads in order to summarise the proposed audit plan which would be used to identify areas for improvement. at the end of the audit, a formal debrief meeting was held with laboratory management, technical staff and key persons from the moh and hospital whose responsibilities affect the smooth functioning of the laboratories. after the baseline audit, a customised quality system implementation plan was developed in order to outline the nonconformities found, recommendations for follow-up actions, responsible persons, timeline for completion and status (table 2). table 2: example of quality systems implementation plan. throughout the programme the laboratories were audited at approximately six-month intervals, which allowed them to monitor their continued progress and update the quality system improvement plans originally developed at the baseline audit. the list of nonconformities found at the previous audit was also comprehensively reviewed and updated to determine the number of completed corrective actions over the period. open nonconformities were assigned for further follow-up by the laboratory and its management. exit audits were conducted using the slipta checklist three months after the last slmta workshop concluded (12 months after baseline). a follow-up audit was then conducted six months later to evaluate the longer-term effectiveness and sustainability of the programme. these audits allowed laboratories to determine their level of progress from the baseline to exit of the slmta training and mentorship programme. slmta workshops the slmta training programme was implemented as a series of three workshops which began in may 2011 and were conducted approximately three months apart (figure 1). a total of 24 participants (three to five per laboratory) from across the five focus laboratories were chosen based on the size of their laboratory and the testing needs of each country. these included staff from the various departments, (i.e., chemistry, blood bank, serology, etc.), as well as the quality manager or designee. participants were required to develop improvement projects and complete them during the hiatus between workshops. the improvement projects were generally chosen based on the areas of nonconformity indicated in the laboratory’s individualised quality systems implementation plan along with the needs of the laboratory at the time. each participant presented a summary of their completed improvement projects at the subsequent workshop, including the baseline data collected, the measure of progress within the study period and the challenges experienced during project execution. final improvement projects were presented orally by each participant and graduation certificates were awarded to them in the presence of officials from the moh and the hospitals in order to highlight the importance of this event to the process of accreditation preparedness. figure 1: laboratory strengthening implementation model for the caribbean region. mentorship for the laboratories each of the engaged laboratories was assigned a mentor to assist in developing and establishing their qms by providing technical assistance and coaching on implementing the improvement projects from the slmta training. three fulltime mentors were used for this activity across the five laboratories. each mentor had at least 10 years of experience in laboratory technology and development of qms. during the first few months of the programme, the mentors spent approximately one week each month embedded in the assigned laboratory. after approximately six months, the length of each mentor’s assignment was increased to two or three weeks, depending on the needs of the laboratories at that time. six-week mentorship action plans were developed to give direction to both the laboratory and the mentor, allowing for measurement of progress over the specific six-week period (table 3). since the mentor was physically on-site for only part of the six-week period, the laboratory had a period of self-management during which time they communicated with the mentor via email, internet conferencing and telephone. all management and technical procedures produced during the assignments were forwarded to the laboratory directors or department directors for final approval. table 3: example of six-week mentorship action plan for a laboratory. results top ↑ at the baseline audits the laboratory scores ranged from 19% to 52%, corresponding to 0 stars (figure 2). scores increased steadily throughout the programme and by 18 months each laboratory had improved, with three of the laboratories more than doubling their baseline scores. one laboratory reached four stars on the five-star scale, two attained three stars and the remaining two laboratories each attained two stars. of this group, one laboratory achieved accreditation through the college of american pathologists (cap) in september 2013; meanwhile three others have applied for accreditation and are preparing for the assessment within the next few months. figure 2: improvement in implementation of the laboratory quality systems and stars attained over 18 months. figure 3 shows the average percentage improvement across the five laboratories for each of the 12 sections of the checklist (i.e., the 12 quality system essentials), measured as the difference between the baseline and follow-up score after 18 months. the greatest improvements were in corrective action (66%), organisation and personnel (55%) and purchasing and inventory (54%). the sections showing the least improvement were process control (18%), occurrence management (25%), internal audits (30%) and equipment (36%). average final absolute scores were > 60% for all areas except occurrence management and internal audits. overall, between 141 and 735 standard operating procedures (sops) were completed and approved for each laboratory over the 18-month period (table 4), leading to an average increase on the checklist of 40% from the baseline score in the area of documents and records (figure 3). table 4: number of standard operating procedures (sops) completed for the 5 laboratories. figure 3: average performance improvement of all laboratories across the 12 sections of the who afro slipta checklist. improvement in each laboratory can also be measured by the change in the number of identified nonconformities (figure 4). nonconformities decreased by more than half during the intervention period. for each laboratory this translated into at least a 50% decrease in outstanding nonconformities over the entire implementation period. figure 4: change in number of nonconformities per laboratory over time. case studies each participant enrolled in the slmta training programme was required to choose, plan and execute at least three improvement projects over the duration of the programme. slmta trainers provided tools, techniques and examples in order to guide participants to design effective projects within their laboratory, whilst mentors provided implementation support. as a result of these projects, tangible improvements were observed in the qms and overall operations of the laboratories. two high-impact projects are presented here as case studies: case study 1 – inventory management laboratory 4 has three store rooms containing hundreds of supplies from various vendors. the baseline audit showed that management of stock was a challenge within this facility, with frequent stock-outs, lack of proper tracking forms in the storage areas and increased borrowing from other laboratories. upon investigation, factors such as unpredictable patient-testing workload, delivery delays and back-order issues consistently affected the supply levels. these issues were exacerbated by the poor record keeping and lack of an organised inventory management system, preventing effective forecasting. a key recommendation to the slmta trainee was to put a system in place to ensure sufficient stock levels of all supplies. hence, an improvement project was designed to enhance inventory management in all areas of the system, with the overall objective to reduce stock-outs to less than 5% within a four-month period. to achieve this objective, all staff were briefed on the project, including their specific roles in the success of the intervention. the following data collection and monitoring tools were developed: expired reagent record log; cardex for storage areas; order form; laboratory inventory card; inventory control colour chart; laboratory loan form; requisition order code form; quotation request form; receiving and inspection investigative checklist; regular receiving and inspection log; refrigerator cardex; section grading card; inventory and usage pattern data collection log; and a laboratory stores task assignment checklist. during the improvement project, 15 quality indicators were monitored (figure 5). the results showed that seven of the 15 areas either maintained or achieved 100% compliance, whilst two other areas achieved 90% and 80% compliance over the baseline results. other areas achieved appreciable improvements (figure 5). overall stock-outs were reduced to 5% as a result of the general improvements in the system. figure 5: improvement in laboratory stock management in laboratory 4. case study 2 – improving documents and records management in the microbiology laboratory laboratory 2 has had problems managing quality system documents and associated manuals in their microbiology section. this has resulted in limited progress toward achieving accreditation and difficulty in training new staff in the department. an improvement project was designed to address document and records management. a team of key organisational individuals was convened to work together on the development of the qms. this critical step helped to gain support for the project throughout the various sections in the department. section leaders had the ultimate responsibility of designating and distributing the assignments within their sections. the documents and records were grouped into four categories: technical sops; management sops; logs and checklists; and equipment (including the equipment list, preventative maintenance logs and sops for each item of equipment). figure 6 depicts the level of improvement in documentation after three months of this intervention. technical sops showed the highest level of improvement, from 0% to 67%, closely followed by equipment documentation, from 0% to 63%; the least improvement was in the management sops. figure 6: improvement in documentation for laboratory 2. discussion top ↑ although diverse in its geography, people, size and economy, the caribbean region shares a common challenge in achieving accreditation of its medical laboratories. previous didactic training programmes introduced laboratory staff to the basic quality management principles and the existence of the iso 15189 standard. despite this knowledge, limited progress was seen. an approach that encompassed slmta training, a stepwise evaluation process and mentorship has resulted in tremendous improvement in the quality systems of five national laboratories in four countries of the region within an 18-month period, one of them having attained accreditation. several factors may have contributed to the successes: early engagement of key stakeholders key to the success of global health interventions is full engagement of decision makers in the process from the beginning. in particular, facilitating meetings of policy makers – permanent secretaries, chief medical officers and top management officials of the hospital – along with technical staff, in order to identify challenges and opportunities to resolve nonconformities was important for this project, since these individuals subsequently provided the laboratories with the support and resources needed to ensure timely improvement of the quality systems. endorsement by top management for laboratory systems strengthening activities has proven to be important for the success of this stepwise approach. an implementation roadmap the process of accreditation can appear to be daunting, as extremely high levels of compliance with the quality requirements are essential for a successful assessment and a passing score. for a laboratory without an effective qms, identifying challenges and developing a quality improvement plan can seem like an insurmountable goal, which can lead to demotivation and subsequent inaction. the use of a stepwise improvement process, along with specialised guidance documents, has been shown to provide laboratory stakeholders with a clearer path toward quality systems improvement and accreditation.1 caribbean laboratory directors and managers emphasised that past laboratory assessments and training did not provide them with a structured roadmap to assist in implementation; as a result, the majority of these laboratories did not initiate the process of qms development and implementation.4 the slipta checklist was used to conduct an initial gap analysis in the participating laboratories, leading to the development of an implementation plan, which provided direction for improving the laboratory qms. this plan outlined the process to be taken and the indicators that would be used to measure tangible progress and outcomes over time. everyone involved, including hospital management, was assigned specific tasks relating to their functions and roles, with key deliverables and solid deadlines. use of the stepwise evaluation method enabled recognition of incremental improvements at each audit throughout the process, providing added motivation to all the staff. the scores achieved at each audit highlighted the status attained and the progress that the laboratories had made in building an effective qms, in eliminating nonconformities and in their readiness for accreditation. structured improvement approach prior approaches to laboratory strengthening in the region focused mainly on mass sensitisation to and training on the iso standards and quality management basics, but not on implementation. in some cases the persons trained had not previously been exposed to the principles of continuous quality improvement, total quality management, or development of a quality system specifically for the laboratory. the slmta programme taught the enrolled laboratories how to change the way they approached quality management and their daily operations. the programme also provided user-friendly tools that allowed staff to work more efficiently, as evidenced by their improved star ratings after 18 months. an important component of the slmta training is the improvement projects developed and implemented by the trainees. this promoted a culture of systematic problem solving and a strategic approach to the application of quality system requirements. these projects and their measureable results served as a tool for the laboratory to advocate with management and policymakers for continued support. with the changing economic priorities and limited resources in these developing countries, it was critical to document the impact of any quality improvement and accreditation preparations, so as to demonstrate for stakeholders that the benefits outweigh the costs.2 in the case of these caribbean laboratories, nonconformities were drastically reduced, with corresponding improvement in each of the quality management systems. for example, a 66% improvement was observed in the laboratories’ ability to perform corrective actions. a similar slmta intervention in lesotho6 reported a 34% improvement in corrective action application over an 11-month period. mentorship according to maruta, rotz and peter, ‘a laboratory mentoring program can be an important way to establish and solidify quality management systems and to help laboratories achieve accreditation goals’.9 the presence of the mentors in this programme served two main purposes. firstly, mentors provided needed technical assistance in order to aid the laboratory in the development and finalisation of the qms documentation. it has been documented that a strong foundation for quality assurance begins with development of a quality manual, sops and test methods, since they serve as a guide for both implementing and enhancing the quality system.10 the mentors played a critical role in bridging the gap between what was learnt in the workshops and what was implemented within the laboratories, drawing the team together to develop a strategy and guiding them to address the existing issues. for example, the majority of laboratory staff initially reported that their quality documents were delayed in the process of development for six or more months. the reduction in nonconformities recorded in these laboratories can be directly linked to the increase in the number of documents developed, completed and implemented as a result of the technical assistance provided by the mentors. secondly, mentors alleviated fears associated with preparation for accreditation and acted as a catalyst for enhanced communication. with the mentors present, communication improved greatly amongst the laboratory staff, laboratory management and medical staff. management was more open to presentations and discussions with the laboratory staff, since these consultations were centered on actual data, nonconformance reports and demonstrated improvement. staff often planned management review meetings whilst the mentor was on site, allowing the mentor to help facilitate communication with upper management and showcase the improvement in the laboratory qms as a result of the interventions. key challenges and recommendations the caribbean region is made up of small island nations with most country populations in the range of hundreds of thousands. ensuring a sufficient number of well-qualified laboratory workers is an ongoing challenge, exacerbated by high levels of attrition as staff that have benefitted from government-supported training leave the public sector for more lucrative jobs in the private sector, either locally or overseas. thus the remaining staff are overworked, reducing the amount of time available for training and quality improvement activities. there is also a shortage of qualified mentors who can provide the needed support to laboratories engaged in quality improvement efforts and accreditation preparation. these personnel challenges limit the laboratories’ opportunities for development of qms and achievement of laboratory accreditation. encouraging governments in the region to prioritise health system–strengthening strategies that lead to staff development and retention would benefit not only laboratories, but the health system overall. one of the main logistical challenges faced in this programme stemmed from the use of mentors based in different countries, who were required to travel by air to provide on-site support. thus, considerable funds needed to be invested and intervention was sometimes delayed because of travel issues. establishment of a cadre of in-country or regional slmta trainers and mentors would build local capacity and help reduce programme costs, especially as the programme expands. the momentum achieved through success of the slmta programme in these five laboratories must now be directed to further improvements in these laboratories, as well as expansion of the programme throughout the region. one of the participating laboratories recently achieved accreditation from cap and three more have subsequently applied for accreditation, as a direct result of the training and technical assistance received in the slmta programme. the remaining laboratory will continue to be monitored by means of slipta audits, whilst preparing actively for accreditation in the near future. introduction and implementation of the slmta programme in the caribbean region has been made possible by funding from the pepfar programme; however, there is now a need to internalise the programme and transition it to local governments and other donors in order to facilitate expansion and ensure sustainability. conclusion quality management interventions in the caribbean over the past 10 years had resulted in few improvements in the overall laboratory quality infrastructure, as evidenced by the low performance scores achieved at baseline audits and the limited number of previously-accredited laboratories in the region. a change of approach was thus needed in order to increase these numbers and put more laboratories on the path to accreditation. implementation of the slmta and mentorship approach in several laboratories in the region has achieved tangible improvements in qms development and overall quality within a very short period. continued improvement in these laboratories and expansion of this programme to other laboratories in the region are recommended. sustained improvement will require government funds to be invested in training resources, including development and establishment of local mentorship programmes. our results strongly support the growing body of evidence indicating that the slmta training programme is an important tool to empower laboratory staff, enhance management competence and achieve observable and measurable results for improved laboratory quality. acknowledgements top ↑ we would like to thank all of the laboratory staff who participated in the slmta programme and who worked diligently on the improvement projects. we thank the mentors for their hard work and persistence with each laboratory. in addition, we would like to acknowledge the technical assistance provided by the african field epidemiology network, the financial support from pepfar and the guidance and technical expertise provided by the cdc. cdc disclaimer: the findings and conclusions in this report are those of the author(s) and do not necessarily represent the views of the us centers for disease control and prevention. this research has been supported by the us president’s emergency plan for aids relief (pepfar) through the us centers for disease control and prevention. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions g.g. (us centers for disease control and prevention, caribbean regional office) was the project leader and wrote the manuscript. f.g. and y.i. (both african field epidemiology network, caribbean office), as well as i.w. (princess margaret hospital, bahamas) and s.b. (ladymeade reference unit, barbados), provided data. k.p. and r.a. (both us centers for disease control and prevention, caribbean regional office) provided comments. t.m. (clinton health access initiative botswana) and n.n. (african field epidemiology network, uganda) provided technical input; and g.a. (us centers for disease control and prevention, caribbean regional office) provided comments and co-wrote the manuscript. references top ↑ 1.gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm2.zeh ce, inzaule sc, magero vo, et al. field experience in implementing iso 15189 in kisumu, kenya. am j clin pathol. 2010;134(3);410–418. http://dx.doi.org/10.1309/ajcpzirkdus5lk2d 3. peter tf, rotz pd, blair dh, et al. impact of laboratory accreditation on patient care and the health system. am j clin pathol. 2010;134(4):550–555. http://dx.doi.org/10.1309/ajcph1skq1hnwghf 4.alemnji ga, branch s, best a, et al. strengthening national laboratory health systems in the caribbean region. glob public health. 2012;7(6):648–660. http://dx.doi.org/10.1080/17441692.2012.670861 5.yao k, maruta t, luman et, nkengasong jn. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(2), art. #194, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i2.194 6.mothabeng d, maruta t, lebina m, et al. strengthening laboratory management towards accreditation: the lesotho experience. afr j lab med. 2012;1(1), art. #9, 7 pages. http://dx.doi.org/10.4102/ajlm.v1i 1.9 7. yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3):401–409. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj 8.maruta t, motebang d, wanyoike j, et al. impact of mentorship on who-afro strengthening laboratory quality improvement process towards accreditation (slipta). afr j lab med. 2012:1(1), art. #6, 8 pages. http://dx.doi.org/10.4102/ajlm.v1i1.6 9.maruta t, rotz p, peter t. setting up a structured laboratory mentoring programme. afr j lab med. 2013;2(1), art. #77, 7 pages. http://dx.doi.org/10.4102/ajlm.v2i1.77 10.abimiku ag. building laboratory infrastructure to support scale-up of hiv/aids treatment, care, and prevention: in-country experience. am j clin path. 2009;131(6):875–886. http://dx.doi.org/10.1309/ajcpelmg6gx6rqsm abstract introduction research method and design results discussion acknowledgements references about the author(s) pamela michelow department of anatomical pathology, university of the witwatersrand and national health laboratory service, johannesburg, south africa amanda sherrin department of anatomical pathology, university of the witwatersrand and national health laboratory service, johannesburg, south africa louise rossouw department of anatomical pathology, university of the witwatersrand and national health laboratory service, johannesburg, south africa samson mohaleamolla department of anatomical pathology, university of the witwatersrand and national health laboratory service, johannesburg, south africa denise evans department of internal medicine, university of the witwatersrand, johannesburg, south africa avril swarts department of medicine, university of the witwatersrand, johannesburg, south africa ntombiyenkosi rakhombe right to care, helen joseph hospital, johannesburg, south africa jennifer s. smith department of epidemiology, university of north carolina, chapel hill, north carolina, united states lineberger comprehensive cancer center, university of north carolina, chapel hill, north carolina, united states cynthia firnhaber department of medicine, university of the witwatersrand, johannesburg, south africa right to care, helen joseph hospital, johannesburg, south africa citation michelow p, sherrin a, rossouw l, et al. performance of the cellslide® automated liquid-based cytology system amongst hiv-positive women. afr j lab med. 2016;5(1), art. #278, 6 pages. http://dx.doi.org/10.4102/ajlm.v5i1.278 original research performance of the cellslide® automated liquid-based cytology system amongst hiv-positive women pamela michelow, amanda sherrin, louise rossouw, samson mohaleamolla, denise evans, avril swarts, ntombiyenkosi rakhombe, jennifer s. smith, cynthia firnhaber received: 03 nov. 2014; accepted: 13 nov. 2015; published: 01 feb. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: many women undergoing cervical screening as part of a national south african screening programme may be positive for hiv. the performance of liquid-based cytology (lbc) on samples from hiv-positive women needs to be determined. objectives: the performance of the cellslide® automated lbc system was evaluated as a possible alternative to conventional cytology in a national cervical cancer screening programme. methods: split samples from 348 hiv-positive women attending an hiv treatment clinic in johannesburg, south africa were examined by conventional cytology and monolayer lbc methods. all samples were stained, examined and reported in the same manner. cytotechnologists were blinded to the conventional smear diagnosis if the lbc smear was screened and vice versa. results: the same percentage of inadequate smears (1.4%) was obtained by conventional cytology and lbc. atypical squamous cells of undetermined significance were observed in 5.2% of conventional smears and 4.0% of lbc smears. low-grade squamous intraepithelial lesions were found in 35.6% of conventional smears and 32.7% of lbc smears. only one conventional smear was categorised as atypical squamous cells – cannot exclude a high-grade lesion, whereas five such cases were identified on lbc. high-grade squamous intraepithelial lesions were seen in 21.6% of conventional smears and 23.3% lbc smears. no invasive carcinoma was identified. conclusion: the performance of the cellslide® lbc system was similar to that of conventional cytology in this population of high-risk hiv-positive women, indicating that it may be introduced successfully as part of a cervical cancer screening programme. introduction cervical cancer is a significant cause of morbidity and mortality in south africa. in 2009, it was the second-most common cancer amongst south african women, with an age-standardised incidence rate of 22.33 per 100 000.1 amongst black south african women, cervical cancer was the most commonly histologically diagnosed cancer in 2009, with an age-standardised incidence rate of 26.19 per 100 000.1 unfortunately, more recent incidence data are not available. from a global perspective, an analysis of 187 countries showed that approximately 200 000 women died from cervical cancer in 2010, of which a significant proportion were women aged between 15 and 49 years in under-resourced nations.2 in south africa, 30.2% of women of reproductive age were hiv-positive in 2010, whereas the rate was 29.5% in 2012.3,4 the burden of cervical cancer and its precursor lesions is intensified amongst hiv-positive women. in a study from johannesburg, hiv-positive women had a higher prevalence of cervix lesions related to human papillomavirus (hpv) compared with hiv-negative women, even after controlling for confounding variables such as age and sexual behaviour.5 this finding reflects the high rate of co-infection of hiv and hpv. such co-infection is often associated with higher prevalence of high-risk hpv infections and increased rates of intraepithelial lesions such as high-grade squamous intraepithelial lesions (hsil), which are often large, multifocal, and have higher recurrence rates. invasive cervical cancer is also more common amongst hiv-positive women, occurring approximately 10 years earlier and with more rapid progression and poorer prognosis, than amongst hiv-negative women.6,7 the national south african guidelines for cervical cancer screening of hiv-positive women recommend screening upon diagnosis of hiv. if the results are negative, follow-up screening every one to three years is recommended. if the initial results show a low-grade lesion (i.e. atypical squamous cells of undetermined significance [ascus] or low-grade squamous intraepithelial lesion [lsil]), a repeat screening is required one year later.8 if a high-grade lesion is found (i.e. hsil or atypical squamous cells – cannot exclude a high-grade lesion [asch]), the patient is referred for further evaluation. suggested screening modalities include conventional and liquid-based cytology (lbc), hpv testing, visual inspection with acetic acid and visual inspection with lugol’s iodine. a number of studies to compare conventional cytology, hpv testing and visual inspection with acetic acid for screening of hiv-positive women in south africa are currently underway.9,10 it is estimated that 80% of south africans use public–sector healthcare facilities, including the national health laboratory service. approximately 1 million screenings using the conventional cervical cytology method were reported by the national health laboratory service in 2013.11 lbc is an alternative method of preparing cervical smears for microscopic examination and is widely used in well-resourced nations. however, although it was developed 15 years ago, it has not been adopted into the south african health sector. several meta-analyses comparing lbc with conventional cytology have reported conflicting results. abulafia et al.12 concluded that a commonly used lbc test was more sensitive and specific compared with conventional cytology for diagnosing cervical dysplasia, whereas arbyn et al.13 and davey et al.14 determined that lbc neither reduces the number of unsatisfactory smears nor improves detection of hsil. karnon et al. found that there is uncertainty regarding the ‘relative effectiveness (and cost-effectiveness) of the two main lbc techniques’.15 advantages of lbc include that screenings for hpv and other sexually transmitted infections, including chlamydial and gonococcal infections, can be performed on the lbc collection fluid, without the need for collecting a separate specimen. hpv testing can be performed on the fluid in the vial, and hpv testing or cytology can be performed on the same sample.16,17 a large percentage of south african women undergoing routine cervical screening may be hiv-positive.3,4 as there is a paucity of literature examining whether lbc can be successfully used to screen for cervical abnormalities in hiv-positive women, the benefit of introducing lbc as part of a national cervical cancer screening programme in south africa is unclear. the aim of this study was to determine whether cellslide® (audit diagnostics, cork, ireland), an automated lbc processing system for the preparation of thin-layer smears, can be used successfully as a screening modality in a high-risk hiv-positive population. research method and design ethical considerations the study protocol was reviewed and accepted by both the university of the witwatersrand (human ethics committee) and the university of north carolina. study design and setting the study was a non-randomised, prospective, observational evaluation. the study population comprised 348 hiv-positive women involved in a cervical cancer screening study in south africa.5 all participants were enrolled consecutively. women aged between 18 and 65 years were recruited from an hiv treatment clinic at a tertiary government hospital in johannesburg, south africa, between november 2009 and august 2011. participants were approached for the study whilst in the hiv clinic awaiting medications or an appointment with a healthcare practitioner. women were ineligible to participate if they were pregnant, had previously undergone a hysterectomy or treatment for cervical neoplasia or cancer, were severely ill or had signs or symptoms suggestive of a sexually transmitted disease. women who had completed treatment for a sexually transmitted disease were eligible. women who were menstruating at the time of study enrolment were asked to return within two weeks to participate. the main reasons that women declined to participate in the study were the fear of losing their place in the queue and time constraints. a cervical fluid sample was taken with a cervical broom. a split-sample method was then used, whereby a conventional cytology smear (pap smear) was prepared by spreading the collected material onto a glass slide and spray fixing immediately, followed by placing the tip of the brush in a vial containing cellslide® preservative solution (audit diagnostics, cork, ireland). both specimens were processed in the cytology unit of the department of anatomical pathology, university of the witwatersrand/national health laboratory service in johannesburg, south africa. the manufacturer’s instructions were used when preparing the cellslide® thin-layer slide. both types of sample were stained, coverslipped, examined under the microscope and reported using the bethesda system for reporting cervical cytology.18 the bethesda system is a widely used cytology reporting system that not only provides guidelines for specimen adequacy but also offers standardised reproducible criteria for cytologic lesions such as ascus, lsil, asch and hsil. the aim of the bethesda system is to minimise inter-observer variability and facilitate communication between the clinician and the laboratory.18 laboratory investigations the conventional and cellslide® cervical smears were examined by different cytotechnologists. the cytotechnologists reporting the cellslide® smears were blinded to the conventional smear diagnosis and vice versa. thirteen cytotechnologists reported out some of the conventional cytology and some of the cellslide® thin-layer slides. cytotechnologists participate in stringent internal quality assurance activities. some of these include all reportedly negative smears undergoing rapid review, all positive smears being evaluated by two technologists and evaluation of each technologist’s ascus : sil ratio. external quality assurance activities employed by this laboratory include the australian rcpa quality assurance proficiency programme and laboratory accreditation by external accreditation bodies. if quality assurance activities identify suboptimal performance by a cytotechnologist, re-training and intense monitoring of the work quality are undertaken. statistical analysis categories were compared using the chi-square test or fisher’s exact test for proportions. we determined the accuracy of cellslide® for identifying the diagnostic category correctly compared to the ‘gold standard’ of conventional cytology (pap smears). the number of positive and negative samples as tested using cellslide® was compared to the number of samples with and without each cervical abnormality of interest (e.g., hsil, asch, lsil, ascus) as determined by conventional cytology. we calculated the sensitivity, specificity, positive predictive value and negative predicative value for each diagnostic category, as well as the corresponding 95% confidence intervals (ci; binomial distribution assumed). the kappa coefficient was used to test for agreement between diagnostic categories for the split sample. kappa values < 0.4 were considered to indicate poor agreement, whereas values of 0.41–0.75 were considered to indicate moderate (fair to good) agreement and values > 0.75 were regarded as indicating excellent agreement.19 as both methods used the same categories within the rating scale, a weighted kappa coefficient was not required. all analyses were performed at a 5% significance level using sas version 9.1 (sas institute, cary, north carolina, united states). results very few inadequate smears were obtained by the respective methods (only five [1.4%] for both conventional cytology and cellslide®) (table 1). a high percentage of abnormal smears were identified with both methods, and a diagnosis of negative for intraepithelial lesion/malignancy (nilm) was found for only 125 (35.9%) by conventional cytology and 129 (37.1%) by cellslide®. lsil was the most frequently diagnosed epithelial abnormality (124 [35.6%] by conventional cytology and 114 [32.7%] by cellslide®). both preparation methods diagnosed a substantial number of hsil cases (75 [21.6%] by conventional cytology and 81 [23.3%] by cellslide®). the cellslide® method diagnosed an additional six cases of hsil (p < 0.001). ascus was diagnosed in 18 samples (5.2%) using conventional cytology and in 14 samples (4.0%) using cellslide® (p < 0.001), whereas asch was diagnosed in only one sample (0.3%) using conventional cytology and in five samples (1.4%) using cellslide® (p < 0.014). no cases of glandular lesions or invasive carcinoma were diagnosed by either method. table 1: results from conventional cytology and cellslide® automated liquid-based cytology amongst hiv-positive women (n = 348) in johannesburg, south africa (november 2009 to august 2011).† twenty cases diagnosed as lsil by conventional cytology were diagnosed as hsil by cellslide®, and 15 cases diagnosed as lsil by cellslide® were diagnosed as hsil by conventional cytology. four cases diagnosed as asch by cellslide® were diagnosed as lsil by conventional cytology. no cases diagnosed as asch by one method (either conventional cytology or cellslide®) were diagnosed as hsil by the other method. three cases diagnosed as ascus by cellslide® were diagnosed as hsil by conventional cytology, but no cases diagnosed as ascus by conventional cytology were diagnosed as hsil by cellslide®. the agreement between the two diagnostic methods was poor for asch (κ = 33.0, 95% ci: 1.4–33.0) (table 2). agreement was also considered poor for ascus, because the kappa value was close to the poor–moderate cut-off and the 95% ci was wide (κ = 41.1, 95% ci: 18.6–62.5). agreement was moderate for lsil and hsil, and excellent for nilm. table 2: level of agreement between conventional cytology and cellslide® for each diagnostic category amongst samples from hiv-positive women (n = 348) in johannesburg, south africa (november 2009 to august 2011). for hsil, cellslide® showed sensitivity of 76.0% (95% ci: 64.8–85.1) and specificity of 91.0% (95% ci: 87.0–94.2), with a false-omission rate < 7%, compared with conventional cytology (table 3). in addition, when compared with conventional cytology, cellslide® showed sensitivity of 89.6% (95% ci: 82.9–94.4) and specificity of 92.2% (95% ci: 87.8–95.4) for nilm, sensitivity of 70.2% (95% ci: 61.3–78.0) and specificity of 87.7% (95% ci: 82.6–91.7) for lsil, and sensitivity of 100% (95% ci: 2.5–100) and specificity of 98.8% (95% ci: 97.1–99.7) for asch. table 3: diagnostic accuracy of cellslide® compared with conventional cytology in samples from hiv-positive women in johannesburg, south africa (november 2009 to august 2011). discussion the results show excellent to moderate agreement between conventional cytology and the automated lbc system cellslide® for diagnosis of nilm, lsil and hsil in this population of hiv-positive women. poor agreement was observed between the two methods for diagnosis of ascus and asch. hpv testing is becoming increasingly popular as a primary cervical screening modality. in south africa, lbc can be efficacious, should hpv testing be implemented. hpv testing and co-testing with either cytology or reflex cytology, if certain high-risk hpv types are found, can be performed on the same vial, without the need for collecting two samples from the same patient. the lbc method may support faster microscope screening and therefore more samples could be screened daily, although not all studies have demonstrated this.12,13,14,15 the lbc method is reported to perform better when using computer-assisted screening devices,12,13,14,15 which is an advantage given the large number of pap smears performed in south african public-sector healthcare facilities. the cost associated with lbc has to be considered. results on the cost-effectiveness of lbc are conflicting, depending on whether studies found improvements in the adequacy or detection of abnormality rates. a study by taylor et al.20 found that a commonly used lbc product reduced the number of inadequate smears but did not improve detection of histology-confirmed disease (cervical intraepithelial neoplasia grade 1 or worse) and therefore concluded that the increased cost does not justify the implementation of lbc. in contrast, a review by cox21 showed that lbc was cost-effective. a study by de bekker-grob et al.22 similarly determined that lbc can be cost-effective as a cervical screening modality, specifically if the cost of lbc exceeded the cost of conventional cytology by less than €3.2, the sensitivity of lbc was at least 3% – 5% greater than conventional cytology and the rate of inadequate smears on conventional cytology was at least 16.2%. only one study has investigated the performance of lbc on samples from hiv-positive women.23 the findings need to be considered critically to inform a decision about introducing lbc in the south african public health sector, as a large percentage of south african women eligible for cervical screening may be hiv-positive.6,7,24 the study by swierczynski et al.23 concluded that conventional cytology and lbc detected the same number of squamous intraepithelial lesions. however, the diagnosis of ascus by lbc was more likely to be associated with a squamous intraepithelial lesion on follow-up compared with diagnoses of ascus by conventional cytology. they also determined that both methods could readily identify infectious organisms. in the current study, the conventional cytology and cellslide® methods detected a similar number of unsatisfactory smears and ascus and lsil diagnoses, but more cases of asch and hsil were diagnosed by cellslide®. furthermore, we found excellent agreement between the two methods for diagnosis of nilm and moderate agreement for diagnosis of lsil and hsil. our finding of poor agreement for diagnosis of ascus and asch highlights the well-described poor intraand inter-observer reproducibility for these epithelial abnormalities.25,26,27 however, it is important to note that the small number of samples in the ascus and asch categories could have compromised the accuracy of the statistical analysis. the results of the current study show that cellslide® has good sensitivity and specificity for nilm. as a screening test, a negative result is useful for determining that the patient does not have the disorder. at this initial screening, cellslide® correctly identified more than 90% of samples that showed no cervical abnormalities. similar to these results, a brazilian study found that the agreement between conventional cytology and lbc was highest in the nilm category.28 the authors further noted that this influenced the agreement rate, as the majority of cervical smears were negative. in comparison, the proportion of samples that showed abnormal cytology exceeded 60% for both preparation methods in the current study. other studies have also documented below-excellent agreement for epithelial abnormalities when comparing conventional cytology to lbc preparations.27,28 factors that influence agreement include the method employed to collect the smear, variations in cellular material between the conventional and lbc samples and the level of experience of the cytotechnologists in interpreting the smears.29,30,31,32,33,34 limitations one of the main limitations of the current study is the small number of samples in some of the epithelial abnormality categories (e.g., ascus, asch) as determined by the bethesda system; these results should be interpreted with caution. another limitation is that cellular morphology is somewhat different on lbc preparations compared with conventional cytology and cytotechnologists face a learning curve when moving from conventional cytology to lbc.30 in addition, training in lbc cytomorphology is recommended to facilitate accurate interpretation of an lbc smear.34 as this study was conducted over only a limited time period, cytotechnologists may not have mastered lbc cytomorphology completely. accurate costing of a cervical smear, whether for conventional cytology or lbc, is a complex problem and beyond the scope of the current study. however, cost is a critical factor and must be considered when deciding whether to move to lbc or continue using conventional cytology. conclusion results obtained with cellslide® were similar to those of conventional cytology in this population of high-risk hiv-positive women. the technique may therefore be used successfully should it be decided to move to lbc. acknowledgements we would like to thank the nurse clinicians at right to care for performing the cervical smears and the cytotechnologists at the national health laboratory service for screening the samples. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. sources of support the study was supported by the university of north carolina center for aids research (p30-ai50410), usaid pepfar (674-a-00-08-00007-00) and the south african research chairs initiative of the department of science and technology (phe za.09.0265). authors’ contributions p.m. (university of the witwatersrand and national health laboratory service) and c.f. (university of the witwatersrand and helen joseph hospital) were the main investigators, designed the study, analysed the results and wrote the manuscript. a.sh., l.r. and s.m. (university of the witwatersrand and national health laboratory service) prepared and examined the cervical smears. a.sw. (university of the witwatersrand) was the data manager. n.r. (helen joseph hospital) enrolled patients in the study, performed cervical smears and was responsible for patient follow-up. d.e. (university of the witwatersrand) and j.s.s. (university of north carolina) performed the statistical analysis and contributed to writing the manuscript. references national cancer registry. cancer in south africa: 2009. full report, national cancer registry [report on the internet]. national health laboratory service; no date [cited 2015 july 18]. available from: http://www.nioh.ac.za/assets/files/ncr_2009_final.pdf. forouzanfar m, foreman k, delossantos a, et al. breast and cervical cancer in 187 countries between 1897 and 2010: a systematic analysis. lancet. 2001;378(9801):1461–1484. http://dx.doi:10.1016/s0140-6736(11)61351-2. national department of health. the 2010 national antenatal sentinel hiv and syphilis prevalence survey in south africa. pretoria: national department of health; 2011. national department of health. the 2012 antenatal sentinel hiv and herpes simplex type-2 prevalence survey in south africa. pretoria: national department of health; 2014 firnhaber c, van le h, pettifor a, et al. association between cervical dysplasia and human papillomavirus in hiv-seropositive women from johannesburg, south africa. cancer causes control. 2010;21(3):433–443. http://dx.doi:10.1007/s10552-009-9475-z pantanowitz l, michelow p. review of human immunodeficiency virus (hiv) and squamous lesions of the cervix. diagn cytopathol. 2011;39(1):65–72. doi: 10.1002/dc.21364 firnhaber c, michelow p. cervical cancer and the human immunodeficiency virus: a review. sa j hiv med. 2009;10(3):23–27. national department of health. clinical guidelines for the management of hiv and aids in adults and adolescents [document on the internet]. south african national department of health; 2010 [cited 2015 march 27]. available from: http://www.who.int/hiv/pub/guidelines/south_africa_art.pdf. firnhaber c, mayisela n, mao l, et al. validation of cervical cancer screening methods in hiv positive women from johannesburg, south africa. plos one. 2013;8(1):e53494. doi: 10.1371/journal.pone.0053494 kelly h, muzah b, sawadogo b, et al. prospective evaluation of cervical screening methods in hiv positive women in africa (harp study): baseline results. paper presented at: eurogin; 2012 july 8–11 july 2012; prague, czech republic. national health laboratory service. annual report 2013–14 [document on the internet]. national health laboratory service; 2014 [cited 2015 july 17]. available from: http://www.nhls.ac.za/assets/files/an_report/nhls_annual_report_2013_2014.pdf. abulafia o, pezzulo j, sherer d. performance of thinprep liquid-based cervical cytology in comparison with conventionally prepared papanicolaou smears: a quantitative study. gynecol oncol. 2003;90(1):137–144. http://dx.doi.org/10.1016/s0090-8258(03)00176-8 arbyn m, bergeron c, klinkhammer p, martin-hirsh p, siebers a, bulten j. liquid compared with conventional cytology: a systematic review and meta-analysis. obstet gynecol. 2008;111(1):167–177. doi: 10.1097/01.aog.0000296488.85807.b3 davey e, barratt a, irwig l, et al. effect of study design and quality on unsatisfactory rates, cytology classifications, and accuracy in liquid-based versus conventional cytology: a systematic review. lancet. 2006;367(9505):122–132. http://dx.doi.org/10.1016/s0140-6736(06)67961-0 karnon j, peters j, platt j, chilcott j, mcgoogan e, brewer n. liquid-based cytology in cervical screening: an updated rapid and systematic review and economic analysis. health technol assess. 2004;8(20):1–78. http://dx.doi.org/10.3310/hta8200 pan q, hu s, guo h, et al. liquid-based cytology and human papillomavirus testing: a pooled analysis using data from 13 population-based cervical cancer screening studies from china. gynecol oncol. 2014;133(2):172–179. doi: 10.1016/j.ygyno.2014.03.008 arbyn m, ronco g, anttila a, et al. evidence regarding human papillomavirus testing in secondary prevention of cancer. vaccine. 2012;30 suppl 5: f88–f99. solomon d, nayar r, editors. the bethesda system for reporting cervical cytology: definitions, criteria and explanatory notes. 2nd ed. new york: springer; 2004. http://dx.doi.org/10.1007/978-1-4612-2042-8 cohen j. a coefficient of agreement for nominal scales. educ psychol meas. 1960;20:37–46. http://dx.doi.org/10.1177/001316446002000104 taylor s, kuhn l, dupree w, denny l, de souza m, wright t. direct comparison of liquid-based and conventional cytology in a south african screening trial. int j cancer. 2006;118(4):957–962. http://dx.doi.org/10.1002/ijc.21434 cox t. liquid-based cytology: evaluation of effectiveness, cost-effectiveness, and application to present practice. j natl compr canc netw. 2004;2(6):597–611. de bekker-grob e, de kok i, bulten j, et al. liquid-based cervical cytology using thinprep technology: weighing the pros and cons in a cost-effectiveness analysis. cancer causes control. 2012;23(8):1323–1331. http://dx.doi.org/10.1007/s10552-012-0011-1 swierczynski s, lewis-chambers s, anderson j, keller j, hinkle d, ali s. impact of liquid-based gynecologic cytology on an hiv-positive population. acta cytol. 2004;48(2):165–172. http://dx.doi.org/10.1159/000326311 firnhaber c, zungu k, michelow p, et al. diverse and high prevalence of human papillomavirus associated with a significant high rate of cervical dysplasia in hiv-infected women in johannesburg, south africa. acta cytol. 2009;53(1):10–17. http://dx.doi.org/10.1159/000325079 grenko r, abendroth c, frauenhoffer e, ruggiero f, zaino r. variance in the interpretation of cervical biopsy specimens obtained for atypical squamous cells of undetermined significance. am j clin pathol. 2000;114(5):735–740. http://dx.doi.org/10.1309/k7c9-x5p0-001b-2hk5 bonvicino a, huitron s, fadare o. papanicolaou test interpretations of “atypical squamous cells, cannot exclude high grade squamous intraepithelial lesion”: an investigation of requisite duration and number of colposcopic procedures to a definitive diagnosis of high-grade dysplasia in routine practice. cancer. 2007;111(6):477–481. http://dx.doi.org/10.1002/cncr.23121 gupta s, sodhani p, chachra k, singh v, sehgal a. outcome of “atypical squamous cells” in a cervical screening programme: implications for follow up in resource limited settings. diagn cytopathol. 2007;35(11):677–680. http://dx.doi.org/10.1002/dc.20719 girianelli v, thuler l. evaluation of agreement between conventional and liquid-based cytology in cervical cancer early detection based on analysis of 2,091 smears: experience at the brazilian national cancer institute. diagn cytopathol. 2007;35(9):545–549. http://dx.doi.org/10.1002/dc.20699 cocchi v, carretti d, fanti s, et al. intralaboratory quality assurance in cervical/vaginal cytology: an evaluation of intercytologist diagnostic reproducibility. diagn cytopathol. 1997;16(1):87–92. http://dx.doi.org/10.1002/(sici)1097-0339(199701)16:1<87::aid-dc19>3.0.co;2-7 stoler m, schiffman m, atypical squamous cells of undetermined significance/low grade squamous intraepithelial lesion triage study (alts) group. interobserver reproducibility of cervical cytologic and histologic interpretations: realistic estimates from the ascus-lsil triage study. jama. 2001;285(11):1500–1505. http://dx.doi.org/10.1001/jama.285.11.1500 chung j, park e, choi d, et al. efficacy assessment of cellslide® in liquid-based gynecologic cytology. gynecol oncol. 2005;99(3):597–602. http://dx.doi.org/10.1016/j.ygyno.2005.06.059 obwegeser j, brack s. does liquid-based technology really improve detection of cervical neoplasia? a prospective, randomized trial comparing the thinprep pap test with conventional pap test including follow-up of hsil cases. acta cytol. 2001;45(5):709–714. http://dx.doi.org/10.1159/000328292 yeoh g, chan k. cell block preparation on residual thinprep sample. diagn cytopathol. 1999;21(6):427–431. http://dx.doi.org/10.1002/(sici)1097-0339(199912)21:6<427::aid-dc12>3.0.co;2-4 iverson d. impact of training on cytotechnologists’ interpretation of gynecologic thin-layer preparations. diagn cytopathol. 1998;18(3):230–235. http://dx.doi.org/10.1002/(sici)1097-0339(199803)18:3<230::aid-dc14>3.0.co;2-l abstract introduction methods results discussion acknowledgements references about the author(s) susan k. musau department of laboratory, maryland global initiatives corporation (mgic), nairobi, kenya christina mwachari department of laboratory, maryland global initiatives corporation (mgic), nairobi, kenya elvis kirui department of strategic information, maryland global initiatives corporation (mgic), nairobi, kenya junghae muthoni laboratory department, centers for disease control, nairobi, kenya taylor lascko center for international health, education, and biosecurity, university of maryland, baltimore, maryland, united states natalia blanco center for international health, education, and biosecurity, university of maryland, baltimore, maryland, united states alash’le abimiku school of medicine, university of maryland, baltimore, maryland, united states emily koech department of laboratory, maryland global initiatives corporation (mgic), nairobi, kenyacenter for international health, education, and biosecurity (ciheb), nairobi, kenya citation musau sk, mwachari c, kirui e, et al. implementing an intensified mentorship approach towards accelerated medical laboratory accreditation in 10 counties in kenya. afr j lab med. 2022;11(1), a1814. https://doi.org/10.4102/ajlm.v11i1.1814 original research implementing an intensified mentorship approach towards accelerated medical laboratory accreditation in 10 counties in kenya susan k. musau, christina mwachari, elvis kirui, junghae muthoni, taylor lascko, natalia blanco, alash’le abimiku, emily koech received: 14 dec. 2021; accepted: 18 mar. 2022; published: 01 july 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: despite kenya’s roll-out of the strengthening laboratory management towards accreditation programme in 2010, most laboratories had not made significant or tangible improvements towards accreditation by 2016. in april 2016, the university of maryland, baltimore enrolled 27 facilities in the standard strengthening laboratory management towards accreditation programme. objective: this study aimed to describe and evaluate the implementation of an intensified mentorship strategy on laboratory accreditation. methods: in october 2017, the university of maryland, baltimore implemented intensive mentorship in 27 hospital laboratories in nairobi, kiambu, meru, embu, muranga, nyeri, laikipia, nyandarua, tharaka-nithi, and kirinyaga counties in kenya. laboratories were paired with competent mentors whose skills were matched to facility gaps. baseline and follow-up assessments were done between april 2016 and march 2019 using the world health organization’s stepwise laboratory quality improvement process towards accreditation checklist and overall scores of the 12 quality system essentials and star ratings (from zero to five, based on scores) used to evaluate the effectiveness of the intensified mentorship. results: in september 2017, 14 laboratories scored zero stars, three scored one star, eight scored two stars, one scored three stars, and one laboratory was accredited. by march 2019, eight laboratories were accredited, five scored four stars, 10 scored three stars, three scored two stars, and only one scored one star. the average score change with the intensified approach was 81.5 versus 53.9 for the standard approach. conclusion: the intensified mentorship strategy resulted in fast-tracked progress towards laboratory accreditation and can be adopted in similar resource-limited settings. keywords: accreditation; slmta; slipta; qms; intensified mentorship; kenya. introduction implementing a laboratory quality management system (qms) is critical to providing reliable results for clinical decision-making.1 inaccurate laboratory results can lead to inappropriate interventions, adversely affecting the credibility of the laboratory, and may invite legal action.2 laboratory accreditation formally recognises both the qms and the technical competence of a laboratory to perform specific tests.1,3 laboratory accreditation is a system of standard procedures that aims to improve laboratory services’ quality, results’ accuracy, and patients’ safety.4,5 most laboratories rely on international quality standards for medical laboratories (international organization for standardization 15189), which specify the requirements of quality and competence to medical laboratories. this set of standards is comprehensive but also resource-intensive.5 resource-limited settings face several challenges when implementing quality standards and practices in laboratories, including financial constraints, human resource capacity, limited physical infrastructure, lack of equipment and equipment maintenance, failure to create and/or implement national laboratory policies, and substandard performance in laboratory quality indicators due to a lack of quality standards.6,7 the strengthening laboratory management towards accreditation (slmta) programme was launched in 2009,8 and adopted in kenya in 2010. strengthening laboratory management toward accreditation is a competency-based programme that uses a series of short courses and work-based learning projects to effect immediate and measurable laboratory improvements while empowering laboratory managers to implement a practical qms to ensure better patient care. a standard slmta training programme spans from 12 to 18 months. it includes three workshops spaced between qms’ application and improvement projects’ implementation. the process is supported by regular supervisory visits or on-site mentoring. audits are conducted at the beginning, midterm, and end of the training using the world health organization’s stepwise laboratory quality improvement process towards accreditation (slipta) checklist to assess strengths, weaknesses, and progress made.9 laboratories scoring three stars and above are eligible to apply to the kenya accreditation service, the sole national accreditation body mandated to offer accreditation services for laboratories in kenya. kenya has progressed tremendously in using the slmta programme to accredit medical laboratories, however, only 13 laboratories had been accredited by 2016 through the slmta programme.10,11 with more than 3000 medical laboratories in kenya offering basic diagnostic services, a considerable gap remains in implementing qms. the university of maryland, baltimore (umb), with funding from the united states president’s emergency plan for aids relief through the united states centers for disease control and prevention (cdc), has worked with kenya’s national and county governments to enhance laboratory systems to support hiv/tuberculosis services. as part of this scope, umb, through the boresha maabara programme, supported and mentored 27 hospital laboratories from 10 selected counties (nairobi, kiambu, meru, embu, muranga, nyeri, laikipia, nyandarua, tharaka and kirinyanga) in implementing continuous quality improvement initiatives, and laboratory qms mentorship towards accreditation. the hospitals laboratories included all cadres, such as county, districts and low-level health centres. the objective of this paper is to describe and evaluate an intensified laboratory qms mentorship strategy, which was introduced by umb to move public health laboratories in kenya to accreditation. methods ethical considerations the implementation protocol was approved by the university of maryland, baltimore institutional review board (hp-00094192) and amref ethics and scientific review committee (p 815/2020). this project was also reviewed in accordance with the cdc human research protection procedures. the institutional review boards gave this protocol a “not human research” determination. only aggregated non-human and non-identifiable data were used for this analysis. progression of mentorship strategy at the boresha maabara programme’s inception in april 2016, 27 facilities were enrolled in the slmta programme. baseline audits using the slipta checklists were conducted in april 2016. key healthcare workers, including laboratory managers and quality assurance officers, were enrolled in three consecutive qms training workshops. identified gaps were addressed through improvement projects, and their progress was evaluated during midterm audits. the standard slmta mentorship approach, as described by makokha et al.,12 was used to support the facilities towards achieving accreditation, including embedding qms mentors and supervisory mentorships in the laboratories for 360 days. using this approach, 15 mentors were assigned to at least two slmta facilities. in addition to laboratory qms mentorship, mentors were involved in supporting continuous quality improvement initiatives for hiv and tuberculosis testing-related services in other facilities. this broad scope of work stretched their workload and time (figure 1). figure 1: comparison between the standard and intensified mentorship structures in university of maryland, baltimore from april 2016 to march 2019, kenya. the boresha maabara programme implemented an intensified mentorship approach, starting october 2017, to accelerate the accreditation process. in this approach, seven mentors were identified and dedicated purely to mentoring the 27 laboratories towards accreditation (figure 1). the approach was overseen by a intensively trained qms coordinator who had technical expertise and laboratory accreditation experience. the qms coordinator led planning, supervising, and training facility teams on qms initiatives. mentorship involved conducting initial audits, reviewing non-conformities for each laboratory, and planning meetings for target setting and mentor pairing to support facilities based on non-conformities. mentor competencies and strengths were matched with the laboratory needs, based on the 12 quality system essentials, and mentorship addressed the 12 quality system essentials for each laboratory to resolve all non-conformities. mentorship was complemented with international organization for standardization 15189:2012, method verification, root cause analysis/corrective action and preventive action, and safety trainings based on the laboratory’s needs. monthly virtual meetings were held to check on the progress of all 27 laboratories, which involved re-strategising and re-identifying the skillsets required in different facilities, including re-assigning mentors to different laboratories, if needed. internal audits were conducted by umb mentors, and external audits by the african society for laboratory medicine and kenya accreditation service. the cdc engaged african society for laboratory medicine to conduct external audits to map slmta laboratories in africa, which allowed additional audits to be conducted before kenya accreditation service assessments for accreditation. data collection and analysis baseline and quarterly audits using the slipta checklist (figure 2) were conducted across the 27 laboratories, starting from april 2016 to march 2019, and scores were assigned based on standard scores allocated to the 12 slipta sections. data were analysed using descriptive statistics and graphical representations of the star ratings. accredited laboratories were allocated the highest slipta score (275) of a five-star rating during the computation. we calculated means, standard deviations, medians, and interquartile ranges for scores. the statistical analysis was performed using stata statistical software release 17 (college station, texas, united states: statacorp llc). figure 2: timeline of the evaluation of 27 laboratories starting april 2016 to march 2019, kenya. results at baseline in april 2016, 23 laboratories scored zero stars, three scored one star, and one was already accredited. after 18 months of implementation of the standard mentorship strategy (april 2016 – september 2017), 14 laboratories scored zero stars, three scored one star, eight scored two stars, one scored three stars and one laboratory was accredited, showing slow improvement. in september 2017, the midterm audit demonstrated the common non-conformities emanated from three slipta sections: management review meetings (section 2), evaluation and audits (section 6), and identification and control of non-conformities (section 10) (figure 3). these non-conformities were addressed throughout the intensified mentorship through targeted trainings on how to convene and conduct management review meetings, root cause analysis, corrective actions, and internal audits. figure 3: spider chart showing mean scores for 10 university of maryland, baltimore-supported laboratories for stepwise laboratory quality improvement process towards accreditation sections audited in september 2017, kenya. eighteen months after implementing the intensified mentorship strategy (october 2017 – march 2019), eight laboratories were accredited, with five scoring four stars, 10 scoring three stars, three scoring two stars and only one laboratory scoring one star (figure 4). seven laboratories were newly accredited following the intensified mentorship, while the number of laboratories with ≥ 3 stars improved by 91%. figure 4: audit star ratings comparison between the standard and intensified mentorship approach for university of maryland, baltimore-supported laboratories from april 2016 to march 2019, kenya. across the evaluation period, the median slipta checklist scores increased. with the standard mentorship approach, the median increased from 79 (interquartile range: 55–117) in april–june 2016 to 148 (interquartile range: 116–191) in july–september 2017. with the intensified mentorship, the median score increased to 225 (interquartile range: 207–275) by january–march 2019. the average change of audit scores while using the standard approach was 53.9. the average change with the intensified approach was 81.5, showing greater score improvement after implementing the intensified approach (table 1). table 1: median scores against the standard mentorship (april 2016 – sep 2017) and the intensified mentorship approaches (oct 2017 – mar 2019) for university of maryland, baltimore-supported laboratories in kenya. discussion the boresha maabara programme focused on intensified mentorship of facilities towards reaching and maintaining accreditation. this mentorship approach allowed facilities to be mentored by mentors with the skills necessary to close identified gaps. mentors were allowed to focus on their areas of expertise, which led to a rapid improvement in laboratory audit scores and fast-tracked the laboratories towards accreditation. the standard mentorship approach, as recommended by the slmta programme, assumes that knowledgeable and competent mentors capable of implementing all 12 quality system essentials independently are available.13 makokha et al. noted that the standard mentorship approach that has embedded mentors handling all quality system essentials did not yield results in kenya as expected, as the majority of the laboratories stagnated with minimal improvements.12 these findings suggest the need to re-strategise effective strategies towards laboratory accreditation.12 each country may need to modify the standard approach and develop a country-specific strategy. quality management system mentors may have varying skill levels across the different slipta sections; therefore, they are naturally more effective mentoring in those areas of expertise. our model of pairing qms mentors based on their skills brought together a well-rounded team to provide mentorship to facilities and to resolve non-conformities quickly. other challenges of the standard approach have been documented, including unstructured and often unscheduled mentorship visits that lacked practical work plans and had inconsistent mentorship strengths; these circumstances led to an inability to implement some quality essentials as required.13 the intensified strategy adopted by umb, led by an experienced qms coordinator, provided structured mentorship visits by qualified mentors with monthly reviews of progress. constant interactions between the qms coordinator and mentors allowed opportunities to learn mentors individual strengths and weaknesses, thereby enabling suitable mentor pairs for a well-rounded team. assigning mentors to facilities based on their skillsets and gaps identified in these facilities resulted in tasks being completed more quickly. eventually, this model not only resulted in strengthened qms in the facilities, but also in improved mentors’ technical capacities as mentors learned from each other. monthly meetings between mentors and the qms coordinator through virtual systems provided a forum for sharing progress and discussing laboratory-related challenges and allowed for follow-up of action items. university of maryland, baltimore’s strategy also emphasised dedicating mentors solely to qms, which allowed them to focus their time and effort towards mentoring assigned facilities towards accreditation. while this approach may appear resource-intensive, it allowed for shorter periods of intense interaction with facilities than the standard mentorship approach, as it enabled non-conformities to be efficiently resolved; the model is actually more beneficial for resource-limited countries.14 in our model, accrediting all facilities was in the best interest of all mentors; these mentors functioned together as a network and depended on each other rather than competing against each other. some challenges experienced in the field included inadequate laboratory staffing in the supported facilities; additional mentor-time spent on training lab personnel on qms, as it is not part of the medical laboratory science training curriculum; lack of sufficient budget allocations for qms activities; and use of substandard equipment failing the verification process. these challenges could be overcome by top management embracing qms, building it as part of the medical laboratory science curriculum in the training institutions, and allocating portions of the budget specifically to qms. these factors could also aid in maintaining the achieved gains over time. limitations the paper is restricted to umb experience, staff, and mentorship structure in 10 kenyan counties (nairobi, kiambu, meru, embu, muranga, nyeri, laikipia, nyandarua, tharaka, and kirinyanga) and is not necessarily representative of the whole country. the audits were also conducted by different people and are subject to inter-personnel variations. conclusion the intensified mentorship strategy, led by an experienced qms coordinator working with qms-dedicated mentor teams, resulted in accelerated progress towards accreditation. this strategy could be adopted to accelerate the process of accreditation in similar countries with proper planning and supervision to ensure success. including top-level management, clinicians, and all laboratory staff in the qms process is also essential for enhancing sustainability. additionally, incorporating qms as part of the medical laboratory science curriculum in the training institutions and allocating portions of the budget specifically for qms could help maintain the achieved gains over time. acknowledgements we wish to acknowledge the support from the university of maryland under which this manuscript was written. we thank all the mentors including andrew mboche, sebastian muoki, kola mbangula, vincent mutava, james njogu, beryl mukhwana and lawrence gitonga who worked tirelessly to ensure that the qms was implemented in the laboratories. we also appreciate the contribution of our program managers alan logendo and patricia rarriw. our gratitude goes to both national and county governments that supported this process. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions s.k.m. was the quality management system coordinator and wrote the article; c.m. was the project director and reviewed the article; e. kirui performed the statistical analysis; j.m. was the cdc activity manager for the grant and reviewed the article; t.l. reviewed the article; n.b. reviewed the article and monitored key timelines; a.a. was a principal investigator to the grant and reviewed the article; and e. koech was the country director overseeing implementation of the grant and critically reviewed the article. sources of support this article has been supported in part by the united states president’s emergency plan for aids relief through the united states centers for disease control and prevention, under the terms of cdc-rfa-gh15-1544. data availability data not available due to ethical/ownership restrictions. disclaimer the findings and conclusions in this article are those of the authors and do not necessarily represent the official position of the funding agencies. references guevara g, gordon f, irving y, et al. the impact of slmta in improving laboratory quality systems in the caribbean region. afr j lab med. 2014;3(2):199. https://doi.org/10.4102/ajlm.v3i2.199 hollensead sc, lockwood wb, elin rj. errors in pathology and laboratory medicine: consequences and prevention. j surg oncol. 2004;88(3):161–181. https://doi.org/10.1002/jso.20125 international organization for standardization (iso): iso 15189:2012 medical laboratories – requirements for quality and competence. 2012. geneva, switzerland. zima t. accreditation of medical laboratories – system, process, benefits for labs. j med biochem. 2017;36(3):231–237. https://doi.org/10.1515/jomb-2017-0025 world health organization. laboratory quality management system: handbook, version 1.1. france: world health organization; 2011. guindo ma, shott jp, saye r, et al. promoting good clinical laboratory practices and laboratory accreditation to support clinical trials in sub-saharan africa. am j trop med hyg. 2012;86(4):573–579. https://doi.org/10.4269/ajtmh.2012.11-0691 elbireer am, opio aa, brough rl, jackson jb, manabe yc. strengthening public laboratory service in sub-saharan africa: uganda case study. lab med. 2011;42(12):719–725. https://doi.org/10.1309/lm2obnyy9d0uxzjo yao k, luman e, authors s, moyo s. evidence from 617 laboratories in 47 countries for slmta-driven improvement in quality management systems. afr j lab med. 2014;3(2):362. https://doi.org/10.4102/ajlm.v3i2.262 yao k, maruta t, luman et, nkengasong jn. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(3):194. https://doi.org/10.4102/ajlm.v3i2.194 slmta. slmta laboratories that have achieved accreditation. 2021. https://slmta.org/accredited-labs/ african society for laboratory medicine (aslm). internationally accredited laboratories in africa. 2017. https://aslm.org/resource/gmap/ makokha ep, mwalili s, basiye fl, et al. using standard and institutional mentorship models to implement slmta in kenya. 2014. afr j lab med. 2014;3(2):220. https://doi.org/10.4102/ajlm.v3i2.220 maruta t, rotz p, peter t. setting up a structured laboratory mentoring programme. afr j lab med. 2013;2(1):77. https://doi.org/10.4102/ajlm.v2i1.77 adane k, girma m. iso 15189 laboratory support … how does iso 15189 laboratory accreditation support the delivery of healthcare in ethiopia? a systematic review. ethiopian j health sci. 2018;29(2):259–264. https://doi.org/10.4314/ejhs.v29i2.13 abstract introduction methods results discussion acknowledgements references about the author(s) annie e. cook department of clinical biochemistry, derriford combined laboratory, university hospitals plymouth national health service trust, derriford hospital, plymouth, united kingdom division of chemical pathology, department of pathology, stellenbosch university and national health laboratory service, tygerberg hospital, cape town, south africa thumeka p. jalavu division of chemical pathology, department of pathology, stellenbosch university and national health laboratory service, tygerberg hospital, cape town, south africa annalise e. zemlin division of chemical pathology, department of pathology, stellenbosch university and national health laboratory service, tygerberg hospital, cape town, south africa citation cook ae, jalavu tp, zemlin ae. audit of amylase and lipase requests in suspected acute pancreatitis and cost implications, south africa. afr j lab med. 2022;11(1), a1834. https://doi.org/10.4102/ajlm.v11i1.1834 original research audit of amylase and lipase requests in suspected acute pancreatitis and cost implications, south africa annie e. cook, thumeka p. jalavu, annalise e. zemlin received: 25 jan. 2022; accepted: 25 may 2022; published: 26 sept. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the internationally accepted criteria for the diagnosis of acute pancreatitis (ap) requires two of the three following features to be present: characteristic abdominal pain, elevated serum amylase and/or lipase enzymes, or consistent imaging results. however, sensitivity and specificity can vary depending on the population and cut-off values used. objective: this study evaluated the suitability of amylase and lipase as first-line diagnostic biomarkers of suspected ap for the local population served by tygerberg hospital, south africa. methods: this retrospective analysis was conducted in june 2019 using all amylase and/or lipase request data from december 2018. patient clinical data were included in sensitivity and specificity analyses of amylase, lipase or dual requests for diagnosis of ap. cost per test data were obtained from the national health laboratory service and used to calculate the total cost of the tests and potential savings. results: sensitivity for lipase was 90.0% compared to 50.0% for amylase. specificity was similar for singular measurements of lipase and amylase. dual measurement of amylase and lipase showed no improvement in sensitivity (83.3%) and only a minor increase in specificity (97.4%) compared with measurement of lipase alone. the estimated savings was r2522.85 ($174.98 usd), with a potential annual cost saving of r84 423.74 ($5855.69 usd). conclusion: lipase was shown to be a more sensitive biomarker compared to amylase for the screening of ap, providing evidence for laboratories to educate local staff and promote improved requesting practices by clinicians. additionally, preventing unnecessary dual requests may reduce costs. keywords: audit; pancreatitis; amylase; lipase; cost. introduction the atlanta symposium in 1992 sought to provide a unified international consensus for the classification of acute pancreatitis (ap). the revised version of the atlanta criteria expands on the previous definition to include a classification of severity, defines imaging morphology and discriminates ap as either intestinal oedematous pancreatitis or necrotising pancreatitis.1 according to the revised atlanta criteria and several other international guidelines, ap diagnosis requires two of the following three criteria to be present: the rapid onset of severe and persistent epigastric pain consistent with ap; serum lipase and/or amylase levels greater than three times the upper limit of normal (uln); or imaging consistent with ap, using contrast-enhanced computed tomography, magnetic resonance imaging or transabdominal ultrasonography.1,2,3,4 the gold standard for diagnosis of ap is often considered to be supporting radiological evidence,5 however, this option is rarely considered suitable as a first-line test, and therefore pancreatic enzyme biomarkers provide an essential criterion for quick and accurate assessment in cases where ap is suspected. currently serum amylase and lipase remain the most commonly requested tests for investigation of ap, which is supported by several international guidelines.1,3,4 the limitations of these biomarkers is that both lipase and amylase can also be raised to levels of greater than three times the uln in non-pancreatic conditions, such as renal disease, appendicitis and cholecystitis.6 liraglutide, a glucagon-like peptide-1 receptor agonist used for the treatment of diabetes, has also been shown to falsely elevate serum amylase and lipase levels.7 additionally, neither enzyme can determine aetiology or severity of ap in adults.8 despite this, lipase is considered to be the preferential diagnostic biomarker.9 this was first suggested in united kingdom guidelines 2005,4 due to the longer half-life of lipase compared with amylase; such that lipase still remains detectable 7–14 days after symptom onset, compared with 3–4 days with amylase.6 lipase is now globally accepted as the superior analyte for the investigation of ap, with many international studies and reviews evidencing its improved sensitivity and specificity compared with that of amylase.5,10,11 sensitivity and specificity can vary depending on the population included and the cut-off values used. the two most common aetiologies of ap internationally are gallstones or alcohol abuse. the most common cause of ap in the tygerberg community where this study was performed is alcohol abuse.11,12 we aimed to determine the relative sensitivity and specificity of lipase compared with amylase for the diagnosis of ap within the local population served by tygerberg hospital for the month of december 2018, and to determine what proportion of these requests were clinically indicated. the recommended test for ap is serum lipase, where available; amylase is used in settings where serum lipase is not available. the practice of requesting both tests (dual testing) in the initial evaluation is not recommended but is seen on a regular basis in our laboratory. this practice is not cost effective; it is therefore important to assess how prevalent it is so that it can be addressed. the final aim, therefore, was to review the absolute numbers of dual versus single amylase and lipase requests for the whole of 2018 and to calculate the potential cost-savings associated through changes in requesting practices since lipase became available in our laboratory. methods ethical considerations ethical approval was obtained to access the clinical data of patients where these analytes had been requested. this was sought in advance of the project start and was granted by the health research ethics committee of the faculty of medicine and health sciences at stellenbosch university (reference number: n19/03/036). patient consent was not obtained as a waiver of consent was awarded by the approving ethics committee. the clinical records of patients were only accessed if they were included in the study and were only accessed by authors of this paper. full anonymity of patients was maintained throughout the study and all data was stored in password protected files. study site this was a retrospective clinical audit of amylase and lipase requesting at tygerberg hospital, cape town, south africa. tygerberg hospital is a tertiary hospital in parow, cape town and provides inpatient and outpatient care to public health-sector users. it is a 1400 bed multidisciplinary teaching hospital affiliated with stellenbosch university. the national health laboratory service (nhls) chemical pathology laboratory at tygerberg hospital provides 24-h diagnostic service. the nhls is the preferred provider of pathology services to the public health sector. data collection pathology information technology provided an anonymised data set for all amylase and lipase requests for 2018 that included the following parameters: patient hospital numbers, ward location, date of birth and the amylase and/or lipase results. data from 1 december 2018 to 31 december 2018 were chosen as an initial subset for analysis and the clinical notes surrounding the dates of the amylase and lipase requests within this month were then individually scrutinised. enterprise content management software (open text ecm, opentext corporation, waterloo, ontario, canada), a web-based electronic patient management system, was used to assess each patient record and to determine whether any consistent imaging was performed, the patient clinical symptoms upon presentation and the final patient diagnosis. a diagnosis of ap was considered confirmed if two of the three atlanta criteria were fulfilled or in most cases, by an explicit statement provided in the clinical notes. requests were only included and analysed where the patient was being managed at tygerberg hospital and the laboratory test(s) had been carried out at the tygerberg nhls laboratory. records were excluded if patients were aged < 13 years old, had a recent history of penetrating or blunt abdominal trauma and where notes were incomplete or unclear. the cost per test data were obtained from the nhls and used to calculate the total cost of the tests over the month of december and potential savings if dual testing were to be eliminated. laboratory analyses samples were analysed for amylase and lipase using an enzymatic colorimetric methodology and performed on the roche® cobas® 6000 analyser (roche diagnostics, mannheim, germany) at the tygerberg hospital nhls laboratory. tygerberg hospital is accredited by the south african national accreditation system that regularly participates in external quality assessment to retain this status. statistical analysis all data used within the study were recorded, sorted and analysed using microsoft excel (microsoft corporation, 2019 [16.0], redmond, washington, united states). the normal ranges used by tygerberg hospital at the time this audit was conducted were 20 u/l – 104 u/l for serum amylase and 13 u/l – 60 u/l for lipase. based on these ranges, patient results for all amylase and/or lipase results for december 2018 were either designated ‘≤ 3uln’ less than or equal to three times the uln; or ‘> 3uln’, if the result was greater than three times the uln. presenting clinical details (if available), final diagnosis and relevant imaging were also recorded and then allocated either ‘yes’, ‘no’ or ‘unknown’ depending on whether they were considered consistent with ap. based on these parameters, amylase and/or lipase were designated either a false positive (fp), false negative (fn), true positive (tp) or true negative (tn) status. the relative sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) were calculated using the following equations: microsoft excel was also used to provide estimated cost savings associated with changes in amylaseand lipase-requesting practices. the sample numbers obtained from the 1 to 31 december dataset were used to extrapolate predicted annual savings. results a total of 268 patients had either a lipase or amylase test (or both) carried out during december 2018 at tygerberg hospital (table 1). forty-six of the 268 requests were excluded from the final data analysis, including duplicate requests (n = 16), requests on patients where notes were incomplete or unclear (n = 10) , patients with a recent history of penetrating or blunt abdominal trauma (n = 9), patients with chronic pancreatitis (n = 9), and patients < 13 years old (n = 2). a total of 222 patients were therefore included in the final data analysis; 12 (5.4%) patients had a final diagnosis of ap. table 1: amylase and lipase results for acute pancreatitis testing obtained from tygerberg hospital, south africa, 1–31 december 2018. specificities were relatively equivocal for single amylase (98.6%) and lipase tests (96.9%) (table 2). sensitivity of amylase showed a significant difference, however, at only 50.0% compared to lipase at 90.0% for the subset of data analysed. dual requesting showed an equivocal specificity of 97.4%, however the sensitivity was calculated to be 83.3%, which was 6.7% less than lipase testing alone. table 2: sensitivity, specificity, positive predictive value, negative predictive value and respective 95% confidence interval for amylase, lipase and dual testing for acute pancreatitis at tygerberg hospital, 1–31 december 2018. in december 2018, lipase was requested singly 151 times compared with amylase, which was requested 16 times (table 3). fifty-five of the 222 patients (25%) had dual testing for amylase and lipase. the price of amylase and lipase tests was r45.87 (south african rand [zar], equivalent to $3.10 united states dollars [usd]) per individual test for the period of december 2018. the cost of dual requests for the month of december was r2522.85 ($174.98 usd); that of unnecessary tests was r733.92 ($50.90 usd) and for those not clinically indicated, r5320.92 ($369.06 usd). the projected potential annual cost saving from all these additional or unnecessary tests is r84 423.74 ($5855.69 usd) (table 3). table 3: amylase and lipase test requests and predictive cost savings, tygerberg hospital, 1–31 december 2018. discussion the aim of this study was to compare the relative sensitivities and specificities of lipase and amylase for the diagnosis of ap across the tygerberg hospital population, and to evaluate this in the context of current requesting practices. the initial hypothesis by the laboratory was that amylase was often requested preferentially to that of lipase; the results of this audit, however, have shown that the opposite is true and that most requests in 2018 were, instead, for lipase. additionally, we examined the cost implications associated with current requesting practices and have estimated significant cost-saving potential with a change to single lipase requesting in place of unnecessary dual requests. lipase and amylase have been shown to have high specificity with respect to the diagnosis of ap,5,10,11 when used at the levels of greater than three times the uln, as recommended in the atlanta criteria.2 in this context, specificity is the ability of these biomarkers to correctly identify patients without ap when they are at levels of less than three times uln as stipulated in the atlanta criteria.2 whilst both biomarkers showed similar specificities from the results of this study, at 98.6% for amylase and 96.9% for lipase, sensitivity between the two analytes differed significantly, which was likely to have been significantly impacted by the small sample size. despite this, the results of this study agreed with the widely accepted international consensus, that lipase is a superior biomarker in cases of suspected ap, with a sensitivity of 90% compared with only 50% for amylase. this study was useful because it provided data based on the local tygerberg hospital population. it is widely acknowledged that the aetiology of ap can have a significant impact on the serum levels of these pancreatic enzymes,11 thus it can provide more relevant data for the local medical community to guide recommendations on appropriate requesting practices. amongst the tygerberg population, alcohol abuse is the primary cause of ap,11,12 which typically exhibits lower levels of amylase and lipase in comparison to the next most common cause of ap, gallstones. this could impact the relative sensitivity and specificity of these analytes according to the levels stipulated in the atlanta criteria.1 the sensitivity and specificity from dual requests showed minimal improvement in specificity when compared to lipase alone, and a reduced sensitivity. there were 513 dual requests for 2018. this represents around 257 unnecessary test requests annually which can be costly and potentially detrimental with respect to reduced sensitivity. a recent 2017 review of nine studies by ismail and bhayana,8 which included studies carried out by chang and chung5 and hofmeyr et al.,11 concluded a negligible difference in sensitivity and specificity from dual testing of lipase and amylase compared with singular testing of lipase.5,8,11 whilst studies by basnayake and ratnam,9 and ismail and bhayana8 have commented that the ratio between amylase and lipase levels could help direct clinicians to the aetiology of ap,8,9 it was generally concluded across several reviews and studies, that dual testing was not a cost-effective option for diagnosis of ap.5,8,11,13 a prospective study by hofmeyr et al.11 concluded that on admission to tygerberg hospital lipase sensitivity was significantly improved at 91% compared with 62% for amylase. the specificity of amylase and lipase testing for this population group were comparable, at 93% and 92%, respectively. the recommendations from this study were to promote lipase as the biomarker of choice locally for the diagnosis of ap.11 a potential strategy for overcoming inappropriate dual requesting and the use of amylase instead of lipase in these patient groups could be to limit or introduce a local procedure for demand management of amylase requests. the largest potential cost-saving identified from this study, however, would be through the promotion of greater clinically led requesting in suspected ap. over half of the requests included within this study were considered inappropriate based on the clinical details and final diagnosis provided in the patient notes. limitations there were several important limitations that should be considered with respect to this study. firstly, and most significantly, only 12 of the 222 patients used for the final data analysis had confirmed ap. despite this small subset of data, sensitivity and specificity of lipase and amylase was consistent with a previous study of these biomarkers within the tygerberg population.11 however, it should be noted that the large sensitivity differences observed between amylase and lipase are likely because of the small sample size used. the second limitation of this study is that requests were only considered clinically relevant if the patient notes mentioned abdominal, epigastric or associated pain or overtly queried ap. it is important to note that there are other disease states where these biomarkers may be requested for use in diagnosis and monitoring, where the patient may not typically present with abdominal pain for example, pancreatic cancer, mumps and cystic fibrosis.14,15 whilst these test requests would be considered clinically relevant, for the purposes of this audit, these cases were not accounted for in the final data and cost analysis. in addition, it is important to note that the main ap aetiology will vary between different population groups and this study only includes the local tygerberg population. another consideration that has not been accounted for as part of this study but may affect the associated sensitivities and specificities of these analytes is assay performance across different analytical platforms and methods. notwithstanding the limitations of this study, even this small dataset supports the existing literature, that lipase shows improved sensitivity when compared with amylase for the diagnosis of ap. ideally, a repeat clinical audit should be conducted to include at least a year of data which would enhance the robustness of these results. indeed, a comparative clinical audit from a united kingdom population, where the most common aetiology of ap differs, would help to demonstrate that lipase is a better marker, regardless of aetiology. the results of this study have helped to better equip the laboratory to inform and promote more clinically led and evidence-based requesting practices amongst local clinicians. whilst these pancreatic enzyme biomarkers can provide an important tool for clinicians trying to detect or eliminate an ap diagnosis, it is also important for requestors to have a full understanding of the potential limitations associated with them. a prompt diagnosis of ap is important for limiting associated complications if left undetected and ultimately improving patient outcomes. conclusion the results of this clinical audit support a growing body of evidence that lipase is superior as a first-line test for suspected ap and that there is little additional clinical value derived from dual requesting of lipase and amylase. despite the small subset of data used within this audit, we have shown that the sensitivity and specificity of lipase and amylase was consistent with a previous study of these biomarkers within the tygerberg population, and that lipase is the superior biomarker in terms of sensitivity. it is therefore recommended that dual requests for amylase and lipase are replaced by singular lipase requests where ap is suspected. acknowledgements this study was conducted at tygerberg hospital by a.e.c. during an elective placement, the travel for which was partially funded through a biochemical society travel grant. we would like to thank mr. w. kleinhans for his help with data acquisition. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.e.z. conceived the study and a.e.z. and t.p.j. supervised a.e.c. a.e.c. and t.p.j. performed the audit and analysed the data. a.e.c. wrote the first draft and all authors contributed to the final version of this article. sources of support a.e.c. received a biochemical society (general travel grant), london, united kingdom. data availability data supporting the findings of this study are available from the corresponding author, a.e.z., on request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references banks pa, bollen tl, dervenis c, et al. classification of acute pancreatitis–2012: revision of the atlanta classification and definitions by international consensus. gut. 2013;62(1):102–111. https://doi.org/10.1136/gutjnl-2012-302779 international association of pancreatology; american pancreatic association. iap/apa evidence-based guidelines for the management of acute pancreatitis. pancreatology. 2013;13(4 suppl. 2):e1–e15. https://doi.org/10.1016/j.pan.2013.07.063 tenner s, baillie j, dewitt j, vege ss. american college of gastroenterology guideline: management of acute pancreatitis. am j gastroenterol. 2013;108(9):1400–1415. https://doi.org/10.1038/ajg.2013.218 johnson cd. uk guidelines for the management of acute pancreatitis. gut. 2005;54(suppl. 3):1–9. https://doi.org/10.1136/gut.2004.057026 chang jwy, chung ch. diagnosing acute pancreatitis: amylase or lipase? hong kong j emerg med. 2011;18(1):20–25. https://doi.org/10.1177/102490791101800104 panteghini m, bais r. serum enzymes. in: rifai n, horvath ar, wittwer ct, editors. tietz textbook of clinical chemistry and molecular diagnostics. 6th ed. maryland heights, mo: elsevier, 2018; p. 404–434. steinberg wm, buse jb, ghorbani mlm, ørsted dd, nauck ma. amylase, lipase, and acute pancreatitis in people with type 2 diabetes treated with liraglutide: results from the leader randomized trial. diabetes care. 2017;40:966–972. https://doi.org/10.2337/dc16-2747 ismail oz, bhayana v. lipase or amylase for the diagnosis of acute pancreatitis? clin biochem. 2017;50(18):1275–1280. https://doi.org/10.1016/j.clinbiochem.2017.07.003 basnayake c, ratnam d. blood tests for acute pancreatitis. aust prescr. 2015;aug;38(4):128–130. butler j. towards evidence based emergency medicine: best bets from the manchester royal infirmary edited by k mackway-jones electrical stimulation and bell’s palsy. emerg med j. 2002;19(5):428–434. https://doi.org/10.1136/emj.19.5.428 hofmeyr s, meyer c, warren bl. serum lipase should be the laboratory test of choice for suspected acute pancreatitis. s afr j surg. 2014;52(3):72–75. https://doi.org/10.7196/sajs.2003 anderson f, thomson sr, clarke dl, loots e. acute pancreatitis: demographics, aetiological factors and outcomes in a regional hospital in south africa. s afr j surg. 2008;46(3):83–86. akhtar a, sarode r, agrawal d. measuring both serum amylase and lipase for acute pancreatitis lowers quality and raises cost. cleve clin j med. 2017;84(9):670–672. https://doi.org/10.3949/ccjm.84a.16103 acb. amylase test [homepage on the internet]. lab tests online uk; 2018 [cited 2019 nov 01]. available from: https://labtestsonline.org.uk/tests/amylase-test acb. lipase test [homepage on the internet]. lab tests online uk; 2018 [cited 2019 nov 01]. available from: https://labtestsonline.org.uk/tests/lipase-test abstract introduction methods results discussion acknowledgements references about the author(s) naseem cassim department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa national priority programme, national health laboratory service, johannesburg, south africa lindi-marie coetzee department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa national priority programme, national health laboratory service, johannesburg, south africa deborah k. glencross department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa citation cassim n, coetzee l-m, glencross dk. modelling cd4 reagent usage across a national hierarchal network of laboratories in south africa. afr j lab med. 2023;12(1), a2085. https://doi.org/10.4102/ajlm.v12i1.2085 original research modelling cd4 reagent usage across a national hierarchal network of laboratories in south africa naseem cassim, lindi-marie coetzee, deborah k. glencross received: 21 sept. 2022; accepted: 15 feb. 2023; published: 15 may 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the national health laboratory service is mandated to deliver cost-effective and efficient diagnostic services across south africa. their mandate is achieved by a network of laboratories ranging from centralised national laboratories to distant rural facilities. objective: this study aimed to establish a model of cd4 reagent utilisation as an independent measure of laboratory efficiency. methods: the efficiency percentage was defined as finished goods (number of reportable results) over raw materials (number of reagents supplied) for 47 laboratories in nine provinces (both anonymised) for 2019. the efficiency percentage at national and provincial levels was calculated and compared to the optimal efficiency percentage derived using pre-set assumptions. comparative laboratory analysis was conducted for the provinces with the best and worst efficiency percentages. the possible linear relationship between the efficiency percentage and call-outs, days lost, referrals, and turn-around time was assessed. results: data are reported for 2 806 799 cd4 tests, with an overall efficiency percentage of 84.5% (optimal of 84.98%). the efficiency percentage varied between 75.7% and 87.7% between provinces, while within the laboratory it ranged from 66.1% to 111.5%. four laboratories reported an efficiency percentage ranging from 67.8% to 85.7%. no linear correlation was noted between the efficiency percentage, call-outs, days lost, and turn-around time performance. conclusion: reagent efficiency percentage distinguished laboratories into different utilisation levels irrespective of their cd4 service level. this parameter is an additional independent indicator of laboratory performance, with no relationship with any contributing factors tested, that can be implemented across pathology disciplines for monitoring reagent utilisation. what this study adds: this study provides an objective methodology to assess reagent utilisation as an independent measure of laboratory efficiency. this model could be applied to all routine pathology services. keywords: hiv; cd4; efficiency; reagents; laboratory. introduction the national health laboratory service (nhls) provides essential laboratory testing to the public health sector in south africa.1 the organisation is mandated to provide cost-effective, reliable diagnostics to all public health facilities, irrespective of size, location, or level of complexity.1 a network of well-placed laboratories, ranging from highly sophisticated and centralised academic facilities to small rural, hospital-based laboratories, provide essential services1 for both communicable (like hiv and tuberculosis) and non-communicable diseases (cancer, diabetes, and cardiovascular diseases).1 laboratory services support a vast testing repertoire, but this study focused on hiv-related diagnostics prescribed by the world health organization and the national treatment guidelines.2,3 people living with hiv are eligible for antiretroviral therapy irrespective of age, cd4 cell count, and clinical stage,2 and it is recommended that antiretroviral therapy is initiated within seven days if there are no clinical contraindications.2 laboratory services play a key role in the baseline clinical evaluation that includes confirming hiv status, screening for tuberculosis, cryptococcal disease, renal insufficiency and determining cd4 count (to assess immune status), among others.2 once patients are on antiretroviral therapy, routine hiv viral load monitoring is required, with cd4 monitoring only recommended for patients on cotrimoxazole preventive therapy.2 cd4 services in south africa are offered through an integrated tiered service delivery model that aims to extend coverage, improve turn-around time (tat), and contain programmatic costs.4,5 since the implementation of the integrated tiered service delivery model, multiple remote areas with existing community laboratories that previously referred samples have been upgraded to include cd4 services, such as in de aar, upington, and tshwaragano. the local cd4 testing in the newly established remote laboratories consequently reduced cd4 testing referrals, decentralising cd4 services, and caused a dramatic reduction in the tat of cd4 test results.6 one way of monitoring instrument performance is through the assessment of tat. turn-around time assessment measures and monitors laboratory testing efficiency and result delivery to support patient care. thus, substantial emphasis is placed on laboratory tat, with each test having a predetermined cut-off target to ensure accurate testing and timely results. various local studies reported a substantial improvement in tat performance across the network of cd4 testing facilities, with continuous monitoring through a weekly tat dashboard ensuring timely intervention where the tat target is unmet.7,8,9,10 for laboratories to meet their tat targets, they must streamline all aspects of their workflow; this includes optimising sample preparation and reagent use. effective reagent utilisation notably saves time and money and can be used as an additional measure of laboratory performance and efficiency. less efficient laboratories can be identified by assessing laboratory cd4 reagent utilisation, corrective practices implemented, and streamlined workflow processes introduced. furthermore, as all cd4 testing procedures are standardised across the nhls, including the adoption of good laboratory practice principles, practices of effective reagent utilisation can offer additional value in the quest for better service efficiencies. ‘six sigma’ is a quality management strategy that aims to improve the quality of processes and focuses on identifying and removing defects.11,12 this strategy includes ‘lean’, a methodology used to improve the efficiency of clinical laboratory procedures.11,12 one of the key factors is to identify the value stream and remove wastage.13 in the delivery of any efficient service, lean manufacturing concepts include the flow of raw material, work in progress, and finished goods, while in the case of this study, laboratory staff, analysers, and information elucidate efficiency.13,14 in a pathology setting, the flow of raw materials relates to the supply of test reagents and associated consumables required to perform a specific test. finished goods in a laboratory environment refer to the verified result delivered to the healthcare worker or originator of the test request. to produce a result, the laboratory requires trained staff to conduct testing on appropriate analysers before test results are verified on the laboratory information system (lis) for reporting. the study assessed laboratories’ optimal use of cd4 test reagents to identify laboratories that need to minimise wastage by improving and refining their workflow. the laboratories utilised national, standardised analyser platforms and protocols (standard operating procedures) and a national lis. this study aimed to model cd4 reagent use across a national network of laboratories, using data for the 2019 calendar year in south africa. a secondary objective was to review national and provincial efficiency of reagent use and reagent usage at the individual laboratory level. in laboratories where efficiency was suboptimal, a further objective was to assess whether there was any correlation between their reagent efficiency percentage and other service efficiency indicators. these service indicators included the number of service call-outs during instrument failure, days lost (as a consequence of instrument downtime), delays due to referrals (where samples are sent to sister laboratories for testing during instrument downtime), and tat performance of the affected laboratory. methods ethical considerations ethical clearance for this study was obtained from the university of the witwatersrand (m220163). only aggregate secondary laboratory data were used; our study did not require the use of any patient identifiers. all specific province and individual laboratory identifiers were removed, and sites were anonymised for this study. the human research ethics committee did not require patient consent. study design the cross-sectional study design assessed the cd4 reagent utilisation of 47 nhls laboratories in south africa for the 2019 calendar year. cd4 testing was performed on the beckman coulter fc500 mpl/cellmek and aquios cl cytometers (beckman coulter, miami, florida, united states) during the studied period. beckman coulter supplied all the cd4 reagents for both cytometers per the national tender agreement.15,16 irrespective of the instrument used, all laboratories utilised the same cd4 panleucogating reagents and national standard operating procedures.15 optimal efficiency percentage calculation the optimal efficiency percentage per 100 tests was calculated using over 18 years of cd4 testing data (from 2004) of the national priority programme.17,18 to determine the optimal efficiency percentage, we assumed an 8-h working day and a 5-day testing week and included error rates, the number of controls used (which count as one test per control tested), days lost (due to instrument downtime), and other indicators (such as electricity outages). data extraction beckman coulter (miami, florida, united states) provided aggregate data on raw materials (test reagent kits) delivered to each laboratory for 2019, and the number of tests provided from each delivery was estimated by multiplying the number of kits delivered by 300 (the number of tests per kit). the variables for this extract included: (1) reagent item number (the company’s reagent kit product identifier), (2) ‘ship-to location’ used for supply chain management (which indicates the laboratory that received the kits), and (3) number of kits delivered. in addition, beckman coulter also provided the number of service call-outs for each laboratory during 2019. in this study, finished goods were defined as the number of cd4 results authorised by qualified technical personnel on the lis (for this study, this process is termed ‘review’). the reported test volumes, identified per laboratory, were extracted by the corporate data warehouse. the specimen-level cd4 variables reported were: (1) episode number, (2) testing laboratory, (3) source laboratory, (4) province, (5) review date, (6) tat (in hours), and (7) referral status (referred or not referred). the source laboratory may not routinely offer cd4 services and would have to refer samples to a testing facility. data preparation the beckman coulter ‘ship-to location’ was matched to the respective equivalent-identified corporate data warehouse testing laboratory, for example ‘bongani hospital’ with ‘welkom’. the raw and finished materials were used to calculate the efficiency percentage (finished goods/raw materials). the analysis was conducted using microsoft excel (microsoft corp., redmond, washington, united states). all provinces and laboratories have been anonymised, for example laboratory 1 (p3), to ensure confidentiality. data analysis the following indicators were reported for each laboratory: (1) efficiency percentage, (2) number of call-outs (calls logged with the supplier helpdesk), (3) days lost, (4) sample referral percentage, (5) samples meeting tat cut-off percentage, and (6) capacity utilisation. microsoft excel was used for the capacity utilisation by calculating the throughput, for example 120 samples per 8-h shift for the aquois flow cytometer, and the test volumes performed for a defined period.15 for this analysis, we divided the annual test volumes by annual capacity, assuming hours worked and the number and type of flow cytometer platform used per laboratory. for each laboratory, the total number of call-outs logged with the beckman coulter call centre was reported. call-outs were categorised as field mentoring, field service visits, or modification type 2 (software or hardware update) reports. in consultation with beckman coulter, call-outs related to non-essential support from the supplier, including activities such as courtesy visits or initial instrument installation, were not deemed to impact service delivery and efficiency and were disregarded. daily laboratory test volumes were analysed to determine the total number of days lost. a lost day was defined as a weekday (tuesday to friday) where fewer than 15 tests were performed. test volumes over weekends, public holidays and mondays were excluded from this analysis.19 monday test volumes were excluded because, historically, there are limited hiv services on this day, resulting in low test volumes.19 the daily test volumes were coded using this threshold as 1 or 0 to determine the number of lost days. the laboratory data were used to determine the percentage of samples that were referred (where the source and testing laboratory were different in the lis). a referral was defined as a sample originating from a different laboratory from where it was tested. referrals involve the transportation of samples with delays that could affect sample stability. all references to referrals in the manuscript relate to the percentage of referred samples. a key laboratory efficiency report is the tat performance for various tests.20 turn-around time assesses laboratory service delivery speed, reliability, and efficiency.20 the nhls agrees to an annual performance plan with the national department of health, which includes key outcomes and outputs.20 the optimal national cd4 reporting tat target for 2019 was 90% of samples tested within 40 h. this tat allows timely baseline laboratory investigations to ensure antiretroviral therapy initiation within seven days.2 all laboratories within the national cd4 network are required to meet the organisation’s cd4 tat targets, which are monitored by measuring the percentage of samples that any given laboratory reports within the 40-h window. for this study, this measurement was also used and is shown as the percentage of samples that met the tat cut-off of 40 h, referred to as tat performance, for comparison to other efficiency parameters reported. a microsoft excel bubble chart was used to report the reagent efficiency percentage, the percentage of samples that met the organisational tat target, and annual raw material cost (in united states dollars). laboratory performance was then categorised as a scatter plot reporting the reagent use efficiency by tat performance. to assess any linear relationship to the percentage reagent efficiency and call-outs, days lost, referrals, and tat performance scatter plots were generated for all laboratories using stata® se (statacorp llc, college station, texas, united states). pairwise correlation coefficients between these variables were also determined. for the top and bottom performing provinces (with the lowest and highest percentage reagent efficiency), the following parameters were tabled for each testing laboratory within these provinces: (1) efficiency percentage, (2) number of call-outs, (3) days lost, (4) percentage of referrals, and (5) percentage of samples that met the tat cut-off. an analysis of the instrument capacity utilisation for these laboratories was also conducted. results we report the data for 2 806 799 cd4 tests performed in 2019 across 47 testing laboratories. there are nine provinces with 3–10 testing laboratories per province. optimal efficiency percentage we assumed an error rate of 8%, four controls per day, 4 days lost per year and 3% for other interruptions for an optimal efficiency percentage. the days lost were calculated using 50 weeks and five working days per week. two weeks were excluded to account for annual public holidays.21 thus, the calculated optimal efficiency percentage was 84.98% (table 1), and indicates that for a typical kit of 300 tests, each laboratory, in an optimal setting, should be able to produce 254 reportable cd4 results. the remaining 15% (46 tests per kit) accounts for other aspects such as daily controls, error rates, and other testing interruptions (i.e. power outages or instrument downtime). table 1: assumptions and calculation of the optimal cd4 reagent efficiency percentage, south africa, 2019. national and provincial analysis a national efficiency percentage of 84.5% was reported across all cd4 laboratories during 2019 (data not shown). the provincial analysis revealed that 53.7% of all finished materials (cd4 results) were generated by two provinces (data not shown). the provincial efficiency percentage ranged from 75.7% (p8) to 87.7% (p2) (figure 1). only two provinces reported an efficiency percentage of ≤ 80% (p8: 75.7% and p7: 79.9%). the percentage of samples that met the tat target ranged from 87.2% (p6) to 98.0% (p7). figure 1: bubble chart assessing provincial cd4 reagent utilisation of nine provinces in south africa for 2019. the efficiency percentage, percentage of samples that met the turn-around time target, and raw materials cost (united states dollars) are reported on the x-axis, y-axis, and as bubble size. laboratory reagent efficiency the laboratory efficiency percentages ranged from 66.1% (laboratory 13 [p3]) to 104.5% (laboratory 22 [p2]) (figure 2). other indicators of laboratory efficiency also varied; the percentage of samples that met the stipulated organisational cd4 tat cut-off varied from 79.5% (laboratory 19 [p4]) to 99.0% (laboratory 3 [p7]). the days lost to testing ranged from 1 day (laboratory 1 [p3]) to 52 days (laboratory 22 [p2]) (figure 2). overall, 21/47 laboratories met the optimal efficiency percentage target (44.7%) compared to 40/47 for tat performance (85.1%). quadrants a and c met the national stipulated tat target (> 90% of reported cd4 results with 40 h). quadrant a (comprising 36.2% or 17/47 laboratories) showed efficient reagent use (≥ 84.98%), while quadrant c (comprising the majority of laboratories, i.e. 53.2% or 25/47 laboratories) showed less efficient reagent usage (< 84.98%). quadrants b and d did not meet the stipulated tat; however, quadrant b (8.5% or 4/47 laboratories) showed efficient use of reagents ≥ 84.98%, while quadrant d (2.1% or 1/47 laboratories) reported < 84.98% reagent efficiency. figure 2: cd4 reagent utilisation of 47 laboratories across nine provinces in south africa for 2019. correlation of reagent efficiency percentage to other factors the scatter plot analysis for all laboratories did not reveal a linear relationship between efficient reagent use and call-outs, days lost, referrals, and tat performance (figure 3). a pairwise correlation coefficient of −1 or +1 would indicate a perfect linear relationship15; however, values of −0.0770, −0.0151, 0.0853 and −0.2596 were reported for these test parameters, confirming very weak correlation (values very close to 0). figure 3: correlation of cd4 reagent use efficiency of 47 laboratories in south africa for 2019. efficiency is plotted with (a) the number of call-outs, (b) the number of days lost, (c) the percentage of referrals, and (d) turn-around time performance. detailed analysis of individual laboratory efficiency for the top and bottom provinces the laboratories in the bottom province (p8) had an efficiency percentage ranging from 67.8% to 85.7% (table 2). only one laboratory in this province met the optimal efficiency percentage (laboratory 20). the number of call-outs for these laboratories varied from 42 to 64; however, days lost to service delivery in these same four sites ranged between 2 and 4 days. the percentage of referred samples tested in these sites also varied from 23.4% to 41.0%. conversely, all four laboratories had tat values ranging from 95.8% to 97.2%. the instrument capacity utilisation was below 85% for all testing sites (table 2). table 2: comparison of the laboratories with highest and lowest cd4 reagent use efficiency, south africa, 2019. in the best-performing province (p2), the reagent use efficiency percentage ranged from 80.7% to 104.9% across three laboratories. a range of 25–34 call-outs and 4–52 days lost was reported. only laboratory 22 reported a ≥ 90% tat performance. instrument capacity utilisation was under 62% for this province. discussion the work investigated how effectively cd4 laboratories used reagents to deliver cd4 services in south africa. the outcomes revealed that the national cd4 network reported a cd4 efficiency percentage of 84.5%, ranging from 75.7% to 87.7% at the provincial level. individual laboratory efficiency percentage went as low as 66.1%. the averaged national performance masked less efficient reagent use at provincial and laboratory levels, evident in the variability of efficiency percentages reported for provinces and laboratories. several laboratories had efficiency percentages below 18.9% of the calculated optimal reagent use percentage. thus, it is essential to assess efficiency at the decentralised level to identify the less efficient performers for appropriate corrective action and intervention. similar masking of laboratory performance was reported for tat in the cd4 testing network.10 one interesting observation was a laboratory with an efficiency reagent use of over 100%. this aberration indicates that some reagents from the previous year may have been used in 2019 (for example, careful provision for contingency and reagents for the 2018 holiday period, which was then used in 2019). diligent stock control will assist in standardising contingency reagent stocks held in laboratories. only 21 sites met the calculated optimal reagent efficiency percentage, indicating that perhaps the optimal efficiency percentage was too optimistic and failed to factor in laboratory testing volume differences. for example, a site with much higher cd4 processing volumes must perform relatively more control tests each day than a site with lower test volumes or that work only an 8-h shift versus two or three 8-h shifts. the detailed analysis for the top and bottom provinces also suggests that in addition to the standardised test platform, the workflow and stock management procedures need to be standardised across laboratories to ensure best practices. other contributing variables that could have affected the laboratory efficiency percentage include varying staff capacity (staff numbers) and expertise. these aspects are especially difficult to assess in smaller, community-type laboratories that typically employ multi-tasking rotations across pathology disciplines to deliver essential pathology services.4,6,7 instrument log data files were unavailable at the time of this study. it was, therefore, impossible to assess the number of repeated tests run, the frequency of controls performed (especially as a proportion of the total volume of tests performed), and the number of invalid runs, all of which could contribute to the lower percentage efficiency, especially in smaller cd4 volume laboratories. this study, however, highlights the value of instrument log data files and the need to collate this information on an organisation’s central data server to facilitate near real-time analysis, including reagent utilisation. other information, including days lost, duplicated testing, and the number of controls used, could also be logged and centrally monitored to identify more systemic problems in laboratories. here, instrument-collected data would be invaluable to identify practices employed in the better-performing laboratories that could be standardised across all testing sites. such an approach would make it easier to introduce corrective interventions and pair provinces with a poor efficiency percentage to their better-performing sister sites. ultimately all efficiency parameters mentioned in this article should ideally be monitored at a centralised level by a national overseeing, harmonising body that uses instrument logs and other relevant data to report on national service efficiency and performance.4,6,7 a comprehensive monitoring and evaluation plan should be developed that stipulates the indicators to be reported. the development of appropriate dashboards reporting reagent utilisation efficiency is needed to monitor performance for continual improvement of the national laboratory system. furthermore, this reagent efficiency model could be extended to other tests across a national laboratory network to improve reagent efficiency across all pathology disciplines.8,9 to accomplish this, a national lis is key to generating the data for real-time monitoring. well-narrated data provide far more granularity than aggregate data systems22 and ensure that data are continually visible from the national to the laboratory level for all tests. in our context, all laboratory data is stored in a data warehouse environment with massive server capacity for all tests in the national network.22 further investigation is required to understand how reagents are used in cd4 laboratories with higher and lower volumes to improve overall reagent usage. interestingly, the lack of linear correlation between reagent usage and other efficiency parameters, including call-outs, days lost, referrals, and tat performance, could indicate more systemic laboratory problems. although beyond the scope of this study, detailed on-site inspection at each laboratory, with full audits of procedures and practices, could address some of these questions. limitations a limitation of this study was the absence of instrument log file data. unfortunately, these data are stored on a local instrument-linked computer. in addition, the optimal efficiency percentage calculated was based on averages of laboratories studied with the assumptions stated in the methods section; outlines suggest this calculated percentage may have been too optimistic and requires further workflow studies. conclusion we reported a model of reagent efficiency across a pathology network with various service tiers. the work emphasises the importance of review at all hierarchical tiers of service, in this instance at the provincial or individual laboratory level, to detect masked poorer levels of efficiency and areas for corrective action. there was no correlation between the reagent use efficiency with days lost, call-outs, referrals, and tat performance, suggesting that although reagent usage and instrument function are both aspects that contribute to overall laboratory efficiency, these are separate issues that need to be monitored individually. underlying factors contributing to reagent wastage should be investigated to inform waste awareness strategies and standardised procedures to minimise missed diagnostic opportunities and cost implications. real-time monitoring of reagent use through instrument log data files can save costs, which is especially relevant in an expenditure-constrained setting. acknowledgements the authors would like to thank beckman coulter for providing expenditure data, and silence ndlovu of the national priority programme of the nhls for extracting the laboratory test volumes. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions n.c. and l.-m.c. developed and executed the research, conducted the data analysis, prepared the first draft and provided editorial input. d.k.g. gave editorial input and was the project leader. sources of support the authors received no financial support for the research, authorship, or publication of this article. data availability the authors do not have permission to share the laboratory data. disclaimer the views expressed in this manuscript are those of the authors and not those of the university of the witwatersrand or the nhls. references national health laboratory service (nhls). annual report 2018/19 johannesburg, south africa [homepage on the internet]: national health laboratory service (nhls). 2019 [cited 2020 feb 19]. available from: https://www.nhls.ac.za/key-documents/annual-reports/ national department of health (ndoh). art clinical guidelines for the management of hiv in adults, pregnancy, adolescents, children, infants and neonates pretoria, south africa [homepage on the internet]: national department of health (ndoh). 2019 [cited 2020 jan 18]. available from: https://www.knowledgehub.org.za/system/files/elibdownloads/2020-05/2019%20art%20guideline%2028042020%20pdf.pdf world health organization (who). consolidated hiv guidelines for prevention, treatment, service delivery & monitoring: recommendations for a public health approach [homepage on the internet]. 2021 [cited 2022 aug 17]. available from: https://apps.who.int/iris/rest/bitstreams/1357089/retrieve glencross dk, coetzee lm, cassim n. an integrated tiered service delivery model (itsdm) based on local cd4 testing demands can improve turn-around times and save costs whilst ensuring accessible and scalable cd4 services across a national programme. plos one. 2014;9(12):e114727. https://doi.org/10.1371/journal.pone.0114727 cassim n, coetzee lm, schnippel k, glencross dk. estimating implementation and operational costs of an integrated tiered cd4 service including laboratory and point of care testing in a remote health district in south africa. plos one. 2014;9(12):e115420. https://doi.org/10.1371/journal.pone.0115420 coetzee lm, cassim n, glencross dk. implementation of a new ‘community’ laboratory cd4 service in a rural health district in south africa extends laboratory services and substantially improves local reporting turn-around time. s afr med j. 2015;106(1):82–87. https://doi.org/10.7196/samj.2016.v106i1.10081 coetzee lm, cassim n, glencross dk. newly implemented community cd4 service in tshwaragano, northern cape province, south africa, positively impacts result turn-around time. afr j lab med. 2022;11(1):1376. https://doi.org/10.4102/ajlm.v11i1.1376 cassim n, coetzee lm, tepper mee, perelson l, glencross dk. timely delivery of laboratory efficiency information, part ii: assessing the impact of a turn-around time dashboard at a high-volume laboratory. afr j lab med. 2020;9(2):948. https://doi.org/10.4102/ajlm.v9i2.948 cassim n, tepper me, coetzee lm, glencross dk. timely delivery of laboratory efficiency information, part i: developing an interactive turn-around time dashboard at a high-volume laboratory. afr j lab med. 2020;9(2):947. https://doi.org/10.4102/ajlm.v9i2.947 coetzee l-m, cassim n, glencross dk. weekly laboratory turn-around time identifies poor performance masked by aggregated reporting. 2020;9(1):a1102. https://doi.org/10.4102/ajlm.v9i1.1102 inal tc, goruroglu ozturk o, kibar f, cetiner s, matyar s, daglioglu g, et al. lean six sigma methodologies improve clinical laboratory efficiency and reduce turn-around times. j clin lab anal. 2018;32(1):e22180. https://doi.org/10.1002/jcla.22180 montgomery dc, woodall wh. an overview of six sigma. int stat rev. 2008;76(3):329–346. https://doi.org/10.1111/j.1751-5823.2008.00061.x hayes kj, reed n, fitzgerald a, watt v. applying lean flows in pathology laboratory remodelling. j health organ manag. 2014;28(2):229–246. https://doi.org/10.1108/jhom-03-2013-0064 black jr. lean production: implementing a world-class system new york, usa [homepage on the internet]: industrial press incorporated; 2008 [cited 2021 april 20]; p. 189. available from: https://books.industrialpress.com/9780831133511/lean-production/ coetzee lm, glencross dk. performance verification of the new fully automated aquios flow cytometer panleucogate (plg) platform for cd4-t-lymphocyte enumeration in south africa. plos one. 2017;12(11):e0187456. https://doi.org/10.1371/journal.pone.0187456 national health laboratory service (nhls). national cd4 count testing programme [homepage on the internet]. 2022 [cited 2022 aug 18]. available from: https://www.nhls.ac.za/priority-programmes/cd4/ glencross d. dual platform panleucogated (plg) cd4+ t cell enumeration. afford cd4. com; 2002. national health laboratory service (nhls). priority programmes [homepage on the internet]. 2022 [cited 2022 dec 19]. available from: https://www.nhls.ac.za/priority-programmes/ drury s, coetzee lm, cassim n, carmona s, stevens ws, glencross dk. using central data warehouse (cdw) reports for monitoring cd4 laboratory workload and related turn-around-time (tat) cape town, south africa [homepage on the internet]. 2012 [cited 2022 aug 20]. available from: https://www.researchgate.net/publication/267756209_using_central_data_warehouse_cdw_reports_for_monitoring_cd4_laboratory_workload_and_related_turn-_around-time_tat?channel=doi&linkid=5459d3fb0cf2cf516483e354&showfulltext=true national health laboratory service (nhls). annual performance plan 2020–2021 johannesburg, south africa [homepage on the internet]: national health laboratory service (nhls). 2020 [cited 2020 feb 19]. available from: https://www.parliament.gov.za/storage/app/media/docs/tpap/642703b7-8ff7-48c8-a758-d4a4292b6b28.pdf the republic of south africa. the public holidays act 36 of 1994 cape town, south africa [homepage on the internet]: republic of south africa; 1994 [cited 2022 aug 19]. available from: https://www.gov.za/sites/default/files/gcis_document/201409/act36of1994.pdf stevens ws, cunningham b, cassim n, gous n, scott le. cloud-based surveillance, connectivity, and distribution of the genexpert analyzers for diagnosis of tuberculosis (tb) and multiple-drug-resistant tb in south africa. mol microbiol 2016:707–718. ajlm 11(1)_2022_contents.indd http://www.ajlmonline.org open access table of contents lessons from the field covid-19 mass testing and sequencing: experiences from a laboratory in western kenya john n. waitumbi, esther omuseni, josphat nyataya, clement masakhwe, faith sigei, allan lemtudo, george awinda, eric muthanje, brian andika, rachel githii, rehema liyai, gathii kimita, beth mutai african journal of laboratory medicine | vol 11, no 1 | a1737 | 22 july 2022 lessons from the field tuberculosis-loop-mediated isothermal amplification implementation in cameroon: challenges, lessons learned and recommendations valerie f. donkeng-donfack, suzanne m. ongoulal, yvonne j. djieugoue, yannick kamdem simo, henri manga, danielle a.d. tollo, edwige m.a. belinga, vincent mbassa, jean l. abena, sara eyangoh african journal of laboratory medicine | vol 11, no 1 | a1792 | 26 august 2022 review article beta-lactamase resistance genes in enterobacteriaceae from nigeria babafela b. awosile, michael agbaje, oluwawemimo adebowale, olugbenga kehinde, ezekiel omoshaba african journal of laboratory medicine | vol 11, no 1 | a1371 | 22 february 2022 review article microbiology laboratories involved in disease and antimicrobial resistance surveillance: strengths and challenges of the central african states passoret vounba, severin loul, ludovic f. tamadea, joël f.d. siawaya african journal of laboratory medicine | vol 11, no 1 | a1570 | 31 march 2022 review article low-level viraemia: an emerging concern among people living with hiv in uganda and across sub-saharan africa nicholus nanyeenya, noah kiwanuka, damalie nakanjako, gertrude nakigozi, simon p.s. kibira, susan nabadda, charles kiyaga, isaac sewanyana, esther nasuuna, fredrick makumbi african journal of laboratory medicine | vol 11, no 1 | a1899 | 20 october 2022 original research impact of pre-covid-19 epidemic preparedness on the trajectory of the pandemic in african countries talkmore maruta, sikhulile moyo african journal of laboratory medicine | vol 11, no 1 | a1571 | 31 march 2022 original research prevalence of alpha and beta haemolysin among blood group o donors in bamenda, cameroon victor n. fondoh, nobert ndzenjempuh, tamunjoh stella, richard m. fondoh, charles n. awasom, rebecca enow-tanjong, egbe p. egbengu, robert leke, njini f.n. rose, denis nsame african journal of laboratory medicine | vol 11, no 1 | a1432 | 19 april 2022 original research red cell distribution width as a surrogate marker of haemoglobinopathies in western kenya benard m. mutua, george sowayi, patrick okoth african journal of laboratory medicine | vol 11, no 1 | a1644 | 29 april 2022 36 42 49 59 69 74 83 89 page i of iv table of contents editorial cop27 climate change conference: urgent action needed for africa and the world lukoye atwoli, gregory e. erhabor, aiah a. gbakima, abraham haileamlak, jean-marie kayembe ntumba, james kigera, laurie laybourn-langton, robert mash, joy muhia, fhumulani m. mulaudzi, david ofori-adjei, friday okonofua, arash rashidian, maha el-adawy, siaka sidibé, abdelmadjid snouber, james tumwine, sahar yassien mohammad, paul yonga, lilia zakhama, chris zielinski african journal of laboratory medicine | vol 11, no 1 | a2080 | 04 november 2022 editorial point-of-care testing: connecting communities in africa and ensuring equity in access to health and diagnostics rajiv t. erasmus african journal of laboratory medicine | vol 11, no 1 | a2072 | 13 december 2022 opinion paper challenges and complexities in evaluating severe acute respiratory syndrome coronavirus 2 molecular diagnostics during the covid-19 pandemic lesley e. scott, lara d. noble, ashika singh-moodley, trish kahamba, diana r. hardie, wolfgang preiser, wendy s. stevens african journal of laboratory medicine | vol 11, no 1 | a1429 | 26 april 2022 opinion paper to break the cycle of covid-19 lockdowns and safely open up economies, we must ensure equitable access to diagnosis and treatment nqobile ndlovu, yenew k. tebeje, renuka gadde, trevor peter african journal of laboratory medicine | vol 11, no 1 | a1650 | 27 may 2022 opinion paper the time to address diagnostic needs in universal health coverage is now: leveraging the scale up of national testing capacity for hiv viral load and sars-cov-2 francesco marinucci, jens dhein african journal of laboratory medicine | vol 11, no 1 | a1685 | 22 june 2022 lessons from the field strengthening laboratory networks in the central africa region: a milestone for epidemic preparedness and response patrick a. njukeng, charles njumkeng, callistus ntongowa, mohammed abdulaziz african journal of laboratory medicine | vol 11, no 1 | a1492 | 19 may 2022 lessons from the field practical tips to using formalin-fixed paraffin-embedded tissue archives for molecular diagnostics in a south african setting barbara s. van deventer, lorraine du toit-prinsloo, chantal van niekerk african journal of laboratory medicine | vol 11, no 1 | a1587 | 23 june 2022 lessons from the field challenges with the pursuit of iso 15189 accreditation in a public health laboratory in ghana seth attoh, francis k.m. tetteh, mary mcaddy, kingsley ackah, richmond kyei, marcus moroti, cynthia boateng, laurinda adusu-donkor, joseph boafo, alhassan yakubu, sarah kwao, emmanuel sarkodie, nana-banyin koranteng, monica a. addo, frederick hobenu, kwasi agyeman-bediako, raymond d. fatchu african journal of laboratory medicine | vol 11, no 1 | a1448 | 19 july 2022 1 4 6 11 14 18 23 29 vol 11, no 1 (2022) issn: 2225-2002 (print) | issn: 2225-2010 (online)african journal of laboratory medicine http://www.ajlmonline.org open access table of contents original research appropriate use of plasma glucose tests for diagnosis of diabetes mellitus in ibadan, nigeria modupe a. kuti, olabisi t. bamidele, chioma t. udeh, bola j. eseile, olajumoke a. ogundeji african journal of laboratory medicine | vol 11, no 1 | a1433 | 29 april 2022 original research high-risk human papillomavirus-associated vulvar neoplasia among women living with human immunodeficiency virus in zambia fred maate, peter julius, stepfanie siyumbwa, leeya pinder, trevor kaile, mulindi mwanahamuntu, groesbeck parham african journal of laboratory medicine | vol 11, no 1 | a1563 | 12 may 2022 original research diagnostic challenges with accurate identification of listeria monocytogenes isolates from food and environmental samples in south africa teena s.m. thomas, juno thomas, karren le roux, sanelisiwe t. duze, faith mkhwanazi, adriano duse african journal of laboratory medicine | vol 11, no 1 | a1482 | 23 may 2022 original research south african study of blast phase chronic myeloid leukaemia: a poor prognostic outlook katherine e. hodkinson, nikki bouwer, jenifer vaughan african journal of laboratory medicine | vol 11, no 1 | a1578 | 31 may 2022 original research enhancing accreditation outcomes for medical laboratories on the strengthening laboratory management toward accreditation programme in kenya via a rapid results initiative ernest p. makokha, raphael o. ondondo, daniel k. kimani, thomas gachuki, frank basiye, mercy njeru, muthoni junghae, marie downer, mamo umuro, margaret mburu, jane mwangi african journal of laboratory medicine | vol 11, no 1 | a1614 | 31 may 2022 original research aetiology of pancytopenia: experience of a south african tertiary academic centre erica-mari nell, zivanai c. chapanduka african journal of laboratory medicine | vol 11, no 1 | a1645 | 31 may 2022 original research newly implemented community cd4 service in tshwaragano, northern cape province, south africa, positively impacts result turn-around time lindi-marie coetzee, naseem cassim, deborah k. glencross african journal of laboratory medicine | vol 11, no 1 | a1376 | 03 june 2022 original research childhood aplastic anaemia with paroxysmal nocturnal haemoglobinuria clones: a retrospective single-centre study in south africa candice l. hendricks, ashen naidoo, rajendra thejpal, nadine rapiti, beverley neethling, yasmin goga, suvarna buldeo african journal of laboratory medicine | vol 11, no 1 | a1537 | 06 june 2022 original research implementation of a customised antimicrobial resistance laboratory scorecard in cameroon, ethiopia and kenya andré trollip, renuka gadde, tjeerd datema, kamau gatwechi, linda oskam, zachary katz, andrew whitelaw, peter kinyanjui, patrick njukeng, dawit a. wendifraw, ibrahimm mugerwa, grace najjuka, nicholas dayie, japheth a. opintan, heidi albert african journal of laboratory medicine | vol 11, no 1 | a1476 | 20 june 2022 97 102 112 118 125 133 141 150 157 original research the application of sigma metrics in the laboratory to assess quality control processes in south africa marli van heerden, jaya a. george, siyabonga khoza african journal of laboratory medicine | vol 11, no 1 | a1344 | 22 june 2022 original research delays in hiv-1 infant polymerase chain reaction testing may leave children without confirmed diagnoses in the western cape province, south africa kamela l. mahlakwane, wolfgang preiser, nokwazi nkosi, nasheen naidoo, gert van zyl african journal of laboratory medicine | vol 11, no 1 | a1485 | 23 june 2022 original research the togo national proficiency test pilot programme for basic clinical chemistry tests kafui c. kouassi, améyo m. dorkenoo, komivi gbada, yaovi-gameli afanyibo, minogblon têko, adjane koura african journal of laboratory medicine | vol 11, no 1 | a1565 | 24 june 2022 original research implementing an intensified mentorship approach towards accelerated medical laboratory accreditation in 10 counties in kenya susan k. musau, christina mwachari, elvis kirui, junghae muthoni, taylor lascko, natalia blanco, alash’le abimiku, emily koech african journal of laboratory medicine | vol 11, no 1 | a1814 | 01 july 2022 original research cost of running a full-service receiving office at a centralised testing laboratory in south africa naseem cassim, neeshan ramdin, sadhaseevan moodly, deborah k. glencross african journal of laboratory medicine | vol 11, no 1 | a1504 | 13 july 2022 original research evaluation of fixed-panel, multicolour clearllab 10c at an academic flow cytometry laboratory in johannesburg, south africa deborah k. glencross, leanne swart, melanie pretorius, denise lawrie african journal of laboratory medicine | vol 11, no 1 | a1458 | 15 july 2022 original research formulation of phage cocktails and evaluation of their interaction with antibiotics in inhibiting carbapenemase-producing klebsiella pneumoniae in vitro in kenya noutin f. michodigni, atunga nyachieo, juliah k. akhwale, gabriel magoma, abdoul-salam ouédraogo, andrew n. kimang’a african journal of laboratory medicine | vol 11, no 1 | a1803 | 18 july 2022 original research occurrence and antimicrobial susceptibility patterns of salmonella species from poultry farms in ibadan, nigeria terese g. orum, olayinka o. ishola, oluwawemimo o. adebowale african journal of laboratory medicine | vol 11, no 1 | a1606 | 20 july 2022 original research therapeutic drug monitoring of phenytoin and valproic acid in critically ill patients at windhoek central hospital, namibia bonifasius s. singu, helen morrison, lydia irengeya, roger k. verbeeck african journal of laboratory medicine | vol 11, no 1 | a1628 | 21 july 2022 166 173 180 186 192 200 210 218 224 page ii of iv http://www.ajlmonline.org open access table of contents original research prevalence of hepatitis b virus core antibodies among blood donors in nigeria: implications for blood safety foluke a. fasola, adeola a. fowotade, adedayo o. faneye, adeyeni adeleke african journal of laboratory medicine | vol 11, no 1 | a1434 | 26 july 2022 original research efficacy of diversely isolated lytic phages against multi-drug resistant enterobacter cloacae isolates in kenya ivy j. mutai, angela a. juma, martin i. inyimili, atunga nyachieo, anthony k. nyamache african journal of laboratory medicine | vol 11, no 1 | a1673 | 11 august 2022 original research establishment of haemoglobin a 2 reference intervals in pretoria, south africa: a retrospective secondary data analysis cailin nieuwenhuizen, tshiphiri netshidzivhani, johan potgieter african journal of laboratory medicine | vol 11, no 1 | a1841 | 12 august 2022 original research hepatitis b virus infection and hbeag positivity among pregnant women in south west uganda naome mugabiirwe, rogers kalyetsi, richard ayella, james obote, frank ssedyabane african journal of laboratory medicine | vol 11, no 1 | a1784 | 18 august 2022 original research bacteriological profile and antibiogram of blood culture isolates from bloodstream infections in a rural tertiary hospital in nigeria oluwalana t. oyekale, bola o. ojo, adewale t. olajide, oluwatoyin i. oyekale african journal of laboratory medicine | vol 11, no 1 | a1807 | 24 august 2022 original research genexpert rollout in three high-burden tuberculosis countries in africa: a review of pulmonary tuberculosis diagnosis and outcomes from 2001 to 2019 victor williams, marianne calnan, bassey edem, chukwuemeka onwuchekwa, chika okoro, christine candari, rhodora cruz, kennedy otwombe african journal of laboratory medicine | vol 11, no 1 | a1811 | 30 august 2022 original research global antimicrobial resistance and use surveillance system on the african continent: early implementation 2017–2019 barbara tornimbene, sergey eremin, reuben abednego, elamin o. abualas, ilhem boutiba, abiodun egwuenu, walter fuller, laetitia gahimbare, susan githii, watipaso kasambara, chileshe lukwesa-musyani, fidy a. miamina, sekesai mtapuri-zinyowera, grace najjuka, olga perovic, bassem zayed, yahaya a. ahmed, maha t. ismail, carmem l. pessoa da silva african journal of laboratory medicine | vol 11, no 1 | a1594 | 31 august 2022 original research effect of sputum quality and role of xpert® mtb/rif assay for detection of smear-negative pulmonary tuberculosis in same-day diagnosis strategy in addis ababa, ethiopia waganeh sinshaw, abebaw kebede, adane bitew, mengistu tadesse, zemedu mehamed, ayinalem alemu, bazezew yenew, misikir amare, biniyam dagne, getu diriba, ephrem tesfaye, dinka f. gamtesa, yeshiwork abebaw, helina mollalign, getachew seid, muluwork getahun african journal of laboratory medicine | vol 11, no 1 | a1671 | 31 august 2022 229 237 246 251 257 264 272 283 original research human herpes virus type-6 is associated with central nervous system infections in children in sudan nada a. abdelrahim, nahla mohamed, magnus evander, clas ahlm, imad m. fadl-elmula african journal of laboratory medicine | vol 11, no 1 | a1718 | 22 september 2022 original research audit of amylase and lipase requests in suspected acute pancreatitis and cost implications, south africa annie e. cook, thumeka p. jalavu, annalise e. zemlin african journal of laboratory medicine | vol 11, no 1 | a1834 | 26 september 2022 original research red blood cell alloimmunisation in multi-transfused patients from an haemodialysis service in burkina faso koumpingnin nebie, salam sawadogo, salifo sawadogo, jérôme koulidiati, habi y.a. lengani, abdoul g. sawadogo, jérôme babinet, mohammed khalloufi, saliou diop, eléonore kafando african journal of laboratory medicine | vol 11, no 1 | a1625 | 26 september 2022 original research comparison of commercial assays and two-step approach to detect clostridioides difficile in south africa sarishna singh, mae newton-foot, pieter nel, colette pienaar african journal of laboratory medicine | vol 11, no 1 | a1809 | 29 september 2022 original research molecular detection of hepatitis b virus genotype e with immune escape mutations in chronic hepatitis b patients on long-term antiviral therapy in jos, nigeria joseph anejo-okopi, edith okeke, pantong m. davwar, chika onwuamah, harris onywera, patience omaiye, mary duguru, ocheme j. okojokwu, otobo i. ujah, bulus jonathan, chima a. george, ramyil s. crown, fiyaktu b. yakubu, judith o. sokei, leona c. okoli, onyemocho audu, seth c. inzaule, isaac o. abah, patricia agaba, oche o. agbaji, atiene s. sagay, claudia hawkins african journal of laboratory medicine | vol 11, no 1 | a1677 | 18 october 2022 original research agreement between xpert and ampfire tests for high-risk human papillomavirus among hiv-positive women in rwanda anthere murangwa, kanan t. desai, julia c. gage, gad murenzi, patrick tuyisenge, faustin kanyabwisha, aimable musafili, gallican kubwimana, leon mutesa, kathryn anastos, hae-young kim, philip e. castle african journal of laboratory medicine | vol 11, no 1 | a1827 | 19 october 2022 original research optimising courier specimen collection time improves patient access to hiv viral load testing in south africa sarah j. girdwood, thomas crompton, naseem cassim, floyd olsen, portia sejake, karidia diallo, leigh berrie, dorman chimhamhiwa, wendy stevens, brooke nichols african journal of laboratory medicine | vol 11, no 1 | a1725 | 25 october 2022 original research practices and barriers to screening for hyperglycaemia in pregnancy among providers of antenatal care in jos, nigeria lucius c. imoh, abdulazis s. longwap, favour e. haruna, oghale j. asieba, joy p. istifanus, joy a. imoh, mathilda e. banwat african journal of laboratory medicine | vol 11, no 1 | a1845 | 31 october 2022 290 296 301 307 313 320 325 331 page iii of iv http://www.ajlmonline.org open access table of contents original research causes of death and post-mortem testing for sars-cov-2 in a tertiary hospital during the covid-19 pandemic in ghana edward asumanu, seth attoh, raymond x. servor, clement laryea, mary mcaddy, fred hobenu, raymond factchu, kwesi agyemang-bediako, edward o. nyarko, godwin k. nyarko, marcus k. moroti, lawrence edusei african journal of laboratory medicine | vol 11, no 1 | a1766 | 23 november 2022 original research malaria an opportunistic infection in hiv/aids patients? – a nigerian experience joseph n. enuma, felix o. sanni, malau b. matur, njab e. jean, tosan erhabor, iheukwumere i. egbulefu african journal of laboratory medicine | vol 11, no 1 | a1842 | 24 november 2022 original research oral human papilloma virus infection among dental clinic attendees in ibadan, nigeria adedayo o. faneye, oyeteju s. babalola, georgina n. odaibo, juwon arotiba, olufemi d. olaleye african journal of laboratory medicine | vol 11, no 1 | a1555 | 25 november 2022 original research impact of rapid centrifugation on routine coagulation assays in south africa reola haripersadh, dashini pillay, nadine rapiti african journal of laboratory medicine | vol 11, no 1 | a1901 | 28 november 2022 original research commercial duraclone panels for extending the repertoire of multicolour immunophenotypic panels in an academic flow cytometry laboratory in south africa leanne swart, melanie pretorius, denise lawrie, deborah k. glencross african journal of laboratory medicine | vol 11, no 1 | a1720 | 29 november 2022 340 348 354 359 366 scientific letter appropriate disposal of waste in the laboratory: neglected but not forgotten christoffel j. opperman, sarishna singh, francois barton african journal of laboratory medicine | vol 11, no 1 | a1786 | 14 july 2022 case study extranodal presentation of a lymphoma with precursor b-cell phenotype and translocation t(8;14) in south africa katherine e. hodkinson, yvonne perner, deborah k. glencross, tracey wiggill, adam botha, janet poole african journal of laboratory medicine | vol 11, no 1 | a1355 | 31 january 2022 brief report retrospective analysis of vitek®2 performance compared to manual broth micro-dilution for colistin susceptibility testing of acinetobacter baumannii complex isolates in south africa vuyolwethu fadana, teena thomas, nina von knorring african journal of laboratory medicine | vol 11, no 1 | a1597 | 28 february 2022 correction corrigendum: higher proportion of non-classical and intermediate monocytes in newly diagnosed multiple myeloma patients in egypt: a possible prognostic marker asmaa m. zahran, hanaa nafady-hego, sawsan m. moeen, hanan a. eltyb, mohammed m. wahman, asmaa nafady african journal of laboratory medicine | vol 11, no 1 | a1713 | 25 march 2022 reviewer acknowledgement african journal of laboratory medicine | vol 11, no 1 | a2096 | 19 december 2022 375 377 382 386 387 page iv of iv  article information authors: talkmore maruta1 katy yao2 nqobile ndlovu3 sikhulile moyo4 affiliations: 1clinton health access initiative, maseru, lesotho2international laboratory branch, division of global hiv/aids, center for global health, us centers for diseases control and prevention, united states 3african field epidemiology network, kampala, uganda 4botswana–harvard aids institute partnerships, botswana–harvard hiv reference laboratory, botswana correspondence to: talkmore maruta postal address: po box 354, harare, zimbabwe dates: received: 18 may 2014 accepted: 02 july 2014 published: 16 sept. 2014 republished: 07 nov. 2014 how to cite this article: maruta t, yao k, ndlovu n, moyo s. training-of-trainers: a strategy to build country capacity for slmta expansion and sustainability. afr j lab med. 2014;3(2), art. #196, 7 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.196 note: article republished with updated references relating the special issue. copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. training-of-trainers: a strategy to build country capacity for slmta expansion and sustainability in this lessons from the field... open access • abstract • introduction • research method and design    • teachback methodology    • the slmta trainer’s toolkit    • slmta training-of-trainers workshop structure    • final participant assessment    • training-of-trainers programme scale-up    • development of the master trainer cadre    • training-of-trainers programme evaluation • results • discussion    • next steps • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: the strengthening laboratory management toward accreditation (slmta) programme uses a training-of-trainers (tot) model to build capacity for programme scale-up. the tot strategy is designed to maximise utilisation of its graduates whilst minimising inconsistencies and ensuring high programme quality during global expansion.objectives: to describe the slmta tot programme approach. methods: the two-week training, led by carefully selected and trained master trainers, enables effective and authentic implementation of the curriculum by its graduates. the teachback methodology used allows participants to practise teaching the curriculum whilst learning its content. a trainer’s toolkit provides all the materials necessary for teaching and must be followed faithfully during training. two surveys were conducted to assess the effectiveness of the tot strategy: one sent to 316 tot graduates in 25 countries and the other sent to the programme leaders in 10 countries. results: by the end of 2013, 433 slmta trainers had been trained who, in turn, taught more than 1900 people to implement slmta in 617 laboratories in 47 countries. ninety-seven percent of the 433 tot graduates and 87% of the 38 master trainers are based in developing countries. ninety-two per cent of the graduates have been utilised at least once in programme implementation and, as of august 2013, 87% of them were still actively involved in programme activities. ninety-seven per cent of the graduates stated that the tot workshop prepared them well for training or other programme tasks. conclusion: the slmta tot strategy is effective in building local capacity for global programme expansion whilst maintaining programme quality. introduction top ↑ the current drive toward laboratory quality improvement and accreditation in resource-limited settings will be hard to sustain without building local capacity. launched in 2009, the strengthening laboratory management toward accreditation (slmta) programme has revolutionised the ability of government medical laboratories in low-resource settings to implement quality management systems and pursue international accreditation.1,2 slmta is an innovative management training programme that employs a series of workshops and improvement projects to realise immediate and measurable improvement.1 within five years of its launch, slmta has been implemented in 617 laboratories from 47 countries in africa, southeast asia, the caribbean and latin america and has been used to train more than 1900 people.2 its rapid expansion is attributed in part to its effective local capacity-building effort using a training-of-trainers (tot) strategy.tot has been applied across many disciplines including education, healthcare, health promotion and disease prevention to provide would-be trainers with the necessary knowledge and skills for training others.3,4 its popularity is because of its cost-effectiveness and the potential for rapid expansion of local capacity.5 additionally, the development and utilisation of local trainers ensure that the curriculum content is culturally relevant and applicable.6 despite these benefits, there are challenges associated with the tot approach. one is the concern over the ‘wastage’ or low utilisation rate of tot graduates; some studies have reported that 30% − 50% of tot trainees did not deliver any training after the tot.5,6 there are also issues with ‘fidelity of implementation’ – the dilution and distortion of core messages and information as the programme is cascaded downstream because trainers fail to adhere to the curriculum and training protocols;5,6 and also perhaps because documentation of the curriculum does not provide sufficient details to ensure standardisation. furthermore, qualifications of tot participants may be inadequate if their selection does not take into account future availability to train and their defined roles in the programme, in addition to technical competency. great variations of tot methodology have been described, ranging from didactic presentations to group discussions and role-play.7 some tot workshops put equal emphasis on curriculum content learning and facilitation skills development whilst others focus exclusively on the former. although there is no consensus regarding the ideal tot model,4 there has been an increasing trend from traditional passive teaching methods toward active methods that involve the students in both the teaching and learning process.8 in this paper, we describe a teachback-based tot model for building in-country and regional capacity in the implementation of laboratory quality management systems and, specifically, the slmta programme. we discuss how the slmta tot strategy ensures high utilisation of tot graduates and maintains the fidelity of implementation whilst facilitating the rapid scale-up and sustainability of the programme. research method and design top ↑ teachback methodology the slmta tot uses the teachback methodology developed by dr gordon pask in 1975.9 teachback has been recognised as an effective method for educating and assessing learning for patients in clinical practice10 and has been adapted to the teaching of training skills to trainers as well. this intensive practice-based training approach requires participants to play the roles of both trainer and participant: they must teach the curriculum at the same time as they are learning the curriculum content. the slmta tot is organised into three phases: (1) tot facilitators, also called master trainers, teach the curriculum whilst modeling the learner-centred, interactive facilitation skills; (2) participants learn the curriculum content and practise teaching it back to other participants; and (3) master trainers provide feedback on the teachback performance.11feedback is the cornerstone of teachback methodology and follows a distinct protocol. it is given immediately at the end of each teachback session after the participants have had a chance to assess their own performance. praise is always offered first, followed by suggestions for improvement. effective feedback is positive, encouraging and specific, building upon each participant’s strengths. comments must be constructive, focusing on areas that can be improved and behaviours that can be changed. master trainers use standard feedback forms to guide them on what to look for during the teachback. participants receive their feedback both orally and on the written feedback forms.11 the slmta trainer’s toolkit the slmta programme provides a training curriculum that embodies the principle of ‘learning by doing’; lectures are limited to only 20% of the total training time whilst 80% is demonstration, discussion, role play, simulation and hands-on practice. the curriculum comprises 44 activities, which teach participants to use more than 100 tools and job aids in performing 66 management tasks and routines. it requires a facilitation style that engages the participants actively in the learning process. each tot participant receives a trainer’s toolkit containing all the information necessary to teach the 44 slmta activities. in addition to the handouts, tools, worksheets and job aids, the toolkit provides detailed preparation instructions and a step-by-step teaching protocol, grounded in adult learning principles, for each activity. during the tot, master trainers and participants are both required to adhere to the protocols prescribed in each activity. the only deviation allowed is their personal examples and stories for illustrating key concepts and creativity in designing the visual aids. this comprehensive toolkit, coupled with the rigorous teachback process, serves to preserve the fidelity of the programme as the participants deliver the training back home. slmta training-of-trainers workshop structure the slmta tot teaches participants to deliver the slmta curriculum effectively and to implement the programme appropriately. the tot workshop lasts two weeks. because of its intensive performance-based nature, the participant-to-facilitator ratio must not exceed eight to one; a typical tot workshop has at least three master trainers for a maximum of 24 participants.to ensure return on investment and a high utilisation rate of the tot graduates, countries are asked to screen their participants carefully using set criteria. ideal candidates are those who have participated in the slmta process and successfully implemented improvement projects in their laboratories. additional qualifications include: (1) availability to train; (2) defined role to implement the programme as designated by the country’s ministry of health; (3) evidence of motivation; (4) excellent training and communication skills; (5) technical laboratory experience in a clinical setting; and (6) proven ability to manage a laboratory successfully. it is critical to balance participants’ need to learn the unconventional curriculum from the master trainers first hand, with sufficient time to practise teaching each other. the 44 slmta activities are thus presented differently, depending on their level of complexity. master trainers typically teach the 25 most difficult activities so as to ensure fidelity, whilst describing (instead of teaching) the 10 least complicated activities to the participants. sixteen activities are assigned to participants to teach back. some of these activities are so important that they are taught initially by master trainers and then assigned to participants for teachback. table 1 lists the 44 activities in the curriculum and how they are presented during the tot. table 1: workshop activities and how they are presented. the 35 activities taught or described by master trainers occur in plenary sessions. the teachback sessions, on the other hand, occur initially in small breakout groups in order to foster a nurturing and less intimidating environment. each breakout group typically contains eight people, who are then further divided into four teams of two. these teams become teachback partners and remain together throughout the workshop for their teachback assignments. each breakout group is led by a master trainer who serves as a mentor and coach for the group. the 16 teachback activities are assigned evenly to the four teams within each breakout group: each team is responsible for teaching back a total of four activities. the first three are done within their small breakout groups and the fourth is done in plenary sessions to give participants the experience of teaching in a large-group setting. to allow time for preparation, teachback does not begin until the end of the first week of the two-week tot workshop. specific times are allocated for coaching, usually at the end of each day. coaching sessions last 30–60 minutes and allow teams to discuss their upcoming teachback assignments with their master trainers. additional coaching sessions are available by appointment with master trainers. teams then prepare for their assignments, often late into the evening. final participant assessment in addition to the feedback given immediately after each teachback presentation, master trainers meet with individual participants privately at the end of the tot and provide assessment orally on their overall performance. a written report is also submitted to the participants and their organisations following the tot. irrespective of the assessment, all participants who meet the 100% participation requirements are allowed to graduate from the course. training-of-trainers programme scale-up beginning in 2009, slmta tots have been conducted at the african centre for integrated laboratory training (acilt) in johannesburg, south africa, enrolling participants from multiple countries. as the slmta programme spreads deeper within countries, more local trainers and implementers are needed. the acilt-based tot, which admits only two to four participants from each country, is no longer sufficient. to meet the demand, 12 countries and/or regions to date (botswana, dominican republic, ethiopia, ghana, kenya, mozambique, nigeria, rwanda, south africa, tanzania, vietnam/cambodia and zimbabwe) have hosted their own tot workshops. prerequisites for an in-country tot include completion of at least one round of the slmta process (three slmta workshops with supervisory visits and audits), as well as a sound country roll-out plan that details how the increased capacity will be used effectively. development of the master trainer cadre slmta’s tot strategy hinges on the availability of master trainers. with the expansion of the tot programmes, the need for master trainers, who facilitate the tot workshops and mentor future trainers, has escalated. starting in 2011, an effort was made to increase the number of master trainers with a focus on building in-country capacity.master trainers have extensive experience in training, coaching and feedback as well as in implementing laboratory quality systems. they play a critical role in ensuring programme fidelity in the rapid scale-up of slmta around the world. there is a stringent selection and grooming process to create these master trainers. to be eligible for consideration, a candidate must be a certified slmta trainer and have implemented at least one round of slmta roll-out in-country. this includes teaching the entire series of slmta workshops, mentoring laboratories to implement improvement projects and conducting follow-up visits after each workshop. the candidate must be available and must be released by their employer to conduct the two-week long tot. in addition, they must be engaged in all preparation tasks leading to the workshop. finally, this candidate must be nominated by a master trainer and approved by his or her country’s slmta programme coordinator or office before being invited to a tot for apprenticeship. master trainer candidates shoulder equal amounts of responsibility in the tot under the watchful eye of master trainers. they participate fully in the pre-tot planning sessions and serve as lead facilitators for activities assigned to them. they coach the teams to prepare for teachback and provide feedback afterwards. candidates must be prepared to re-teach activities whenever a team fails to deliver their teachback activity effectively. they also provide input to the final assessment of each participant’s performance (both oral and written). in turn, they receive coaching and feedback from the master trainers throughout the tot. at the end of the workshop, master trainers provide feedback and recommendations. because of the apprenticeship model used to develop new master trainers, each tot workshop can accommodate up to three master trainer candidates, with each master trainer mentoring one candidate. training-of-trainers programme evaluation an online survey was conducted in august 2013 in order to assess the effectiveness of the tot strategy on building country capacity for programme scale-up. it included both openand closed-ended questions to collect data on tot graduates’ utilisation rates and on how well the tot prepared them for programme implementation using a four-point likert scale (extremely well; well; not sure; and not at all). the survey was sent to all 316 slmta tot graduates from 25 countries who attended one of the 14 regional or in-country tot workshops held between november 2009 and march 2013. one hundred and seventeen more recent tot graduates were not surveyed because they would not yet have had enough time to deliver any slmta training.to verify the tot graduates’ survey data, a second survey was sent to the programme leaders in all 10 countries that had held a local tot before march 2013 and therefore had large trainer populations. these countries’ slmta programme leaders were asked to provide data on the number of trainers that are still involved in slmta activities. results top ↑ between november 2009 and november 2013, 8 regional and 11 local tot workshops have been conducted, yielding a total of 433 trainers (figure 1). to expand the programme in non-english speaking countries, vietnam, mozambique and the dominican republic each hosted a tot dedicated to producing vietnamese-, portugueseand spanish-speaking trainers, respectively. to address the shortage of francophone trainers, two french-language tots are being planned by cameroon and cote d’ivoire. figure 1: cumulative number of slmta trainers, master trainers and tots 2009-2013. figure 2: global distribution of slmta master trainers. before 2011, there were only two master trainers, both based in the united states. as of the end of 2013, there were a total of 38 master trainers: 27 in africa (71%), seven (18%) in the americas (including two in latin america) and four in southeast asia (11%) (figures 1 and 2). the language portfolio of the master trainers now includes french (n = 5), portuguese (n = 5), vietnamese (n = 4) and spanish (n = 2), in addition to english. of the 316 tot graduates from november 2009 to march 2013, 195 (62%) returned the survey. of the respondents, 160 (82%) have delivered at least one slmta training. an additional 19 (10%) are still involved in slmta programme activities such as mentoring and coordination, yielding a 92% utilisation rate. for the remaining 8% (n = 15) of the tot graduates, reasons for non-involvement included being too busy, not being chosen to train, not being released by supervisors and changing jobs (figure 3). figure 3: results from the tot graduates survey. of the 160 respondents who had facilitated slmta training(s), 59% (n = 94) stated that the tot prepared them extremely well and 36% (n = 58) well. the remaining 5% (n = 8) of the respondents were not sure about the effectiveness of the tot, viewed it as being not at all helpful, or did not respond. the 19 respondents who had been involved only in non-training aspects of programme implementation stated that the tot had prepared them extremely well (n = 13; 69%) or well (n = 6; 31%). all 10 country programme leaders returned the survey. current involvement rates of their local tot graduates (as of august 2013) ranged from 62% to 100%, with an overall 87% (n = 197) of tot graduates still being involved in slmta activities (table 2). table 2: results from the country programme leader survey. discussion top ↑ since slmta tot began in 2009, the slmta programme has expanded rapidly1,2 and many countries have rolled out the programme independently with little or no outside assistance. evidence suggests that the slmta tot strategy has been effective in building local capacity to accelerate programme expansion in multiple regions of the world,2 with 97% of the tot graduates and 87% of the master trainers based in developing countries. today, these cadres of master trainers, trainers and slmta-trained laboratory managers serve as the local champions of laboratory quality and activists in a global network that has kept the slmta ‘grassroots movement’ going.high utilisation rates of the tot graduates have been observed, at 92%. however, the relatively low response rate (62%) on the part of the tot graduates may limit the generalisation of this observation. the low response rate can most likely be attributed to the short survey period, which lasted only two weeks, as well as the fact that an online survey was used which required access to the internet. in the second survey, however, all 10 countries’ programme leaders responded and they reported an average 87% utilisation rate, which is similar to the results from the first survey. fidelity of implementation is also critical as the programme expands and the cadre of trainers multiplies. the quality of training is more difficult to evaluate and was not measured directly in this study. however, evidence suggests that there has been no deterioration of implementation quality with programme expansion, as average audit improvement scores for laboratories implementing slmta in 2011–2013 were the same as those for laboratories implementing slmta during its initial year (2010), at 24%.2 additionally, tot participants reported overwhelmingly that the training was effective in preparing them to implement the programme. several key elements have been built into the slmta tot in an effort to ensure continued quality: (1) the slmta training toolkit comprehensively standardises training protocols and content, assisting trainers to teach the information consistently; (2) a highly structured and demanding tot programme ensures that trainers are fully capable and confident in their training abilities; (3) a prescribed intensive implementation process fosters commitment, understanding and collaboration; and (4) a rigorous process is used for selecting and grooming master trainers, who are the gatekeepers for the quality of future trainers. a collaborative south-to-south network has emerged. eighty-seven per cent of the 38 master trainers reside in 15 developing countries across africa, southeast asia and latin america. these highly-skilled master trainers are often invited to conduct tot workshops in countries other than their own. notably, the depth of the african master trainer pool allowed the export of two master trainers to assist latin america when it held its first regional slmta tot in the summer of 2013. furthermore, most in-country tots provide a few slots for participants from other countries. this fosters networking and experience sharing, as well as assisting countries who are not ready to conduct their own tot but do not want to wait for the next available tot at acilt. there are many examples where countries with slmta experience are aiding those without sufficient training capacity: rwandan trainers travelled to burundi to deliver workshops; namibian participants went to zimbabwe to be trained; the dominican republic enrolled participants from six central american countries alongside its own people; and the african field epidemiology network, headquartered in uganda, delivered slmta training in the caribbean region. the globally-standardised slmta curriculum and common implementation process facilitate cross-border and cross-region technical assistance. next steps casual observers of the programme have noted that the current geographic distribution of the master trainers does not reflect the size and maturity of country programmes (table 3). of the seven countries with the largest number of laboratories enrolled in slmta,2 three (ethiopia, uganda and tanzania) have no master trainers, whilst kenya, with the most rounds of implementation (six), has only one master trainer. lacking master trainers does not necessarily correspond to the quality of a country’s programme, since slmta is implemented by trainers rather than master trainers; nevertheless, going forward it seems prudent that these countries should be encouraged to build a pool of local master trainers. this will not only reduce costs for future tots by eliminating the need to bring in international master trainers, but will also motivate in-country trainers to be involved in more programme activities so they may be eligible to become master trainers. table 3: number of master trainers in countries with the most enrolled slmta laboratories. conclusion top ↑ slmta has helped transform the laboratory landscape in resource-limited countries worldwide.1 through the careful execution of a deliberate tot strategy, the programme has focused on building local capacity to accelerate the breadth and depth of programme spread. rapid programme scale-up has been achieved with continued high quality results,2 using a strategy of centralised and in-country tots, in-country workshops and implementation at the laboratory level. strict maintenance of training and qualification standards has been key to the success of the strategy. the growing pool of motivated and skilled local implementers will be critical with regard to sustaining the on-going quality improvement and accreditation drives in resource-limited settings.skills acquired through the slmta tot, such as programme planning, mentoring and training facilitation, will reach beyond slmta to make participants become more effective managers and mentors. moreover, it has been suggested that slmta be ‘adapted for clinical settings in developing countries, with a goal towards overall hospital accreditation’.1 based on the success of the slmta tot model, we would recommend that other programmes consider adopting the model to build local capacity for rapid programme expansion whilst maintaining programme quality. acknowledgements top ↑ the authors would like to thank dr barbara mckinney, anna murphy and philip rotz for their significant contribution in the development of the slmta toolkit and the design of the slmta tot workshop. the authors also extend their gratitude to all the slmta master trainers and trainers for their tireless effort in building country capacity with regard to implementing the slmta programme. thanks also go to acilt staff members for their on-going support for workshop logistics and preparations.this research has been supported by the president’s emergency plan for aids relief (pepfar) through the us centers for disease control and prevention. the findings and conclusions in this paper are those of the authors and do not necessarily represent the views of the us centers for disease control and prevention. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions t.m. (clinton health access initiative) implemented the programme and wrote the manuscript. k.y. (us centers for diseases control and prevention) designed and implemented the slmta tot training programme, collected and analyzed the data and substantially revised the manuscript. n.n. (african field epidemiology network) implemented the programme and reviewed the manuscript. s.m. (botswana–harvard hiv reference laboratory), reviewed the article and assisted in development of the data analysis plan. references top ↑ 1.yao k, luman et. slmta collaborating authors. evidence from 617 laboratories in 47 countries for slmta-driven improvement in quality management systems. afr j lab med. 2014. in press.2.yao k, maruta t, luman et, nkengasong jn. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014. in press. 3.usaid. training of trainers manual: conflict transformation and peacebuilding in rwanda [document on the internet]. c2008 [cited 2013 april 01]. available from: http://pdf.usaid.gov/pdf_docs/pnadm806.pdf 4.ray ml, wilson mm, wandersman a, et al. using a training-of-trainers approach and proactive technical assistance to bring evidence based programs to scale: an operationalization of the interactive systems framework’s support system. am j community psychol. 2012;50(3–4):415–427. http://dx.doi.org/10.1007/s10464-012-9526-6 5.unicef. evaluation of unicef learning strategy to strengthen staff competencies for humanitarian response 2002–2004. new york: united nations children’s fund; 2005. 6.hiner ca, mandel bg, weaver mr, et al. effectiveness of a training-of-trainers model in a hiv counseling and testing program in the caribbean region. hum resour health. 2009;7:11. available from: http://dx.doi.org/10.1186/1478-4491-7-11 http://www.human-resources-health.com/content/7/1/11 7.pearce j, mann mk, jones c, et al. the most effective way of delivering a train-the-trainers program: a systematic review. j contin educ health prof. 2012;32(3):215–226. http://dx.doi.org/10.1002/chp.21148 8.yolsal n, bulut a, karabey s, et al. development of training of trainers programmes and evaluation of their effectiveness in istanbul, turkey. med teach. 2003;25(3):319–324. http://dx.doi.org/10.1080/0142159031000092779 9.pask g. the cybernetics of evolutionary process and of self-organising systems. 3rd intern conf on cybernetics. namur: gauthier-villars; 1961. 10.white m, garbez r, carroll m, et al. is ’teach-back’ associated with knowledge retention and hospital readmission in hospitalized heart failure patients? j cardiovasc nurse. 2013;28(2):137–146. http://dx.doi.org/10.1097/jcn.0b013e31824987bd 11.us centers for disease control and prevention, national center for hiv, std and tb prevention, global aids program training team. train-up with teachback! [dvd] produced in partnership with international training and education center on hiv (i-tech) and caribbean hiv aids regional training network (chart). article information authors: philip h. fortgens1,2 fierdoz omar1,2 affiliations: 1department of clinical laboratory sciences, division of chemical pathology, university of cape town, south africa2national health laboratory service, groote schuur hospital, south africa correspondence to: philip fortgens postal address: division of chemical pathology, university of cape town/national health laboratory service, groote schuur hospital, anzio road, observatory, cape town, 7925 dates: received: 09 dec. 2011 accepted: 20 sep. 2013 published: 03 dec. 2013 how to cite this article: fortgens ph, omar f. cardiac troponin t quantitative assay failure as a result of antibody interference. afr j lab med. 2013;2(1), art. #23, 3 pages. http://dx.doi.org/10.4102/ ajlm.v2i1.23 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. cardiac troponin t quantitative assay failure as a result of antibody interference in this oorspronklike navorsing: wiskundeonderrig... open access • abstract • introduction • research method and design    • case    • interference experiments • results • discussion • conclusion • acknowledgements    • competing interest    • authors’ contributions • references abstract top ↑ background: immunoassays are prone to interference by various substances which may cause inaccurate results. this type of interference is difficult to detect analytically.objective: a case of cardiac troponin t quantitative reader (roche diagnostics) assay failure was detected and investigated in order to ascertain the likely cause. method: patient whole blood was mixed with cardiac troponin t-positive blood, patient and control sera were denuded of immunoglobulin g by protein a-affinity chromatography and patient sera were mixed with mouse serum. samples were analysed on a cardiac troponin t quantitative reader. results: a mixture of patient whole blood and cardiac troponin t-positive blood resulted in assay failure; removal of immunoglobulin g from patient sera reversed the cardiac troponin t assay failure; the addition of mouse serum as a heterophile antibody blocking agent had no effect. conclusion: it is proposed that the interference resulting in assay failure may not be because of a heterophile antibody, but rather a result of a circulating autoantibody to cardiac troponin t, which may compete with antibody assay reagents for binding sites. introduction top ↑ a consensus document released by the european society of cardiology, the american college of cardiology, the american heart association and the world heart federation task force in 2012 has proposed cardiac troponin (ctn) as the preferred biomarker for myocardial necrosis because of its superlative myocardial tissue specificity and high clinical sensitivity.1 furthermore, ctn has also been shown to have value for the prediction of adverse cardiovascular events in patients presenting with acute coronary syndrome.2 cardiac troponin t (ctnt) appears to be an important marker of coronary heart disease, mortality and risk of heart failure in a healthy population without manifest cardiovascular disease.3measurement of cardiac troponins is achieved by immunoassay. despite extensive experience with this methodology, however, immunoassays are occasionally subject to interfering substances that compromise their accuracy – indeed, it is estimated that antibody interference affects approximately one in 2000 immunoassay results.4 we report a novel case of assay failure using the cardiac troponin t quantitative reader (roche diagnostics). research method and design top ↑ case a 61-year-old female, with a history of ischaemic heart disease and hypertension, presented to the emergency unit on two occasions 12 days apart with chest discomfort. repeated attempts by the diagnostic laboratory to obtain ctnt measurements failed, as reflected by the absence of a positive control line on test strips (cardiac troponin t quantitative reader, roche diagnostics; figure 1). as the creatinine kinase level was within normal limits (26–140 u/l) at both visits and the myoglobin was normal (7–64 ng/l) when measured at the second visit, the patient was discharged with follow-up. figure 1: absence of a control line on the roche cardiac troponin t quantitative test strip. interference experiments antibody interference was suspected and the following investigation was thus performed. prior ethics approval was not obtained as the investigation would lead to improvement in this patient’s management. firstly, a 1:1 mixture of the patient’s sample and a recently-assayed anonymous sample positive for ctnt (both heparinised whole bloods), was assayed for ctnt.5 secondly, patient and control plasma samples were depleted of immunoglobulin g (igg) using protein a-affinity chromatography.6 these samples were analysed for ctnt prior to and after igg depletion. the cardiac troponin t quantitative reader is a lateral flow immunoassay, utilising the sandwich principle on a test strip with two murine monoclonal anti-ctnt antibodies.7 thirdly, in order to exclude the presence of interfering human anti-mouse antibodies (hama), mouse serum was added to the patient plasma (1:4) and the mixture was incubated for one hour at room temperature, following which the ctnt was measured. lastly, to determine whether the automated ctnt assay on the roche elecsys e170 analyser was subject to the same interference, dilutions of a known ctnt-positive plasma sample mixed with the patient plasma were assayed for ctnt. results top ↑ the mixture of whole blood patient sample and a ctnt-positive specimen inhibited the formation of the control line on the ctnt reagent strip, supporting our suspicion of an interfering substance. whilst only the control sample elicited a control line prior to igg depletion (ctnt < 0.03 ng/ml), both the patient and control samples elicited control lines after igg depletion (ctnt < 0.03 ng/ml), suggesting that igg was the interfering substance. test-strips contain hama-blocking antibodies,7 but despite the presence of additional blocking agent (mouse serum), the control line did not develop, which suggested strongly that the interfering igg was not an hama (table 1). dilutions of a known ctnt-positive plasma sample with the patient plasma showed a linear response when assayed for ctnt on the roche elecsys e170 analyser, suggesting that this platform is not subject to the same autoantibody interference. table 1: cardiac troponin t quantitative test strip performance. discussion top ↑ there is increasing use of ctn measurement in the diagnosis of myocardial injury and any inaccuracy with regard to measured values is more likely to have serious clinical impact than would be the case with immunoassays for many other analytes. several immunochemical interferences have been well described, including heterophile antibodies,8 rheumatoid factor9 and circulating troponin autoantibodies.10,11 recent studies have shown that 15.9% of samples from cohorts of normal blood donors were positive for autoantibodies to troponin t or troponin i and 10.9% were positive for both.12 autoantibody–antigen macrocomplexes have been well described for a number of biomarkers such as salivary amylase,13 creatine kinase,14 aspartate aminotransferase15 and lactate dehyrogenase,16 but there is no evidence to suggest that these complexes contribute to a pathological process. in contrast, ctn autoantibodies have been shown to contribute to the progression toward heart failure in mice and a similar process may occur in humans.17 furthermore, it has been proposed that the reduced clearance of immunoglobulin–cardiac troponin complexes from the circulation may result in increased levels of measured troponins.18 this phenomenon does not, therefore, represent assay interference, but rather an analytically-true result, albeit misleading. this study shows that the removal of igg from patient serum reverses the ctnt assay failure and that the addition of mouse serum as a heterophile antibody-blocking agent has no effect. this, together with the quantifiable result of the myoglobin assay (roche diagnostics), which uses the same test principle, species of monoclonal antibody and reader as the ctnt assay, suggests that the interference may not be because of a heterophile antibody, but rather because of an autoantibody to ctnt. such an antibody may compete with the mouse anti-ctnt–gold complex for binding to immobilised ctnt on the test-strip, which normally serves as a test control. in the case of the myoglobin assay, the immobilised control antigen (myoglobin) is different, which may explain why it is unaffected. an autoantibody could also compete with reagent anti-ctnt antibodies for binding sites, thereby preventing the development of a test line (figure 2). there have been several reports of positive interference in the ctnt assay by heterophilic antibodies with the cardiac troponin t quantitative test7 and the elecsys e1708 automated platform but, to the best of our knowledge, this is the first report of ctnt assay failure on the former platform. we conclude that the failure of this assay is a result of putative autoantibodies to ctnt. assay failure also allowed for the immediate detection of potential interference, but the majority of cases of interference remain undetected at the analytical level. it is crucial, therefore, that any discrepancies between results and the clinical picture be addressed by clinicians with the laboratory, to avoid further potentially-invasive and expensive investigations. figure 2: possible effect of blocking human anti-cardiac troponin t antibodies on the roche cardiac troponin t quantitative lateral flow immunoassay. conclusion top ↑ a case of assay failure was detected in the cardiac troponin t quantitative reader. immunoassays are subject to interference and it is important to investigate anomalous results to exclude such assay interference. after removing igg from patient serum and re-assaying the ctnt, assay failure was found to reverse. this case highlights the fact that immunoassay interference remains a persistent problem and vigilance is encouraged in order to minimise its impact. furthermore, in the case of troponins, interference may be caused by circulating autoantibodies to ctn. acknowledgements top ↑ we thank noel markgraaf for mouse serum, roche diagnostics for the elecsys e170 troponin t assay kit and dr judy king for her critical appraisal of the manuscript.this work was submitted as a presentation at the federation of south african pathology societies 50th annual congress, september 2010 and as a poster at the international federation of clinical chemistry and laboratory medicine (ifcc) worldlab and euromedlab congress, berlin, 2011. competing interest the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions f.o. (university of cape town/nhls) initiated the study, contributed to project design and critically revised the manuscript. p.h.f. (university of cape town/nhls) contributed to study design, performed the experiments and wrote the manuscript. references top ↑ 1. thygesen k, alpert js, jaffe as, et al. on behalf of the joint esc/accf/aha/whf task force for the universal definition of myocardial infarction. third universal definition of myocardial infarction. eur heart j. 2012;33:2551–2567. http://dx.doi.org/10.1093/eurheartj/ehs184, pmid:22922414 2. aldous sj, florkowski cm, crozier ig, et al. high sensitivity troponin outperforms contemporary assays in predicting major adverse cardiac events up to two years in patients with chest pain. ann clin biochem. 2011;48(pt 3):249–255. http://dx.doi.org/10.1258/acb.2010.010220, pmid:21441393 3. saunders jt, nambi v, de lemos ja, et al. cardiac troponin t measured by a highly sensitive assay predicts coronary heart disease, heart failure, and mortality in the atherosclerosis risk in communities study. circulation. 2011;123(13):1367–1376. http://dx.doi.org/10.1161/circulationaha.110.005264, pmid:21422391, pmcid:pmc3072024 4. levinson ss, miller jj. towards a better understanding of heterophile (and the like) antibody interference with modern immunoassays. clin chim acta. 2002;325(1–2):1–15. http://dx.doi.org/10.1016/s0009-8981(02)00275-9 5. chan ao, chan jp, choi kl, et al. a patient with an increased troponin level without evidence of ischaemic cardiac injury. hong kong med j. 2004;10(4):277–279. pmid:15299174 6. selby c. interference in immunoassay. ann clin biochem. 1999;36(pt 6):704–721. pmid:10586307 7. white gh, tideman pa. heterophilic antibody interference with cardiac t quantitative rapid assay [letter]. clin chem. 2002;48(1):201–202. pmid:11751561 8. shayanfar n, bestmann l, schulthess g, et al. false-positive cardiac troponin t due to assay interference with heterophilic antibodies [letter]. swiss med wkly. 2008;138(31–32):470. pmid:18690561 9. onuska kd, hill sa. effect of rheumatoid factor on cardiac troponin i measurement using two commercial measurement systems [letter]. clin chem. 2000;46(2):307–308. pmid:10657400 10. bohner j, von pape kw, hannes w, et al. false-negative immunoassay results for cardiac troponin i probably due to circulating troponin i autoantibodies [letter]. clin chem. 1996;42(12):2046. pmid:8969651 11. legendre-bazydlo la, haverstick dm, kennedy jl, et al. persistent increase of cardiac troponin i in plasma without evidence of cardiac injury. clin chem. 2010;56(5):702–705. http://dx.doi.org/10.1373/clinchem.2009.138164, pmid:20427735 12. adamczyk m, brashear rj, mattingly pg. coprevalence of autoantibodies to cardiac troponin i and t in normal blood donors [letter]. clin chem. 2010;56(4):676–677. http://dx.doi.org/10.1373/clinchem.2009.138099, pmid:20093555 13. berk je, kizu h, wilding p, et al. macroamylasemia: a newly recognized cause for elevated serum amylase activity. n engl j med. 1967;277(18):941–946. http://dx.doi.org/10.1056/nejm196711022771801, pmid:4167531 14. lee kn, csako g, bernhardt p, et al. relevance of macro creatine kinase type 1 and type 2 isoenzymes to laboratory and clinical data. clin chem. 1994;40(7 pt 1):1278–1283. pmid:8013099 15. krishnamurthy s, korenblat km, scott mg. persistent increase in aspartate aminotransferase in an asymptomatic patient. clin chem. 2009;55(8):1573–1575. http://dx.doi.org/10.1373/clinchem.2008.120782, pmid:19638492 16. fujita k, takeya c, saito t, et al. macro lactate dehydrogenase: an ldh–immunoglobulin m complex that inhibits lactate dehydrogenase activity in a patient’s serum. clin chim acta. 1984;140(2):183–195. http://dx.doi.org/10.1016/0009-8981(84)90343-7 17. wu ah. cardiac troponin: friend of the cardiac physician, foe to the cardiac patient? circulation. 2006;114(16):1673–1675. http://dx.doi.org/10.1161/circulationaha.106.652123, pmid:17043176 18. pettersson k, eriksson s, wittfooth s, et al. autoantibodies to cardiac troponin associate with higher initial concentrations and longer release of troponin i in acute coronary syndrome patients. clin chem. 2009;55(5):938–945. http://dx.doi.org/10.1373/clinchem.2008.115469, pmid:19264856 article information authors: siyem c. nkwawir1 nakeli n. batumani1 talkmore maruta2 charles n. awasom1 affiliations: 1bamenda regional hospital laboratory, cameroon2african society for laboratory medicine, ethiopia correspondence to: siyem nkwawir postal address: po box 818, regional hospital bamenda, north west region, cameroon dates: received: 27 may 2014 accepted: 20 aug. 2014 published: 03 nov. 2014 how to cite this article: nkwawir sc, batumani nn, maruta t, awasom cn. from grass to grace: how slmta revolutionised the bamenda regional hospital laboratory in cameroon. afr j lab med. 2014;3(2), art. #203, 6 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.203 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. from grass to grace: how slmta revolutionised the bamenda regional hospital laboratory in cameroon in this original research... open access • abstract • introduction • research methods and design    • phase 1: preparation    • phase 2: slmta programme implementation    • phase 3: evaluation • results • discussion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: public health laboratories form the foundation on which today’s clinical laboratory practice in cameroon is built. the advent of the strengthening laboratory management toward accreditation (slmta) programme in 2009 empowered the bamenda regional hospital laboratory (brhl) to improve its working culture, practices and management.objectives: to evaluate the results of slmta implementation at brhl and discuss lessons learned. method: in 2010, the slmta programme was rolled out in cameroon to improve laboratory quality management systems in five laboratories, including brhl. three workshops were conducted (the first centralised, the remaining two on-site at each laboratory) and improvement projects were implemented after each workshop with the assistance of mentors. audits were used in order to evaluate performance and to identify areas for further improvement. results: brhl had the lowest score (18%) amongst the cohort at the baseline audit and the highest (81%) at the official stepwise laboratory quality improvement process towards accreditation (slipta) audit conducted in august 2013 by the african society for laboratory medicine. improvements were observed in each of the 12 quality system essentials; improvement was especially noteworthy in the areas of facilities and safety, and purchasing and inventory. staff investment and pride in the quality of laboratory services increased. conclusion: brhl’s remarkable improvement was achieved with a combination of slmta training activities, intensive on-site mentorship and the collective focus of all laboratory staff. the experience at bamenda hospital illustrates what can be achieved when a laboratory successfully harnesses the energy of its staff and implements changes to improve the quality of services in a transformation taking them from grass to grace. introduction top ↑ the need to improve health outcomes regionally has led health systems and governments in africa toward evidence-based medicine, which requires access to high-quality information such as laboratory tests for decision making.1 given the emphasis on reliable data, increased attention has been focused on medical laboratories and their quality of services. in 2008, at a meeting in yaoundé, cameroon, organised by the world health organization’s regional office for africa (who afro), leaders from several african countries recognised the critical role of public health laboratories in disease control and prevention and resolved to strengthen public health laboratories throughout the region.2 in the following year, who afro launched a stepwise laboratory accreditation preparation scheme, later called the stepwise laboratory quality improvement process towards accreditation (slipta).3 slipta provides a framework for benchmarking progress using an audit checklist based on international organization for standardization (iso) 15189:2007 requirements. at the same meeting, the strengthening laboratory management toward accreditation (slmta) programme was launched in order to help support laboratory quality improvement. slmta is a laboratory management training programme which follows a multi-workshop implementation model4 and supports the development of the 12 quality system essentials (qses) of the clinical and laboratory standards institute (clsi) laboratory quality management system guidelines.5cameroon is a central african country with a population of approximately 20 million people living in 10 regions.6 bamenda is the headquarters of the northwest region, which has a population of approximately 1.8 million.6 bamenda regional hospital, a 400-bed public health hospital inaugurated in 1956, serves as the referral hospital for the 19 district hospitals of the northwest region. the field of laboratory medicine in cameroon has developed in several phases, starting with the paramedic ‘medical field unit’ introduced before independence in 1961. these laboratory medicine professionals received minimal training; their primary role was field identification of contagious diseases such as varicella, measles, yaws, smallpox, leprosy and tuberculosis. in most cases, they used basic identification techniques in mobile units.7 in 1968, a decree by the ministry of health led to the establishment of an organisational structure within public hospitals and health units.8 the hospital’s chief pharmacist became the laboratory head, under the supervision of the hospital director or a medical doctor who had specialised qualifications in microbiology, biochemistry, haematology or serology. at the time, laboratory services were either free or offered at minimal cost to the patient.8 two decades later, in 1990, a subsequent decree established organisational structures within private clinical laboratories.9 these statutes were designed to ensure the widespread provision of affordable laboratory services. they did not, however, address the quality of those services. in cameroon, the public health sector, in particular, has been characterised by a laissez-faire approach, with little concern for client satisfaction, limited specialised skills and mismanagement of both human and financial resources.10,11,12 in an effort to strengthen laboratory management so as to achieve rapid laboratory quality improvement, the slmta programme was launched in cameroon in october 2010. the bamenda regional hospital laboratory (brhl) was selected as one of five laboratories in the initial slmta cohort. laboratory personnel and other hospital staff participated in a series of training activities and quality improvement projects in an effort to enhance laboratory management, provide reliable patient services and create a lasting culture of quality. this article discusses slmta implementation at brhl, as well as results achieved and lessons learned. research methods and design top ↑ phase 1: preparation the us centers for disease control and prevention (cdc) served as the sponsor for slmta implementation in cameroon, with global health systems solutions (ghss) as the implementing partner. five laboratories (yaoundé central hospital laboratory, brhl, buea regional hospital laboratory, douala laquintinie hospital laboratory and laboratoire d’analyse medicales du centre yaoundé) were selected based on commitment from hospital laboratory management, appointment of a quality officer and the availability of participants who would maintain the same job responsibilities through the duration of the programme.4prior to slmta implementation, local capacity for auditors, trainers, and mentors was developed. in 2009, seven cameroonian auditors were trained to evaluate laboratory quality management systems (qms). five cameroonian laboratory personnel completed the slmta training-of-trainers course. in 2010, an experienced mentor and instructor from the clinton health access initiative (chai), lesotho, trained 11 cameroonian laboratory mentors. along with training on international standards, mentors were taught to build a sustainable culture of quality by transferring their knowledge and expertise to bench technologists. during the planning stages, cdc and ghss organised an advocacy meeting with hospital directors and laboratory managers of the five laboratories to ensure their buy-in and commitment to laboratory improvement. brhl further engaged hospital management for support in implementing qms, through meetings and active involvement in the laboratory improvement process. several hospital managers were also invited to attend the on-site slmta trainings, which helped encourage their support and collaboration. additionally, all hospital staff members were briefed on the laboratory improvement efforts. phase 2: slmta programme implementation the 12 slmta modules were taught in a series of three workshops, each lasting four days, over a period of eight months (table 1). the five in-country slmta trainers facilitated the programme. table 1: slmta workshop topics, bamenda regional hospital laboratory. the first workshop was conducted in mutengene, cameroon (october 2010) for staff from the five targeted laboratories, including four staff members from brhl. this workshop focused on the cross-cutting, productivity management, quality assurance, and documents and records management modules. following the first workshop, the slmta coordinators and trainers decided that it would be more effective and practical to hold the remaining two slmta training workshops at each of the enrolled laboratories so as to allow for more personnel to be trained. therefore, brhl’s second and third workshops were conducted on-site at the bamenda regional hospital. to maximise impact, 17 staff members attended the training, including the heads of the blood bank, reception and customer service, haematology, biochemistry, microbiology, serology and parasitology, as well as the safety officer, equipment officer, quality officer, laboratory director and hospital director. the 13 participants who missed the first centralised workshop were given materials to review. after each workshop, participants implemented improvement projects based on areas of weakness in the laboratory. all laboratory staff were involved in the improvement projects, including those who did not attend the training. following the training, ghss mentors were deployed to the five targeted laboratories, spending extended periods of time on site. the structured peer-to-peer, side-by-side mentorship approach was used to impart knowledge and expertise from the mentor to the laboratory staff following set guidelines.13,14 two additional mentors were assigned to brhl, alternating supervisory visits and working with all staff members to implement a change in culture and address gaps. iso 15189, the slipta checklist and the slmta toolkit were the primary tools used for follow-up. the mentors helped staff members address areas requiring improvement by examining the slmta toolkit, developing improvement projects, repeating activities from the training as needed, and then measuring improvement achieved using the checklist. phase 3: evaluation a series of audits using the slipta checklist were conducted and scores were calculated for each of the 12 qses so as to pinpoint problem areas and guide the development of improvement projects. overall scores were calculated as a percentage of the maximum possible points. to benchmark progress, these scores were also broken into ‘star’ levels, with zero stars corresponding to < 55%, one star 55% – 64%, two stars 65% – 74%, three stars 75% – 84%, four stars 85% – 94% and five stars 95% – 100%.the baseline audit was conducted 11 months prior to slmta implementation (november 2009). interim audits were conducted at various points (october 2010 before workshop 1; june 2011 before workshop 2; december 2011 after workshop 3; and february 2012). the exit audit was conducted in september 2012. these audits were performed by locally-trained auditors who were neither slmta trainers nor mentors for the brhl staff. finally, in august 2013, the african society for laboratory medicine (aslm) conducted an official slipta audit. results top ↑ brhl’s performance progressed steadily from 18% (zero stars) at baseline, where it was ranked last amongst the laboratories in its cohort, to 85% (four stars) in an interim audit eight months after completion of the three workshops, but then dropped to 67% (two stars) at the exit audit seven months later (figure 1). at the official aslm audit, the laboratory scored 81% (three stars), and was ranked first amongst the four laboratories audited, with the others scoring 60% – 72%. figure 1: figure 1: progress of the bamenda regional hospital laboratory in quality management systems as measured by the stepwise laboratory quality improvement process towards accreditation (slipta) checklist from november 2009 to august 2013. improvement was observed in each of the 12 qses measured in the slipta checklist (figure 2). from baseline to the last interim audit in february 2012, the most substantial improvements were in facilities and safety (93 percentage points), and purchasing and inventory (80 percentage points), whilst the least improved area was internal audit (6 percentage points). from the last interim to the exit audit, there were noted decreases in three areas: facilities and safety (37 percentage points), corrective action (33 percentage points), and equipment (27 percentage points). at the official aslm audit, the strongest areas were client management and customer service (100%), process control and internal and external quality assessment (94%), purchasing and inventory (93%) and facilities and safety (93%); whilst the weakest areas were corrective action (42%), management reviews (53%) and documents and records (56%). figure 2: performance of the bamenda regional hospital laboratory (%) across the 12 quality system essentials at baseline, last interim, exit and official aslm audit. improvement projects were selected and implemented after each slmta workshop (table 2). after each audit, the summary of non-conformities and recommendations was reviewed by the quality team and additional improvement projects were assigned, depending on what was feasible at the time, available resources and the seriousness of the non-conformity. table 2: a summary of improvement projects, bamenda regional hospital laboratory, cameroon. discussion top ↑ brhl’s substantial improvement was achieved by means of a combination of training activities, intensive on-site mentorship and collective focus of all laboratory staff. the experience at brhl illustrates what can be achieved when a laboratory successfully harnesses the energy of its staff and implements changes in order to improve the quality of services.engaging the management of bamenda hospital was key to ensuring financial support and sufficient human resources for the quality improvement process. the hospital director attended several of the slmta trainings, engaged directly with the mentor on quality management issues and increased the frequency of his visits to the laboratory. with management buy-in, the laboratory implemented infrastructure renovations and gained financial support for the purchase of reagents. since slmta completion, meetings between clinicians and laboratory staff have continued on a regular basis so as to enable laboratory staff to inform and consult with those they now see as critical clients and partners. because of these meetings, corrective actions have been easier to implement, customer satisfaction surveys have been facilitated and trust has been built between these users and providers of laboratory services. for brhl patients, the improvements have resulted in better service delivery because of procedure standardisation, fewer recalls of specimens, a safer environment, more reliability of testing and the ability to provide feedback on services offered. cameroon was the first country to decentralise slmta training,15 conducting the second and third workshops on-site at each enrolled laboratory. moving the slmta trainings to the facility allowed more staff to be trained and helped encourage universal participation and enthusiasm amongst all staff. whilst there was resistance to change in the beginning, by the third training there was a strong zeal to increase the laboratory’s star rating, as can be seen in the exit audit results. staff members became optimistic as the scores started to rise; their determination to improve the laboratory’s ratings was evidenced by their willingness to work extra hours, including weekends and public holidays. one young female laboratorian in the microbiology department said, ‘slmta has made us more ambitious. i now look forward to progressing in this profession and being able to help people of my community’. initially, the laboratory implemented what was possible at the time, rather than waiting for the ideal situation to arise. the quick successes of initial improvement projects bolstered staff confidence and inspired the desire for success, helping the laboratory sustain motivation for the more difficult projects. these successes also helped garner additional technical and financial assistance for further changes. whilst some improvement projects dealt with day-to-day activities, others sought to address larger challenges and attain more long-term goals. one noteworthy improvement project was to reorganise the reception area, which resulted in the creation of multiple reception sites, increased confidentiality and reduced turnaround time for test results. better organisation is a common theme in slmta training; one elderly female participant noted, ‘it has even extended to my home. now all my cupboards in the kitchen are labelled. all junk i removed’. laboratory staff members have reported a change in the culture of the laboratory. one commented that ‘our patients have ceased to be patients; they are now our clients’. the hospital director agreed, saying, ‘if i travel, i am now confident that work still continues, even in my absence’. he even took a leading role in directing improvement projects, personally supervising the closing of gaps in preparation for the official aslm audit. he met twice daily with quality team members for a period of three weeks; daily tasks were identified every morning and achievements and bottlenecks were reported every evening. a full-time mentorship approach allowed the on-site mentor to participate in daily activities in the laboratory and provide targeted training and guidance regarding the implementation of various aspects of the qms. the slmta-trained mentors helped build a working culture that emphasised quality and a sustainable qms. their continued presence over time helped them understand the local culture and practices, enabling them to design a tailored approach to assisting the laboratory in quality improvements. by working side-by-side with staff as peer coaches and role models, the mentors gained acceptance by laboratory personnel and accelerated implementation of quality requirements. the laboratory's performance increased steadily through slmta implementation, but the dramatic drop in the exit audit score 15 months after the last workshop highlights the importance of focusing on sustainability. staff had viewed the attainment of improved scores as the end of the struggle, rather than as part of an ongoing process of continuous quality improvement, requiring the maintenance of a new culture of quality. for example, client management and customer satisfaction, purchasing and inventory, process control and occurrence management all improved substantially from baseline to the last interim audit, but then declined once the slmta programme had ended and focus was lost. after the drop in score at the exit audit, the laboratory staff redoubled their efforts and results for these areas increased substantially once again by the official aslm audit. one area that did not improve after the exit audit was documents and records. whilst documentation improved from 6% at baseline to 72% at the last interim audit, it then dropped to 56% at exit where it remained at the official audit. the noted improvement resulted from the development and authorisation of many new policies and procedures, including a quality manual. however, by the time of the exit audit, the laboratory had begun to experience new challenges associated with the expanded documentation structure; the laboratory was overwhelmed by the number of documents that needed review and the ongoing maintenance required to retrieve documents, archive obsolete documents and communicate new changes. this experience highlights the need to plan ahead and to consider not only the development but also the maintenance of new systems. a new quality culture is growing at brhl as staff have embraced the need for continuous improvement and excellence in patient care. however, there is now a critical need to develop components outside the reach of the laboratory’s control: updated national guidelines for policies and procedures, professional boards for licensure, an up-to-date electronic database for proper document control and management, a hospital improvement programme to keep pace with improvements in the laboratory, regular audits of the qms and qualified service engineers. though the ultimate goals of reaching five stars and international accreditation have not yet been realised at brhl, the measurable improvement to date is a catalyst for greater achievements as the laboratory continues its journey from grass to grace. acknowledgements top ↑ the authors wish to acknowledge the commitment of the bamenda regional hospital management. we would also like to acknowledge the implementing partner (ghss), the sponsors (cdc) and all persons who supported slmta implementation at bamenda hospital. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions s.c.n. (brhl) wrote the manuscript. n.n.b. (brhl) was quality officer at the time and provided valuable data. t.m. (aslm) performed critical reviews and analyses. the article was conceived of and supervised by c.n.a. (brhl). references top ↑ 1. world health organization’s regional office for africa. resolution afr/rc59/r4: policy orientations on the establishment of centres of excellence for disease surveillance, public health laboratories, food and medicines regulation. in: final report: 59th session of the who regional committee for africa. brazzaville, republic of the congo: world health organization’s regional office for africa, 2009; p. 12–13.2.world health organization’s regional office for africa. resolution afr/rc58/r2: strengthening public health laboratories in the who african region: a critical need for disease control. in: final report: 58th session of the who regional committee for africa. yaounde, republic of cameroon: world health organization’s regional office for africa, 2008; p. 11–13. 3. world health organization regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region (with checklist) [document on the internet]. c2011 [cited 2013 may 31]. available from: http://www.afro.who.int/index.php?option=com_docman&task=doc_download&gid=8642&itemid=2593 4.yao k, mckinney b, murphy a, rotz p, wafula w, sendagire h, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010 sep;134(3):401-9. pubmed pmid: 20716796. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj 5.clinical and laboratory standards institute. application of a quality management system model for laboratory services; approved guidelines – third edition. clsi document gp26-a3 [isbn 1-56238-553-4]. clinical and laboratory standards institute; 2004. 6.republic of cameroon. 3e rgph. la population du cameroun en 2010 [cameroon’s population in 2010] [document on the internet]. c2010 [cited 2014 sep 09]. available from: http://www.statistics-cameroon.org/downloads/la_population_du_cameroun_2010.pdf 7.awasum ss. evaluation of medical laboratory practice and training in anglophone cameroon; 2002 (unpublished presentation: 1st national scientific conference of medical laboratory technologists, ‘creating professional awareness in the new millennium’, 3–4 may 2002). 8.decree no. 68-df-419 of 15th october 1968: establishing the structural organization and the organic functioning of the hospital and health facilities in cameroon. government printers; 1968. 9.federal republic, cameroon. decree no 90-1465 of 9 november 1990: to lay down the organization and functioning of private clinical laboratories. government printers; 1990. 10.agendia a. cameroon’s free fall health system: two videos shock the world [page on the internet]. c2010 [cited 2014 sep 07]. available from: http://agendia.jigsy.com/entries/health/cameroon%e2%80%99s-free-fall-health-system-two-videos-shock-the-world 11.achanyi-fontem j. cameroon government x-rays health sector plan for 2012 [page on the internet]. c2008 [cited 2014 sep 07]. available from: http://www.leffortcamerounais.com/2008/03/cameroon-govern.html 12.the world justice project. cameroon healthcare access program [page on the internet]. c2008 [cited 2014 sep 09]. available from: http://worldjusticeproject.org/opportunity-fund/cameroon-healthcare-access-program-1 13.maruta t, motebang d, wanyoike j, et al. impact of mentorship on who-afro strengthening laboratory quality improvement process towards accreditation (slipta). afr j lab med. 2012;1(1), art. #6, 8 pages. http://dx.doi.org/10.4102/ajlm.v1i1.6 14.maruta t, rotz p, trevor p. setting up a structured laboratory mentoring programme. afr j lab med. 2013;2(1), art. #77, 7 pages. 15.ndasi j, dimite l, mbome v, et. al. decentralised facility-based training as an alternative model for slmta implementation: cameroon experience. afr j lab med. 2014;3(2), art. #231, 6 pages. http://dx.doi.org/10.4102/ajlm.v3i2.231 article information authors: elizabeth t. luman1 katy yao1 john n. nkengasong1 affiliations: 1international laboratory branch, division of global hiv/aids, center for global health, us centers for disease control and prevention, atlanta, georgia, united states correspondence to: elizabeth luman postal address: 1600 clifton road, atlanta, ga 30333, united states dates: received: 18 sept. 2014 accepted: 21 sept. 2014 published: 03 nov. 2014 how to cite this article: luman et, yao k, nkengasong jn. a comprehensive review of the slmta literature part 1: content analysis and future priorities. afr j lab med. 2014;3(2), art. #265, 11 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.265 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. a comprehensive review of the slmta literature part 1: content analysis and future priorities in this review article... open access • abstract • introduction • research methods and design • results and discussion    • literature search results    • previous experience with quality management systems    • the drive for action    • implementation    • capacity building    • partnership    • on-site support and mentorship    • success factors    • challenges    • limitations to the study    • feedback on the slmta programme • future directions    • progression (continued improvement in slmta laboratories)    • saturation (additional laboratories within countries that have implemented slmta)    • expansion (implementation in additional countries)    • extension (adapting slmta for implementation beyond the laboratory)    • transformation (impact on health systems and patient care) • acknowledgements    • competing interests    • authors’ contributions    • cdc disclaimer • references abstract top ↑ background: since its introduction in 2009, the strengthening laboratory management toward accreditation (slmta) programme has been implemented widely throughout africa, as well as in the caribbean, central and south america, and southeast asia.objective: we compiled results from local, national and global studies to provide a broad view of the programme and identify directions for the future. the review consists of two companion papers; this paper focuses on content analysis, examining various thematic components of the slmta programme and future priorities. methods: a systematic literature search identified 28 published articles about implementing the slmta programme. results for various components of the slmta programme were reviewed and summarised. results: local and national studies provide substantial information on previous experiences with quality management systems; variations on slmta implementation; building human resource capacity for trainers, mentors and auditors; the benefits and effectiveness of various types of mentorship; the importance of management buy-in to ensure country ownership; the need to instill a culture of quality in the laboratory; success factors and challenges; and future directions for the programme. conclusions: local, national and global results suggest that the slmta programme has been overwhelmingly successful in transforming laboratory quality management. there is an urgent need to move forward in four strategic directions: progression (continued improvement in slmta laboratories), saturation (additional laboratories within countries that have implemented slmta), expansion (implementation in additional countries), and extension (adapting slmta for implementation beyond the laboratory), to lead to transformation of overall health systems and patient care. introduction top ↑ the strengthening laboratory management toward accreditation (slmta) programme is a structured quality improvement curriculum designed to achieve immediate and tangible advances in health service delivery.1 the central feature of this intervention is its emphasis on the ‘how-to’ of implementing quality management systems (qms) by translating the ‘what’ (concepts, principles, theories, guidelines and standards) into practical behaviours, laboratory practices and daily routines, through hands-on practice during training and improvement projects in trainees’ home laboratories. the importance of practical training dedicated to building management capacity has been well deliberated in a series of working papers published by the world health organization (who) on strengthening leadership and management in low-income countries, titled ‘making health systems work’.2,3,4,5in the five years since its introduction in 2009, the slmta programme has been implemented widely throughout the developing world.1 in december 2012, slmta country coordinators and implementers gathered at a two-day symposium in cape town, south africa to discuss their experiences and lessons learned. it was evident that a substantial amount of programmatic expertise had been gained. a supplemental issue of the african journal of laboratory medicine (ajlm) was commissioned in order to document and share successes and challenges, summarize laboratoryand country-specific analyses, and publish global programme data. this systematic literature review aims to compile existing fragmented results into a comprehensive report, to provide a broad view of the programme and to identify directions for the future. because of the large volume of information collected, the review has been published in two parts. part 1 focuses on content analysis, examining various thematic components of the slmta programme and future priorities. part 2, published separately, compiles the quantitative data reported in the publications, examines scores and indicators, and uses meta-analysis to augment the results.6 research methods and design top ↑ a comprehensive search of electronic bibliographic databases was performed, including medline and the directory of open access journals, using the keyword ‘slmta’. slmta country programme leaders and partner agencies were contacted so as to identify additional sources. we included all published and in-press studies that discussed the slmta programme. the majority of the search results were in-press manuscripts being prepared for the supplemental issue of ajlm focusing on the slmta programme, called ‘transforming the quality of laboratory medicine through the strengthening laboratory management toward accreditation program’; authors of this review coordinated the issue. results and discussion top ↑ literature search results we identified 28 published manuscripts focusing on the slmta programme,1,7– 33 including 26 published concurrently with this article,1,7-20,22-31,33one previously-published article summarising the development and methodology of the slmta programme32 and one previously-published study regarding slmta implementation in lesotho21 (table 1). six papers presented experiences from a single laboratory7,10,17,26,28 or a single hospital,9 14 presented data from two to 45 laboratories within a single african country,8,12,– 15,18,– 22,25,29– 31 one discussed activities in two southeast asian countries,23 one covered data from four countries in the caribbean region11 and six had a general or global focus.1,16,23,24,32,33 in total, these studies included detailed information on slmta implementation in 211 laboratories in 18 countries. table 1: characteristics of published slmta studies. previous experience with quality management systems a recent study summarised the scarcity of accreditation in public laboratories throughout sub-saharan africa.34 however, the results from the slmta laboratories paint an even more problematic picture – not only are laboratories in developing countries not meeting the international quality standards needed for accreditation, but the level of quality management is extremely low. several authors reported that their laboratories had no prior experience with qms,7,10,17,22 with one study reporting that prior to slmta, ‘the idea of qms was entirely new to most laboratory staff in the selected facilities, where a culture of quality has been lacking’.22 the low level of baseline audit scores across the programme – 84% of laboratories did not reach even one star on the who regional office for africa’s (afro) stepwise laboratory quality improvement process towards accreditation (slipta) five-star scale and the mean baseline audit score was 39% – seems to confirm this assessment.33 these findings are supported by a study in kampala, uganda, which found that only 5% of audited laboratories in the city had reached one star on the slipta quality scale.35previous experience in establishing qms was limited in these countries and what had been done was largely reported to be ineffective. one study of slmta implementation in the caribbean region reported that ‘past laboratory assessments and training did not provide them with a structured roadmap to assist in implementation; as a result, the majority of these laboratories did not initiate the process of qms development and implementation’.11 another reported that ‘prior to the introduction of slmta, several trainings and quality improvement initiatives had been implemented in hospitals and laboratories in ethiopia, but little improvement was noted’.12 in botswana, there had been ‘slow progress in implementing qms’, and authors noted that ‘previous training of healthcare workers has focused on general management topics rather than identifying tangible tasks to bring about change, making the training difficult to apply in the laboratory’.28 the drive for action several authors report that implementation of slmta came after years of neglect of laboratories. in many cases, these circumstances improved with the advent of the us president’s emergency plan for aids relief (pepfar) in 2003,7,11,14,15,22,27 which emphasised the importance of quality laboratory tests and infused much-needed capital into the laboratory systems.36 other international programmes also played a key role, such as the global fund to fight aids, tuberculosis and malaria37 and the global health initiative.3838 concurrently, several regional and global policy statements called for strengthening public medical laboratories: the maputo declaration, which called on ‘national governments to support laboratory systems as a priority’;39 the lyon statement on the need for laboratory qms and accreditation of national reference laboratories;40 and the yaoundé resolution, in which who afro recognised the need to further strengthen public health laboratories and to encourage member states ‘to develop or strengthen comprehensive national laboratory policies’.41authors noted that also during this timeframe, countries began to develop five-year laboratory strategic plans – kenya14,15 and ethiopia12,13 in 2005; botswana20,28 and lesotho21 in 2008; zimbabwe30 in 2010; and ghana25 and namibia19 in 2012. these plans called for laboratory strengthening and development of qms, some specifying accreditation goals. for example, the laboratory strategic plan in botswana called for implementation of qms in all laboratories by 2014 and accreditation of four district-level laboratories by 2013 and two national-level laboratories by 2014.20 in kenya, the ministry of health set a goal to accredit all national and regional level public laboratories10,14 and established a national accreditation steering committee to coordinate accreditation activities. the rwanda ministry of health aimed to enroll all 48 central and district hospital laboratories in the accreditation preparation process,29 and the ministry of health in mozambique established a national laboratory policy, which outlined their commitment to implement qms and to pursue accreditation for reference and provincial hospital laboratories.18 the focus on laboratory quality improvement led these countries to implement the slmta programme in order to help them improve their quality management. implementation the standard slmta implementation model includes three workshops followed by periods of several months for laboratories to implement improvement projects, usually with onsite support and mentorship.1 the slmta programme is evaluated through audits based on the slipta checklist.1several countries have customised slmta implementation to meet their needs. in cameroon, slmta workshops were decentralised and conducted on site, allowing many more staff to participate in training.1,22,26 the slmta team in vietnam developed an electronic tool for slipta audits so as to improve timeliness and accuracy of audit results and reduce language barriers.23 in rwanda, performance-based financing was used in one cohort, in which payment was provided to laboratories based on slipta audit scores to incentivise continuous quality improvement.29 in several programmes, non-laboratory personnel participated in the slmta training, including hospital managers and administrators9,22,26 as well as clinicians.13,22,29 several countries have established departments or workgroups dedicated to the implementation of quality management. for example, zimbabwe’s national quality assurance program established a training and mentorship department;30 kenya’s ministry of health created a national accreditation steering committee to coordinate laboratory accreditation activities;14 and mozambique established a national laboratory technical working group to build a framework for a national laboratory quality improvement programme, to lead and coordinate its implementation and to monitor and maintain results.18 individual facilities also established quality improvement programmes. for example, a hospital in cameroon developed a quality improvement task force to coordinate quality improvement efforts.9 one laboratory in kenya formed a tiered accreditation team structure, including a management team, quality assurance team and section teams, to run the programme and lead the laboratory to international accreditation.10 capacity building with the rapid expansion of the slmta programme, the need to build capacity for more slmta trainers, mentors and auditors has been identified.1,16 maruta et al. summarise global efforts to develop trainers and master trainers using a training-of-trainers (tot) strategy with teach-back methodology.16 the tot course is an intensive two-week training course taught by master trainers, in which candidate trainers learn to teach the 44 activities in the slmta curriculum (through a combination of skills learning, practice and feedback) and to follow set guidelines for programme implementation. as of the end of 2013, 433 trainers and 38 master trainers have been produced, and the vast majority (97% and 87%, respectively) are based in developing countries. tot courses have been held in botswana, dominican republic, ethiopia, ghana, kenya, mozambique, nigeria, rwanda, south africa, tanzania, vietnam and zimbabwe and have been conducted in english, portuguese, spanish and vietnamese.critical considerations include ensuring that tot graduates are utilised effectively and that fidelity of implementation is maintained as the programme expands. the maruta et al. study found a 92% utilization rate of tot graduates, with 97% of participants reporting that the tot trained them either well or extremely well for implementing slmta.16 furthermore, global data suggest that training quality has been maintained, as the 132 laboratories that implemented slmta during the first year (2010) had the same mean improvement (24 percentage points) as the 170 laboratories that implemented slmta in subsequent years (2011–2013).33 the development of mentors and auditors has not been summarised globally, although several studies report country-specific efforts. in cameroon, 11 mentors and seven auditors have been trained to support programme scale-up.33 in rwanda, 17 local mentors were trained to roll out the slmta programme.29 in mozambique, 15 auditors were trained18 and in ghana, 15 mentors and 11 auditors were trained.25 several papers discussed national plans to train additional mentors and auditors.8,18,19,25,26,29 one critical consideration for auditor training is to ensure high qualifications and consistency between them. several studies discussed the variability of auditor expertise and reliability as a serious limitation of both the programme and their reported results.8,29,33 partnership the rapid and widespread expansion of the slmta programme could not have occurred without the active participation of an extensive network of partners. these various partners have been instrumental in all aspects of programme development and implementation (table 2). primarily funded through pepfar1 and developed under the leadership of the us centers for disease control and prevention (cdc), ministries of health have implemented the programme with support from international organisations, institutions and private companies, parastatal organisations and local non-governmental organisations. one study focused on the use of partnership to implement slmta in 15 laboratories in ghana, concluding that ‘building in-country capacity through local partners is a sustainable model for improving service quality in resource-constrained countries’ and that ‘local partners, when supported and managed adequately, can achieve great results at a reasonable cost’.25 table 2: partners contributing to the slmta programme as reported by published studies. table 2 continues: partners contributing to the slmta programme as reported by published studies. on-site support and mentorship on-site support and mentorship are key components of the slmta programme, as mentors are expected to provide in-depth support after workshops to assist laboratory personnel in implementing changes.32 support and mentorship models have ranged from no site visits or mentorship,8,20 occasional visits,14 periodic visits of several days to several weeks,8,10, 16, 28, 29, 30 to embedded mentors working full time in their assigned laboratories for extended periods11, 14, 18, 22, 25, 26, 29, 30 (table 3). kenya has piloted a novel mentorship model, ‘twinning’ public laboratories with local state-of-the-art research laboratories. this institutional mentorship approach holds promise not only for laboratory improvement, but also for fostering long-standing sustainable partnerships between public health and research laboratories.15 table 3: mentorship models reported by slmta studies. well-planned scientific studies are lacking with regard to the effectiveness of mentorship overall or the relative effectiveness of various models. all of the slmta-related studies conducted to date have serious methodological flaws, primarily the lack of random assignment to mentorship models, lack of control groups and small sample sizes. despite these limitations, several post-hoc analyses comparing results in mentored laboratories to those in non-mentored laboratories,8,20 as well as intensive mentorship to less intensive mentorship,15,29 concur that mentorship appears to be beneficial (table 3). these findings agree with an earlier study that found that slipta scores increased in four lesotho laboratories after mentorship, although no control laboratories were used on which to base a comparison.42 despite the lack of solid scientific evidence, there is a general belief that mentorship is an important component of the slmta programme and a critical factor for success. success factors numerous factors have been identified by authors as being critical to the success of slmta implementation. at the facility level, many authors reported the importance of engaging hospital and senior management from the beginning so as to ensure their buy-in and ownership of the programme7,12, 17, 20, 25, 26, 28, 29, 30 and to promote institutionalisation and thus sustainability.18 many also noted the importance of a strong commitment and team spirit amongst the laboratory staff7,9, 10, 18, 20, 28, 29 and a willingness to build a culture of quality17,26 and problem solving.20 the various components of the slmta programme – including the how-to guidance provided by the workshops;7,10, 17, 28, 32 mentorship7, 10, 11, 25, 30 and supervisory visits7, 12, 18, 21, 32 to keep laboratories on track; improvement projects;20, 26, 32 and ensuring that both staff11, 17, 32 and leadership17 are accountable and motivated – were viewed as critical. in addition, authors noted that it is essential to measure what has been accomplished through audits using the slipta checklist10, 11, 17, 18, 21, 32 and analysis of other key indicators.28 these data are powerful means to recognise and motivate staff, determine further actions needed17 and provide information as an advocacy tool.16 finally, ongoing communication with hospital management21 and clinical staff9, 17, 26, 28 is critical so as to ensure continued focus on patient care and support for future activities.at the country level, it is critical to ensure clear commitment and ownership within the ministry of health in order to improve laboratory quality at all levels, including development of a national laboratory policy and strategic plan, establishment of a laboratory technical working group and provision of financial support.1, 21, 25 early engagement of key stakeholders and partners,11,21, 25 followed by effective communication and continuous advocacy for laboratory quality,9, 12, 18 were identified as important components of success, as were the development of a programme implementation plan9, 10, 11 that includes human resource needs for trainers, auditors and mentors;1 a plan to ensure sufficient geographic coverage through careful site selection;1, 18 and establishment of specific programme goals.18 challenges challenges at both the facility and programme levels were identified, as well as those beyond the scope of the programme that affect the programme. at the facility level, common concerns surrounded the difficulty of engaging non-slmta-trained laboratory staff,1, 10, 12, 13 as well as hospital management and regional health bureaus1, 12, 13 and ensuring harmonisation with other hospital improvement programmes.12 it was noted that behavioural change requires time, commitment and consistent support.32 also documented were difficulties in: providing sufficient site support;1, 12 ensuring that staff understand the requirements of qms8 and the international organization for standardization (iso) 15189 standard;8, 10, 12 how to conduct internal audits;25 and the importance of establishing quality in laboratory testing.22 implementing a qms is a difficult process; particularly noted were the challenges of: balancing the requirements of multiple functions within a laboratory;20 establishing root causes of nonconformities;8, 14 equipment maintenance and outages;13, 22 development of method validation;10 space shortages;10 document review and maintenance;7, 20, 26 insufficient time to implement improvement projects;21 and the general concern that the entire process required more time and resources than anticipated.13at the programme level, the shortage of well-trained mentors11, 13, 15, 25 was a common concern, as were the lack of trainers22 and auditors.23 in addition, it was noted that mentors and other implementers have competing duties, since they are generally not dedicated solely to slmta activities,18, 30 which may lead to suboptimal utilisation rates of trainees.16 furthermore, language and communication barriers amongst mentors, trainers and auditors can be a challenge,23,25 exacerbating the shortages. also reported were higher-level challenges that had an impact on programme implementation. the most-commonly reported challenge was that of staff attrition.10, 11, 13, 17, 20, 21, 29, 31, 32 one study of programme implementation in the caribbean region reported the following: [e]nsuring a sufficient number of well-qualified laboratory workers is an ongoing challenge, exacerbated by high levels of attrition as staff that have benefitted from government-supported training leave the public sector for more lucrative jobs in the private sector, either locally or overseas. thus the remaining staff are overworked, reducing the amount of time available for training and quality improvement activities.11 in a study from rwanda, the only laboratory whose scores decreased from baseline to exit audit lost both their quality officer and laboratory manager during the programme;29 and a laboratory in kenya found that patient complaints increased as a result of high staff turnover.17 shumba et al. suggest several strategies to reduce staff attrition, including encouraging ministries of health and supervisors to agree not to reassign trained staff for a period of time, providing financial incentives to participants at the completion of slmta and using binding contracts in which participants agree to remain on the job for a specified period.31 decentralised training may also help reduce the effect of attrition; in cameroon, the authors concluded that, ‘[i]n the decentralised model, the majority of laboratory staff are trained to implement qms, reducing the impact of attrition of a few trained staff members’.22 in addition, studies reported a general shortage of qualified laboratory staff, especially staff with qms expertise.11,12,22, 25, 29 also noted was a lack of quality manuals, guidelines and procedures12 to provide clear direction, as well as a lack of national strategic plans22 to define stakeholders and facilitate coordination with partners.13 these issues, when coupled with institutional bottlenecks,22,25 slow procurement processes10 and lack of or limited accreditation preparation budgets,12,25 further hamper improvement efforts. finally, the existing low level of quality management in developing countries33 suggests that much work is needed to ensure sufficient quality of laboratory services to provide for public and personal health needs. limitations to the study this review is subject to several limitations. primarily, the results may not be representative of the programme as a whole, or a comprehensive account of all laboratories’ experiences. for example, whilst these studies mentioned some 30 partners who helped to develop or implement the slmta programme, others were undoubtedly missed. feedback on the slmta programme overall, the published literature was strongly in favour of the slmta programme, with investigators and public health officials reporting satisfaction with the results. nkengasong and birx suggest that ‘with the introduction of slmta, the prospects of implementing sustainable quality-assured laboratory medicine seem to be a reality in developing countries’.24 other investigators agreed, saying that slmta ‘was found to be a practical option that yielded positive results for strengthening laboratories’20 and also that ‘[t]he tremendous improvement… shows that slmta coupled with mentorship is an effective, user-friendly, flexible and customisable approach to implementation of laboratory qms’.11a study of attitudes of health professionals in ethiopia found that laboratory professionals had a supportive perception of slmta, whilst some hospital chief executive officers ‘were more sceptical of slmta and raised concerns regarding programme costs and the prolonged process associated with implementation’.13 a hospital director in cameroon disagreed, saying that: slmta is an invaluable tool for every lab director, every hospital manager and health policy maker because of its value in ensuring quality improvement within the laboratory and its potential in contributing to strengthening the entire health system at little or no cost.9 future directions top ↑ the slmta programme is expanding rapidly and authors have identified an urgent need to sustain the gains and move forward in four strategic directions to lead to transformation of overall health systems and patient care (figure 1). figure 1: future directions of the slmta programme. progression (continued improvement in slmta laboratories) several authors have discussed the need to ensure that laboratories sustain gains made and continue to move forward.8,11, 17, 20, 26, 27, 29 yao et al. point out that quality improvement should be seen as an ongoing journey and that slmta provides the tools needed to ensure better patient care;1 ntshambiwa et al. concur that slmta has ‘helped lay a firm foundation for further advancements in patient care’.28 rwanda’s first two slmta cohorts not only sustained their results at a surveillance audit a year after slmta completion, but increased their scores by a median of 10 percentage points.29 globally, 92 laboratories have completed surveillance audits; 62% further increased their score.33 nkrumah contends that indigenous capacity building is critical in order to ensure sustainability,25 whilst maruti et al. focus on the need to change the laboratory culture by establishing universal rules, teaching staff the principles and techniques of quality improvement, continually reinforcing the behaviours by integrating them into daily routines and engaging hospital stakeholders.17 audu et al. argue that ‘sustainability is a common concern for any improvement programme; once the intense focus of implementation ceases, special efforts and continued supervision are required so as to ensure that old habits do not return’.8 maina et al. also focus on post-slmta sustainability, identifying internal audits and corrective action as catalysts for continued improvement.14 noble et al. commend the achievements made by laboratories to date, cautioning:but to them we put forward this challenge: whilst it is a great achievement to reach a level of success where the requirements of accreditation are met, the true accomplishment is reaching the point where the level of quality is an everyday practice and expectation, and the laboratory is ‘accreditation-ready’ over and over. when the inevitable slips and mistakes occur in laboratories that are accreditation-ready, the processes of error detection, correction and improvement, and progress back to quality, must occur quickly, smoothly and sustainably.27 as the slmta programme matures, studies measuring the long-term sustainability of results and examining factors associated with continued progress will be critical for ensuring the enduring impact of the programme. saturation (additional laboratories within countries that have implemented slmta) several countries have established formal plans for country-wide qms implementation, using the slmta programme as the central improvement tool.18,25, 29 many have already implemented second (13 countries), third (six countries) and further cohorts (three countries) as they expand slmta nationally; of the 21 countries that began slmta in 2010–2011, 16 (76%) have thus far implemented additional cohorts.33 kenya has conducted six rounds of slmta, whilst lesotho has reached near saturation, with slmta implemented in 18 of its 19 laboratories.33several authors discuss the next steps toward achieving greater saturation within countries. most common amongst these is the need to develop additional local trainers, mentors and auditors,1,11, 13, 16, 22, 23, 25, 30 as well as educating the general workforce through pre-service training.24 guevara et al. suggest that ‘there is now a need to internalise the programme and transition it to local governments and other donors in order to facilitate expansion and ensure sustainability’,11 whilst lulie et al. argue that further efforts are needed to ‘decentralise responsibility from the government to the management at their facilities’.13 it is evident that not all laboratories in a country’s health system need to be accredited; however, all laboratories must maintain a culture of continuous quality improvement. nkengasong and birx discuss the need for countries to identify a ‘tipping point’ or threshold of laboratories that must be accredited in order to establish this culture and ‘increase confidence in quality-assured laboratory medicine for evidence-based patient management’.24 once accreditation goals are defined in national laboratory strategic plans, a slmta implementation plan can be developed with clear priorities to help guide laboratories in the tiered health system to achieve their goals. masamha et al. suggest that, in addition to accreditation, countries could track the progress of quality systems with indicators such as: [the] number of provinces with dedicated quality management officers; percentage of laboratories audited in the previous 12 months; percentage of audited laboratories demonstrating improvement as measured by the slipta checklist; and percentage of laboratories implementing external proficiency testing for select services.18 expansion (implementation in additional countries) slmta implementation started with 11 countries in 2010 and spread to an additional 10 in 2011, 15 in 2012 and 11 in 2013.33 because pepfar is the primary funding source, slmta to date has been rolled out largely in pepfar-supported countries43 (43 of the 47 countries implementing slmta are pepfar-funded, 91%). as of the end of 2013, 75% of the 57 pepfar-supported countries have implemented slmta, with most of the remaining countries located in asia.to date, slmta has been implemented in 38% of low-income countries and 26% of lower-middle-income countries, based on world bank classifications.44 further expansion beyond pepfar-supported countries to other lowand lower-middle-income countries will require the identification of additional global partners to provide funding and implementation support. extension (adapting slmta for implementation beyond the laboratory) to take full advantage of the benefits of improved laboratory quality, improvements will need to be made outside the scope of the laboratory as well. in her commentary, the laboratory director for namibia’s ministry of health and social services (mohss) explains:as quality improvements become institutionalised in hospital laboratories, it is becoming evident that entire hospital systems are in dire need of similar quality improvement programmes. the namibia mohss calls on international agencies to develop and adapt programmes such as slipta and slmta for use throughout hospital systems so as to ensure continuous quality patient care.19 along the same lines, eno et al. report on the experience of one hospital that adapted the slmta tool for wider implementation, inspired by successful implementation of the programme in their laboratory.9 results were encouraging, with ‘steady improvement in service delivery’; reduced patient wait times, infection rates and stillbirths; and increased hospital revenue. the authors concluded that ‘[s]uch a programme has the potential to impact positively on hospital quality systems; consideration should be made for development of a formal slmta-like programme for hospital quality improvement … expanding the strengthening laboratory management toward accreditation programme into one for strengthening hospital management toward accreditation’.9 transformation (impact on health systems and patient care) as slmta continues to grow, it has the potential to have a profound and lasting impact on health systems and patient care. in a report on the impact of pepfar, the institute of medicine concluded that:pepfar’s laboratory efforts have had a fundamental and substantial impact on laboratory capacity in countries. this laboratory infrastructure and capacity has been, and can continue to be, leveraged to improve the functioning of countries’ entire health systems.36 there is a growing movement toward establishing a culture of quality at all levels of service in order to care for patients,24 including not only the laboratory but the pharmacy, clinics, maternity, surgical rooms and others. as quality improves, there is a need to measure the impact on patient outcomes through well-defined and rigorous programme evaluation. such data will provide local, national and global decision makers with the evidence needed to justify expenditures and implement the most appropriate solutions for their given situations. acknowledgements top ↑ we would like to thank the lead authors of the in-press papers for allowing us to examine their results prior to publication, making it possible to publish this review simultaneously with their work: linda andiric, rosemary audu, laura eno, thomas gachuki, giselle guevara, tilahun hiwotu, adino lulie, robert maina, ernest makokha, talkmore maruta, phidelis maruti, jessina masamha, mary mataranyika, kelebeletse mokobela, juliana ndasi, thuong nguyen, bernard nkrumah, siyem nkwawir, michael noble, keoratile ntshambiwa, innocent nzabahimana, phoebe nzombe and edwin shumba. special thanks go to philip rotz, lee schroeder, bethanie rammer and penny smorenburg for their valuable feedback in manuscript revision. this research has been supported by pepfar through the cdc. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions e.t.l (cdc, atlanta) analysed the data and wrote the manuscript. k.y. (cdc, atlanta) and j.n.n (cdc, atlanta) provided substantial input to the revision of the manuscript. cdc disclaimer the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the cdc. references top ↑ 1. yao k, maruta t, luman et, nkengasong jn. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(2), art. #194, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i2.1942. egger d, travis p, dovlo d, et al. strengthening management in low-income countries. making health systems work: working paper no. 1 who/hss/healthsystems/2005.1 [document on the internet]. c2005 [cited 2014 oct 04]. available from: http://www.who.int/management/working_paper_1_en_opt.pdf 3. egger d, ollier e. managing the health millennium development goals – the challenge of management strengthening: lessons from three countries: report on an international consultation on strengthening health leadership and management in low-income countries, 29 january – 1 february 2007, accra, ghana. making health systems work: working paper no. 8 who/hss/healthsystems/2007.1 [document on the internet]. c2007 [cited 2014 oct 23]. available from: http://www.who.int/management/working_paper_8_en_opt.pdf 4. waddington c, egger d, travis p, et al. towards better leadership and management in health: report on an international consultation on strengthening health leadership and management in low-income countries, 29 january – 1 february 2007, accra, ghana. making health systems work: working paper no. 10. who/hss/healthsystems/2007.3 [document on the internet]. c2007 [cited 2014 oct 22]. available from: http://www.who.int/management/working_paper_10_en_opt.pdf 5. egger d, ollier e, tumusiime p, j. b. strengthening management in low-income countries: lessons from uganda. making health systems work: working paper no. 11. who/hss/healthsystems/2007.4 [document on the internet]. c2007 [cited 2014 oct 23]. available from: http://www.who.int/management/working_paper_11_en_opt.pdf 6. luman et, yao k, nkengasong jn. slmta: a comprehensive review of the slmta literature part 2: measuring success. afr j lab med. 2014;3(2), art. #276, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i2.276 7. andiric lr, massambu cg. one laboratory’s progress toward accreditation in tanzania. afr j lab med. 2014;3(2), art. #202, 4 pages. http://dx.doi.org/10.4102/ajlm.v3i2.202 8. audu ra, onubogu cc, nwokoye nn, et al. improving quality in national reference laboratories: the role of slmta and mentorship. afr j lab med. 2014;3(2), art. #200, 7 pages. http://dx.doi.org/10.4102/ajlm.v3i2.200 9. eno lt, asong t, ngale e, et al. driving hospital transformation with slmta in a regional hospital in cameroon. afr j lab med. 2014;3(2), art. #221, 5 pages. http://dx.doi.org/10.4102/ajlm.v3i2.221 10. gachuki t, sewe r, mwangi j, et al. attaining iso 15189 accreditation through slmta: a journey by kenya’s national hiv reference laboratory. afr j lab med. 2014;3(2), art. #216, 9 pages. http://dx.doi.org/10.4102/ajlm.v3i2.216 11. guevara g, gordon f, irving y, et al. the impact of slmta in improving laboratory quality systems in the caribbean region. afr j lab med. 2014;3(2), art. #199, 9 pages. http://dx.doi.org/10.4102/ajlm.v3i2.199 12. hiwotu tm, ayana g, mulugeta a, et al. laboratory system strengthening and quality improvement in ethiopia. afr j lab med. 2014;3(2), art. #228, 6 pages. http://dx.doi.org/10.4102/ajlm.v3i2.228 13. lulie ad, hiwotu tm, mulugeta a, et al. perceptions and attitudes toward slmta amongst laboratory and hospital professionals in ethiopia. afr j lab med. 2014;3(2), art. #233, 6 pages. http://dx.doi.org/10.4102/ajlm.v3i2.233 14. maina rn, mengo dm, mohamud ad, et al. progressing beyond slmta: are internal audits and corrective action the key drivers of quality improvement? afr j lab med. 2014;3(2), art. #222, 7 pages. http://dx.doi.org/10.4102/ajlm.v3i2.222 15. makokha ep, mwalili s, basiye fl, et al. using standard and institutional mentorship models to implement slmta in kenya. afr j lab med. 16. maruta t, yao k, ndlovu n, moyo s. training-of-trainers: a strategy to build country capacity for slmta expansion and sustainability. afr j lab med. 2014;3(2), art. #196, 7 pages. http://dx.doi.org/10.4102/ajlm.v3i2.196 17. maruti pm, mulianga ea, wambani ln, et al. creating a sustainable culture of quality through the slmta programme in a district hospital laboratory in kenya. afr j lab med. 2014;3(2), art. #201, 5 pages. http://dx.doi.org/10.4102/ajlm.v3i2.201 18. masamha j, skaggs b, pinto i, et al. working toward a sustainable laboratory quality improvement programme through country ownership: mozambique’s slmta story. afr j lab med. 2014;3(2), art. #253, 6 pages. http://dx.doi.org/10.4102/ajlm.v3i2.253 19. mataranyika mn, beukes lk. view from the top: involvement of namibia’s health ministry in laboratory quality improvement. afr j lab med. 2014;3(2), art. #195, 2 pages. http://dx.doi.org/10.4102/ajlm.v3i2.195 20. mokobela ko, moatshe mt, modukanele m. accelerating the spread of laboratory quality improvement efforts in botswana. afr j lab med. 2014;3(2), art. #207, 6 pages. http://dx.doi.org/10.4102/ajlm.v3i2.207 21. mothabeng d, maruta t, lebina m, et al. strengthening laboratory management towards accreditation: the lesotho experience. afr j lab med. 2012;1(1), art. #9, 7 pages. http://dx.doi.doi: 10.4102/ajlm.v1i1.9 22. ndasi j, dimite l, mbome v, et al. decentralised facility-based training as an alternative model for slmta implementation: the cameroon experience. afr j lab med. 2014;3(2), art. #231, 6 pages. http://dx.doi.org/10.4102/ajlm.v3i2.231 23. nguyen tt, mckinney b, pierson a, et al. slipta e-tool improves laboratory audit process in vietnam and cambodia. afr j lab med. 2014;3(2), art. #219, 5 pages. http://dx.doi.org/10.4102/ajlm.v3i2.219 24. nkengasong jn, birx d. quality matters in strengthening global laboratory medicine. afr j lab med. 2014;3(2), art. #239, 4 pages. http://dx.doi.org/10.4102/ajlm.v3i2.239 25. nkrumah b, van der puije b, bekoe v, et al. building local human resources to implement slmta with limited donor funding: the ghana experience. afr j lab med. 2014;3(2), art. #214, 7 pages. http://dx.doi.org/10.4102/ajlm.v3i2.214 26. nkwawir sc, batumani nn, maruta t, awasom cn. from grass to grace: how slmta revolutionised the bamenda regional hospital laboratory in cameroon. afr j lab med. 2014;3(2), art. #203, 6 pages. http://dx.doi.org/10.4102/ajlm.v3i2.203 27. noble ma, martin r, ndihokubwayo j-b. making great strides in medical laboratory quality. afr j lab med. 2014;3(2), art. #256, 2 pages. http://dx.doi.org/10.4102/ajlm.v3i2.256 28. ntshambiwa k, ntabe-jagwer w, kefilwe c, samuel f, moyo s. translating a national laboratory strategic plan into action through slmta in a district hospital laboratory in botswana. afr j lab med. 2014;3(2), art. #209, 5 pages. http://dx.doi.org/10.4102/ajlm.v3i2.209 29. nzabahimana i, sebasirimu s, gatabazi jb, et al. innovative strategies for a successful slmta country programme: the rwanda story. afr j lab med. 2014;3(2), art. #217, 6 pages. http://dx.doi.org/10.4102/ajlm.v3i2.217 30. nzombe p, luman et, shumba e, et al. maximising mentorship: variations in laboratory mentorship models implemented in zimbabwe. afr j lab med. 2014;3(2), art. #241, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i2.241 31. shumba e, nzombe p, mbinda a, et al. weighing the costs: implementing the slmta programme in zimbabwe using internal versus external facilitators. afr j lab med. 2014;3(2), art. #248, 6 pages. http://dx.doi.org/10.4102/ajlm.v3i2.248 32. yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3):401–409. 33. yao k, luman et, slmta collaborating authors. evidence from 617 laboratories in 47 countries for slmta-driven improvement in quality management systems. afr j lab med. 2014;3(2), art. #262, 11 pages. http://dx.doi.org/10.4102/ajlm.v3i2.262 34. schroeder lf, amukele t. medical laboratories in sub-saharan africa that meet international quality standards. am j clin pathol. 2014;141(6):791–795. 35. elbireer am, jackson jb, sendagire h, et al. the good, the bad, and the unknown: quality of clinical laboratories in kampala, uganda. plos one. 2013:8(5);e64661. 36. committee on the outcome and impact evaluation of global hiv/aids programs implemented under the lantos-hyde act of 2008; board on global health; board on children, youth, and families. evaluation of pepfar. washington, dc: the national academies press; 2013 [available from: http://www.nap.edu/catalog.php?record_id=18256] 37. the global fund to fight aids, tuberculosis and malaria [homepage on the internet]. n.d. [cited 2014 aug 28]. available from: http://www.theglobalfund.org/en/ 38. us department of health and human services. global health initiative [page on the internet]. n.d. [cited 2014 aug 28]. available from: http://www.globalhealth.gov/global-programs-and-initiatives/global-health-initiative/ 39. world health organization regional office for africa. the maputo declaration on strengthening of laboratory systems [document on the internet]. c2008 [cited 2013 jan 02]. available from: http://www.who.int/diagnostics_laboratory/maputo-declaration_2008.pdf 40. world health organization. joint who–cdc conference on health laboratory quality systems. who/hse/ihr/lyo/2008.3 [document on the internet]. c2008 [cited 2013 jan 03]. available from: http://www.who.int/ihr/lyon/report20080409.pdf 41. world health organization regional office for africa. who report calls for strengthening public health laboratories [page on the internet]. c2008 [cited 2014 jul 21]. available from: http://www.afro.who.int/en/media-centre/pressreleases/item/679-who-report-calls 42. maruta t, motebang d, mathabo l, et al. impact of mentorship on who-afro strengthening laboratory quality improvement process towards accreditation (slipta). afr j lab med 2012;1(1), art. #6, 8 pages. 43. the united states president’s emergency plan for aids relief. countries [page on the internet]. n.d. [cited 2014 aug 28]. available from: http://www.pepfar.gov/countries/ 44. the world bank. country and lending groups [page on the internet]. c2014 [cited 2014 aug 28]. available from: http://data.worldbank.org/about/country-and-lending-groups abstract introduction methods results discussion acknowledgements references about the author(s) waganeh sinshaw tuberculosis research unit/national tuberculosis reference laboratory, ethiopian public health institute, addis ababa, ethiopia department of microbiology, immunology and parasitology, school of medicine, addis ababa university, addis ababa, ethiopia department of medical laboratory science, college of health sciences, addis ababa university, addis ababa, ethiopia abebaw kebede tuberculosis research unit/national tuberculosis reference laboratory, ethiopian public health institute, addis ababa, ethiopia department of microbial, cellular, and molecular biology, college of natural and computational sciences, addis ababa university, addis ababa, ethiopia adane bitew department of medical laboratory science, college of health sciences, addis ababa university, addis ababa, ethiopia mengistu tadesse tuberculosis research unit/national tuberculosis reference laboratory, ethiopian public health institute, addis ababa, ethiopia zemedu mehamed tuberculosis research unit/national tuberculosis reference laboratory, ethiopian public health institute, addis ababa, ethiopia ayinalem alemu tuberculosis research unit/national tuberculosis reference laboratory, ethiopian public health institute, addis ababa, ethiopia bazezew yenew tuberculosis research unit/national tuberculosis reference laboratory, ethiopian public health institute, addis ababa, ethiopia misikir amare tuberculosis research unit/national tuberculosis reference laboratory, ethiopian public health institute, addis ababa, ethiopia biniyam dagne tuberculosis research unit/national tuberculosis reference laboratory, ethiopian public health institute, addis ababa, ethiopia getu diriba tuberculosis research unit/national tuberculosis reference laboratory, ethiopian public health institute, addis ababa, ethiopia ephrem tesfaye tuberculosis research unit/national tuberculosis reference laboratory, ethiopian public health institute, addis ababa, ethiopia dinka f. gamtesa tuberculosis research unit/national tuberculosis reference laboratory, ethiopian public health institute, addis ababa, ethiopia yeshiwork abebaw tuberculosis research unit/national tuberculosis reference laboratory, ethiopian public health institute, addis ababa, ethiopia helina mollalign tuberculosis research unit/national tuberculosis reference laboratory, ethiopian public health institute, addis ababa, ethiopia getachew seid tuberculosis research unit/national tuberculosis reference laboratory, ethiopian public health institute, addis ababa, ethiopia muluwork getahun tuberculosis research unit/national tuberculosis reference laboratory, ethiopian public health institute, addis ababa, ethiopia citation sinshaw w, kebede a, bitew a, et al. effect of sputum quality and role of xpert® mtb/rif assay for detection of smear-negative pulmonary tuberculosis in same-day diagnosis strategy in addis ababa, ethiopia. afr j lab med. 2022;11(1), a1671. https://doi.org/10.4102/ajlm.v11i1.1671 note: additional supporting information may be found in the online version of this article as online supplementary 1. original research effect of sputum quality and role of xpert® mtb/rif assay for detection of smear-negative pulmonary tuberculosis in same-day diagnosis strategy in addis ababa, ethiopia waganeh sinshaw, abebaw kebede, adane bitew, mengistu tadesse, zemedu mehamed, ayinalem alemu, bazezew yenew, misikir amare, biniyam dagne, getu diriba, ephrem tesfaye, dinka f. gamtesa, yeshiwork abebaw, helina mollalign, getachew seid, muluwork getahun received: 08 july 2021; accepted: 24 may 2022; published: 31 aug. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: there is limited information on the performance of the xpert® mtb/rif test for diagnosis of smear-negative pulmonary tuberculosis (snpt) and rifampicin resistance (rr) in the same-day diagnosis approach. the effects of sputum quality and other factors affecting the xpert performance are also under-investigated. objective: this study aimed to determine the performance of the xpert® mtb/rif test for detection of snpt and rr in the same-day diagnosis strategy and the effect of sputum quality and other factors on its performance. methods: a cross-sectional study was conducted from august 2017 to january 2018 across 16 health facilities in addis ababa, ethiopia. two spot sputum samples were collected from 418 presumptive snpt patients, tested with xpert® mtb/rif, then compared to tuberculosis culture. additionally, culture isolates were tested for rr by bactec mgit™ 960 drug susceptibility testing (dst) and mtbdrplus version 2. results: the xpert® mtb/rif test detected 24 (5.7%) snpt cases, with a sensitivity of 92.3% (75.9% – 97.9%) and specificity of 99.2% (97.8% – 99.7%) compared with tuberculosis culture. xpert® mtb/rif also detected three (11.58%) rr strains with 100.0% concordance with bactec mgit™ 960 dst and mtbdrplus results. three blood-stained snpt samples were positive by xpert (30.0%), which was 6.9 times higher compared to salivary sputum (odds ratio: 6.9, 95% confidence interval: 1.36–34.96, p = 0.020). conclusion: the performance of the xpert® mtb/rif to detect snpt and rr in same-day diagnosis is high. however, snpt positivity varies among sputum qualities, and good sample collection is necessary for better test performance. keywords: smear-negative pulmonary tuberculosis; xpert® mtb/rif test; sputum quality; tuberculosis culture; diagnostic performance. introduction smear-negative pulmonary tuberculosis (snpt) occurs when a presumptive pulmonary tuberculosis (ptb) patient tests negative by acid-fast bacilli microscopy but tests positive by more accurate diagnostic techniques.1 it is one of the most problematic issues in tuberculosis diagnosis.2 patients with snpt contributed 17.3% – 41.0% of community tuberculosis transmission in vancouver, canada, from january 1995 to march 19993 and 13.0% in the netherlands from 1996 to 2004.4 also, 55.4% of belarus tuberculosis cases were smear-negative but culture-positive in 2012.5 in ethiopia, bacteriologically confirmed snpt prevalence was reported as high as 23.9% in 2007.6 although snpt is assumed to be less contagious and have lower mortality compared to smear-positive tuberculosis, 50.0% – 71.0% of snpt patients develop active tuberculosis disease.3,4 these snpt patients also harbour a high proportion of drug-resistant tuberculosis strains. the prevalence of smear-negative multidrug-resistant (mdr) tuberculosis among presumptive ptb patients enrolled with other patients was 47.0% in belarus in 20125 and 11.5% in ethiopia august 2017 to january 2018.7 mortality due to snpt is also substantial, especially among the immunocompromised. in mozambique, the mortality of hiv co-infected patients reached 55.8%.8 thus, early detection with sensitive diagnostic tools that simultaneously detect drug-resistant tuberculosis is critical. however, diagnosis of snpt and smear-negative drug-resistant tuberculosis is challenging in lowand middle-income countries (lmics) like ethiopia,9 mainly due to a lack of sensitive diagnostic tools. in most lmics, smear microscopy, which has low sensitivity, is still the first line of diagnosis. the xpert® mtb/rif test is a cartridge-based, fully automated dna testing platform that, in less than 2 h, simultaneously detects tuberculosis and mutations conferring rifampicin resistance (rr).10 the technology detects rr using five probes targeting mutations in the rpob region of the mycobacterium tuberculosis genome11,12: rpob mutations are responsible for rr in over 99.5% of rr strains.10 the xpert® mtb/rif test currently resolves the challenges of initial smear microscopy in tuberculosis case detection, which improves the clinical management of tuberculosis cases.13,14 in addition, the xpert® mtb/rif is advantageous over smear microscopy and m. tuberculosis culture by its higher sensitivity, simultaneous detection of rr, shorter turnaround time (tat) (2 h), and a minimal safety requirement.15 by comparison, culture-based rr confirmation takes more than two weeks to get results.16 nevertheless, various factors impact the xpert® mtb/rif’s performance, such as the type of specimen,17 sputum quality,18,19,20 sample collection and diagnosis strategy,21,22,23 and clinical characteristics of patients.17,24,25 since 2012, more than 314 genexpert® instruments have been installed in ethiopia. also, the diagnostic strategy changed from spot-morning-spot, which required three samples collected over two days, to same-day diagnosis (spot-spot diagnosis), which requires two samples collected on the same day.26 however, information is lacking on the performance of the xpert® mtb/rif test to diagnose snpt, smear-negative rr tuberculosis, the effects of sputum quality and other factors in the same-day tuberculosis diagnosis approach in addis ababa, ethiopia. therefore, this study aimed to determine the performance of the xpert® mtb/rif test to diagnose snpt and rr against conventional tuberculosis culture and drug susceptibility testing (dst) in the spot-spot diagnostic strategy. it also intended to determine the snpt positivity in sputum samples with varying quality and the effect of sputum quality and other factors on xpert® mtb/rif test performance. methods ethical considerations the study was reviewed and approved by the institutional review board of the ethiopian public health institute (ref. no. ephi/613/535) and the departmental research and ethics review committee of the department of medical laboratory science, addis ababa university (ref. no. mls/364/17). written informed consent from the participants and assent from the guardians of participants less than 18 years of age were obtained. permission was requested with a legal letter and received from participanting health facilities. unique patient identifiers were used instead of patient names to conceal patient identity. the laboratory testing results and other patient information were locked. laboratory results were reported to the clinicians who ordered the test for further patient management. study setting and design this cross-sectional study was conducted from august 2017 to january 2018 in addis ababa, ethiopia. addis ababa is the federal capital of ethiopia and has a population of more than three million.27 the study was conducted in 16 systematically selected governmental and private directly observed treatment short-course tuberculosis sites. these sites participate in blind rechecking, and most participate in the on-site supervision programme by the addis ababa city administration health research and laboratory service. data collection socio-demographic information, clinical presentations, comorbidities, and other factors were collected using a structured questionnaire by trained clinicians through interviews. the questionnaire was piloted following the on-site training of the data collectors. patient enrollment a total of 418 presumptive snpt patients who were negative for two consecutive spot sputum smears were enrolled. adults and children with presumptive ptb who visited the sites during the study period were eligible. however, those taking anti-tuberculosis drugs for more than one week or unable to submit two spot sputum samples were excluded. trained laboratorians gave patients detailed instructions on sputum collection. each presumptive snpt patient collected two sputum spot ≥ 3 ml samples into sterile 50 ml falcon tubes. sputum samples were transported with a triple packaging system to the national tuberculosis reference laboratory (ntrl) of the ethiopian public health institute (ephi) for laboratory investigation. sputum samples were stored at 2 °c – 8 °c until transported by triple packaging system for safety, with a thermometer inside for temperature monitoring. the transportation from study sites to ntrl takes less than one hour since it is within the city. samples were submitted to ntrl within a maximum of two days. macroscopic sputum quality was evaluated and categorised as blood-stained, purulent, mucoid, and salivary based on the global laboratory initiative (gli) guidance.28 laboratory investigations the two consecutive spot sputum samples collected from each patient were pooled into one and homogenised. afterwards, each sputum pool was split into two; one for xpert® mtb/rif test (cepheid, sunnyvale, california, united states) and the other for culture testing on bactec™ mgit™ 960 system (becton-dickinson and company, sparks, maryland, united states) and in laboratory-made lowenstein jensen (lj). drug susceptibility testing (phenotypic and genotypic) was performed in the ntrl of ephi, as explained in sinshaw et al., 2019.7 quality assurance and quality control the sterility and performance of lj media-manufactured in-house were verified before use for testing using controls by randomly selecting some prepared lj medium, putting it in the lj incubator and monitoring for any contaminant growth. if no growth was observed within 56 days, it was considered as sterile or safe for use. likewise, a new lot or batch of bactec™ mgit™ 960 media was also verified. each of the culture and identification procedures was performed in a certified class ii biosafety cabine. preventive maintenance of the equipment, temperature monitoring, and instrument operation checks were performed. in each batch of sample cultured, reagent sterility and process contamination check control were incorporated based on the ntrl standard procedures. a proficiency testing scheme continuously monitored all study test methods. also, the ntrl is international organization for standardization 15189 accredited by the ethiopian national accreditation office for xpert® mtb/rif. data entry and analysis double data entry was perfomed on epidata statistical software version 3.0 (epidata association, odense, denmark). the clean data were transferred to and analysed using ibm statistical package for social sciences software version 20.0 (chicago, illinois, united states). data entry and cleaning were performed by the ntrl data manager. the characteristics of the study participants were analysed using descriptive statistics. the sensitivity and specificity of the xpert® mtb/rif test were calculated at 95% confidence intervals (ci). pearson chi-square, fisher’s exact test, or binary logistic regression was used to determine the associations between dependent and independent variables; variables with a p ≤ 0.2 were selected for multivariable analysis. the strength of associations was measured by odds ratios, and a p < 0.05 was taken as statistically significant. results socio-demographic and clinical characteristics of study participants most of the participants (231; 55.3%) were female. the average participant age was 36 years (standard deviation [s.d.] ± 18). most of the participants (257, 61.8%) had completed some schooling (grades 1–12), while 112 (26.9%) were uneducated, and more than half of the participants (218; 52.3%) were married. cough was the leading symptom (413; 98.8%), followed by fever (255; 61.2%). of the 225 participants interviewed or laboratory tested, 57 (25.3%) were positive for hiv (table 1 and table 2). table 1: socio-demographic characteristics of the participants and their association with snpt detection by xpert mtb/rif test in addis ababa, ethiopia, from august 2017 to january 2018. table 2: clinical manufestations and behavioural characteristics of the presumptive snpt patients in addis ababa, ethiopia, from august 2017 to january 2018. sputum quality and performance characteristics of xpert® mtb/rif test salivary sputum was the leading sample quality (147, 35.2%), while blood-stained sputum was the least (10; 2.4%) sputum quality submitted (table 3). the majority of the sputum submitted (310; 74.3%) had 3 ml – 5 ml volume, while 16.8% had 6 ml – 7 ml and 8.9% had 8 ml – 9 ml volume. table 3: positivity of xpert mtb/rif assay in different sputum sample qualities in addis ababa, ethiopia, from august 2017 to january 2018. the majority of the positive xpert® mtb/rif tests, 14 (51.9%), were low and very low in bacillary load. there were 20 (4.8%) unsuccessful xpert mtb/rif results (sum of errors, invalids and no results). the most unsuccessful results were errors (15; 3.6%) (table 4). table 4: semi-quantitative bacilli dna quantification and unsuccessful xpert mtb/rif test results in addis ababa, ethiopia, from august 2017 to january 2018. the error code 5007 was the most common (table 5). mucoid sputum accounted for seven (46.7%) errors, while purulent sputum accounted for five (33.3%). the other three (20.0%) errors were from salivary sputum. table 5: xpert mtb/rif test post-run analysis error results and possible causes in addis ababa, ethiopia, from august 2017 to january 2018. the xpert® mtb/rif test detected three rr cases. two of the rr cases were caused by probe e (codons 529–533) missing mutations and one by probe b (codons 511–518) missing mutation. sputum m. tuberculosis positivity rate mycobacterium tuberculosis positivity rate was 6.1% for salivary sputum and 4.2% for mucoid sputum. however, the m. tuberculosis positivity rate was higher (30.0%) in blood-stained sputum. blood-stained sputum was 6.9 times more m. tuberculosis positive than salivary sputum (95% ci: 1.36–34.96; odds ratio [or]: 6.9; p = 0.02), while purulent sputum was the second most positive (7.7%) (table 3). detection of snpt and rr by xpert® mtb/rif test of the 418 smear-negative presumptive ptb patients enrolled in this study, 27 (6.5%; 95% ci: 4.10–8.81) were m. tuberculosis positive by xpert® mtb/rif. on the other hand, 26 (6.4%; 95% ci: 4.00–8.74) were culture-positive. twenty-four (5.7%; 95% ci: 3.51% – 7.97%) sputum samples were positive by both xpert® mtb/rif and tuberculosis culture (lj and mgit) (figure 1). figure 1: xpert® mtb/rif assay performance reference to the mgit and lj mycobacterium tuberculosis culture in addis ababa, ethiopia, from august 2017 to january 2018. five samples were discordantly positive by both methods; one sample was lj culture-positive but mgit culture-negative. on the other hand, four samples were mgit culture-positive, two of which were lj culture-negative, while the other two were lj culture contaminated. besides, of the two xpert® mtb/rif negative culture-positive samples, one was mgit negative and the other mgit positive, but both were positive by lj solid culture. moreover, three sputum samples were xpert® mtb/rif positive but culture-negative. thus, the diagnostic sensitivity and specificity of the xpert® mtb/rif test relative to tuberculosis culture was 92.3% (75.9% – 97.9%) and 99.2% (97.8% – 99.7%). the overall diagnostic accuracy was 98.8% (97.2% – 99.5%) (figure 1). three (11.54%) rr snpt cases were detected by the xpert® mtb/rif test; two of the rr detected were from a new case and one from previously treated cases. these strains were later confirmed as mdr by genotype mtbdrplus version 2 and bactec™ mgit™ 960 dst. most of the snpt cases were detected from men (95% ci: 1.29–11.44; or: 3.85; p = 0.015]) (table 1). patients with weight loss had 4.05 times more risk of being diagnosed with snpt, in 95% ci (p = 0.007) compared to those without weight loss (supplementary table 1). discussion smear microscopy is still a front-line tuberculosis diagnostic tool in developing countries like ethiopia.1,21,29 however, because of its low sensitivity, smear microscopy misses many tuberculosis cases, resulting in delayed diagnosis and treatment initiation.15,30,31 therefore, this study evaluated the effect of sputum quality and xpert® mtb/rif performance for snpt detection in same-day diagnosis. sputum quality has effects on the detection of ptb.19,20,32,33 however, many studies do not consider attributes of sputum quality in mtb testing by the xpert® mtb/rif test.34,35 in the present study, the m. tuberculosis positivity by xpert® mtb/rif significantly varied across sputum qualities. the majority of sputum, 147 (35.2%), submitted was salivary with a positivity rate of 6.1%. although the blood-stained sample was the least submitted, the positivity was very high at 30.0%. in 95% ci, blood-stained samples showed 6.9 (p = 0.020) times more positive than salivary. many laboratories reject blood-stained sputum, assuming that it brings unreliable xpert results due to polymerase chain reaction inhibition; however, this study revealed the highest snpt positivity in blood-stained sputum. contrary to the present study, a study showed that the xpert result is only valid at less than 2.0% blood contamination of the sputa. if sputum contamination with blood is beyond 5.0%, the result will be unreliable and absolute inhibition occurs at 20.0% blood contamination.18 however, studies found that patients who are mtb positive in a blood sample have a higher risk of death.36 therefore, we are missing the most important sputum quality. the current finding is different from a study in uganda, where snpt was more in salivary sputum and lowest in blood-stained sputum.20 this might be as a result of the fact that the majority of the ugandan study participants were hiv positive and most of them had a very low cd4 count (≤ 200 cells/µl). the second highest snpt positivity, 7.7%, was diagnosed from purulent sputum. although the difference is not statistically significant, blood-stained sputum showed greater positivity than purulent sputum, whereas purulent sputum is considered the best sputum for mtb detection. the difference in macroscopic sputum appearance significantly varied for mtb positivity by xpert® mtb/rif test, implying a need for proper sputum collection, similar to other reports.20,33,34 in ethiopia, the sputum sample collection strategy for mtb diagnosis was changed from spot-morning-spot to spot-spot (same-day diagnosis) in 2017.26 same-day diagnosis stops multiple visits to the health facilities by the patient to submit sputum and receive a result. however, it is 2.8% less sensitive with a lower dropout rate than the conventional -spot-morning-spot strategy.21 in the spot-morning-spot strategy, three smear slides are made. the first slide made from the first spot sputum, the second slide made from the morning sputum and the third slide from the second spot sputum.22 this change increases the possibilities of being smear-negative. in the spot-spot approach, in the current study, xpert® mtb/rif assay detected an extra 24 (5.7%) snpt and three rr strains in comparison with smear microscopy. the ability of the xpert® mtb/rif test to detect snpt and rr is high in this study which might be because the missed morning sputum increased the snpt cases. the xpert® mtb/rif testing was performed on the direct sputum before culture to evaluate the performance of the xpert® mtb/rif in the peripheral health facilities where direct sputum only is used for xpert® mtb/rif testing. sputum processing, including decontamination, neutralisation, and pellet concentration before culture, is only possible in the tuberculosis culture reference laboratories. the diagnostic sensitivity of the xpert® mtb/rif test in this study was 92.3%, while specificity was 99.2% reference to tuberculosis culture. the present study revealed higher sensitivity and specificity than the uganda report20 because most of uganda’s study participants were hiv-positive and used a spot-morning-spot diagnostic approach. another justification might be tuberculosis prevalence is higher in ethiopia. similarly, the present study revealed a very high sensitivity compared to a study in jigjiga, ethiopia: 48.5% for smear-negative ptb.37 likewise, the current study showed higher sensitivity and specificity relative to a review conducted in liverpool, united kingdom, in 2013, which was 67.0% – 74.0% pooled sensitivity and 99.0% pooled specificity.38 however, the sensitivity of the xpert® to diagnose snpt showed considerable variability in different studies.39,40,41 the possible reasons for the variabilities in sensitivity and specificity might be differences in study design, study population, sample collection strategy, study period, study area or location, tuberculosis prevalence, comorbidity and laboratory performance. the three (11.54%) rr strains detected by the xpert® mtb/rif test in this study concords 100.0% with genotype mtbdr plus version 2 and phenotypic bactec™ mgit™ 960 dst results. in addition, studies reported high xpert® mtb/rif detection performance: 95.0% – 97.0% sensitivity and 98.0% – 99.0% specificity, for culture positives.38 the findings in this study are commendable since they facilitate early tuberculosis diagnosis and universal access to dst,42 which are pivotal to the endtb strategy. furthermore, the world health organization approved that tuberculosis diagnostic technologies such as xpert® mtb/rif need to be scaled up to become the first line of diagnosis. with the scale-up, patients will benefit from early diagnosis and initiation of treatment. more than half of the positive xpert® mtb/rif results (14; 51.9%) had a low or very low bacillary load, implying that snpt patients have a paucibacillary load. twenty (4.8%) xpert® mtb/rif results were unsuccessful (the sum of errors, invalids and no results), which is lower than a report from nigeria.43 the nigeria study did not include only presumptive smear-negative snpt cases. most of our unsuccessful results were due to error results (15; 3.6%), but in the nigerian study, these were considered invalid results.43 the error rate in the present study was higher than the gli recommendation (< 3.0%),44 but lower than a report from addis ababa, which reported 8.9%.45 the discrepancy in the study reports might result from the difference in the testers’ expertise and experience, study population, sample type, and study period. the error code 5007 was the most common (12; 2.9%), often caused by viscous sputum or wrong sample volume, improper filling of cartridge reaction tube, bubbles, or probe integrity issues. errors are a loss of valuable time and money for the patients and the laboratory. thus, patient training on quality sputum collection and laboratory staff refresher training may minimise these errors. of the three rr cases detected by the xpert® mtb/rif test, two were due to probe e (codons 529–533) missing, which is the most frequent type of mutation in the rpob region of the mycobacteria.46,47 the other was probe b missing (codons 511–518; mutation). limitations the current study did not explain the performance of the xpert® mtb/rif test in the same-day diagnosis to diagnose smear-negative rr as it did not include enough rr cases, which is a limitation of the study. conclusion the performance of the xpert® mtb/rif test to detect snpt in spot-spot samples and rapid detection of rr were high. however, the diagnostic performance of the test significantly varied across different sputum qualities. thus, good patient instruction or training and close follow-up on sputum sample collection are essential for getting quality sputum for better xpert® mtb/rif yield. we recommend testing sputum samples by xpert® mtb/rif irrespective of sputum quality. acknowledgements we acknowledge the ethiopian public health institute for their material and technical support of the study. we are also thankful for the study site staff and study participants. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions w.s. designed and conceptualised the study, performed the data analysis and interpretation of the results, and drafted the manuscript. a.k., a.a. and a.b. reviewed and edited the manuscript. a.k. was also involved in the study’s conceptualisation. z.m. performed the research methods, data analysis, and interpretation of results. e.t., m.t., b.y., m.a., b.d., g.d., a.a., h.m., y.a., g.s., m.g. and w.s. performed the laboratory analysis and reporting, site supervision, data collection, and data entry. d.f.g was responsible for data entry, data curation and analysis. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability all of the data are available from the corresponding author, w.s. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated organisations of the authors. references world health orgnization. improving the diagnosis and treatment of smear negative pulmonary and extra-pulmonary tuberculosis among adults and adolescents. recommendations for hiv-prevalent and resource-constrained settings [homepage on the internet]. 2007 [cited 2017 may 17]. available from: http://www.who.int çalışkan t, kaya h. smear-negative pulmonary tuberculosis. tubercle. 1980;61(2):113–115. https://doi.org/10.1016/0041-3879(80)90021-5 hernández-garduño e, cook v, kunimoto d, elwood rk, black wa, fitzgerald jm. transmission of tuberculosis from smear negative patients: a molecular epidemiology study. thorax. 2004;59(4):286–290. https://doi.org/10.1136/thx.2003.011759 tostmann a, kik sv, kalisvaart na, et al. tuberculosis transmission by patients with smear-negative pulmonary tuberculosis in a large cohort in the netherlands. clin infect dis. 2008;47(9):1135–1142. https://doi.org/10.1086/591974 harries ad, bianchi l, jensen pm, et al. high time to use rapid tests to detect multidrug resistance in sputum smear-negative tuberculosis in belarus. public health action. 2014;4(4):243–248. https://doi.org/10.5588/pha.14.0069 keflie tss, ameni g. microscopic examination and smear negative pulmonary tuberculosis in ethiopia. pan afr med j. 2014;19(162):1–10. https://doi.org/10.11604/pamj.2014.19.162.3658 sinshaw w, kebede a, bitew a, et al. prevalence of tuberculosis, multidrug resistant tuberculosis and associated risk factors among smear negative presumptive pulmonary tuberculosis patients in addis ababa, ethiopia. bmc infect dis. 2019;19(1):641. https://doi.org/10.1186/s12879-019-4241-7 lisboa m, fronteira i, colove e, nhamonga m, martins mdro. time delay and associated mortality from negative smear to positive xpert mtb/rif test among tb/hiv patients: a retrospective study. bmc infect dis. 2019;19(1):1–10. https://doi.org/10.1186/s12879-018-3656-x parsons lm, somoskövi á, gutierrez c, et al. laboratory diagnosis of tuberculosis in resource-poor countries: challenges and opportunities. clin microbiol rev. 2011;24(2):314–350. https://doi.org/10.1128/cmr.00059-10 world health organization. xpert mtb/rif implementation manual. geneva: who, 2014; p. 1–35. reddy r, alvarez-uria g. molecular epidemiology of rifampicin resistance in mycobacterium tuberculosis using the genexpert mtb/rif assay from a rural setting in india. hindawi j pathog. 2017;2017:1–5. https://doi.org/10.1155/2017/6738095 kaur r, jindal n, arora s, kataria s. epidemiology of rifampicin resistant tuberculosis and common mutations in rpob gene of mycobacterium tuberculosis: a retrospective study from six districts of punjab (india) using xpert mtb/rif assay. j lab physicians. 2016;8(2):96–100. https://doi.org/10.4103/0974-2727.180789 world health organization. rapid communication: molecular assays as initial tests for the diagnosis of tuberculosis and rifampicin resistance. geneva: who; 2020. lombardi g, di gregori v, girometti n, tadolini m, bisognin f, dal monte p. diagnosis of smear-negative tuberculosis is greatly improved by xpert mtb/rif. plos one. 2017;12(4):1–10. https://doi.org/10.1371/journal.pone.0176186 world health organization. implementing tuberculosis diagnostics policy framework. geneva: who/htm/tb; 2015. foundation for innovative new diagnostics. mgittm procedure manual for bactectm mgit 960tm tb system. geneva: foundation for innovative new diagnostics (find), 2006; p. 3–52. theron g, peter j, calligaro g, et al. determinants of pcr performance (xpert mtb/rif), including bacterial load and inhibition, for t.b. diagnosis using specimens from different body compartments. sci rep. 2014;4:1–10. https://doi.org/10.1038/srep05658 aninagyei e, ayivor-djanie r, attoh j, dakorah mp, ginko mn, acheampong do. molecular detection of mycobacterium tuberculosis in blood stained sputum samples using genexpert pcr assay. diagn microbiol infect dis. 2021;100(3):115363. https://doi.org/10.1016/j.diagmicrobio.2021.115363 orina f, kiptoo m, mwangi m, et al. effect of sputum quality on xpert® mtb/rif results in the detection of mycobacterium tuberculosis from persons presumed to have tuberculosis in eaphln project operational research study sites in kenya. afr j health sci. 2014;32(6):446–458. meyer aj, atuheire c, worodria w, et al. sputum quality and diagnostic performance of genexpert mtb/rif among smear-negative adults with presumed tuberculosis in uganda. plos one. 2017;12(7):1–12. https://doi.org/10.1371/journal.pone.0180572 world health organization. same-day diagnosis of tuberculosis by microscopy: policy statement. vol. 47. geneva: who, 2011; p. 1–6. van deun a, salim ah, cooreman e, et al. optimal tuberculosis case detection by direct sputum smear microscopy: how much better is more? int j tuberc lung dis. 2002;6(3):222–230. kirwan de, gilman rh. same-day diagnosis and treatment of tuberculosis. lancet infect dis. 2013;13(2):102–104. https://doi.org/10.1016/s1473-3099(12)70270-0 world health orgnization. implementing tuberculosis policy framework tuberculosis policy framework. geneva: who, 2015; p. 1–47. campos lc, vieira rocha mv, cunha willers dm, silva dr. characteristics of patients with smear-negative pulmonary tuberculosis (tb) in a region with high tb and hiv prevalence. plos one. 2016;11(1):1–8. https://doi.org/10.1371/journal.pone.0147933 federal democratic republic of ethiopia ministry of health and ethiopian public health institute. afb smear microscopy manual. 4th ed. addis ababa: moh; 2017. ethiopian central statistical agency. central statistical agency population projections for ethiopia 2007–2037. addis ababa: csa; 2013. global laboratory initiative (gli). laboratory diagnosis of tuberculosis by sputum microscopy [homepage on the internet]. 2013 [cited 2017 jun 16]; p. 1–88. a publication of the global laboratory initiative a working group of the stoptb partnership. adelaide. available from: http://www.stoptb.org/wg/gli/assets/documents/tbmicroscopyhandbook_final.pdf federal democratic republic of ethiopia ministry of health. national guidelines for tb, dr-tb and leprosy in ethiopia. 6th ed. addis ababa: moh; 2018. world health organization. the global plan to stop tb 2011–2015. geneva: who; 2006, p. 1–91. ngabonziza jcs, ssengooba w, mutua f, et al. diagnostic performance of smear microscopy and incremental yield of xpert in detection of pulmonary tuberculosis in rwanda. bmc infect dis. 2016;16(1):1–7. https://doi.org/10.1186/s12879-016-2009-x orikiriza p, nyehangane d, white lf, kim s, bonnet m, fennelly kp. effect of previous treatment and sputum quality on diagnostic accuracy of xpert mtb/rif. int j tuberc lung dis. 2017;21(4):389–397. https://doi.org/10.5588/ijtld.16.0785 yoon sh, lee nk, yim jj. impact of sputum gross appearance and volume on smear positivity of pulmonary tuberculosis: a prospective cohort study. bmc infect dis. 2012;12(1):1. https://doi.org/10.1186/1471-2334-12-172 ho j, marks gb, fox gj. the impact of sputum quality on tuberculosis diagnosis a systematic review. int j tuberc lung dis. 2015;19(5):537–544. https://doi.org/10.5588/ijtld.14.0798 ho j, nguyen ptb, nguyen ta, et al. the role of macroscopic sputum quality assessments to optimise sputum testing for tuberculosis. int j tuberc lung dis. 2016;20(3):319–322. https://doi.org/10.5588/ijtld.15.0620 feasey na, banada pp, howson w, et al. evaluation of xpert mtb/rif for detection of tuberculosis from blood samples of hiv-infected adults confirms mycobacterium tuberculosis bacteremia as an indicator of poor prognosis. j clin microbiol. 2013;51(7):2311–2316. https://doi.org/10.1128/jcm.00330-13 geleta da, megerssa yc, gudeta an, akalu gt, debele mt, tulu kd. xpert mtb/rif assay for diagnosis of pulmonary tuberculosis in sputum specimens in remote health care facility. bmc microbiol. 2015;15(1):1–6. https://doi.org/10.1186/s12866-015-0566-6 steingart kr, schiller i, horne dj, pai m, boehme cc, dendukuri n. xpert® mtb/rif assay for pulmonary tuberculosis and rifampicin resistance in adults. cochrane database syst rev. 2014;2014(1):1–32. https://doi.org/10.1002/14651858.cd009593.pub3 williamson da, basu i, bower j, freeman jt, henderson g, roberts sa. an evaluation of the xpert mtb/rif assay and detection of false-positive rifampicin resistance in mycobacterium tuberculosis. diagn microbiol infect dis. 2012;74(2):207–209. https://doi.org/10.1016/j.diagmicrobio.2012.06.013 lawn sd, brooks sv, kranzer k, et al. screening for hiv-associated tuberculosis and rifampicin resistance before antiretroviral therapy using the xpert mtb/rif assay: a prospective study. plos med. 2011;8(7):e1001067. https://doi.org/10.1371/journal.pmed.1001067 vassall a, van kampen s, sohn h, et al. rapid diagnosis of tuberculosis with the xpert mtb/rif assay in high burden countries: a cost-effectiveness analysis. plos med. 2011;8(11):e1001120. https://doi.org/10.1371/journal.pmed.1001120 world health organiztion. implementing the end t.b. strategy: the essentials [homepage on the internet]. geneva: world health organiztion; 2015 [cited 2021 feb 28]; p. 1–130. available from: https://www.who.int/tb/publications/2015/end_tb_essential.pdf?ua=1 gidado m, nwokoye n, nwadike p, et al. unsuccessful xpert® mtb/rif results: the nigerian experience. public health action. 2018;8(1):2–6. https://doi.org/10.5588/pha.17.0080 global laboratory initiative (gli). gli practical guide to t.b. laboratory strengthening [homepage on the internet]. 2017 [cited 2021 feb 21]; p. 130. available from: www.stoptb.org/wg/gli kebede a, beyene d, yenew b, et al. monitoring quality indicators for the xpert mtb/rif molecular assay in ethiopia. plos one. 2019;14(11):e0225205. https://doi.org/10.1371/journal.pone.0225205 alemu a, tadesse m, seid g, et al. does xpert® mtb/rif assay give rifampicin resistance results without identified mutation? review of cases from addis ababa, ethiopia. bmc infect dis. 2020;20(1):1–6. https://doi.org/10.1186/s12879-020-4817-2 ochang ea, udoh ua, emanghe ue, et al. evaluation of rifampicin resistance and 81-bp rifampicin resistant determinant region of rpob gene mutations of mycobacterium tuberculosis detected with xpert mtb/rif in cross river state, nigeria. int j mycobacteriol. 2016;5(2016):s145–s146. https://doi.org/10.1016/j.ijmyco.2016.09.007 abstract introduction methods results discussion acknowledgements references about the author(s) caroline a. fernando department of medical laboratory sciences, faculty of allied health sciences, general sir john kotelawala defence university, werahera, sri lanka deklanji t. dissanayake department of medical laboratory sciences, faculty of allied health sciences, general sir john kotelawala defence university, werahera, sri lanka uththara i. hewamana department of medical laboratory sciences, faculty of allied health sciences, general sir john kotelawala defence university, werahera, sri lanka shyamini rathnaweera department of medical laboratory sciences, faculty of allied health sciences, general sir john kotelawala defence university, werahera, sri lanka wickrama a. samanthilake department of quality management, national blood center, narahenpita, sri lanka ranga tudugala department of radiography and radiotherapy, faculty of allied health sciences, general sir john kotelawala defence university, werahera, sri lanka kithsiri b. jayasekara department of medical laboratory sciences, faculty of allied health sciences, general sir john kotelawala defence university, werahera, sri lanka kumudu kuruppu department of quality management, national blood center, narahenpita, sri lanka citation fernando ca, dissanayake dt, hewamana ui, et al. alternative methods for calculating percentage haemolysis of red cell concentrates in peripheral blood banks in sri lanka. afr j lab med. 2023;12(1), a1987. https://doi.org/10.4102/ajlm.v12i1.1987 original research alternative methods for calculating percentage haemolysis of red cell concentrates in peripheral blood banks in sri lanka caroline a. fernando, deklanji t. dissanayake, uththara i. hewamana, shyamini rathnaweera, wickrama a. samanthilake, ranga tudugala, kithsiri b. jayasekara, kumudu kuruppu received: 16 june 2022; accepted: 17 nov. 2022; published: 23 feb. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: haemolysis – one of the major limiting factors of red cell concentrate quality – must be measured as a quality-monitoring requirement. according to international quality standards, percentage haemolysis must be monitored in 1.0% of red cell concentrates produced monthly and maintained under 0.8%. objective: this study assessed three alternative methods for determining plasma haemoglobin concentration in peripheral blood banks that lack a plasma or low haemoglobin photometer – the gold-standard method – in sri lanka. methods: a standard haemolysate was prepared using an unexpired whole blood pack of normal haemoglobin concentration. a concentration series from 0.1 g/dl to 1.0 g/dl was prepared by diluting portions of standard haemolysate with saline. the alternative methods, namely visual haemoglobin colour scale, spectrophotometric calibration graph, and standard haemolysate capillary tube comparison, were designed using this concentration series and were used to test red cell concentrates received at the quality control department of the national blood center, sri lanka, from february 2021 to may 2021. results: a strong correlation was observed between the haemoglobin photometer method and the alternative methods (r = ~0.9). based on the linear regression model, the standard haemolysate capillary tube comparison method was the best of the three alternative methods (r2 = 0.974). conclusion: all three alternative methods are recommended for use in peripheral blood banks. the standard haemolysate capillary tube comparison method was the best model. keywords: blood banks; capillary tube comparison; haemoglobin colour scale; percentage haemolysis; red cell concentrate. introduction red cell concentrate (rcc) transfusion is essential to treat patients with anaemia and bleeding disorders, as well as in emergencies where there is severe blood loss during surgery or an accident.1 several clinical manifestations can occur in patients due to inappropriate blood transfusion. therefore, to ensure patient safety, rcc for transfusion must always be of good quality.1,2 quality of rcc depreciates with storage time, and haemolysis, the destruction of red blood cells, is one of its quality indicators.3,4 the morphology of red blood cells changes with an increase in osmotic fragility, resulting in deformity and rupture.5 haemolysis can occur due to the use of inappropriate methods during blood collection, processing,6 transportation, handling, and storage.7 upon rupture of the red blood cells, haemoglobin is released into the plasma, leading to a colour change8 that is indicative of haemolysis and can be detected in the supernatant plasma of centrifuged rcc.9 however, this does not totally define whether or not the blood pack is suitable for transfusion. rather, a quality parameter referred to as percentage haemolysis is used. according to international quality standards and those governed by the sri lankan national blood transfusion service quality control (qc) unit, rccs with percentage haemolysis greater than 0.8% towards the end of the storage period are unsuitable for transfusion.10,11 to calculate the percentage haemolysis, plasma haemoglobin concentration is required. the plasma or low haemoglobin photometer (lhbp) provides accurate results on the plasma haemoglobin concentration.11 the lhbp method relies on the oxidation of haemoglobin to haemoglobin by sodium nitrite, and the subsequent conversion of haemoglobin to hemiglobinazide by sodium azide, leading to a colour change that is measured by the photometric principle.12,13 presently, in the government sector of sri lanka, an lhbp is only available at the national blood center (nbc) because it is difficult to afford them at all other peripheral blood banks (pbbs). all pbbs in sri lanka face challenges in identifying haemolysed blood packs. when component storage refrigerators at these pbbs are subjected to temperature fluctuations or any malfunction, rccs are sent to the nbc for quality checks. when it is difficult to send the entire batch of rccs, they are discarded without quality assessment, leading to huge losses because the cost of a blood component includes the high costs of collection, preparation, testing, etc. in some pbbs, at times when blood collection rates are high and usage is low, excess blood packs are transported to other pbbs or to the nbc to be used before expiry. in such cases, the receiving pbbs have no means of assessing the quality of the rccs received. currently, pbbs only carry out visual detection of haemolysis in rccs before transfusion, and any rccs suspected of haemolysis are sent to the nbc to determine the percentage haemolysis. also, as a quality-monitoring procedure, pbbs send 1% of the monthly production of rcc to the nbc to detect haemolysis. however, possible mechanical damage to rccs due to poor transport facilities, difficulty in maintaining cold chain, high costs, and the time and labour requirements make this impractical, especially in emergencies. these problems are faced by many developing and low-income countries in the world, thus necessitating the establishment of alternative methods to detect haemolysis. this was an experimental study to design easy, reliable, and cost-effective methods for determining plasma haemoglobin concentration at pbbs where no lhbp is available. we introduce three alternative methods – visual haemoglobin colour scale (cs), spectrophotometric calibration graph (scg), and standard haemolysate capillary tube comparison (sctc) – for the determination of plasma haemoglobin concentration, which is required when calculating the percentage haemolysis of rccs. we also compare the performance, accuracy, and reliability of these methods to the gold-standard method, lhbp. methods ethical considerations ethical clearance was obtained from the research review committee of national blood transfusion services, narahenpita, sri lanka (ethical clearance number: nbts/moic/etru/co/2021/011). the ethical approval was obtained in a written format before beginning the study upon submission of a study proposal. all rccs used in this study were those obtained from voluntary blood donors and randomly selected and sent to the qc department at the national blood center, sri lanka, for haemolysis testing. therefore, no individual donor consent was required. permission to use the qc data of rccs received at the qc department was given by the senior medical laboratory technologist of the department. a barcode system was maintained on all blood components and no donor details were disclosed to the investigators of the study. raw data were stored by principal investigators on password-protected computers with restricted access. study setting this was a pilot experimental study conducted at the qc department of nbc, sri lanka, from february to may 2021. the nbc is the central hub and largest blood bank of the national blood transfusion service of sri lanka under the government sector. all other pbbs belonging to the national blood transfusion service in the country maintain a close link with the nbc for its services and their monthly quality checks. sampling the three developed alternative methods were used to determine the percentage haemolysis of rccs received at the qc department of the nbc between february and may 2021. red cell concentrates that were clotted, had leaky or physically damaged packs, or whose labels lacked necessary information were excluded from this study. the plasma haemoglobin concentrations of 68 rccs were measured using the lhbp method (standard method) and the three developed alternative methods (cs, scg and sctc methods), and their results were compared statistically. preparation of haemolysate the haemolysate was prepared using an unexpired, healthy whole blood pack. the whole blood was added into a sterile 50 ml falcon™ conical centrifuge tube (thermo fisher scientific, waltham, massachusetts, united states). to remove the plasma and buffy coat, the whole blood was centrifuged at 3500 rotations per minute (rpm) for 10 min using a thermo scientific™ labofuge™ 400 centrifuge (thermo fisher scientific, waltham, massachusetts, united states). afterwards, the supernatant containing the plasma and buffy coat was removed and discarded using a disposable samco™ fine tip transfer pipette (thermo fisher scientific, waltham, massachusetts, united states). the remaining blood was washed three times with equal volumes of 9 g/l nacl to ensure the complete removal of plasma, leukocytes, and platelets. the blood samples were mixed thoroughly between washes and the saline supernatant was carefully removed after centrifugation. fifty millilitres of distilled water and 50 ml of toluene (for red blood cells lysis) were added to the washed blood and homogenised using a mechanical shaker (~180 rpm) for 1 h (table orbital laboratory shaker flat platform-tos-4030p, mrc ltd; laboratory instruments, holon, israel). the mixture was stored at 4 ºc for 24 h – 48 h to allow the lipid and cell debris to form a semisolid surface between the toluene and the lysate. then after 48 h, the mixture was centrifuged at 3500 rpm for 10 min to remove the lysate layers. the lysate was pooled in a clean plain tube and centrifuged at 3500 rpm for 20 min. a sterile syringe was used to aspirate the required volume of lysate from the bottom into a clean sterile container, leaving the top 90% to be discarded. 5.35 ml of glycerol was added as a preservative to 12.5 ml of the lysate obtained, maintaining a 3:7 ratio. the broad-spectrum antibiotics amikacin sulphate (500 mg/2 ml; two drops) and gentamicin (80 mg/2 ml; two drops) were also added to prevent bacterial contamination. the lysate was then dispensed into clean sterile bottles, tightly capped, and stored at 4 ºc until the preparation of the concentration series. the preparation of the standard haemolysate was carried out according to the protocol mentioned in dacie and lewis’s practical hematology,14 but with modifications tailored to the laboratory setting and available resources. preparation of standard haemolysate concentration series the haemolysate stock was diluted using normal saline to prepare a standard haemolysate concentration series with haemoglobin concentrations of 0.1 g/dl, 0.2 g/dl, 0.3 g/dl, 0.4 g/dl, 0.5 g/dl, 0.6 g/dl, 0.7 g/dl, 0.8 g/dl, 0.9 g/dl and 1.0 g/dl. the haemolysate concentrations were prepared in clean 10 ml test tubes. a sysmex automated haematology analyser kx-21 (sysmex corporation, kobe, japan) was used to measure the haemoglobin concentration of the haemolysate stock, while the hemocue® lhbp (standard method) (hemocue, inc., brea, california, united states) was used to measure the lower haemoglobin concentrations of the series since low haemoglobin values (plasma haemoglobin) cannot be measured using a haematology analyser. visual haemoglobin colour scale method each concentrate (0.1 g/dl – 1.0 g/dl) was aspirated into five haematocrit capillary tubes (globe scientific inc., mahwah, new jersey, united states), which were then arranged in ascending order. high-quality photographs of each concentrate in the haematocrit capillary tubes were taken using the nikon d7500 camera (nikon inc., melville, new york, united states) under the same lighting conditions, from the same angle, and at the same place. colours from the photographs were used to design the visual haemoglobin cs using adobe® photoshop cc2017 software (2016, adobe inc., san jose, california, united states). the cs was then printed on 230 gsm (grams per square metre) glossy photo paper (printery company ltd., hong kong, china) using a three-colour laser printer (hewlett-packard colour laserjet pro mfp m281fdw, hewlett-packard company, palo alto, california, united states). the gloss lamination of the prepared cs protects it from mechanical damage and colour changes. this paper was also chosen because it was readily available in sri lanka at a low cost and can easily be printed. spectrophotometric calibration graph method the absorbance values of the standard haemolysate concentration series (0.1 g/dl to 1.0 g/dl) were measured for eight consecutive days using an enzyme-linked immunosorbent assay reader (bio-rad model 680, hercules, california, united states) at 450 nm. using microsoft excel software (2018, microsoft corporation, redmond, washington, united states), the average absorbance was plotted against the supernatant haemoglobin concentration values to prepare the scg. standard haemolysate capillary tube comparison method a portion of each concentrate from the standard haemolysate concentration series (0.1 g/dl – 1.0 g/dl) was aspirated into clean haematocrit capillary tubes, which were then anchored onto the concentration series holder made of hardboard with a white surface (figure 1). figure 1: standard haemolysate capillary tube comparison method developed at the quality control department of the national blood center, sri lanka, september 2020 – may 2021. sample preparation plasma obtained from rcc packs (suspected of haemolysis and received at the nbc qc laboratory, sri lanka) was used to determine plasma haemoglobin concentration by the standard method (lhbp) and the alternative methods (cs, scg and sctc). five millilitres of blood from the rcc was transferred into a clean test tube and centrifuged at 3500 rpm for a few seconds to separate the plasma. application of alternative methods visual haemoglobin colour scale method to estimate plasma haemoglobin for percentage haemolysis calculation, plasma from an rcc tubing is aspirated into a haematocrit capillary tube and visually compared to the scale (figure 2). the corresponding plasma haemoglobin value of the closest matching colour on the cs is selected and used in the percentage haemolysis calculation with the following equation: figure 2: visual haemoglobin colour scale printed on 230 gsm (grams per square metre) glossy laminated photo paper using a three-colour laser printer. the scale was developed at the quality control department of the national blood center, sri lanka, september 2020 – may 2021. spectrophotometric calibration graph method the absorbance values of the standard concentrations obtained on eight consecutive days were plotted against the plasma haemoglobin values of the standard concentration series (figure 3). also, a linear relationship between the mean plasma haemoglobin values and the mean absorbance values of the standard concentration series obtained on eight consecutive days was plotted to prepare the scg (figure 4). the resulting linear equation was used to determine the plasma haemoglobin values of rcc packs with unknown plasma haemoglobin concentrations: figure 3: plasma haemoglobin values of the standard concentration series versus the absorbance values obtained on eight consecutive days at the quality control department of the national blood center, sri lanka, september 2020 – may 2021. figure 4: spectrophotometric calibration graph developed at the quality control department of the national blood center, sri lanka, september 2020 – may 2021. the scg method requires access to an enzyme-linked immunosorbent assay reader, which is available in most pbbs in sri lanka. in sri lanka, pbbs are required to compute an scg and derive a linear equation using the standard concentration series issued to them by the nbc. the prepared scg can be used in subsequent measurements. also, in the absence of the graph, the linear equation obtained from the graph could be used to get a rough estimate of the plasma haemoglobin concentration. anyone who does not have sufficient knowledge of graphs can use the linear equation. data analysis statistical analyses were performed using ibm® spss® (version 20.0) statistical software (2011, ibm corp., armonk, new york, united states). the dataset was assumed to be normally distributed, and descriptive statistics such as mean, standard deviation, range, median, variance, and 95% confidence intervals were computed. we also determined the pearson product-moment correlations between the plasma haemoglobin values obtained using the different methods (lhbp vs cs, lhbp vs scg, lhbp vs sctc, cs vs scg, cs vs sctc, and scg vs sctc). thereafter, simple linear regression models were built based on the plasma haemoglobin values obtained when measured by the lhbp method (gold standard) (response variable – y-axis) and the alternative methods (explanatory variable – x-axis). results were considered statistically significant if p was 0.001 or less, with a 95% confidence interval. data obtained from the 68 rcc packs were filtered such that plasma haemoglobin values less than 0.1 g/dl or greater than 1.0 g/dl were not considered for statistical analysis. therefore, only results obtained from 46 rccs were considered (n = 46). since the measurable range of the developed alternative methods (cs, scg and sctc) was from 0.1 g/dl to 1.0 g/dl (that includes only the clinically significant range), any plasma haemoglobin value outside this range cannot be considered for statistical analysis purposes. results the mean plasma haemoglobin concentration of the rccs as determined by the lhbp method was 0.535 (standard deviation ± 0.277) (table 1). the mean plasma haemoglobin concentrations obtained using the cs, scg and sctc methods were 0.513 (standard deviation ± 0.274), 0.645 (standard deviation ± 0.286), and 0.530 (standard deviation ± 0.280). table 1: plasma haemoglobin concentrations (g/dl) of red cell concentrates received at the quality control department of the national blood center, sri lanka, february 2020 – may 2021. the correlation coefficient (r) between the lhbp and cs methods was 0.986 (p < 0.001), suggesting a statistically significant strong correlation between both methods (table 2). there were also statistically significant strong correlations between the lhbp and scg methods (r = 0.962; p < 0.001), the lhbp and sctc methods (r = 0.987; p < 0.001), the cs and scg methods (r = 0.937; p < 0.001), the cs and sctc methods (r = 0.982; p < 0.001), and between the scg and sctc methods (r = 0.939; p < 0.001). table 2: correlation between the plasma haemoglobin values determined using different methods at the quality control department of the national blood center, sri lanka, september 2020 – may 2021. all three alternative methods followed a simple linear regression. the sctc method had the highest coefficient of determination (r2) value (sctc = 0.974; cs = 0.972; scg = 0.926) (table 3). the sctc method also had the highest beta value (sctc = 0.987; cs = 0.986; scg = 0.962). all models based on the three methods were statistically significant (p < 0.001). table 3: linear regression for plasma haemoglobin values determined by the plasma or low haemoglobin photometer and three alternative methods developed at the quality control department of the national blood center, sri lanka, september 2020 – may 2021. discussion in this study, we developed three alternative methods for the determination of plasma haemoglobin concentration needed to calculate the percentage haemolysis of rccs in pbbs with limited resources. all three alternative methods correlated strongly with the gold-standard method, and they required only a very small volume of supernatant to determine the plasma haemoglobin concentrations of rcc samples. however, statistically, the sctc method gave the best results, having the highest coefficient of determination (r2 = 0.974; when the value of r2 is closer to 1, the model is considered more reliable) and the highest beta value (0.987; the degree of change in haemoglobin concentration measured by lhbp for every unit increase in haemoglobin concentration measured by the sctc method). nevertheless, the cs method was much simpler and practically easier to use than the sctc and scg methods. one limitation of the scg method is the excessive time consumption compared to the lhbp method. this is because each pbb must prepare its own scg graph and obtain the linear equation as absorbance values depend on the enzyme-linked immunosorbent assay reader used. one limitation of the sctc method is the difficulty in handling the 10 capillary tubes. to overcome that, a standard haemolysate capillary tube holder was developed. also, the sctc method consumed more time than the lhbp method. in 1995, a similar colour chart called the ‘haemoglobin colour scale’ was prepared as a simple alternative to assess anaemia and was intended for use when a haemoglobinometer was unavailable or impractical to use in the field.15 another colour chart was also previously developed for the quantitative estimation of methaemoglobinaemia in patients with propanil poisoning in rural areas and in hospitals with limited resources.11,16 as the cs is a visual method, results may vary with the eyesight of individuals, lighting, level of training, etc.17 another study introduced a world health organization haemoglobin cs17 for the diagnosis of anaemia in primary healthcare settings in low-income countries, and a meta-regression analysis was done to assess the impact of variables such as light source, level of training, population type, type of reference test, use of same or different samples for reference and colour scale testing, and the anaemia prevalence on the results.15,17 daylight coming over the shoulder of the observer was found to be the most appropriate light source for colour matching (r2 = 0.9386). inter-observer variation was measured by comparing the means of each group (r2 = 0.9500 for nurses, r2 = 0.9570 for laypeople, r2 = 0.9592 for students). the need for training was also statistically demonstrated (without training r2 = 0.8867; after training r2 = 0.9734). there was, however, no statistically significant difference between the reference method and the world health organization cs (f ratio 1.0198 at v = 99), and anaemia (haemoglobin < 12 g/dl) was efficiently diagnosed in 87% of cases.15 such a meta-analysis must be performed to further validate the methods developed in this study using larger rcc sample sizes. future statistical studies must be performed to prove that the three alternative methods introduced agree with the gold-standard method (lhbp) for detecting low plasma haemoglobin concentrations. apart from a simple linear regression analysis that estimates the linear relationship between plasma haemoglobin values determined by different methods, a level-of-agreement statistical analysis needs to be performed on the collected dataset to determine the degree of concordance. the stability of the standard haemolysate concentration series (used in the sctc method) must also be investigated to evaluate the stability of the colour references, and a repeated measures analysis of variance must be performed. limitations there are limited studies on methods to detect haemolysis in rcc packs. therefore, related articles were not found to compare our findings. furthermore, the preparation of a standard cs requires quality equipment like high-quality cameras, printers, and scanners. access to such high-quality technology and equipment was limited, and the technology and equipment used in this study may have affected the quality of the prepared cs. with better technological equipment, more accurate colours could be printed. also, a decrease in the number of blood donors due to the coronavirus disease 2019 pandemic resulted in the limited availability of rcc for haemolysis testing and method validation. another limitation of the study was that we evaluated the stability of the standard haemolysate series for only 8 days, which was a short time. a significant change in colour or absorbance after 8 days could have affected the results obtained using the scg and sctc methods. conclusion in this study, there was a strong and statistically significant correlation between the lhbp method (gold-standard method) and the three alternative methods (cs, scg and sctc) for the estimation of plasma haemoglobin concentrations. all three alternative methods were found to be suitable for the estimation of plasma haemoglobin concentrations, which is required for the calculation of percentage haemolysis in rccs. the three alternative methods can be introduced as cost-effective, and easy-to-use methods in pbbs with limited facilities. further validation of all methods is required. acknowledgements the authors are extremely grateful to dr lakshman edirisinghe, director, national blood transfusion service, mrs y.v.a.l. mangalika, acting superintendent medical laboratory technologist, national blood transfusion service, mr manuranga jayalath, medical laboratory technologist, quality control laboratory, national blood center, and nursing officer ms m.d. udula for the support given to carry out our research activities. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions c.a.f., d.t.d., u.i.h., and s.r. performed the experimental procedures and contributed to data analysis and writing and editing the manuscript. w.a.s. and s.r. developed the concept and performed the experimental analysis. r.t. contributed to statistical analysis of the data. k.b.j. and k.k. supervised the project and engaged in reviewing and editing the manuscript. all authors have read and agreed to the published version of the manuscript. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the authors confirm that the data supporting the findings of this study are available within the article. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references balentine jr. why would you need a blood transfusion? [homepage on the internet]. medicinenet.com; 2022 [cited 2022 mar 17]. available from: https://www.medicinenet.com/blood_transfusion/article.htm harris jc, crookston kp. blood product safety [homepage on the internet]. ncbi.nlm.nih.gov; 2021 [cited 2022 may 12]. available from: https://www.ncbi.nlm.nih.gov/books/nbk539826/ arif s, yadav n, rehman s, mehdi g. study of hemolysis during storage of blood in the blood bank of a tertiary health care centre. indian j hematol blood transfus. 2016;33(4):598–602. https://doi.org/10.1007/s12288-016-0769-5 garcía-roa m, vicente-ayuso mdc, bobes am, et al. red blood cell storage time and transfusion: current practice, concerns and future perspectives. blood transfus. 2017 may;15(3):222–231. https://doi.org/10.2450/2017.0345-16 aubron c, nichol a, cooper d, bellomo r. age of red blood cells and transfusion in critically ill patients. ann intensive care. 2013;3(1):2. https://doi.org/10.1186/2110-5820-3-2 sawant r, jathar s, rajadhyaksha s, kadam p. red cell hemolysis during processing and storage. asian j transfus sci. 2007;1(2):47–51. https://doi.org/10.4103/0973-6247.33446 choudhury n, mathur a. visual detection of hemolysis in a blood bag before issue. asian j transfus sci. 2011;5(1):61. https://doi.org/10.4103/0973-6247.76013 sowemimo-coker s. red blood cell hemolysis during processing. transfus med rev. 2002;16(1):46–60. https://doi.org/10.1053/tmrv.2002.29404 gautam r, oh j, marques m, dluhy r, patel r. characterization of storage-induced red blood cell hemolysis using raman spectroscopy. lab med. 2018;49(4):298–310. https://doi.org/10.1093/labmed/lmy018 hess j, sparrow r, van der meer p, acker j, cardigan r, devine d. blood components: red blood cell hemolysis during blood bank storage: using national quality management data to answer basic scientific questions. transfusion. 2009;49(12):2599–2603. https://doi.org/10.1111/j.1537-2995.2009.02275.x shihana f, dawson a, buckley n. a bedside test for methemoglobinemia, sri lanka. bull world health organ. 2016;94(8):622–625. https://doi.org/10.2471/blt.15.158147 cardigan r, smith k. evaluation of the hemocue plasma hemoglobin analyser for assessing haemolysis in red cell concentrates during storage. vox sanguinis. 2002;82(2):76–79. https://doi.org/10.1046/j.0042-9007.2001.00149.x hudson-thomas m, bingham kc, simmons wk. an evaluation of the hemocue for measuring haemoglobin in field studies in jamaica. bulletin of the world health organization. 1994;72(3):423–426. bain b, dacie j, lewis s. dacie and lewis practical haematology. edinburgh: churchill livingstone; 2012. lewis s, stott g, wynn k. an inexpensive and reliable new hemoglobin colour scale for assessing anaemia. j clin pathol. 1998;51(1):21–24. https://doi.org/10.1136/jcp.51.1.21 shihana f, dissanayake d, buckley n, dawson a. a simple quantitative bedside test to determine methemoglobin. ann emerg med. 2010;55(2):184–189. https://doi.org/10.1016/j.annemergmed.2009.07.022 marn h, critchley j. accuracy of the who hemoglobin colour scale for the diagnosis of anaemia in primary health care settings in low-income countries: a systematic review and meta-analysis. lancet global health. 2016;4(4):e251–e265. https://doi.org/10.1016/s2214-109x(16)00005-x article information authors: thuong t. nguyen1 barbara mckinney2 antoine pierson3 khue n. luong4 quynh t. hoang5 sandeep meharwal6 humberto m. carvalho7 cuong q. nguyen8 kim t. nguyen9 kyle b. bond5 affiliations: 1national institute of hygiene and epidemiology (nihe), vietnam 2mayo clinic, united states 3integrated quality laboratory services (iqls), france 4vietnam administration for medical services (vams), vietnam 5us centers for disease control and prevention (cdc), vietnam 6united states agency for international development (usaid) deliver project, indonesia 7substance abuse and mental health services administration (samhsa), united states 8fhi360, vietnam 9french department, hanoi university of science and technology, hanoi, vietnam correspondence to: kyle bond postal address: 1600 clifton road, atlanta, ga 30329-4018, united states dates: received: 06 sept. 2014 accepted: 22 sept. 2014 published: 03 nov. 2014 how to cite this article: nguyen tt, mckinney b, pierson a, luong kn, et al. slipta e-tool improves laboratory audit process in vietnam and cambodia. afr j lab med. 2014;3(2), art. #219, 5 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.219 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. slipta e-tool improves laboratory audit process in vietnam and cambodia in this lessons from the field... open access • abstract • introduction • research methods and design    • development of the e-tool • results • discussion • acknowledgements    • competing interests    • authors’ contributions    • cdc disclaimer • references abstract top ↑ background: the stepwise laboratory quality improvement process towards accreditation (slipta) checklist is used worldwide to drive quality improvement in laboratories in developing countries and to assess the effectiveness of interventions such as the strengthening laboratory management toward accreditation (slmta) programme. however, the paper-based format of the checklist makes administration cumbersome and limits timely analysis and communication of results.development of e-tool: in early 2012, the slmta team in vietnam developed an electronic slipta checklist tool. the e-tool was pilot tested in vietnam in mid-2012 and revised. it was used during slmta implementation in vietnam and cambodia in 2012 and 2013 and further revised based on auditors’ feedback about usability. outcomes: the slipta e-tool enabled rapid turn-around of audit results, reduced workload and language barriers and facilitated analysis of national results. benefits of the e-tool will be magnified with in-country scale-up of laboratory quality improvement efforts and potential expansion to other countries. introduction top ↑ the stepwise laboratory quality improvement process towards accreditation (slipta) checklist was established by the world health organization’s regional office for africa (who afro) and partners to assess the level of a laboratory’s compliance with the international organization for standardisation (iso) 15189 standard.1 this checklist is the standardised tool used to assess the effectiveness of the strengthening laboratory management toward accreditation (slmta) training programme2 through audits at the start (baseline) and end (exit) of the programme. audits are conducted using the paper-based slipta checklist containing 111 major questions (and numerous, related sub-questions) divided into 12 sections; as of 2012 only the english version was available. to date, the checklist has been used in 617 laboratories implementing slmta in 47 countries.3 data collected using the slipta checklist reveal the current state of a laboratory’s quality management systems and identify gaps in the 12 quality system essential areas defined by the clinical and laboratory standards institute.4 audit results are used to develop action plans and guide the selection of quality improvement activities for the slmta programme so as to help laboratories move toward national or international accreditation.the slmta programme in vietnam and cambodia began in 2010 when representatives from each country participated in a two-week training-of-trainers workshop. after stakeholder engagement, governmental approval and, in the case of vietnam, planning with international partners, baseline audits were conducted in seven laboratories in cambodia in june of 2011 using the standard paper-based slipta checklist. auditors and implementing leadership from vietnam noted that using the paper-based checklist created several challenges. cumbersome audit and administrative processes meant that auditors were required to bring blank paper-based checklists and, in the case of exit or follow-up audits, previously-completed checklists to review prior recommendations and scores. scores for all 111 questions had to be calculated and/or graphed manually for each report in each round of audits. additionally, communication opportunities were lost; data analysis was inefficient and delayed; reports were not standardised; and there were difficulties in combining data for regionalor country-level management. as a result of the observed difficulties, prior to performing the baseline audits in vietnam, ministry of health (moh) laboratorians supporting slmta in vietnam set out to develop an electronic tool (e-tool) for collection and use of audit data. this paper describes the development of the slipta checklist e-tool and discusses its benefits for slmta implementation. research methods and design top ↑ development of the e-tool development of the slipta checklist e-tool began with analysis of the current audit workflow. next, experts from the moh and us centers for disease control and prevention’s vietnam office (cdc-vietnam) gathered background information about the structure, scoring strategy and other aspects of the slipta checklist from auditors and other stakeholders. the tuberculosis electronic laboratory assessment tool (tb-lat), a microsoft® excel-based e-tool developed previously by integrated quality laboratory services and used globally for tuberculosis assessment,5 was modified by the slmta management team within the vietnam hospital administration system so as to incorporate the slipta checklist. the structure of the checklist was retained. during the development of the e-tool, input was sought from the moh and cdc-vietnam laboratorians working as slmta trainers and auditors in vietnam.the vietnam slmta team pilot tested the e-tool in early 2012 in parallel with the paper-based checklist in a provincial public health laboratory. the e-tool was then used for the baseline audits of a mix of 12 public health, hospital and hiv laboratories in vietnam in may 2012. the tool was further enhanced in an iterative process of feedback and improvement. exit audits of the first round of slmta-supported laboratories in both cambodia (january 2013) and vietnam (june to july 2013) provided additional opportunities to refine the tool and validate macro formulae. results top ↑ the baseline audit in cambodia using the paper-based slipta checklist had several limitations. audits required at least one-and-a-half days, including two hours to manually fill out the 44-page slipta checklist and calculate site scores in order to create the reports. although audit teams gave verbal reports at the individual laboratory summation conferences immediately following the audits, final written reports to the laboratories were compiled remotely after the audit and delivered by mentors at their next visit, sometimes delayed as long as two weeks. because the existing paper-based checklist is in a pdf or microsoft® word format, multi-site data were not easily manipulated, reducing the analytical value. additionally, the country team scanned and saved all final paper documents, requiring significant administrative time and costs for electronic data storage.the e-tool6 addresses several issues related to ease of use during the audit process and solutions for data management (table 1). drop-down response selections enable automatic scoring, easily accounting for non-applicable questions. additional features of the electronic format include: automatic linkage of information; pre-programmed calculations to compare baseline and exit audits; and pre-set data check limits so as to help improve scoring accuracy. all comments entered in each of the 12 sections are automatically pulled to the end of the report to complete the recommendation section. summary pages visually highlight the accomplishments and remaining gaps to be addressed, utilising clear colour-coded graphs. table 1: challenges of the paper-based slipta checklist, and solutions provided by the slipta e-tool. in addition to improved functionality of data at the laboratory-level, the e-tool improves usability for country-level programme management. the ability to merge data easily from multiple laboratories affords an accurate analysis with robust statistical indicators, including means and medians, minimum and maximum values, interquartile ranges, standard deviations of means and coefficients of variation at both indicator and section levels. compiling results electronically for multiple laboratories allows the remaining gaps to be addressed by appropriate improvement projects in the slmta programme and shared with moh leadership (figure 1). figure 1: example of e-tool data output for baseline (row 1) and exit audit (other rows) for section 1 (documents and records) compiled from 12 laboratories in vietnam, 2013. data collected from all laboratories can be collated and used to assess countrywide gaps and initiate corrective actions. figure 2 presents a sample e-tool report, which combines results from all 12 participating laboratories in vietnam. baseline audits showed major gaps in four sections: internal audit (0%), corrective action (0%), occurrence management and process improvement (0%) and documents and records (22%) (figure 2a). section-level reports break out details of each question. for example, the section 1 issues pertained predominantly to the development of manuals and standard operating procedures (sops) (figure 2b). additional training was conducted on sops for the laboratory system, lectures on development of manuals and internal audit were included in the third slmta workshop and improvement projects related to these gaps were organised. exit audit results showed substantial increases in the scores of these sections (100%, 57%, 87% and 88%, respectively) (figure 2a), as well as in the subsection scores for section 1 (figure 2c). the clear visual summary of results provided by the e-tool report facilitated timely development of an action plan to address the issues. figure 2: visualised results for 12 laboratories in vietnam using the slipta e-tool. (2a) spider plot of combined baseline (may 2012) and exit (june to july 2013) audit median data for all 12 laboratory sites. (2b) computer screen shot of baseline audit data for section 1 (documents and records) of the slipta checklist with bar-chart results. colours indicate results that are acceptable (green), unacceptable (red) or partially compliant (yellow). (2c) computer screen shot of exit audit data for section 1 (documents and records) of the slipta checklist with bar-chart results. discussion top ↑ the newly-developed slipta e-tool improved the audit process, enhanced communication of audit results and ensured timely usability of the data collected in vietnam and cambodia.7 the e-tool received positive feedback from in-country auditors for reducing workload. because auditors are highly-trained limited resources, they must be utilised as efficiently and effectively as possible. vietnam and cambodia have only 10 to 12 qualified auditors in each country. as laboratory quality improvement scales up to include the more than 1700 public laboratories in vietnam8 and 82 public laboratories in cambodia,9 increasing the efficiency of the audit process will become even more critical.the ability to leave a one-page printed summary in vietnamese for each laboratory was a substantial improvement over the paper tool. the communication benefits of providing same-day, on-site language-appropriate results to laboratorians and administrators encourage engagement from leadership in the improvement process. furthermore, presenting reliable results in a graphical format leads to better understanding of problems and use of data for improvement. as with the tb-lat e-tool, this first version of the slipta e-tool has capacity for data exporting; however, some minor issues have been identified when transferring data between various versions of microsoft® excel. improvements to the tool should continue, based on feedback from end-users. in addition, studies are needed to provide a formal evaluation of the tool, including time and cost savings, improved accuracy and user preferences. potential benefits of the e-tool expand beyond improved auditing. the tool incorporates auditing instructions for each slipta question, which will enhance learning and consistency for laboratories performing internal audits. furthermore, as additional auditors are trained, the e-tool can be used for evaluation and validation of their competency and standardisation of audit criteria. moreover, when unlocked, the electronic checklist can be customised and expanded to address additional laboratoryor countryspecific data needs. the slipta checklist e-tool is now available for use by other countries and can easily be customised as needed; for example, optional languages can be added for local settings. widespread use of the tool will allow development of a database for slmta programme managers at the global level in order to evaluate systematic gaps in laboratory quality and to enhance overall planning, implementation and sharing of results. acknowledgements top ↑ the authors would like to extend their sincere thanks to dr katy yao, cdc, for her strong support and invaluable contribution to and review of the design of the slipta e-tool, as well as to dr elizabeth luman, cdc, for her patience and professional guidance in development of the manuscript. finally, the authors would like to thank all end-users of the slipta e-tool for their invaluable feedback and contribution.this project has been supported by the us president’s emergency plan for aids relief (pepfar) through the cdc under the terms of cooperative agreement project number ps001928 (vietnam administration for medical services) and ps001285 (american society for clinical pathology). competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions t.t.n. (nihe) led the design of the e-tool, analysis/ interpretation of data and led the manuscript writing/ revision. b.m. (mayo clinic) participated in implementation of the audit, interpretation of data and supported the writing and revision of the manuscript. a.p. (iqls) led the design and review of the manuscript. k.n.l. (vams) was responsible for implementation of the slmta programme in vietnam and supported introduction of the e-tool. q.t.h. (cdc, vietnam), s.m. (usaid, indonesia), c.q.n. (fhi360, vietnam) and k.t.n. (hust, vietnam) participated in the design and review of the e-tool. h.m.c. (samhsa) was responsible for coordination of the implementation work and participated in the design of the e-tool. k.b.b. (cdc, vietnam) directed the e-tool introduction and supported the writing of the manuscript, as well as being responsible for the programme implementation and revision of the manuscript. cdc disclaimer the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the cdc. references top ↑ 1. world health organization’s regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region [document on the internet]. c2013 [cited 2014 sep 20]. available from: http://www.afro.who.int/en/clusters-a-programmes/hss/blood-safety-laboratories-a-health-technology/blt-highlights/3859-who-guide-for-the-stepwise-laboratory-improvement-process-towards-accreditation-in-the-african-region-with-checklist.html2.yao k, maruta t, luman et, et al. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(1), art. #194, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i1.194 3.yao k, luman et, slmta collaborating authors. evidence from 617 laboratories in 47 countries for slmta-driven improvement in quality management systems. afr j lab med. 2014;3(2), art. #262, 11 pages. http://dx.doi.org/10.4102/ajlm.v3i2.262 4.clinical and laboratory standards institute. application of a quality management system model for laboratory services; approved guidelines – third edition. [clsi document gp26-a3]. clinical and laboratory standards institute; 2004. 5.integrated quality laboratory services (iqls). tb-lat tuberculosis electronic laboratory assessment tool [document on the internet]. c2013 [cited 2014 apr 28]. available from: http://www.iqls.net/docs/tb-lat_presentation.pdf 6.thuong nt, pierson a, hoang qtt, et al. vietnam slmta e-tool [document on the internet]. c2013 [cited 2014 apr 28]. available from: http://aslm.org/aslm2012/images/docs/speaker_presentations/sunday_2_dec/symposium_overview_day1/2b_-_vietnam_slmta_e-tool.pdf 7.carvalho hm. slmta in vietnam – the roadmap for laboratory quality. vietnam: us centers for disease control and prevention; 2012 (unpublished). 8.ministry of health of vietnam. laboratory qms in vietnam – roads towards improvement. report presented at the symposium on quality management for medical laboratories. bangkok, thailand; 2011 march 17–18. 9.kingdom of cambodia, ministry of health. appendix 17: complementary package of activities for referral hospitals 2006–2010. in: national guidelines on complementary package of activities for referral hospital development from 2006 to 2010. 2nd ed., 2006; p. 128–130. abstract introduction methods results discussion acknowledgements references about the author(s) neema camara epidemiology and disease control section, ministry of health, dodoma, united republic of tanzania nyambura moremi department of bacteriology, national public health laboratory, dar es salaam, united republic of tanzania janneth mghamba epidemiology and disease control section, ministry of health, dodoma, united republic of tanzania eliudi eliakimu health quality assurance unit, ministry of health, dodoma, united republic of tanzania edwin shumba african society for laboratory medicine, addis ababa, ethiopia pascale ondoa african society for laboratory medicine, addis ababa, ethiopia beverly egyir department of bacteriology, noguchi memorial institute for medical research, college of health sciences, university of ghana, legon, ghana citation camara n, moremi n, mghamba j, et al. surveillance of antimicrobial resistance in human health in tanzania: 2016–2021. afr j lab med. 2023;12(1), a2053. https://doi.org/10.4102/ajlm.v12i1.2053 review article surveillance of antimicrobial resistance in human health in tanzania: 2016–2021 neema camara, nyambura moremi, janneth mghamba, eliudi eliakimu, edwin shumba, pascale ondoa, beverly egyir received: 08 aug. 2022; accepted: 09 mar. 2023; published: 22 may 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: antimicrobial resistance (amr) surveillance plays an important role in early detection of resistant strains of pathogens and informs treatments decisions at local, regional and national levels. in 2017, tanzania developed a one health amr surveillance framework to guide establishment of amr surveillance systems in the human and animal sectors. aim: we reviewed amr surveillance studies in tanzania to document progress towards establishing an amr surveillance system and determine effective strengthening strategies. methods: we conducted a literature review on amr studies conducted in tanzania by searching google scholar, pubmed, and the websites of the tanzania ministry of health and the world health organization for articles written in english and published from january 2012 to march 2021 using relevant search terms. additionally, we reviewed applicable guidelines, plans, and reports from the tanzanian ministry of health. results: we reviewed 10 articles on amr in tanzania, where studies were conducted at hospitals in seven of tanzania’s 26 regions between 2012 and 2019. nine amr sentinel sites had been established, and there was suitable and clear coordination under ‘one health’. however, sharing of surveillance data between sectors had yet to be strengthened. most studies documented high resistance rates of gram-negative bacteria to third-generation cephalosporins. there were few laboratory staff who were well trained on amr. conclusion: important progress has been made in establishing a useful, reliable amr surveillance system. challenges include a need to develop, implement and build investment case studies for the sustainability of amr surveillance in tanzania and ensure proper use of third-generation cephalosporins. what this study adds: this article adds to the knowledge base of amr trends in tanzania and progress made in the implementation of amr surveillance in human health sector as a contribution to the global amr initiatives to reduce amr burden worldwide. it has highlighted key gaps that need policy and implementation level attention. keywords: surveillance; antimicrobial resistance; covid-19; one health; tanzania; africa. introduction antimicrobial resistance (amr) is a global public health threat with extensive social, health and economic impacts.1,2 globally, it accounts for about 700 000 deaths annually.3 antimicrobial resistance threatens the lives of 10 million people and an economic loss of up to $100 trillion (united states dollar [usd]) per year by 2050, if there are no effective interventions.3 it is estimated that the magnitude of the amr burden falls on lowand middle-income countries.3 antimicrobials are the mainstay of modern medicine; without them, medical procedures, including surgeries, joint replacements, and treatments, such as cancer chemotherapy, could become too risky to be undertaken as healing would take a long time and become costly.3 in 2015, the world health assembly, through its 68th session, adopted the global action plan on amr to ensure the treatment and prevention of infectious diseases with quality-assured, safe, and effective medicines available.4 the global action plan outlines five strategic objectives, which are: (1) to improve awareness and understanding of amr; (2) to strengthen knowledge through surveillance and research; (3) to reduce the incidence of infection; (4) to optimise the use of antimicrobial agents; and (5) to ensure sustainable investment in countering amr.4 to support the implementation of the global action plan, during the same year, the world health organization (who) launched the global amr surveillance system (glass), the first global collaborative effort to standardise amr surveillance.5 the glass provides a standardised approach for collecting, analysing, and sharing amr data and documents the status of existing or newly developed national amr surveillance systems.5 in 2016, tanzania developed the national action plan for amr (2017–2022) following the who and global health security agenda joint external evaluation recommendation. subsequently, a holistic one health amr surveillance framework was developed to guide the establishment of amr surveillance systems in the human, animal and environmental health sectors. the country is bordered by more than eight countries, which poses a high risk of pathogen importation into the country. in addition, several socioeconomic, demographic and environmental factors also facilitate the emergence and spread of microorganisms; thus, robust health systems are paramount for detecting, responding, and mitigating the effects of the resistant microbes. in this current global coronavirus disease 2019 (covid-19) pandemic situation, where scientists are struggling to find an effective treatment for covid-19, antibiotics have been widely used to manage covid-19, either to treat covid-19 itself or co-infections.6 in fact, recent studies have shown the rampant use of antibiotics by most covid-19 patients without bacterial co-infections.7 this paper reviewed amr surveillance studies and documents the progress made in establishing the amr surveillance system in the human health sector in tanzania and provides recommendations for strengthening it. the literature review was essential to contextualise the amr situation in the past decade and the need to strengthen amr surveillance. methods data collection we searched google scholar, pubmed, and websites of the tanzania ministry of health and who written in english and published from january 2012 to march 2021. we used the search terms: ‘antimicrobial resistance’, ‘bacterial resistance’, ‘antibiotic resistance’; ‘amr surveillance’, or ‘surveillance’ or ‘cross-section’ or ‘review’; and ‘tanzania’. all words were searched together, and, in some instances, two of the three words were used. we reviewed guidelines, plans, and reports from the ministry of health to describe the tanzania amr surveillance system’s objective, surveillance sites, data collection, reporting, analysis, interpretation, and dissemination, coordination of amr surveillance activities, and funding of amr surveillance. setting and structure of healthcare system in tanzania the united republic of tanzania comprises tanzania’s mainland and the semiautonomous islands of zanzibar, and it lies on the east african coast. the tanzania 2012 population census was 44 928 923.8 tanzania mainland has 26 administrative regions, 139 districts and 184 councils. the council divides into divisions, then wards, and streets/villages. the local government authorities (or councils) are the most important administrative and implementation units for public services. health services in tanzania are decentralised into three categories: district (primary level), regional (secondary level), and zonal and national hospital (tertiary level). the district level provides primary health care services through dispensaries at the village level, health centres at the ward level, and level 1 hospital at the council level.9 dispensaries provide preventive and curative out-patient services. in contrast, health centres admit patients and sometimes provide surgical services. council hospitals provide healthcare to referred patients and provide medical and basic surgical services. regional referral hospitals provide specialist medical care. zonal and national hospitals offer advanced (super specialist) medical care and are teaching hospitals for medical, paramedical, and nursing training.9 public, private and faith-based organisations health facilities, private pharmacies, and accredited drug dispensing outlets provide pharmaceutical services.10,11 results antimicrobial resistance trends of priority pathogens in tanzania a total of 10 articles on amr in tanzania were retrieved and reviewed. these studies were conducted at either the zonal or regional referral hospitals between 2012 and 2019. four of the 10 studies were conducted at kilimanjaro christian medical centre in the kilimanjaro region, three at bugando medical centre in mwanza region, two at muhimbili national hospital in dar es salaam region, and one study each at maweni regional referral hospital in kigoma region, musoma regional referral hospital in mara region, sumbawanga regional referral hospital in rukwa region, st. benedict ndanda hospital in mtwara region, bagamoyo district hospital in pwani region, sekou toure regional referral hospital, nyamagana district hospital, and sengerema district designated hospital in mwanza region (figure 1). blood, pus and wound swabs, and urine were the most common laboratory samples analysed (table 1). all of the studies performed antimicrobial susceptibility testing (ast) using the disk-diffusion method per the clinical laboratory standards institute guidelines.12 the most frequently isolated microorganisms from blood were staphylococcus aureus, klebsiella pneumoniae and escherichia coli; and from pus, pseudomonas aeruginosa (table 1). s. aureus resistance to clindamycin ranged between 33.3% to 68.4% and erythromycin between 35.6% to 76.3%, while resistance to cotrimoxazole was 82.6% and ampicillin was 100%.13,14,15,16 the studies reported low rates of resistance to cefoxitin (27.3%), tetracycline (34.9%), cotrimoxazole (26.5%) and ceftriaxone (11.1%).14,17,18 prevalence of methicillin-resistant s. aureus decreased from 41.2% in 2013 to 9.5% in 2015, but rose to 66.7% in 2018.14,15 k. pneumoniae was resistant to ampicillin (100%), cotrimoxazole (96.3%), ceftriaxone (95.7%), amoxicillin/clavulanate (94.6%), ceftazidime (90.9%), gentamycin (86.4%) and cefepime (75.6%).13,16,19 compared to other gram-negatives, e. coli was more resistant to ampicillin, amoxicillin-clavulanic acid, gentamycin, tetracycline, ciprofloxacin, amikacin, third-generation cephalosporins (ceftazidime and ceftriaxone) and cefepime.,13,15,16,19,20 notably, p. aeruginosa was resistant to cefepime (93.8%).13 overall, most studies documented high resistance rates of gram-negative bacteria to third-generation cephalosporins.17,21,22 figure 1: antimicrobial resistance study locations and sentinel surveillance sites, august 2021, tanzania. table 1: antimicrobial resistance trends of glass priority pathogens in tanzania, 2012–2021. progress in implementation of antimicrobial resistance surveillance coordination in 2018, tanzania took the first step of developing a one health national amr surveillance framework to guide the establishment of amr surveillance programmes in the country. the framework provides a holistic approach to monitor trends of infections and resistance that will inform standard treatment guidelines that support best practices for patient care, and links amr information from the human, animal and environmental health sectors.23 the objectives of amr surveillance are to routinely collect, analyse, and interpret quality amr data to generate evidence on trends and the burden of the who priority pathogens, and monitor amr interventions’ effectiveness. the country also established a national multi-sectoral coordinating committee (mcc) to oversee and coordinate all amr-related activities in all sectors. the chief medical officer of the ministry of health and the director of veterinary services, ministry of livestock and fisheries alternate as co-chair of the committee. the committee is composed of representatives from the human, animal and environmental health sectors, as well as livestock and food production stakeholders, and those from medical and agricultural universities. the who, food and agriculture organization, united states centres for disease control and prevention, management science for health and world organization for animal health, are also represented in the mcc. there are designated national amr focal points from animal and human sectors that form part of the mcc secretariat, as well as four multisectoral technical working groups on (1) awareness, effective communication and education; (2) knowledge, surveillance, research and sustainable investments; (3) sanitation, hygiene and infection prevention and control; and (4) antimicrobial use stewardship. the mcc and surveillance twg meets at least once every quarter of the year. the whole coordination structure operates under the ‘one health’ and whole-of-government approach. antimicrobial resistance surveillance system tanzania started with laboratory-based amr surveillance in healthcare settings, as laboratory-based surveillance is the most efficient amr burden determination method.24,25 in the first phase of the national tanzania amr surveillance there were two laboratory levels: coordinating laboratory and site (sentinel/participating) laboratories. as of 2022, there are a total of nine amr sentinel sites which are active and functional. the sentinel sites include muhimbili national hospital, mbeya zonal referral hospital, bugando medical centre, kilimanjaro christian medical centre, mnazi mmoja hospital in zanzibar, temeke regional referral hospital in dar es salaam region, morogoro regional referral hospital in morogoro region, maweni regional referral hospital in kigoma region, and benjamin mkapa hospital in dodoma region (figure 1). the national health laboratory (nhl) is the national coordinating laboratory, and its primary roles include: developing amr national standard operating procedures; training, mentoring and supervising sentinel laboratories; conducting external quality assurance and monitoring internal quality assurances done by sentinel laboratories; and compiling, aggregating, analysing, visualising and disseminating national amr surveillance data to the national mcc and the glass. on the other hand, sentinel laboratories isolate and identify organisms; perform susceptibility tests; store isolates as per national standardised operating procedures; produce and share timely antibiograms with clinicians; and conduct internal quality assurances. antimicrobial resistance surveillance involves the routine collection of blood and urine specimens from in and out-patients with clinical signs and symptoms attending the hospitals. clinicians decide whether to take samples for microbiological culture based on the patient’s clinical assessment. presently, the participating laboratories employ phenotypic methods for pathogen identification and disk diffusion for ast. the ast is a laboratory procedure to identify effective antimicrobial agents that kill or prevent the growth of isolated pathogens recovered from samples of individual patients.26 antimicrobial susceptibility testing results guide clinicians and service providers to decide on target therapy, preserve drugs, and evaluate treatment services.26 notably, continuous surveillance for resistance patterns is crucial due to the mutations in bacterial dna.26 the ast is performed and interpreted according to international guidelines such as the clinical and laboratory standards institute guidelines. the ast results are categorised into either susceptible (s) or non-susceptible, which include intermediate (i) and resistant (r) according to clinical breakpoints defined by clinical and laboratory standards institute. patient clinical data, including infection origin (community or hospital), age, gender and admission types (out-patient, in-patient, general ward or intensive care unit) are collected regardless of culture positivity or negativity. infection origin are categorised as hospital-acquired (specimen from an in-patient admitted for > 2 days) or community-acquired (specimen from an out-patient or in-patient admitted for ≤ 2 days).23 clinical data and ast positive culture results are recorded in the reporting forms and entered into the laboratory information system and the world health organization network (whonet), a freely available system for capturing, analysing and sharing amr data in a standardised format. data import into whonet can be semi-automated using the add-on baclink software (who collaborating centre for surveillance of antimicrobial resistance, boston, massachusetts, united states), which allows for import from other data sources, for example, text files exported from a laboratory information management system (lims) or directly from a laboratory instrument.27 however, whonet intentionally provides only a solution for basic laboratory specimen management and result reporting and does not have comprehensive lims functionality.27 laboratory departments communicate ast results immediately to clinicians as well as the infection prevention and control and amr teams for appropriate treatment and control programs in the local setting. target pathogens for monitoring and reporting as per the national and who priorities include e. coli, k. pneumoniae, acinetobacter baumannii, s. aureus, neisseria meningitidis, streptococcus pneumoniae, salmonella spp., shigella spp., pseudomonas spp. and neisseria gonorrhoeae. data analysis, interpretation and dissemination the participating laboratories must clean, collate, analyse, and create site-specific bacterial antibiograms every month. in addition, annual amr surveillance reports are shared with the relevant clinical departments and hospital committees to increase hospital and community amr awareness, inform treatment policies at the health facility, and encourage continued participation in the surveillance system. antimicrobial resistance data from surveillance sites are centrally stored and managed at the nhl. the nhl conducts data quality checks, analysis, and visualisation, generates official amr reports, and provides long-term data storage. antimicrobial resistance surveillance reports, including trends and resistant pathogen prevalences, are generated at least twice a year and disseminated to stakeholders after approval by the amr surveillance technical working group and the national mcc. at the same time, the clean amr data set is transmitted to the glass (figure 2). the amr surveillance data guides strategies and policies for combating amr. it also provides opportunities for in-depth scientific research that generates additional knowledge on amr. however, sharing of surveillance data among sectors is yet to be strengthened, that is, environment, health and animal. tanzania started reporting amr data to glass in june 2021. figure 2: antimicrobial resistance data flow in tanzania (9 january 2022). quality assurance and standards all nine sentinel sites participate in the external quality assurance which is being supported by nhl and african society for laboratory medicine under the project called external quality assessment for africa. external quality assurance is done twice a year for all sites and involves most of the glass priority pathogens, including e. coli, k. pneumoniae, a. baumannii, s. aureus, n. meningitidis, s. pneumoniae and salmonella spp. as per the amr surveillance framework, all isolates are to be stored at −70 °c for future studies. isolates with unusual, unexpected, or indeterminate resistance patterns are sent to the nhl for confirmatory testing and ast. also, every 10th isolate from each site is sent to nhl for quality assessment. funding funds to run the nine amr surveillance sentinel laboratories are contributed by the government of the united republic of tanzania, the fleming fund, and the united states agency for international development fund under the infectious disease diagnostic and surveillance project. therefore, there is a need to establish a sustainable funding mechanism for amr activities in tanzania. discussion this review has shown that the most frequently isolated microorganisms from blood were s. aureus, k. pneumoniae and e. coli; from urine, e. coli; and from pus, p. aeruginosa. most studies documented high resistance rates of gram-negative bacteria to third-generation cephalosporins. importantly, significant progress has been made in establishing amr surveillance; nine sentinel sites across tanzania have been established and are generating data and there are suitable and clear coordination structures and platforms for multisectoral engagement and collaboration under one health. however, sharing of surveillance data between sectors is yet to be strengthened. there are also few laboratory staff well trained on amr practices. studies in tanzania reveal increasing bacterial resistance to third-generation cephalosporins. in tanzania, ceftriaxone is reportedly prescribed excessively and inappropriately in hospital settings,28 and this may explain the observed third-generation cephalosporins resistance trends. if this trend continues, clinicians will resort to broad-spectrum antibiotics, such as carbapenem, which are the last resort according to the tanzania treatment guideline.29,30 if such a situation occurs, effective, quality, and affordable healthcare provisions, the core fundamentals for universal health coverage, will be far from being realised. progress made in establishing the amr surveillance system in the country is commendable. a total of nine sentinel sites have been established. there are suitable coordination structures and platforms for multisectoral engagement and collaboration under the ‘one health’.31 the amr sentinel surveillance sites are representative and provide amr trends and burden data. amr surveillance system has helped to standardise routine microbiological cultures and ast in hospitals, particularly those participating in amr surveillance according to global standards (mcc meeting minutes of 11 may 2021, unpublished). however, the facilities still face challenges while implementing amr surveillance, including a lack of interoperability between the sentinel site laboratory information system and whonet to enable automatic data transfer between the two systems. the double entry of the same information in two different systems exhausts and overworks the laboratory staff. a lack of fit-for-purpose lims and open-source lims software with technical standards and functionality for amr surveillance is a particular concern in lowand middle-income countries.27 although whonet is a functional and useful repository for microbiological data with capabilities for standardised data sharing, it lacks full lims functionality.27 thus, there is an urgent need for investment in laboratory information technology infrastructure and data management systems that can capture high-quality laboratory and patient management data. there are few well-trained laboratory staff at the sentinel sites for data analysis and the production of antibiograms, which can be shared with the clinicians and amr teams to inform on the appropriate treatment and measures to tackle amr at the hospital or community. at the surveillance sites, frequent stock out of ast reagents and analysis, inadequate resources, and poor laboratory infrastructure for phenotypic and genotypic analysis is commonplace. although amr surveillance receives much support from the government as per human resources and infrastructure, there are also funds from donors. a sustainability plan is essential to prevent over-dependence on donors over time. these challenges are unique in tanzania and have also been reported elsewhere in africa.32,33 however, despite the challenges, amr surveillance is still ongoing in the country. sharing of surveillance data between sectors is yet to be strengthened. antimicrobial resistance is a broad and complex issue affecting the animal, human and environmental sectors; thus, a multisectoral and multidisciplinary combat approach is needed. antimicrobials are also widely used in animals for treatment and growth promoters. in addition, evidence suggests that antimicrobial use in animals contributes significantly to the development of amr in humans,34 necessitating a comprehensive and coordinated amr surveillance system that can continuously share amr data between sectors to inform public health interventions. this study has some limitations. the review was based on reports only. we did not seek additional inputs and insights from amr stakeholders through a standardised questionnaire or interview. as such some comprehensive views and perspectives may have been missed out. the amr trends in tanzania presented here should be interpreted with caution as the review was only based on 10 surveillance articles. conclusion tanzania is currently implementing amr surveillance in nine hospitals, and reporting of amr data to glass has commenced. there are well-established amr coordination mechanisms at health facilities and national levels to effectively implement and utilise the amr information. although there are challenges affecting implementation, the current amr surveillance system in place is useful, reliable and capable of better performance. there is a need to develop, implement and build an investment case study for the sustainability of amr surveillance in tanzania. we recommend that the government creates a fit-to-purpose laboratory information system with functionality able to link with other systems; develops a mechanism for sustainable financing for laboratory infrastructure development and continuous supply of reagents, commodities, and laboratory materials; and invests hugely in building human capacities for bacterial identification and ast and data analysis, interpretation and utilisation. acknowledgements the authors would like to acknowledge the government of tanzania and partners for the efforts to implement amr surveillance activities. also, we appreciate authors of the surveillance articles and contributors of the amr reports, minutes, guidelines, and frameworks which informed this writing. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions n.c., e.s., p.o. and b.e. were involved in the conceptualization of the project. n.c. and b.e, were involved in the writing of an initial draft, reviewing and editing the manuscript. n.c., n.m., j.m, e.e., e.s., p.o., b.e. reviewed and edited the manuscript. all authors read and approved the final version of the manuscript. ethical considerations this paper has no ethical concerns as it does not have human or animal subjects or data, and as such ethical approval from an ethics review board was not required. sources of support this work was part of the activities of a fellowship (surveillance in human health) workplan, which was supported by the fleming fund through the fellowship scheme in tanzania. data availability the authors confirm that the data supporting the findings of this study are available within the article. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references world health organization, editor. antimicrobial resistance: global report on surveillance. world health organization; geneva, 2014. howell l, world economic forum, risk response network. global risks 2013. world economic forum; geneva, 2013. o’neill j. tackling drug-resistant infections globally: final report and recommendations: the review on antimicrobial resistance. world bank; 2016 (cited 2020 april 10). available from: https://amr-review.org/sites/default/files/160525_final%20paper_with%20cover.pdf world health organization. 2015. global action plan on antimicrobial resistance [homepage on the internet]. world health organization. c2015 (cited 2020 september 13). available from: https://apps.who.int/iris/handle/10665/193736 world health organization. global antimicrobial resistance surveillance system (glass) report: early implementation 2016–2017. world health organization; geneva, 2017. iwu cj, jordan p, jaja if, iwu cd, wiysonge cs. treatment of covid-19: implications for antimicrobial resistance in africa. pan afr med j. 2020;35(suppl 2):119. https://doi.org/10.11604/pamj.supp.2020.35.2.23713 rawson tm, moore lsp, zhu n, et al. bacterial and fungal co-infection in individuals with coronavirus: a rapid review to support covid-19 antimicrobial prescribing. clin infect dis. 2020;71(9):2459–2468. https://doi.org/10.1093/cid/ciaa530 united republic of tanzania. population and housing census 2012: population distribution by administrative areas. dar es salaam: national bureau of statistics; 2013. hokororo j, eliakimu e, ngowi r, et al. report of trend for compliance of infection prevention and control standards in tanzania from 2010 to 2017 in tanzania mainland. microbiol infect dis. 2021; 5(3):1–10. minzi om, manyilizu vs. application of basic pharmacology and dispensing practice of antibiotics in accredited drug-dispensing outlets in tanzania. drug healthc patient saf. 2013;5:5–11. https://doi.org/10.2147/dhps.s36409 poyongo bp, sangeda rz. pharmacists’ knowledge, attitude and practice regarding the dispensing of antibiotics without prescription in tanzania: an explorative cross-sectional study. pharmacy. 2020;8(4):238. https://doi.org/10.3390/pharmacy8040238 clinical and laboratory standards institute (clsi). performance standards for antimicrobial susceptibility testing. 27th ed. clsi supplement m100. pennsylvania: clinical and laboratory standards institute; 2017. mikomangwa wp, bwire gm, kilonzi m, et al. the existence of high bacterial resistance to some reserved antibiotics in tertiary hospitals in tanzania: a call to revisit their use. infect drug resist. 2020;13:1831–1838. https://doi.org/10.2147/idr.s250158 kazimoto t, abdulla s, bategereza l, et al. causative agents and antimicrobial resistance patterns of human skin and soft tissue infections in bagamoyo, tanzania. acta trop. 2018;186:102–106. https://doi.org/10.1016/j.actatropica.2018.07.007 mnyambwa np, mahende c, wilfred a, et al. antibiotic susceptibility patterns of bacterial isolates from routine clinical specimens from referral hospitals in tanzania: a prospective hospital-based observational study. infect drug resist. 2021;14:869–878. https://doi.org/10.2147/idr.s294575 seni j, mwakyoma aa, mashuda f, et al. deciphering risk factors for blood stream infections, bacteria species and antimicrobial resistance profiles among children under five years of age in north-western tanzania: a multicentre study in a cascade of referral health care system. bmc pediatr. 2019;19(1):32. https://doi.org/10.1186/s12887-019-1411-0 kumburu hh, sonda t, mmbaga bt, et al. patterns of infections, aetiological agents and antimicrobial resistance at a tertiary care hospital in northern tanzania. trop med int health. 2017;22(4):454–464. https://doi.org/10.1111/tmi.12836 mauki ii, william jd, mlay hl, et al. etiologies of bloodstream infection and antimicrobial resistance: a cross sectional study among patients in a tertiary hospital, northern tanzania. east afr sci. 2021;3(1):104–111. https://doi.org/10.24248/easci-d-20-00012 kiponza r, balandya b, majigo mv, matee m. laboratory confirmed puerperal sepsis in a national referral hospital in tanzania: etiological agents and their susceptibility to commonly prescribed antibiotics. bmc infect dis. 2019;19(1):690. https://doi.org/10.1186/s12879-019-4324-5 pratap fn. prevalence and antimicrobial resistance patterns of extended spectrum beta-lactamase producing e. coli in human isolates at kilimanjaro medical centre, moshi, tanzania [homepage on the internet]. thesis. 2019 [cited 2021 jun 15]. available from: http://repository.costech.or.tz//handle/123456789/15023 moremi n, claus h, mshana se. antimicrobial resistance pattern: a report of microbiological cultures at a tertiary hospital in tanzania. bmc infect dis. 2016;16(1):756. https://doi.org/10.1186/s12879-016-2082-1 shabhay aa, horumpende p, mujuni m, et al. antibiotic resistance in aerobic bacterial isolates from infected diabetic foot ulcers in north eastern tanzania: an urgent call to establish a hospital antimicrobial stewardship committee. preprint 2021 jun 8 [cited 2021 jun 15]. https://doi.org/10.21203/rs.3.rs-569062/v1 united republic of tanzania. national antimicrobial resistance framework. dar es salaam: united republic of tanzania; 2018. mendelson m, matsoso mp. the world health organization global action plan for antimicrobial resistance. s afr med j. 2015;105(5):325–325. https://doi.org/10.7196/samj.9644 perovic o, schultsz c. stepwise approach for implementation of antimicrobial resistance surveillance in africa. afr j lab med. 2016;5(3):7. https://doi.org/10.4102/ajlm.v5i3.482 bayot ml, bragg bn. antimicrobial susceptibility testing [homepage on the internet]. statpearls publishing; 2021 [cited 2021 mar 07]. available from: http://www.ncbi.nlm.nih.gov/books/nbk539714/ turner p, rupali p, opintan ja, et al. laboratory informatics capacity for effective antimicrobial resistance surveillance in resource-limited settings. lancet infect dis. 2021;21(6):e170–e174. https://doi.org/10.1016/s1473-3099(20)30835-5 sonda tb, horumpende pg, kumburu hh, et al. ceftriaxone use in a tertiary care hospital in kilimanjaro, tanzania: a need for a hospital antibiotic stewardship programme. plos one. 2019;14(8):e0220261. https://doi.org/10.1371/journal.pone.0220261 mushi mf, mshana se, imirzalioglu c, bwanga f. carbapenemase genes among multidrug resistant gram-negative clinical isolates from a tertiary hospital in mwanza, tanzania. biomed res int. 2014;2014:e303104. https://doi.org/10.1155/2014/303104 ssekatawa k, byarugaba dk, wampande e, ejobi f. a systematic review: the current status of carbapenem resistance in east africa. bmc res notes. 2018;11(1):629. https://doi.org/10.1186/s13104-018-3738-2 frumence g, mboera leg, sindato c, et al. the governance and implementation of the national action plan on antimicrobial resistance in tanzania: a qualitative study. antibiotics. 2021;10(3):273. https://doi.org/10.3390/antibiotics10030273 kariuki s, keddy kh, antonio m, okeke in. antimicrobial resistance surveillance in africa: successes, gaps and a roadmap for the future. afr j lab med. 2018;7(2):924. https://doi.org/10.4102/ajlm.v7i2.924 ndihokubwayo jb, yahaya aa, desta at, et al. antimicrobial resistance in the african region: issues, challenges and actions proposed. african health monitor. 2023;(16):4. carrique-mas jj, choisy m, van cuong n, thwaites g, baker s. an estimation of total antimicrobial usage in humans and animals in vietnam. antimicrob resist infect control. 2020;9(1):16. https://doi.org/10.1186/s13756-019-0671-7 article information authors: michael l. kasusse1,2 nazarius m. tumwesigye1 steven aisu3 joseph k.b. matovu1,2 rhoda wanyenze1,2 affiliations: 1makerere university school of public health, kampala, uganda 2maksph-cdc fellowship program, makerere university school of public health, kampala, uganda 3central public health laboratories, ministry of health uganda, kampala, uganda correspondence to: michael kasusse email: lkasussem@yahoo.com postal address: po box 32060, kampala, uganda dates: received: 27 aug. 2014 accepted: 15 aug. 2015 published: 30 nov. 2015 how to cite this article: kasusse ml, tumwesigye nm, aisu s, matovu jkb, wanyenze r. effectiveness of the credit-line approach for support of cd4 equipment functionality in northern uganda. afr j lab med. 2015;4(1), art. #234, 5 pages. http://dx.doi.org/10.4102/ajlm.v4i1.234 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. effectiveness of the credit-line approach for support of cd4 equipment functionality in northern uganda in this original research... open access • abstract • introduction • methods • results • discussion    • limitations    • recommendations    • conclusion • acknowledgements    • funding sources    • competing interests    • authors’ contributions • references abstract top ↑ background: improving laboratory service delivery requires a functioning logistics and supply system. uganda’s ministry of health uses the credit-line approach to provide laboratory supplies including commodities for cd4 test equipment. objectives: we examined the effectiveness of the credit-line approach in improving laboratory service delivery by using the functionality of cd4 test equipment as a proxy indicator. method: a cross-sectional survey was conducted at 7 level-three health centres (hc iiis), 18 level-four health centres (hc ivs), and 10 hospitals in 15 districts of mid-northern uganda, including the lango (17 facilities) and acholi sub-regions (18 facilities), between july 2013 and august 2013. functionality, was determined through selfand interviewer-administered questionnaires. the chi-squared test was used to assess differences in functionality by sub-region, facility type, and equipment type. results: a total of 38 cd4 test analysers were assessed. of these, 26 (68%) were functional. in hospitals, 85% of cd4 analysers were functional, in hc ivs, 67% were functional and in hc iiis, 43% were functional. the differences did not reach statistical significance. in the lango sub-region, 72% of analysers were functional; in the acholi sub-region, 65% were functional. non-functionality was mainly due to lack of reagents and cartridges, as well as low staffing levels of laboratory technicians with the skills necessary to operate the equipment. conclusion: the credit-line approach supported the functionality of cd4 equipment in the surveyed facilities. however, there is a need to address issues of staffing and availability of reagents to enhance the functionality of cd4 equipment and improve patient care, especially at hc iiis. introduction top ↑ until 2003, the ministry of health (moh) of uganda used a ‘push’ system to guarantee supply of medical commodities, especially in areas affected by natural disasters or areas such as northern uganda, which was affected by a 20-year-long civil war. in the push system, the authorised supplier determines the types and quantities of supplies to be issued to health facilities.1 inconsistencies remained between the needs of the user areas and the medical items supplied. in many health facilities, there were frequent stock outs of supplies (including reagents for cd4 equipment) and large quantities of expired items (including cartridges for cd4 equipment).2 as a consequence, this led to non-functioning equipment and patients missing cd4 monitoring. the moh introduced a ‘pull’ system in 2003 to overcome the challenges of frequent stock outs and expired items. this system required health facilities to determine the types and quantities of medical items that they needed.3,4 an assessment of the performance of the pull system to improve the availability of equipment and reduce expiration of medical supplies showed that the pull system improved the availability of medical supplies, but did not address challenges such as inadequate training of staff, lack of transport and inadequate funding.5 in order to address the challenges of funding and transport, the moh introduced the ‘credit-line’ approach in 2009 at the government-owned national medical stores supported by development partners, including the united states centers for disease control and prevention, uganda; the clinton foundation health access initiative, uganda; unitaid; and global fund, amongst others. of the funding available, 80% was allocated to public health facilities and 20% to private not-for-profit health facilities.6 this approach requires that, during every cycle of two or four months, each health facility is allocated a financial vote, known as a credit line, from which to draw an equivalent of equipment and supplies. the moh’s central public health laboratories (cphl), the laboratory activities unit, identifies a list of supplies, recommends specifications, quantifies and makes forecasts that cater to the credit line for each facility. laboratory commodities and supplies, including cd4 equipment commodities, are procured and distributed or transported to the districts by the national medical stores for public facilities and by a private, not-for-profit store, the joint medical store, for public, not-for-profit facilities. each health facility is tasked with placing orders, picking up the supplies from the districts, maintaining consumption data and sending the data to cphl for use in making other forecasts.6 however, the role of this approach in supporting the functionality of facilities’ cd4 equipment has not been evaluated in northern uganda. laboratory monitoring of hiv patients determines eligibility for anti-retroviral treatment (art), which slows down the progression of hiv to aids, in addition to monitoring the efficacy of art after initiation.7 it has been shown that, compared with viral load, cd4 count is a better predictor of clinical progression of hiv to aids and is a better guide for initiation of art.8,9 in low-income countries, laboratory monitoring of patients on art remains controversial because of ongoing resource limitations.10 in addition, unreliable or inaccurate testing leads to unnecessary costs in areas that already experience shortages. this, in turn, leads to the perception that laboratory testing is unhelpful or that it could compromise patient care.11 in developing countries, cd4 testing is common in urban areas, where patients can undertake multiple visits to clinics. the expansion of art programmes into rural areas created a need for point-of-care (poc) cd4 testing in order to overcome logistical barriers in the timely dissemination of test results and initiation to care programmes.12 rapid, reliable and affordable poc cd4 tests are not yet widely available.13,14 some of the poc cd4 test technologies on the market or in development include: pointcare® now; cyflow® minipoc; pima™ cd4; coulter cd4 count kit; dynal® t4 quant kit; daktari cd4; mbio® cd4 system; visitect® cd4; and zyomyx®. some of these are being used in the northern region of uganda.15 poc cd4 testing is an efficient intervention to reduce pre-treatment loss to follow up, because it enables clinics to stage patients rapidly on site, so that more patients are determined to be eligible and initiated on art.16 the aim of diagnostic poc testing is to minimise the wait time to obtain a test result, allowing clinicians and patients to make a quick clinical decision. in resource-limited settings, the benefits of poc testing outweigh its costs by focusing on the relevant clinical outcomes.17 there is also a need for laboratory follow-up of hiv patients in resource-limited settings, if appropriate cd4 test equipment is used. the auto 40 system, which uses thermoresistant reagents, is one example of a cd analyser that is appropriate for such settings.18 the moh’s credit-line approach is intended to ensure adequate supply of such equipment and associated reagents for clinical monitoring of patients with hiv. we evaluated the effectiveness of the credit-line approach for improving laboratory service delivery to healthcare facilities in northern uganda. facilities were surveyed to determine the functionality of their cd4 equipment as a proxy indicator of the effectiveness of the approach, including reasons for non-functionality. various factors, such as facility type and staffing levels, were examined to suggest ways of improving laboratory service delivery in the country. methods top ↑ as part of the national laboratory supplies quantification and verification exercise, between 01 july 2013 and 02 august 2013, the moh’s cphl conducted a cross-sectional survey at health facilities in the lango and acholi sub-regions of mid-northern uganda. the lango sub-region includes eight districts and the acholi sub-region, seven districts. facilities in the study area included the following types ordered by complexity of service delivery: private for-profit; private not-for-profit and government or public clinics, including level-two health centres, which are closest to communities and offer basic health services; level-three health centres (hc iiis); level-four health centres (hc ivs) which offer more complex services similar to hospitals; general hospitals; and regional referral hospitals. at least one government laboratory facility was included for each of the 15 districts of mid-northern uganda (figure 1). laboratories in an hc iv facility were selected preferentially, because the moh plans to transform hc ivs into laboratory hubs. figure 1: area of survey: acholi and lango sub-regions of uganda. depending on the availability of the laboratory managers (or ‘in charges’), semi-structured questionnaires were either self-administered by the laboratory managers or interviewer-administered to the available laboratory staff by the research team. the research team, which was composed of three cphl technical staff, including a team leader and a driver, travelled to each facility to distribute and collect the questionnaires. the questionnaires gathered the following information: the type and location of cd4 equipment and functionality; reasons for non-functionality; and staffing levels for each cadre, including laboratory technologists, laboratory technicians, laboratory assistants and microscopists, by facility. completed questionnaires were first reviewed in the field by the research team to ensure completeness and accuracy. the questionnaires had an optional telephone contact field and collectors contacted the respondents to validate information that was not clear. the statistical package for the social sciences statistical software (version 17.0; spss inc., chicago, il 2008) was used for data entry and analysis. the chi-squared method was used to evaluate whether there was a statistically-significant difference between the functionality of cd4 equipment by sub-region, facility level or equipment type. the outcome variable for determining the effectiveness of the credit-line approach was whether the cd4 equipment at each facility was functional. functional was defined as cd4 equipment that was capable of carrying out cd4 tests. the credit-line approach was considered to be effective for facilities with functional cd4 test equipment. we also evaluated whether facility level, distribution and type of cd4 equipment or the staffing levels by facility affected the presence of functional cd4 equipment. p-values below the conventionally accepted significance level of 0.05 (or 5%) were considered to be statistically significant. results top ↑ a total of 16 self-administered questionnaires were completed by the managers of laboratory facilities. laboratory managers were absent at 19 laboratory facilities, at which the research team conducted interviewer-administered questionnaires with available staff. thus, of the 68 public facilities in the lango and acholi sub-regions, a total of 35 facilities were assessed (table 1), including 7 hc iiis, 18 hc ivs and 10 hospitals in the lango (17 facilities) and acholi (18 facilities) sub-regions. these 35 facilities included 38 cd4 analysers – 18 in the lango sub-region and 20 in the acholi sub-region. pima was a predominant type of cd4 equipment and the majority of cd4 analysers were in hc iv facilities (18 of 38; 48%). overall, 26 of the 38 (68%) cd4 analysers were functional (table 2). although the lango sub-region had fewer cd4 analysers than the acholi region, a higher percentage were functional (72% in lango vs. 65% in acholi). similarly, although there were more cd4 analysers at hc iv facilities, hospitals had a higher percentage of functional cd4 equipment (67% in hc iv facilities vs. 85% in hospitals); hc iiis had the lowest percentage of functional cd4 analysers (43%). there were no significant differences in the functionality of cd4 equipment by sub-region, facility level or equipment type. non-functionality of cd4 equipment was mainly because of the lack of reagents or cartridges (table 3). other reasons given included expired cartridges, unpredictable power sources and lack of controls. low staffing levels were reported amongst the various laboratory cadres with the skills necessary to operate the equipment (table 4). the lowest levels were observed amongst laboratory technologists at hospitals (6 of 31 positions filled; 19%) and laboratory assistants at both hc iii (4 of 12 positions filled; 33%) and hc iv facilities (19 of 70 positions filled; 27%). table 1: distribution of cd4 analysers at healthcare facilities surveyed in northern uganda, july–august 2013. discussion top ↑ this assessment of the credit-line approach for providing laboratory supplies (including commodities for the functionality of cd4 equipment) to health facilities found that 68% of cd4 equipment was functional, despite the low staffing levels of laboratory cadres by facility level. staffing issues related to provision of commodities and supplies still play a role in the effectiveness of the credit-line approach, especially in areas with a history of war and natural disasters.3,4,5 despite persistent resource constraints,10 poc cd4 testing is being implemented successfully in northern uganda to overcome logistical barriers to the timely dissemination of test results and initiation to care programmes.12 table 2: functionality of cd4 equipment by region, facility level and equipment type. limitations the credit-line approach in uganda is being supported by developing partners to provide commodities and supplies to public and private not-for-profit health laboratories for the functioning of cd4 equipment.6 this approach excludes specialised testing at high-level reference laboratories, which play a very important role in outbreaks and disease surveillance in the country. when interpreting our results, readers should also consider the lack of baseline data to use as a comparison between the push and pull systems with the credit-line approach; the fact that the selection of laboratories included in the analysis was not random; and that the intended respondents (laboratory managers) were not available for more than half of the surveyed laboratories where interviewer-administered questionnaires were instead conducted with laboratory staff members. recommendations there is a need to address issues of staffing and availability of reagents to enhance the functionality of cd4 equipment and improve patient care, especially at hc iiis. staffing issues related to the functionality of cd4 equipment used in the clinical monitoring of hiv patients, such as provision of commodities and supplies, should be regarded as highly important in the effectiveness of the credit-line approach. such issues may include recruitment, as well as retention and training. there is also a need to include specialised facilities in the credit-line approach systems for meaningful laboratory services in uganda. conclusion the credit-line approach supports the functionality of cd4 equipment used in the clinical monitoring of hiv patients in areas with a history of war and natural disasters, such as northern uganda. staffing issues related to provision of commodities and supplies play a role in the effectiveness of the credit-line approach. table 3: reasons for non-functionality of cd4 equipment. table 4: staffing levels of facilities by cadre. acknowledgements top ↑ the authors appreciate the support provided by mr wilson nyegenye (laboratory logistics coordinator of the moh’s cphl) in conceiving, designing and carrying out the survey. the authors also acknowledge dr olico okui of monitoring and evaluation technical assistance at the makerere university school of public health, for reviewing the drafts, as well as mr. william lali (coordinator quality assurance, cphl) for providing technical input on issues concerning laboratory work during the manuscript’s correction stage. funding sources the authors would also like to acknowledge the moh’s cphl and the maksph-cdc fellowship program for providing the financial and material support to conduct the survey. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions m.l.k. (makerere university school of public health, maksph-cdc fellowship program) was responsible for the project design, survey, statistical calculations and writing the manuscript. n.m.t. (makerere university school of public health) was responsible for scientific and conceptual contributions to the manuscript. s.a. (cphl, moh) was the project leader. j.k.b.m. (makerere university school of public health, maksph-cdc fellowship program) provided contextual contributions on the subject of the article and r.w. (makerere university school of public health, maksph-cdc fellowship program) was responsible for the accuracy and validation of all medical information provided in the manuscript; and for proof reading and aligning the manuscript with the author guidelines. references top ↑ world health organization. analysing disrupted health sectors: a modular manual [document on the internet]. c2009 [cited 2014 jun 23]. available from: http://www.who.int/hac/techguidance/tools/disrupted_sectors/adhsm.pdf. muyingo s, david v, olupot g, et al. baseline assessment of drug logistics systems in twelve dish-supported districts and service delivery points (sdps) (draft report). delivery of improved services for health (dish) project for united states agency for international development; 2000; p. 15–34. uganda malaria control programme, ministry of health. uganda malaria control strategic plan 2005/06–2009/10 [document on the internet]. c2005 [cited 2014 jun 23]. available from: http://www.eac.int/health/index.php?option=com_docman&task=doc_download&gid=52&itemid=144. ministry of health (republic of uganda). annual health sector performance report. financial year 2003/2004 [document on the internet]. c2004 [cited 2014 jun 23]. available from: http://www.health.go.ug/docs/ahspr.pdf. tumwine y, kutyabami p, odoi ra, kalyango j. availability and expiry of essential medicines and supplies during the ‘pull’ and ‘push’ drug acquisition systems in a rural ugandan hospital. trop j pharm res. 2010;9(6):557–564. ministry of health (republic of uganda). quantification of national laboratory equipments, reagents and other supplies for 2013–2015. ministry of health; 2013. gilks cf, crowley s, ekpini r, et al. the who public-health approach to antiretroviral treatment against hiv in resource-limited settings. lancet. 2006;368(9534):505–510. pmid: 16890837, http://dx.doi.org/10.1016/s0140-6736(06)69158-7 world health organization. consolidated guidelines on the use of antiretroviral drugs for treating and preventing hiv infection. recommendations for a public health approach [document on the internet]. c2013 [cited 2013 dec 05]. available from: http://www.who.int/hiv/pub/guidelines/arv2013/download/en/. gibb dm, mugyenyi p. sustainable and cost-effective monitoring of patients on art. lancet. 2014;2(1):e4–e5. pmid: 25104633, http://dx.doi.org/10.1016/s2214-109x(13)70081-0 boyer s, march l, kouanfack c, et al. monitoring of hiv viral load, cd4 cell count, and clinical assessment versus clinical monitoring alone for antiretroviral therapy in low-resource settings (stratall anrs 12110/esther): a cost-effectiveness analysis. lancet infect dis. 2013;13(7):577–586. pmid: 23602084, http://dx.doi.org/10.1016/s1473-3099(13)70073-2 petti ca, polage cr, quinn tc, ronald ar, sande ma. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377–382. pmid: 16392084, http://dx.doi.org/10.1086/499363 mtapuri-zinyowera s, chideme m, mangwanya d, et al. evaluation of the pima point-of-care cd4 analyzer in vct clinics in zimbabwe. j acquir immune defic syndr. 2010;55(1):1–7. pmid: 20622679, http://dx.doi.org/10.1097/qai.0b013e3181e93071 anderson da, crowe sm, garcia m. point-of-care testing. curr hiv/aids rep. 2011;8(1):31–37. pmid: 21184203, http://dx.doi.org/10.1007/s11904-010-0067-z zachariah r, reid sd, chaillet p, massaquoi m, schouten ej, harries ad. viewpoint: why do we need a point-of-care cd4 test for low-income countries? trop med int health. 2011;16(1):37–41. pmid: 21371207, http://dx.doi.org/10.1111/j.1365-3156.2010.02669.x boyle ds, hawkins kr, steele ms, singhal m, cheng x. emerging technologies for point-of-care cd4 t-lymphocyte counting. trends biotechnol. 2012;30(1):45–54. pmid: 21798607, http://dx.doi.org/10.1016/j.tibtech.2011.06.015 jani iv, sitoe ne, alfai er, et al. effect of point-of-care cd4 cell count tests on retention of patients and rates of antiretroviral therapy initiation in primary health clinics: an observational cohort study. lancet. 2011;378(9802):1572–1579. http://dx.doi.org/10.1016/s0140-6736(11)61052-0 drain pk, hyle ep, noubary f, et al. diagnostic point-of-care tests in resource-limited settings. lancet infect dis. 2014;14(3):239–249. pmid: 24332389, http://dx.doi.org/10.1016/s1473-3099(13)70250-0 dieye tn, diaw pa, daneau g, et al. evaluation of a flow cytometry method for cd4 t cell enumeration based on volumetric primary cd4 gating using thermoresistant reagents. j immunol methods. 2011;372(1–2):7–13. pmid: 21835181, http://dx.doi.org/10.1016/j.jim.2011.07.012 reviewer acknowledgement open accesshttp://www.ajlmonline.org page 1 of 1 we would like to take this opportunity to thank all reviewers for the african journal of laboratory medicine, volume 2: abiy b. ambaye ahmed mohamed andrew thaiyah atunga nyachieo barbara marston beldinah r. ochola burton w. wilcke charles g. massambu chin-yih ou christophe longuet david turgeon debola olayinka dennis ellenberger diane waku edward kamau elizabeth luman eric opiyo fengxiang gao fredrick n. nindo gajendran sivakumar georges dahourou heather alexander helen perry henry s. limula jack nyamongo jane mwangi jean l. sankale jemal ali jesse kwiek john n. nkengasong john sorkin kapila bhowan keith p. klugman larry westerman lawrence barker linda de gouveia linda oskam luc kestens michael aidoo miguel e. quiñones-mateu milijaona radrianarivelojosia musau wakabongo naomi maina olumide ogundahuns pascale ondoa patty wilkins paul klatser paula fernandes polyxeni potter sam kariuki samoel khamadi sanon souleymane seema meloni segundo r. leon sharon martin sheba gitta stephania k. deme tjeerd datema tom shinnick walter r. campos william lali zilma rey should names have inadvertently been excluded from this list the publisher apologises and undertakes to amend the exclusion in the next issue. ajlm african journal of laboratory medicine in an effort to facilitate the selection of appropriate 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performing this task. chantal parkins submissions@ajlmonline. org tel: +27 (0)21 975 2602 fax: +27 (0)21 975 4635 91 article information how to cite this article: how to cite: the supplement coordinators and guest editors. information for action: ajlm’s special issue on transforming the quality of laboratory medicine through the strengthening laboratory management toward accreditation programme. afr j lab med. 2014;3(2), art. #279, 1 page. http://dx.doi.org/10.4102/ ajlm.v3i2.279 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. information for action: ajlm’s special issue on transforming the quality of laboratory medicine through the strengthening laboratory management toward accreditation programme in this foreword... open access this inaugural special issue of the african journal of laboratory medicine spotlights the critical need for continuous quality improvement in laboratories and the impressive global expansion and impact of one programme aimed to make such improvements a reality: the strengthening laboratory management toward accreditation (slmta) programme. slmta that teaches laboratory managers how to implement practical quality management systems in resource-limited settings using available resources. with a series of short courses and work-based improvement projects supported by site visits and mentoring, slmta is designed to achieve rapid, measureable improvement in laboratories. slmta was launched in 2009, and has since been implemented in some 617 laboratories in 47 countries worldwide. this special issue provides a comprehensive collection of programme results, including a detailed description of the slmta methodology and variations thereof, analyses of global programme data, and a summary of the progress made in developing a cadre of indigenous slmta trainers to facilitate global scale-up. a two-part comprehensive review of the literature summarizes the qualitative and quantitative results, and identifies strategic directions for the future of the programme. equally, or perhaps more important, are the numerous reports of countryand laboratory-level experiences providing in-depth lessons learned from ground-level implementers. from these papers we learn about factors contributing to success in implementing a quality management system, as well as pitfalls to beware of, and glean practical insights from the slmta pioneers who have gained first-hand experience striving for continuous quality improvement. these articles focus on navigating the balance between country ownership and effective partnership, using innovative incentives to accelerate improvements, building local human resources, and developing a culture of quality in laboratories that is sustainable. other articles relate experiences, such as continuing on to the goal of international accreditation, and addressing the need to extend the slmta programme beyond the laboratory to all parts of the health system. the remaining articles focus on specific facets of the programme, including the impact of mentorship, whether establishing in-country training facilitators is less expensive than using international trainers, and the pros and cons of conducting centralized versus facility-based training. others explore how to engage local resources, such as research laboratories and partners, and the benefit of using electronic tools to streamline the audit process. until now, little has been published on this groundbreaking programme, as efforts thus far have been focused on implementation rather than evaluation. this collection contains a vast wealth of information from a programmatic and observational point of view. the more complicated work of rigorous programme evaluation – for example, studies using randomized interventions and control groups; formal calculations of cost-effectiveness; and assessment of the health impact of laboratory quality improvement – remains to be done. by combining these studies into a single collection, we hope to assist readers with assimilation of the results into a meaningful understanding of the slmta programme and its implementation. whether used by individual laboratories to improve quality of services, by country-level managers and partners to guide slmta implementation, or by ministry of health leaders and other decision-makers as a source of evidence for large-scale planning, the data and insights of those who have successfully implemented the programme provide a wealth of knowledge and information for evidence-based decision-making to ensure continuous quality improvement for better patient care and public health outcomes. the supplement coordinators and guest editors abstract introduction methods results discussion acknowledgements references about the author(s) alicia naidoo department of medical microbiology, rk khan laboratory, national health laboratory service, durban, south africa department of medical microbiology, school of laboratory medicine and medical sciences, college of health sciences, university of kwazulu-natal, durban, south africa afsana kajee department of medical microbiology, school of laboratory medicine and medical sciences, college of health sciences, university of kwazulu-natal, durban, south africa department of medical microbiology, inkosi albert luthuli central hospital, national health laboratory services, durban, south africa nomonde r. mvelase department of medical microbiology, rk khan laboratory, national health laboratory service, durban, south africa department of medical microbiology, school of laboratory medicine and medical sciences, college of health sciences, university of kwazulu-natal, durban, south africa khine swe swe-han department of medical microbiology, school of laboratory medicine and medical sciences, college of health sciences, university of kwazulu-natal, durban, south africa department of medical microbiology, inkosi albert luthuli central hospital, national health laboratory services, durban, south africa citation naidoo a, kajee a, mvelase nr, swe-han ks. antimicrobial susceptibility of bacterial uropathogens in a south african regional hospital. afr j lab med. 2023;12(1), a1920. https://doi.org/10.4102/ajlm.v12i1.1920 original research antimicrobial susceptibility of bacterial uropathogens in a south african regional hospital alicia naidoo, afsana kajee, nomonde r. mvelase, khine swe swe-han received: 12 apr. 2022; accepted: 13 dec. 2022; published: 03 mar. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: urinary tract infections are common bacterial infections affecting millions worldwide. although treatment options for urinary tract infections are well established, with ciprofloxacin long considered one of the antibiotics of choice, increasing antibiotic resistance may delay the initiation of appropriate therapy. while this increase in antimicrobial resistance has been demonstrated in multiple studies around the world, there is a dearth of information from developing countries. objective: this study aimed to describe the antimicrobial susceptibility patterns of commonly isolated bacterial uropathogens in a south african hospital. methods: antimicrobial susceptibility data of isolates obtained from urine specimens at the rk khan hospital, a regional hospital in kwazulu-natal, south africa, between january 2018 and december 2020 were retrieved from the hospital’s laboratory information system and analysed to determine the differences in resistance rates between the most frequently isolated bacterial uropathogens. results: of the 3048 bacterial urinary pathogens isolated between 2018 and 2020, escherichia coli (1603; 53%) was the most common, followed by klebsiella spp. (437; 14%). both e. coli and klebsiella spp. showed high rates of resistance to amoxicillin/clavulanic acid (29.8% and 42.3%) and ciprofloxacin (37.7% and 30.4%). nitrofurantoin resistance was low among e. coli (6.2%) but high among klebsiella spp. (61.3%). conclusion: e. coli and klebsiella spp. in this study were highly resistant to amoxicillin/clavulanic acid and ciprofloxacin, two of the frequently prescribed oral treatment options. what this study adds: this study highlights the importance of regular local antimicrobial resistance surveillance to inform appropriate empiric therapy. keywords: antimicrobial susceptibility patterns; uropathogens; urinary pathogens; antibiotic resistance; urinary oral treatment. introduction although the urinary tract has multiple mechanisms in place to keep it sterile, urinary tract infections (utis) are one of the most common bacterial infections affecting nearly 150 million people worldwide and amounting to more than $6 billion united states dollars in healthcare costs, with approximately 95% of all utis occurring because of periurethral contamination by enteric uropathogens.1,2 international studies indicate that due to the high incidence of utis and the widespread practice of empirical treatment, the rate of antibiotic prescriptions is also increasing.3 as antibiotic use is known to be the main driver of the evolution of resistance, the increasing use of antibiotics in the treatment of utis has contributed to the alarming increase in antimicrobial resistance among frequently isolated urinary pathogens, thus further limiting treatment options, particularly with orally administered drugs.2,4,5,6 escherichia coli, klebsiella spp., and enterococcus spp., which are all members of the normal gut flora, are the most typically isolated organisms from urine, with e. coli being the most frequently isolated.7,8 empiric treatment options for utis are well established globally, and these include ciprofloxacin, amoxicillin/clavulanic acid, nitrofurantoin, fosfomycin, and cephalosporins.9,10 however, studies conducted locally and internationally demonstrate that an increase in the use of most of these drugs has caused a surge in antimicrobial resistance and a concomitant increase in treatment failures.2,11 this exerts greater pressure on an already overburdened healthcare system. antimicrobial resistance surveillance studies thus need to be conducted regularly to identify bacterial uropathogens and their antimicrobial susceptibility patterns to guide the empiric treatment of utis, which is a crucial part of antimicrobial stewardship. therefore, this study aimed to determine the prevalence of bacterial uropathogens and their antimicrobial susceptibility patterns to assist antimicrobial stewardship in a regional hospital in kwazulu-natal, south africa. methods ethical considerations ethics approval was granted by the university of kwazulu-natal biomedical research ethics committee, with permit number brec/00001578/2020. informed patient consent was not required by the university of kwazulu-natal ethics committee as this was a retrospective study and there was no patient interaction. patient privacy and confidentiality of data were protected in accordance with the declaration of helsinki. study design and setting a retrospective study was performed by retrieving three years of laboratory data from 01 january 2018 to 31 december 2020 at the national health laboratory services, rk khan hospital, durban, kwazulu-natal, south africa. the 543-bed hospital ranks as both a regional and district hospital and serves many internal and external clinics. data, which included patient location (inpatient and outpatient) and isolated uropathogens, were collected from the laboratory information system and captured onto a microsoft excel 365 spreadsheet, version 2205 (microsoft corporation, redmond, washington, united states). interpreted antimicrobial susceptibility data (‘resistant’, ‘intermediate’, ‘sensitive’) for the retrieved isolates were also extracted from the laboratory information system. inclusion criteria data on all positive urine bacterial cultures processed between january 2018 and december 2020 were retrieved from the laboratory information system. we then determined the distribution of bacterial uropathogens and analysed the antimicrobial susceptibility patterns of the two most common enterobacterales, namely e. coli and klebsiella spp. considering the study setting and the current literature, enterococcus faecalis, enterococcus faecium, group b streptococcus, and staphylococcus spp. were also chosen for analysis as they are the most common gram-positive bacterial causes of uti. antibiotics analysed in the study were based on the recommended empiric therapy as per the south african standard treatment guideline.9 exclusion criteria as this study was on bacterial uropathogens, all data on yeasts were excluded. to exclude duplicate entries, we used the following patient variables: patient name, surname, date of birth, and organism name. the organism name was included to remove duplicate entries from the same patient with the same organism. laboratory analysis urine specimens were processed according to the laboratory’s routine standard operating procedure. bacterial identification and antimicrobial susceptibility testing were carried out using the vitek® 2 automated system (biomerieux, marcy l’ètoile, rhône-alpes, france). as cephalothin was not part of the antibiotics tested on the vitek® 2 n255 card, cephalothin susceptibility was analysed using the kirby bauer disk diffusion method. for gram-negative bacteria, the antibiotics analysed were amikacin, amoxicillin/clavulanic acid, cefotaxime or ceftriaxone, ceftazidime, cephalothin, ciprofloxacin, gentamicin, meropenem, nitrofurantoin, piperacillin/tazobactam and trimethoprim/sulfamethoxazole. for the gram-positive bacteria, the antibiotics analysed include ampicillin, cloxacillin, penicillin, and vancomycin. results for cefotaxime and ceftriaxone were reported together as they have the same mechanism of action and resistance patterns and were therefore used interchangeably at rk khan hospital. all antimicrobial susceptibility results were interpreted using the clinical and laboratory standards institute guidelines.12 an extended-spectrum beta-lactamase producer was defined as an organism that was resistant to all cephalosporins, while carbapenem-resistant enterobacterales were defined as isolates that were resistant to any carbapenem. data analysis we determined the antimicrobial resistance rates of the most commonly isolated gram-negative (e. coli and klebsiella spp.) and gram-positive bacteria (e. faecalis, e. faecium, group b streptococcus, and staphylococcus spp.). additionally, for e. coli, we compared the antibiotic resistance rates between strains obtained from inpatients and outpatients and analysed the trends in antibiotic resistance rates over the three-year study period (2018–2020). we also determined the statistical significance of differences observed in the resistance rates between e. coli and klebsiella spp., as well as differences in the resistance rates of e. coli strains isolated from inpatients and outpatients. these statistical comparisons were done using the chi-square test, where a p-value less than 0.05 was considered statistically significant. the p-values for the pairwise comparisons were adjusted using the bonferroni correction method (r statistical computing software, version 3.6.3, r core team, auckland, new zealand). results from january 2018 to december 2020, the national health laboratory services rk khan laboratory analysed 3804 urine samples from patients with suspected utis. after removing duplicate data and data on yeasts, data on a total of 3048 bacterial isolates were included in this study. escherichia coli (n = 1603; 53%) was the most frequent urinary pathogen isolated, followed by klebsiella spp. (n = 437; 14%), including 398 (13%) klebsiella pneumoniae and 39 (1%) klebsiella oxytoca (figure 1). enterobacter spp. (n = 97; 3.2%) (including 82 [2.7%] enterobacter cloacae and 15 [0.5%] enterobacter aerogenes), pseudomonas aeruginosa (n = 61; 2%), acinetobacter baumannii (n = 84; 3%), and other gram-negative bacilli (n = 189; 6%) made up a further 14%. enterococcus faecalis (n = 372; 12%) was the most frequent gram-positive organism isolated, followed by e. faecium (n = 109; 4%), group b streptococcus (n = 55; 2%), and staphylococcus spp. (n = 41; 1%). figure 1: distribution of the 3048 bacterial urinary pathogens isolated at the rk khan hospital, durban, kwazulu-natal, south africa, january 2018 – december 2020. as e. coli and klebsiella spp. made up the majority of the gram-negative bacilli isolated, it was important to determine their antimicrobial resistance rates. the total number of cephalothin susceptibility results differed from the other antibiotics because cephalothin susceptibility was determined manually and the result was not always entered into the laboratory information system. similarly, the total number of results was different for each antimicrobial tested due to missing results linked to random terminations that can sometimes occur during automated antimicrobial susceptibility testing with the vitek® 2 instrument. e. coli demonstrated low levels of resistance to meropenem (0.56%; n = 9), amikacin (2.9%; n = 47), nitrofurantoin (6.2%; n = 99) and piperacillin/tazobactam (5.3%; n = 85) (figure 2). a very high level of resistance was noted in recommended oral treatment options, namely trimethoprim/sulfamethoxazole (61%; n = 988), ciprofloxacin (38%; n = 605) and amoxicillin/clavulanic acid (30%; n = 478). similarly, klebsiella spp. strains were highly resistant to nitrofurantoin (61%; n = 268), trimethoprim/sulfamethoxazole (48%; n = 208), amoxicillin/clavulanic acid (42%; n = 185), and ciprofloxacin (30%; n = 133). twenty-six percent of e. coli (n = 424) isolates were extended-spectrum beta-lactamase producers and 0.6% (n = 9) were carbapenem-resistant. among the klebsiella spp. isolates, 41.8% (n = 184) were extended-spectrum beta-lactamase producers and 5% (n = 22) were carbapenem-resistant. figure 2: antimicrobial susceptibility rates of e. coli and klebsiella spp. isolated from patients with suspected urinary tract infections at the rk khan hospital, durban, kwazulu-natal, south africa, january 2018 – december 2020. the total number of isolates is different for each antimicrobial due to missing results linked to random terminations that can sometimes occur during automated antimicrobial susceptibility testing with the vitek® 2 instrument. overall, klebsiella spp. was significantly more resistant than e. coli to all the tested antimicrobials except cephalothin (e. coli – 56% vs klebsiella spp. – 50%), ciprofloxacin (38% vs 30%), and trimethoprim/sulfamethoxazole (61% vs 48%). for all comparisons except cephalothin (p = 0.03) and ciprofloxacin (p = 0.005), the p-values were less than 0.001. among the gram-positive bacteria, e. faecium isolates showed high levels of resistance to both ampicillin (83.5%; n = 91) and penicillin (83.5%; n = 91) (table 1). conversely, e. faecalis isolates had low rates of resistance to ampicillin (0.8%; n = 3) and penicillin (2.4%; n = 9). in addition, the staphylococcus spp. isolates had high resistance rates to ampicillin (n = 38; 92.7%) and penicillin (n = 38; 92.7%). vancomycin was the most effective antibiotic against e. faecium (97.2% susceptibility) and staphylococcus spp. (100% susceptibility), while ampicillin and penicillin were most effective against group b streptococci (100% susceptibility). table 1: antimicrobial susceptibility patterns of commonly isolated urinary gram-positive cocci at the rk khan hospital, durban, kwazulu-natal, south africa, january 2018 – december 2020. for analysis of inpatient and outpatient data, only e. coli was studied as it constituted more than half of the bacterial pathogens. five hundred and eleven e. coli isolates (32%) were isolated from inpatients, while 1092 (68%) were from outpatients (table 2). low level of resistance was observed in both inpatient and outpatient wards for meropenem (0.8%; n = 4 and 0.1%; n = 1), amikacin (3.1%; n = 16 and 2.8%; n = 31), piperacillin/tazobactam (5.5%; n = 28 and 4.7%; n = 51) and nitrofurantoin (6.3%; n = 32 and 5.5%; n = 60). however, there were high rates of resistance to trimethoprim/sulfamethoxazole (inpatients: 69.7%; n = 355, outpatients: 57.6%; n = 625), ciprofloxacin (inpatients: 34.7%; n = 177, outpatients: 39%; n = 426), and amoxicillin/clavulanic acid (inpatients: 32.9%; n = 168, outpatients: 28.4%; n = 310). the prevalence of extended-spectrum beta-lactamases and carbapenem resistance in e. coli was higher among inpatients (31.0%; n = 158 and 0.8%; n = 4) compared to outpatients (24%; n = 261 and 0.1%; n = 1). table 2: antimicrobial susceptibility patterns of e. coli isolated from inpatient and outpatient wards at the rk khan hospital, durban, kwazulu-natal, south africa, january 2018 – december 2020 there were no statistically significant differences in resistance rates to recommended oral antibiotics, namely amoxicillin/clavulanic acid (p = 0.07), ciprofloxacin (p = 0.10) and nitrofurantoin (p = 0.54) between the e. coli isolates from inpatients and outpatients. however, for trimethoprim/sulfamethoxazole, e. coli isolates from inpatient wards were significantly more resistant than those from outpatient wards (p < 0.001). of the 1603 e. coli strains isolated between 2018 and 2020, 625 (39.1%) strains were isolated in 2018, 539 (33.6%) were isolated in 2019, and 439 (27.4%) were isolated in 2020, demonstrating slight decreases in prevalence over the study period (figure 3). amikacin and meropenem resistance rates, while low, increased slightly between 2018 (2.4% and 0.5%) and 2020 (3.6% and 0.9%). conversely, the e. coli isolates had higher resistance rates to the more commonly prescribed oral antibiotics, but these rates decreased steadily between 2018 and 2020 (cephalothin – 63.9% vs 49.8%; ciprofloxacin – 42.9% vs 30.4%; amoxicillin/clavulanic acid – 36.6% vs 22.4%). figure 3: antimicrobial susceptibility trends of e. coli isolated from patients with suspected urinary tract infections at the rk khan hospital, durban, kwazulu-natal, south africa, january 2018 – december 2020. the total number of isolates is different for each antimicrobial due to missing results linked to random terminations that can sometimes occur during automated antimicrobial susceptibility testing with the vitek® 2 instrument. over the three years (2018–2020), there were no statistically significant differences in resistance rates to amikacin (p = 0.50), meropenem (p = 0.75), nitrofurantoin (p = 0.16), piperacillin/tazobactam (p = 0.34), and trimethoprim/sulfamethoxazole (p = 0.2). in contrast, resistance rates to amoxicillin/clavulanic acid, ceftazidime, cephalothin and ciprofloxacin decreased significantly between 2018 and 2020 (p < 0.001). the e. coli isolates also demonstrated a significant decrease in resistance to cefotaxime or ceftriaxone (p = 0.04) and gentamicin (p = 0.02). discussion in this study, we found that e. coli was the most frequently isolated uropathogen at the rk khan hospital, durban, kwazulu-natal, south africa, between 2018 and 2020, followed by klebsiella spp. and e. faecalis. we also found that the commonly identified urinary pathogens had relatively high resistance rates to the widely administered antibiotics for uti treatment. our findings are consistent with those of previous local and international studies.13,14 a study conducted among patients with community-acquired utis in india showed that e. coli (55.1%), e. faecalis (15.8%), and k. pneumoniae (13.7%) were the most prevalent uropathogens isolated.13 similarly, a six-year (2011–2016) study conducted in the obstetric departments of six public-sector hospitals in durban, kwazulu-natal, south africa, showed that e. coli (54.2%) was the most common uropathogen, followed by k. pneumoniae (12.9%).14 in 2010, the infectious diseases society of america and the european society of microbiology and infectious disease published a clinical practice guideline outlining their recommendations for the treatment of uncomplicated utis, with nitrofurantoin, fosfomycin, trimethoprim/sulfamethoxazole, and ciprofloxacin recommended as the drugs of choice.6 similarly, in the south african standard treatment guideline, published in 2019, fosfomycin, ciprofloxacin, amoxicillin/clavulanic acid, and nitrofurantoin are also considered as empiric oral treatment options for utis.9 in their guidelines, both the infectious diseases society of america and the european society of microbiology and infectious disease recommend that ciprofloxacin should be used as an empiric antibiotic therapy provided that resistance rates remain below 10%.6 it was therefore disconcerting to discover that, in our study, both e. coli and klebsiella spp. showed high resistance rates against ciprofloxacin and amoxicillin/clavulanic acid. over 30% of the e. coli and klebsiella spp. isolates were resistant to ciprofloxacin and amoxicillin/clavulanic acid. in contrast, while only 6.2% of e. coli were resistant to nitrofurantoin, klebsiella spp. showed a high rate (61.3%) of nitrofurantoin resistance. although nitrofurantoin is still an option for treating utis caused by e. coli, our study suggests that it is ineffective against the majority of klebsiella spp. infections, leaving physicians with limited empiric oral therapy alternatives. similar to our findings, in a 9-year (2011–2019) study conducted in europe,15 resistance rates to ciprofloxacin and amoxicillin/clavulanic acid exceeded the 10% threshold for both e. coli (42.1% and 14.3%) and klebsiella spp. (68.7% and 38.8%). although the e. coli nitrofurantoin resistance rate (4.8%) in our study was below this 10% threshold, the resistance rate for klebsiella spp. (46.0%) far exceeded the threshold. these high rates of resistance to recommended oral empiric therapy are of major concern as this could lead to increased treatment failure rates. as our study was laboratory-based and the relevant information was not available on the laboratory information system, we were unable to distinguish between community-acquired and hospital-acquired utis. we, therefore, examined the antimicrobial patterns of e. coli isolates from the inpatient wards versus the outpatient wards. although resistance rates were generally higher (between 1% and 12% difference) among inpatients than outpatients, these differences were not statistically significant, except for trimethoprim/sulfamethoxazole. a japanese study done in 2020 showed that the differences in e. coli resistance rates between hospital-acquired and community-acquired utis were between 5% and 10%.16 according to the south african public healthcare system, new patients do not directly present to hospitals. instead, they are initially seen at local clinics and only referred to hospitals if required.9 as a result, patients who are seen at hospitals are either known hospital patients with chronic diseases or patients who need a higher level of care than the primary healthcare offered in the clinics. this may explain the high resistance rates in frequently isolated urinary bacteria in our study. it is however interesting to note that in a local study conducted in kwazulu-natal, south africa, between 2011 and 2016, lower levels of resistance to recommended empiric oral antimicrobials were reported among e. coli isolates from pregnant, but otherwise healthy, individuals who were generally not on antimicrobial therapy.14 despite these rates being lower than those of our study, they are still above the empirical antibiotic resistance threshold of 10% recommended by the infectious diseases society of america and the european society of microbiology and infectious disease. limitations because this was a retrospective study, the reliability of the data presented depended on the accuracy of the recording of the initial patient results. the resistance rates in this study may have been overrepresented as clinicians are unlikely to request susceptibility testing for patients that respond to empiric therapy. also, due to a lack of clinical information, we were unable to determine the difference between hospital-acquired and community-acquired infections. conclusion our study revealed that e. coli was the most predominant urinary pathogen isolated and that antimicrobial resistance levels in commonly isolated uropathogens remain elevated, especially against frequently prescribed oral antimicrobials. our findings indicate that the current empiric therapy used in the treatment of utis would fail in a large proportion of affected individuals, thus highlighting the need for more research to evaluate alternate oral drugs such as fosfomycin, which is not in use in the public sector despite being recommended in the south african standard treatment guideline.9 acknowledgements we would like to acknowledge the management and staff of rk khan laboratory (national health laboratory services) as well as the academic affairs and research office (national health laboratory services) for all their assistance while completing this project. competing interests the authors declare that they have no financial or personal relationship that may have inappropriately influenced them in writing this article. authors’ contributions a.n. was involved in conceptualisation and implementation of the research project, data collection, data analysis, literature review, writing of the first draft, writing, and editing of all subsequent drafts and the writing of the final draft. a.k. was responsible for editing the first and second drafts. n.r.m. contributed to conceptualisation of the research project and provided input on all drafts, editing and analysis. k.s.s.-h. provided input on implementation of the project and editing the first and final drafts. sources of support this research did not receive a grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the authors confirm that the data supporting the findings of this study are available within the article. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references van schoor j. urinary tract infection in women. s afr fam pract. 2016;58(1):6–10. https://doi.org/10.4102/safp.v58i1.4434 raja ns. oral treatment options of patients with urinary tract infections caused by extended spectrum beta-lactamase (esbl) producing enterobacteriaceae. j infect public health. 2019;12(6):843–846. https://doi.org/10.1016/j.jiph.2019.05.012 schmiemann g, kniehl e, gebhardt k, matejczyk mm, hummers-pradier e. the diagnosis of urinary infection. deutsches ärzteblatt int. 2010;107(21):361–367. https://doi.org/10.3238/arztebl.2010.0361 zur wiesch pa, kouyos r, engelstädter j, regoes rr, bonhoeffer s. population biological principles of drug-resistance evolution in infectious diseases. lancet infect dis. 2011;11(3):236–247. https://doi.org/10.1016/s1473-3099(10)70264-4 read af, woods rj. antibiotic resistance management. evol med public health. 2014;28(1):147. https://doi.org/10.1093/emph/eou024 gupta k, hooton tm, naber kg, et al. international clinical practice guidelines for the treatment of acute uncomplicated cystitis and pyelonephritis in women: a 2010 update by infectious diseases society of america and the european society for microbiology and infectious diseases. clin infect dis. 2011;52:e103–e102. https://doi.org/10.1093/cid/ciq257 flores-mireles al, walker jn, caparon m, hultgren sj. urinary tract infections: epidemiology, mechanisms of infection and treatment options. nat rev microbiol. 2015;13:269–284. https://doi.org/10.1038/nrmicro3432 elmanama aa, alreqeb aa, kalloub hk, et al. bacterial etiology of urinary tract infection and their antimicrobial resistance profiles. j al azhar university-gaza (nat sci). 2018;20:81–98. national department of health, south africa. essential drugs programme. hospital level (adults) standard treatment guidelines and essential medicines list. 5th ed. pretoria: ndoh; 2019. tan cw, chlebicki mp. urinary tract infections in adults. singapore med j. 2016;57(9):485–490. https://doi.org/10.11622/smedj.2016153 lewis da, gumede lye, van der hoven la, et al. antimicrobial susceptibility of organisms causing community acquired urinary tract infections in gauteng province, south africa. s afr med j. 2013;103:377–381. https://doi.org/10.7196/samj.6722 clinical & laboratory standards institute. performance standards for antimicrobial susceptibility testing 29th ed. wayne, pa: m100; 2019. gupta s, kapur s, padamavathi dv. comparative prevalence of antimicrobial resistance in community acquired urinary tract infection cases from representative states of northern and southern india. j clin diagn res. 2014;8:9–12. bhola p, mvelase nr, balakrishna y, milisana kp, swe swe-han k. antimicrobial susceptibility patterns of uropathogens isolated from pregnant woman in kwazulu natal province: 2011–2016. s afr med j. 2020;110(9):872–876. https://doi.org/10.7196/samj.2020.v110i9.14468 hrbacek j, cermak p, zachoval r. current antibiotic resistance trends of uropathogens in central europe: survey from a tertiary hospital urology department 2011–2019. antibiotics. 2020;9(9):630. https://doi.org/10.3390/antibiotics9090630 kanda n, hashimoto h, sonoo t, et al. gram-negative organisms from patients with community acquired urinary tract infections and associated risk factors for antimicrobial resistance: a single centre retrospective observational study in japan. antibiotics (basel). 2020;9(8):438. https://doi.org/10.3390/antibiotics9080438 acknowledgements references about the author(s) francesco marinucci abbott gmbh, wiesbaden, germany jens dhein abbott gmbh, wiesbaden, germany citation marinucci f, dhein j. the time to address diagnostic needs in universal health coverage is now: leveraging the scale up of national testing capacity for hiv viral load and sars-cov-2. afr j lab med. 2022;11(1), a1685. https://doi.org/10.4102/ajlm.v11i1.1685 opinion paper the time to address diagnostic needs in universal health coverage is now: leveraging the scale up of national testing capacity for hiv viral load and sars-cov-2 francesco marinucci, jens dhein received: 28 july 2021; accepted: 01 mar. 2022; published: 22 june 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the goals of universal health coverage (uhc) are to ensure that all people can access the quality health services that they need, to protect people from public health threats and to avoid catastrophic health expenditure.1 the range of essential services under uhc is country-specific and depends on factors such as the existing health system, the burden of different diseases and the resources available. as countries embark on defining their guaranteed package of services, the existing laboratory network represents an extraordinary opportunity to use the already existing equipment at its install base and full capacity across programmes, and to expand the laboratory testing menu in the uhc package. this article focuses on diagnostics and describes the reasons why flexible and high-throughput molecular platforms are critical to ensure access to quality testing and to improve diagnostic service coverage in the context of uhc initiatives. integrated models of service delivery are instrumental for ensuring impactful and sustainable uhc interventions. horizontal integration links healthcare professionals operating at the same level of care and ensures multiple diagnostic tests are available in the same laboratory tier. the focus of vertical integration is to increase coordination among testing sites and to improve communication within the different tiers of the laboratory network (figure 1). as a result of the laboratory network optimisation, the laboratory system can fulfil the multiple needs of patients in more efficient and sustainable ways.2 figure 1: vertical and horizontal integration of diagnostic services across the laboratory network allows all patients to access the same testing menu regardless of their entry point to the health system. most testing has been carried out in central laboratories serving a large network of health facilities. unfortunately, the role of laboratory-based platforms that allow the expansion of laboratories’ menus of assays (polyvalence) on the same analyser across different public health programmes has been undervalued, resulting in inadequate investments in vertical integration. this lack of investment has exacerbated the persistent issues related to communications between health facilities with the consequence of long turn-around times to get actionable results back to clinicians. instead of strengthening the infrastructural gaps, the focus of different vertical programmes switched to bringing diagnostic services closer to patients, which increased the divides within laboratory networks. the development of diagnostics that can generate results in primary healthcare settings has favoured the decentralisation of diagnostic services in several areas, including tuberculosis, hiv, viral hepatitis and malaria, with the main result being that diagnostic capacity is now closer to the patients.3 global and national guidelines accompanied this transition to lower-level health facilities; however, many programmes for major diseases in lowand middle-income countries are still organised in vertical silos and lack the vision of combining platforms allowing menu expansion in different tiers of the laboratory network.4 combined vertical and horizonal integration of diagnostic services across diseases has the potential to bring numerous benefits. for patients this results in more comprehensive care and services, added convenience due to time savings and reduced out-of-pocket expenses. for programmes, this results in cost savings from a more efficient use of resources, earlier treatment initiation and hence reallocation of resources to additional services. lastly, this would increase the resilience of the health system to adapt to the onset of new diseases and public health threats that happen unexpectedly, such as outbreaks and epidemics. the recent coronavirus disease 2019 (covid-19) pandemic exacerbated the need for expanded diagnostic capacity in such situations and the importance of a quick response time to address the increased demand for testing.5 a significant number of countries, including lowand middle-income countries, have built up their infrastructure in a short time. diagnostics manufacturers responded to the pressing need for covid-19 testing with rapid development and the launch of new covid-19 tests. the increased awareness of the importance of diagnostic testing provides the opportunity to look at other diseases that can be eliminated and where diagnostics plays a key role. tuberculosis, hiv, cervical cancer and viral hepatitis, although treatable, are still underdiagnosed in many high burden countries. considering these concerns, over the years the world health organization has released several global strategies to end these diseases6 (figure 2). covid-19 has demonstrated the advantages and limitations of current technologies to diagnose infections. the polymerase chain reaction is regarded as the gold standard for the detection of severe acute respiratory syndrome coronavirus 2 as a result of its high sensitivity for specific genetic sequences. polymerase chain reaction-based techniques amplify this genetic material through multiple amplification cycles allowing the detection of small quantities of pathogens. over the last 17 years many countries have scaled up their hiv viral load testing capacity with financial support from the united states president’s emergency plan for aids relief and the global fund. the covid-19 pandemic has pushed countries to further scale up their polymerase chain reaction testing capacities for severe acute respiratory syndrome coronavirus 2 by installing automated and high-throughput molecular diagnostic systems. many of these systems have random access capability and allow the use of multiple polymerase chain reaction assays on the same platform. while demand for hiv viral load testing will remain stable in the near future, the demand for severe acute respiratory syndrome coronavirus 2 testing will likely decline, leaving molecular diagnostic testing platforms free and available for testing of other disease markers. with the equipment, reagents, skilled personnel, connectivity to the laboratory information system, training, and the technical support already in place, such automated platforms may help to achieve meeting the targets defined in the global strategies and uhc initiatives (figure 2). previous limitations would be mitigated since the increased level of automation reduces complexity and makes it easier for laboratories to perform testing. random access systems eliminate the need for the sorting and batching of samples, provide continuous loading capability, and ready-to-use reagents. larger on-board assay storage capacities and random access allow testing of low, medium and high-volume assays side-by-side. testing capabilities could be rapidly adapted in response to increasing demand during a pandemic or in response to seasonal changes caused by respiratory viruses.7 short sample-to-answer turn-around times allow same day reporting of results, with some of the molecular diagnostic platforms providing capability for sample prioritisation (true random access function) without disrupting routine sample processing.8 menus comprising multiplex polymerase chain reaction assays allow differentiation of several pathogens in a single test, such as for respiratory or sexually transmitted infections. multiplex assays enable laboratories to process more tests on demand in a given period of time. this conserves important testing materials that may be in short supply during an outbreak situation, and it saves sample volume that can be used for additional analyses. the addition of middleware solutions to the automated molecular diagnostic platforms could provide auto-verification capabilities that automate data transfer, improve turn-around time, reduce manual labour and the potential for human error. centralised statistics and consolidated reports provided from the laboratories can give public health officials information for their ongoing surveillance programmes to control the spread of diseases and to monitor programme successes. taken together, the hiv viral load and the covid-19-driven uptake of molecular diagnostic platforms with expanded menus of assays provides a unique opportunity for laboratories to scale up molecular testing for hiv, viral hepatitis, tuberculosis, respiratory viruses, and high-risk human papillomavirus. the high automation level would enable laboratories to become more efficient in delivering high-quality results to support global health strategies in high burden countries. furthermore, the flexibility of such platforms, both in terms of volumes and testing menus, place them at the forefront of regional initiatives for coordinated outbreak response and other initiatives of concern to public health.9 simply increasing the number of shifts in the laboratory allows increases in the number of samples run on the platform. this flexibility is key for countries’ ability to rapidly cope with an increased demand for testing due to outbreaks and epidemics. the capability to use laboratory-developed tests on automated molecular diagnostic platforms can further strengthen the laboratories’ capabilities to respond in a timely manner to newly emerging diseases or variants. figure 2: link between global health strategies and automated molecular platforms. while enabling scalability to react in an outbreak situation, an expanded menu of assays is a strong basis to continue expanding uhc packages. countries can leverage existing sample referral networks, such as for hiv viral load testing, to serve multiple disease programmes by using the current infrastructure to move different specimen types from peripheral sites to the testing laboratory.10 the incremental cost-effectiveness of adding assays to the same high-throughput molecular diagnostic platform and the transportation of multiple specimens to the same laboratory would provide better use of scarce resources.11 this is particularly effective for infections like viral hepatitis c and human papillomavirus, where related diseases progress slowly, and access to results in a follow up visit does not impact clinical management. if required, results can be reported rapidly in situations where immediate clinical decisions must be made.8 good coordination across multiple programmes is the foundation for more integrated testing to combine the advantages of both point-of-care tests and high-throughput molecular diagnostic platforms. ideally, vertical and horizontal integration should occur simultaneously as part of the laboratory network optimisation supported by united states president’s emergency plan for aids relief, the african society for laboratory medicine and the africa centres for disease control and prevention, with the ultimate goal of deploying laboratory-based platforms and point-of-care technologies where they are needed the most. high-throughput molecular diagnostic platforms, when used at full capacity, allow better use of resources across the laboratory network, which is central for achieving more people-centred care. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions f.m. and j.d. contributed equally to the conceptualisation and writing of the manuscript. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references kieny mp, bekedam h, dovlo d, et al. strengthening health systems for universal health coverage and sustainable development. bull world health organ. 2017;95(7):537–539. https://doi.org/10.2471/blt.16.187476 ondoa p, ndlovu n, keita ms, et al. preparing national tiered laboratory systems and networks to advance diagnostics in africa and meet the continent’s health agenda: insights into priority areas for improvement. afr j lab med. 2020;9(2):1103. https://doi.org/10.4102/ajlm.v9i2.1103 parsons lm, somoskovi a, lee e, et al. global health: integrating national laboratory health systems and services in resource-limited settings. afr j lab med. 2012;1(1):11. https://doi.org/10.4102/ajlm.v1i1.11 considerations for adoption and use of multidisease testing devices in integrated laboratory networks [homepage on the internet]. world health organization information note 2017. who/htm/tb/2017.06. [cited 2021 may 07]. available from: https://www.who.int/tb/publications/2017/considerations_multidisease_testing_devices_2017/en/ ondoa p, kebede y, loembe mm, et al. covid-19 testing in africa: lessons learnt. lancet microbe. 2020;1(3):e103–e104. https://doi.org/10.1016/s2666-5247(20)30068-9 pai m, walia k, boehme cc. essential medicines and essential diagnostics: a package deal. lancet public health. 2019 oct;4(10):e492. https://doi.org/10.1016/s2468-2667(19)30165-3 venkatesan p. covid-19 diagnostics-not at the expense of other diseases. lancet microbe. 2020;1(2):e64. https://doi.org/10.1016/s2666-5247(20)30041-0 obermeier m, pacenti m, ehret r, et al. improved molecular laboratory productivity by consolidation of testing on the new random-access analyzer alinity m. j lab med. 2020;44(6):319–328. https://doi.org/10.1515/labmed-2020-0102 amukele t. africa cdc: establishing integrated surveillance and laboratory networks for rapid disease detection and response, control, prevention, and clinical care in africa. afr j lab med. 2017;6(1):a638. https://doi.org/10.4102/ajlm.v6i1.638 fonjungo pn, alemnji ga, kebede y, et al. combatting global infectious diseases: a network effect of specimen referral systems. clin infect dis. 2017;64(6):796–803. https://doi.org/10.1093/cid/ciw817 williams j, edgil d, wattleworth m, et al. the network approach to laboratory procurement and supply chain management: addressing the system issues to enhance hiv viral load scale-up. afr j lab med. 2020;9(1):a1022. https://doi.org/10.4102/ajlm.v9i1.1022 article information authors: jessina masamha1 beth skaggs2 isabel pinto3 ana paula mandlaze4 carolina simbine4 patrina chongo4 leonardo de sousa1 solon kidane5 katy yao6 elizabeth t. luman6 eduardo samogudo4 affiliations: 1center for global health (cgh), division of global hiv/aids (dgha), us centers for disease control and prevention (cdc), maputo, mozambique2cgh, division of global disease detection and emergency response, cdc, atlanta, united states 3central laboratory department, ministry of health, maputo, mozambique 4national institute of health, ministry of health, maputo, mozambique 5association of public health laboratories, silver spring, united states 6cgh, dgha, cdc, atlanta, united states correspondence to: jessina masamha postal address: cdc, 7th floor jat complex, 267 zedequias manganhela avenue, maputo, mozambique dates: received: 10 sept. 2014 accepted: 21 sept. 2014 published: 03 nov. 2014 how to cite this article: masamha j, skaggs b, pinto i, et al. working toward a sustainable laboratory quality improvement programme through country ownership: mozambique’s slmta story. afr j lab med. 2014;3(2), art. #253, 6 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.253 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. working toward a sustainable laboratory quality improvement programme through country ownership: mozambique’s slmta story in this original research... open access • abstract • introduction • research methods and design    • institutionalisation of the slmta programme    • implementation • results • discussion    • challenges    • conclusion • acknowledgements    • competing interests    • authors’ contributions    • cdc disclaimer • references abstract top ↑ background: launched in 2009, the strengthening laboratory management toward accreditation (slmta) programme has emerged as an innovative approach for the improvement of laboratory quality. in order to ensure sustainability, mozambique embedded the slmta programme within the existing ministry of health (moh) laboratory structure.objective: this article outlines the steps followed to establish a national framework for quality improvement and embedding the slmta programme within existing moh laboratory systems. methods: the moh adopted slmta as the national laboratory quality improvement strategy, hired a dedicated coordinator and established a national laboratory quality technical working group comprising mostly personnel from key moh departments. the working group developed an implementation framework for advocacy, training, mentorship, supervision and audits. emphasis was placed on building local capacity for programme activities. after receiving training, a team of 25 implementers (18 from the moh and seven from partner organisations) conducted baseline audits (using the stepwise laboratory quality improvement process towards accreditation [slipta] checklist), workshops and site visits in six reference and two central hospital laboratories. exit audits were conducted in six of the eight laboratories and their results are presented. results: the six laboratories demonstrated substantial improvement in audit scores; median scores increased from 35% at baseline to 57% at exit. it has been recommended that the national tuberculosis reference laboratory apply for international accreditation. conclusion: successful implementation of slmta requires partnership between programme implementers, whilst effectiveness and long-term viability depend on country leadership, ownership and commitment. integration of slmta into the existing moh laboratory system will ensure durability beyond initial investments. the mozambican model holds great promise that country leadership, ownership and institutionalisation can set the stage for programme success and sustainability. introduction top ↑ reliable laboratory services and networks are fundamental components of well-functioning health systems and are essential for patient management, disease detection and control, surveillance and outbreak investigations.1 however, in most resource-poor countries, mozambique included, access to quality laboratory services is limited,2 resulting in delayed detection of outbreaks and lengthy or inaccurate diagnoses that may compromise patient care and outcomes. laboratory quality improvement efforts are intended to strengthen laboratory services, leading to advances in the overall health system and the health of the nation.the national laboratory network in mozambique consists of reference laboratories and clinical laboratories. clinical laboratory services are integrated into a tiered national health service (nhs) that comprises central, provincial and district hospitals, as well as health centres (figure 1). of the more than 1300 health facilities in the nhs, only 314 (24%) have laboratories; approximately 30% of these offer only microscopy and rapid test services. whilst strides have been made to improve laboratory infrastructure, a great number of health facilities and laboratories remain under-resourced in areas such as electricity, water supply, physical environment and equipment. in addition to constrained infrastructure, laboratories are affected by frequent stock outs of reagents and consumables, equipment breakdowns, insufficient numbers of qualified laboratory staff and a lack of laboratory quality systems. figure 1: tiered ministry of health laboratory network in mozambique. addressing gaps in the laboratory network is a critical objective for laboratory systems strengthening and a top priority for the government of mozambique.3 consequently, the ministry of health (moh) drafted a national laboratory policy that defines the governance structure for the national network in order to ensure consistent provision of quality laboratory services for clinical diagnosis, research and surveillance.3 the national laboratory policy outlines the ministry’s commitment to the goals of implementing quality management systems (qms) and pursuing national or international accreditation for its reference, central and provincial hospital laboratories.3 with these goals in mind, in 2010 the moh in mozambique implemented strengthening laboratory management toward accreditation (slmta), an innovative training and mentorship programme for continuous quality improvement aimed at improving laboratory qms and preparation for accreditation.4 in addition to slmta, the moh adopted the world health organization regional office for africa's (who afro) stepwise laboratory quality improvement process towards accreditation (slipta), a framework for measuring and recognising quality levels in public health laboratories in developing countries.5 slipta incorporates a checklist to audit laboratory performance and a scoring system to determine a laboratory’s level on the pathway toward achieving accreditation to the international organization for standardization (iso) 15189 standard. the moh incorporated slmta and slipta into the existing health system infrastructure in order to overcome the challenges met by some externally-supported programmes that failed to last after support ended.6 often, externally-supported programmes are viewed as short-term projects and therefore change is either resisted or temporary; desirable outcomes are either not achieved or not maintained. institutionalisation, careful planning, local capacity building, country ownership and strong leadership have been described as being key ingredients for sustainable programmes.7 this article outlines the steps followed by the mozambican moh to establish a national framework for quality improvement and embed the slmta programme within existing moh laboratory systems. results from the initial cohort of participating laboratories are presented along with insights into strategies to establish programme sustainability. research methods and design top ↑ institutionalisation of the slmta programme in 2011, a national laboratory technical working group (twg) consisting of key moh personnel and partners was established to build a framework for a national laboratory quality improvement programme. the heads of reference laboratory services and clinical laboratory services within the moh were appointed as co-chairs. the twg developed a slmta implementation plan, which included training, mentorship, supervision and audits. roles and responsibilities for each stakeholder were defined. the moh created a logistics and administrative position under which the slmta coordinator was hired and a slipta focal person was appointed. the moh coordinated financing for programme implementation from its various partners. the twg met weekly to coordinate and monitor the implementation of slmta. to ensure maximum programme integration and success, the twg felt it was critical for slmta to be accepted as a national moh programme within the laboratory network rather than as an externally-implemented project. to localise the programme, the slmta training tool kit was translated into portuguese, locally-relevant implementation and advocacy strategies were developed and a portuguese acronym, fogela (fortalecer a gestão de laboratórios para acreditação), was created. moh twg members were assigned leading roles in advocacy, planning and implementation. prior to implementation, the slmta programme was introduced by twg co-chairs through presentations at an annual national health directors meeting hosted by the moh. various members of the twg introduced the programme at other meetings with key health leaders, including provincial health directors and medical directors in the provinces.the slmta programme in mozambique was designed to be implemented in a phased, hierarchical approach in which top-tier laboratories (national reference laboratories and central hospital laboratories) were prioritised for enrolment in the first cohort. after gaining experience implementing the programme in their own laboratories, trained managers and quality officers would themselves become a resource for training, mentoring and supervising laboratories enrolled in succeeding cohorts. after demonstrating competence in slmta implementation, provincial quality focal points would lead implementation at lower laboratory tiers within their provinces. to ensure feasibility, a small number of laboratories would be enrolled in successive years, prioritising provincial hospital laboratories. this phased approach was seen as essential for the development of slmta into a sustainable moh programme that could keep pace with human and financial resource needs and maintain programme results. country ownership requires sufficient institutional capacity to define and implement a strategy.7 the need for trainers, mentors, supervisors and auditors was identified, and training was conducted in order to build the various skills required by moh staff to successfully roll out and sustain the slmta programme. in december 2010, in collaboration with a global public healthcare foundation, mozambique trained 15 auditors using the who afro auditor training curriculum. the following year, six of the auditors who were also twg members were trained as slmta trainers, four from the moh and two from the us centers for disease control and prevention (cdc) office in mozambique. these slmta trainers also received training in mentorship and supervision and, as part of the twg, oversaw programme implementation in the first cohort of laboratories. there was little in-country experience in implementing qms at the inception of slmta. thus, three expatriate laboratory professionals with experience in qms development were contracted as mentors and trained by an experienced slmta mentor in the provision of guidance and support. in 2012, in preparation for programme decentralisation and expansion, mozambique conducted the first portuguese-language slmta training-of-trainers (tot) course, which included 18 laboratory professionals from mozambique and three from angola. mozambique now has a team of 25 slmta trainers, including four master trainers (two from the moh) capable of conducting tots. implementation eight laboratories were enrolled in the first slmta cohort, including six reference laboratories and two central hospital laboratories. in january 2011, newly-trained auditors were paired with the experienced consultant auditors to conduct baseline audits for the enrolled laboratories using the slipta checklist. the checklist is divided into 12 sections. laboratory quality is benchmarked on a zeroto five-star scale, awarded based on a percentage of the total score: < 55% assigned zero stars, 55% – 64% one star, 65% – 74% two stars, 75% – 84% three stars, 85% – 94% four stars and ≥ 95% five stars.5the slmta curriculum was then presented during three 3–5-day workshops conducted four months apart. three key staff members from each laboratory who were mandated to lead the implementation of quality improvement in their laboratory participated in the workshops: the laboratory manager, the quality officer and one head of section. throughout the programme, mentors worked with the laboratory staff, spending sixto eight-week periods embedded in a laboratory, followed by an eight-week absence. selected improvement projects such as documentation, equipment management, inventory management, quality control and safety were implemented following each workshop. two follow-up supervisory visits were conducted in each laboratory during these periods in order to provide technical assistance and to monitor the implementation process. the first visit was to support data collection for the selected improvement projects and to review laboratory action plans; the second visit six to eight weeks later was to monitor progress. supervision reports were shared with participating laboratories and feedback on progress and challenges was shared with laboratory and hospital management. in march 2012, after completion of the slmta curriculum, locally-trained auditors, supported by expert international auditors, conducted exit audits of the laboratories. summaries from audit findings, corresponding scores and laboratory star ratings were shared with laboratory management and staff, hospital directors and provincial health department officials. results top ↑ all eight laboratories completed the three slmta workshops and the assigned improvement projects within the implementation period. of the eight laboratories, six had complete exit audit data; the remaining two reference laboratories had some missing data and were excluded from the analyses. the six laboratories with complete scores demonstrated overall improvement after 12 months of implementation (figure 2). at baseline, all laboratories began with zero stars (median score 35%, range 14% – 50%). at exit, scores for all laboratories improved (median exit score 57%, range 44% – 76%; improvements 7–53 percentage points). one laboratory remained at zero stars (though its score increased from 14% to 44%), three laboratories were at one star, one laboratory was at two stars and one laboratory had reached three stars. the areas of greatest average improvement (> 40 percentage points) were client management and customer service, corrective action, purchasing and inventory and management reviews (figure 3). the areas of least improvement (< 15 percentage points) were information management, equipment, facilities and safety and internal audit. figure 2: baseline, exit and official slipta audit results and star ratings for the first cohort* of laboratories based on the slipta checklist. mozambique slmta programme, january 2011 to june 2013. figure 3: average performance of six laboratories enrolled in first slmta cohort in mozambique across the 12 sections of the slipta checklist before and after slmta implementation as measured by baseline and exit audits (january 2011 to march 2012). the best-performing laboratory at the exit audit was the national tuberculosis (tb) reference laboratory, which attained three stars (76%). this laboratory had an overall improvement of 53 percentage points across all 12 checklist sections, with improvements of ≥ 80 percentage points in corrective action and purchasing and inventory. at the end of the programme, three of the six laboratories were officially enrolled into the who afro slipta programme for review by auditors from the african society for laboratory medicine. the scores from the official audits were slightly higher than exit audit results, at 58% for nampula central hospital, 72% for the cellular immunology reference laboratory and 79% for the national tb reference laboratory (figure 2). the national tb reference laboratory was recommended to apply for international accreditation. a work plan was developed to address gaps identified and a target set for the laboratory to have the accreditation audit in august 2014 with the portuguese accreditation institute based in portugal. discussion top ↑ through slmta, mozambique has begun to make substantial progress in laboratory quality improvement and has developed a plan to ensure that continued progress is both feasible and sustainable. the progress made would be short-lived without country ownership and leadership, both of which are fostered by institutionalisation of the programme. the moh made a sustained effort to take ownership of the slmta programme as part of their overarching strategy to improve laboratory quality systems, embedding it within the moh structure in order to ensure that planning, advocacy and implementation were led and championed by moh personnel. as a result, laboratory management and staff viewed slmta as a permanent moh programme, rather than as a ‘one-off’ event that would soon disappear.moh leadership not only influenced perception of the programme but also staff commitment. laboratory personnel were wholly committed to the programme, working creatively and beyond designated work hours in order to implement assigned improvement projects. in one laboratory, the quality manager and laboratory staff contributed money to purchase file folders for laboratory documents. in another, laboratory personnel paid for tokens to reward staff who made outstanding contributions to quality improvement projects. in fact, so much excitement was generated over the programme that managers of laboratories yet to be enrolled in slmta wanted to know when their laboratories would be considered for future cohorts. the moh led advocacy efforts, such as introduction of the programme and presentation of results at key strategic forums, which were aimed at promoting senior-level ownership, support and commitment crucial for the success of the programme. these efforts led to widespread buy-in and interest in the programme amongst provincial health and hospital directors as well as key moh decision makers. the provincial and hospital directors received regular updates during routine slmta supervisory visits and showed a keen interest in knowing how their laboratories were progressing and what they could do to support the laboratory staff. hospital directors began to visit the laboratories to follow up on projects and to ask ‘how many stars’ their laboratories had now achieved. these hospital directors commented on the increased interaction with the laboratories and improved understanding of their institutions’ quality indicators; and became involved in resolving problems that were beyond the laboratories' control. for example, when one laboratory showed decreased customer satisfaction as a result of staff shortages, the hospital director engaged the provincial health director to request additional laboratory staff at her facility. as guided by the implementation plan, expansion of the slmta programme is underway. in october 2012, a second slmta cohort was launched. in keeping with the tiered strategy, laboratories from central, provincial, general and rural hospitals were eligible to apply and 10 laboratories were selected. as slmta roll-out continues, the laboratory twg has set programme implementation goals based on the country’s capacity and available financial, human and material resources. the moh continues to lead all aspects of programme planning and implementation and, since 2012, approximately 30% of funding for the programme has been incorporated into annual moh budgets. mozambique has built a foundation for a sustainable laboratory quality improvement programme; however, to guarantee longevity, critical aspects of the national laboratory system must be strengthened. establishing a national laboratory policy is essential with regard to sustaining the improvements achieved through the slmta programme.1 whilst mozambique has drafted such a policy, it has not yet been given official approval, limiting the moh’s ability to drive the implementation of qms and enforce adherence to quality standards across the laboratory network. without such a formal policy, continued and sustained progress cannot be mandated systematically. mozambique has set a goal of accreditation for its six reference laboratories and 10 central and provincial hospital laboratories. however, international accreditation is not a goal that is achievable for all laboratories in the network. thailand has successfully implemented a model to accredit laboratories according to national standards that vary by laboratory level.8 to ensure continuous quality improvement through expansion of slmta to lower-tier laboratories, mozambique may consider developing a similar framework that outlines national standards for quality laboratory services, against which rural hospital and health centre laboratories could be monitored and evaluated. once a policy is in place, clear measures must be established to assess progress and effectiveness, as well as to sustain laboratory strengthening efforts. nkengasong et al. suggest using the indicator ‘number of laboratories accredited in the public health sector’ in order to track the progress of quality systems implementation for a national laboratory network.9additional indicators could include: number of provinces with dedicated quality management officers; percentage of laboratories audited in the previous 12 months; percentage of audited laboratories demonstrating improvement as measured by the slipta checklist; and percentage of laboratories implementing external proficiency testing for select services. beyond system structures and policy, human resources are a critical requirement for the slmta programme. yao et al. recommend an investment in human resources and allowance of time for dedicated personnel to successfully implement and sustain slmta activities so as to achieve the goal of laboratory accreditation.4 as mozambique continues to work toward increasing coverage of slmta, it is critical to develop local training and mentoring capacity. significant upfront investment in local capacity building will keep long-term costs down and ensure sufficient reach and uptake of the national programme. for example, in zimbabwe, shumba et al. found that it cost less over the long term to engage and train local quality managers as auditors, trainers and mentors than to import international consultants to do the job.10 minimizing costs is essential because programmes that require unacceptable levels of resource commitment are unsustainable.6 challenges mozambique has invested in developing a large implementation team that has been trained adequately for the task. in fact, in addition to conducting in-country training, mozambican-based trainers have since assisted with slmta training in angola and peru; whilst its master trainers have supported tots in kenya, ethiopia and the dominican republic. however, with the exception of the slmta logistics coordinator, none of these programme implementers work in an official capacity for the slmta programme. because of competing priorities, slmta activities may not always be completed adequately, as evidenced by missing exit audit data for two laboratories in the first cohort. within the laboratories, quality managers have bench responsibilities that compete with improvement projects and continued maintenance of the qms. to be successful, the terms of reference for quality managers must be revised to include adequate time for implementation of the laboratory’s qms. although the initial plan was to groom local mentors from the first cohort of laboratories for subsequent cohorts, it has been a challenge to secure time for these now ‘more experienced’ staff members to leave their day-to-day work and provide mentorship to newly-enrolled laboratories. staffing moh quality departments with personnel dedicated to maintaining the daily operations of the quality improvement programme may address some of these challenges. conclusion successful implementation of the slmta programme requires partnership; however effectiveness and long-term viability depend on country leadership, ownership and commitment. the moh has collaborated with cdc and various partners to implement the slmta programme, whilst at the same time assuming leadership for the process, including establishment of the structures necessary for long-term programme success. the mozambican model of institutionalisation of slmta into existing moh laboratory systems demonstrates that country leadership, commitment and ownership can provide a strong platform for sustainability. by taking advantage of the current influx of laboratory resources to strengthen existing structures, build essential human resource capabilities and establish guiding strategies and policies, the mozambique laboratory quality improvement programme is designed to be sustainable and is positioned to achieve the goal of providing quality laboratory services across the country. acknowledgements top ↑ this programme has been supported by the us president’s emergency plan for aids relief (pepfar) through the cdc under the terms of cooperative agreement number u2gps12985. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions j.m. (cdc, mozambique) supported the planning and implementation of the programme and wrote the manuscript. b.s. (cdc, atlanta) provided oversight for the programme and substantial input into the writing of the manuscript. i.p. (central laboratory department, moh), a.p.m., c.s. and p.c. (all national institute of health, moh), l.s. (cdc, mozambique) and s.k. (association of public health laboratories) supported the planning and implementation of the programme. k.y. and e.t.l. (cdc, atlanta) substantially revised the manuscript. e.s. (national institute of health, moh) championed the institutionalisation of the programme, led its implementation and reviewed the manuscript. cdc disclaimer the findings and conclusions in this report are those of the author and do not necessarily represent the official position of the cdc. references top ↑ 1. gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2011;134(3):393–400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm2. olmsted ss, moore m, meili rc, et al. strengthening laboratory systems in resource-limited settings. am j clin pathol. 2011;134(3):374–380. http://dx.doi.org/10.1309/ajcpdqosb7qr5glr 3. ministry of health mozambique. national laboratory policy; 2012 (unpublished). 4. yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2011;134(3):401–409. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj 5. world health organization’s regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region (with checklist) [document on the internet]. c2011 [cited 2013 may]. http://www.afro.who.int/en/clusters-a-programmes/hss/blood-safety-laboratories-a-health-technology/blt-highlights/3859-who-guide-for-the-stepwise-laboratory-improvement-process-towards-accreditation-in-the-african-region-with-checklist.html 6. swerrison h. understanding the sustainability of health programs and organisational change. a paper for the victorian quality council [document on the internet]. c2007 [cited 2013 may]. available from: http://www.health.vic.gov.au/qualitycouncil/downloads/sustainability_paper.pdf [url no longer available]. 7. world bank. country ownership [page on the internet]. c2008 [cited 2013 may]. available from: http://web.worldbank.org/archive/website01013/web/0__co-89.htm 8. wattanasri n, manoroma w, viriyayudhagorn s. laboratory accreditation in thailand: a systemic approach. am j clin pathol. 2011;134(4):534–540. http://dx.doi.org/10.1309/ajcpzyy19wmkmazt 9.nkengasong j. a shifting paradigm in strengthening laboratory health systems for global health: acting now, acting collectively, but acting differently. am j clin pathol. 2011;134(3):359–360. http://dx.doi.org/10.1309/ajcpy5asuejyq5rk 10.shumba e, nzombe p, mbinda a, et al. weighing the costs: implementing the slmta programme in zimbabwe using internal versus external facilitators. afr j lab med. 2014;3(2), art. #248, 6 pages. http://dx.doi.org/10.4102/ajlm.v3i2.248 abstract introduction methods results discussion acknowledgements references about the author(s) reola haripersadh department of haematology, national health laboratory services, inkosi albert luthuli central hospital, durban, south africa department of haematology, school of laboratory medicine, university of kwazulu-natal, durban, south africa dashini pillay department of haematology, national health laboratory services, inkosi albert luthuli central hospital, durban, south africa department of haematology, school of laboratory medicine, university of kwazulu-natal, durban, south africa nadine rapiti department of haematology, national health laboratory services, inkosi albert luthuli central hospital, durban, south africa department of haematology, school of laboratory medicine, university of kwazulu-natal, durban, south africa citation haripersadh r, pillay d, rapiti n. impact of rapid centrifugation on routine coagulation assays in south africa. afr j lab med. 2022;11(1), a1901. https://doi.org/10.4102/ajlm.v11i1.1901 original research impact of rapid centrifugation on routine coagulation assays in south africa reola haripersadh, dashini pillay, nadine rapiti received: 24 mar. 2022; accepted: 10 aug. 2022; published: 28 nov. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the recommendation for coagulation blood samples is to centrifuge at 4000 revolutions per minute (rpm) for 15 min to produce platelet-poor plasma before analysis. rapid centrifugation, defined as centrifuging samples at higher speeds for shorter durations, could potentially reduce turn-around times (tat), provided sample integrity is maintained. objective: this study assessed the impact of rapid centrifugation on routine coagulation assay results. methods: blood samples were collected from volunteers at inkosi albert luthuli central hospital and king edward viii hospital, durban, kwazulu-natal, south africa, from september to november 2021. samples were centrifuged using method a, the current standard (4000 rpm/15 min), method b (4000 rpm/10 min), method c (5000 rpm/10 min) and method d (5000 rpm/5 min). platelet count, prothrombin time, activated partial thromboplastin time, thrombin time (tt), fibrinogen and d-dimer levels were analysed and results from methods b, c and d compared to reference method a. results: platelet-poor plasma was obtained from all samples (n = 60) using methods a and b, and from 33/60 (55%) samples using methods c and d. differences between method a and methods c and d for normal prothrombin time, normal d-dimer and abnormal tt results were statistically significant. prothrombin time results correlated strongly across all methods, while tt and d-dimer results correlated poorly. activated partial thromboplastin time and fibrinogen results showed no significant differences across all methods. conclusion: rapid centrifugation at 4000 rpm/10 min (method b) showed results consistent with the reference method. this method could potentially reduce the overall tat for routine coagulation assays. keywords: rapid centrifugation; coagulation assays; platelet-poor plasma; pre-analytic; clinical and laboratory standards institute guidelines; haemostasis. introduction laboratories are expected to provide accurate and reliable results within defined turn-around times (tats) to facilitate the diagnosis, management and prognostication of patients.1,2 there are ongoing attempts to improve tats as this will directly impact patient management.3,4 coagulation assays are some of the most commonly ordered urgent and routine investigations.1,2 the recommended sample preparation method for coagulation testing is the centrifugation of whole blood to obtain platelet-poor plasma (ppp), defined as plasma with a platelet count of < 10 × 109/l, which minimises interference by the platelet phospholipid surface, thereby preventing the activation of clotting factors.5 obtaining ppp from whole blood requires the application of specific centrifugal forces over a given period.6,7 minimal centrifugation times for routine coagulation assays vary across laboratories, often based on local observations.8 the clinical and laboratory standards institute guidelines recommend that whole blood collected in tri-sodium citrate tubes be centrifuged at 4000 revolutions per minute (rpm) for 15 min at room temperature to produce ppp.9 strategies such as rapid centrifugation that are designed to reduce specimen processing times are being pursued as they could potentially reduce tat in laboratories, eliminate the need for batch processing of samples and free up resources.10 as centrifugation time can be a bottleneck in coagulation testing, there is a need to determine the impact of rapid centrifugation on the laboratory workflow and tat.1,7,9 several studies have shown that centrifuging samples at higher speeds for shorter durations to obtain ppp has no significant effect on sample integrity and the accuracy of coagulation assay results.11,12,13,14 the primary goal of this study was to assess the impact of rapid centrifugation on the accuracy of routine coagulation test results. methods ethical considerations the study was approved by the biomedical research ethics committee of the university of kwazulu-natal, south africa (brec/00002366/2021). informed consent was obtained from each study participant and a unique study number was allocated to samples to ensure anonymity. the research data was stored electronically on password-protected devices and was only accessible by the researchers. study design and setting this study was conducted at the inkosi albert luthuli central hospital and king edward viii hospital, durban, kwazulu-natal, south africa, from september 2021 to november 2021. patients older than 18 years were included in the study. samples were excluded if there were insufficient blood volumes, or if clots, fibrin strands, or haemolysis were observed. sixty-two participants were included in the study (table 1). two samples were excluded from the data analysis due to tube breakage and one sample was treated as an outlier due to an abnormally high d-dimer result (35.2 mg/l) for method a.15 from each participant, venous blood samples were collected into four 3.2% sodium citrate tubes (bd vacutainer 0.109 m/3.2 citrate, becton dickinson, new jersey, united states) labelled a, b, c and d, each representing one of the four centrifugation methods. blood samples were collected and transported to the laboratory at room temperature (20 °c – 25 °c). table 1: demographics and clinical diagnoses of study participants, inkosi albert luthuli central hospital and king edward viii hospital, durban, kwazulu-natal, south africa, september 2021 – november 2021. centrifugation samples were centrifuged using two table-top consul 22r (ortoaltresa®, madrid, spain) instruments. the first instrument (instrument 1) had a maximum rotor speed of 4000 rpm and required a 5 ml tube, while the second instrument (instrument 2) had a maximum rotor speed of 9000 rpm and required a 50 ml tube (table 2). a pilot experiment revealed that decanting the sample from the 5 ml citrate tube into the larger 50 ml tube before centrifugation resulted in a very thin supernatant plasma layer that was difficult to remove with a pipette. placing the 5 ml citrate tube directly into the 50 ml tube resulted in the risk of tube breakage, therefore, cotton wool was used as a buffer between the two tubes. samples were centrifuged within 4 h of phlebotomy. table 2: speed and time specifications for centrifugation methods, inkosi albert luthuli central hospital and king edward viii hospital, durban, kwazulu-natal, south africa, september 2021 – november 2021. method a was the clinical and laboratory standards institute-recommended reference method for centrifugation in the laboratory (4000 rpm for 15 min). methods b (4000 rpm/10 min), c (5000 rpm/10 min) and d (5000 rpm/5 min) were compared to method a.9,16,17,18 a literature search confirmed that centrifuging samples at 4000 rpm for 5 min did not produce ppp and therefore this method was excluded from the study.19,20 samples for methods a and b were centrifuged on instrument 1, while samples for methods c and d were centrifuged on instrument 2. the supernatant plasma was transferred to an empty tube using a plastic pipette.16,21 all samples were tested on the sysmex® cs5100 (siemens healthcare gmbh, marburg, germany) automated coagulation analyser with reagents from siemens healthcare products gmbh (marburg, germany). the reagents included innovin® (prothrombin time [pt] – clotting assay); actin® fsl (activated partial thromboplastin time [aptt] – clotting assay); test thrombin® (thrombin time [tt] – clotting assay); dade® thrombin reagent (fibrinogen – clotting assay) and innovance® (d-dimer – immunoassay). the study samples were only analysed when the commercial controls were within the predetermined limits.16,22 an automated platelet count was performed on every sample using the sysmex® xn 3000 automated haematology analyser (siemens healthcare gmbh, marburg, germany) by flow cytometry based on principles of light scattering. this was done to assess the percentage of samples that produced a platelet count of < 10 × 109/l in each of the four methods. data analysis capturing of results, statistical tests and construction of bland altman plots were done using microsoft® excel 2016 (microsoft®, redmond, washington, united states). the ep evaluator software version 8 (informer technologies inc, los angeles, california, united states) and stata version 17 (statacorp®, college station, texas, united states) were used for statistical analysis. for descriptive statistics, numerical data were summarised as means, medians, standard deviations or percentages.23,24 the quality of the data was assessed using an acceptable correlation coefficient (r) value of > 0.975. the correlation coefficient (r) was used to assess the linear relationship between the reference method and methods b, c and d.25,26 the paired student t-test was used to assess the statistical significance of differences between samples. the level of statistical significance was set at a p-value < 0.05. percentage variations were compared with the current desirable quality specifications for bias and were derived from westgard biological variation.20,26 results platelet-poor plasma was produced in all samples (n = 60) centrifuged at 4000 rpm for 15 and 10 min (methods a and b) (table 3). for methods c and d, ppp was produced in 55% (33/60) of the samples, and 45% (27/60) of the samples had a platelet count between 11 and 90 × 109/l. table 3: platelet counts, prothrombin time, activated partial thromboplastin time, thrombin time, fibrinogen and d-dimer levels using four centrifugation methods, inkosi albert luthuli central hospital and king edward viii hospital, durban, kwazulu-natal, south africa, september 2021 – november 2021. forty-nine (49/60; 82%) samples had a normal pt level (method a: mean [seconds] = 10.71 s, median [seconds] = 10.6 s; method b: mean = 10.69 s, median = 10.5 s; method c: mean = 10.62 s, median = 10.5 s; method d: mean = 10.62 s, median = 10.5 s). eleven (11/60; 18%) samples had an abnormal prothrombin result (method a: mean = 19.26 s, median = 15.6 s; method b: mean = 19.22 s, median = 15.1 s; method c: mean = 19.25 s, median = 15.5 s; method d: mean = 19.30 s, median = 15.4 s). there were statistically significant differences for method c (p = 0.002) and method d (p = 0.005) in the normal pt group, with results being lower than the pt results for methods a and b. despite there being statistically significant differences in the normal pt results for methods c and d, the pt results obtained using all three test methods correlated strongly with results obtained using the reference method (r ≥ 0.99) (figure 1). figure 1: comparison of prothrombin time results between the reference centrifugation method (method a) and methods b, c and d, inkosi albert luthuli central hospital and king edward viii hospital, durban, kwazulu-natal, south africa, september 2021 – november 2021. linear regressions for: (a) method a vs method b, (b) method a vs method c, (c) method a vs method d. thirty-eight (38/60; 63%) of the samples had normal aptt values (method a: mean [seconds] = 28.26 s, median [seconds] = 27.9 s; method b: mean = 27.97 s, median = 27.6 s; method c: mean = 28.02 s, median = 27.7 s; method d: mean = 28.16 s, median = 27.7 s). twenty-two (22/60; 37%) samples had abnormal aptt results (method a: mean = 37.48 s, median = 24.1 s; method b: mean = 37.27 s, median = 24.4 s; method c: mean 37.44 s, median = 24.3 s; method d: mean = 37.19 s, median = 24.2 s). there were no statistically significant differences in the aptt results obtained using method a versus methods b, c and d; aptt results from the three processing methods strongly correlated with the results of the reference method (r > 0.975). forty-six (46/60; 77%) samples had normal tt levels (method a: mean [seconds] = 17.47 s, median [seconds] = 17.5 s; method b: mean = 16.69 s, median = 17.3 s; method c: mean = 17.37 s, median = 17.3 s; method d: mean = 17.34 s, median = 17.5 s). fourteen (14/60; 23%) had abnormal tt levels (method a: mean = 16.72 s, median = 15.6 s; method b: mean = 16.69 s, median = 15.6 s; method c: mean = 16.57 s, median = 15.5 s; method d: mean = 16.46 s, median = 15.6 s). abnormal tt levels were significantly lower among samples analysed using method d compared to methods a, b and c. all three methods correlated poorly (r = 0.92–0.93) with the reference method (figure 2). figure 2: comparison of thrombin time results between the reference centrifugation method (method a) and methods b, c and d, inkosi albert luthuli central hospital and king edward viii hospital, durban, kwazulu-natal, south africa, september 2021 – november 2021. linear regressions for: (a) method a vs method b, (b) method a vs method c, (c) method a vs method d. forty-seven (47/60; 78%) samples had normal fibrinogen levels (method a: mean [g/l] = 3.41, median [g/l] = 3.63; method b: mean = 3.46, median = 3.61; method c: mean = 3.52, median = 3.64; method d: mean = 3.45, median = 3.57). thirteen (13/60; 22%) samples had abnormal fibrinogen levels (method a: mean = 5.28, median = 5.31; method b: mean = 5.18, median = 5.43; method c: mean = 5.44, median = 5.60; method d: mean = 5.37, median = 5.40). the results of all three test methods correlated strongly with the reference method. nineteen (19/59; 32%) samples had normal d-dimer levels (method a: mean [mg/l] = 0.20, median [mg/l] = 0.19; method b: mean = 0.27, median = 0.19; method c: mean = 0.21, median = 0.19; method d: mean = 0.21, median = 0.20). forty (40/59; 62%) samples had abnormal d-dimer levels (method a: mean = 1.5, median = 0.58; method b: mean = 1.49, median = 0.57; method c: mean = 1.44, median = 0.56; method d: mean = 1.47, median = 0.60). d-dimer levels were significantly lower for methods c (p = 0.04) and d (p = 0.02) when compared to method a. method d correlated strongly with the reference method, while methods b and c correlated poorly with the reference method (r < 0.975) (figure 3). a single sample result for method a (35.2 mg/l) had an extreme outlying value when compared with the results from methods b, c and d (< 0.36 mg/l). figure 3: comparisons of d-dimer results between the reference centrifugation (method a) and methods b, c and d, inkosi albert luthuli central hospital and king edward viii hospital, durban, kwazulu-natal, south africa, september 2021 – november 2021. linear regressions for: (a) method a vs method b, (b) method a vs method c, (c) method a vs method d. discussion our study found that centrifuging samples at 4000 rpm for 10 min yielded ppp in 100% of samples compared to centrifugation at 5000 rpm for 10 min and 5 min, which only yielded ppp in 55% of samples. the higher centrifugation speed caused an increase in platelet count in 45% of cases. this study has shown that method b (4000 rpm/10 min) is superior to methods c and d as it did not have a significant impact on the coagulation assay results. hence, method b could be an alternate method of processing samples for coagulation tests. a study by barnes et al showed that 10 min was the minimum centrifugation time required to consistently meet the recommendations for ppp.27 this differed from a previous study by sultan et al., which reported that the majority of the 46 samples tested produced ppp when centrifuged at 5000 rpm for 5 min.12 although ppp is recommended for coagulation testing, studies suggest that coagulation test results are not affected by platelet counts of > 10 × 109/l and that samples with platelet counts of up to 199 × 109/l produce similar results to samples with ppp.28 in our study, platelet counts differed significantly between the samples centrifuged at 4000 rpm (method a and b) and those processed at 5000 rpm (method c and d); however, there were no statistically significant differences between method a and methods b, c and d in results for the aptt and fibrinogen assays, as well as for the abnormal pt, normal tt and abnormal d-dimer assays. this is similar to the findings of previous studies conducted in 2002 and 2011.19,27 statistically significant differences were observed in the normal pt and normal d-dimer assay results when centrifuged at 5000 rpm compared to the reference method. abnormal tt levels were significantly lower when measured with method d compared to method a; however, this was not the case for method c which was also a 5000-rpm method. although the results for normal pt were found to be statistically significantly different between the reference method and methods c and d, the differences in the actual mean, median and standard deviation values between the groups were minimal. when combined, the normal and abnormal pt results showed a good correlation between the different methods (r = 0.99) and these results are similar to those discovered in a previous study by azlin et al.19 there were statistically significant differences in the abnormal tt (method d) and normal d-dimer values (methods c and d) when compared to the reference method; however, there was minimal variation in the mean, standard deviation and median values. furthermore, the tt results showed poor correlation (r = 0.92–0.93) for all three methods when compared to the reference method. these findings suggest that the variation of the raw data may not be clinically significant as the clinical management of patients in our setting would not be altered. the aptt assay is usually more sensitive to platelet contamination than the pt assay.19 this was not observed in our study as the results of the aptt and fibrinogen assays showed no significant differences and correlated strongly between the test methods and the reference method. a single d-dimer result from method a had an outlying value when compared with the results from methods b, c and d. it was confirmed that the sample was collected following standard practice guidelines, labelled correctly and processed using the standard operating procedure of the laboratory.29 processing errors can occur during the pre-analytical phase8,30; however, a transcription error was excluded in this case. owing to limited sample availability and repeat sampling not being possible, method a could not be rerun.30,31 our study results encourage further research on rapid centrifugation of coagulation samples to verify the reliability of the results and explore the potential benefits it could have in a clinical laboratory setting. these findings could have practical applications and serve as a basis for additional research to establish local centrifugation protocols in laboratories.19 limitations the results of this study (sample size = 60) need to be validated with a larger case-control study. a larger number of healthy individuals (controls) should be included. the pt, tt and fibrinogen assays had low abnormal sample numbers ranging from 20% to 30% and the tt results did not reflect extreme abnormal ranges. participants were recruited on a voluntary basis; therefore, some assays showed a bias in the normal-to-abnormal ratios. conclusion this study demonstrates that method b is superior to methods c and d as it produced results that were most consistent with those obtained using the reference method. methods c and d produced statistically significant differences in results for the pt, tt and d-dimer assays. we show that the centrifugation of whole blood samples in 5 ml citrate tubes at 4000 rpm for 10 min is suitable for routine coagulation testing. this rapid centrifugation method provides consistent and reliable results and could potentially reduce the overall tat. these findings may assist experts in revising the current recommendations for the centrifugation of coagulation samples. acknowledgements the authors wish to thank: rookaya mahomed – senior technologist, national health laboratory services inkosi albert luthuli central hospital, for the processing of the study samples; catherine connolly – statistician university of kwazulu-natal, for assisting with the statistical analysis of the results; national health laboratory services haematology laboratory, inkosi albert luthuli central hospital, staff for the technical assistance; inkosi albert luthuli central hospital and king edward viii hospital: department of clinical haematology staff for their assistance during the sample collection process. competing interests the authors wish to declare that they have no personal or financial interests that may have had an influence on the writing of this article. authors’ contributions r.h. conceived and presented the idea of the study to d.p. r.h. and d.p. developed the theory, methodology and protocol for this study. n.r. reviewed the work and advised on the study design and implementation of the research. d.p. supervised and n.r. co-supervised the project. all authors contributed to the analysis of the results and to the writing of the final manuscript. sources of support the authors acknowledge the receipt of financial support for the research from the national health laboratory services department of haematology, inkosi albert luthuli central hospital. data availability the data generated during the study is available on request from the corresponding author, r.h., due to privacy/ethical restrictions. disclaimer the views expressed in this article are those of the authors and not of the institution (university of kwazulu-natal) or the funder (national health laboratory services). references rajashekar r, krishnamurthy v, murthy doreswamy s. does platelet contamination really affect the coagulation tests? a prospective study. int j hematol res. 2016;2(3):155–159. https://doi.org/10.17554/j.issn.2409-3548.2016.02.38 gürsoy o, tür fç, aksay e, et al. the impact of coagulation testing on patient management in the emergency department. acta medica mediterr. 2015;31(3):597–602. khan ih, savarimuthu s, leung mst, harky a. the need to manage the risk of thromboembolism in covid-19 patients. j vasc surg. 2020 sep;72(3):799–804. https://doi.org/10.1016/j.jvs.2020.05.015 kander t. coagulation disorder in covid-19. lancet haematol. 2020;3026(20):30217–30218. https://doi.org/10.1016/s2352-3026(20)30218-0 mendelsohn ee, solum no, brosstad f. effects of platelets and platelet-derived material on the activated partial thromboplastin time (cephotest®) coagulation test. scand j clin lab invest. 2005;65(4):321–332. https://doi.org/10.1080/00365510510025719 steindel sj, novis da. using outlier events to monitor test turnaround time. arch pathol lab med. 1999;123(7):607–614. https://doi.org/10.5858/1999-123-0607-uoetmt minder ei, schibli a, mahrer d, nesic p, plüer k. effects of different centrifugation conditions on clinical chemistry and immunology test results. bmc clin pathol. 2011 may;11(6):2–15. https://doi.org/10.1186/1472-6890-11-6 roberts t, smith m, roberts b. observations on centrifugation: application to centrifuge development. clin chem. 1999;45(11):1889–1897. https://doi.org/10.1093/clinchem/45.11.1889 clinical and laboratory standards institute. collection, transport, and processing of blood specimens for testing plasma-based coagulation assays and molecular hemostasis assays. approved guideline h21-a5. 5th ed. wayne, pa: clsi, 2009; p. 5–15. kao c-h, shu l-c, yen w-h. evaluation of a high-speed centrifuge with rapid preparation of plasma for coagulation testing to improve turnaround time. j biomed lab sci [serial online]. 2010;22(1):23–28. [cited 2020 jul 20]. available from: http://www.labmed.org.tw/upfiles/issues/20104810530.pdf pappas aa, palmer sk, meece d, fink lm. rapid preparation of plasma for coagulation testing. arch pathol lab med. 1991 aug;115(8):816–817. sultan a. five-minute preparation of platelet-poor plasma for routine coagulation testing. east mediterr heal j. 2010;16(2):233–236. https://doi.org/10.26719/2010.16.2.233 lippi g, salvagno gl, montagnana m, guidi gc. preparation of a quality sample: effect of centrifugation time on stat clinical chemistry testing. lab med. 2007;38(3):172–176. https://doi.org/10.1309/d8tjcaruw575cxyh boudaoud l, divaret g, marie p, bezeaud a. rapid centrifugation for routine coagulation testing. ann biol clin (paris). 2006;64(4):315–317. dickinson b. bd vacutainer® evacuated blood collection system [homepage on the internet]. nj: bd; 2005 [cited 2021 jun 19]. available from: www.bd.com/vacutainer/pdfs/blood_collection_tubes_products_insert_vdp40035.pdff mahomed r, singh s, singh r. haematology coagulation procedure manual. gpl4330 version 3. durban, 2019; p. 1–35. dickinson b. recommended centrifugation speeds and times for bd vacutainer blood collection tubes. [homepage on the internet]. east rutherford, nj. [cited 2021 jun 19]. available from: http://www.bd.com/vacutainer/faqs bt lab systems. convert g-force (rcf) to rpm for lab centrifuges [homepage on the internet]. geno technology inc; 2018 [cited 2020 jul 20]. available from: https://blog.btlabsystems.com/blog/centrifuge-g-force azlin i, hafiza a, rz a, rk a, nh h. effect of higher centrifugation speed and shortened centrifugation time on prothrombin and activated thromboplastin time. med health. 2011;6(1):68–72. lippi g, salvagno gl, montagnana m, manzato f, guidi gc. influence of the centrifuge time of primary plasma tubes on routine coagulation testing. blood coagul fibrinol. 2007;18(5):525–528. https://doi.org/10.1097/mbc.0b013e3281eec945 hinz ar, boehm m. technical guide on efficient sample preparation for clinical diagnosis [homepage on the internet]. vol. 1, no. 1. waltham, ma: thermo fisher scientific inc., 2016; p. 1–8 [cited 2020 jul 20]. available from: https://assets.thermofisher.com/tfs-assets/led/application-notes/clinical-centrifuge-sample-prep-application-note-ancfgclinicalbench.pdf suchsland j, friedrich n, grotevendt a, et al. optimizing centrifugation of coagulation samples in laboratory automation. clin chem lab med. 2014;52(8):1187–1191. https://doi.org/10.1515/cclm-2014-0038 morgan cj, aban i. methods for evaluating the agreement between diagnostic tests. j nucl cardiol. 2016;23(3):511–513. https://doi.org/10.1007/s12350-015-0175-7 briggs c, culp n, davis b, d’onofrio g, zini g, machin sj. icsh guidelines for the evaluation of blood cell analysers including those used for differential leucocyte and reticulocyte counting. int j lab hematol. 2014;36(6):613–627. https://doi.org/10.1111/ijlh.12201 swartz m, hanes h, shim j, stevens p, sholander a. hematology validation plan. equ52-01. baltimore, md: smile, 2020; p. 1–6. westgard j. desirable biological variation database specifications [homepage on the internet]. 2019 [cited 2019 jul 28]. available from: https://www.westgard.com/biodatabase1.htm barnes pw, eby cs, lukoszyk m. residual platelet counts in plasma prepared for routine coagulation testing with the beckman coulter power processor. lab hematol. 2002;8(4):205–209. carroll we, wollitzer ao, harris l, ling mc, whitaker wl, jackson rd. the significance of platelet counts in coagulation studies. j med. 2001;32(1–2):83–96. dhingra n, diepart m, dziekan g, et al. who guidelines on drawing blood: best practices in phlebotomy. geneva: who press, world health organization, 2010; p. 1–105. favaloro ej, dorothy m, lippi g. pre-analytical variables in coagulation testing associated with diagnostic errors in hemostasis. lab med. 2012;43(2):1–10. https://doi.org/10.1309/lm749bqetkypypvm richard am, marian a. sources and solutions for spurious test results in coagulation. int j lab hematol. 2019 feb;41(suppl 1):162–169. https://doi.org/10.1111/ijlh.12989 abstract introduction methods results discussion acknowledgements references about the author(s) naome mugabiirwe department of medical laboratory, kyazanga health centre iv, lwengo, uganda rogers kalyetsi department of medical laboratory science, faculty of medicine, mbarara university of science and technology, mbarara, uganda richard ayella department of medical laboratory, kyazanga health centre iv, lwengo, uganda james obote department of medical laboratory, kyazanga health centre iv, lwengo, uganda frank ssedyabane department of medical laboratory science, faculty of medicine, mbarara university of science and technology, mbarara, uganda citation mugabiirwe n, kalyetsi r, ayella r, obote j, ssedyabane f. hepatitis b virus infection and hbeag positivity among pregnant women in south west uganda. afr j lab med. 2022;11(1), a1784. https://doi.org/10.4102/ajlm.v11i1.1784 original research hepatitis b virus infection and hbeag positivity among pregnant women in south west uganda naome mugabiirwe, rogers kalyetsi, richard ayella, james obote, frank ssedyabane received: 09 nov. 2021; accepted: 10 may 2022; published: 15 aug. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: hepatitis b virus is a public health burden in uganda, yet little is known about its epidemiology in pregnancy. objective: this study aimed at determining the prevalence and associated risk factors of hepatitis b virus infection among pregnant women attending antenatal care at the kyazanga health centre iv in lwengo district, uganda. methods: this cross-sectional study was conducted from april 2021 to june 2021 and analysed qualitative data that were collected using a structured in-person questionnaire. aseptically collected blood specimens were screened for hepatitis b virus infection using an immunochromatographic rapid diagnostic test kit. participants who were positive for the hepatitis b surface antigen (hbsag) were further screened for hepatitis b envelope antigen (hbeag) using commercial rapid diagnostic test kits. results: out of 384 pregnant women studied, eight tested positive for hbsag. this gave a prevalence of 2.1% (95% confidence interval: 1.0% – 4.1%); 5/8 (62.5%) were positive for hbeag. none of the variables studied were significantly associated with hbsag positivity among pregnant women. conclusion: hepatitis b viral infection is still a public health challenge in pregnant women with possible risk for vertical transmission to their babies in the study area. we recommend routine screening for hepatitis b virus in pregnancy in addition to strengthening current strategies aimed at controlling and preventing hepatitis b infection spread and transmission. keywords: risk factors; hepatitis b; pregnant women; prevalence; uganda. introduction hepatitis b is caused by hepatitis b virus (hbv) infection. hepatitis b virus belongs to the family hepadnaviridae and is highly hepatotropic, causing acute and chronic liver diseases including cirrhosis and hepatocellular carcinoma.1 hepatitis b virus is estimated to have caused 820 000 deaths worldwide2 by contributing to chronic hepatitis and hepatocellular carcinoma.3 the prevalence of hbv infection in sub-saharan africa is reported to range from 5% to 20%,4 with perinatal transmission estimate rates ranging from 1% to 5%.4 uganda is known to be a highly endemic area of the infection with an estimated national prevalence of 10.0%.5 the regional distribution of disease varies, with the highest prevalence in the northern region; prevalence is 19% in the northwest and 25.0% in the northeast.5 the national prevalence of hbv infection among women attending antenatal care was 4.1% in the year 2018.6 studies done in uganda around 2018 estimated the prevalence of hepatitis b virus infection among pregnant women attending antenatal care to be 2.9% (95% confidence interval: 1.58% – 5.40%) at mulago national referral hospital in central uganda,5 11.8% at two hospitals in northern uganda7 and 3.1% at mbarara regional referral hospital in western uganda.6 some risk factors for hepatitis b infection include tattooing or use of contaminated sharp instruments, reuse of needles, intravenous drug use, percutaneous as well as mucosal exposure to infected blood or any other body fluids such as saliva, vaginal or seminal fluids.5 mothers who are positive for hepatitis b surface antigen (hbsag) have a 10% to 40% risk of passing the infection onto their newborn babies and the risk increases to 90% among mothers who are positive for the hepatitis b envelope antigen (hbeag).8 babies who are infected perinatally have a 90% risk of developing chronic infection.9 antiviral therapy during late pregnancy and immunisation of babies exposed to hbv within the first 12 h of life may reduce mother-to-child transmission by 75% to 90% and reduce risk of developing chronic infection.10,11 the perinatal transmission risk is 70% to 90% for hbeag-positive mothers, 25% for hbeag-negative/anti-hbe-negative mothers and 12% for mothers who are positive for anti-hbe while negative for hbeag.5 a number of studies have been conducted in south western uganda on the epidemiology of hbv infection, though little data exists on prevalence and associated risk factors among pregnant women attending antenatal care at the kyazanga health centre iv (hciv). therefore, this study aimed to determine the prevalence and risk factors associated with hbv infection among pregnant women attending antenatal care at kyazanga hciv, lwengo district, south western uganda. methods ethical considerations ethical approval was sought from the department of medical laboratory science, faculty research committee faculty of medicine mbarara university of science and technology (must/mls/030). permission was also obtained from the district health officer, lwengo district and the office of medical officer health sub-district, kyazanga health centre iv. informed consent was obtained from all study participants. participants were identified with numbers, not names, for prevention of breach of confidentiality. all completed questionnaires were archived in the department of medical laboratory science under lock and key. the generated computer database was kept inaccessible for non-study persons; for study personnel, access was restricted with a password. study design and setting this cross-sectional study was conducted between april 2021 and june 2021 at the kyazanga hciv antenatal clinic located in kyazanga town council, lwengo district in south western uganda. the kyazanga hciv is a public health facility which serves buktoto west, including referrals from five other low-level health facilities. sample size, study population and sampling strategy we calculated a sample size of 384 participants based on a presumed 50% prevalence rate of hbv6 using an allowable standard error of ±0.05 at 95% level of confidence. the study population included pregnant women who attended the antenatal clinic during the study period. we used a systematic random sampling method to recruit all participants. individuals were selected at regular intervals, where every second pregnant woman who arrived at the clinic was selected from the sampling frame. data collection with the help of clinic nurses, we administered an in-person structured questionnaire to collect socio-demographic data (age, place of residence, level of education, primary [completed primary school year seven], secondary [completed senior school year six], or tertiary [reached any tertiary institution]), participant’s place of birth and gravidity (primigravida [first pregnancy], multigravida [2–4 pregnancies], grand multigravida [≥ 5 pregnancies]) and risk factors for hbv (history of blood transfusion, history of abortion or miscarriage, history of hospital admission, history of tooth extraction, history of body piercing, history of unprotected sexual intercourse, history of sharing sharp materials and history of injection drug abuse) from study participants. laboratory testing immediately after consent and completion of the questionnaire, three millilitres (3 ml) of venous blood was drawn aseptically by venipuncture from the mid-cubital vein, into ethylenediaminetetraacetic acid-vacutainers. specimens were labelled using identification numbers (codes) and immediately taken to the laboratory for centrifugation (3000 rpm for 5 min) to separate plasma from blood cells. sd bioline hbsag immunochromatographic rapid diagnostic test kits (abbott diagnostics korea inc, gihueng-gu, yongin-si, republic of korea) were used for qualitative detection of hbsag in serum from each study participant. plasma was added to the sample pad on the hbsag test strip and timed for 15 min for the reaction to occur. results were then read, interpreted and recorded. positive samples were then tested for the hbeag, using the commercial sd bioline hbeag testing kit (abbott diagnostics korea inc, gihueng-gu, yongin-si, republic of korea) to show the level of infectivity and risk of infection. all laboratory testing was carried out within the health unit laboratory, following standard operating procedures as well as manufacturer’s instructions. quality assurance and control the questionnaire was pre-tested and validated on five pregnant women attending antenatal care at the kyazanga hciv. each batch of the rapid dipstick tests was pre-tested with positive and negative controls (known positive and known negative plasma samples) for quality assurance. positive and negative controls were run along with each batch of samples. the sensitivity and specificity of the rapid test strip were 98.84% and 98.94%, respectively. data management and analysis data were entered into a microsoft excel spread sheet (microsoft office professional plus 2013, version 15.0.4675.1003, microsoft inc, redmond, washington, united states) and then imported into stata 13 (statacorp llc, college station, texas, united states) software for analysis. demographic data were presented in the form of frequencies and percentages. prevalence was presented as percentages and using pie charts. associations between risk factors and hepatitis b were determined using regression analysis and a p-value of < 0.05 was considered to be statistically significant. results socio-demographic characteristics of pregnant women attending antenatal clinic at kyazanga health centre iv we recruited 384 pregnant women attending the antenatal clinic at kyazanga hciv from april 2021 to june 2021 (table 1). the participants’ mean age was 27 years, with a standard deviation of 7.34 years. participants’ ages ranged from 17 to 48 years, with the majority aged between 26 and 28 years. most study participants were from urban areas (51.0%), had completed a primary seven level of education (65.0%), had been born in a hospital setting (61.2%) and were multigravidas (54.2%). table 1: demographic characteristics of participants attending the antenatal clinic at kyazanga health centre iv, south western uganda, between april 2021 and june 2021. prevalence of hepatitis b virus infection of the 384 participants, eight participants tested positive for hbsag for an overall prevalence of viral hepatitis of 2.1% (table 2). of the eight hbsag-positive participants, five tested positive for hbeag (62.5% of hbsag-positive participants). of the eight participants who tested positive for hbsag, 75.0% (6/8) were aged 24–35 years, 25.0% (2/8) were aged younger than 24 years (table 3). additionally, 62.5% (5/8) lived in an urban setting, and 37.5% (3/8) lived in a rural setting. a majority (62.5%; 5/8) had completed a primary seven level of education, 25.0% (2/8) had attained secondary education, 12.5% (1/8) had tertiary education. most participants had been born at home (62.5%; 5/8), while 37.5% (3/8) had been born at the hospital. most participants 75.0% were multigravida and 25.0% were primigravida. among women who were hbsag-positive, 50.0% (4/8) had a history of blood transfusion, 62.5% (5/8) had a history of sharing sharp instruments, all had a history of unprotected sexual intercourse, none had any history of drug abuse by injection, 87.5% (7/8) had a history of hospital admission, 37.5% (3/8) had a history of abortion or miscarriage, 62.5% (5/8) had a history of tooth extraction, 50.0% (4/8) had multiple sexual partners and none had any history of body piercing. table 2: overall prevalence of hbsag and hbeag positivity among participants attending antenatal at kyazanga health centre iv, south western uganda, between april 2021 and june 2021. table 3: prevalence of hbv infection by risk factor among pregnant women attending kyazanga health centre iv, south western uganda, between april 2021 and june 2021. risk factors for hepatitis b virus infection on univariate analysis, place of birth (odds ratio: 1.06; p = 0.018), a history of blood transfusion (odds ratio: 1.037; p = 0.043) and a history of hospital admission (odds ratio: 1.03; p = 0.04) were associated with hbv infection (table 4). on multivariate analysis, no variable was significantly associated with hbv infection. table 4: risk factors predisposing pregnant women to hbv at kyazanga health centre iv, south western uganda, between april and june 2021. discussion prevalence of hbv infection we report the prevalence of hbsag at 2.1% among pregnant women attending antenatal care at kyazanga hciv, lwengo district, uganda. the epidemiology of hbv infection can be described in terms of the prevalence of hbsag positivity in a population. this can be broadly classified as high (> 8.0% hbsag prevalence), intermediate (2.0% – 7.0% hbsag prevalence) and low (< 2.0% hbsag prevalence).12 this study reports intermediate endemicity of hbsag and high prevalence of hbeag of 62.5% (5/8) among the hbsag-positive women, which indicates a high risk of perinatal transmission. in comparison to other studies done within uganda, the prevalence reported in this study is comparable to infection levels reported at mbarara regional referral hospital of 2.5% in 201813 and 3.12% in 2019.6 the finding of our study is also comparable to 2.9% prevalence which was reported at mulago regional referral hospital.5 this similarity may be because the same risk group was studied and the tests used to ascertain hbv infection were similar (i.e., rapid tests). however, this study’s result does not correlate with the reported prevalence of 11.8% in two hospitals located in northern uganda where similar diagnostic methods were used.7 this high prevalence of hepatitis b in northern uganda could be due to a difference in socio-demographic factors like level of education. a majority of people in northern uganda are said to have not gone beyond primary seven.14 within the east african countries, this study closely correlates with the finding of 3.8% in mbagathi district in kenya in 2014,15 3.8% at nyamagana district hospital in mwanza, tanzania in 2014,16 3.9% in dar es salaam in 201417 and 3.9% in rwanda in 2018.18 it is lower than the reported prevalence of hbsag at khartoum teaching hospital, sudan, 7.5% in 2010,19 and juba teaching hospital, south sudan, 11.0% in 2012.20 this intra-regional variation could be due to geographical variation, sample size and laboratory methods used for diagnosis. the prevalence of hbeag positivity of 62.5% among hbsag-positive pregnant women reported in this study is higher than the prevalence reported by a study in northern uganda at 14.9%.7 hbsag-positive mothers who test positive for hbeag are known to be highly infectious as the virus is actively replicating.21 therefore, they have a 40.0% to 90.0% risk of transmitting the hbv infection to their babies at birth.22 however, our study did not test for anti-hbe or anti-hbc antibody levels, which would further help predict the risk of perinatal transmission in these women. risk factors for hbv infection none of the risk factors studied had statistically significant associations with hbsag positivity. this concurs with studies done in buea in cameroon in 2012,23 and benin city in nigeria in 2009.24 however, not all of the risk factors considered by those studies were assessed in the current study. we also report here that there was a high prevalence of hbsag among pregnant women aged 24–35 years, although the association with hbsag positivity was not stastically significant; this agrees with a study done in egypt between 2010 and 2011.25 this observation of increasing age with hbsag infection can be attributed to increased likelihood of contracting hbv during each cycle of pregnancy. however, this contrasts with findings from a study done by bayo and his colleagues in 2012, who reported a high prevalence of hbv infection among pregnant women aged 20 years old or less in northern uganda.7 the current study also found no association between participants’ history of blood transfusion and hbsag positivity. this agrees with several studies conducted in various places, including mbarara regional referral hospital in uganda in 201813 and 2019,6 in egypt between 2010 and 2011,25 in mulago, uganda between 2018 and 2019,5 and juba teaching hospital, south sudan in 2012.20 this finding contrasts with studies done in khartoum, sudan, in 2010 which found a positive association between participant’s history of blood transfusion and hbsag positivity.19 this observation may be due to implementation of good blood screening strategies for transfusion-transmissible infections in blood bank donations, reducing incidence of infection by blood transfusion. this study showed a high prevalence of hbsag positivity among the multigravida pregnant women, although the association was not statistically significant. this contrasts with a study done in mwanza, tanzania, in 2014, which showed that multigravidity is associated with hbsag.16 it is thought that chances of exposure to hbv increase as one progresses through the cycle of pregnancy from conception to child birth, and the same happens as the gravidity rises from primagravidity to multigravidity. history of hospital admission was not found to be significantly associated with hbv infection. this contrasts with a study from egypt between 2010 and 2011, which found a significant association between hospital admission and hbv infection, since hospital admission may expose pregnant women to surgical procedures which can lead to hbv infection.25 neither a history of abortion nor history of multiple sexual partners were significantly associated with hbsag positivity in this study. however other studies have reported significant association of hepatitis b infection with history of abortion and multiple sexual partners.26,27 this may be due to increases in sexual activity and sexual contact creating more opportunities for acquiring hbv infection this needs further study to test the level of this significance in this area. no significant association with hbv infection was found with a history of injection drug abuse, body piercing or body tattooing among these women. this is in agreement with a study conducted in southwestern nigeria in 2013.28 however, these factors are considered to increase the chances of becoming infected with hbv.29 the lack of a significant association may be attributed to the fact that practices of drug abuse by injection and tattooing are uncommon in this study setting. recommendations in view of high hbeag positivity among hbsag-positive pregnant women, there is need for routine screening of pregnant women for hbsag and hbeag to predict the risk of perinatal transmission and strengthen immunisation strategies for babies at risk. there is also a need to adapt treatment and prevention strategies to achieve the world target of free communities without hbv infection. limitations this study did not explore all the risk factors that may predispose pregnant women to hbv. for example, information about a history of jaundice, vulvular ulcerations, history of sexually transmitted infection, and a family history of hbv were not collected. the self-reported risk factor data used in this study could be subject to recall bias. conclusion hepatitis b virus was found to have intermediate endemicity among the pregnant women attending antenatal care at kyazanga hciv. however, this is not too low to cause morbidity and mortality among the population. none of the proposed risk factors were significantly associated with hbv infection; explanations for this observation need to be explored in future. acknowledgements we acknowledge the staff and administrators of kyazanga health centre iv for accepting our study and giving us the opportunity to explore the world of science today, thank you so much. we also acknowledge the department of medical laboratory science under the leadership of the head of department, mr rugera simon peter, and the faculty of medicine under the leadership of faculty dean, associate professor getrude n. kiwanuka. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions n.m., r.k., r.a., j.o. and f.s. have contributed substantially to this work and met the criteria for authorship. n.m., r.a. and j.o. conceived and developed the idea. they also performed data collection and prepared the first draft of the manuscript. r.k. and f.s. supervised the entire project and approved the final version. sources of support this research project was funded exclusively by the authors. this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the datasets used during the current study are available from the corresponding author, n.m., on reasonable request. disclaimer the views and opinions expressed in this artice are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references kao j-h, chen d-s. global control of hepatitis b virus infection. lancet infect dis. 2002;2(7):395–403. https://doi.org/10.1016/s1473-3099(02)00315-8 who. fact sheet on hepatitis b. geneva; world health organisation; 2019. kamenya t, damian dj, ngocho js, philemon rn, mahande mj, msuya se. the prevalence of hepatitis b virus among hiv-positive patients at kilimanjaro christian medical centre referral hospital, northern tanzania. pan afr med j. 2017;28:275. https://doi.org/10.11604/pamj.2017.28.275.11926 kiire c, group ars. hepatitis b infection in sub-saharan africa. vaccine. 1990;8:s107–s112. https://doi.org/10.1016/0264-410x(90)90229-f kayondo sp, byamugisha jk, ntuyo p. prevalence of hepatitis b virus infection and associated risk factors among pregnant women attending antenatal clinic in mulago hospital, uganda: a cross-sectional study. bmj open. 2020;10(6):e033043. https://doi.org/10.1136/bmjopen-2019-033043 hillary a, julius m, joseph n, et al. prevalence and factors associated with hepatitis b surface antigen positivity among women receiving antenatal care at mbarara regional referral hospital. j trop dis. 2019;7:321. https://doi.org/10.35248/2329-891x.19.7.321 bayo p, ochola e, oleo c, mwaka ad. high prevalence of hepatitis b virus infection among pregnant women attending antenatal care: a cross-sectional study in two hospitals in northern uganda. bmj open. 2014;4(11). https://doi.org/10.1136/bmjopen-2014-005889 alter mj. epidemiology of hepatitis b in europe and worldwide. j hepatol. 2003;39:64–69. https://doi.org/10.1016/s0168-8278(03)00141-7 islam m, ahmed r, kabria s, et al. clinical significance of serum hbeag among hbsag positive patients. faridpur med coll j. 2010;5(2):50–52. https://doi.org/10.3329/fmcj.v5i2.6821 choisy m, keomalaphet s, xaydalasouk k, quet f, latthaphasavang v, buisson y. prevalence of hepatitis b virus infection among pregnant women attending antenatal clinics in vientiane, laos, 2008–2014. hepat res treat. 2017;2017:1284273. https://doi.org/10.1155/2017/1284273 maclean b, hess rf, bonvillain e, et al. seroprevalence of hepatitis b surface antigen among pregnant women attending the hospital for women & children in koutiala, mali. s afr med j. 2012;102(1). maclachlan jh, cowie bc. hepatitis b virus epidemiology. cold spr harbor perspect med. 2015;5(5):a021410. https://doi.org/10.1101/cshperspect.a021410 derick m, davis kl, morris m, samuel o, rebecca w, benson o. prevalence and associated risk factors of hepatitis b viral infection among pregnant women accessing antenatal care at mbarara regional referral hospital, south west, uganda. int j trop dis health. 2018;32(2):1–8. https://doi.org/10.9734/ijtdh/2018/44572 the world bank group. the uganda poverty assessment eeport 2016. washington, dc: the world bank; 2016. ngaira jam, kimotho j, mirigi i, osman s. prevalence, awareness and risk factors associated with hepatitis b infection among pregnant women attending the antenatal clinic at mbagathi district hospital in nairobi, kenya. pan afr med j. 2016;24:315. https://doi.org/10.11604/pamj.2016.24.315.9255 mirambo mm, mbena pb, mushi mf, et al. prevalence of hepatitis b surface antigen among pregnant women attending antenatal clinic at nyamagana district hospital mwanza, tanzania. tanzania j health res. 2016;18(1). https://doi.org/10.4314/thrb.v18i1.10 manyahi j, msigwa y, mhimbira f, majigo m. high sero-prevalence of hepatitis b virus and human immunodeficiency virus infections among pregnant women attending antenatal clinic at temeke municipal health facilities, dar es salaam, tanzania: a cross sectional study. bmc pregn childbirth. 2017;17(1):1–6. https://doi.org/10.1186/s12884-017-1299-3 makuza jd, rwema jot, ntihabose ck, et al. prevalence of hepatitis b surface antigen (hbsag) positivity and its associated factors in rwanda. bmc infect dis. 2019;19(1):1–10. https://doi.org/10.1186/s12879-019-4013-4 abuelgasim mh, baraka mbk. prevalence of hepatitis b infection among pregnant women at khartoum teaching hospital, sudan. j us china med sci. 2015;12(2):58–63. https://doi.org/10.17265/1548-6648/2015.02.003 kirbak als. sero-prevalence for hepatitis b virus among pregnant women attending antenatal clinic in juba teaching hospital, republic of south sudan. pan afr med j. 2017;26:72. https://doi.org/10.11604/pamj.2017.26.72.11410 stevens ce, toy pt, tong mj, et al. perinatal hepatitis b virus transmission in the united states: prevention by passive-active immunization. jama. 1985;253(12):1740–1745. https://doi.org/10.1001/jama.1985.03350360066020 stevens ce, beasley rp, tsui j, lee w-c. vertical transmission of hepatitis b antigen in taiwan. n engl j med. 1975;292(15):771–774. https://doi.org/10.1056/nejm197504102921503 frambo aab, atashili j, fon pn, ndumbe pm. prevalence of hbsag and knowledge about hepatitis b in pregnancy in the buea health district, cameroon: a cross-sectional study. bmc res notes. 2014;7(1):1–7. https://doi.org/10.1186/1756-0500-7-394 ugbebor o, aigbirior m, osazuwa f, enabudoso e, zabayo o. the prevalence of hepatitis b and c viral infections among pregnant women. n am j med sci. 2011;3(5):238. https://doi.org/10.4297/najms.2011.3238 mortada e-s, mohamed mf, hamdi msed, ehab m, khamiss ss, el-karaksy h. prevalence of hepatitis b virus infection among egyptian pregnant women – a single center study. int j trop dis health. 2013;3(2):157–168. https://doi.org/10.9734/ijtdh/2013/3276 chernet a, yesuf a, alagaw a. seroprevalence of hepatitis b virus surface antigen and factors associated among pregnant women in dawuro zone, snnpr, southwest ethiopia: a cross sectional study. bmc res notes. 2017;10(1):1–5. https://doi.org/10.1186/s13104-017-2702-x tanga at, teshome ma, hiko d, fikru c, jilo gk. sero-prevalence of hepatitis b virus and associated factors among pregnant women in gambella hospital, south western ethiopia: facility based cross-sectional study. bmc infect dis. 2019;19(1):1–7. https://doi.org/10.1186/s12879-019-4220-z anaedobe cg, fowotade a, omoruyi ce, bakare ra. prevalence, socio-demographic features and risk factors of hepatitis b virus infection among pregnant women in southwestern nigeria. pan afr med j. 2015;20:406. https://doi.org/10.11604/pamj.2015.20.406.6206 zenebe y, mulu w, yimer m, abera b. sero-prevalence and risk factors of hepatitis b virus and human immunodeficiency virus infection among pregnant women in bahir dar city, northwest ethiopia: a cross sectional study. bmc infect dis. 2014;14(1):1–7. https://doi.org/10.1186/1471-2334-14-118 article information authors: robyn marshall1,2 jenifer vaughan1,2 ria david1,3 elise schapkaitz1,2 sergio carmona1,2 tracey wiggill1,2 affiliations: 1department of molecular medicine and haematology, university of the witwatersrand, johannesburg, south africa 2national health laboratory service, johannesburg, south africa 3division of medical oncology, department of internal medicine, university of the witwatersrand, johannesburg, south africa correspondence to: robyn marshall email: dr.rmarshall@gmail.com postal address: university of the witwatersrand medical school, room 3b22, 7 york road, johannesburg, south africa dates: received: 11 dec. 2014 accepted: 18 aug. 2015 published: 23 nov. 2015 how to cite this article: marshall r, vaughan j, david r, schapkaitz e, carmona s, wiggill t. primary plasma cell leukaemia in a 22-year-old woman: a case report. afr j lab med. 2015;4(1), art. #289, 5 pages. http://dx.doi.org/10.4102/ajlm.v4i1.289 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. primary plasma cell leukaemia in a 22-year-old woman: a case report in this case studies... open access • abstract • introduction • ethical considerations    • potential benefits and hazards    • recruitment procedures    • informed consent    • data protection • case presentation • management and outcome • discussion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ introduction: primary plasma cell leukaemia is a rare and highly aggressive disease that is commonly diagnosed a decade earlier than multiple myeloma, at a median age of 55 years. however, it has also been described in younger patients, as documented in this case report. it often presents with hepatosplenomegaly and lymphadenopathy, whilst the presence of bony lesions are less-commonly seen when compared to multiple myeloma. case presentation: this report describes the case of a young woman who presented with symptoms of anaemia and a history of menorrhagia. on further careful examination, she was found to have additional signs and symptoms and was later diagnosed with primary plasma cell leukaemia. management and outcome: on admission, the patient received supportive care measures, including blood products. at diagnosis, a specific chemotherapy regimen was commenced; however, this failed to induce remission. the decision to continue with supportive care only was made and the patient died seven months later. discussion: this case study is presented because of its rarity, the young age of the patient at presentation and the unusual clinical and laboratory findings. persistent anaemia unresponsive to standard treatment should raise the index of suspicion and further investigations directed to exclude malignancies should be considered. introduction top ↑ plasma cell leukaemia (pcl) is a rare and aggressive plasma cell dyscrasia,1 which is divided into primary and secondary subtypes. the distinction from multiple myeloma (mm) and other plasma cell dyscrasia is based on the presence of a circulating peripheral blood plasma cell count of > 20% or > 2 × 109/l.2,3 primary pcl presents de novo in the leukaemic phase and comprises 60% of all pcl.3,4 it has a median age of diagnosis of 55 years, a decade earlier than mm and secondary pcl, with the symptoms often mimicking those of acute leukaemia.5 prognostic parameters include a low serum albumin; hypercalcaemia and elevated β2 microglobulin, serum lactate dehydrogenase and serum c-reactive protein; an absolute peripheral blood plasma cell count of > 4 × 109/l; thrombocytopenia and an increased percentage of s-phase plasma cells; advanced age (age at diagnosis of 60 years or older) and poor performance status (eastern cooperative oncology group grade of ≥ 2),6,7 based on a patient's ability to perform the normal activities of daily living. specific cytogenetic results associated with lower overall survival rates include a complex karyotype, hypodiploidy, as well as several deletions and translocations.6 ethical considerations top ↑ consent was obtained from the patient with ethical clearance from the university of the witwatersrand human research ethics committee. the ethics clearance number is m130269. potential benefits and hazards there were no risks to the subject involved in this case report; and no potential physical or psychological dangers were anticipated. there was no perceived benefit to this patient. no information that could identify the patient has been published and the authors have endeavoured to maintain the patient's anonymity. it is hoped that other patients with a similar presentation of severe anaemia and a serious underlying condition may be clinically managed more quickly. recruitment procedures as this was a case study that highlighted a rare and interesting case, the patient was requested to provide consent to make use of her clinical information, including examination findings, special investigation results and treatment strategies applied. entitlement to withdraw consent would lapse once the case report was submitted for publication. informed consent the patient was requested to provide written consent to allow for all or any part of this material collected (other than unique patient identifiers) to appear as an abstract, a case study, or an article in a journal, and any other works or products, in any form or medium. data protection the patient's name has not been published with the material and the authors have endeavoured to assure anonymity. however, it is understood that despite the best efforts of the authors, the possibility that someone, for example, members of the patient's family or the healthcare staff, may recognise the patient from the images and/or the accompanying text. all data collected for this case report was available only to the authors of the case report and no other party had access to any of the patient's individual identifiers. case presentation top ↑ a 22-year-old female patient presented at a peripheral clinic in september 2012 with a history of heavy menses after receiving medroxyprogesterone acetate for contraceptive purposes in july 2012. at this time she complained of fatigue, dizziness, lower back pain and poor appetite; iron supplements were prescribed. in november 2012 she presented at a local hospital complaining of severe back pain and persistent vaginal bleeding. her past medical history revealed that this had been ongoing for more than one month. on admission she was found to have no significant lymphadenopathy or hepatosplenomegaly. she was also tachycardic and an echocardiogram revealed a functional ejection systolic murmur. a radiographic skeletal survey showed multiple lytic lesions (figure 1). all pertinent laboratory investigations are summarised in table 1. figure 1: radiographic skeletal survey showing multiple lytic lesions although no pathological fractures were present. (a) the lesions involved the ribs, the shoulders bilaterally and the spine. (b) the pelvis and the upper femora also reveal lytic lesions. a number of baseline and definitive special investigations were performed (table 1). a bone marrow investigation revealed hypercellular marrow with extensive infiltration by a population of abnormal plasma cells similar to that described in the peripheral blood (figure 2a, 2b and 2c). the infiltrate was positive for immunohistochemical stains, including cd38 (figure 2d), cd56 and mum-1, but was negative for cyclin-d1, cd20 and cd45. fewer than 20% of cells were positive for ki-67. immunophenotypic analysis revealed a population of ~60% – 70% large, more complex cells that expressed bright cd38, moderate cd138 and bright aberrant cd56 with dim kappa light-chain restriction. figure 2: bone marrow aspirate and trephine samples of primary plasma cell leukaemia. (a) tumour cells were variable in size, ranging from small to intermediate. the nuclei were rounded and eccentric and a peri-nuclear hoff was also noted (giemsa staining. magnification: 50×). (b) occasional, more primitive binucleate forms were also observed (giemsa staining. magnification: 50×). (c) the chromatin appeared clumped but had a more primitive appearance in the larger cells with occasional nucleoli (giemsa staining. magnification: 50×). (d) normal haemopoiesis was mostly displaced by the extensive diffuse infiltrate of cd38 positive cells (anti-cd38 staining. magnification: 40×). fluorescence in situ hybridisation analysis revealed that the cells were positive for a deletion at 13q14.3, showed trisomy of chromosome 18 and loss of the fibroblast growth factor receptor (fgfr3) gene. chromosomal analysis of the bone marrow cultures revealed a complex karyotype (table 1). features of note were numerical aberrations, including monosomy of chromosomes 12, 13 and 14 and trisomy of chromosome 18. structural aberrations included partial deletion of chromosome 1p and an extra chromosome 1 with a partial deletion on the p-arm, leading to an additional copy of the 1q region. there was a translocation involving chromosomes 3 and 14, leading to rearrangement of the immunoglobulin heavy chain locus (igh@) gene. terminal deletion of chromosome 4p confirmed the deletion of the fgfr3 gene. table 1: summary of relevant laboratory investigations performed on this patient. management and outcome top ↑ on admission, the patient was stabilised and given fresh frozen plasma, platelets and packed cells. with confirmation of the diagnosis, the patient was started on a chemotherapy regimen which included: vincristine 3.6 mg/m2 intravenously on days 1–4 (d1–d4), doxorubicin 9 mg/m2 intravenously on d1–d4, dexamethasone 40 mg orally on d1–d4, dexamethasone 40 mg orally on d9–d12, dexamethasone 40 mg orally on d17–d20. she received one cycle of therapy; however, this induction failed to induce remission. discussion top ↑ de novo pcl is a rare disease and presentation in patients aged younger than 40 years is exceptionally rare. to the best of our knowledge, only two case reports of such patients, in whom primary pcl presented at aged 30 and 21 years, have been reported in the literature.8,9 as is often reported, this patient presented with symptoms suggestive of an acute leukaemia. however, some of her presenting symptoms would more commonly be seen in secondary pcl and mm, including the presence of bone pain and lytic lesions. there was no evidence of extra-medullary deposits and there was an absence of hepatomegaly, splenomegaly and lymphadenopathy. there was no evidence of a pleural effusion and no renal dysfunction, which are commonly described in primary pcl.5,7,10,11 in addition to some of the unusual clinical features noted, atypical laboratory features were also found, including the presence of bright cd56 expression on flow cytometric assessment and the absence of cd20 on immunohistochemical staining.10 cytogenetic abnormalities are a common feature of pcl, with 70% of patients with primary pcl and 100% of patients with secondary pcl presenting with abnormal karyotypes.5 a recent study that provided a genomic characterisation of patients with primary pcl revealed a significant overlap with characteristics seen in mm, although tp53 deletions, complex karyotypes, hypodiploidy and igh@ translocations were more frequently present in pcl.4,12,13 these igh@ translocations were identified in 87% of primary pcl cases, del(13q) in 74% of cases and del(17p) in 35% of cases.4 in addition, abnormalities in chromosome 1 are frequent in pcl, particularly 1q21 amplification and del(1p), a deletion that has been associated with shorter overall survival.14,15 both monosomy 13 and trisomy 18 are common in pcl, with monosomy 13 occurring in up to 85% of cases and trisomy 18 in 43%.7,16 all of the aberrations described above were part of this patient's karyotype, except for the tp53 deletions. monosomy 12 and 14 have also been reported in primary pcl in the form of case reports.17 poor prognostic factors in primary pcl include both clinical and laboratory parameters.6,7 of these, the case study patient presented with a performance status score of ≥ 2, an absolute peripheral blood plasma cell count of > 4 × 109/l, thrombocytopenia, diffuse marrow infiltration, specific cytogenetic abnormalities, including a complex karyotype, elevated lactate dehydrogenase, elevated β2 microglobulin and hypercalcaemia. induction therapy failed to induce remission. survival is known to be poor in this category of plasma cell dyscrasia, with 28% of patients dying in the first month following diagnosis.10 the average survival for primary pcl is 11.2 months.5 in general, treatment is aimed at improving quality of life and prolonging survival. therapy initiation should begin promptly and aim for rapid disease control in an attempt to prevent early death.11 chemotherapy regimens have previously been based on those used for mm, with no specific standard protocol available. of these, intensive multidrug regimens with an alkylating agent as a base have been used with limited success and more recently bortezomib-based regimens are recommended, followed by autologous stem cell transplantation, if feasible.6,10 allogeneic transplantation can be considered in younger patients.6 our patient received one cycle of induction therapy with a modified vincristine, doxorubicin, dexamethasone regimen, as bortezomib therapy was not available. with the regimen provided, she never obtained complete remission and autologous transplant was not possible. she subsequently received palliative care and died in hospital seven months after the initial diagnosis. this case was presented because of its rarity and the young age of the patient at presentation, as well as its unusual laboratory and clinical features. this was an unexpected diagnosis and, not surprisingly, the diagnosis was not made at first presentation. a history of recent contraceptive use, menorrhagia and a resultant iron deficiency was not an uncommon finding. however, additional symptoms were also noted and the patient had no improvement, despite iron supplementation. a high index of suspicion, together with early referral and the use of basic first line investigations, such as differential count, should be advocated. the inaccessibility of the recommended drug therapy was also likely to have affected survival and diminished chances for possible stem-cell transplant. it is vital to make an early diagnosis of haematological and other malignancies in our setting where treatment options are already limited and delayed diagnosis may negatively impact prognosis. acknowledgements top ↑ karyotyping and fluorescence in situ hybridisation analyses were performed in the somatic cell genetics unit, department of molecular medicine and haematology, university of the witwatersrand, johannesburg, south africa. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions r.m. (university of the witwatersrand and national health laboratory service) was involved with the concept and design, data collection and writing of the article and takes on the overall responsibility for the article. j.v. and e.s. (university of the witwatersrand and national health laboratory service) were responsible for data collection and critical revision of the article. s.c. and t.w. (university of the witwatersrand and national health laboratory service) were responsible for critical revision and final approval of the article. r.d. (university of the witwatersrand) was responsible for data collection and critical revision of the article. references top ↑ dimopoulos ma, palumbo a, delasalle kb, alexanian r. primary plasma cell leukaemia. br j haematol. 1994;88(4):754–759. pmid: 7819100. kyle ra, maldonado je, bayrd ed. plasma cell leukemia. report on 17 cases. arch intern med. 1974;133(5):813–818. pmid: 4821776. international myeloma working group.criteria for the classification of monoclonal gammopathies, multiple myeloma and related disorders: a report of the international myeloma working group. br j haematol. 2003;121(5):749–757. pmid: 12780789. mosca l, musto p, todoerti k, et al. genome-wide analysis of primary plasma cell leukemia identifies recurrent imbalances associated with changes in transcriptional profiles. am j hematol. 2013;88(1):16–23. pmid: 23044976, http://dx.doi.org/10.1002/ajh.23339 tiedemann re, gonzalez-paz n, kyle ra, et al. genetic aberrations and survival in plasma cell leukemia. leukemia. 2008;22(5):1044–1052. pmid: 18216867, http://dx.doi.org/10.1038/leu.2008.4 van de donk nw, lokhorst hm, anderson kc, richardson pg. how i treat plasma cell leukemia. blood. 2012;120(12):2376–2389.pmid: 22837533, http://dx.doi.org/10.1182/blood-2012-05-408682 garcia-sanz r, orfão a, gonzález m, et al. primary plasma cell leukemia: clinical, immunophenotypic, dna ploidy, and cytogenetic characteristics. blood. 1999;93(3):1032–1037. pmid: 9920853. jain d, singh t, akhila l, ghosh n. primary plasma cell leukemia in a 30-year-old woman. indian j pathol microbiol. 2008;51(3):456–457. pmid: 18723997. raj rs, najeeb s, aruna r, pavithran k, thomas m. primary plasma cell leukemia occuring in the young. indian j cancer. 2003;40(3):116–117. pmid: 14716116. albarracin f, fonseca r. plasma cell leukemia. blood rev. 2011;25(3):107–112.pmid: 21295388, http://dx.doi.org/10.1016/j.blre.2011.01.005 fernández de larrea c, kyle ra, durie bg, et al. plasma cell leukemia: consensus statement on diagnostic requirements, response criteria and treatment recommendations by the international myeloma working group. leukemia. 2013;27(4):780–791. pmid: 23288300, http://dx.doi.org/10.1038/leu.2012.336 chiecchio l, dagrada gp, white he, et al. frequent upregulation of myc in plasma cell leukemia. genes chromosomes cancer. 2009;48(7):624–636. pmid: 19396865, http://dx.doi.org/10.1002/gcc.20670 lorsbach rb, hsi ed, dogan a, fend f. plasma cell myeloma and related neoplasms. am j clin pathol. 2011;136(2):168–182. pmid: 21757591, http://dx.doi.org/10.1309/ajcpenj68ffbriyb chang h, qi x, yeung j, reece d, xu w, patterson b. genetic aberrations including chromosome 1 abnormalities and clinical features of plasma cell leukemia. leuk res. 2009;33(2):259–262. pmid: 18676019, http://dx.doi.org/10.1016/j.leukres.2008.06.027 chang h, yeung j, xu w, ning y, patterson b. significant increase of cks1b amplification from monoclonal gammopathy of undetermined significance to multiple myeloma and plasma cell leukaemia as demonstrated by interphase fluorescence in situ hybridisation. br j haematol. 2006;134(6):613–615. pmid: 16889615, http://dx.doi.org/10.1111/j.1365-2141.2006.06237.x avet-loiseau h, daviet a, brigaudeau c, et al. cytogenetic, interphase, and multicolor fluorescence in situ hybridization analyses in primary plasma cell leukemia: a study of 40 patients at diagnosis, on behalf of the intergroupe francophone du myélome and the groupe français de cytogénétique hématologique. blood. 2001;97(3):822–825. pmid: 11157506, http://dx.doi.org/10.1182/blood.v97.3.822 taniwaki m, nishida k, takashima t, et al. nonrandom chromosomal rearrangements of 14q32.3 and 19p13.3 and preferential deletion of 1p in 21 patients with multiple myeloma and plasma cell leukemia. blood. 1994;84(7):2283–2290. pmid: 7919347. about the author(s) marli van heerden national health laboratory service, johannesburg, south africa faculty of health sciences, charlotte maxeke johannesburg academic hospital, university of the witwatersrand, johannesburg, south africa jaya a. george national health laboratory service, johannesburg, south africa faculty of health sciences, charlotte maxeke johannesburg academic hospital, university of the witwatersrand, johannesburg, south africa siyabonga khoza national health laboratory service, johannesburg, south africa faculty of health sciences, charlotte maxeke johannesburg academic hospital, university of the witwatersrand, johannesburg, south africa citation van heerden m, george ja, khoza s. corrigendum: the application of sigma metrics in the laboratory to assess quality control processes in south africa. afr j lab med. 2023;12(1), a1996. https://doi.org/10.4102/ajlm.v12i1.1996 note: doi of original article published: https://doi.org/10.4102/ajlm.v11i1.1344 correction corrigendum: the application of sigma metrics in the laboratory to assess quality control processes in south africa marli van heerden, jaya a. george, siyabonga khoza published: 15 may 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. in the published article, van heerden m, george ja, khoza s. the application of sigma metrics in the laboratory to assess quality control processes in south africa. afr j lab med. 2022;11(1):a1344. https://doi.org/10.4102/ajlm.v11i1.1344, there was an error. the incorrect sigma metric calculation was stated. the correction has now been made on page 2 in the methods section, under data collection and analysis, paragraph 4 and should read: the original paragraph: sigma metrics were calculated for each analyte on two levels as follows: the revised and updated paragraph: sigma metrics were calculated for each analyte on two levels as follows: the authors apologise for this error. the correction does not change the study’s findings of significance or overall interpretation of the study’s results or the scientific conclusions of the article in any way. abstract introduction methods results discussion acknowledgements references about the author(s) hyerim kim department of laboratory medicine, pusan national university hospital, busan, republic of korea jongmin kim department of laboratory medicine, pusan national university hospital, busan, republic of korea citation kim h, kim j. effect of delayed sample draw after blood collection for haemoglobin test in south korea. afr j lab med. 2023;12(1), a2008. https://doi.org/10.4102/ajlm.v12i1.2008 note: additional supporting information may be found in the online version of this article as online supplementary document 1 brief report effect of delayed sample draw after blood collection for haemoglobin test in south korea hyerim kim, jongmin kim received: 08 july 2022; accepted: 06 jan. 2023; published: 28 mar. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract between april and may 2022, 10 healthy adult non-patients were recruited from pusan national university hospital. venous blood drawn into a syringe was transferred into test tubes with a zero-to-45-minute delay. the transfer was done sequentially in two positions with the syringe and the needle adaptor end (1) heading downwards and (2) heading upwards. haemoglobin levels gradually increased over time in position 1 transfer while they gradually decreased in position 2. therefore, blood must be transferred quickly from a syringe to a tube for reliable test results. what this study adds: our findings confirm that delays between blood collection and transfer can affect haemoglobin levels. keywords: haemoglobin; blood specimen collection; erythrocyte count; specimen handling; phlebotomy. introduction blood collection is necessary since numerous tests are performed using blood specimens. inadequate blood sampling may lead to inaccurate results and mislead the clinician; therefore, collecting blood according to best practice guidelines is essential. current guidelines presented by the world health organization, clinical and laboratory standards institute, and european federation of clinical chemistry and laboratory medicine-latin america confederation of clinical biochemistry provides evidence-based recommendations for the stepwise process of adequate blood sampling.1,2,3 however, there is no detailed guidance on the time interval between collecting blood in the syringe and transferring blood to the test tube when using the needle and syringe system. since these guidelines recommend using vacuum extraction instead of the needle and syringe system, there seems to be no need for guidance on transferring blood to the test tube. nevertheless, the needle and syringe system is most commonly used in blood sampling,2 and delay in transferring blood to the test tube following sampling can occur, which may alter the results of blood tests, for instance haemoglobin levels. however, to the best of our knowledge, studies on the effect of delayed blood transfer to the test tube after phlebotomy on haemoglobin level results are lacking. we were asked to evaluate the effect of delayed blood transfer from the syringe to the test tube after receiving complaints of erroneous haemoglobin level changes in several patients (table 1). the physicians observed unanticipated changes in haemoglobin, such as a significant decline in haemoglobin in the absence of relevant events such as bleeding, surgery, or trauma. an incorrect result of haemoglobin level may mislead the clinician in monitoring the patient’s overall health and diagnosing haematologic diseases, including anaemia. the errors can be divided into pre-preanalytical, preanalytical, analytical, postanalytical, and post-postanalytical phases.4 up to 68% of errors are pre-preanalytical,4 including the well-known sample quality factors: haemolysis, cryoglobulin, lipaemia, and clotting issues.5,6 therefore, possible errors in all test phases were carefully examined. after excluding all potential causes of error, we found that all reported cases were sampled using the needle and syringe system and assumed that a delay between blood collection and blood transfer to the test tube may have resulted in a change in haemoglobin levels. this assumption is based on the hypothesis that the sedimentation of red blood cells occurred during the prolonged storage of sampled blood in syringes. red blood cell sedimentation is well known, as in the erythrocyte sedimentation rate (esr) test.7 however, red blood cell sedimentation in a phlebotomy setting and its effect on haemoglobin test results is understudied. thus, we conducted this study to evaluate the effect of delayed blood sample transfer on the haemoglobin level by analysing samples with different time delays under two conditions: drawing blood from the sedimented bottom portion or the non-sedimented upper portion of the syringe. table 1: reported cases of erroneous haemoglobin levels, south korea, april 2022 may 2022. methods ethical considerations an application for full ethical approval was made to the institutional review board of pusan national university hospital. the ethics approval number is 2204-004-113. all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards. written informed consent was obtained from all individual participants involved in the study. the collected personal data was saved on a password-protected computer that only the authors had access to. participant recruitment and data collection between april 2022 and may 2022, 10 healthy adult non-patients were recruited at pusan national university hospital, busan, south korea, using a recruiting poster. the volunteers were eligible for inclusion if they were aged between 20 and 60 years and weighed over 50 kg. each participant was asked a series of prepared questions to determine their eligibility and collect information about them, such as their gender, age, and medical history. volunteers were excluded if they were pregnant, had a history of a blood clotting disorder, or had inadequate venous blood collected. blood collection a trained laboratory technician collected blood from the veins in the antecubital fossa, once in each arm. first, blood was collected using a vacuum extraction system (multiple sample blood collection 21g needle and quick release needle holder, becton, dickinson and company, new jersey, united states). next, 3 ml of blood for the k2 ethylenediaminetetraacetic acid (edta) tube and 2.7 ml for the sodium citrate tube (becton, dickinson and company, new jersey, united states) were collected, which we defined as control samples. on the opposite arm, 3 ml of blood was collected using a needle and syringe system with a 24g winged butterfly needle (jms(k) medical supply, seoul, republic of korea) into ten 10 ml syringes (jung rim medical industrial, chungcheongbuk-do, republic of korea). the winged butterfly needle was used instead of the original needle connected to the syringe to minimise the number of venepunctures performed per participant. the syringes were placed horizontally on the ground without assistive devices. one ml of blood was transferred into two 3 ml k2 edta tubes at 0, 5, 10, 15, 20, 25, 30, 35, 40, and 45 min in two positions in the following order: (1) syringe vertically positioned with needle adaptor end heading downwards and (2) syringe vertically positioned with needle adaptor end heading upwards (figure 1). these edta tubes were defined as the test samples. figure 1: illustration of needle and syringe positioning during blood transfer, south korea, april 2022 – may 2022. (a) position 1, syringe vertically positioned with needle adaptor end heading downwards. (b) position 2, syringe vertically positioned with needle adaptor end heading upwards. outcome assessment the activated partial thromboplastin and prothrombin times were determined using sodium citrate samples in cs-5100 (sysmex corporation, kobe, japan), esr in edta tubes using test1 (alifax, padova, italy), and complete blood count in edta tubes using xn-9000 (sysmex corporation, kobe, japan). for test samples, complete blood count was examined, and the changes in haemoglobin levels were analysed to evaluate the effect of delayed blood transfer. statistical analysis the kruskal-wallis test was used to analyse haemoglobin levels over time. the kolmogorov-smirnov test and the shapiro-wilk test were adopted to test normality. all statistical analyses were performed by spss® 22 for windows (spss inc., chicago, illinois, united states). a p-value of less than 0.05 was considered statistically significant. the line graphs were generated using microsoft excel, and illustrations using microsoft powerpoint (microsoft corporation, redmond, washington, united states). results the baseline characteristics of 10 participants, three male and seven female (online supplementary table 1), show that none had comorbidities. the haemoglobin levels of the control samples were consistent with the 0 min sample of the test sample. in addition, the control samples’ prothrombin times, activated partial thromboplastin, and esr results were within the normal range except for those of two participants, which presented mildly elevated esr. among the control sample results, including esr, activated partial thromboplastin, prothrombin times, and international normalised ratio, none seemed to be correlated with the amount of haemoglobin change over time. the results of samples transferred while the needle adaptor end faced downwards had an increase in haemoglobin levels with time (figure 2, p < 0.001). the average haemoglobin level increased from 13.9 g/dl at 0 min to 20.2 g/dl at 45 min. compared to the 0 min sample, the changes appeared to be statistically significant at 35 min (p = 0.006), 40 min (p = 0.001), and 45 min (p < 0.001). figure 2: changes of haemoglobin level (g/dl) over delayed blood transfer in position, south korea, april 2022 – may 2022. (a) represents the 10 cases of position 1, and (b) shows the increase of average haemoglobin levels of 10 cases (p < 0.001, kruskal-wallis test). statistically significant changes between 0 min and 35 min (p = 0.006), 0 min and 40 min (p = 0.001), 0 min and 45 min (p < 0.001), 5 min and 35 min (p = 0.005), 5 min and 40 min (p = 0.001), 5 min and 45 min (p < 0.001), 10 min and 35 min (p = 0.011), 10 min and 40 min (p = 0.01), 10 min and 45 min (p < 0.001), 15 min and 35 min (p = 0.05), 15 min and 40 min (p = 0.008), and 15 min and 45 min (p = 0.002) were observed in the post hoc test. gradual decreases in haemoglobin levels were observed in samples transferred with the needle adaptor end facing up (figure 3, p < 0.001). the average haemoglobin level dropped from 14.2 g/dl at 0 min to 6.2 g/dl at 45 min. when compared with 0 min, the changes appeared to be statistically significant in 35 min (p = 0.033), 40 min (p = 0.006), and 45 min (p = 0.001). figure 3: change in haemoglobin level (g/dl) over delayed blood transfer in position 2, south korea, april 2022 – may 2022. (a) represents the 10 cases of position 2, and (b) shows the decrease of average haemoglobin levels of 10 cases (p < 0.001, kruskal-wallis test). statistically significant changes between 0 min and 35 min (p = 0.033), 0 min and 40 min (p = 0.006), 0 min and 45 min (p = 0.001), 5 min and 40 min (p = 0.013), 5 min and 45 min (p = 0.001), 10 min and 40 min (p = 0.02), 10 min and 45 min (p = 0.002), and 15 min and 45 min (p = 0.008) were observed in the post hoc test. discussion the findings of this study confirm that the interval between blood collection and blood transfer can affect haemoglobin levels. regardless of needle position, the delay between the blood draw and the blood transfer led to incorrect haemoglobin levels. when the syringe was positioned vertically with the needle adaptor end heading upwards when transferring, the haemoglobin levels dropped with time. on the other hand, the haemoglobin levels increased with time when transferring blood with the needle adaptor end heading downwards. we believe that the transfer delay may have induced sedimentation of the sample transferring the supernatant, that is, needle adaptor end heading upwards, resulting in low haemoglobin levels and transferring the lower part of sedimentation, that is, needle adaptor end heading downwards, results in increased haemoglobin levels. limitations there are several limitations. firstly, as all participants were between 31 and 41 years, the study samples may not represent the general population. the previously reported erroneous cases (table 1) were relatively older patients, aged from 38 to 81. secondly, blood transfer with the needle pointing downwards was performed after transferring blood with the needle pointing downwards; thus, the results of the former position may have been affected by the inversion. thirdly, we used a 24g butterfly needle instead of the original 21g needle connected to the 10 ml syringe, which is one of our institution’s general settings. therefore, the difference in needle gauges can be associated with erroneous factors, such as haemolysis.8,9 however, all 10 samples were obtained in the same position with the same needle. the needle change divulges the effects of delayed blood transfer. lastly, in this study, only 1 ml of blood was transferred into 3 ml edta tubes to minimise the amount of blood sampling per participant. however, a study reported that acceptable complete blood count values could be obtained with as little as 1 ml of blood in 4 ml k2 edta tubes.10 therefore, we believe that the method of this study using 1 ml of blood in 3.0 ml k2 edta tubes can be considered relevant. conclusion in conclusion, the findings of this study show that delayed blood transfer after venepuncture may lead to changes in haemoglobin levels; thus, blood should preferably be transferred directly into test tubes after collection. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions h.k. conceived of the presented idea and designed the experiments. j.k. performed the experiments, analysed data, and wrote the manuscript with support from h.k. sources of support this work was supported by a clinical research funding from pusan national university hospital in 2021. data availability the authors confirm that the data supporting the findings of this study are available within the article. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references institute cls. gp41: procedures for collection of diagnostic blood specimens by venipuncture; approved guideline. 7th ed. wayne, pa: clinical and laboratory standards institute; 2007. world health organization. who guidelines on drawing blood: best practices in phlebotomy. who; 2010 [cited 2022 june 13]. available from: http://whqlibdoc.who.int/publications/2010/9789241599221_eng.pdf simundic am, bölenius k, cadamuro j, et al. joint eflm-colabiocli recommendation for venous blood sampling. clin chem lab med. 2018;56(12):2015–2038. https://doi.org/10.1515/cclm-2018-0602 zandecki m, genevieve f, gerard j, godon a. spurious counts and spurious results on haematology analysers: a review. part ii: white blood cells, red blood cells, haemoglobin, red cell indices and reticulocytes. clin lab haematol. 2007;29(1):21–41. https://doi.org/10.1111/j.1365-2257.2006.00871.x de la salle b. preand postanalytical errors in haematology. int j lab hematol. 2019;41(s1):170–176. https://doi.org/10.1111/ijlh.13007 richard a, mcpherson mrp. henry’s clinical diagnosis and management by laboratory methods. 24th ed. st. louis, mo: elsevier; 2021. daniel b, yuan cs. effects of dietary supplements on coagulation and platelet function. thromb res. 2005;117(1):49–53. https://doi.org/10.1016/j.thromres.2005.04.017 lippi g, salvagno gl, montagnana m, brocco g, cesare guidi g. influence of the needle bore size used for collecting venous blood samples on routine clinical chemistry testing. clin chem lab med. 2006;44(8):1009–1014. https://doi.org/10.1515/cclm.2006.172 sharp mk, mohammad sf. scaling of hemolysis in needles and catheters. ann biomed eng. 1998;26(5):788–797. https://doi.org/10.1114/1.65 xu m, robbe va, jack rm, rutledge jc. under-filled blood collection tubes containing k2edta as anticoagulant are acceptable for automated complete blood counts, white blood cell differential, and reticulocyte count. int j lab hematol. 2010;32(5):491–497. https://doi.org/10.1111/j.1751-553x.2009.01211.x article information authors: juliana ndasi1 laura dimite2 victor mbome3 charles awasom4 elive ngale1 sidney akuro1 ewane leonard1 omotayo bolu2 terence asong2 patrick njukeng1 judith shang2 affiliations: 1global health systems solutions, cameroon 2us centers for disease control and prevention (cdc), cameroon 3buea regional hospital, south west region, cameroon 4bamenda regional hospital, north west region, cameroon correspondence to: juliana ndasi postal address: po box 732, limbe, cameroon dates: received: 22 aug. 2014 accepted: 15 sept. 2014 published: 03 nov. 2104 how to cite this article: ndasi j, dimite l, mbome v, et al. decentralised facility-based training as an alternative model for slmta implementation: the cameroon experience. afr j lab med. 2014;3(2), art. #231, 6 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.231 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. decentralised facility-based training as an alternative model for slmta implementation: the cameroon experience in this original research... open access • abstract • introduction • research methods and design    • selection of laboratories    • preparation    • slmta implementation and supplemental training    • slmta implementation and supplemental training • results • discussion    • limitations of the study    • conclusion • acknowledgements    • competing interests    • authors’ contributions    • cdc disclaimer • references abstract top ↑ background: the strengthening laboratory management toward accreditation (slmta) programme is designed to build institutional capacity to help strengthen the tiered laboratory system. most countries implement the slmta three-workshop series using a centralised model, whereby participants from several laboratories travel to one location to be trained together. objectives: we assessed the effectiveness and cost of conducting slmta training in a decentralised manner as compared to centralised training. methods: slmta was implemented in five pilot laboratories in cameroon between october 2010 and october 2012 by means of a series of workshops, laboratory improvement projects and on-site mentorship. the first workshop was conducted in the traditional centralised approach. the second and third workshops were decentralised, delivered on-site at each of the five enrolled laboratories. progress was monitored by repeated audits using the stepwise laboratory quality improvement process towards accreditation (slipta) checklist. results: audit scores for all laboratories improved steadily through the course of the programme. median improvement was 11 percentage points after the first (centralised) training and an additional 24 percentage points after the second (decentralised) training. the estimated per-laboratory cost of the two training models was approximately the same at us$21 000. however, in the decentralised model approximately five times as many staff members were trained, although it also required five times the amount of trainer time. conclusion: decentralised slmta training was effective in improving laboratory quality and should be considered as an alternative to centralised training. introduction top ↑ international organization for standardization (iso) 15189 accreditation is viewed worldwide as the gold-standard mark of competence for clinical laboratories. however, the process of achieving international accreditation is labour-intensive, complex and expensive, making it challenging even for the best-resourced laboratories.1 these difficulties are magnified in resource-limited settings, where laboratories struggle to maintain staff levels and competence, basic infrastructure, equipment and supplies.1,2,3,4,5 as a result, few laboratories in sub-saharan africa are accredited and no laboratory in cameroon has been accredited to international standards.6 large-scale public health programmes, such as the us president’s emergency plan for aids relief (pepfar), have highlighted gaps in laboratory services, emphasising the urgent need for quality improvement.7 the world health organization’s regional office for africa (who afro) has responded by launching the stepwise laboratory quality improvement process towards accreditation (slipta) scheme, which is a phased approach to quality improvement.8 training and mentoring in laboratory management have been identified as being critical for the implementation of quality management systems (qms).9 however, many training programmes fail to result in measurable changes in laboratory practices because they focus more on theory and generic management topics than on practical aspects that can lead to direct implementation. they also lack follow-up with trainees to assist with application of knowledge into practice.10 the strengthening laboratory management toward accreditation (slmta) programme is an innovative, taskand competency-based training programme that aims to address deficiencies in laboratory quality through a series of workshops, improvement projects and mentoring.11 the slmta three-workshop series is typically conducted in a central location that is logistically convenient for all laboratories in the training cohort. centralised training allows many laboratories to be trained simultaneously and provides opportunities for laboratory networking and inter-facility knowledge-sharing. however, a centralised model can be expensive because of venue hiring and participant travel. some have argued that decentralised training can be more sensitive to the needs of the trainees and tied to specific organisational or project goals, as trainers are able to respond rapidly to the needs of the audience and revise the training approach based on their feedback.12 in addition, decentralised training has been shown to improve relationships between local and central authorities and to increase institutional capacity.13,14 in an effort to improve laboratory quality, cameroon began slmta implementation in 2010, with a first cohort of five laboratories. the initial training workshop was conducted in a centralised location. for the remaining two workshops, the programme shifted to a decentralised model, with facility-based training. the objective of this study is to compare the results and cost of decentralised training versus centralised training for the establishment of a qms in five laboratories in cameroon. research methods and design top ↑ selection of laboratories in 2009, four public hospital laboratories were selected by the ministry of public health, cameroon to enrol in the slmta programme: buea regional hospital laboratory (burhl), bamenda regional hospital laboratory (brhl), laquintinie hospital laboratory douala (lhld) and the yaoundé central hospital laboratory (ychl). in august 2010, a private laboratory, laboratoire d’analyses médicales du centre (lamc), was added to the four selected public laboratories in order to improve the link between the public and private sectors, which is essential for building sustainable national laboratory systems in resource-limited countries3 (table 1). table 1: laboratories included in cameroon’s first cohort of the strengthening laboratory management toward accreditation programme. preparation global health systems solutions (ghss) – a local implementing partner – and the cameroon office of the us centers for disease control and prevention (cdc) led slmta implementation. in preparation, all technical staff members from ghss and the laboratory team of cdc-cameroon underwent several training courses: (1) good clinical laboratory practice, provided by the south african national accreditation system (sanas); (2) use of iso 15189 in internal audits and laboratory assessments toward accreditation, provided by sanas; (3) slmta training, provided by cdc-cameroon staff; and (4) laboratory mentorship, provided by a clinton health access initiative (chai) mentor. to build capacity at the five selected slmta laboratories and to ensure sustainability, two employees from each laboratory were appointed as on-site mentors and trained for five days by a chai mentor on qms, mentoring techniques, the 12 quality system essentials (qses), iso 15189 and conducting laboratory audits using the slipta checklist. slmta implementation and supplemental training in october 2010, five participants from each of the five selected laboratories travelled to a central location in mutengene, south west region, cameroon, for the first five-day slmta training workshop. the second and third trainings of five days each were held on-site in each laboratory, from february to march 2011 and june to july 2011, respectively. a catch-up training was provided to personnel who missed the initial centralised training. the number of personnel trained per site ranged from 12 to 24 persons, including laboratory managers and clinicians. in addition to slmta training, the following centralised supplemental training courses were conducted for two employees from each laboratory: laboratory biosafety and biosecurity; development of standard operating procedures; internal audit; and use of a basic laboratory information system. most of these supplemental trainings were done at the end of the three slmta training workshops. a mentorship model that embeds a mentor within the daily routine of a laboratory for an extended period with a defined engagement schedule was used in these laboratories. these embedded mentors provided on-site coaching and guided the laboratories toward international accreditation by ensuring the implementation of improvement projects. in addition to embedded mentors, visiting mentors conducted two site visits following each workshop. laboratory improvement projects are an integral part of the slmta programme, and were assigned to participants after each workshop. in subsequent workshops, participants presented their improvement projects and shared results and lessons learned. these sessions offered an opportunity for participants to learn from each other and facilitated the formation of a peer-learning network. evaluation the slipta checklist was used to evaluate the laboratories’ progress, strengths and weaknesses. this checklist contains 12 sections (a total of 111 items) for a total of 258 points.15 slipta checklist scores are categorised into star levels, with < 55% corresponding to zero stars, 55% – 64% one star, 65% – 74% two stars, 75% – 84% three stars, 85% – 94% four stars and 95% – 100% five stars. a ghss staff member trained by who afro as an auditor conducted baseline audits of the four public laboratories between november and december 2009, and the fifth, private laboratory in august 2010. who afro-trained in-country auditors conducted four intermediate audits, just before the second and third slmta training workshops and after the third slmta workshop, in order to evaluate progress, identify gaps and develop action plans to close existing gaps. costs in us dollars to implement slmta training workshops were estimated for the centralised and decentralised models. for centralised training, we assumed that five people per laboratory would attend the three workshops. for decentralised training, we assumed that 24 participants would attend each on-site workshop. for both models, we assumed four trainers would be needed and that each workshop would last five days. costs included lodging, per diem, land transport to the training venue, training materials for all participants, food and venue hiring (for centralised training only). we did not include salary or time missed from work for participants or trainers, nor other components of slmta implementation such as improvement projects, mentorship and audits, which would not be affected by training location. trainer days were estimated for each model based on one travel day and five training days per workshop. results top ↑ at baseline audit, the five laboratories scored a median of 23%, all at zero stars. median scores increased steadily to 34% at the first intermediate audit, 58% at the second, 66% at the third and 68% at the fourth; they remained at 68% for the exit audit. thus, there was a median total improvement of 45 percentage points. after 24 months of the slmta programme, two laboratories attained one star, two attained two stars and one attained three stars based on the audit scores. lamc had the largest improvement of 69 percentage points (figure 1). figure 1: performance of five cameroon laboratories over 24 months of slmta implementation as measured by the slipta checklist. the median improvement from the first slmta training to the second (intermediate audits 1 and 2) was 11 percentage points. after the first decentralised training, median scores improved an additional 24 percentage points. from the final training to exit, median scores improved 10 more percentage points. there was substantial variability in the timing of improvements. for example, lamc had their greatest improvement between the baseline and first intermediate audit, whilst ychl had their greatest improvement between the second intermediate audit and exit; lhld’s score decreased slightly from the second intermediate audit to exit (figure 1). all five laboratories improved their scores in each of the 12 qses. internal audit had the highest percentage average improvement (61 percentage points), followed by corrective action (55 percentage points) and documents and records (53 percentage points). improvements from the baseline audit for five of the qses were greatest after the first slmta training, whilst seven of the qses improved most after the second slmta training (figure 2). figure 2: average performance of the five laboratories measured at baseline, first and second intermediate, and exit audits. average marks are expressed as a percentage of the total for each of the 12 quality system essentials sections of the slipta checklist. the estimated cost of the workshop portion of slmta implementation for the two models is presented in table 2. with the centralised training model, it would cost approximately $105 610 to hold the three slmta workshops for 25 participants from five laboratories ($21 122 per laboratory). if the workshops were decentralised and conducted in-house in each laboratory, the total cost would be approximately $107 400 to train 120 participants from the five laboratories ($21 480 per laboratory). centralised training would require 72 trainer days; decentralised training would require 360 trainer days. table 2: estimated cost in us dollars (usd) of conducting centralised versus decentralised slmta training workshops. discussion top ↑ laboratory scores improved steadily throughout the two-year programme, with all laboratories reaching at least the one-star level. improvements after the decentralised second slmta workshop were twice as large as those after the centralised first workshop. the total estimated cost of the centralised and decentralised training models was about the same. however, in the decentralised model, approximately five times as many staff were trained as compared with the centralised model, whilst the centralised model required one-fifth the amount of trainer time as the decentralised model. decentralised workshops allowed more staff to participate in the training, facilitating shared understanding of the importance of quality improvement and the plan to achieve it. hospital managers and clinicians were able to participate in the training alongside laboratory managers, improving clinician–laboratory interactions and providing them an opportunity to understand the potential for long-term improvement. on-site training enabled the use of familiar facilities to conduct interactive activities; slmta concepts could easily be shared amongst laboratory staff and any site-specific non-compliance could be discussed during the workshops. finally, on-site workshops allowed the course to be tailored to the needs of the individual laboratories, with all workshop discussions related to site-specific challenges and solutions. on the other hand, centralised training fosters communication between laboratories, helping to build important networks. participants in centralised training can learn from the experiences of other laboratories and get feedback on what did and did not work for them. two critical aspects to consider when implementing slmta are cost and manpower. in this study, we found that centralised and decentralised training cost roughly the same amount, at approximately $21 000 per laboratory. savings made in the decentralised model for reduced costs for per diem, lodging, transport and venue hire were offset by the increased cost of trainers, training materials and food for the expanded group of participants. however, the two models have important consequences regarding manpower. experience from other countries implementing slmta has suggested that staff attrition, especially through reassignment to other laboratories within the ministry of health system or to employment in private laboratories, is one of the critical challenges facing sustainability of results after slmta completion.16 when only a few staff members from each laboratory are trained, their departure has a pronounced effect on institutional memory and new staff must receive intensive training in order to continue the qms work. in the decentralised model, the majority of laboratory staff are trained to implement qms, reducing the impact of attrition of a few trained staff members. on the other hand, decentralised training requires far more trainer time, as the full series of workshops is conducted at each laboratory. the shortage of qualified trainers throughout africa has been well noted.17 usually, countries use trainers who are borrowed from their normal laboratory duties for the slmta training weeks. but when those weeks increase geometrically from three per cohort to three per laboratory, the feasibility of borrowing trainers is questionable. globally, the median cohort size for slmta training has been 10 laboratories, with cohorts ranging from one to 27 laboratories.18 the logistical and manpower issues associated with decentralised training could quickly escalate in larger programmes, such that national laboratory programmes may need to consider adding staff dedicated to implementing slmta if decentralised training is desired. several challenges were faced in the implementation of the slmta programme in cameroon. the first challenge was that governmental bureaucracy caused delays in project implementation. this problem was exacerbated by the lack of a national laboratory strategic plan to define overall goals and stakeholders. this plan has now been developed and is pending adoption. another major challenge was the lack of personnel trained and skilled in quality laboratory practices in selected facilities and insufficient numbers of local slmta trainers. this challenge is being resolved by recent training of three more slmta trainers and a plan to conduct in-country slmta training-of-trainers in the near future. the concept of qms was entirely new to most laboratory staff in the selected facilities where a culture of quality has been lacking. as the staff undertook the training modules and understood the benefits of quality improvement, they became more cooperative and committed. finally, in a system without biomedical engineers, there were difficulties with equipment maintenance. most of the laboratory equipment is not available in the cameroonian markets; this, coupled with the high cost of import duties in cameroon, made equipment procurement and maintenance very costly. this challenge is being addressed with an on-going improvement project on equipment maintenance and calibration. limitations of the study whilst the greater median improvement in scores after decentralised training suggests that it may have been more effective than centralised training, these results should be viewed in light of study limitations. most importantly, this was an observational study of the natural progress of a programme; thus, there were no control laboratories on which to base a comparison. the difference in changes over time could be as a result of several factors, including timing of specific improvement projects undertaken after each workshop and variability in mentorship support. furthermore, the pattern was not consistent amongst all five laboratories in the cohort; whilst three laboratories improved more after the second training than after the first, lamc had its greatest improvement after the first and ychl after the third. the immediate improvement in lamc could possibly be because it is a private laboratory with few administrative bottlenecks that are common in larger public health facilities. additional operational studies randomising cohorts to centralised versus decentralised training would provide more solid evidence of the relative effectiveness of these strategies. conclusion quality laboratory systems are essential for providing patient care and global health. a competency-based programme such as slmta can assist public health laboratories in resource-limited settings to improve the quality of their services. the success of any programme depends on its sustainability. the lack of a national laboratory strategic plan, along with inadequate government funds and the absence of policies for equipment procurement and maintenance were major challenges faced by laboratories in cameroon and other resource-limited settings and may continue even in the post-accreditation period. training of facility-based mentors will help ensure continuous quality improvement, sustainability and country ownership. although the challenges were many, slmta implementation successfully improved laboratory quality, ensuring better laboratory services and patient care. whether to conduct slmta trainings using a centralised or decentralised model will depend on situation-specific factors; however, decentralised training should be considered to widen the reach of the training within the laboratories. cameroon intends to use decentralised training for future slmta cohorts. acknowledgements top ↑ this programme was financed with funds from pepfar through a cooperative agreement with cdc. it was implemented in collaboration with cdc’s office in cameroon, with technical support from cdc’s division of global hiv/aids laboratory team. we thank the minister of public health for the guidance and commitment to facilitating the implementation of these activities. we appreciate the collaborations of ministry staff, especially the regional delegates, directors of hospitals and all the laboratory staff that worked closely together in this programme. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions j.n. (global health systems solutions) was the project coordinator. l.d. (cdc, cameroon) was the activity manager for this project. v.m. (buea regional hospital) and c.a. (bamenda regional hospital) are hospital directors and were implementers of the project. e.n., s.a. and e.l. (all global health systems solutions) were laboratory mentors for this project. o.b. (cdc, cameroon) facilitated implementation of the programme by advocating for it to the ministry of health. t.a. (cdc, cameroon) was a laboratory coach and mentor. p.n (global health systems solutions) was principal investigator for this project. j.s. (cdc, cameroon) was the project officer. cdc disclaimer the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the cdc. references top ↑ 1.olmsted ss, moore m, melli rc, et al. strengthening laboratory systems in resource-limited settings. am j clin pathol 2010;134(3):374–380. http://dx.doi.org/10.1309/ajcpdqosb7qr5glr 2.nkengasong jn. a shifting paradigm in strengthening laboratory health systems for global health: acting now, acting collectively, but acting differently. am j clin pathol. 2010;134(3):359–360. http://dx.doi.org/10.1309/ajcpy5asuejyq5rk 3.nkengasong jn, nsubuga p, nwanyanwu n, et al. laboratory systems and services are critical in global health: time to end the neglect? am j clin pathol. 2010;134(3):368–373. http://dx.doi.org/10.1309/ajcpmpsinq9brmu6 4.justman je, stephania kd, amiliar t, et al. developing laboratory systems and infrastructure for hiv scale-up: a tool for health systems strengthening in resource-limited settings. j acquir immune defic syndr. 2009;52(suppl 1):s30–s33. http://dx.doi.org/10.1097/qai.0b013e3181bbc9f5 5.gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm 6.schroeder l, amukele t. medical laboratories in sub-saharan africa that meet international quality standards. am j clinpathol. 2014;141(6):791–795. http://dx.doi.org/10.1309/ajcpq5ktkagsscfn 7.the president’s emergency plan for aids relief. planning and reporting: next generation indicators reference guide. version1.1 [document on the internet]. c2009 [cited 2013 mar]. available from: http://www.pepfar.gov/documents/organization/81097.pdf 8.world health organization. kigali host the launch of a program to accelerate national laboratory service capacity building towards accreditation in the african region [document on the internet]. c2009 [cited 2014 oct 05]. available from: http://www.who.int/hiv/amds/diagnostics/amds_kigali_pr_lab.pdf 9.yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3):401–409. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj 10.mothabeng d, maruta t, lebina m, et al. strengthening laboratory management towards accreditation: the lesotho experience. afr j lab med. 2012;1(1), art. #9, 7 pages. 11.yao k, maruta t, luman et, nkengasong jn. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(1), art. #194, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i1.194 12.oakes k. grand central training, part two: industrial experts provide their thoughts on moving to centralized training [document on the internet]. c2005 [cited 2014 mar 14]. available from: http://www.astdcascadia.org/conference/2005/pdf_documents/oakes_article_2.pdf 13.lawrence sh. centralization and decentralization: the compunications connection [document on the internet]. c1983 [cited 2013 mar]. available from: http://www.pirp.harvard.edu/pubs_pdf/lawrenc/lawrenc-i83-2.pdf 14.fida office of evaluation. evaluación del programa en el país [country program evaluation]. report no. 2322-ar [document on the internet]. c2010 [cited 2014 oct 14]. available from: http://www.ifad.org/evaluation/public_html/eksyst/doc/country/pl/argentina/argentina_cpe.pdf 15.world health organization’s regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region (with checklist) [document on the internet]. c2012 [cited 2013 jun]. available from: http://www.afro.who.int/en/clusters-a-programmes/hss/blood-safety-laboratories -a-health-technology/blt-highlights/ 3859-who-guide-for-the-stepwise-laboratory-improvement-process-towards-accreditationin-the-african-region-with-checklist.html 16.luman et, yao k, nkengasong jn. a comprehensive review of the slmta literature part 1: content analysis and future priorities. afr j lab med. 2014;3(2), art. #265, 11 pages. http://dx.doi.org/10.4102/ajlm.v3i2.265 17.maruta t, yao k, ndlovu n, moyo s. training-of-trainers: a strategy to build country capacity for slmta expansion and sustainability. afr j lab med. 2014;3(2), art. #196, 7 pages. http://dx.doi.org/10.4102/ajlm.v3i2.196 18.yao k, luman et, slmta collaborating authors. evidence from 617 laboratories in 47 countries for slmta-driven improvement in quality management systems. afr j lab med. 2014;3(2), art. #262, 11 pages. http://dx.doi.org/10.4102/ajlm.v3i2.262 abstract introduction methods results discussion acknowledgements references about the author(s) jenipher g. mwakyabala department of microbiology and immunology, weill bugando school of medicine, catholic university of health and allied sciences, mwanza, united republic of tanzania conjester i. mtemisika molecular biology laboratory, central pathology laboratory, bugando medical centre, mwanza, united republic of tanzania stacy mshana department of microbiology and immunology, weill bugando school of medicine, catholic university of health and allied sciences, mwanza, united republic of tanzania adam a. mwakyoma department of clinical microbiology, kilimanjaro christian medical centre, moshi, united republic of tanzania vitus silago department of microbiology and immunology, weill bugando school of medicine, catholic university of health and allied sciences, mwanza, united republic of tanzania citation mwakyabala jg, mtemisika ci, mshana s, mwakyoma aa, silago v. characterisation of genes encoding for extended spectrum β-lactamase in gram-negative bacteria causing healthcare-associated infections in mwanza, tanzania. afr j lab med. 2023;12(1), a2107. https://doi.org/10.4102/ajlm.v12i1.2107 brief report characterisation of genes encoding for extended spectrum β-lactamase in gram-negative bacteria causing healthcare-associated infections in mwanza, tanzania jenipher g. mwakyabala, conjester i. mtemisika, stacy mshana, adam a. mwakyoma, vitus silago received: 30 oct. 2022; accepted: 27 jan. 2023; published: 12 apr. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract healthcare-associated infections (hcais) caused by extended spectrum β-lactamase-producing gram-negative bacteria (esbl-gnb) increase morbidity and mortality. this cross-sectional study characterised esbl genes (blactx-m, blatem and blashv) among 30 ceftriaxone-resistant gnb causing hcais between january 2022 and july 2022 by multiplex polymerase chain reaction assay at the zonal referral hospital in mwanza, tanzania. twenty-five (83.3%) had at least one esbl gene, of which 23/25 (92.0%) carried the blactx-m gene. seventy-two percent (18/25) of the gnb-esbl isolates carried more than one esbl gene, of which the majority (88.8%; n = 16/25) carried the blactx-m and blatem genes. extended spectrum β-lactamase genes, particularly blactx-m, are common among ceftriaxone-resistant gnb causing hcais. what this study adds: this study revealed the distribution of genes (blactx-m, blatem and blashv) coding for esbl production among ceftriaxone resistant gnb causing hcais however, all esbl producing gnb were susceptible towards ceftriaxone-sulbactam indicating that ceftriaxone-sulbactam may be empirically prescribed for treating patients with hcais. keywords: beta lactamases; extended spectrum beta-lactamase; gram-negative bacteria; healthcare-associated infections; multiplex pcr assay. introduction healthcare-associated infections (hcais), also referred to as nosocomial infections, are infections acquired by patients while receiving healthcare services from ≥ 48 h after admission to a healthcare facility.1,2,3 admission into intensive care units increases the risk of acquiring hcais due to (1) chronic diseases which lower body immunity; (2) surgical procedures which interfere with natural body defenses; and (3) medical invasive devices such as urinary catheters, central lines and intubators, which provide bacteria with direct entry into bodily tissues.4 escherichia coli, klebsiella aerogenes, enterobacter spp., acinetobacter baumannii and pseudomonas aeruginosa are the most common gram-negative bacteria (gnb) known to cause hcais.5,6,7,8 healthcare-associated infections are associated with significant increased cost of healthcare services, days of hospitalisation and mortality.9 healthcare-associated infections caused by multidrug-resistant bacteria phenotypes, such as extended spectrum β-lactamase-producing gnb (esbl-gnb), further exaggerate morbidity and mortality. at the study site in mwanza, tanzania, the prevalence of hcais in surgical site infections ranges from 10% to 26%.5,9,10 staphylococcus aureus accounts for nearly one-third of these, of which about 16% to 19% are methicillin resistant.5,9 on the other hand, only one study reported 13% of implicating gnb showed esbl phenotypes.9 to date, the distribution of esbl genes among esbl-gnb phenotypes causing hcais is not clearly known. this study unravels the distributions of esbl genes (blactx-m, blatem and blashv) among ceftriaxone-resistant gnb causing hcais at a zonal referral hospital in mwanza, tanzania. methods ethical considerations this study received ethical approval from the joint catholic university of health and allied sciences and bugando medical centre research ethics and review committee. the study approval number is crec: 2368/2022. all participants voluntarily provided written informed consent before being enrolled in the study. unique identification numbers were used to ensure confidentiality. laboratory results were communicated in a timely manner to attending doctors in order to guide rational therapy. study design, population, setting and duration this was a cross-sectional laboratory-based study of ceftriaxone-resistant gnb isolated from different hcais between january 2022 and july 2022 (unpublished data) at bugando medical centre – a zonal referral hospital located in mwanza, tanzania. the bacterial isolates, which had been archived in 20% glycerol stocks stored in a –40 °c freezer in the microbiology laboratory as part of a biorepository, were recovered for this study in july 2022. the duration of archive ranged from 1 to 6 months before recovery for molecular characterisation of esbl genes. clinical information related to each isolate, namely ward or clinic of origin, sample type, bacterial species name, and susceptibility towards third-generation cephalosporins, notably ceftriaxone, was extracted from the laboratory database. laboratory procedures were conducted in microbiology research laboratory and molecular biology research laboratory at the catholic university of health and allied sciences located at bugando medical centre in mwanza, tanzania. definition of healthcare-associated infection in the current study, hcai was defined as an infection that a patient develops after 48 h of hospital admission, while receiving healthcare for another disease or condition.11 laboratory procedure recovery of cro-r-gnb causing healthcare-associated infections and phenotypic detection of esbl production ceftriaxone-resistant gnb causing hcais were recovered by sub-culturing on plates of macconkey agar with salt (mca; himedia, mumbai, india). plates were incubated aerobically at 35 °c ± 2 °c for 20 h – 24 h followed by phenotypic detection of esbl production and dna extraction for multiplex polymerase chain reaction (pcr) assay. the disk combination method (dcm) from the clinical and laboratory standards institute12 was used for phenotypic detection of esbl production among recovered ceftriaxone-resistant gnb. dna extraction from 5 to 10 fresh colonies (≤ 24 h) of ceftriaxone-resistant gnb on plates of mca were used for dna extraction. a protocol for dna extraction from gnb by qiamp min dna extraction kit (qiagen, wuerzburg, germany) was used according to manufacturer’s instructions. dna samples were stored at −20 °c. multiplex pcr assay a multiplex pcr assay described by monstein et al.13 was used for amplification and detection of esbl genes (blactx-m, blashv, and blatem). briefly, 2 µl of each dna sample was added into a pcr reaction tube containing hotstartaq dna polymerase master mix (new england biolabs; hitchin, hertfordshire, united kingdom) and a set of primers (table 1), resulting in a final pcr reaction volume of 25 µl. the thermal cycler (t100™, bio-rad, kaki-bukit, singapore) was run with the following conditions: initial denaturation at 95 °c for 5 min; 30 cycles of denaturation at 94 °c for 30 s, annealing at 56 °c for 30 s, and extension at 72 °c for 1 min; and a final extension at 72 °c for 10 min. products were detected by using a 1% agarose gel with tris-acetate-edta buffer stained with safeviewtm dna stain (abm; richmond, british colombia, canada) and visualised under ultraviolet light. table 1: sequences of primers used for multiplex polymerase chain reaction assays for extended spectrum β-lactamase genes, bugando medical centre, mwanza, tanzania, january 2022 – july 2022. data management and analysis quantitative data were descriptively analysed by using microsoft excel (microsoft office; redmond, washington, united states) and stata version 15.0 (statacorp llp; college station, texas, united states; https://www.stata.com/stata15/). results a total of 30 ceftriaxone-resistant gnb causing hcais were recovered during this study period. most of the recovered bacteria were e. coli 43.3% (n = 13). the majority of ceftriaxone-resistant gnb were isolated from the burn unit (40%; n = 12), and from pus/pus swab samples (56.6%; n = 17). by dcm, all (100%; n = 30) ceftriaxone-resistant gnb had positive esbl phenotypes. multiplex pcr assay revealed that about 83.3% (n = 25) had at least one esbl gene, of which the majority (92.0%; n = 23) harboured the blactx-m gene. out of 25 gnb carrying esbl genes, 18 (72.0%) carried multiple genes; of these, 88.8% (n = 16) carried blactx-m and blatem genes (table 2 and figure 1). five isolates with negative pcr were e. coli (n = 3), isolated from pus/pus swab samples in the burn unit, and acinetobacter spp. (n = 2), one isolated from a urine sample in the medical ward and the other isolated from a pus/pus swab sample from the neonatal intensive care unit (table 3). figure 1: molecular characterisation of extended spectrum β-lactamase genes by multiplex polymerase chain reaction assay, bugando medical centre, mwanza, tanzania, january 2022 – july 2022. table 2: description of ceftriaxone-resistant gnb recovered for multiplex polymerase chain reaction amplification and detection of extended spectrum β-lactamase genes, bugando medical centre, mwanza, tanzania, january 2022 – july 2022. table 3: results of disk combination method and multiplex pcr assay, and distributions of esbl genes, bugando medical centre, mwanza, tanzania, january 2022 – july 2022. discussion the current study characterised the proportions and distributions of esbl genes (blactx-m, blatem, and blashv) among ceftriaxone-resistant gnb which were isolated from different hcais between january 2022 and july 2022 at a tertiary zonal referral hospital in mwanza, tanzania. the majority of ceftriaxone-resistant gnb were recovered from the burn unit, from patients with burn injuries who were prone to infections because of the breached skin barrier.14 moreover, e. coli accounted for the majority of recovered bacterial species, suggesting the patients’ own gut flora as an endogenous source of infection.3 however, e. coli can also be acquired from exogenous sources, such as contaminated inanimate surfaces, whenever hospital environmental cleaning and decontamination are poor.15 this study observed that all ceftriaxone-resistant gnb had positive esbl phenotypes by dcm, even though four out of five (83.3%) esbl phenotypes had at least one esbl gene on multiplex pcr assay. our findings are similar to a study by silago et al., conducted in mwanza, tanzania, in 2020, which reported a proportion of 93.3% esbl among gnb isolated from the hospital environment and hospitalised patients at the same setting.16 our findings are, however, higher than a study by said et al., which was conducted in 2021 in mwanza, tanzania, which reported that about 65.9% of gnb colonising children, of whom the majority were not hospitalised, harboured esbl genes at the same setting.17 therefore, the difference in study populations between the studies may explain the difference observed. similar to previous studies published in 2020 and 2021 in mwanza and in 2021 in morogoro, tanzania,16,17,18 the majority of ceftriaxone-resistant gnb were harbouring the blactx-m gene. the predominance of blactx-m may be a result of successful dissemination by conjugative epidemic plasmids, which facilitates its horizontal and vertical transmission.16,19,20,21 five confirmed esbl phenotypes by dcm did not harbour any of the three esbl genes (blactx-m, blatem, and blashv) by multiplex pcr assay. this observation is in line with previous studies conducted from the same setting, mwanza, tanzania, in 2020 and 2021.16,17 the isolates may be harbouring other esbl families which are non-cefotaximase-munich beta-lactamase (non-ctx-m), non-temoniera beta-lactamase (non-tem), and non-sulfhydryl reagent variable beta-lactamase (non-shv), such as oxacillinase beta-lactamase, pseudomonas extended resistant, vietnam extended-spectrum β-lactamase, tlahuica indianand guiana-extended spectrum families.22 limitations the small sample size of ceftriaxone-resistant gnb isolates obtained for this study is a weakness but did not affect the interpretation of the results. conclusion extended spectrum β-lactamase genes, to be specific blactx-m, are common among ceftriaxone-resistant gnb causing hcais. therefore, rational management of patients with hcais, guided by culture and sensitivity, is warranted. acknowledgements authors appreciate the support from microbiology research laboratory and molecular biology laboratory of the catholic university of health and allied sciences. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions v.s. and a.a.m. conceptualised the idea of the manuscript; j.g.m. and s.m. retrieved laboratory data, recovered bacteria isolates, and performed laboratory procedures; j.g.m. and c.i.m. interpreted and analysed data; c.i.m. wrote the first draft of the manuscript, which was critically reviewed by all co-authors who also approved the final manuscript. v.s. supervised protocols and every step of this research. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the data that support the findings made in this study can be made available from the corresponding author, c.i.m., on request. disclaimer the views expressed in this study are those of the authors and are not an official position of the affiliation institutes of the authors. references danasekaran r, mani g, annadurai k. prevention of healthcare-associated infections: protecting patients, saving lives. int j community med public health. 2014;1(1):67–68. peleg ay, hooper dc. hospital-acquired infections due to gram-negative bacteria. n engl j med. 2010;362(19):1804–1813. https://doi.org/10.1056/nejmra0904124 nagarjuna d, mittal g, dhanda rs, verma pk, gaind r, yadav m. faecal escherichia coli isolates show potential to cause endogenous infection in patients admitted to the icu in a tertiary care hospital. new microbes new infect. 2015;7:57–66. https://doi.org/10.1016/j.nmni.2015.05.006 world health organization. report on the burden of endemic health care-associated infection worldwide. world health organization: geneva, switzerland. 2011. mawalla b, mshana se, chalya pl, imirzalioglu c, mahalu w. predictors of surgical site infections among patients undergoing major surgery at bugando medical centre in northwestern tanzania. bmc surg. 2011;11(1):1–7. https://doi.org/10.1186/1471-2482-11-21 agarwal s. study of postoperative wound infection. indian j surg. 1972;34:314–320. nichols rl. surgical wound infection. am j med. 1991;91(3):s54–s64. https://doi.org/10.1016/0002-9343(91)90344-w kotisso b, aseffa a. surgical wound infection in a teaching hospital in ethiopia. east afr med j. 1998;75(7):402–405. mpogoro fj, mshana se, mirambo mm, kidenya br, gumodoka b, imirzalioglu c. incidence and predictors of surgical site infections following caesarean sections at bugando medical centre, mwanza, tanzania. antimicrob resist infect control. 2014;3(1):1–10. https://doi.org/10.1186/2047-2994-3-25 moremi n, claus h, vogel u, mshana se. surveillance of surgical site infections by pseudomonas aeruginosa and strain characterization in tanzanian hospitals does not provide proof for a role of hospital water plumbing systems in transmission. antimicrob resist infect control. 2017;6(1):1–8. https://doi.org/10.1186/s13756-017-0216-x haque m, sartelli m, mckimm j, abu bakar m. health care-associated infections – an overview. infect drug resist. 2018 nov 15;11:2321–2333. https://doi.org/10.2147/idr.s177247 clsi. performance standards for antimicrobial susceptibility testing. 32nd ed. clsi supplement m100. clinical and laboratory standards institute: berwyn, pennsylvania. 2022. monstein hj, ostholm-balkhed a, nilsson mv, nilsson m, dornbusch k, nilsson le. multiplex pcr amplification assay for the detection of blashv, blatem and blactx-m genes in enterobacteriaceae. apmis. 2007;115(12):1400–1408. https://doi.org/10.1111/j.1600-0463.2007.00722.x church d, elsayed s, reid o, winston b, lindsay r. burn wound infections. clin microbiol rev. 2006;19(2):403–434. https://doi.org/10.1128/cmr.19.2.403-434.2006 odoyo e, matano d, georges m, et al. ten thousand-fold higher than acceptable bacterial loads detected in kenyan hospital environments: targeted approaches to reduce contamination levels. int j environ res public health. 2021;18(13):6810. https://www.mdpi.com/1660-4601/18/13/6810 silago v, kovacs d, samson h, et al. existence of multiple esbl genes among phenotypically confirmed esbl producing klebsiella pneumoniae and escherichia coli concurrently isolated from clinical, colonization and contamination samples from neonatal units at bugando medical center, mwanza, tanzania. antibiotics. 2021;10(5):476. https://doi.org/10.3390/antibiotics10050476 said mm, msanga dr, mtemisika ci, et al. extended spectrum β-lactamase producing lactose fermenting bacteria colonizing children with human immunodeficiency virus, sickle cell disease and diabetes mellitus in mwanza city, tanzania: a cross-sectional study. trop med infect dis. 2022;7(8):144. https://doi.org/10.3390/tropicalmed7080144 moremi n, silago v, mselewa eg, et al. extended-spectrum β-lactamase blactx-m-1 group in gram-negative bacteria colonizing patients admitted at mazimbu hospital and morogoro regional hospital in morogoro, tanzania. bmc res notes. 2021;14(1):1–7. https://doi.org/10.1186/s13104-021-05495-x xia s, fan x, huang z, et al. dominance of ctx-m-type extended-spectrum β-lactamase (esbl)-producing escherichia coli isolated from patients with community-onset and hospital-onset infection in china. plos one. 2014;9(7):e100707. https://doi.org/10.1371/journal.pone.0100707 rossolini g, d’andrea m, mugnaioli c. the spread of ctx-m-type extended-spectrum β-lactamases. clin microbiol infect. 2008;14:33–41. https://doi.org/10.1111/j.1469-0691.2007.01867.x zhuo c, li xq, zong zy, zhong ns. epidemic plasmid carrying bla ctx-m-15 in klebsiella penumoniae in china. plos one. 2013;8(1):e52222. https://doi.org/10.1371/journal.pone.0052222 paterson dl, bonomo ra. extended-spectrum β-lactamases: a clinical update. clin microbiol rev. 2005;18(4):657–686. https://doi.org/10.1128/cmr.18.4.657-686.2005 article information authors: kelebeletse o. mokobela1 mpho t. moatshe1 mosetsanagape modukanele2 affiliations: 1ministry of health, botswana2us centers for disease control and prevention, botswana correspondence to: mosetsanagape modukanele postal address: po box 90, gaborone, botswana dates: received: 26 june 2014 accepted: 15 sept. 2014 published: 03 nov. 2014 how to cite this article: mokobela ko, moatshe mt, modukanele m. accelerating the spread of laboratory quality improvement efforts in botswana. afr j lab med. 2014;3(2), art. #207, 6 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.207 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. accelerating the spread of laboratory quality improvement efforts in botswana in this original research... open access • abstract • introduction • research methods and design    • the slmta programme    • additional training and mentorship in selected laboratories    • evaluation    • results • discussion    • conclusion • acknowledgements    • competing interests    • authors’ contributions    • cdc disclaimer • references abstract top ↑ background: in 2002, the ministry of health (moh) of botswana began its journey toward laboratory accreditation in an effort to enhance the quality of laboratory services. after a difficult start, the moh recognised the need for a more practical and sustainable method for change that could be implemented nationally; they therefore adopted the strengthening laboratory management toward accreditation (slmta) programme.objective: this study describes the process and lessons learned in implementing slmta and the role of supplemental training and mentoring so as to achieve botswana’s national laboratory quality improvement goal. methods: eight laboratories were enrolled into the slmta programme in 2010, which included a series of workshops and improvement projects conducted over nine months. four of these laboratories received supplementary training and focused mentorship from the botswana bureau of standards (bobs). laboratory performance was measured at baseline and exit using the world health organization regional office for africa’s stepwise laboratory quality improvement process towards accreditation (slipta) checklist. one laboratory did not receive an exit audit and was thus excluded from the analysis. results: an 18 percentage-point improvement was observed when comparing the median baseline score (53%) to the median exit score (71%) for the seven laboratories. laboratories that received additional training and mentorship from bobs improved 21 percentage points, whilst non-bobs-mentored laboratories improved eight percentage points. hospital management buy-in and strong laboratory staff camaraderie were found to be essential for the positive changes observed. conclusion: slmta facilitated improvements in laboratory quality management systems, yielding immediate and measurable results. this study suggests that pairing the slmta programme with additional training and mentorship activities may lead to further increases in laboratory performance; and that slmta is a practical approach to extending quality improvement to moh laboratories. introduction top ↑ laboratory quality management systems (qms) provide a strong foundation for promoting excellence in laboratory services that support fundamental components of effective healthcare systems.1in many resource-limited countries, laboratories lack robust quality systems, as they have historically been afforded low priority and few resources. this situation has led to poor-quality patient care and health outcomes, as well as loss of revenue resulting from inefficient and redundant processes. however, in recent years, an increased focus has been placed on the delivery of quality services as governments have moved toward initiating improvements in laboratory services.1,2in 2002, the botswana ministry of health’s (moh’s) laboratory services developed a five-year work plan with the goal of accrediting 16 laboratories, which resulted in the initial introduction of qms in select laboratories. six years later, limited improvements in the quality of laboratory management and service were noted because of high workload, inadequate staffing and poor infrastructure, amongst other factors. by 2011, after nine years of implementation and extensive partner and consultant support, four laboratories had attained international accreditation. whilst this accomplishment was commendable, it had been clear for some time that this approach was too costly (as consultants from outside botswana were employed) and too slow to be a sustainable option for long-term quality improvement on a national level; botswana needed a more viable strategy. in 2008, the moh developed a five-year (2009–2014) national laboratory strategic plan,3which called for implementation of qms in all laboratories by 2014 and accreditation of four district-level and two national-level laboratories by 2013 and 2014, respectively. the strategic plan directed country laboratory qms activities by incorporating a mentoring approach for laboratory accreditation. shortly after initiating this plan, the moh adopted the strengthening laboratory management toward accreditation (slmta) programme so as to catalyse the operation of the strategic plan and provide a platform to promote quality management of laboratories. in accordance with the strategic plan, key laboratories throughout the country were identified to participate in the slmta programme, which consists of a comprehensive management framework, training and mentoring toolkit, and a multi-workshop implementation model.4 whilst the slmta programme formed the cornerstone of botswana's laboratory improvement strategy, the moh theorised that combining slmta with additional qms training and targeted mentoring might achieve superior results.5 therefore, additional training and mentorship were offered to four top-priority laboratories designated as future centres of excellence. this article describes the process and lessons learned in implementing slmta and the role of supplemental training and mentoring so as to achieve botswana's national qms and accreditation goals. research methods and design top ↑ the slmta programme the moh enrolled eight national, regional, district and primary level laboratories throughout botswana in the slmta programme, beginning in august 2010. profiles of each laboratory (a to h) are listed in table 1. implementation followed the standard slmta process with a series of three workshops delivered over a period of nine months.5a total of 24 laboratory staff, including laboratory managers, quality officers and section heads, participated in the training. table 1: profiles of laboratories enrolled in the botswana slmta programme, 2010. each workshop was followed by a period of three months to allow participants to implement improvement projects. laboratory staff members were allowed to choose improvement projects that were relevant to their local environment and priorities. each laboratory was encouraged to involve all laboratory staff members in improvement project implementation. after each workshop, two follow-up visits were conducted by moh/slmta trainers in order to provide further training and coaching on improvement projects. the trainers spent one day in each laboratory. the visits included meetings with hospital management so as to create awareness and solicit support for the laboratory improvement process. additional training and mentorship in selected laboratories four of the eight laboratories (e, f, g and h) had been recently relocated to new facilities designated as centres of excellence in medical specialties. these facilities received additional training by the botswana bureau of standards (bobs), a certified international organization for standardization (iso) training organisation. training focused on understanding the auditing and documentation requirements for iso 15189. bobs also provided extra mentoring to these four laboratories from april to june 2011; monthly visits lasted one week in each laboratory. bobs provided mentorship on system documentation, covering the development of quality manuals, standard operating procedures (sops) and other quality documents as required by the iso standard. bobs mentors, together with the laboratory staff, conducted a gap analysis and developed a work plan with deliverables for the mentee laboratories. once a task from the work plan was completed (e.g., writing a quality manual), the laboratory would share it with the mentor who would then make corrections. the mentor provided guidance on the document’s layout, as well as on interpretation of different clauses of the iso standard. after finalising the documents, the mentor assisted with implementation. the laboratory and the mentor established a relationship that allowed both parties to communicate via email or telephone, as needed, between visits. evaluation slmta in-country trainers conducted audits in order to measure laboratory improvements using the world health organization regional office for africa’s stepwise laboratory quality improvement process towards accreditation (slipta) checklist.6the slipta checklist is organised around the 12 quality system essentials (qses).7 for each qse, a score is obtained by calculating the points that a laboratory has received from each item on the checklist. an overall score is used to rate laboratories on a zeroto five-star rating scale, with a score of < 55% as zero stars, 55% – 64% as one star, 65% – 74% as two stars, 75% – 84% as three stars, 85% – 94% as four stars and ≥ 95% as five stars. slipta was used for both the baseline (july 2010) and exit (november 2011) audits.from july 2012 to february 2013, bobs mentors conducted trial assessments in their laboratories using the south african national accreditation system’s (sanas) checklist in order to determine the laboratories’ readiness for accreditation by sanas, a key regional accreditation body for southern africa.9this checklist yields qualitative results rather than a numerical score and highlights areas of focus for accreditation preparation. one of the eight laboratories (laboratory h) did not receive an exit audit as a result of a schedule conflict and therefore is not included in the slipta data analysis in this article; it did, however, participate in the trial assessment using the sanas checklist. results at baseline, four of the seven laboratories had a zero-star rating, two had one star and one had two stars. at exit, two laboratories remained at zero stars, four laboratories were rated at two stars and one laboratory was rated at three stars. the median slipta audit scores were 53% at baseline and 71% at exit, a median increase of 18 percentage points. the three bobs-mentored laboratories improved by 21 percentage points from a median score of 53% at baseline to 74% at exit, whilst the non-bobs-mentored laboratories increased eight percentage points from a median score of 49% at baseline to 57% at exit. the greatest improvements were in two of the bobs-mentored laboratories (e and g), which gained 27 percentage points each. among non-bobs-mentored laboratories, one (d) had a slight decrease in score, whilst the others each increased by three to 15 percentage points (figure 1). figure 1: slipta scores and star levels at the baseline and exit audits, botswana slmta programme 2010–2011. comparing the median performance scores at baseline with those at exit for each qse across the seven laboratories, the greatest improvement was in occurrence management and process improvement (40 percentage points), management reviews (25 percentage points), client management and customer service (25 percentage points), documents and records (20 percentage points) and internal audit (20 percentage points). corrective action, however, had a 13 percentage-point drop from baseline to exit (figure 2). figure 2: mean qse scores at the baseline and exit audits, botswana slmta programme 2010-2011 (n = 7). figure 3a shows the performance of the three bobs-mentored laboratories (e, f and g) in the 12 qse sections of the slitpa checklist. for these laboratories, the greatest gains were in the areas of occurrence management and process improvement (50 percentage points), internal audit (40 percentage points), client management and customer service (38 percentage points) and documents and records (36 percentage points). for the non-bobs-mentored laboratories (a, b, c and d), documents and records (26 percentage points) and client management and customer service (25 percentage points) had the greatest improvement (figure 3b). none of the qse scores dropped for bobs-mentored laboratories; for non-bobs laboratories, however, scores dropped in three areas: corrective action (25 percentage points); information management (4 percentage points); and process control and internal and external quality assessment (3 percentage points). figure 3: mean qse scores of (a) bobs-mentored laboratories (n = 3) and (b) non-bobs-mentored laboratories (n = 4) at the baseline and exit audits, botswana slmta programme 2010-2011. in the trial assessment using the sanas checklist after the exit audit in the bobs-mentored laboratories (e, f and g), the laboratories had made additional improvements in many areas (table 2). for example, quality manuals were now complete, internal audits were executed and safety procedures were in place in most of the audited laboratories. some notable deviations included: incomplete finalisation of critical documents; out-of-date and incomplete equipment services and calibrations; and incomplete external quality assessment (eqa) programmes. following this audit, laboratory (e) and laboratory (h) were encouraged to apply for international accreditation. table 2: qualitative results from trial assessment using the sanas checklist in bobs-mentored laboratories enrolled in the botswana slmta programme 2010-2011. discussion top ↑ the introduction of the slmta programme in hospitals in botswana was found to be a practical option that yielded positive results for strengthening laboratories. all but one laboratory demonstrated improvements and, as a whole, laboratories improved in all qses except corrective action.supplemental mentorship and training may have contributed to the success amongst bobs-mentored laboratories, which showed greater median improvements in the slipta audit results. however, it is important to note that the small number of laboratories and lack of random assignment to bobs mentorship limits the ability to draw definitive conclusions regarding this comparison. other studies have also noted that supplementing slmta with additional training and mentoring may be beneficial.9,10,11our findings, when joined with these others, suggest that combining slmta with other strategies in a qms programme may lead to synergistic improvement. one of the keys to success of the roll-out of slmta in the laboratories was strong staff commitment and involvement. during the training sessions, staff involvement was cultivated by the formation of teams that brainstormed improvement projects and outlined specific implementation tasks. this practice fostered a culture of problem solving and boosted confidence amongst laboratory staff, who felt empowered to implement improvement projects previously considered beyond their capability. these projects were developed by the trainees and responsibility was shared across the laboratory team. laboratories showed improvements in areas in which they had previously struggled, such as internal audit and management reviews. some laboratories also improved their inventory control systems and anecdotally reported decreased turnaround times. support and buy-in from hospital management for slmta was another critical component of the quality improvement process. as understanding and ownership of the process increased amongst hospital management, managers became more willing to assist laboratories in improvement projects that required additional funds or involvement from multiple hospital departments. for example, some projects required infrastructure modifications; hospital management provided resources for sink relocations, trunks to hold electrical cords and facility painting. one hospital hired extra temporary staff to assist with phlebotomy duties whilst laboratory staff concentrated on quality improvement activities. as a result, laboratory staff morale and commitment improved. three laboratories had minimal improvements or decreased scores. laboratory a, the national health laboratory, serves multiple functions: as a national reference laboratory, a procurement agency and a training agency. balancing the requirements of these diverse functions was a unique challenge that will require resolution before tangible results can be achieved. laboratory f had initially shown improvements, but had difficulties in getting documents reviewed and authorised by laboratory management prior to the exit audit. in laboratory d, both of the slmta-trained staff members transferred to other facilities during the implementation of the programme, which impacted negatively on performance. limited progress was observed across all the laboratories in the areas of equipment; facilities and safety; and organisation and personnel, indicating that further work is needed. these areas may require greater funding and management involvement at higher levels, which the moh is attempting to address. for example, throughout the country medical equipment maintenance and calibration programmes are weak and national eqa programmes and laboratory information systems do not meet the needs of laboratories. as the five-year national laboratory strategic plan comes to an end, the moh is in the process of launching a follow-up plan for 2015 to 2019. this plan seeks to address remaining challenges and to identify ways of maintaining and increasing the improvement that was achieved in all participating laboratories. one new approach will be to use the many documents (e.g., sops and safety manuals) developed during the slmta programme as the standard national documents for distribution to all moh laboratories. in addition, to ensure sustainability and continuous quality improvement amidst reduced donor funding, an additional 18 local mentors have been trained to help accelerate the spread of laboratory quality improvement efforts. conclusion the effort for widespread improvement of laboratory services in botswana has gained momentum in the past few years following the introduction of the slmta programme. the programme was well received by staff for its practicality and measurable impact, as improvement was demonstrated in most of the enrolled laboratories. whilst positive gains have been achieved, progress still needs to be made in struggling areas to minimise system disruptions in the laboratory and improve the national service programmes that support them. this study adds to the growing body of evidence that a combined strategy of slmta plus targeted training and mentorship can lead to further effectiveness of a qms programme and higher-quality laboratory service delivery. acknowledgements top ↑ we thank the moh for continued support in the laboratory endeavour to attain accreditation. we also acknowledge the us centers for disease control and prevention (cdc) for providing funding for the programme through the us president’s emergency plan for aids relief (pepfar). thanks also go to the bobs staff and management as implementing partners for this programme. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions k.o.m. (moh) provided input to the manuscript. m.t.m. (moh) drafted the manuscript. m.m. (cdc, botswana) provided input to the manuscript and is the corresponding author. cdc disclaimer the findings and conclusions in this report are those of the authors and do not necessarily reflect the official position of the cdc. references top ↑ 1. petti c, polage cr, quinn tc, et al. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377382.2. gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm 3. botswana ministry of health. health sector laboratory strategic plan (hslsp): 2009–2014 (unpublished); n.d. 4. yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol.2010;134(3):401–409. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj 5. maruta t, motebang d, mathabo l, et al. impact of mentorship on who-afro strengthening laboratory quality improvement process towards accreditation (slipta). afr j lab med 2012;1(1), art. #6, 8 pages. 6. world health organization regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region (with checklist) [document on the internet]. c2011 [cited 2014 may 14]. available from: http://www.afro.who.int/en/clusters-a-programmes/hss/blood-safety-laboratories-a-health-technology/blt-highlights/3859-who-guide-for-the-stepwise-laboratory-improvement-process-towards-accreditation-in-the-african-region-with-checklist.html 7. clinical and laboratory standards institute. application of a quality management system model for laboratory services; approved guidelines – third edition. clsi document gp26-a3 [isbn 1-56238-553-4]. wayne, pa: clinical and laboratory standards institute; 2004. 8. south african national accreditation system [homepage on the internet]. n.d. [cited 2013 jun 12]. available from: http://www.home.sanas.co.za/ 9. audu ra, onubogu cc, nwokoye nn, et al. improving quality in national reference laboratories: the role of slmta and mentorship. afr j lab med. 2014;3(2), art. #200, 7 pages. http://dx.doi.org/10.4102/ajlm.v3i2.200 10. nzabahimana i, sebasirimu s, gatabazi jb, et al. innovative strategies for a successful slmta country programme: the rwanda story. afr j lab med. 2014;3(2), art. #217, 6 pages. http://dx.doi.org/10.4102/ajlm.v3i2.217 11. makokha ep, mwalili s, basiye fl, et al. using standard and institutional mentorship models to implement slmta in kenya. afr j lab med. 2014;3(2), art. #220, 8 pages. http://dx.doi.org/10.4102/ajlm.v3i2.220 abstract introduction description of laboratory development building blocks building on the foundation discussion acknowledgements references about the author(s) kirtika patel department of immunology, moi university, eldoret, kenya r. matthew strother oncology department, university of otago, christchurch, new zealand francis ndiangui department of pathology, moi teaching and referral hospital, eldoret, kenya david chumba department of pathology, moi university college of health sciences, eldoret, kenya william jacobson department of pathology, indiana university school of medicine, indianapolis, indiana, united states cecelia dodson histology laboratory, indiana university health, indianapolis, indiana, united states murray b. resnic department of pathology, brown university, providence, rhode island, united states randall w. strate department of pathology, indiana university school of medicine, indianapolis, indiana, united states james w. smith department of pathology, indiana university school of medicine, indianapolis, indiana, united states citation patel k, strother rm, ndiangui f, et al. development of immunohistochemistry services for cancer care in western kenya: implications for lowand middle-income countries. afr j lab med. 2016;5(1), a187. http://dx.doi.org/10.4102/ajlm.v5i1.187 lessons from the field development of immunohistochemistry services for cancer care in western kenya: implications for lowand middle-income countries kirtika patel, r. matthew strother, francis ndiangui, david chumba, william jacobson, cecelia dodson, murray b. resnic, randall w. strate, james w. smith received: 07 may 2014; accepted: 03 dec. 2015; published: 04 may 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: cancer is becoming a major cause of mortality in lowand middle-income countries. unlike infectious disease, malignancy and other chronic conditions require significant supportive infrastructure for diagnostics, staging and treatment. in addition to morphologic diagnosis, diagnostic pathways in oncology frequently require immunohistochemistry (ihc) for confirmation. we present the experience of a tertiary-care hospital serving rural western kenya, which developed and validated an ihc laboratory in support of a growing cancer care service. objectives, methods and outcomes: over the past decade, in an academic north-south collaboration, cancer services were developed for the catchment area of moi teaching and referral hospital in western kenya. a major hurdle to treatment of cancer in a resource-limited setting has been the lack of adequate diagnostic services. building upon the foundations of a histology laboratory, strategic investment and training were used to develop ihc services. key elements of success in this endeavour included: translation of resource-rich practices to a resource-limited setting, such as using manual, small-batch ihc instead of disposableand maintenance-intensive automated machinery, engagement of outside expertise to develop reagent-efficient protocols and supporting all levels of staff to meet the requirements of an external quality assurance programme. conclusion: development of lowand middle-income country models of services, such as the ihc laboratory presented in this paper, is critical for the infrastructure in resource-limited settings to address the growing cancer burden. we provide a low-cost model that effectively develops these necessary services in a challenging laboratory environment. introduction cancer is a leading cause of mortality in lowand middle-income countries (lmics), already accounting for more deaths than tuberculosis, hiv and/or aids and malaria combined.1,2 according to the international agency for research on cancer there were more than 600 000 new cancer cases and more than half a million cancer deaths in africa in 2008, with a projected doubling by 2030.3 the need to develop an infrastructure for cancer research and care in lmics has been recognised by various researchers.4,5,6 a core component of cancer infrastructure is adequate pathology services. the breast health global initiative provides excellent resource-stratified guidelines for the development of pathology services.7 however, development of pathology services in lmics faces a number of challenges, including insufficient funds, lack of skilled personnel at all levels (technicians to pathologists), unavailable equipment and unreliable supply chains for consumables.8,9,10 despite these challenges, there have been a number of approaches to solving the deficits in lmic pathology services. three primary themes characterise these efforts: the development of systems to export specimens, either preor post-processing, for reading and diagnosis by out-of-country pathologists; services offered in-country through volunteer pathologists from other countries, and the use of telepathology.11 however, there are shortfalls with each of these approaches. specimen export faces challenges with acquisition of export permits, postage costs and risk of specimen loss during transit. in addition, long turnaround times hamper appropriate patient care.12 volunteer pathology services have difficulty identifying sufficient pathologists who have time available for travel and service provision, loss of income for the pathologist and the cost of airfare, visas, and room and board. once arrived, these experts often face a learning curve as they work with older and frequently ill-maintained equipment.12 telepathology, although promising, is presently slowed by inadequate bandwidth and the cost of required equipment.12 each of these approaches provides key elements to the successful development of pathology services for cancer care in resource-limited settings. these approaches depend on the development of sufficient local infrastructure and expertise. in the absence of development of local expertise, inadequate sample preservation and preparation can render a well-resourced pathology laboratory inadequate for diagnosis. in this paper, we describe our effort to combine all three of these approaches, along with investment in local resources and extensive local training, to develop pathology services in a resource-limited setting for immunohistochemistry (ihc), which is essential for reliable cancer diagnosis, prognosis and therapeutic decision-making.13 this process required extensive collaboration, investments in training and the local infrastructure, as well as coordination with telepathology resources and volunteer pathologists. description of laboratory development the academic model providing access to healthcare programme the academic model providing access to healthcare (ampath) programme links the resources of the moi teaching and referral hospital (mtrh), kenya’s second referral hospital, with the expertise and creativity of the moi university school of medicine (musom) and a consortium of north american academic medical centres (indianna university, brown university, duke university, lehigh valley hospital allentown, pennsylvania, university of notre dame, providence portland medical center, purdue university, university of massachusetts, medical school, university of utah, school of medicine, university of toronto, faculty of medicine and university of california, san francisco). the collective goal is to find sustainable solutions to many aspects of health and disease in western kenya.14,15 ampath-oncology evolved to deliver appropriate care to cancer patients presenting to mtrh and found that a major impediment to a successful cancer care programme was that pathology was limited only to morphologic diagnosis. building blocks according to world bank data, in 2014, the population of kenya was 44.86 million people (http://data.worldbank.org/country/kenya#; accessed: 21 april 2016.). at present, there are only two tertiary-care facilities in kenya, one serving nairobi and eastern kenya, and mtrh, which serves the rift valley and western kenya. mtrh serves a catchment area that includes half of kenya’s population – approximately 20 million people. to start building an ihc programme, a first step was an assessment of the existing resources at mtrh, musom and ampath. there were four general pathologists and six laboratory technicians serving in the department of pathology who provided diagnostic surgical pathology services to this population. pathologists have a masters in medicine (mmed) qualification and are employed by mtrh and musom, whereas technicians are registered members of the kenya technicians and technologist board and hold a certificate, diploma or degree. the surgical pathology laboratory at mtrh performs grossing services and processes tissue with a semi-automated tissue processor. final diagnosis is based on morphologic assessment with haematoxylin and eosin staining. clinical personnel at mtrh frequently have co-appointments at musom. the university also has non-clinical faculty involved in extensive laboratory and research endeavours. relevant to ihc services, the department of immunology had 10 staff members, was responsible for the training of medical, dental, nursing, physical therapy and environmental health students, and had 100 m2 of laboratory space for training. within the department of immunology, one faculty member with extensive prior experience with ihc was identified, having trained and worked in the united kingdom, and was a certified assessor for the united kingdom national external quality assurance scheme. ampath, which had been operational since 2001, had also put some infrastructure in place. prior investment in mtrh pathology included a tissue processor, tissue embedding station, convection oven, microtome, tissue section flotation water bath and microscopes. a critical contribution was maintaining service contracts and reagent agreements with a local company representative (dako, nairobi, kenya), which services ihc equipment and stocks reagents. this allowed for functional equipment and easy access to reagents, a frequent issue in resource-limited settings. in addition, ampath had extensive connections to pathologists through its large number of research projects and linked university programmes in north america. several pathologists had travelled to kenya to establish digital pathology training resources for musom. the combined resources available for development of ihc are presented in table 1. table 1: building blocks: existing resources within musom, mtrh, and ampath, western kenya, before 2009. building on the foundation the development of the ihc service required several key components to be coordinated, purchased or developed: the physical housing of an ihc laboratory, the coordination of equipment, the organisation of the service, training of personnel and ongoing quality assessments. physical housing: discussions between all partners (mtrh, musom and ampath) identified space in the existing histopathology laboratory for ihc services. renovations were required for temperature control (to ensure reagent stability and process reproducibility), air flow (to ensure safety for technicians using solvents for slide preparation) and back-up power supplies (for both the air conditioning unit and the reagent storage refrigerators). coordination of equipment: the equipment of the ihc laboratory itself was procured through repurposing of local equipment, donations and direct purchases through grants and donations. table 2 outlines the equipment and consumables required for the ihc laboratory, along with the sources of these items. table 2: strategic investments to develop immunohistochemistry laboratory services, western kenya, 2009–2012. organisation of service: administrative structures had to be established between partners to facilitate personnel management, establish processes for requesting ihc and reporting results, and perform administrative tasks such as ordering reagents. a laboratory director was assigned from the musom department of immunology and charged with overseeing daily operations and reporting to the head pathologist within mtrh’s histopathology department. management of the laboratory’s finances (both expenditures and billing) was routed through an office for research and sponsored projects maintained by all three partners. training of personnel: four histopathologists, four technicians and two doctoral students in immunology were trained both through short group workshops conducted by the local ihc expert and representatives from dako, the company providing ihc reagents (nairobi, kenya), and through one-on-one instruction taught by the consortium of north american universities. personnel were selected for training from amongst available staff at mtrh who were willing to take on the extra ihc training duties. skills developed included manual staining and use of the autostainer. pathologists were also trained on the importance of ihc in diagnosis and prognosis, and sessions with surgeons emphasised the importance of quick fixation for good ihc results. protocols were developed to ensure that specimen transport from the operating theatre to the histopathology laboratory occurred in a timely manner. quality assessment: following initial competency assessments by north american experts, protocols were developed to ensure ongoing quality and reproducibility. methods used to maintain high quality included: written standard operating procedures, inclusion of positive and negative controls in all staining batches and use of electronic image transfer for consultation with north american pathologists. in addition, during routine site visits by the north american pathologists, a selection of ihc slides were assessed and stained slides were sent to reference laboratories at indiana university and the university of california, san francisco. further, the ihc laboratory was enrolled in the united kingdom national external quality assessment scheme, an external quality assessment scheme, for validation and quality assurance/quality control of staining and reporting. the current immunohistochemistry programme: operations and outcomes operations establishing a functioning ihc service started at the level of tissue acquisition. standard operating procedures were designed to ensure that specimens are immediately placed in formalin, that the time of collection is recorded and that the attending surgeons make radial cuts across larger specimens to ensure adequate penetration of formalin. schedules were developed for specimen transport to the laboratory and rapid grossing, followed by processing and embedding into paraffin within 24 hours of resection. a major quality improvement effort was required to ensure 3–5-µm sectioning and ultimately required gross analysis is performed by a pathologist rather than a technologist. the lead pathologist must request specific ihc stains following haematoxylin and eosin staining and reporting. for the ihc laboratory, one pathologist is assigned for all aspects of ihc analysis, including reporting and dispatch of results. antigen retrieval for ihc is performed with a pt link machine (pre treatment link, dako north america, inc., carpentaria, california, united states). antigen retrieval is achieved by using high-ph tris-edta (ph 8.0) (dako k8004) and a low-ph citrate buffer (ph 6.0) (dako k8005). for example, high-ph antigen retrieval is used for oestrogen and progesterone receptors, whereas a low-ph antigen retrieval buffer is used for her2. the dako envision flex (dako, k8000 denmark) kit is used for immunostaining. endogenous peroxide is blocked by dako hydrogen peroxide (dako sm801 k8002). ready-to-use primary antibodies are used and staining is performed as per the manufacturer’s (dako ir/is series) instructions. envision flex mouse linker (k 8021) is used as a secondary antibody. slides are than labelled with envision flex linked with horseradish peroxidase (hrp) (dako sm802) and the staining is visualised by using the substrate dab chromogen mix (dako sm 803). slides are counterstained with envision flex heamatoxyline (dako k8008), dehydrated and cover slipped, and examined under the microscope. two tris buffer washes of 5 min each are performed between all steps. known positive and negative controls are also stained for all runs for quality assurance. other immunostains used to report other tumours are shown in table 3. the allred scoring system is used to score the slides for the hormones.16 the other immunostains are reported as percentage positive or negative in the tumours. most stains are performed manually, although used infrequently at this time, a donated autostainer (dako autostainer plus, dako north america, inc., carpentaria, california) is maintained for large-batch staining (48 slides). table 3: current productivity of the mu/mtrh/ampath immunohistochemistry laboratory, western kenya, 2009–2012. once the ihc staining is complete, all staff pathologists have an opportunity to review the slides for interpretation. three pathologists from indiana university visited the laboratory for a two-week period twice a year and one pathologist from brown university visited the laboratory once during the inception period. these visits provided continuous training. as per protocol, ihc slides are read together with the pathologist, ihc laboratory director and technologists. all ihc results are recorded in a logbook for technical purposes and in a file dedicated to ihc, with the matched control slide results for future reference. all slides are catalogued, dated and stored for easy reference. paraffin blocks are also catalogued and stored, according to standard laboratory practices. owing to constrained resources, scanning slides for electronic storage and subsequent distribution was not practical. consequently, a photomicroscope was put in place to photograph slides in the mtrh ihc laboratory. we are adapting this approach to capture images using a mobile phone, likely the best approach in a resource-poor laboratory. outcomes from 2006 to 2012, there was increased demand for basic histopathology – 1200 cases in 2006 rising to 6500 cases by 2012 – largely related to the clinical oncology programme. along with this increased service demand, immunostaining provided an invaluable adjunct to diagnosis of cancer. to date, the ihc laboratory performed 406 immunostains. before 2009, the turnaround time for results was at least three weeks, whereas after 2012 the turnaround time decreased to one week. patients were treated as per the results obtained. the types of other immunostains performed and the method for external validation are shown in table 3. box 1: lessons learned. discussion recent studies in sub-saharan africa have suggested improving pathology services to be key in cancer control, treatment and research.17,18 building local capacity for ihc in lmics has several tangible benefits: rapid turnaround time, improved pre-treatment diagnostic accuracy and capability for more rapid treatment initiation. further, it avoids the risk of specimen loss associated with the use of remote laboratory facilities. currently, the mtrh ihc laboratory is the only laboratory of its kind in a public institution in kenya. however, similar efforts are ongoing in malawi, which has already proven to be a robust platform for providing cancer care to its patients and for research.19 currently our laboratory performs oestrogen, progesterone and her2 staining for breast cancer treatment following guidelines,20,21 differentiation of lymphomas using a basic panel of antibodies,22,23 research-based diagnostics for kaposi’s sarcoma (ks) using latency-associated nuclear antigen (lana-1) and wilm’s tumour. this work helps to facilitate treatment of patients, avoid treatment of clinically ambiguous presentations of disease as cancer24 and more efficiently utilise limited treatment resources. however, in developing ihc capacity several issues were highlighted in pathology in kenya. it quickly became clear that reliable ihc results were dependent upon good morphology, which, in turn, relied upon proper handling and fixation of tissues.25 identifying simple issues, such as replacing non-standardised fixatives with neutral buffered formalin and standardising fixation, avoided issues of masked antigen, which were found to be important to allow interpretation of stains.19 in addition, investment in skills development of technologists was required to allow thin (3−5 μm), reproducible sections of paraffin-embedded tissues to be cut for ihc and with regard to the use and repair of the microtome. the ihc staining technique is a new technique and can be performed manually or with the use of an autostainer and the pt link machines for antigen retrieval at our institution. several technologists had to be trained on the principles of the ihc technique, methodology and the use of equipment for ihc. retention of highly qualified personnel after training is an ongoing concern, owing both to personnel leaving for better employment and to internal procedures whereby technologists are rotated regularly across clinical laboratories within mtrh, a mechanism designed to ensure equity amongst technologists. this approach, common in resource-constrained settings, required specific negotiation with hospital administration and allowed ongoing skills development. pathologists and technical staff should be trained locally, where the resources and equipment are relevant. training should be continuous and immunostaining should be performed at least once a week for maintaining knowledge, skills and technical ability. training of local pathologists and technologists by experts from other institutions (locally-based and internationally-based) should be encouraged. under ideal circumstances, there should be ongoing support from experts for troubleshooting, quality assurance and external review of slide evaluation and reporting. this process required extensive collaboration, investments in training and the local infrastructure and coordination with telepathology resources and volunteer pathologists. however, a notable lesson learnt is that not all resource-rich practices can translate to the low-resource environment. ihc performed manually was less costly and equally effective compared with the use of an autostainer. autostaining used considerable amounts of reagents and solvents and frequent mechanical failures were experienced owing to power fluctuations. similarly, we found that a microwave or a pressure cooker instead of expensive antigen retrieval equipment was adequate for good ihc staining in western kenya. given the high cost of servicing, we recommend manual batch processing of tissues for staining as the standard methodology in a resource-poor setting. small amounts of manual staining can be performed in slide storing boxes instead of having to use expensive humidifying chambers. diaminobenzidine (dab), which is a potential carcinogen, is used as a chromogen during ihc staining. gloves and a face mask have to be worn when handling dab. the dab waste at our institution was diluted with water to a concentration of 0.9 mg/ml before being discarded in the hazardous waste container. the ihc laboratory is air-conditioned so there is a constant exchange of air and the temperature is maintained at 20 °c. all the stages that involved solvents such as alcohol and xylene (i.e. dewaxing, dehydrating and cover slipping) are performed in the histology laboratory, which is equipped with a fume hood.26 consumables and reagents (e.g. alcohol, xylene, disposable plastic pipettes, pipette tips, cover slips and charged slides) remain a challenge to acquire and, relative to developed settings, are quite expensive. further, there can be substantial delays in receiving purchased quality reagents. we are continuing to negotiate local purchase versus importing supplies from our international partner institutions, where competitive pricing, quality assurance and reliable delivery may outweigh the drawbacks of international importation. despite our obvious cost constraints, we have selected more expensive ready-to-use polymer-based reagents for the following reasons: procedural simplification as a result of bound secondary antibody and a detection system which minimises errors, no reliance on avidin and biotin for conjugation and the potential for high background readings, and longer shelf stability.20 our experience has been that the additional cost of buying ready-to-use reagents, which are more expensive ($200 united states dollars [usd] for 200 primary antibody tests) than the undiluted reagents ($200 usd for 300 tests), is outweighed by the benefits of obtaining consistent results, with fewer chances of repetition, in a lmic setting. conclusion researchers working in sub-saharan africa have stressed the urgent need for improvement of cancer therapy by improving pathological diagnostic capabilities as part of capacity building efforts in low-resource settings.27 the global task force on expanded access to cancer care and control in developing countries has recommended improving cancer diagnosis and pathology expertise.28 local capacity in pathology and laboratory services therefore has to be improved. opportunities for collaborative models involving regional and global partners should be employed as pathology services develop in lmics.29 ultimately, this multi-modal approach resulted in an efficient lmic pathology service performing basic immunostains with the resources and expertise to facilitate expansion. our experience illustrates that it is possible to establish extended pathology services efficiently with the help of partnership and collaboration in kenya, a lmic. development of lmic models of services such as this is critical to resource-constrained settings seeking to address the growing burden in cancer prevention, care and research. acknowledgements competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. sources of support this work was supported in part by the national council of science and technology ncst/5/003/call2/09 and pfizer grant. an ihc autostainer and a pt link machine for antigen retrieval were donated by indiana university. authors’ contributions k.p. carried out the work described and conceived and wrote the manuscript. r.m.s. had oversight of ampath-oncology, helped coordinate the international expertise and resources, and contributed to conception and writing of the manuscript, f.n. was the pathologist in charge of breast cancers and d.c. was the pathologist in charge of other tumours. w.j. was a visiting pathologist and c.d. helped in setting up the ihc laboratory and provided technical support. as visiting pathologists, m.b.r. provided advice for ihc diagnosis and r.w.s. provided technical support and advice in diagnoses. j.w.s. helped with coordination and acquiring instrumentation and reagents for the ihc laboratory. references jemal a, bray f, center mm, ferlay j, ward e, forman d. global cancer statistics. ca cancer j clin. 2011;61(2):69–90. http://dx.doi.org/10.3322/caac.20107 kanavos p. the rising burden of cancer in the developing world. ann oncol. 2006;17(suppl 8):viii15–viii23. ferlay j, shin h-r, bray f, forman d, mathers c, parkin dm. estimates of worldwide burden of cancer in 2008: globocan 2008. int j cancer. 2010;127(12):2893–2917. http://dx.doi.org/10.1002/ijc.25516 lingwood rj, boyle p, milburn a, ngoma t, arbuthnott j, mccaffrey r, et al. the challenge of cancer control in africa. nat rev cancer. 2008;8(5):398–403. http://dx.doi.org/10.1038/nrc2372 kerr dj. introduction. ann oncol. 2006;17(suppl 8):viii5–viii5. cantreat international. scaling up cancer diagnosis and treatment in developing countries: what can we learn from the hiv/aids epidemic? ann oncol. 2010;21(4):680–682. http://dx.doi.org/10.1093/annonc/mdq055 shyyan r, masood s, badwe ra, errico km, liberman l, ozmen v, et al. breast cancer in limited-resource countries: diagnosis and pathology. breast j. 2006;12(s1):s27–s37. http://dx.doi.org/10.1111/j.1075-122x.2006.00201.x adeyi oa. pathology services in developing countries—the west african experience. arch pathol lab med. 2011;135(2):183–186. benediktsson h, whitelaw j, roy i. pathology services in developing countries: a challenge. arch pathol lab med. 2007;131(11):1636–1639. stalsberg h, awuah b, ibarra ja, nsiah-asare a. re-establishing a surgical pathology service in kumasi, ghana. cancer. 2008;113(s8):2338–2346. http://dx.doi.org/10.1002/cncr.23830 garrett l. the challenge of global health. foreign affairs. 2007;86(1):14–38. hoenecke h, lee v, roy i. pathologists overseas: coordinating volunteer pathology services for 19 years. arch pathol lab med online. 2011;135(2):173–178. shah aa, frierson hf, cathro hp. analysis of immunohistochemical stain usage in different pathology practice settings. am j clin pathol. 2012;138(6):831–836. http://dx.doi.org/10.1309/ajcpagvtckdxkk0x einterz rm, kelley cr, mamlin jj, van reken de. partnerships in international health. the indiana university-moi university experience. infect dis clin north am. 1995;9(2):453–455. einterz rm, kimaiyo s, mengech hnk, khwa-otsyula bo, esamai f, quigley f, et al. responding to the hiv pandemic: the power of an academic medical partnership. acad med j assoc am med coll. 2007;82(8):812–818. http://dx.doi.org/10.1097/acm.0b013e3180cc29f1 mohsin sk, weiss h, havighurst t, clark gm, berardo m, roanh ld, et al. progesterone receptor by immunohistochemistry and clinical outcome in breast cancer: a validation study. mod pathol. 2004;17(12):1545–1554. http://dx.doi.org/10.1038/modpathol.3800229 mpunga t, tapela n, hedt-gauthier bl, milner d, nshimiyimana i, muvugabigwi g, et al. diagnosis of cancer in rural rwanda: early outcomes of a phased approach to implement anatomic pathology services in resource-limited settings. am j clin pathol. 2014;142(4):541–545. http://dx.doi.org/10.1309/ajcpypdes6z8eley african pathologists’ summit working groups. proceedings of the african pathologists summit; march 22-23, 2013; dakar, senegal: a summary. arch pathol lab med. 2015;139(1):126–132. http://dx.doi.org/10.5858/arpa.2013-0732-cc gopal s, krysiak r, liomba ng, horner m-j, shores cg, alide n, et al. early experience after developing a pathology laboratory in malawi, with emphasis on cancer diagnoses. plos one. 2013;8(8):e70361. http://dx.doi.org/10.1371/journal.pone.0070361 wolff ac, hammond meh, schwartz jn, hagerty kl, allred dc, cote rj, et al. american society of clinical oncology/college of american pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. arch pathol lab med. 2007;131(1):18–43. hammond meh, hayes df, dowsett m, allred dc, hagerty kl, badve s, et al. american society of clinical oncology/college of american pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer. j clin oncol. 2010;28(16):2784–2795. http://dx.doi.org/10.1200/jco.2009.25.6529 leoncini l, raphael m, stein h. burkitt lymphoma. in: who classification of tumours of haematopoietic and lymphoid tissues. 4th ed. lyon: international agency for research on cancer; 2008; p. 262–264. naresh kn, ibrahim hah, lazzi s, rince p, onorati m, ambrosio mr, et al. diagnosis of burkitt lymphoma using an algorithmic approach – applicable in both resource-poor and resource-rich countries. br j haematol. 2011;154(6):770–776. http://dx.doi.org/10.1111/j.1365-2141.2011.08771.x cheuk w, wong koy, wong csc, dinkel je, ben-dor d, chan jkc. immunostaining for human herpesvirus 8 latent nuclear antigen-1 helps distinguish kaposi sarcoma from its mimickers. am j clin pathol. 2004;121(3):335–342. http://dx.doi.org/10.1309/b8tc0lbvh8xy5mfv paavilainen l, edvinsson a, asplund a, hober s, kampf c, pontén f, et al. the impact of tissue fixatives on morphology and antibody-based protein profiling in tissues and cells. j histochem cytochem. 2010;58(3):237–246. http://dx.doi.org/10.1369/jhc.2009.954321 ramos-vara ja. technical aspects of immunohistochemistry. vet pathol. 2005;42(4):405–426. http://dx.doi.org/10.1354/vp.42-4-405 soliman as, schairer c. considerations in setting up and conducting epidemiologic studies of cancer in middleand low-income countries: the experience of a case-control study of inflammatory breast cancer in north africa in the past 10 years. cancer med. 2012;1(3):338–349. http://dx.doi.org/10.1002/cam4.36 knaul fm, frenk j, shulman l for the global taskforce on expanded access to cancer care and control in developing countries. closing the cancer divide: a blueprint to expand access in low and middle income countries [report on the internet]. boston ma: harvard global equity initiative; c2011 [cited 2015 oct 24]. available from: http://gtfccc.harvard.edu/fs/docs/icb.topic1063570.files/ccd_report_111027.pdf. roberts dj, wilson ml, nelson am, adesina am, fleming ka, milner d, et al. the good news about cancer in developing countries – pathology answers the call. lancet. 2012;379(9817):712. http://dx.doi.org/10.1016/s0140-6736(12)60306-7 reviewer acknowledgement open accesshttp://www.ajlmonline.org page 1 of 1 we would like to take this opportunity to thank all of those who provided scientific and logistical support for this issue of the african journal of laboratory medicine: should names have inadvertently been excluded from this list the publisher apologises and undertakes to amend the exclusion in the next issue. ajlm african journal of laboratory medicine in an effort to facilitate the selection of appropriate peer reviewers for the african journal of laboratory medicine, we ask that you take a moment to update your electronic portfolio on 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linda de gouveia stephania k. deme alpha diallo dennis ellenberger ifeoma enweani nwadiuto e. esiobu jessie n. githanga esther de gourville jean m. heraud john kagira edward kamau phyllis kanki reba kanungo samoel khamadi segundo r. leon henry s. limula john f. nahabedian jack nyamongo bryan nyary beldinah r. ochola brenda okech eric opiyo wellington oyibo julius oyugi milijaona radrianarivelojosia zilma rey abdoulaye sarr ritu shrivastava andrew thaiyah john thuita musau wakabongo larry westerman article information authors: elise schapkaitz1 dashini pillay2 affiliations: 1department of molecular medicine and haematology, charlotte maxeke johannesburg academic hospital, national health laboratory system complex and university of witwatersrand, south africa 2department of haematology, national health laboratory services and university of kwazulunatal, south africa correspondence to: elise schapkaitz email: elise.schapkaitz@nhls.ac.za postal address: po box 28985, sandringham 2131, south africa dates: received: 27 june 2014 accepted: 30 june 2015 published: 31 aug. 2015 how to cite this article: schapkaitz e., pillay d. prolonged storage-induced changes in haematology parameters referred for testing. afr j lab med. 2015;4(1), art. #208, 8 pages. http://dx.doi.org/10.4102/ajlm.v4i1.208 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. prolonged storage-induced changes in haematology parameters referred for testing in this original research... open access • abstract • introduction • research method and design    • ethical considerations    • materials and setting    • design       • blood sampling and laboratory methods       • laboratory testing       • analyses • results    • storage at room temperature    • storage at 4 °c – 8 °c • discussion • limitations of the study • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: referral of samples for the work-up of haematological disorders from remote laboratories can result in a delay in analysis. objective: the stability of the full blood count (fbc), differential count (diff), reticulocyte and peripheral blood smear (pbs) morphology during extended storage was evaluated. methods: forty blood samples stored in ethylenediaminetetraacetic acid (edta) were analysed on an advia® 120 haematology analyser. the samples (25% abnormal; 75% normal) were stored at room temperature (rt) and at 4 °c – 8 °c. analysis of samples stored at rt was performed every 12 hours for two days. analysis of samples stored at 4 °c – 8 °c was performed at 12 hours and subsequently every 24 hours for seven days. results: fbc parameters (red cell count, haemoglobin) and diff parameters (percentages of basophils, lymphocytes and monocytes) were stable for at least 48 hours when stored at rt. platelets were only stable for 12 hours and the white cell count was stable for 36 hours when stored at rt. storing samples at 4 °c – 8 °c significantly increased the stability of most parameters, in particular, mean cell volume and percentage of reticulocytes. however, diff parameters were associated with lower stability at 4 °c – 8 °c. pbs morphology was compromised prior to 12 hours whether stored at rt or at 4 °c – 8 °c. conclusion: this study provides evidence that blood samples stored in edta at 4 °c – 8 °c for seven days are suitable for testing on the advia® 120 analyser for the fbc and percentage of reticulocyte parameters. however, storage at 4 °c – 8 °c is not a solution for samples referred for diff and pbs morphology review. introduction top ↑ pre-analytical variables, such as storage time and temperature, affect the measurement of laboratory parameters collected in ethylenediaminetetraacetic acid (edta).1,2 laboratory staff need to be aware of the changes that occur during storage in their specific setting in order to decide whether to accept or reject samples that are too old to obtain reliable results. accurate measurement of full blood count (fbc), differential count (diff) and reticulocyte parameters, as well as peripheral blood smear (pbs) morphology, are essential for the correct interpretation of haematology results. it is recommended that traditional fbc parameters such as red cell count (rcc), white cell count (wcc), haemoglobin and platelet count be analysed 24 hours after sample collection when stored at room temperature (rt).3,4,5 however, parameters useful for diagnosis and monitoring of haematological disorders, such as mean cell volume (mcv), reticulocyte and pbs morphology, are unreliable after 12 hours.5 osmotic swelling of red cells during storage at rt affects volume-dependant variables and results in misclassification of a microcytic anaemia as normocytic and, similarly, a normocytic anaemia as macrocytic.6 reticulocytes mature into red cells after 24 hours in circulation. the clinical and laboratory standards institute (clsi) recommends that samples stored at rt should be analysed for reticulocytes within six hours of collection.7 it is further recommended that pbs for morphologic analysis be prepared within four hours, prior to the onset of edta-induced changes in red and white cell morphology.8,9,10 with centralisation of laboratory services, it is not always feasible to meet these deadlines. large academic laboratories are commonly faced with the scenario where a sample collected on friday is not received in the laboratory for analysis until monday morning. recent studies indicate that longer storage durations are acceptable when samples are stored at 4 °c – 8 °c.3,5,6,11,12 however, information on stability beyond 72 hours is limited.6 furthermore, these studies are small, specific to the haematology analyser, and the definition of stability used is not standardised. the aim of this study was to evaluate the stability of the fbc, diff, reticulocyte and pbs morphology during extended storage at rt and at 4 °c – 8 °c in order to determine laboratory criteria for storage time and temperature for specimens referred for the work-up of haematological disorders from remote laboratories. research method and design top ↑ ethical considerations the study was approved by the human research ethics committee of the university of the witwatersrand (m090688). all tests were done as part of routine diagnostic workups and no additional blood samples were taken from the participants for this study. materials and setting this study was conducted at the main haematology laboratory of the charlotte maxeke johannesburg academic hospital (cmjah), national health laboratory service complex, johannesburg, south africa. forty blood samples, representative of the patient population (25% abnormal and 75% normal specimens), that were left over after routine testing were selected from the haematology workload. only samples collected in edta (becton-dickinson, oxford, united kingdom) vials with adequate volume (> 4 ml) received within two hours of collection were included. samples with results that indicated partial aspiration were excluded from the final analysis. design blood sampling and laboratory methods blood samples for evaluation of fbc, diff and reticulocyte parameters were collected in k2edta tubes (1.5–2.2 mg of dipotassium edta dihydrate per millilitre of blood). the parameters were analysed with the laboratory's advia® 120 automated haematology analyser (siemens healthcare diagnostics, inc, tarrytown, new york, united states). cells were counted and sized by light scatter technology using white light for white cells and laser light for red cells and platelets. haemoglobin was measured by the conventional cyanmethaemoglobin method. the six-part differential analysis was performed in two channels. cells in the peroxidase channel were measured by peroxidase staining intensity and light scatter. cells in the basophil/lobularity channel were measured by dual laser light scatter, nuclear density and lobulation index. reticulocytes were stained with oxazine 750. the films were spread on the advia® autoslide slide maker and stained on the hema-tek® 2000 slide stainer (siemens healthcare diagnostics inc, tarrytown, new york, united states). laboratory testing the samples were analysed within two hours of collection (time zero) at rt. samples were aliquoted into two sets; one was stored at rt (18 °c – 24 °c) and and the other at 4 °c – 8 °c. analyses of samples stored at rt were performed after 12, 24, 36 and 48 hours of storage. analyses of samples stored at at 4 °c – 8 °c were performed after 12, 24, 36, 48, 72, 96, 120, 144 and 168 hours of storage. a manual diff was performed on pbs of five samples stored at rt and at 4 °c – 8 °c. reviews were performed at 12, 24, 36 and 48 hours. the pbs was first examined for the presence of edta-induced changes, including red cell spherocytes, echinocytes, sphero-echinocytes, increased rouleaux formation, degeneration of neutrophils and lobulation of lymphocyte nuclei,10 because these changes preclude an accurate manual diff. analyses data were captured from the analyser printouts on excel™ spreadsheets (microsoft office excel™ 2007, redmond, washington, united states) and analysed using statistica 9.1 software (statsoft, tulsa, oklahoma, united states). the mean percentage difference from the value at time zero was calculated and tabulated.13 stability of a parameter was defined in relation to the precision of the advia® 120 analytical method. acceptable limits were defined in accordance with the royal college of pathologists of australasia external quality assurance annual review for 2013.14 the coefficients of variation (% cv) for the parameters were as follows: wcc 3.4%, rcc 1.8%, haemoglobin 1.8%, haematocrit 2.4%, platelet count 2.4% and percentages of neutrophils 0.9%, lymphocytes 4.9%, monocytes 5.6%, eosinophils 16.7%, basophils 55% and reticulocytes 8.1%. a parameter was considered stable, when its difference was smaller than 1% cv for the assessed method.5 results top ↑ storage at room temperature the wcc was stable until 36 hours after collection and showed a significant decrease at 48 hours after collection (figure 1). a significant increase in the percentages of neutrophils and eosinophils was observed at 24 hours and 36 hours, respectively. red cell parameters including rcc, haemoglobin, mean cell haemoglobin (mch) and red cell distribution width (rdw) were stable for at least 48 hours after collection when stored at rt and were not significantly affected by storage temperature. in contrast, other rcc measurements, including haematocrit, mcv, mean cell haemoglobin content (mchc) and the percentage of reticulocytes, were not stable after storage at rt for 48 hours after collection. after rt storage for 24 hours, a significant increase in mcv, as well as a decrease in the percentage of reticulocytes, was observed. analysis of platelet stability showed platelets were stable for 12 hours and significantly decreased at 24 hours after collection. at rt storage, the stability of the mean platelet volume (mpv) was less than 12 hours as a result of artificial platelet swelling. at rt storage, the stability of the manual diff was also less than 12 hours. the percentages of neutrophils and monocytes showed significant increases, whereas percentages of lymphocytes and eosinophils showed significant decreases at 12 hours after collection. the slides examined contained too few basophils to obtain reliable results for basophil stability. edta-induced changes were noted at 24 hours after collection, which precluded a manual diff. figure 1: stability analysis (mean percent difference) at room temperature (18 °c – 24 °c) storage at 4 °c – 8 °c the wcc was stable at 4 °c – 8 °c until 48 hours after collection (figure 2). a significant decrease in the percentage of neutrophils was observed at 72 hours after collection. the percentages of eosinophils, basophils and monocytes were not stable when stored at 4 °c – 8 °c and showed significant increases at 12, 24 and 48 hours, respectively. compared with rt storage, we observed improved stability of rcc parameters when stored at 4 °c – 8 °c. haematocrit, mcv and mchc were stable until 168 hours when stored at 4 °c – 8 °c. the percentage of reticulocytes was stable until 120 hours after collection and showed significant decreases thereafter. platelets were stable until 96 hours after collection when stored at 4 °c – 8 °c. mpv showed a significant increase at 24 hours after collection. as a result of the presence of edta-induced changes prior to 12 hours after collection, a manual diff could not be performed. figure 2: stability analysis (mean percent difference) at 4 °c – 8 °c. discussion top ↑ routine tests such as the fbc, diff, reticulocyte and pbs morphology are commonly referred to the cmjah haematology laboratory as part of the diagnostic work-up for haematological disorders. in large academic laboratories, where aged samples make up a significant proportion of the workload, the storage time and temperature of samples must be taken into consideration. the findings of this study performed on edta samples add to the evidence that stability varies according to storage time and temperature. according to the findings of this study, fbc parameters, namely rcc, haemoglobin, mch and rdw, and diff parameters, namely percentages of basophils, lymphocytes and monocytes, were least affected by storage temperature and time and can be analysed until 48 hours after sample collection when stored at rt. it is recommended that traditional fbc parameters be analysed 24 hours after sample collection when stored at rt.3,4,5 however, in this study, platelets were only stable until 12 hours after collection when stored at rt. the stability of the wcc was also found to be shorter than other studies, which have recommended analysis up to 48 hours after collection when stored at rt.5,11,15 in this study, the wcc was stable only until 36 hours after collection when stored at rt. in this study, the mcv was stable until 12 hours after collection. imeri et al. found that mcv increased significantly after 4–10 hours, regardless of the haematology analyser;5 whereas other studies have indicated a longer stability of up to 24 hours for mcv at rt.3,11,15 these discordant results may be attributed to the different statistical methods used in these studies for the evaluation of stability, which limits accurate comparison. in this study, the percentage of reticulocytes was stable at rt until 12 hours after collection, which concurs with current recommendations.3,5 however, the stability of reticulocyte parameters has been found to be longer at rt on other haematology analysers, such as the coulter® lh 750, sysmex xe-2100™ and cell-dyn sapphire™.5,11 according to wiegand et al., the oxazine 750 on the advia® haematology analysers may not be sufficient to detect more mature reticulocytes.16 imeri et al. conducted a three-way comparison study of the coulter® lh750, sysmex xe-2100™ and advia® haematology analysers that further illustrated how the stability of many haematology parameters depends on the analytical method used.5 thus, the findings of this study are specific to advia® haematology analysers, which currently represent 60% of haematology analysers in south africa. therefore, the findings of this study have widespread local implications. however, clsi do recommend that ‘laboratories should assess fbc stability in their specific settings’.17 storage of samples at 4 °c – 8 °c for seven days increased the stability of most parameters. fbc parameters, namely wcc, platelet count, haematocrit, mcv and mchc, as well as diff parameters, namely percentages of neutrophils and reticulocytes, were more stable when stored at 4 °c – 8 °c. however, some diff parameters, namely percentages of eosinophils, basophils and monocytes, had lower stability. changes were present on pbs morphology prior to 12 hours after collection when stored at either rt or at 4 °c – 8 °c. this precluded assessment of dysplastic morphological features. it is currently recommended that pbs be prepared within a few hours for assessment of haematological disorders, in particular for the presence of dysplastic features.9,10,18 limitations of the study top ↑ a limitation of this study is that pbs morphology was not evaluated prior to 12 hours after collection (i.e., at four and six hours). as such, a manual differential could not be performed. furthermore, this study was performed under optimal conditions on inpatient samples that were received in the laboratory within two hours of collection. samples referred for testing are often subject to variation in temperature during collection and transport. conclusion top ↑ in conclusion, this study provides evidence regarding the viability of blood samples collected in edta vials and stored at rt and at 4 °c – 8 °c. samples that have been stored at 4 °c – 8 °c for seven days are suitable for testing on the advia® 120 analyser for fbc and reticulocyte parameters. however, this is not a solution for samples referred for diff or pbs morphology review. acknowledgements top ↑ we thank celeste mcpherson and the laboratory staff of the cmjah national health laboratory service complex for their technical assistance. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions e.s. (charlotte maxeke johannesburg academic hospital) was the author of the manuscript and also performed data entry and analysis. d.p. (national health laboratory services, university of kwazulu-natal) was responsible for data collection, data entry and manuscript review. references top ↑ queen e, ifeanyi oe, chinedum ok. the effect of storage on full blood count in different anticoagulant. iosr jdms. 2014;3(9):128–131. http://dx.doi.org/10.9790/0853-1392128131 guder wg. preanalytical factors and their influence on analytical quality specifications. scand j clin lab invest. 1999;59(7):545–549. http://dx.doi.org/10.1080/00365519950185328 bourner g, dhaliwal j, sumner j. performance evaluation of the latest fully automated hematology analyzers in a large, commercial laboratory setting: a 4-way, side-by-side study. lab hematol. 2005;11(4):285–297. http://dx.doi.org/10.1532/lh96.05036 cohle sd, saleem a, makkaoui de. effects of storage of blood on stability of hematologic parameters. am j clin pathol. 1981;76(1):67–69. imeri f, herklotz r, risch l, et al. stability of hematological analytes depends on the hematology analyser used: a stability study with bayer advia 120, beckman coulter lh 750 and sysmex xe 2100. clin chim acta. 2008;397(1–2):68–71. http://dx.doi.org/10.1016/j.cca.2008.07.018 ashenden m, clarke a, sharpe k, et al. stability of athlete passport parameters during extended storage. int j lab hematol. 2013;35(2):183–192. http://dx.doi.org/10.1111/ijlh.12014 national committee for clinical laboratory standards. methods for reticulocyte counting (flow cytometry and supravital dyes). approved guideline. h44-a. wayne, pa: nccls; 1997. vives-corrons jl, briggs c, simon-lopez r, et al. effect of edta-anticoagulated whole blood storage on cell morphology examination. a need for standardization. int j lab hematol. 2014;36(2):222–226. http://dx.doi.org/10.1111/ijlh.12170 zini g. stability of complete blood count parameters with storage: toward defined specifications for different diagnostic applications. int j lab hematol. 2014;36(2):111–113. http://dx.doi.org/10.1111/ijlh.12181 antwi-baffour s, quao e, kyeremeh r, et al. prolong storage of blood in edta has an effect on the morphology and osmotic fragility of erythrocytes. int j biomed sci eng. 2013;1(2):20–23. http://dx.doi.org/10.11648/j.ijbse.20130102.11 hedberg p, lehto t. aging stability of complete blood count and white blood cell differential parameters analyzed by abbott cell-dyn sapphire hematology analyzer. int j lab hematol. 2009;31(1):87–96. http://dx.doi.org/10.1111/j.1751-553x.2007.01009.x lippi g, salvagno gl, solero gp, et al. stability of blood cell counts, hematologic parameters and reticulocytes indexes on the advia a120 hematologic analyzer. j lab clin med. 2005;146(6):333–340. http://dx.doi.org/10.1016/j.lab.2005.08.004 international council for standardization in haematology. guidelines for the evaluation of blood cell analysers including those used for differential leucocyte and reticulocyte counting and cell marker applications. clin lab haematol. 1994;16(2):157–174. rcpa. rcpa quality assurance programs [homepage on the internet]. n.d. [cited 2015 apr 02]. available from: http://www.rcpaqap.com.au. gulati gl, hyland lj, kocher w, et al. changes in automated complete blood cell count and differential leukocyte count results induced by storage of blood at room temperature. arch pathol lab med. 2002;126(3):336–342. wiegand g, effenberger-klein a, weber r, et al. potential pitfalls of comparative measurements of reticulocytes with flow cytometry and microscopy in prematures and infants. clin chem lab med. 2004;42(10):1150–1154. http://dx.doi.org/10.1515/cclm.2004.234 clinical and laboratory standards institute. validation, verification, and quality assurance of automated hematology analyzers, approved standard – 2nd ed. h26-a2, vol. 30(14). wayne, pa: clsi; 2010. brunning rd, orazi a, germing u, et al. myelodysplastic syndromes/neoplasms, overview. in: swerdlow sh, campo e, harris nl, et al., editors. who classification of tumours of haematopoietic and lymphoid tissues.lyon: iarc, 2008; p. 88–93. abstract introduction methods results discussion acknowledgements references about the author(s) immaculate s. dlamini department of chemical pathology, faculty of laboratory medicine, national health laboratory service, durban, south africa department of chemical pathology, faculty of laboratory medicine, university of kwazulu-natal, durban, south africa verena gounden department of chemical pathology, faculty of laboratory medicine, national health laboratory service, durban, south africa department of chemical pathology, faculty of laboratory medicine, university of kwazulu-natal, durban, south africa nareshni moodley department of chemical pathology, faculty of laboratory medicine, national health laboratory service, durban, south africa department of chemical pathology, faculty of laboratory medicine, university of kwazulu-natal, durban, south africa citation dlamini is, gounden v, moodley n. evaluation of tumour marker utilisation and impact of electronic gatekeeping in the province of kwazulu-natal, south africa. afr j lab med. 2023;12(1), a2027. https://doi.org/10.4102/ajlm.v12i1.2027 original research evaluation of tumour marker utilisation and impact of electronic gatekeeping in the province of kwazulu-natal, south africa immaculate s. dlamini, verena gounden, nareshni moodley received: 20 july 2022; accepted: 13 apr. 2023; published: 30 june 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: inappropriate testing remains a high healthcare cost driver. tumour marker tests are more expensive than routine chemistry testing. implementing test demand management systems like electronic gatekeeping (egk) has reportedly decreased test requests. objective: this study aimed to describe the appropriateness of tumour marker tests, carcinoembryonic antigen, alpha foetal protein, prostate-specific antigen, carbohydrate antigen 19-9, cancer antigen 15-3, cancer antigen 125, and human chorionic gonadotropin, and determine the effectiveness of the egk used in the public health sector in kwazulu-natal, south africa. methods: tumour marker test data for the kwazulu-natal province were extracted from the national health laboratory service central data warehouse for 01 january 2017 – 30 june 2017 (pre-egk) and 01 january 2018 – 30 june 2018 (post-egk implementation). questionnaires were sent to the clinicians in the regional hospitals ordering the most tumour marker tests to assess ordering practices. in addition, we assessed monthly rejection reports to determine the effect of the egk. results: the egk minimally reduced tumour marker requests or associated costs (1.4% average egk rejection rate). an overall 18% increase in the tumour marker tests occurred in 2018. the data suggest inappropriate tumour marker test utilisation, particularly for screening. conclusion: the introduction of egk as a test demand management had little impact on tumour marker test requests and costs. continuous education and reiteration of indications for tumour marker test use are required. what this study adds: this study demonstrates the ineffectiveness of egk in tumour marker orders, and provides some insight as to why these markers are being ordered, which is important in trying to decrease inappropriate ordering of these tests. keywords: tumour marker; demand management; electronic gatekeeping; minimum retesting interval; cost reduction. introduction serum tumour markers are biochemical markers released by tumour cells directly or indirectly as a source or effect of malignant development. tumour markers are a less invasive tool than a biopsy and are used to increase or decrease the clinical suspicion of a developing new or secondary cancer, detect the recurrence or progression of cancer, monitor response to treatment, and identify a specific therapeutic modality. ideally, requesting and testing a tumour marker should allow for effective patient management, when appropriately performed, and reduce unnecessary redundant costs.1 therefore, the measurement of non-invasive serum tumour markers has been pursued to expedite early diagnosis and detection of cancer aimed at reducing cancer morbidity and mortality. however, the diagnostic sensitivity and specificity of most currently available serum tumour markers are limited.2 the inappropriate use of serum tumour markers has been reported.3,4,5 the improved analytical sensitivity and specificity of high-output automated platforms have increased accessibility to the use of tumour markers and increased the use of serum tumour markers.6 however, the progression in instrumentation has been incongruent with the adoption of evidence-based guidelines to guide the appropriate use of tumour markers.7 the cost of inappropriate testing of tumour markers indirectly affects patient safety, depending on the management strategies initiated based on the results reported.8 increased healthcare costs with decreased healthcare budgets have forced laboratories to develop strategies to reduce and prevent inappropriate testing.9 demand management is one strategy that focuses on ensuring appropriate requests while ensuring quality care to the patient. laboratories have adopted several strategies to limit test demand, such as requiring requesting physicians to have a predetermined educational level, redesigning test request forms, and physical and electronic-based gatekeeping and reflex testing.9 most serum tumour markers are not recommended as first-line rule-in or rule-out tests for cancer, but rather for detecting tumour recurrence and monitoring treatment. thus, demand management strategies have been developed.9,10 some strategies have been validated for serum tumour marker testing,11 such as the minimum retesting interval (mri). the mri strategy stipulates the minimum time before repeating a test based on the test’s properties, such as clinical indication.12,13 the south african national department of health, in conjunction with the national health laboratory service, which serves the south africa public health sector facilities, have used the mri strategy, termed electronic gatekeeping (egk), to manage test demand since 2017. electronic gatekeeping was introduced to limit healthcare spending on ‘unnecessary’ laboratory investigations. the criteria for selecting the mri were based on a combination of literature and consensus agreement between expert clinicians representing the department of health in each region, and expert pathologists. electronic gatekeeping implementation studies have demonstrated that egk is an effective cost-saving tool for several laboratory tests. in 2010, tygerberg hospital management and the national health laboratory service conducted a pilot project in cape town, south africa, to identify the number of egk-rejected and egk-restored (i.e., approved for analysis) tests, the costs saved, and the impact test rejections. the study concluded that the egk was an effective and sustainable demand management tool. they found that most rejected tests were not restored, revealing the inappropriateness of those test requests. the use of egk did not appear to negatively impact patient care but was an effective cost-saving tool.14 however, an academic hospital in gauteng province, south africa, reported that egk test demand management does not dramatically influence requesting behaviour or save costs. they reported an unchanged monthly percentage of egk-held tests over a 22-month retrospective study period, suggesting that a solitary demand management strategy is not as effective as anticipated or as demonstrated in other studies.15 both the cape town and gauteng studies only reviewed the effect of routine chemistry testing demand, not tumour marker testing. to date, no study has reviewed the requesting nature of tumour markers in the south african public health sector. in the south african public healthcare sector, all laboratory and other diagnostic costs are borne by the state. tumour markers were chosen as they are one of the most highly requested tests in the chemistry laboratory, are more costly to process, and are thus charged at a higher rate than the more routine general chemistry testing. at the time of the study, the national health laboratory service chemical pathology laboratory at laboratory a provided tumour marker testing services for most patients in the public sector covering the entire province of kwazulu-natal, except a more northern region, where testing is provided by laboratory b, a national health laboratory service laboratory, which provides a smaller tumour marker test repertoire. the serum tumour markers that were assessed during this audit were carcinoembryonic antigen, alpha foetal protein (afp), prostate-specific antigen (psa), carbohydrate antigen 19-9 (ca 19-9), cancer antigen 15-3 (ca 15-3), cancer antigen 125 (ca 125), and human chorionic gonadotropin (hcg). disorders with high afp include hepatocellular carcinoma, hepatoblastoma, non-seminomatous testicular germ cell tumours of the embryonal carcinomas, cancers of the pancreas, lung, and gastric, and non-malignant processes such as acute viral hepatitis, liver cirrhosis, and obstructive jaundice.2,16,17 carcinoembryonic antigen is a tumour marker for gastrointestinal cancers, but it is also elevated in breast, lung and liver cancers, and non-malignant conditions like heavy smoking, bronchitis, gastritis, duodenal ulcer, liver diseases, pancreatitis, and colorectal polyposis.16,18 human chorionic gonadotropin is produced by embryonal tissue1 but is used as a tumour marker in seminomatous and non-seminomatous testicular tumours, ovarian germ cell tumours, the gestational hydatid form mole, choriocarcinoma, and non-testicular teratomas.19 carbohydrate antigen 19-9 is normally synthesised by the pancreas, biliary ductal cells, gastric, colon, and endometrial and salivary epithelia. it is mainly used to prognosticate and monitor response to interventions in patients with pancreatic and gastrointestinal cancer.1 increased ca 125 values most often are associated with epithelial ovarian cancer, although levels can also be increased in other malignancies, such as breast, endometrial, cervix, peritoneal, uterus, lung, pancreas, hepatocellular and non-hodgkin’s lymphoma and multiple benign disorders, which include pregnancy, endometriosis, uterine fibroids, pancreatitis, normal menstruation, pelvic inflammatory disease, and cirrhosis of the liver.1 cancer antigen 15-3 levels have been reported to be useful to prognosticate in breast cancer patients,20 but elevations of ca 15-3 levels are also seen in other malignancies, including pancreatic, lung, ovarian, colon, and liver cancer as well as benign breast and liver conditions.1 prostate-specific antigen aids in the diagnosis, risk assessment, and monitoring of prostate carcinoma, but it is also elevated in non-malignant conditions like acute urinary retention, benign prostatic hyperplasia, prostatitis, and urinary tract infection.1 this study aimed to describe the tumour marker requesting practices across different levels of healthcare in the province of kwazulu-natal, south africa, assess the effect of egk on these requesting practices and, lastly, determine via questionnaire the rationale for tumour marker requesting by the clinicians at the highest ordering facilities. methods ethical considerations ethics approval for this study was obtained from university of kwazulu-natal biomedical research ethics committee (number be035/18). written informed consent was received from the participating clinicians. data were collected on a password-protected computer and the primary investigator was the only person with access to it. patients were identified by hospital numbers and their identities were not revealed. questionnaires were anonymised and identified by numbers allocated to the specific sites. survey respondents were assured raw data would remain confidential and would not be shared. data collection the national health laboratory service central data warehouse reposits all test results generated by national health laboratory service laboratories. we extracted from the national health laboratory service central data warehouse all tumour marker tests performed in public healthcare facilities in kwazulu-natal province, south africa. the data extracted included requests made 6 months pre-egk (01 january 2017 to 30 june 2017) and 6 months post-egk implementation (01 january 2018 to 30 june 2018). the data consisted of results from laboratory a and b national health laboratory service chemical pathology laboratory. laboratory a offers the following tumour marker tests: carcinoembryonic antigen, afp, psa, ca 19-9, ca 15-3, ca 125, and hcg, whereas laboratory b offers all the above tests except ca 19-9. both laboratories analysed tumour markers on the siemens advia centaur xp (siemens, tarrytown, new york, united states). the mri rules that were in use for egk per tumour marker test were as follows: hcg: 1 day; afp: 1 month; psa: previous result abnormal = 1 month and previous result normal = 1 year; carcinoembryonic antigen: 1 month; ca 125: 1 month; ca 19-9: 1 month; and ca 15-3: 1 month; where: 1 day is 12 h, 1 month is 21 days, and 1 year is 322 days since daily tests are not performed at the same time each day and a repeat visit within a set interval (eg. week or month) may happen before the exact interval has passed. the monthly egk rejections reports were assessed from 01 january 2018 to 30 june 2018, to determine the number of requests rejected by gatekeeping in the included laboratories. the top five highest requesting units from all healthcare facilities over the period of the study were identified and chosen as the sites for questionnaire distribution. written informed consent and the questionnaire (adapted from mcginley and kilpatrick21) were hand delivered and obtained from clinicians from these selected facilities. the questionnaire identified who was ordering tumour markers and why. responses were collated in microsoft excel (microsoft corporation, redmond, washington, united states) for further evaluation. data analysis statistical analyses were performed using medcalc r version 18.11 (medcalc software, mariakerke, belgium) and microsoft excel 2016 (microsoft corporation, redmond, washington, united states). data were assessed for normality using the shapiro-wilk test. normal data were presented as mean ± standard deviation. the monthly average rejection rate was calculated. costing was done using on the national health laboratory service state price list 2017. results a total of 38 615 tumour marker tests for the specified analytes were processed during the 6-month pre-egk introduction period (01 january 2017 to 30 june 2017), while 45 567 tumour markers requests were processed in the post-egk implementation period (01 january 2018 to 30 june 2018). in 2018, there was an 18% increase in tumour marker tests processed. the most ordered tumour marker was psa (41.1% of tested tumour markers in 2017, and 38.4% in 2018), while the least ordered was ca 15-3 (< 3% of tested tumour markers in both 2017 and 2018) (table 1). table 1: summary of tumour markers processed in kwazulu-natal, south africa, 01 january 2017 to 30 june 2018. the majority of samples for psa, afp, carcinoembryonic antigen, ca 19-9, ca 15-3 and ca 125 had normal results (defined as results within the reference interval) in both years of the study period. in contrast, hcg had the most abnormal results (outside of reference interval) for both years in the study period, with 90.1% (2783/3088) abnormal results for 2017, and 92.1% (3771/4092) for 2018. clinical history data provided on the laboratory information system via the central data warehouse demonstrated that there were no clinical histories recorded for most samples (n = 21 299, 65%). a minority of samples (n = 1535, 5%) had a history of cancer documented on the request forms. nine percent (n = 2927) of requests indicated suspicion of malignancy or screening as request reason (figure 1). figure 1: clinical details for tumour marker test requests retrieved from laboratory information system in kwazulu-natal, south africa, 01 january 2018 – 30 june 2018. based on the national health laboratory service state price list 2017, the cost of normal results was markedly higher than abnormal results for both study periods. for example, from 01 january 2017 to 30 june 2017, normal results cost 3 995 553.00 south african rand (r) ($218 409.51 united states dollars [usd]), while abnormal results cost r1 210 461.00 ($66 167.61 usd). in the same period in 2018, normal results cost r4 764 052.00 ($260 418.09 usd) and abnormal results cost r1 181 010.00 ($64 557.73) (figure 2). figure 2: cost of resulted normal and abnormal tumour markers test requests in (a) 2017 and (b) 2018 in kwazulu-natal, south africa. all cost amounts are shown in south african rand (zar). most test requests were received from the outpatient departments or non-oncology clinics of the respective healthcare facilities. however, test requests from the oncology wards and clinics were the lowest. the tertiary academic hospitals made the fewest requests, followed by primary healthcare facilities. requests from district hospitals increased in 2018 by 58.7% to overtake regional hospitals as the main requestors (table 2). table 2: distribution of samples processed per location for tumour marker testing in kwazulu-natal, south africa, 01 january 2017 – 30 june 2018. the egk rejected an average of 95 tumour marker test requests per month from 01 january 2018 to 30 june 2018 with a total of 570 tests rejected over this period. additionally, during the same 6-month period, no egk-rejected tumour marker tests were restored. the total cost of tumour marker rejected test requests was r78 043.86 in 2018 (table 3). table 3: electronic gatekeeping rejection rate and costs saved in kwazulu-natal, south africa, 01 january 2018 – 30 june 2018. clinician questionnaire findings we reviewed 22 responses from the 37 questionnaires distributed (59% response rate). most respondents were from surgical departments (n = 24; 64%), followed by medical (n = 9; 24%), with the remainder (n = 3; 9%) being from general outpatient clinics or unspecified. participants consisted predominantly of junior staff (interns) and non-specialist medical officers. ninety-five percent (n = 21) of respondents indicated that their facility had no dedicated oncology unit or clinics. a further 91% (n = 20) of the participants were unaware of any local or international tumour marker test request guidelines for clinical practice. participants indicated the following as consequent actions to an abnormal tumour marker result: imaging studies (n = 20, 91%), 63% (n = 14) included biopsy, referral (n = 11, 50%), requesting another tumour marker test (n = 3, 14%), and 9% (n = 2) included repeating the tumour marker test. most respondents requested tumour markers to query suspected tumours; over 20% (n = 4) indicated their use to detect tumour sources (figure 3). figure 3: reasons for requesting tumour marker testing per questionnaire respondents, june 2018, in kwazulu-natal, south africa. discussion tumour markers are some of the more expensive clinical chemistry tests. based on the national health laboratory service test pricing for the period 2017/2018, the total cost of tumour marker testing for the two periods reviewed was more than r10 million ($546 631.50 usd). all rejected tumour marker tests were estimated to cost r78 043.86 ($4266.12 usd) in 2018. in the public sector in south africa, the cost of laboratory testing is paid by the department of health (state), with no cost to the patient. the egk rejects test requests before payment and laboratory testing, hence no refund is made on rejected requests. previous reports from 1997–2012 state that 20% – 50% of laboratory tests are inappropriate or are not evidence-based practices.22,23 pema, kiabilua and pillay (gauteng, south africa, in 2018),15 and smit, zemlin and erasmus (tygerberg, western cape, south africa, in 2015)14 reported significant cost reductions through egk of requests. however, the test requests reviewed were smaller-volume tests. our findings showed that the number of tumour marker tests rejected by the egk rules was minimal. fewer than 20 tests were rejected on average, per month, for each of the tumour markers apart from psa. this low rejection rate suggests that appropriate test ordering, per the test’s correct clinical requirement and guidelines, would be the most effective way of controlling inappropriate tumour marker test requests. appropriate test ordering practice requires education on and routine reiteration of appropriate request guidelines, and the development and implementation of national testing guidelines. the lack of continuous clinician education is a reported driver of inappropriate testing.24,25 education and continuous reiteration of best practices are especially important in the non-academic centres, where there are more generalists than specialists managing patients. the high request numbers from district health facilities support the fact that education regarding tumour marker utilisation is most needed in non-academic centres; however, district hospitals represent most hospitals servicing the population (n = 37) versus regional hospitals (n = 13).26 as evidenced by the respondents on the questionnaire, a lot of clinicians were requesting tumour markers to screen for malignancy. our findings may also be an indication that the egk rules require further review and are not strict enough to achieve sufficient demand management. these rules could include limiting requests to only two tumour marker tests on one visit or admission to dissuade panel screening. however, stricter rules may not be possible for hcg, as it is also a test for normal pregnancy and pregnancy-related disorders (for example ectopic pregnancy) and serial measurements are critical. human chorionic gonadotrophin was frequently requested for younger patients, probably because the laboratory information system could not distinguish between malignancy-related and pregnancy-related hcg testing. additionally, germ cell tumours in which hcg concentrations may be increased are more frequently seen in younger adults and adolescents. this is the first study, to the authors’ knowledge, that examines the use of tumour markers in sub-saharan healthcare facilities. the findings of this study suggest that repeat testing represents a small fraction of the cost associated with tumour marker requests and that inappropriate requests (use of all tumour markers as screening tests) are likely resulting in test overuse and associated increased healthcare costs. the introduction of egk has made little or no impact on the number and cost of tumour marker tests performed. while there are no national consensus guidelines for the utilisation of tumour markers in south africa, international guidelines or best practice documents are available to guide clinicians to order tests appropriately.27,28,29 we recommend developing local and national tumour marker ordering guidelines for all levels of healthcare. focused education at the undergraduate level and continuous professional development regarding appropriate utilisation of laboratory tests including tumour markers is also required. greater involvement of pathologists in spreading appropriate utilisation awareness and coaching of junior doctors is also essential. the increasing demands on limited healthcare resources and funding necessitate careful management of testing to ensure optimal patient care while managing costs. limitations one of the limitations of this study was that access to histology results was not available. thus, tumour marker test results could not be confirmed by tumour biopsy results. furthermore, the small number of questionnaires distributed may not be representative of the clinician cohort. additionally, we did not sample clinicians from different healthcare facility levels. furthermore, due to the geographical limitations, lack of internet availability, and other limited resources, we restricted questionnaire distribution to facilities within kwazulu-natal. in addition to that, hcg results were not separated into pregnant versus non-pregnant due to missing clinical records on many samples. lastly, the effects of interventions to improve clinician knowledge of tumour marker requests were not assessed. conclusion it appears that many clinicians do not appropriately request and utilise tumour marker tests and there is no guideline for tumour test ordering and result interpretation. the egk barely reduced tumour marker requests and costs as there was an increase in costs and testing numbers, despite egk implementation. education of doctors, stricter egk rules, and additional demand management measures may be required to make a noticeable demand difference. acknowledgements the authors would like to acknowledge the national health laboratory services laboratory information system (trak care) central data warehouse for the data retrieval, and the clinicians at the department of health hospitals who completed the study questionnaires. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article, except where otherwise indicated. authors’ contributions i.s.d., v.g. and n.m. confirmed they have contributed to the intellectual content of this paper and have met the following four requirements: (1) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (2) drafting or revising the article for intellectual content; (3) final approval of the published article; and (4) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. sources of support this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability raw data were extracted from the national health laboratory service central data warehouse. costing was done using 2017 pricing at the national health laboratory service. figures with raw data include: figure 2, figure 3, table 1, table 2 and table 3. testing data may be made available on reasonable request from the corresponding author, n.m. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references sturgeon c. tumour markers. in: rifai n. tietz textbook of clinical chemistry and molecular diagnostics. chapter 31. st louis, missouri: elsevier, 2018; pp. 436–478. malati t. tumour markers: an overview. indian j clin biochem. 2007;22(2):17–31. https://doi.org/10.1007/bf02913308 ntaios g, hatzitolios a, chatzinikolaou a, et al. an audit of tumour marker utilisation in greece. eur j intern med. 2009;20(3):e66–e69. https://doi.org/10.1016/j.ejim.2008.07.026 ferraro s, mozzi r, panteghini m. tumour marker ordering: do not lose control: a prospective clinical trial. am j clin pathol. 2015;144(4):649–658. https://doi.org/10.1309/ajcpnzapjrb3t6kk al-mughales ja, alahwal ms. inappropriate practice in tumour marker requests at a university hospital in western saudi arabia: a 3-year retrospective study. int j biol markers. 2020;35(4):35–43. https://doi.org/10.1177/1724600820971305 lee p, jain so, matthew r, et al. diagnosis and management of cancer using serologic and other body fluid markers. henry’s clinical diagnosis and management by laboratory methods. 23rd ed. philadelphia, pa: saunders elsevier, 2016; pp. 1508–1524. schrohl as, holten-andersen m, sweep f, et al. tumour markers: from laboratory to clinical utility. mol cell proteomics. 2003;2(6):378–387. https://doi.org/10.1074/mcp.r300006-mcp200 lui jk, ogunsua aa, hatch sc, liebmann j, scully g. the diagnostic utility of tumour markers: a teachable moment. am j med. 2015;128(5):e9–10. https://doi.org/10.1016/j.amjmed.2014.10.063 fryer aa, smellie ws. managing demand for laboratory tests: a laboratory toolkit. j clin pathol. 2013;66(1):62–72. https://doi.org/10.1136/jclinpath-2011-200524 sturgeon cm, hoffman br, chan dw, et al. national academy of clinical biochemistry laboratory medicine practice guidelines for use of tumour markers in clinical practice: quality requirements. clin chem. 2008;54(8):e1–e10. https://doi.org/10.1373/clinchem.2007.094144 mrazek c, haschke-becher e, felder tk, keppel mh, oberkofler h, cadamuro j. laboratory demand management strategies an overview. diagnostics (basel). 2021;11(7):1141. https://doi.org/10.3390/diagnostics11071141 lang t. minimum retesting intervals in practice: 10 years experience. clin chem lab med. 2021;59(1):39–50. https://doi.org/10.1515/cclm-2020-0660 lang t, bernie c. g147_national minimum retesting intervals in pathology [homepage on the internet]. march 2021 [cited july 2021]; version 2. available from: https://www.rcpath.org. smit i, zemlin ae, erasmus rt. demand management: an audit of chemical pathology test rejections by an electronic gate-keeping system at an academic hospital in cape town. ann clin biochem. 2015;52(pt 4):481–487. https://doi.org/10.1177/0004563214567688 pema ak, kiabilua o, t.s. pillay ts. demand management by electronic gatekeeping of test requests does not influence requesting behaviour or save costs dramatically. ann clin biochem. 2018;55(2):244–253. https://doi.org/10.1177/0004563217707980 novakovic s. tumour markers in clinical oncology. radiol oncol. 2004;38(2):73–83. zhang j, chen g, zhang p, et al. the threshold of alpha-fetoprotein (afp) for the diagnosis of hepatocellular carcinoma: a systematic review and meta-analysis. plos one. 2020;15(2):e0228857. https://doi.org/10.1371/journal.pone.0228857 wang hy, hsieh ch, wen cn, wen yh, chen ch, lu jj. cancers screening in an asymptomatic population by using multiple tumour markers. plos one. 2016;11(6):e0158285. https://doi.org/10.1371/journal.pone.0158285 foley kf. using human chorionic gonadotropin as a tumour marker. clinical laboratory news 2021 april [cited 2021 july]. available from: https://www.aacc.org/cln/articles/2021/april/using-human-chorionic-gonadotropin-as-a-tumor-marker tomlinson ip, whyman a, barrett ja, kremer jk. tumour marker ca15-3: possible uses in the routine management of breast cancer. eur j cancer. 1995 jun;31a. mcginley pj, kilpatrick es. tumour markers: their use and misuse by clinicians. ann clin biochem. 2003;40(pt 6):643–647. https://doi.org/10.1258/000456303770367234 zhi m, ding el, theisen-toupal j, whelan j, arnaout r. the landscape of inappropriate laboratory testing: a 15-year meta-analysis. plos one. 2013;8:e78962. https://doi.org/10.1371/journal.pone.0078962 morris tf, ellison tl, maysoon m, althawadi si, heppenheimer m. demand management and optimisation of clinical laboratory services in a tertiary referral centre in saudi arabia. ann. saudi med. 2018;38:299–304. https://doi.org/10.5144/0256-4947.2018.299 naugler c. a perspective on laboratory utilisation management from canada. clin chim acta. 2014;427:142–144. https://doi.org/10.1016/j.cca.2013.09.022 liu z, abdullah a, baskin l, lewis g, kelter g, naugler c. an intervention to reduce laboratory utilisation of referred-out tests. lab med. 2012;43:164–167. https://doi.org/10.1309/lmspkpzex602noks kzn department of health website [homepage on the internet]. [cited 2021 july 20]. available from: http://www.kznhealth.gov.za sharma s. tumour markers in clinical practice: general principles and guidelines. indian j of paediatr oncol. 2009;30(1):1–8. https://doi.org/10.4103/0971-5851.56328 sturgeon c. practice guidelines for tumour marker use in the clinic. clin chem. 2002;48(8):1151–1159. https://doi.org/10.1093/clinchem/48.8.1151 locker gy, hamilton s, harris j, et al. asco 2006 update of recommendations for the use of tumour markers in gastrointestinal cancer. j clin oncol;24(33):5313–5327. https://doi.org/10.1200/jco.2006.08.2644 abstract introduction methods results discussion conclusion acknowledgements references about the author(s) oluwalana t. oyekale department of medical microbiology and parasitology, faculty of medicine and health sciences, afe babalola university, ado ekiti, nigeria department of medical microbiology and parasitology, federal teaching hospital, ido-ekiti, nigeria bola o. ojo department of medical microbiology and parasitology, federal teaching hospital, ido-ekiti, nigeria adewale t. olajide department of surgery, faculty of medicine and health sciences, afe babalola university, ado ekiti, nigeria department of surgery, federal teaching hospital, ido-ekiti, nigeria oluwatoyin i. oyekale department of radiology, federal teaching hospital, ido-ekiti, nigeria citation oyekale ot, ojo bo, olajide at, oyekale oi. bacteriological profile and antibiogram of blood culture isolates from bloodstream infections in a rural tertiary hospital in nigeria. afr j lab med. 2022;11(1), a1807. https://doi.org/10.4102/ajlm.v11i1.1807 original research bacteriological profile and antibiogram of blood culture isolates from bloodstream infections in a rural tertiary hospital in nigeria oluwalana t. oyekale, bola o. ojo, adewale t. olajide, oluwatoyin i. oyekale received: 01 dec. 2021; accepted: 25 may 2022; published: 24 aug. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: bloodstream infections (bsis) are a cause of significant morbidity and mortality requiring urgent antibiotic treatment. however, there is widespread antibiotic-resistance from the bacterial causes, necessitating regular surveillance for drug-resistant bacteria and their antibiograms. objective: this study isolated and identified various bacterial causes of bsis, determined their antibiotic susceptibility patterns, and determined the best empirical treatment for cases of bsi in the setting. methods: a cross-sectional study was carried out at the federal teaching hospital, ido-ekiti, nigeria between june 2020 and february 2021 on 177 blood culture samples from cases of bsi. identification of isolated bacteria and antibiotic susceptibility testing of the isolates were carried out following the standard protocol. results: culture positivity in this study was 19.2%. no significant difference was seen in culture positivity between male and female participants (p = 0.97). gram-negative enteric bacteria were predominantly isolated (67.6%), including escherichia coli (29.4%) and klebsiella aerogenes (20.6%). staphylococcus aureus was the most common gram-positive bacterium isolated (23.5%). three (37.5%) s. aureus isolates were methicillin-resistant. all isolates were sensitive to meropenem, and 97.1% were sensitive to imipenem; other sensitivity patterns were: ceftazidime (85.3%), ciprofloxacin (79.4%), ofloxacin (79.4%), and gentamicin (76.5%). there was low sensitivity to ampicillin (32.4%) and cotrimoxazole (38.2%). all gram-positive isolates, including methicillin-resistant s. aureus, were sensitive to vancomycin. conclusion: regular surveillance of isolate sensitivity patterns, formulation of hospital antibiotic policies based on existing data and compliance with treatment guidelines will promote rational antibiotic use and reduce resistance among bacteria. keywords: antibiotic-resistance; antibiotic sensitivity; bacterial isolates; blood culture; bloodstream infections. introduction bloodstream infections (bsis), which range from self-limiting bacteraemia to an outright life-threatening septicaemia, are some of the most common healthcare-associated infections globally.1 bacteraemia is simply described as the presence of viable bacteria in the blood, while septicaemia connotes systemic manifestations caused by bacteria or their toxins in blood. septicaemia constitutes a significant cause of morbidity and mortality, requiring prompt assessment, diagnosis, and antibiotic treatment. bloodstream infections account for up to 9% – 11% of hospital-acquired infections in the developed countries of europe and the united states, while higher prevalence of up to 19% has been recorded from lowand middle-income countries of the world.2,3 a european study had estimated that bsi patients spend an additional 6.0–11.5 days in hospital compared to other patients and the cost of management associated with bsi ranges from $8000 united states dollars (usd) to $56 000 usd.4 the risk factors for bsis include the use of healthcare devices such as: peripheral and central venous catheters on patients; age (elderly patient, neonates); and premorbid medical conditions of patients, such as diabetes mellitus, malignancies, renal failure, burns, and prior hospitalisation.5 the mortality rate from bloodstream infections ranges from 4.0% to 41.5% depending on severity, age, sex, and other risk factors. infections due to antibiotic-resistant strains of bacteria present with a significantly higher morbidity and mortality.6 blood culture remains the gold standard in the laboratory diagnosis and identification of bloodstream pathogens; however, bacteria are not isolated in many cases of bsi.7 blood culture positivity rates among patients with bsi in developing countries range from 9.2% to 44.0%. there is a paucity of data from nigeria on bsi as a result of a poor surveillance system for the associated pathogens, thus depriving us of any useful antibiotic policy or treatment guidelines for these infections.8,9 the few studies carried out in nigeria on bsis were mainly among neonates and younger children. ogunkunle et al., iregbu et al., and uzodima et al., all from nigeria, reported blood culture positivity rates of 19.0%, 22.0% and 35.0%, respectively, among suspected cases of bsi.10,11,12 numerous bacteria have been associated with causation of bsis including gram-negative bacteria: escherichia coli, pseudomonas species (spp.), klebsiella spp., serratia spp., salmonella spp. and enterobacter spp.; and gram-positive bacteria: staphylococcus spp., streptococcus spp. and enterococcus spp.13,14 however, recent findings suggested an upsurge in bsis caused by multidrug-resistant bacteria, including the members of the enterobacteriaceae family and other gram-negative bacteria, such as klebsiella spp., pseudomonas spp., acinetobacter spp. and citrobacter spp., most of which are extended spectrum beta-lactamase (esbl) producers, and also some gram-positive bacteria, including methicillin-resistant staphylococcus aureus (mrsa) and the vancomycin-resistant enterococci.15,16 the carbapenem antibiotics remain the antibiotic agents of choice in the management of emerging esbl-producing gram-negative bacteria, while vancomycin is the mainstay in treatment of mrsa.17 however, of particular concern is the recent emergence of increasing resistance of some gram-negative bacteria to carbapenems through the production of enzymes, carbapenemases.18 this trend of multidrug resistance, especially among the gram-negative bacteria causing bsis, has created a very serious therapeutic dilemma, especially in the management of intensive care unit patients, since it leads to fewer therapeutic options, use of more expensive drugs, increased hospital stay, and increased morbidity and mortality.19 bearing in mind this trend of antibiotic-resistant bacterial agents of bsis and the known fact that antibiotic-resistance patterns vary with geographical locations, regular surveillance and documentation of blood culture isolates and their antibiogram is imperative in formulating an antibiotic policy and identifying the best empirical antibiotic therapeutic options for different scenarios of bsis in each hospital environment.20 this will encourage ‘rational use’ of antibiotics and reduce the tendency towards increasing antibiotic-resistance. there have been random reports of multiple antibiotic-resistant isolates causing a therapeutic dilemma among patients with bsis in our hospital with occasional attendant case fatalities in the past years. also, there was no existing antibiotic policy or treatment guideline for bsi management in our centre, as there was poor or non-existent surveillance for these multiple antibiotic-resistant pathogens. these factors necessitated this study. the objectives of this study are: to isolate and identify different bacterial causes of bsis; to determine the antibiotic susceptibility patterns of isolated bacteria; and to suggest the best empirical treatment of bsis in different scenarios in the hospital. methods ethical considerations ethical approval (protocol number: erc/2020/10/16/431a) for the study was obtained from the human research and ethics committee, federal teaching hospital, ido-ekiti, nigeria. written informed consent was also obtained from all participants prior to inclusion in the study. for underage participants, consent was sought and obtained from the parent/guardian. participants were de-identified by encoding their names with research numbers determined based on the units from which participants were recruited. study design and hospital setting this cross-sectional study was conducted at the department of medical microbiology and parasitology of federal teaching hospital, ido-ekiti, a rural southwestern nigerian teaching hospital. the hospital is a tertiary health facility which serves as a referral centre to other primary and secondary healthcare facilities in ekiti state, nigeria. it is a 290-bed hospital with many modern facilities for healthcare. study population and sampling method using a simple random sampling technique, a total of 177 clinically diagnosed bsi patients were recruited into the study, thus a total of 177 blood culture samples from different hospital units were received and investigated at the medical microbiology laboratory of the hospital between june 2020 and february 2021. patients’ clinical history and other relevant details were recorded in a predesigned form. blood sample collection and processing a set of two venous blood samples from two different sites were collected 30 minutes apart from each participant with suspected bsi, following strict aseptic precautions before commencement of antibiotic treatment. each set consisted of 8 ml – 10 ml of venous blood from adults, 2 ml of venous blood from neonates and 2 ml – 5 ml from other paediatric patients. blood samples were immediately inoculated into bact/alert® (biomérieux, inc., durham, north carolina, united states) aerobic blood culture bottles (adult or paediatric bottles, as necessary). these bottles were immediately incubated in a bact/alert® 3d automated blood culture analyser (biomérieux, inc., durham, north caroline, united states). all bact/alert-positive broths were immediately brought out for subculture onto blood agar (oxoid, wade road, basingstoke, united kingdom) and macconkey agar (oxoid, basingstoke, united kingdom). for those bottles without positive signals, blind subculture onto blood agar and macconkey agar was done on days 2, 5 and 7 of incubation. the inoculated blood agar and macconkey agar plates were incubated at 37 °c for 18 hours – 24 hours. any bacterial growth on agar plates was identified using colonial morphology, gram staining, and conventional biochemical tests using standard laboratory protocols.21 antibiotic susceptibility testing of isolated bacteria was performed on mueller-hinton agar (oxoid, basingstoke, united kingdom) using the modified kirby-bauer disc diffusion method. the results were interpreted as sensitive or resistant using the guidelines of the clinical and laboratory standard institute.22 the following antibiotics were tested on all identified isolates: imipenem (10 μg), meropenem (10 μg), ampicillin/sulbactam (20/10 μg), gentamicin (10 μg), ciprofloxacin (5 μg), ofloxacin (5 μg), cefriaxone (30 μg), ceftazidime (30 μg), amoxycillin/clavulanate (20/10 μg), cefixime (5 μg), and ampicillin (10 μg). all antibiotic discs were from oxoidtm (oxoid, wade road, basingstoke, united kingdom). all gram-negative bacteria isolates were tested for esbl production by the double disc synergy test using ceftazidime (30 μg) and ceftazidime/clavulanate (30/10 μg) discs, while the cefoxitin disc diffusion method was used to identify mrsa.22 all s. aureus isolated, including mrsa and coagulase-negative s. aureus (cons), were tested against vancomycin using etest® (biomérieux, inc., durham, north carolina, united states). inclusion and exclusion criteria all blood samples of patients with suspected bsi without a history of antibiotic medication prior to sample collection were included, while blood samples of bsi patients with a history of antibiotic medication prior to sample collection were excluded. data entry and analysis data were entered into microsoft excel 2017 (microsoft corporation, redmond, washington, united states) and data analysis was done using the statistical package for social sciences version 20.0 (ibm corp., armonk, new york, united states). results were presented in tables and expressed as frequencies and percentages. association between variables was tested using chi-square and/or fisher’s exact tests, as appropriate. statistical significance was accepted at p ≤ 0.05. results the age range of participants was 4 days to 87 years (mean: 23.29 ± 26.58 years) (table 1). of the 177 suspected cases of sepsis and other bloodstream-related infections that were investigated, 102 (57.6%) of the patients were male, and 75 (42.4%) were female. there were 27 (26.5%) male patients and 15 (20.0%) female patients below the age of one month (neonates), and 15 (14.7%) male patients and 23 (30.7%) female patients older than 50 years. table 1: age distribution of bloodstream infection study participants in relation to gender at the federal teaching hospital, ido-ekiti, nigeria, between june 2020 and february 2021. only 34 (19.2%) of the total blood culture broths were culture-positive for bacteria (table 2). culture positivity was highest among neonates (aged < 1 month; 23.8%) and lowest among patients aged 6–17 years (12.9%). culture positivity was higher among female patients (20.0%) compared to male patients (9.5%) aged 6–17 years, but no significant difference was seen (p = 0.57). no significant difference was seen in the overall isolation rate between male and female patients (p = 0.97). table 2: frequency of culture-positive broths by age group among bloodstream infection cases at the federal teaching hospital, ido-ekiti, nigeria, between june 2020 and february 2021. the most commonly isolated bacteria were e. coli (10/34, 29.4%), s. aureus (8/34, 23.5%), and k. aerogenes (7/34, 20.6%) (table 3). gram-negative bacteria species were the most commonly isolated (23/34 isolates, 67.6%) (table 4). gram-negative bacteria were isolated at a higher rate among neonates < 1 month (8/42 neonates, 19.0%) compared to other participants older than one month (15/135 participants, 11.1%) but no significant difference was seen in the rate of isolation (p = 0.18). the isolation rate was higher among neonates (10/42 neonates, 23.8%) compared to other participants older than one month (24/135 participants, 17.8%), but no significant difference was found in the isolation rate (p = 0.39). table 3: frequency of isolates from culture-positive broths among bloodstream infection cases by age group at the federal teaching hospital, ido-ekiti, nigeria, between june 2020 and february 2021. table 4: comparison of gram-positive and gram-negative isolates from bloodstream infection cases between neonates and other participants at the federal teaching hospital, ido-ekiti , nigeria, between june 2020 and february 2021. all (100.0%) of the isolates were sensitive to meropenem, 33/34 isolates (97.1%) were sensitive to imipenem, 29/34 isolates (85.3%) were sensitive to ceftazidime, 27/34 isolates (79.4%) were sensitive to ciprofloxacin and ofloxacin, and 26/34 isolates (76.5%) were sensitive to gentamicin (table 5). very poor sensitivity was observed for ampicillin (11/34 isolates, 32.4%) and cotrimoxazole (13/34 isolates, 38.2%). none of the gram-negative enteric bacteria isolated were esbl producers. three (37.5%) of the eight s. aureus isolated were mrsa. all of the isolated cons (3/3 isolates, 100%) and s. aureus (8/8 isolates, 100%), including the mrsa, were sensitive to vancomycin. table 5: antibiotic susceptibility pattern of isolates from bloodstream infection cases at the federal teaching hospital, ido-ekiti, nigeria, between june 2020 and february 2021. discussion the low culture positivity rate of 19.2% seen in this study is similar to findings in some previous studies. deku et al. reported a culture positivity rate of 13.1% in ghana and gupta et al. reported 16.5% in north india.23,24 lower rates have been reported in other studies: khanal et al. and gohel et al. reported a culture positivity rate of 10.3% and 9.2%, respectively, from bsi cases in india.8,25 a higher rate of 44.0% has been reported by khanal et al. in another prospective study on patients with infective endocarditis in india.9 most similar studies conducted in nigeria and africa focused mainly on neonatal and childhood bsis, and data on adult bsis is scanty. of those studies on neonatal bsis, some have reported similar culture positivity rates to the 23.8% seen among neonates in this study: ogunkunle et al. (19.0%) and iregbu et al. (22.0%), both in nigeria, while higher isolation rates were reported by sorsa in ethiopia (29.3%), uzodimma et al. in nigeria (35.0%) and el-din et al. in egypt (40.7%).10,11,12,26,27 factors determining rate of culture positivity include the methods or technique used in isolating bacteria, volume and the number of blood samples collected for the culture; also in addition, prior use of antibiotics before sample collection will affect the likelihood of culture positivity.28 in this study, adequate volumes of blood samples were collected before the commencement of antibiotic treatment, and patients with history of antibiotic medication prior to sample collection were excluded from the study, yet, prior self-medication at home or use of antibiotics at peripheral health facilities before transfer to our centre could not be totally ruled out. this coupled with the fact that some of these patients clinically diagnosed as bsi may actually have been suffering from other conditions mimicking bsi (and not bsi), thus accounting for the low culture positivity seen. gram-negative enteric bacteria (67.6%) were the predominant isolates in this study, of which e. coli (29.4%) was the most commonly isolated. staphylococcus aureus (23.5%) and cons (8.8%) were the only gram-positive bacteria isolated. similar findings have been reported by earlier studies where gram-negative bacteria were the predominant isolates from cases of bsi.24,28 however, some studies have reported s. aureus or cons as the most commonly isolated bacterial pathogen from cases of bsi.15,23,25,29,30,31 generally, there is wide variability in the pathogens isolated from cases of bsi in different settings. gram-positive bacteria were the most common cause of sepsis prior to the advent of antibiotics in the 1950s, but gram-negative bacteria became the most predominant after the introduction of antibiotics from the 1960s to 1980s. however, from the 1980s, gram-positive bacteria, most commonly staphylococcus spp., were thought to cause more than 50% of cases of sepsis.32,33 in hospital patients, there is a higher chance of hospital-selected gram-negative bacteria causing bsis due to the instruments and procedures carried out on these patients. this may account for the preponderance of gram-negative bacteria isolates in this study. predominant isolation of gram-negative enteric bacteria among neonates in this study contrasted with some previous studies on neonatal bsis: ogunkunle et al., sorsa and el-din et al. all reported gram-positive bacteria as the most commonly isolated pathogens from cases of neonatal bsis.10,26,27 this contrasting result may be because of the small number of neonates examined in this study compared to those other studies that focused mainly on neonates. iregbu et al., however, reported isolation of gram-positive and gram-negative bacteria in equal proportion from cases of neonatal bsis in abuja, nigeria.11 all of the cons and s. aureus, including the three mrsa isolated in this study, were sensitive to vancomycin. resistance of cons and mrsa to vancomycin is of immense challenge to the treatment of diseases caused by these strains because of associated persistent infections, vancomycin treatment failure and a generally poor clinical outcome. all of the isolates in this study were sensitive to meropenem, and 97.1% were sensitive to imipenem. these carbapenems represent the last resort, in antibiotic treatment, for most facilities in the less-developed world, where newer antibiotics are out of reach of the majority, especially for esbl-producing gram-negative bacteria.17 favourable sensitivity patterns were also seen in this study to other antibiotics, including ceftazidime (85.3%), ofloxacin (79.4%), ciprofloxacin (79.4%), and gentamicin (76.5%). the poor sensitivity pattern seen with these isolates to cotrimoxazole (32.4%), ampicillin (38.2%), amoxycillin-clavulanate (64.7%), ampicillin-sulbactam (67.6%) and ceftriaxone (67.6%) is undoubtedly a result of irrational antibiotic use. some of these drugs are regularly abused by patients even without prescription while others are indiscriminately used in our health facilities, resulting in the generation of resistance to these antibiotics. formulation of antibiotic policy on bsis from this data and compliance with treatment guidelines are of paramount importance, not only to save patients’ lives, but also to reduce further resistance generated by bacteria to more antibiotics. for the empirical treatment of bsis in this setting, a combination of ceftazidime with gentamicin is recommended in children less than 16 years of age, while a combination of ciprofloxacin/ofloxacin with gentamicin is recommended in the older age-groups. these three antibiotics demonstrated favourable sensitivity patterns against most bacteria isolated in this study; their combination in treatment of bsis will not only produce a synergistic effect, but will also reduce the rate at which isolates develop resistance to individual antibiotics. meropenem and imipenem should be reserved for cases not amenable to the aforementioned combination therapy. it is advisable that meropenem or imipenem should also be used in combination with other classes of antibiotics with a good sensitivity profile to the isolated pathogen, such as gentamicin, to reduce the speed at which bacteria generate resistance to these valuable drugs. in cases of bsi due to mrsa, vancomycin is the recommended treatment of choice in this setting. limitations the method of antibiotic sensitivity testing employed for all antibiotics (except vancomycin) tested against isolates in this study was limited to disc diffusion sensitivity testing. additional determination of the minimum inhibitory concentration of the different antibiotics which would have supplied quantitative data on the susceptibility pattern of isolates to these antibiotics was desired, but the study was limited by supply of funds. similarly, due to limited funding, we were unable to carry out the molecular characterisation of the resistant genes among the mrsa isolated. moreover, this study involved only our centre, which may have limited the relevance of data generated as regards antibiotic policy formulation and determination of treatment guideline for managing bsis throughout nigeria and other african countries, a multicentre study is desirable in future. conclusion gram-negative bacteria were predominantly isolated from cases of bsi. isolates demonstrated good sensitivity to meropenem, imipenem, ceftazidime, ciprofloxacin/ofloxacin, and gentamicin. regular surveillance of isolate sensitivity patterns, formulation of hospital antibiotic policies based on existing data, and compliance with treatment guidelines will promote rational antibiotic use and reduce resistance generation among bacteria. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions o.t.o. was responsible for conceptualisation of the study, design, definition of intellectual content, manuscript preparation, data collection, statistical analysis, and manuscript review. b.o.o. and a.t.o. were involved in data collection, literature search, experimental studies, data acquisition, manuscript, editing, and manuscript review. o.i.o. was involved in the literature search, data acquisition, manuscript editing, and manuscript review. sources of support the study was funded by all four authors. data availability the raw data from the study are available upon request from the corresponding author, o.t.o. disclaimer the views and opinions expressed in the article are those of the authors and do not necessarily reflect the critical policy or position of any affiliated agency or authors. references diekema dj, beekman se, chapin kc, morel ka, munson e, doern gv. epidemiology and outcome of nosocomial and community onset bloodstream infection. j clin microbiol. 2003;41(8):3655–3660. https://doi.org/10.1128/jcm.41.8.3655-3660.2003 report on the burden of endemic health care-associated infection worldwide [homepage on the internet]. geneva: world health organization; 2011 [cited 2021 oct 28]. available from: https://apps.who.int/iris/handle/10665/80135 suetens c, latour k, karki t, et al. prevalence of healthcare-associated infections, estimated incidence and composite antimicrobial resistance index in acute care hospitals and long-term care facilities: results from two european point prevalence surveys, 2016 to 2017. euro surveill. 2018;23(46):1800516. https://doi.org/10.2807/1560-7917.es.2018.23.46.1800516 cassini a, colzani e, pini a, et al. impact of infectious diseases on population health using incidence-based disability-adjusted life years (dalys): results from the burden of communicable diseases in europe study, european union and european economic area countries, 2009 to 2013. euro surveill. 2018;23:17-00454. https://doi.org/10.2807/1560-7917.es.2018.23.16.17-00454 sainfer a, bevin c, jianfang l, elaine l. prevalence and risk factors for bloodstream infections present on hospital admission. j infect prev. 2018;19(1):37–42. https://doi.org/10.1177/1757177417720998 christaki e, giamarellos-bourboulis ej. the complex pathogenesis of bacteremia: from antimicrobial clearance mechanisms to the genetic background of the host. virulence. 2014;5(1):57–65. https://doi.org/10.4161/viru.26514 paolucci m, landini mp, sambri v. how can the microbiologist help in diagnosing neonatal sepsis? int j pediatr. 2012;2012:120139. https://doi.org/10.1155/2012/120139 gohel k, jojera a, soni s, gang s, sabnis r, desai m. bacteriological profile and drug resistance patterns of blood culture isolates in a tertiary care nephrourology teaching institute. biomed res int. 2014;2014:153747. https://doi.org/10.1155/2014/153747 khanal b, harish bn, sethuraman kr, srinivasan s. infective endocarditis: report of prospective study in an indian hospital. trop doct. 2002;32(2):83–85. https://doi.org/10.1177/004947550203200208 ogunkunle to, abdulkadir mb, katibi os, bello so, raheem ra, olaosebikan r. pediatric blood culture isolates and antibiotic sensitivity pattern in a nigerian tertiary hospital. niger j med. 2020;29(2):261–264. https://doi.org/10.4103/njm.njm_55_20 iregbu kc, olufumilayo y, elegba oy, babaniyi ib. bacteriological profile of neonatal septicaemia in a tertiary hospital in nigeria. afr health sci. 2006;6(3):151–154. uzodimma cc, njokanma f, ojo o, falase m, ojo t. bacterial isolates from blood cultures of children with suspected sepsis in an urban hospital in lagos: a prospective study using bactec blood culture system. internet j pediatr neonatol [serial online]. 2013;16:1–8. available from: http://www.ispub.com ahmed d, nahid ma, sami ab, et al. bacterial etiology of bloodstream infections and antimicrobial resistance in dhaka, bangladesh, 2005–2014. antimicrob resist infect control. 2017;6:2. https://doi.org/10.1186/s13756-016-0162-z rolston kv, yadegarynia d, kontoyiannis dp, raad ii, ho dh. the spectrum of gram-positive bloodstream infections in patients with hematologic malignancies, and the in vitro activity of various quinolones against gram-positive bacteria isolated from cancer patients. int j infect dis. 2006;10(3):223–230. https://doi.org/10.1016/j.ijid.2005.05.007 rani nv, gopal k, narendra mv, et al. a retrospective study on blood stream infections and antibiotic susceptibility patterns in a tertiary care teaching hospital. int j pharm sci [serial online]. 2012 [cited 2021 aug 16];4:543–548. corpus id: 251044. available from: https://www.semanticscholar.org/paper/a-retrospective-study-on-blood-stream-infections-in-vanitharani-gopal/82b5b8979427f4472293928d6a53e7b528f5c8a9 ndugulile f, jureen r, harthug s, urassa w, langeland n. extended spectrum β‑lactamases among gram‑negative bacteria of nosocomial origin from an intensive care unit of a tertiary health facility in tanzania. bmc infect dis. 2005;5:86. https://doi.org/10.1186/1471-2334-5-86 vardakas kz, tansarli gs, rafailidis pi, falagas me. carbapenems versus alternative antibiotics for the treatment of bacteraemia due to enterobacteriacea producing extended spectrum beta lactamases: a systemic review and meta-analysis. j antimicrob chemother. 2012;67(12):2793–2803. https://doi.org/10.1093/jac/dks301 codjoe fs, donkor es. carbapenem resistance: a review. med sci (basel). 2017;6:1. https://doi.org/10.3390/medsci6010001 howard dh. the global impact of drug resistance. clin infect dis. 2003;36(suppl_1):s4–s10. https://doi.org/10.1086/344656 jadhav s, gandham n, paul r, misra rn. bacteriological profile of septicaemia and antimicrobial susceptibility of isolates from tertiary care hospital in india. res j pharm biol chem sci [serial online]. 2012 [cited 2021 sept 22];3(4):1100–1108. available from: https://www.researchgate.net/publication/286817962 collee, j.g., miles, r.s. and watt, b. tests for the identification of bacteria. in: collee, j.g., marmion, b.p., fraser, a.g. and simmons, a., eds., mackie & mccartney practical medical microbiology, 14th edition, churchill livingstone, new york, 1996; p. 131–151. clinical and laboratory standard institute. performance standards for antimicrobial susceptibility testing: twenty-fourth informational supplement [homepage on the internet]. clsi document m100-s27.wayne, pa: clinical and laboratory standards institute; 2014 [cited 2021 sept 26]. available from: https://www.nih.org.pk deku jg, dakorah mp, lokpo sy, et al. the epidemiology of bloodstream infections and antimicrobial susceptibility patterns: a nine-year retrospective study at st. dominic hospital, akwatia, ghana. j trop med. 2019;2019:6750864. https://doi.org/10.1155/2019/6750864 gupta s, kashyap b. bacteriological profile and antibiogram of blood culture isolates from a tertiary care hospital of north india. trop j med res. 2016;19(2):94–99. https://doi.org/10.4103/1119-0388.185426 khanal lk. bacteriological profile of blood culture and antibiogram of the bacterial isolates in a tertiary care hospital. int j health sci res [serial online]. 2020 [cited 2021 nov 28];10(8):10–14. available from: https://www.ijhsr.org/ijhsr_vol.10_issue.8_a_aug2020/3.pdf sorsa a. epidemiology of neonatal sepsis and associated factors implicated: observational study at neonatal intensive care unit of arsi university teaching and referral hospital, south east ethiopia. ethiop j health sci. 2019;29(3):333–342. https://doi.org/10.4314/ejhs.v29i3.5 shehab el-din em, el-sokkary mm, bassiouny mr, hassan r. epidemiology of neonatal sepsis and implicated pathogens: a study from egypt. biomed res int. 2015;2015:509484. https://doi.org/10.1155/2015/509484 lee a, mirrett s, reller lb, weinstein mp. detection of bloodstream infections in adults: how many blood cultures are needed? j clin microbiol. 2007;45(11):3546–3548. https://doi.org/10.1128/jcm.01555-07 reddy ea, shaw av, crump ja. community acquired bloodstream infections in africa: a systematic review and meta-analysis. lancet infect dis. 2010;10(6):417–432. https://doi.org/10.1016/s1473-3099(10)70072-4 mia ar, zerin t. antibiogram of blood culture isolates of patients from a hospital in dhaka, bangladesh. matrix sci med. 2020;4(1):1–5. https://doi.org/10.4103/mtsm.mtsm_4_19 sangita km, tomar r, saha nk. bacteriological profile and antibiogram of blood culture isolates from a tertiary care hospital. int j med sci innov res [serial online]. 2019;4(6):187–192. available from: http://www.ijmsir.com polat g, ugan ra, cadirci e, halici z. sepsis and septic shock: current treatment strategies and new approaches. eurasian j med. 2017;49(1):53–58. https://doi.org/10.5152/eurasianjmed.2017.17062 martins gs. sepsis, severe sepsis and septic shock: changes in incidence, pathogens and outcomes. expert rev anti infect ther. 2012;10(6):701–706. https://doi.org/10.1586/eri.12.50 article information authors: caroline mangare1 amos mbugua1 peter maturi2 jamila rajab2 rainer blasczyk3 hans-gert heuft3 affiliations: 1jomo kenyatta university of agriculture and technology, nairobi, kenya 2kenyatta national hospital/university of nairobi, department of hematology and blood transfusion, nairobi, kenya 3institute for transfusion medicine, hannover medical school, hannover, germany correspondence to: caroline mangare email: caroleunice2000@gmail.com postal address: 54128 00200 nairobi, kenya dates: received: 28 jan. 2015 accepted: 15 aug. 2015 published: 25 sept. 2015 how to cite this article: mangare c, mbugua a, maturi p, et al. red cell alloand autoimmunisation in transfused sickle cell and cancer patients in kenyatta national hospital, nairobi, kenya. afr j lab med. 2015;4(1), art. #297, 7 pages. http://dx.doi.org/10.4102/ajlm.v4i1.297 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. red cell alloand autoimmunisation in transfused sickle cell and cancer patients in kenyatta national hospital, nairobi, kenya in this original research... open access • abstract • introduction • methods    • setting and design    • data and sample collection    • laboratory investigations    • immunohaematological testing    • statistical analyses    • ethical considerations • results    • patient data    • serological results    • rbc alloantibody identification    • red cell autoimmunisation    • comparison of combined rbc alloand autoimmunisation in sickle cell versus cancer patients    • healthy donor phenotypes • discussion    • limitations    • conclusion • trustworthiness    • reliability and validity • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: currently, no data are available on the prevalence of red blood cell (rbc) antibody formation amongst kenyan patients with multiple transfusion needs, such as patients with sickle cell disease (scd) or haematological malignancies (hm) and solid (sm) malignancies. objectives: we determined the prevalence and specificities of rbc alloantibodies and autoantibodies in two patient groups with recurrent transfusion demands at kenyatta national hospital, nairobi, kenya. method: between february and august 2014, 300 samples from scd, hm and sm patients were collected and screened for alloantibodies. samples from 51 healthy blood donors were screened for irregular antibodies and phenotyped. results: amongst the 228 patients with viable samples (scd, n = 137; hm, n = 48; sm, n = 43), the median transfusion frequency was two to three events per group, 38 (16.7%) were rbc immunised and 32 (14.0%) had a positive direct antiglobulin test. we identified specific alloantibodies in six patients (2.6%). four of these six were scd patients (2.9%) who had specific rbc alloantibodies (anti-cw, anti-m, anti-cob, anti-s); amongst hm patients one had anti-k and one had anti-lea. rbc autoantibody prevalence was 3.1% (7/228). amongst the healthy blood donors, the ror, ccd.ee and r2r, ccd.ee phenotypes accounted for 82% of the rhesus phenotypes and all were kell negative. conclusion: the numbers of transfusions and the rates of rbc alloantibodies are low and the most important rbc alloantibody-inducing blood group antigens are relatively homogeneously distributed in this population. a general change in the kenyatta national hospital pre-transfusion test regimen is thus not necessary. the current transfusion practice should be reconsidered if transfusion frequencies increase in the future. introduction top ↑ blood transfusion constitutes an important supportive modality in the management of patients with sickle cell disease (scd) and cancer, because of longer periods of treatment and increased survival rates. scd is the most prevalent haematologic genetic disease in kenya1 and cancer is an increasingly important challenge for the kenyan public health system.2 red blood cell (rbc) alloand autoimmunisation often develop as a result of transfusions with allogeneic blood and occur because of the response of recipients’ immune systems to foreign rbc antigens from donors.3 some of the facets involved in these immunological reactions are: recipient age; sex; history of pregnancy; number of blood units transfused; and diagnosisand treatment-related impairment to the recipient's immune system.4,5 rbc alloimmunisation is associated with clinical complications, such as morbidity resulting from acute and delayed haemolytic transfusion reactions. the former can mimic a sickle cell crisis. furthermore, alloimmunisation creates difficulties for laboratories, including expensive and time-consuming laboratory workups to determine compatible blood, especially for cases with multiple alloantibodies (alloab). alloabs can become undetectable over time and/or be boostered as an anamnestic response after another transfusion. rbc alloab against incompatible rbc in an allogeneic bone marrow transplant may require procedures for rbc reduction.6,7 the development of alloab has been associated with that of autoantibodies (autoab),8,9 which can shorten the lifespan of recipients’ own rbcs and/or transfused rbcs and potentially cause haemolysis. because of this, these patients may require several transfusions and may need interventions, such as drugs to suppress the immune system and/or splenectomy.9 these challenges need to be considered when handling patients who are likely to be transfusion-dependent, as well as those who could benefit from haematopoietic stem cell transplantations. decreasing the risk of rbc alloimmunisation by implementing strategies to avoid allogeneic blood transfusions (e.g., erythropoietin administration in cancer patients) or extensive phenotypic matching of rbc blood group antigens, such as the rhesus, kell, duffy, kidd and mns blood groups, has been advocated previously.10,11 however, this is costly and impractical in many health settings, particularly in developing countries. studies conducted on the frequency of rbc alloimmunisation in different patient populations have reported rates of 1% to 6% in occasionally transfused patients and up to 30% in poly-transfused patients.12 in europe and the united states, alloimmunisation rates of 5% to 36% have been reported amongst transfused scd patients.13 currently, there are minimal data from africa regarding transfusion-dependent rbc allo-/autoimmunisation. the few existing studies have reported varied results. a ugandan study3 recently reported an rbc alloimmunisation prevalence rate of 6.1% amongst 428 scd patients. an investigation conducted in egypt amongst 42 scd patients reported an alloimmunisation rate of 21.4%,14 whereas amongst 130 tunisian thalassaemia patients, rbc alloimmunisation was 7.7% and 40% of these patients developed rbc autoantibodies.15 in a study of 108 ugandan patients with malignancies, alloimmunisation was reported at a frequency of 8.3%.16 there are no data on the prevalence of rbc alloab/autoab formation amongst kenyan patients, where pretransfusion testing is limited to abo/rhesus d group typing and crossmatching only. as there is no routine pre-transfusion rbc antibody screening or identification, this study sought to determine the prevalence and specificities of rbc alloabs and autoabs amongst two different groups of transfusion recipients at kenyatta national hospital (knh), nairobi, kenya. in addition, we screened samples from blood donors for irregular antibodies and phenotyped them for abo, rhesus and kell antigens in order to determine whether there is alloimmunisation in the general population served by knh. methods top ↑ setting and design using a cross-sectional design, scd, haematological malignancy (hm) and solid malignancy (sm) patients attending haematology and oncology clinics at knh were approached between february and august 2014 and invited to participate in the study. to be eligible for the study, participants had to be knh patients with scd, hm or sm who had received at least one allogeneic blood transfusion; 300 patients met the inclusion criteria. samples from 51 healthy blood donors of african ancestry from knh's blood bank were obtained for limited rbc antigen phenotyping. the kenya national blood transfusion policy defines the criteria for healthy donors as those who are aged 18–65 years; weigh more than 50 kg; have a minimum haemoglobin of 12 g/dl; have normal blood pressure (systolic 120–129 mmhg, diastolic 80–89 mmhg) and a pulse rate of 60–100 beats per minute.17 data and sample collection after obtaining informed consent, 2–4 ml of blood was drawn from patients into ethylenediaminetetraacetic acid tubes for laboratory investigations. patients’ notes were reviewed for: demographic characteristics; recipient age; sex; diagnosis; history of pregnancy; and transfusion history and indications. the number of blood components, units transfused and transfusion episodes were recorded. healthy blood donor samples were collected from donors who gave consent and met the healthy donor criteria. documentation of patient ethnicity is a routine requirement in kenyan medical records or clinical data. laboratory investigations plasma and rbcs were separated within two hours after collection and the plasma was frozen whilst the red cells were stored at 2 °c – 6 °c. rbcs were preserved by adding a drop of citrate phosphate dextrose anticoagulant. the samples were then shipped on dry ice at a controlled temperature to the institute for transfusion medicine at hannover medical school, hannover, germany for immunohaematological analysis. immunohaematological testing the bio-rad id gel card system (diamed-id®; bio-rad laboratories, diamed gmbh, cressier, switzerland) was used with both untreated and papain-treated rbc reagents. plasma samples were screened for the presence of rbc alloab by use of a standard three-cell panel of reagent group o rbcs using nacl gel cards at room temperature for alloabs with low thermal range and low ionic strength saline (liss) gel cards (liss/coombs) at 37 °c for warm-reacting alloabs. for samples that showed agglutination, subsequent antibody identification was carried out with at least one 11-cell group o rbc panel (usually bio-rad, switzerland). in instances without immediate determination of alloab specificity, additional cell panels (e.g., an 11-cell [grifols inc., los angeles, california, united states] and/or a 16-cell panel [sanquin, plesmanlaan, amsterdam, netherlands]) were used. if a patient's plasma sample showed agglutination of reagent screening cells, an autocontrol was also performed by reacting the patient's rbcs with his or her own plasma. positive autocontrols were further evaluated by means of a poly-/monospecific direct antiglobulin test (dat). dat was performed using monoclonal gel cards consisting of anti-igg, anti-iga, anti-igm, anti-c3c and anti-c3d. from samples that were positive with at least one of these antiglobulins, an acid eluate was prepared. the eluate was screened using the standard three-cell panel. those that were positive were then analysed for specificity using 11-cell panels containing these antigens: d, c, e, c, e, k, k, fya, fyb, jka, jkb, lea, leb, p1, m, n, s and s. in addition, donor blood was screened for irregular antibodies using a panel of three screening cells and then phenotyped for abo, rhesus (c, c, d, e, e) and kell antigens. plasma samples of 40 blood donors were screened for rbc alloab. patients were considered to be alloimmunised if antibodies to one or more rbc antigens could be identified, whilst autocontrol and dat screening remained negative. patients with a positive autocontrol, a positive dat and a reactive acid eluate were considered to be sensitised to have autoabs to rbcs. in cases with a positive autocontrol and a positive dat, but a non-reactive acid eluate, a non-specific loading of the rbc surface with immunoglobulins was assumed. ‘immune’ antibodies are formed after immunisation through pregnancy and/or previous transfusions. ‘naturally-occurring’ antibodies are formed as a result of exposure to environmental agents similar to red cell antigens, such as bacteria. statistical analyses statistical software packages were used: excel 5.0 (microsoft, redmond, california, united states 1993) for data management and statistical package for the social sciences 12.0 (spss inc., chicago, illinois, united states 2003) was used for analysis. student's t-test was used for variables with normal distribution. categorical variables of possible associations between rbc alloimmunisation and sex, units of blood transfusion, diagnosis of scd or solid and haematological malignancy were compared using the chi-squared test. groups were assumed to differ significantly when the probability level was less than 0.05. ethical considerations ethical approval was obtained from knh/university of nairobi ethics review committee. both oral and written informed consent was obtained from patients or their guardians. donors completed a questionnaire provided by the blood bank services and signed a consent form. results top ↑ patient data of the samples from 300 patients who met the inclusion criteria, 72 samples could not be evaluated for the following reasons: insufficient sample because of leakage during shipment (n = 40); samples breaking in the centrifuge whilst processing (n = 20); and lack of proper labeling (n = 12). a total of 228 samples were analysed, including 137 from scd patients, 48 from hm patients and 43 from sm patients, with a median number of two to three transfusions per group (table 1). of these, 117 (51.3%) were women, of whom 22 (18.8%) had a history of pregnancy. overall, the mean age at the time of blood transfusion was aged 17.2 years (range: 1–93). cancer patients, in particular sm patients, were significantly older than scd and hm patients (p < 0.001). indeed, the majority of patients were children aged 16 years or younger (n = 159; 70%); 14% were aged three years or younger (figures 1 and 2). there were no significant differences in the female to male ratios between the groups. figure 1: transfused sickle cell disease patients by age, sex and seropositivity at kenyatta national hospital, nairobi, kenya, 2014. figure 2: transfused cancer patients by age, sex and seropositivity at kenyatta national hospital, nairobi, kenya, 2014. table 1: characteristics of transfused sickle cell disease and cancer patients at kenyatta national hospital, nairobi, kenya, 2014. patients received abo⁄rhesus d compatible and non-leucocyte-depleted whole blood units (n = 532), packed rbc transfusions (n = 85) and platelet transfusions (n = 68), totaling 685 units of blood in 593 transfusion events (i.e., 1 transfusion unit per transfusion episode or a mean of 2.7 transfusion units per patient). of the scd patients, 90% were transfused because of severe anaemia – haemoglobin less than 5–6 g/dl according to world health organization guidelines.18 transfusion in malignancy patients was mainly a result of anaemia caused by intensive chemotherapy, by the disease process and/or surgical interventions. all cancer patients were receiving chemotherapy at the time of enrollment into the study. hm patients had the following types of malignancies: acute lymphoblastic leukaemia (n = 15), chronic lymphoblastic leukaemia (n = 10), hodgkin's lymphoma (n = 9), multiple myeloma (n = 6), acute myeloid leukaemia (n = 5) and chronic myeloid leukaemia (n = 3). sm patients had the following types of malignancies: abdominal tumour (n = 1), bladder (n = 2), breast (n = 8), cervical (n = 4), colon (n = 3), hepatocellular (n = 2), mouth (n = 1), nasopharyngeal (n = 1), pancreatic (n = 2), prostate (n = 4), rectum (n = 4), rhabdomyosarcoma (n = 4), sinonasal tumour (n = 1), squamous cell carcinoma (n = 2) and stomach cancer (n = 4). the p-value for the number of blood units transfused was 0.004, which was statistically significant as cancer patients received more transfusions. serological results the overall prevalence of rbc immunisation was 16.7%, with 38 of the 228 patients testing positive for antibody screening. the prevalence of rbc immunisation amongst scd patients was 20.4% (28 of 137 patients) and amongst malignancy patients, 11.0% (dat+, n = 8 plus alloab+, n = 2; altogether 10 out of 91 patients). rbc alloantibody identification only 38 patients were positive for the antibody screening, and rbc alloantibodies were detected in only 6 of 228 patients (2.6%) (table 2). the rate of alloab formation amongst scd patients was 2.9% (4 of 137) and 4.2% (2 of 48) amongst hm patients, whereas the prevalence amongst sm patients for alloab identification was 0. the specificities of the alloabs from the scd patients were anti-cw, anti-s, anti-cob (probably immune in nature) and anti-m (probably naturally occurring). in addition, there was one anti-k (immune) and one anti-lea (natural) in the two hm patients, whereas the sm group showed no rbc alloimmunisation. the rate of alloimmunisation was 6.14% for men versus 8.33% for women; the difference was not statistically significant (p = 0.25). table 2: serological results of transfused sickle cell disease and cancer patients at kenyatta national hospital, nairobi, kenya, 2014. red cell autoimmunisation of the 228 patients, 32 patients (14.0%) presented a positive dat (table 2). fifty per cent of these patients (16 of 32) were positive for anti-igg alone, whereas 18.8% (6 of 32) showed reactions to anti-igg plus anti-c3c or c3d. out of the subset of 21 igg-positive patients, the acid eluate was reactive in seven, thereby indicating a true rbc autoab prevalence of 3.1% for this population of patients (7 of 228) and 33.3% (7 of 32) amongst the dat-positive patients. rbc autoab prevalence was 5.1% (7 of 137) amongst scd patients, whereas there were no rbc autoabs amongst patients with malignancies. moreover, we observed a few cases (3 of 32) with isolated igm or iga reactivity. the majority (24 of 32) of the dat-positive reactions with anti-igm and anti-iga were observed in the scd group. eighteen per cent (24 of 137) of the scd group were dat positive compared with 8.8% (8 of 91) in the hm/sm group. comparison of combined rbc alloand autoimmunisation in sickle cell versus cancer patients the prevalence of rbc immunisation (demonstration of an immune alloab and a positive dat) amongst scd patients was 19.7% (27 of 137) versus 9.9% (9 of 91). immune alloabs were found in 2.2% (3 of 137) of the scd patients versus 1.1% (1 of 91) of the patients with malignancies. with one exception (polyspecific in eluate, but auto-anti-e in serum), these autoabs showed polyspecificity only. we also performed a comparison for demographic and transfusion variables between patients with and without serological reactivity (table 3). we did not find a significant link between patients’ sex, age or number of units of blood transfused and the positivity of the antibody screening. table 3: characteristics of immunised and non-immunised transfused sickle cell and cancer patients at kenyatta national hospital, 2014. healthy donor phenotypes amongst the blood donor samples, there were no serological peculiarities. fifty-one donors were phenotyped for the rhesus antigens c/c, d, e/e and for the antigen k (table 4). of these, 29 donors (57%) showed the rh phenotype ccd.ee, the other phenotypes were ccd.ee (n = 13), ccd.ee (n = 5), ccddee (n = 2) and single cases of ccddee (n = 1) and ccd.de (n = 1). none of the 51 donors were kell positive. table 4: rhesus and kell phenotypes amongst 51 healthy kenyan blood donors at kenyatta national hospital, 2014. discussion top ↑ the risk of alloimmunisation is a concern that needs to be addressed and managed, especially amongst patients requiring multiple blood transfusions, such as those with scd and malignances. this investigation sought to determine the magnitude of rbc immunisation and to identify antibodies amongst two transfused patient groups. in this study, we detected a significant proportion of patients with some degree of rbc autoimmunisation, as shown by a positive dat in 14.0% of the patients, seven of whom had true rbc autoantibodies. eighteen per cent of scd patients were dat positive compared with 8.8% of hm/sm patients. in contrast, rbc alloab formation was low, at only 2.6%. moreover, the specificities of the demonstrated alloabs do not occur often in daily laboratory results. anti-cw and anti-s are comparatively rare rhand mns antibodies, respectively; and anti-cob is a very rare rbc alloab specificity of the colton system. the one example of anti-k that we detected was the only common rbc alloab specificity. other common rbc alloab specificities, such as anti-d, anti-e, anti-c, anti-c, belonging to the rhesus system, or those of the kell system (other than anti-k), the duffy or the kidd blood group systems, were not found in our study population. in this study, the frequency of alloimmunisation across all patients was determined to be 2.6%; and the rate of alloab formation was 2.2% amongst patients with malignancies and 2.9% amongst scd patients. there may be several reasons for these unexpected findings. firstly, the total numbers of transfusions and the numbers of transfusion events were low, never exceeding the mean values of three transfusion events per patient. this was particularly true for the scd patients, who are known to be at high risk for rbc alloab formation.3,10,19 however, scd patients showed the lowest values for transfused rbc units per patient and transfusion events per patient in our study. the rbc alloimmunisation rate of 2.9% amongst our scd patients is comparable to a study in a jamaican cohort,19 where the rate was 2.6% amongst 115 transfused scd patients and 1.6% amongst the total number of 190 patients. however, this rate differs considerably from that reported in a ugandan study of 428 scd patients, where the prevalence rate was 6.1%.3 although the mean number of transfusions was three blood units in all of these studies, 21 of the 26 alloimmunised patients in the ugandan study had received up to 10 blood units. this is marginally higher than the maximum number (n = 8) of transfusions observed in our study. our scd patients received a mean of 2.4 units of transfusions, hm patients received 4.7 units and sm patients received 3.0 units. therefore, all groups were exposed to minimal antigenic challenge. numerous studies have reported that the rate of rbc alloimmunisation increases with the number of transfusions.3,10,19,20,21,22 this could explain the low alloimmunisation rate amongst our study participants compared with their counterparts in developed countries who received more transfusions. secondly, many other studies have reported higher percentages of rbc alloimmunisation in haemoglo­binopathies, such as scd or thalassaemia, including uganda3 (scd, alloab 6.1% amongst 428 patients), tunisia21 (scd and thalassaemia, alloab 7.8% amongst 309 patients), italy23 (thalassaemia, alloab 5% amongst 1435 patients) and brazil24 (scd, alloab 9.9% amongst 828 patients). these studies included a significantly higher number of patients; thus, the relatively low number of patients in our study might be a second limiting factor. the low rates in our study also differ from studies conducted in populations where there is high heterogeneity between donors and patients. in a study by rosse et al.,10 involving 1814 scd patients with an rbc alloimmunisation rate of 18.6%, the donors were of european-american ancestry and the scd patients were of african-american ancestry. thirdly, patients in our study were predominantly children aged 16 years or younger (n = 159; 70%), 14% were aged ≤ 3 years. studies of paediatric patients have reported lower rbc alloimmunisation rates. aygun et al.25 and sarnaik et al.21 concluded that children with scd who were hypertransfused had a lower frequency of alloimmunisation as compared with adults.21 another study involving 167 paediatric and 62 adult scd patients supported this observation, where the rates of alloand autoimmunisation in children and adults were 29% and 8%; 47% and 9.7%, respectively.26 other authors advocate that transfusion started when patients are young (aged 1–3 years) may induce immune tolerance against alloimmunisation.9 the fact that 14% of our study patients were aged ≤ 3 years could have contributed to the low rate of rbc alloab formation that we observed. fourth, the prevalence of rbc alloimmunisation amongst our cancer patients was low (2.2%), with only two hm patients and no alloabs amongst sm patients. this is lower than that in the ugandan study, where the rate was 8.3% amongst cancer patients. shahida et al.22 studied 150 cancer patients who had at least five transfusions and found the prevalence rate of alloabs to be 6%. in a study by seyfried and walewska11 of 1502 multi-transfused patients, the overall incidence of alloabs was 5.7%, with the lowest rate found amongst patients with lymphoproliferative syndromes (1.8%).11 of note, all the cancer patients in our study were undergoing chemotherapy at the time of transfusion. it has been observed that patients with progressive malignancies undergoing intensive chemotherapy tend to have a low antibody formation response to foreign antigens.26,27,28 finally, a majority (57%) of our donor population expressed the rh formula of ccd.ee, which could partly explain why we did not find rbc alloantibodies directed against highly immunogenic antigens such as d or e. a study by badjie et al.,29 conducted amongst 800 donors from various ethnic groups, found the prevalence of the ccd.ee phenotype to be 81.9% in east africa and a study by baby et al.30 found a prevalence of 67.9% in west africa (mali). these results suggest that a large proportion of donors – exceeding 50% – and transfusion recipients in africa share equal rh phenotypes, so that rh antibodies may be less frequently induced than in other parts of the world. this view is also supported by the low numbers for the ‘rr’ (rhesus-d negative) phenotype amongst our donor group (only 4%). this phenomenon might be also true for kell antibody formation, as we found no kell positive individuals amongst our donors. it has been reported that more than 98% of black africans are kell negative.30,31 we found a positive dat in 32 (14.0%) patients, with a subgroup of seven igg warm autoabs, which can induce significant clinical autoimmune haemolysis. we did not seek information about the presence of autoimmune haemolytic anaemia in these patients, because this can be clinically asymptomatic and the reaction can be masked by the severity of the underlying disease and lack of adequate post-transfusion records. limitations it has been reported that 25% of alloantibodies become undetectable within a median of 10 months of follow-up, which may lead to the underestimation of the prevalence of antibodies formed.10,32 this can result in a patient receiving rbcs and consequently experiencing a secondary immune response that may compromise the benefit of the following transfusion.33 because this was a cross-sectional study, some rbc alloantibodies might have been missed, since they have been reported to disappear with time.32,34 other factors that might also be responsible for the disparity in results include: the fact that the majority of the study patients were children; low mean of transfused units; inability to meet optimal transfusion needs for these patient groups; and the frequency of testing. conclusion in this study, we observed a low rate of rbc alloimmunisation amongst both scd and cancer patients. the low numbers of transfusions and transfusion events that are currently being applied at knh and the relatively homogeneous distribution of rh-/k-rbc alloantigens amongst kenyan donors provide an explanation for the low alloab frequency amongst kenyan transfusion recipients. at the current stage of the kenya health care system, routine antibody screenings or extended rbc antigen matching do not seem to be justified, as the relatively homogenous rbc alloantigen distribution of kenyan blood donors provides at least some protection from immune rbc alloab formation. however, with improvements in health care, more scd and haemato-oncology patients are likely to receive a more intensive transfusion treatment, which could lead to an increased risk of rbc alloimmunisation. therefore, further development of the healthcare system in kenya will require a thorough reconsideration of the pretransfusion laboratory practice, in particular, if transfusion frequencies increase and/or donor groups change. trustworthiness top ↑ this study reflects the findings obtained from laboratory testing and analysis as observed by the technical group. reliability and validity the experimental design and procedures used in this study are reliable and valid as they have been used previously in other studies, most of which are cited in this article. the results of the experiments in this article were obtained using specimens collected in various clinics at kenyatta national hospital, kenya and were analysed using standard procedures in hannover, germany. acknowledgements top ↑ the authors thank the staff of the hematology and oncology clinics and blood transfusion unit at knh, kenya for their help in recruiting patients and donors respectively. the authors also thank knh research and programs department for their monetary support. the authors thank the staff at the institute for transfusion medicine, hannover medical school, hannover, germany, for providing laboratory reagents, space and technical and medical advice. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions c.m. (jomo kenyatta university of agriculture and technology) was the project leader, developed the proposal, obtained ethical clearance for the study, collected and shipped specimens, performed most of the experiments and developed the manuscript. a.m. (jomo kenyatta university of agriculture and technology) made conceptual contributions. p.m. and j.r. (kenyatta national hospital/university of nairobi) were responsible for experimental and project designs. h.-g.h. and r.b. (hannover medical school) provided laboratory space and reagents and performed some of the experiments. all authors were responsible for polishing and approving the final manuscript. references top ↑ aluoch jr, aluoch lh. survey of sickle disease in kenya. trop geogr med. 1993;45(1):18–21. strother rm, asirwa fc, busakhala nb, et al. the evolution of comprehensive cancer care in western kenya. journal of cancer policy. 2013;1(1–2): e25–e30. http://dx.doi.org/10.1016/j.jcpo.2013.04.001 natukunda b, schonewille h, ndugwa c, et al. red blood cell alloimmunization in sickle cell disease patients in uganda. transfusion. 2010;50(1):20–25. http://dx.doi.org/10.1111/j.1537-2995.2009.02435.x klein hg, anstee dj. immunology of red cells. in: klein hg, anstee dj, editors. mollison's blood transfusion in clinical medicine. 11th ed. oxford: blackwell publishing, 2005; p. 48–113. http://dx.doi.org/10.1111/j.1537-2995.2007.01433.x bauer mp, wiersum-osselton j, schipperus m, et al. clinical predictors of alloimmunization after red blood cell transfusion. transfusion. 2007;47(11): 2066–2071. http://dx.doi.org/10.1111/j.1537-2995.2007.01433.x de la rubia j, arriaga f, andreu r, et al. development of non-abo rbc alloantibodies in patients undergoing allogeneic hpc transplantation. is abo incompatibility a predisposing factor? transfusion. 2001;41(1):106–110. http://dx.doi.org/10.1046/j.1537-2995.2001.41010106.x franchini m, gandini g, aprili g. non-abo red blood cell alloantibodies following allogeneic hematopoietic stem cell transplantation. bone marrow transplant. 2004;33(12):1169–1172. http://dx.doi.org/10.1038/sj.bmt.1704524 ahrens n, pruss a, mayer b, et al. association between alloantibody specificity and autoantibodies to red blood cells. transfusion. 2008;48(1):20–24. singer st, wu v, mignacca r, et al. alloimmunization and erythrocyte autoimmunization in transfusion-dependent thalassemia patients of predominantly asian descent. blood. 2000;96(10):3369–3373. rosse wf, gallagher d, kinney tr, et al. transfusion and alloimmunization in sickle cell disease. blood. 1990;76(7):1431–1437. seyfried h, walewska i. analysis of immune response to red blood cell antigens in multitransfused patients with different diseases. mater med pol. 1990;22(1): 21–25. norol f, nadjahi j, bachir d, et al. [transfusion and alloimmunization in sickle cell anemia patients] article in french. transfus clin biol. 1994;1(1):27–34. http://dx.doi.org/10.1016/s1246-7820(05)80054-0 talano ja, hillery ca, gottschall jl, et al. delayed hemolytic transfusion reaction/hyperhemolysis syndrome in children with sickle cell disease. pediatrics. 2003;111(6 pt 1);e661–e665. http://dx.doi.org/10.1542/peds.111.6.e661 aly r, el-sharnoby mr, hagag aa. frequency of red cell alloimmunization in patients with sickle cell anemia in an egyptian referral hospital. transfus apher sci. 2012;47(3):253–257. http://dx.doi.org/10.1016/j.transci.2012.07.014 guirat-dhouib n, mezri m, hmida h, et al. high frequency of autoimmunization among transfusion-dependent tunisian thalassaemia patients. transfus apher sci. 2011;45(2):199–202. http://dx.doi.org/10.1016/j.transci.2011.08.003 natukunda b, schonewille h, van de watering l, et al. prevalence and specificities of red blood cell alloantibodies in transfused ugandans with different diseases. vox sang. 2010;98(2):167–171. http://dx.doi.org/10.1111/j.1423-0410.2009.01241.x fhi360. guidelines for the appropriate use of blood and blood products. second edition april 2004 [document on the internet]. c2004 [20 april 2014]. available from: http://www.fhi360.org/sites/default/files/media/documents/guidelines%20for%20the%20appropriate%20use%20of%20blood%20and%20blood%20products.pdf world health organization. worldwide prevalence of anaemia 1993–2005: who global database on anaemia [document on the internet]. c2008 [24 april 2014]. available from: http://whqlibdoc.who.int/publications/2008/9789241596657_eng.pdf olujohungbe a, hambleton i, stephens l, et al. red cell antibodies in patients with homozygous sickle cell disease: a comparison of patients in jamaica and the united kingdom. br j haematol. 2001;113(3):661–665. http://dx.doi.org/10.1046/j.1365-2141.2001.02819.x hmida s, mojaat n, maamar m, et al. red cell alloantibodies in patients with haemoglobinopathies. nouv rev fr hematol. 1994;36(5):363–366. sarnaik s, schornack j, lusher jm. the incidence of development of irregular red cell antibodies in patients with sickle cell anemia. transfusion. 1986;26(3): 249–252. http://dx.doi.org/10.1046/j.1537-2995.1986.26386209381.x mohsin s, amjad s, amin h, et al. red cell alloimmunization in repeatedly transfused cancer patients. journal of rawalpindi medical college (jrmc). 2013;17(2):219–222. sirchia g, zanella a, parravicini a, et al. red cell alloantibodies in thalassemia major: results of an italian cooperative study. transfusion. 1985; 25(2):110–112. http://dx.doi.org/10.1046/j.1537-2995.1985.25285169198.x murao m, viana mb. risk factors for alloimmunization by patients with sickle cell disease. braz j med biol res. 2005;38(5):675–682. http://dx.doi.org/10.1590/s0100-879x2005000500004 aygun b, padmanabhan s, paley c, et al. clinical significance of rbc alloantibodies and autoantibodies in sickle cell patients who received transfusions. transfusion. 2002;42(1):37–43. http://dx.doi.org/10.1046/j.1537-2995.2002.00007.x schonewille h, haak hl, van zijl am. alloimmunization after blood transfusion in patients with hematologic and oncologic diseases. transfusion. 1999;39(7): 763–771. http://dx.doi.org/10.1046/j.1537-2995.1999.39070763.x lichtiger b, huh yo. autologous blood deposit and transfusion in cancer patients. current issues in transfusion medicine. 1992;1(1):10–14. quijada jg. post-transfusion alloimmunization (letter). br j haematol. 1996; 95(3):573–574. badjie ks, tauscher cd, van buskirk cm, et al. red blood cell phenotype matching for various ethnic groups. immunohematology. 2011;27(1):12–19. baby m, fongoro s, cissé m, et al. [frequency of red blood cell alloimmunization in polytransfused patients at the university teaching hospital of point g, bamako, mali] article in french. transfus clin biol. 2010;17(4):218–222. http://dx.doi.org/10.1016/j.tracli.2010.06.026 m’baya b, mfune t, mogombo e, et al. the prevalence of red-cell antigens and antibodies in malawi. transfus med. 2010;20(3):196–199. http://dx.doi.org/10.1111/j.1365-3148.2009.00985.x schonewille h, haak hl, van zijl am: rbc antibody persistence. transfusion. 2000;40(9):1127–1131. http://dx.doi.org/10.1046/j.1537-2995.2000.40091127.x natukunda b. red blood cell alloimmunization and antigen matching in sickle cell disease – the african perspective. isbt science series. 2012;7(1):129–133. http://dx.doi.org/10.1111/j.1751-2824.2012.01572.x reverberi r. the persistence of red cell alloantibodies. blood transfus. 2008; 6(4):225–234. article information authors: peter n. fonjungo1 mulu girma2 zenebe melaku3 teferi mekonen1 amilcar tanuri3 bereket hailegiorgis3 belete tegbaru2 yohannes mengistu1 aytenew ashenafi4 wubshet mamo5 tesfay abreha3 gudetta tibesso2 artur ramos6 gonfa ayana2 richard freeman7 john n. nkengasong6 solomon zewdu4 yenew kebede1 almaz abebe2 thomas a. kenyon1 tsehaynesh messele2 affiliations: 1center for disease control and prevention (cdc), addis ababa, ethiopia2ethiopian health and nutrition research institute (ehnri), addis ababa, ethiopia 3icap, columbia university, addis ababa, ethiopia 4john hopkins university tsehai program, addis ababa, ethiopia 5university of washington, itech program, addis ababa, ethiopia 6center for global health, centers for disease control and prevention (cdc), atlanta, usa 7clinton hiv/aids access initiative (chai), addis ababa, ethiopia correspondence to: peter fonjungo postal address: po box 1014, entoto road, addis ababa, ethiopia dates: received: 20 mar. 2012 accepted: 05 oct. 2012 published: 22 may 2013 how to cite this article: how to cite this article: fonjungo pn, girma m, melaku z, et al. field expansion of dna polymerase chain reaction for early infant diagnosis of hiv-1: the ethiopian experience. afr j lab med. 2013;2(1), art. #31, 7 pages. http://dx.doi.org/10.4102/ ajlm.v2i1.31 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. field expansion of dna polymerase chain reaction for early infant diagnosis of hiv-1: the ethiopian experience in this lessons from the field... open access • abstract • introduction • materials and methods    • project area    • approach to project implementation • results    • laboratory infrastructure and training    • scale-up of laboratory services    • quality of dbs dna pcr testing • ethical considerations • discussion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: early diagnosis of infants infected with hiv (eid) and early initiation of treatment significantly reduces the rate of disease progression and mortality. one of the challenges to identification of hiv-1-infected infants is availability and/or access to quality molecular laboratory facilities which perform molecular virologic assays suitable for accurate identification of the hiv status of infants.method: we conducted a joint site assessment and designed laboratories for the expansion of dna polymerase chain reaction (pcr) testing based on dried blood spot (dbs) for eid in six regions of ethiopia. training of appropriate laboratory technologists and development of required documentation including standard operating procedures (sops) was carried out. the impact of the expansion of eid laboratories was assessed by the number of tests performed as well as the turn-around time. results: dna pcr for eid was introduced in 2008 in six regions. from april 2006 to april 2008, a total of 2848 infants had been tested centrally at the ethiopian health and nutrition research institute (ehnri) in addis ababa, and which was then the only laboratory with the capability to perform eid; 546 (19.2%) of the samples were positive. by november 2010, ehnri and the six laboratories had tested an additional 16 985 hiv-exposed infants, of which 1915 (11.3%) were positive. the median turn-around time for test results was 14 days (range 14−21 days). conclusion: expansion of hiv dna pcr testing facilities that can provide quality and reliable results is feasible in resource-limited settings. regular supervision and monitoring for quality assurance of these laboratories is essential to maintain accuracy of testing. introduction top ↑ the joint united nations programme on hiv and/or aids (unaids) has estimated that in 2009 between 230 000 and 510 000 children under the age of 15 years became infected with hiv worldwide, with over 90% of these new infections occurring in sub-saharan africa, and mainly through mother-to-child-transmission.1 disease progression is aggressive in the first months of life in infants who acquired the infection in utero or around the time of delivery. if left untreated, almost half of these children will die before turning two years of age,2 and 75% by the age of five years. most of these deaths in children with hiv could have been avoided through early infant diagnosis (eid) and provision of effective care and treatment. interventions like the use of antiretroviral (art) drugs by infected pregnant women, safe delivery practices and safe infant feeding have helped reduce the risk of transmission to infants (from 40% to 5−10%).3,4,5,6 despite the availability of these life-saving interventions, some exposed infants still get infected. in 2003, art services were initiated in ethiopia and by 2005 free art services started with the support of the president’s emergency plan for aids relief (pepfar) and the global fund to fight aids, tuberculosis and malaria (gfatm).7 furthermore, these services have been rolled out to regional health facilities at both hospital and health centre levels. the total number of patients on art as of february 2010 was estimated at 179 183,8 including 10 496 children. one of the major challenges to expansion of art to children is limited or inadequate hiv diagnostic capacity. early identification of infants infected with hiv followed by prompt art treatment can help reduce morbidity and mortality. the need for early initiation of treatment in infants infected with hiv was emphasised in a south african cohort study that showed that 55% and 85% of hiv-infected infants had their cd4 cell count reduced to less than 25% by three and six months after birth, respectively.9 another study conducted in south africa showed that eid and early initiation of antiretroviral therapy helped to reduce mortality by 76% and hiv progression by 75%.10 other studies have shown improved outcomes when early art treatment is started in children.11,12 thus, laboratory diagnosis plays a critical role in increasing access of hiv-exposed infants to testing, and eventual timely and effective treatment and care. in adults, hiv is diagnosed by testing for the presence of antibodies to hiv in blood. in infants, antibody testing is inadequate, as persistent passively acquired maternal antibodies in the infant may yield false-positive results for up to 18 months or longer.13,14 it is important to provide accurate diagnostic services for identification of infants infected with hiv. because of its high sensitivity and specificity, dna polymerase chain reaction (pcr) has been widely used for diagnosis of hiv amongst exposed infants.15,16,17,18 the technology allows for pcr to be performed using a small spot from a dried blood spot (dbs) sample, as well as identification of infection from birth.19 additionally, this molecular test has been successfully established in several resource-limited settings to increase access to treatment.20,21,22,23 ethiopia has a tiered laboratory network, which is a hierarchical or ladder-like system with the national reference laboratory at the top followed by regional, referral and/or specialised hospital laboratories, then district and health centre laboratories. the tiered laboratory network reflects the structure through which health services are delivered to the population, and is under the coordination of the ethiopian health and nutrition research institute (enhri), which is the technical arm of the ministry of health, with responsibility and oversight of the country’s public health laboratories. ehnri has developed a comprehensive national laboratory master plan for integrated diseases. it has been used to coordinate and guide partners and stakeholders in the implementation of strategic laboratory objectives. the importance of the master plan in health system strengthening has previously been described.24 until october 2007, ehnri, which hosts ethiopia’s national hiv reference laboratory, was the only facility performing dna pcr for early infant diagnosis. ehnri was able to serve the entire country with the implementation of a sample referral system which allowed for dbs to be transported and tested, and returned results to the different regions of the country. ethiopia consists of about 1 127 127 square kilometers of land area, ranking it tenth in africa in terms of land size.25 the challenge was to scale up laboratory capacity for dna pcr in selected regions of ethiopia in order to provide better and more adequate coverage. here we report on the experience of expansion of dna pcr testing capabilities for early infant diagnosis to six different sites in ethiopia with a substantial increase in the numbers of hiv-exposed infants tested. materials and methods top ↑ project area the project was carried out in ethiopia, a country with a large land area situated in the horn of africa and divided into nine administrative regions and two metropolitan city administrations, addis ababa and dire dawa. hiv prevalence is estimated at 2.1%.26 there are an estimated 64 813 children under the age of 14 years living with hiv and/or aids, 75 420 hiv positive pregnant women per year with an estimated 14 138 new infections occurring in children annually.26 the decentralisation of dna pcr laboratories for eid was envisaged for six different sites to cover a greater geographic population. approach to project implementation in 2006, the national implementation plan for eid of hiv infection was developed by ehnri with the support of the us centers for disease control and prevention (cdc) and its partner, columbia university’s international center for aids care and treatment program (icap). the dbs-based dna pcr technology using the amplicor hiv-1 monitor v1.5 manual assay (roche diagnostics, indianapolis indiana, usa) was evaluated and eid services established at the national hiv reference laboratory of ehnri. dna pcr testing of dbs specimens at the national hiv reference laboratory started in april 2006. because of the vastness of the country and the time taken to transport dbs samples to the only eid laboratory at ehnri, there was the need to decentralise dna pcr testing for eid to the regions. in 2007, a joint assessment between cdc ethiopia, ehnri and university partners was conducted on the facilities in the identified regions. prior to this, a site assessment checklist was developed and used to assess potential sites. the joint site visit assessed state of infrastructure, personnel capacity, water, electricity and inventory, and provided recommendations. technical inputs were provided in the design of the laboratories, including physical separations of pre-pcr and post-pcr rooms. university partners (john hopkins, columbia university and university of washington) refurbished the laboratories in six regions for carrying out dna pcr for eid. cdc ethiopia and ehnri developed and purchased a comprehensive list of laboratory equipment, reagents and consumables. all sites had generators to serve as back-up for power outages. standard operating procedures were developed and distributed to all six sites. training materials were developed by ehnri in collaboration with cdc ethiopia and icap-columbia university and used at each site. at least two laboratory technologists were trained per site on dna pcr for eid using dbs. following training, laboratory technologists were independently assessed for accurately performing the test. two methods were used for assessing staff competency: the direct observational method was employed where the technologists were closely observed by a mentor as they performed assays independently per the sops and corrective actions provided where needed. additionally, each technologist was provided with six dbs of known results (blinded dbs specimen) and asked to perform assays unsupervised. as part of maintaining quality of testing in each laboratory, yearly refresher trainings were conducted, followed by challenging trainees with blind dbs specimens. additional trainings were provided for selected facilities based on the needs identified. also, to control for quality of testing in the laboratory, any two or more consecutive enzyme-linked immunosorbent assay (elisa) wells samples that were positive were each repeated from the same dbs card sample on a different day. ehnri served as a quality assurance laboratory for retesting the first 100 dbs specimens for each of the newly established sites. a comprehensive eid supply list was developed and laboratory technologists were trained in inventory management to ensure availability of supplies and thus avoid disruption of services as a result of shortage of one or several commodities. dna pcr testing started at the addis ababa regional laboratory in april 2008, and at the mekelle, adama and harar regional laboratories in may 2008, whilst at bahir dar and hawassa it started in june 2008. analysis of dbs tested for all sites was done up to november 2010. all six laboratories were enrolled in the cdc atlanta external quality assessment programme to monitor laboratory performance for dna pcr testing. proficiency testing (pt) panels were sent three times per year to each testing site. significant stages in the development and expansion of laboratory services for dna pcr for eid are presented here (figure 1). figure 1: milestones in expansion of laboratory services for early hiv infant diagnosis in ethiopia. in 2010, the postal service was introduced to transport dbs samples from referring sites to eid laboratories as well as delivering results back to referring sites. prior to initiation of sample referral using the postal service, a memorandum of understanding was signed between the ethiopian postal services enterprise and ehnri that included 565 facilities referring infant dbs samples. postal workers were trained in transportation of dbs, filling in of dbs samples transport log forms indicating date and time of pick up and drop off from dbs referring sites and eid laboratories, respectively. before the implementation of postal services in 2010, dbs samples and results were transported by an assigned facility staff member and/or university partners.the laboratory turn-around time was established with the help of the eid laboratory logbook. each dbs sample collected from an infant was accompanied by an eid request form from the referring site. the laboratory technologists recorded in the eid laboratory log book the date the dbs sample was received at the laboratory. when eid results are ready, the date the results are released or returned from the laboratory is recorded and signed for by a university partner or postal worker for the return of results to the referring site. results top ↑ laboratory infrastructure and training the six regional laboratories identified for expansion were strategically located around the country and included (1) bahir dar for the amhara region, (2) mekelle for tigray region, (3) hawassa for the southern nations, nationalities and people’s (snnp) region, (4) adama for the oromia region, (5) harar for the harari and (6) somali regions and addis ababa for addis ababa city administration (figure 2). by june 2008, all six identified regional reference laboratories had been completely renovated and well equipped with the instruments, software and laboratory consumables required for performing hiv-1 dna pcr. figure 2: regionalisation of early infant diagnosis. the laboratory technologists were trained in laboratory biosafety, dbs specimen reception and/or rejection using criteria developed by cdc,27 accurately performing and interpreting test results, and reporting results back to referral sites. a total of 12 laboratory technologists (2 per site) were successfully trained and certified competent on-site for hiv-1 dna pcr testing. the laboratory technologists each scored 100% in their competency assessment when challenged with six blind specimens. that is, they correctly interpreted test kit controls, in-house controls, as well as all blind dbs specimens. the modest number of laboratory technologists trained was aimed at ensuring that testing services would be minimally disrupted should one technologist be absent. sample referral testing documentation and mechanisms to enable specimens to be transferred from referring sites to laboratories performing dna pcr as well as reporting results back to referring sites were fully established. scale-up of laboratory services prior to april 2008, only prior to decentralisation the national hiv reference laboratory at ehnri, located in addis ababa, was performing dna pcr for eid (figure 2). from 2006 to april 2008, the national hiv reference laboratory had tested a total of 2848 infants’ dbs samples, of which 546 (19.2%) were positive and the results were sent back to referring sites (figure 3). the dbs of hiv-exposed infants from prevention of mother-to-child transmission services and diagnostic specimens for clinical symptoms were tested. the results obtained for all laboratories that tested and provided infant dbs results up to november 2010 are shown in figure 3. of the 2209 tested for bahir dar, 278 (12.6%) were positive; 330 (7.5%) of 4369 were positive for addis ababa; 123 (10.1%) of 1222 were positive for hawassa; 594 (13.1%) of 4525 positive for adama; 117 (11.6%) of 1007 tested positive for harar; and 214 (13.9%) of 1542 tested positive for mekelle (figure 3). following decentralisation, ehnri tested 2110 specimens, of which 259 (12.3%) were positive (figure 3, ehnri). figure 3: proportion of dried blood spot (dbs) tested positive by site. although the number of specimens tested at ehnri declined, ehnri was still serving as a quality assurance laboratory for the newly established sites and testing specimens received from non-governmental organisation facilities, military and police hospitals, afar, gambella and benishangul gumuz regions. of the total 19 832 dbs specimens tested from all testing sites between april 2006 and november 2010, 2461 (12.4%) were positive. at the end of 2007, 26 clinical sites were collecting dbs samples. a total of 565 active facilities are currently sending infant samples to these testing laboratories. together with the establishment of these new collection sites, there was a substantial increase in the number of samples tested following decentralisation of laboratory services from 2008 and each year thereafter (figure 4). figure 4: the total number of dried blood spot (dbs) specimens tested and the number positive by year between 2006 and 2010. before decentralisation (april 2006 to march 2008), all the dbs samples were tested only at ehnri laboratory and the median turn-around time was 13 days (range 1−59 days). after decentralisation (april 2008 to november 2010), the median turn-around time at ehnri was 12 days (range 1-63 days). after decentralisation, the other six regional laboratories in the country had a median turn-around time of 14 days (range 14−21 days). quality of dbs dna pcr testing in addition to routinely conducting cdc atlanta ’in-house’ dbs controls and test kit controls to validate performance of assays, all laboratories performed satisfactorily by scoring 100% in all pt sessions with the cdc pt panel programme. the quality assurance results on the first 100 dbs specimens for each of the newly established sites was concordance between results of ehnri and those of the newly established laboratories. the repeat testing of any two or more consecutive positive samples showed concordance between the initial and the repeated test results. ethical considerations top ↑ because this was a programme impact assessment of expansion of dna pcr laboratory for eid, no ethical approval was required. discussion top ↑ in this project, we have demonstrated the feasibility of laboratory expansion of dna pcr testing for eid in a resource-limited setting. following the expansion of testing facilities and the establishment of new collection sites, there was an increase in the number of infant dbs samples tested. early diagnosis of hiv-infected infants can enable faster access to treatment and, in combination with other indicators, can contribute to monitoring the success in programmes for the prevention of mother-to-child transmission. hiv dna pcr is recommended as the gold standard for virologic diagnosis of hiv in infants. however, issues with the high expense associated with establishing a virologic testing laboratory, technical complexities as well as the need to ensure good quality control and assurance in a resource-constrained setting have been raised. this has led to the establishment of a few facilities in centralised areas in resource-limited countries.the commitment of the government and partners was key to the rapid and successful regional expansion of eid molecular technology. the expansion of the laboratory diagnostics services is in line with one of the strategic objectives of the master plan developed by ehnri. this further demonstrates the importance of developing a master plan with clear strategic objectives that serves as a rallying and coordinating tool for implementing laboratory programmes amongst several laboratory stakeholders as well as evaluation of the implementation of the master plan. there was also the need to quickly establish good laboratory practice in these new facilities and to maintain quality of testing, considering the technical complexities of such molecular methods and the fact that infants may only have the opportunity for a single hiv test to determine their hiv infection status. proper on-site training and competency assessment coupled with yearly refresher training and regular joint supportive site visits by ehnri and partners have been important to maintaining quality testing. this has also been reflected in the satisfactory performance (100%) of all the facilities in cdc external quality assessment through pt panels. following decentralisation of dna pcr testing sites and scaling up of the number of collection sites, we observed a significant increase in the number of infants tested with dbs without an increase in the turn-around time from when specimens are received at the laboratory to return of results from the laboratory. with decentralisation, we observed that turn-around time of results to referring sites was a median of 14 days. this reflects a strong and functional sample and results referral system for expediting results. however, because of the low number of dbs specimens and the need for batch testing, it still took harar and hawassa up to three weeks to return results. who guidelines recommend that hiv virologic tests be used to diagnose hiv infection in infants and children up to the age of 18 months.29 additionally, the guidelines emphasise post-natal follow up of infants with unknown or uncertain hiv exposure in order to have their hiv exposure status ascertained and that hiv-infected infants be put immediately on art. although 90% of children living with hiv acquired their infection through mother-to-child transmission, relatively few have access to testing, treatment and care.30 there are several challenges to implementing early treatment for hiv-infected infants, including the diagnosis of hiv-infected infants, lack of rapid diagnostic capabilities for infants, weak sample referral testing systems, under-developed quality controls, limited trained personnel, inadequate clinical systems to follow up with patients, lack of integration amongst health services, and exogenous social factors. addressing these challenges is likely to improve and maintain quality of testing and lead to increase uptake of hiv-infected infants to receiving treatment. direct comparison of the number of dbs tested at the different sites is difficult. the number of infants tested at the new facilities varied greatly, with bahir dar, addis ababa and adama each having tested about two or more times as many as hawassa or harar. the observed difference could partly be explained by the population size and hiv prevalence of these regions, as well as the robustness of their pmtct programme and clinical practices. of the nine regions, bahir dar and adama in amhara and oromiya regions, respectively, account for 23% and 36%, respectively, of the general population. also, the hiv prevalence of the two regions is 2.7% and 1.5% for amhara and oromiya, respectively.26 the city of addis ababa site also showed an increase in dbs testing but accounts for only 3% of the population. however, the city of addis ababa has a relatively higher hiv prevalence of 7.5%.26 by contrast, hawassa in the snnp region, which accounts for about 20% of the population, tested only 1222 dbs specimens. together with the harar and mekelle sites, they tested fewer dbs specimens. these low numbers could be due to challenges with coordination of pediatric hiv care and treatment, pmtct, and laboratory services in these regions. despite the successful scale-up of laboratory diagnostic services, significant challenges still persist and pose a problem for the provision of more comprehensive services for diagnosis and treatment of children. for example, the need for proper integration or linkages between programmes (e.g., laboratory and clinicians), identification of pregnant women, enrolling and scaling-up of the pmtct programme. furthermore, it has been difficult to determine the number of infants testing positive who received their test results and who received treatment and care. it is important to put in place a system for tracking dbs-positive results to ensure those infants receive treatment as well as for negative pcr results, to ensure that uninfected infants who continue to be exposed to hiv receive a final diagnosis. similarly, improving the system of follow-up for infants and their caretakers is critical to avoid delays in initiating treatment. the rapid scale-up of laboratory services to perform dna pcr for eid and treatment of infected children is an essential component to child survival and successful outcomes of pmtct programmes. proper joint planning, implementation, commitment and coordination involving stakeholders have made rapid scale-up possible. continuous monitoring of these new sites is essential, and adhering to all aspects of quality would ensure that accurate results are provided for prompt intervention. acknowledgements top ↑ the authors thank the regional health bureau heads, regional laboratory heads and laboratory technnologists from bahir dar regional laboratory (amhara region), mekelle regional laboratory (tigray region), hawassa regional laboratory (snnp), adama regional laboratory (oromiya region), harar regional laboratory (harari and somali regions) and addis ababa regional laboratory (addis ababa city administration) for their helpful assistance in facilitating and ensuring infrastructure renovations and trainings were carried out properly and in a timely manner. we are grateful to jelaludin ahmed for providing the map of ethiopia. competing interests the authors declare that they have no financial or personal relationship(s), which may have inappropriately influenced them in writing this article. authors’ contributions p.n.f. (cdc ethiopia), t.m. (ehnri), z.m. (icap, columbia university), y.k. (cdc ethiopia), g.t. (ehnri), m.g. (ehnri) and y.m. (cdc ethiopia) conceived and designed the study. a.t. (icap, columbia university), b.h. (icap, columbia university), a.r. (cdc atlanta) and b.t. (ehnri) validated dbs technology for eid at ehnri hiv reference laboratory. p.n.f. (cdc ethiopia), t.m. (cdc ethiopia), y.k. (cdc ethiopia), b.t. (ehnri), y.m. (cdc ethiopia), b.h. (icap, columbia university), g.a. (ehnri), r.f. (chai ethiopia), a.a. (ehnri) coordinated implementation and rollout of the program centrally. t.m. (cdc ethiopia), m.g. (ehnri), p.n.f. (cdc ethiopia), b.h. (icap, columbia university), r.f. (chai ethiopia), a.a. (john hopkins university tsehai program) and s.z. (john hopkins university tsehai program) were involved in rollout of dna pcr laboratories in addis ababa and hawassa. t.m. (cdc ethiopia), m.g. (ehnri), b.h. (icap, columbia university), r.f. (chai ethiopia), t.a. (icap, columbia university) and z.m. (icap, columbia university) were involved in rollout of dna pcr laboratories in adama and harar. t.m. (cdc ethiopia), m.g. (ehnri), p.n.f. (cdc ethiopia), b.h. (icap, columbia university), r.f. (chai ethiopia), w.m. (university of washington, itech program) were involved in rollout of dna pcr laboratories in mekelle and bahir dar. t.a.k. (cdc ethiopia) and j.n.n. (cdc atlanta) contributed original ideas, ongoing discussions on all drafts. all co-authors provided critical feedbacks on all drafts. references top ↑ 1. unaids. unaids report on the global aids epidemic 2010. [unaids global aids report web site]. [cited 2011 apr 4] available from http://www.unaids.org/globalreport/global_report.htm 2. newell ml, coovadia h, cortina-borja m, et al. mortality of infected and uninfected infants born to hiv-infected mothers in africa: a pooled analysis. lancet. 2004;364:1236–1243. 3. bulterys m, fowler mg, van rompay kk, kourtis ap. prevention of mother-to-child transmission of hiv-1 through breast-feeding: past, present, and future. j infect dis. 2004;189:2149–2153. 4. becquet r, ekouevi dk, viho i, et al. acceptability of exclusive breast-feeding with early cessation to prevent hiv transmission through breast milk, anrs 1201/1202 ditrame plus, abidjan, côte d’ivoire. j acquir immune defic syndr. 2005;40:600–608. 5. dabis f, bequet l, ekouevi dk, et al. field efficacy of zidovudine, lamivudine and single-dose nevirapine to prevent peripartum hiv transmission. aids. 2005;19:309–18. 6. leroy v, ekouevi dk, becquet r, et al. 18-month effectiveness of short-course antiretroviral regimens combined with alternatives to breastfeeding to prevent hiv mother-to-child transmission. plos one. 2008;3(2):e1645. 7. assefa y, jerene d, lulseged s, ooms g, van damme w. rapid scale-up of antiretroviral treatment in ethiopia: successes and system-wide effects. plos med. 2009;6:1–4. 8. federal ministry of health, ethiopia. federal hiv/aids prevention and control office. monthly hiv care and art update. [updated 2010 feb; cited 2012 sep 19]. available from http://www.etharc.org/resources/download/finish/42/347 9. mphatswe w, blanckenberg n, tudor-williams g, et al. high frequency of rapid immunological progression in african infants infected in the era of perinatal hiv prophylaxis. aids. 2007;21:1253–1261. 10. cotton mf, gibb dm, et al. early antiretroviral therapy and mortality among hiv-infected infants. n engl j med. 2008;359:2233–2244. 11. kumarasamy n, venkatesh kk, devaleenol b, poongulali s, mothi sn, solomon s. safety, tolerability and effectiveness of generic haart in hiv-infected children in south india. jtrop pediatr. 2009;55:155–159. 12. raguenaud me, isaakidis p, zachariah r, et al. excellent outcomes among hiv+ children on art, but unacceptably high pre-art mortality and losses to follow-up: a cohort study from cambodia. bmc pediatr. 2009;20:9:54. 13. rakusan ta, parrott rh, sever jl. limitations in the laboratory diagnosis of vertically acquired hiv infection. j acquir immune defic syndr. 1991;4:116–121. 14. gutierrez m, ludwig da, khan ss, et al. has highly active antiretroviral therapy increased the time to seroconversion in hiv exposed but uninfected children? clin infect dis. 2012;55(9):1255–1261. 15. bremer jw, lew jf, cooper e et al. diagnosis of infection with human immunodeficiency virus type 1 by a dna polymerase chain reaction assay among infants enrolled in the women and infants’ transmission study. j pediatr. 1996;129:198–207. 16. beck ia, drennan kd, melvin aj, et al. simple, sensitive, and specific detection of human immunodeficiency virus type 1 subtype b dna in dried blood samples for diagnosis in infants in the field. j clin microbiol. 2001;39:29–33. 17. sherman gg, stevens g, jones sa, horsfield p, stevens ws. dried blood spots improve access to hiv diagnosis and care for infants in low-resource settings. j acquir immune defic syndr. 2005;38:615–617. 18. sherman gg, cooper pa, coovadia ah, et al. polymerase chain reaction for diagnosis of human immunodeficiency virus infection in infancy in low resource settings. pediatr infect dis j. 2005;24:993–997. 19. fischer a, lejczak c, lambert c, et al. simple dna extraction method for dried blood spots and comparison of two pcr assays for diagnosis of vertical human immunodeficiency virus type 1 transmission in rwanda. j clin microbiol. 2004;42:16–20. 20. creek tl, sherman gg, nkengasong j, et al. infant human immunodeficiency virus diagnosis in resource-limited settings: issues, technologies, and country experiences. am j obstet gynecol. 2007;197(suppl 2):64–71. 21. stevens w, sherman g, downing r, et al. role of the laboratory in ensuring global access to arv treatment for hiv-infected children: consensus statement on the performance of laboratory assays for early infant diagnosis. open aids j. 2008;2:17–25. 22. rollins n, mzolo s, moodley t, esterhuizen t, van rooyen h. universal hiv testing of infants at immunization clinics: an acceptable and feasible approach for early infant diagnosis in high hiv prevalence settings. aids. 2009;23:1851–1857. 23. stevens ws, noble l, berrie l, sarang s, scott le. ultra-high-throughput, automated nucleic acid detection of human immunodeficiency virus (hiv) for infant infection diagnosis using the gen-probe aptima hiv-1 screening assay. j clin microbiol. 2009;47:2465–2469. epub 2009 may 27. 24. nkengasong jn, mesele t, orloff s, et al. critical role of developing national strategic plans as a guide to strengthen laboratory health systems in resource-poor settings. am j clin pathol. 2009;131:852–857. 25. joinafrica.com. area of african countries. [joinafrica.com web site]. [cited 2012 sep 19] available from http://www.joinafrica.com/country_rankings/area_africa.htm 26. federal ministry of health ethiopia. single point prevalence. june 2007. [cited 2012 sep 19] available from http://www.etharc.org/resources/healthstat/nationalfactsheet/11-nationalfactsheet2007 27. dried blood spot (dbs) acceptance and rejection criteria job aid. [women, children, and hiv web site]. [cited 2012 sep 19] available from http://www.womenchildrenhiv.org/wchiv?page=ch-09-03-eid 28. parsons lm, shanmugam v, ou cy, nkengasong j. proficiency testing for dbs-based infant diagnostics using dna pcr. paper presented at: who-cdc informal consultation to review the performance of the dna pcr assay for early hiv-1 diagnosis in pmtct programs in different countries with diverse hiv-1 subtypes. may 2006; entebbe, uganda. 29. who recommendations on the diagnosis of hiv infection in infants and children. 2010. [who hiv/aids web site]. [cited 2012 sep 19] available from http://www.who.int/hiv/pub/paediatric/diagnosis/en/index.html 30. scale up of hiv-related prevention, diagnosis, care and treatment for infants and children. 2008. [who hiv/aids web site]. [cited 2012 sep 19] available at: http://www.who.int/hiv/pub/paediatric/framework_2008/en/index.html abstract introduction methods results discussion conclusion acknowledgements references about the author(s) sarah j. girdwood health economics and epidemiology research office, department of internal medicine, school of clinical medicine, faculty of health sciences, university of the witwatersrand, johannesburg, south africa department of medical microbiology, amsterdam university medical center, amsterdam, the netherlands thomas crompton right to care, johannesburg, south africa naseem cassim department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa national health laboratory service, johannesburg, south africa floyd olsen national health laboratory service, johannesburg, south africa portia sejake national health laboratory service, johannesburg, south africa karidia diallo centers for disease control and prevention, pretoria, south africa leigh berrie centers for disease control and prevention, pretoria, south africa dorman chimhamhiwa right to care, johannesburg, south africa wendy stevens department of molecular medicine and haematology, faculty of health sciences, university of the witwatersrand, johannesburg, south africa national health laboratory service, johannesburg, south africa brooke nichols health economics and epidemiology research office, department of internal medicine, school of clinical medicine, faculty of health sciences, university of the witwatersrand, johannesburg, south africa department of medical microbiology, amsterdam university medical center, amsterdam, the netherlands department of global health and development, school of public health, boston university, boston, massachusetts, united states citation girdwood sj, crompton t, cassim n, et al. optimising courier specimen collection time improves patient access to hiv viral load testing in south africa. afr j lab med. 2022;11(1), a1725. https://doi.org/10.4102/ajlm.v11i1.1725 note: additional supporting information may be found in the online version of this article as online supplementary document 1 original research optimising courier specimen collection time improves patient access to hiv viral load testing in south africa sarah j. girdwood, thomas crompton, naseem cassim, floyd olsen, portia sejake, karidia diallo, leigh berrie, dorman chimhamhiwa, wendy stevens, brooke nichols received: 02 sept. 2021; accepted: 24 may 2022; published: 25 oct. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: south africa uses a courier network for transporting specimens to public laboratories. after the daily collection of specimens from the facility by the courier, patients not yet attended to are unlikely to receive same-day blood draws, potentially inhibiting access to viral load (vl) testing for hiv patients. objective: we aimed to design an optimised courier network and assess whether this improves vl testing access. methods: we optimised the specimen transport network in south africa for 4046 facilities (november 2019). for facilities with current specimen transport times (n = 356), we assessed the relationship between specimen transport time and vl testing access (number of annual vl tests per antiretroviral treatment patient) using regression analysis. we compared our optimised transport times with courier collection times to determine the change in access to same-day blood draws. results: the number of annual vl tests per antiretroviral treatment patient (1.14, standard deviation: 0.02) was higher at facilities that had courier collection after 13:36 (the average latest collection time) than those that had their last collection before 13:36 (1.06, standard deviation: 0.03), even when adjusted for facility size. through network optimisation, the average time for specimen transport was delayed to 14:35, resulting in a 6% – 13% increase in patient access to blood draws. conclusion: viral load testing access depends on the time of courier collection at healthcare facilities. simple solutions are frequently overlooked in the quest to improve healthcare. we demonstrate how simply changing specimen transportation timing could markedly improve access to vl testing. keywords: hiv viral load; scale-up; patient access; south africa; specimen transport. introduction the national health laboratory service (nhls) is the largest diagnostic pathology service provider in south africa, providing laboratory and related public health services to over 80% of the population.1 through a national network of laboratories, the nhls is responsible for supporting the national and provincial health departments in the delivery of healthcare and manages a courier network for transporting specimens from healthcare facilities to centralised testing laboratories. couriers collect specimens from all health facilities at least once per day and more frequently at high-volume facilities. however, anecdotal evidence suggests that after specimens have been collected by the courier from primary healthcare (phc) facilities for the day, patients that are still in the queue are unlikely to have their blood drawn that day, potentially inhibiting access to essential diagnostics. in the case of hiv patients specifically, if the patient is unable to return the following day, viral load (vl) testing could be delayed until the next clinical visit or when the patient is required to collect their next antiretroviral treatment (art) medication – potentially 2–6 months later – due to inadequate cold-storage facilities at phc facilities for blood samples. viral load testing is the recommended method for monitoring hiv patients on art. south africa, through the nhls, currently operates a highly centralised national network that conducted more than five million vl tests at 16 laboratories in 2018.2 despite this, currently, 16% – 31% of hiv-positive south africans have not had a vl test done within the guideline-recommended window (six months after art initiation and at 12-month intervals thereafter).3,4 patients with unsuppressed vl for extended periods without clinical action are at risk of increased morbidity, mortality, and/or onward transmission of hiv.5 improving access to vl testing would not only enhance patient-centred care but could result in higher levels of viral suppression.6 we aimed to design an optimised courier network that could provide expanded service access to all diagnostics available at phc facilities within the costs of the current system. we then aimed to assess the impact of the optimised courier network on vl testing access. methods ethical considerations this project was approved by the university of the witwatersrand human research ethics committee (medical) (hrec) as an additional project within the integrated laboratory data analysis for care programme (study approval number: hrec m160978). additional ethics approval was received from the center for global health, united states centers for disease control and prevention in atlanta (study approval number: cgh 2019-224). as this study involved the retrospective analyses of de-identified, aggregated data collected as part of routine care, no patient consent was sought. although sensitive information was not collected, all data extracted from the nhls were anonymised, aggregated by facility, and transferred to a secure server. patients or the public were not involved in the design, conduct, reporting, or dissemination plans of this research. study design we extended a previously described geospatial cost model to optimise the specimen transport network in south africa.7 courier routing was optimised for 4046 public facilities in south africa to improve turnaround times of specimen transport from the health facility to the laboratory. using data for a sample of phc facilities for which we had current sample transportation times (n = 356), we then used regression analysis to assess the relationship between current specimen transport time and vl testing access. finally, we compared our specimen transport times from the optimised model with current courier collection times for the subset of facilities (n = 356) to determine the percentage change in access to same-day blood draws. network optimisation for healthcare facilities data from all public sector healthcare facilities were extracted from nhls’s corporate data warehouse for a 12-month period (january 2018 to december 2018) and used in the model. extracted data included the number and type of tests (e.g. vl) requested per site, the location of the site, and the laboratory where specimens were sent to be tested. data from 4046 healthcare facilities from the nhls corporate data warehouse were included in the optimisation. these 4046 facilities accounted for 98% of the total test volumes that were couriered during the study period. facilities and laboratories in the corporate data warehouse were matched to the district health information system so that coordinate data from the district health information system could be used to locate the facilities and laboratories. data quality assessments were conducted on the coordinates to ensure accuracy, and missing coordinate data were sourced from internal implementing partner sources, as well as from google searches. the optimised system enhanced the functioning of the current specimen routing network by incorporating factors that would improve specimen validity and would be better aligned with patient-centred care (online supplementary text 1).7 the cost of the current system was calculated using nhls expenditure on contracted couriers. the contracted courier rate per kilometre was applied to the distance outputted by the routing optimisation model to estimate the cost of the optimised system. the optimisation of the specimen transport system was conducted using the arcgis pro network analyst tool (environmental systems research institute, redlands, california, united states) for vehicle routing problems, which optimises a set of transport routes taking into account the expected specimen volumes, distance from the laboratory to the facility, drive times, and other key constraints identified by stakeholders (online supplementary text 2).8 a 2019 tomtom (environmental systems research institute, redlands, california, united states) routable street data set was sourced from environmental systems research institute for use in the routing analyses. relevant outputs from the optimisation included facility name, corresponding route name, time of arrival at the facility by the courier, and the number of vl tests requested by that phc facility. using this output, it was possible to determine the average latest time that a courier would collect specimens at a facility per day. change in viral load access for a subset of facilities to assess whether the optimised specimen transport network improved vl access, we first determined whether facilities that historically had earlier courier collection times had lower vl test volumes compared to facilities with later courier collection times. we then quantified the additional number of patients that would be expected to access vl testing due to the expanded access brought about by delayed courier specimen collection times. to determine the current latest time at which a health facility is serviced by the courier network, we obtained data on current courier routes and the expected time of arrival at each facility by the courier. unfortunately, arrival time was not well documented and was only available for a subset of facilities in two provinces, gauteng and western cape (n = 497). from this sample, correctional service facilities and hospitals (n = 57), as well as facilities that did not have any patients on art or could not be linked to the district health information system (n = 80), were removed. data from the district health information system were used to determine the number of patients on art at the remaining 360 facilities (as of december 2018). finally, to determine the number of annual vl tests requested by each facility in 2018, the 360 facilities were matched to the nhls data from the corporate data warehouse. four facilities were not matched, resulting in a total sample of 356 phc facilities. data analysis using stata software (version 15.0, college station, texas, united states), we performed an unadjusted regression analysis to assess the relationship between current specimen transport time at the 356 phc facilities and vl testing access, defined as the number of annual vl tests performed per patient on art.9 the independent variable in the regression used the current average specimen transport time as a cut-off, with facilities categorised based on whether their average latest specimen transport time was before or after this cut-off. we also repeated the regression analysis, adjusting for the size of the phc facility in terms of the number of patients on art.10 a statistical significance cut-off of 5% was used. we estimated the time distribution of blood draws for patients at phc facilities based on data from an unpublished time-in-motion study on wait times conducted at an hiv outpatient facility in johannesburg in 2014.11 using this distribution, it was possible to estimate the number of additional patients that would be able to access a blood draw at different times in the day, depending on the arrival time of the courier. sensitivity a sensitivity analysis assessed the impact of changing the average latest courier time of the current system on the relationship between the time of the last courier collection for the day and the number of vl tests requested per patient on art. for each time point (representing the average latest time of courier pick-up at a facility), we determined whether there was a statistically significant difference in the average number of vl tests per patient on art between facilities whose last courier collection for the day occurred before that point versus those that had courier pick-ups after that point. in addition, we assessed how a different assumption regarding the distribution of patient flow (a more uniform distribution) at a phc facility would change our results in terms of patient access to same-day blood draws. results network optimisation the optimised network included 1179 courier routes, involved 136 000 km of driving per day, and serviced the 4046 facilities that are part of the nhls national network at least once per day. the cost to operate the current system is estimated to be $283 488 united states dollars (usd) per week, and the optimised system costs 1.3% less at $279 901 usd per week. through specimen network optimisation, the average latest time that patients can access blood draw for laboratory services was delayed to 14:48 from 14:15. in addition, with optimisation, an estimated 85% of specimens arrive at the laboratory by 16:00. impact of optimised network on viral load access in our sample of facilities for which we had complete current specimen transport times, the weighted average latest time an individual can access a blood draw (weighted by the vl volumes at the facility) is currently 13:36. under half (141/356; 40%) of the phc facilities had an average latest courier pick-up time before 13:36, while the average latest courier pick-up time at 215/356 (60%) facilities was after 13:36. the average number of annual vl tests conducted per patient on art was higher at facilities with courier pick-up after 13:36 (1.14; standard deviation: 0.02) compared to those with courier pick-up before 13:36 (1.06; standard deviation: 0.03). the number of vl tests conducted per patient on art was higher (by approximately 0.07) among facilities with courier collection after 13:36 compared to facilities with courier collection before 13:36, and the regression coefficient was significant (p < 5%), even when adjusted for phc facility size (table 1). table 1: relationship between average latest courier specimen collection time and average viral load tests per antiretroviral treatment patient† at public sector healthcare facilities in south africa, january 2018 to december 2018. data from an unpublished time-and-motion study on wait times show that the timing of blood draws was skewed to the right, with only 17% of patients getting a blood draw after 12:00 (figure 1).11 the median time for a blood draw was 09:42 (confidence interval: 08:50–11:15) and, on average, patients waited just under 3.5 h from the time that they arrived at the phc facility until a blood draw was conducted. for our sample of facilities in gauteng and the western cape, we compared the current average latest time for specimen pick-up of 13:36 to the optimised model transport time of 14:35. based on the distribution of the timing of blood draws, we estimated a 6% increase in patient access to same-day blood draws. with the assumption of a uniform patient distribution over time (i.e. a constant flow of patients for each hour that a health facility is open), the estimated patient access to same-day blood draw increased from 6% to 13%. figure 1: hourly distribution of patient blood draws at an outpatient clinic, johannesburg, south africa, 2014. in a sensitivity analysis, facilities that are serviced by a courier earlier in the day are more likely to have a significantly lower number of vl tests conducted per patient on art (figure 2). this was true until approximately 14:45, beyond which it was less likely that there would be a significant difference between the number of vl tests conducted per patient on art at the facilities. figure 2: relationship between time of courier collection and viral load access at public sector healthcare facilities in gauteng and western cape provinces, south africa, december 2019. discussion through the optimisation of the specimen transportation network, the estimated average time of specimen collection was delayed from 14:15 to 14:48 for the entire network of public facilities (n = 4046), and from 13:36 to 14:35 for the subset of facilities for which we had complete data (n = 356). this is an improvement over the current system as it alleviates the constraint faced by patients and facility staff to quickly collect blood samples before the courier arrives for the final time daily. earlier specimen arrival enables laboratory staff to meet the daily processing demands as laboratory spikes and workflow could be smoothed out and specimens processed as quickly as possible. based on our findings, delaying courier collection by an hour can increase patient access to same-day blood draws by 6% – 13% across the gauteng and western cape provinces, depending on the patient flow distribution at the phc facilities. this simple supply-side intervention to improve the logistics around specimen collection could improve access to vl testing and, consequently, increase vl test volumes and reduce the cost of specimen transportation. improving vl testing access in accordance with national guidelines is critical to attaining south africa’s goal of high levels of viral suppression among hiv patients to meet the last 95% of the hiv targets of the joint united nations programme on hiv and aids.12 one study conducted in four provinces in south africa found that only 69% of patients who were due for a vl test had a vl recorded in an electronic register, and only 24% of those who did not have a vl recorded had a blood draw recorded in their file.3 this suggests that a lack of blood draws, and not necessarily a failure of the result making it back to the phc facility, could be a major contributor to the lack of vl results. furthermore, another study5 observed a large number of missing vl test results for patients with a previously unsuppressed vl – patients at greatest risk for hiv morbidity, mortality and onward transmission.5 in another study conducted in the western cape, 84% had a vl test done when it was due.4 optimising the transportation for vl testing is only one mechanism to improve patient access to vl testing. there are alternative strategies that allow for blood to be drawn at any time of the day. if dried specimens (e.g. dried blood spots or dried plasma spots) were used for vl testing, for which they currently are not, specimens could be taken at any time during the day, even after the courier has picked up the specimens for the last time that day.13 further, should specimen stability be maintained beyond the current recommended limits,14 and should guidelines be adjusted to reflect this, this would also be an effective mechanism to increase access to vl testing. point-of-care tests could also be used, when it is cost-effective, to perform vl testing on demand.15,16 furthermore, where space and resources allow, additional cold-storage facilities could be made available at facilities where current cold storage is inadequate so that after the last courier collection for the day, blood samples can be collected and refrigerated until the next day. to our knowledge, this is the first paper to model the relationship between specimen transport collection times and vl access in a national vl programme. this analysis relies on innovative geospatial routing optimisation and primary data on laboratory and facility location matched to programmatic art data.17 although this paper has focused on the impact on vl access, these results are generalisable to other diagnostics where same-day transport is required for specimen integrity. limitations this study has several limitations. first, we sampled a subset of facilities in only two, predominately urban, provinces out of the nine provinces in south africa. nevertheless, while a more representative sample set may have produced different results, this would primarily have been driven by the current average latest time of specimen collection by couriers, which we varied in the sensitivity analysis. the relationship between specimen transport time and vl access would only become insignificant if the average latest time of specimen transport was later than 14:45, which is unlikely to be the case across the network. second, this project was conducted in south africa with an extensive well-functioning specimen referral network that ensures each facility is serviced at least once per day.18 extrapolation of these findings to other countries would require a consideration of the extent of their specimen referral networks. however, for less-developed specimen referral networks, the impact of optimising specimen collection or increasing the frequency of transport such that each facility is serviced at least once a day might result in larger patient access benefits. third, we relied on the patient flow distribution at one phc facility, where the median time of blood draw was 09:42 in the morning, to quantify the patient benefits of increased access. however, for phc facilities with later median times of blood draw (e.g., with a more uniform distribution of patients throughout the day), the benefits in terms of increased patient access of delaying the courier pick-up time would be greater. fourth, we did not quantify the patient benefits of receiving same-day blood draws. between 6% and 13% of patients requiring a blood draw for vl testing would not need to return to the phc facility another day, potentially reducing transport costs/opportunity costs of a missed day of work. patient scheduling and flow may also improve with later specimen transport as facility staff will be aware that there is more time to send patients to the blood draw queue and can thus book patients in for later times in the day, ultimately reducing the waiting time for patients. finally, while this project has primarily addressed phc facilities that predominantly operate during the week and close at 16:00, future research could look at the value of couriers collecting specimens after hours and on weekends at facilities that operate 24 h and/or on weekends. the benefits in terms of increased patient access to vl testing could even be larger as these facilities could potentially offer phlebotomy services to patients after hours and on weekends, where currently there are none. conclusion simple solutions are frequently overlooked in our quest to improve healthcare. delaying specimen pick-up times at phc facilities by an hour can improve patient access to vl testing at no additional cost. this has implications beyond vl testing and extends to all laboratory diagnostics, especially for specimens whose integrity can be compromised without refrigeration. acknowledgements the authors would like to thank the national health laboratory service for their valuable input with acquiring the data. the results from this manuscript have been presented at the conference on retroviruses and opportunistic infections 2020. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions w.s., k.d., l.b., and b.n. conceived the project. s.j.g., b.n. and, t.c. and n.c. acquired and analysed data for the model. s.j.g. and b.n. developed the model. s.j.g. and b.n. interpreted model results. s.j.g. and b.n. wrote the first draft of the manuscript. s.j.g., t.c., n.c, f.o., p.s., k.d., l.b., d.c., w.s. and b.n. read and approved the final manuscript. sources of support this work has been supported by the united states president’s emergency plan for aids relief (pepfar) through the united states centers for disease control and prevention under the terms of coag 5 nu2ggh001631-04-00: strengthening the delivery and expanding access to quality laboratory services and enhancing healthcare worker and laboratory safety in the republic of south africa under pepfar: protocol number 2019-224. the findings and conclusions in the report are those of the authors and do not necessarily represent the official position of the funding agencies. data availability there are restrictions to the availability of the data used to perform the analysis presented in this manuscript. requests for the underlying data used in this manuscript need to be directed to the national health laboratory service. (https://www.nhls.ac.za/; contact person: portia sejake portia.sejake@nhls.ac.za) disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references national health laboratory service. about us [cited 2022 feb 11]. available from: https://www.nhls.ac.za/about-us/ world health organization. chapter 4.1: clinical guidelines: antiretroviral therapy. in: consolidated guidelines on the use of antiretroviral drugs for treating and preventing hiv infection [homepage on the internet]. 2016 [cited 2022 sept 22]. available from: https://nationalgovernment.co.za/entity_annual/1714/2018-national-health-laboratory-service-(nhls)-annual-report.pdf pascoe s, huber a, murphy j, et al. identifying gaps in viral load monitoring: results from an evaluation of viral load reporting at primary health care facilities in south africa. in aids 2018 abstract book: aids 2018 22nd international aids conference – amsterdam. 2018. euvrard j, schulz t, hilderbrand k, et al. how accurately do routinely reported hiv viral load suppression proportions reflect progress towards the 90-90-90 target in the population on antiretroviral treatment in khayelitsha, south africa? s afr med j. 2019 mar 1;109(3):174–177. https://doi.org/10.7196/samj.2019.v109i3.13456 murphy ra, court r, maartens g, sunpath h. second-line antiretroviral therapy in sub-saharan africa: it is time to mind the gaps. aids res hum retroviruses. 2017;33(12):1181–1184. https://doi.org/10.1089/aid.2017.0134 drain pk, dorward j, violette lr, et al. point-of-care hiv viral load testing combined with task shifting to improve treatment outcomes (stream): findings from an open-label, non-inferiority, randomised controlled trial. lancet hiv. 2020;7(4):e229–e237. https://doi.org/10.1016/s2352-3018(19)30402-3 nichols be, girdwood sj, crompton t, et al. impact of a borderless sample transport network for scaling up viral load monitoring: results of a geospatial optimization model for zambia. j int aids soc. 2018;21(12):e25206. https://doi.org/10.1002/jia2.25206 esri. arcgis pro. network analyst tool. release 10.7. redlands, ca: environment systems research institute (esri); 2020. statacorp. stata statistical software: release 15. college station, tx: statacorp lp; 2017. fox mp, brennan at, nattey c, et al. delays in repeat hiv viral load testing for those with elevated viral loads: a national perspective from south africa. j int aids soc. 2020;23(7):e25542. https://doi.org/10.1002/jia2.25542 long l, batiancila r, girdwood s, majuba p, rosen s, lince-deroche n. booked appointments system: an evaluation of the acceptability and feasibility of booked appointments in a large hiv clinic in johannesburg, south africa [serial online]. 2022 [cited 2020 dec]. available from: https://www.medrxiv.org/content/10.1101/2022.06.30.22277110v1 unaids. fast-track. ending the aids epidemic by 2030 [homepage on the internet]. 2014 [cited 2020 nov 3]. available from: https://www.unaids.org/en/resources/documents/2014/jc2686_wad2014report nichols be, girdwood sj, shibemba a, et al. cost and impact of dried blood spot versus plasma separation card for scale-up of viral load testing in resource limited settings. clin infect dis. 2019;70(6):1014–1020. https://doi.org/10.1093/cid/ciz338 hardie d, korsman s, ameer s, vojnov l, hsiao n-y. reliability of plasma hiv viral load testing beyond 24 hours: insights gained from a study in a routine diagnostic laboratory. plos one. 2019;14(7):e0219381. https://doi.org/10.1371/journal.pone.0219381 girdwood s, crompton t, sharma m, et al. cost-effectiveness of adoption strategies for point of care hiv viral load monitoring in south africa. eclinicalmedicine. 2020;28:100607. https://doi.org/10.1016/j.eclinm.2020.100607 girdwood sj, nichols be, moyo c, crompton t, chimhamhiwa d, rosen s. optimizing viral load testing access for the last mile: geospatial cost model for point of care instrument placement. plos one. 2019;14(8):1–13. https://doi.org/10.1371/journal.pone.0221586 nichols k, girdwood sj, inglis a, et al. bringing data analytics to the design of optimized diagnostic networks in lowand middle-income countries: process, terms and definitions. diagnostics. 2021;11(1):22. https://doi.org/10.3390/diagnostics11010022 glencross dk, coetzee lm, cassim n. an integrated tiered service delivery model (itsdm) based on local cd4 testing demands can improve turn-around times and save costs whilst ensuring accessible and scalable cd4 services across a national programme. plos one. 2014;9(12):e114727. https://doi.org/10.1371/journal.pone.0114727 abstract introduction research method and design results discussion limitations acknowledgements references about the author(s) kerusha govender department of virology, inkosi albert luthuli central hospital, national health laboratory service, durban, kwazulu-natal, south africa department of virology, school of laboratory medicine and medical sciences, university of kwazulu-natal, south africa raveen parboosing department of virology, inkosi albert luthuli central hospital, national health laboratory service, durban, kwazulu-natal, south africa department of virology, school of laboratory medicine and medical sciences, university of kwazulu-natal, south africa ntombizandile siyaca department of virology, inkosi albert luthuli central hospital, national health laboratory service, durban, kwazulu-natal, south africa pravikrishnen moodley department of virology, inkosi albert luthuli central hospital, national health laboratory service, durban, kwazulu-natal, south africa department of virology, school of laboratory medicine and medical sciences, university of kwazulu-natal, south africa citation govender k, parboosing r, siyaca n, moodley p. dried blood spot specimen quality and validation of a new pre-analytical processing method for qualitative hiv-1 pcr, kwazulu-natal, south africa. afr j lab med. 2016;5(1), art. #349, 6 pages. http://dx.doi.org/10.4102/ajlm.v5i1.349 original research dried blood spot specimen quality and validation of a new pre-analytical processing method for qualitative hiv-1 pcr, kwazulu-natal, south africa kerusha govender, raveen parboosing, ntombizandile siyaca, pravikrishnen moodley received: 03 aug. 2015; accepted: 12 dec. 2015; published: 25 feb. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: poor quality dried blood spot (dbs) specimens are usually rejected by virology laboratories, affecting early infant diagnosis of hiv. the practice of combining two incompletely-filled dbs in one specimen preparation tube during pre-analytical specimen processing (i.e., the two-spot method) has been implemented to reduce the number of specimens being rejected for insufficient volume. objectives: this study analysed laboratory data to describe the quality of dbs specimens and the use of the two-spot method over a one-year period, then validated the two-spot method against the standard (one-spot) method. methods: data on hiv-1 pcr test requests submitted in 2014 to the department of virology at inkosi albert luthuli central hospital in kwazulu-natal province, south africa were analysed to describe reasons for specimen rejection, as well as results of the two-spot method. the accuracy, lower limit of detection and precision of the two-spot method were assessed. results: of the 88 481 specimens received, 3.7% were rejected for pre-analytical problems. of those, 48.9% were rejected as a result of insufficient specimen volume. two health facilities had significantly more specimen rejections than other facilities. the two-spot method prevented 10 504 specimen rejections. the pearson correlation coefficient comparing the standard to the two-spot method was 0.997. conclusions: the two-spot method was comparable with the standard method of pre-analytical specimen processing. two health facilities were identified for targeted retraining on specimen quality. the two-spot method of dbs specimen processing can be used as an adjunct to retraining, to reduce the number of specimens rejected and improve linkage to care. introduction early diagnosis and treatment of hiv-positive infants significantly reduce hiv disease progression and mortality. early infant diagnosis of hiv-positive infants is a critical step in the timely initiation of antiretroviral therapy (art).1,2 despite these substantial benefits, achieving early infant diagnosis is challenging in resource-limited settings. problems in the pre-analytical stages of specimen collection and processing lead to poor quality specimens and laboratory rejections.3 a meta-analysis of studies between 2001 and 2012 showed that 33.9% of hiv-exposed infants in sub-saharan africa are lost to follow up by three months of age.4 early infant diagnosis in south africa occurs within a prevention of mother-to-child transmission continuum of care. qualitative hiv-1 dna and rna pcr testing is done on dried blood spot (dbs) specimens collected from hiv-exposed infants between six weeks and 18 months of age, using the cobas® ampliprep/cobas® taqman® (cap/ctm) hiv-1 qualitative test (roche® molecular systems, inc., branchburg, new jersey, united states). the current south african national laboratory guideline allows for testing of one completely-filled dbs per assay.5 there have been significant successes in the prevention of mother-to-child transmission programme. however, challenges have been identified in south africa. only 70.4% of hiv-exposed south african infants were tested for hiv by two months of age in 2011, reflecting a lack of implementation of policies in the field.6 the department of virology (dov) in kwazulu-natal province in south africa provides early infant diagnosis services for the province. laboratory data from kwazulu-natal show a reduction in mother-to-child transmission of hiv which reflects national trends.7 however, there is a challenge of identifying and linking every hiv-exposed infant to care, which contributes to a significant number of hiv-positive infants not commencing art in a timely manner.8 laboratory specimen rejections are an essential part of a laboratory quality management system, allowing the technologist to optimise accuracy of results. however, this practice has also been shown to adversely impact patient care as it can lead to abandonment of the test request.9,10 specimen rejection may play a role in the failure to link hiv-exposed infants to care. the challenges with dbs specimens in our setting have not been described. in the standard method of pre-analytical specimen processing, one complete dbs is inserted into one specimen preparation tube. at the dov, the current practice for dbs with blood spots that are smaller than the recommended size is to use two incomplete dbs and combine them in one specimen preparation tube during pre-analytical specimen processing (i.e., the two-spot method). specimen preparation is followed by pcr testing for hiv-1 using the cap/ctm analyser. the two-spot method is based on the hypothesis that the quality of these specimens may be adversely affected by the small volume of blood they contain. by maximising the amount of blood eluted from each specimen, the correct volume for the pcr can be obtained and a good quality laboratory result can be produced. neither the two-spot method nor the impact of this method in reducing laboratory rejections have been described in the scientific literature. the objectives of this study were to analyse dov laboratory data to describe trends in dbs specimen quality, particularly relating to specimens with insufficient volume. in addition, we sought to describe the use of the two-spot method in routine hiv pcr diagnosis. finally, we performed laboratory experiments to validate the two-spot method. research method and design ethical considerations the university of kwazulu-natal biomedical research ethics committee (brec) approved this research under the approval classes bca-143/09 and bca 256/010. setting the study was conducted in the dov at inkosi albert luthuli central hospital in kwazulu-natal province, south africa. the dov is an iso 15189:2007 accredited laboratory within an academic quaternary-level hospital. the dov does early infant diagnostic testing of dbs using hiv-1 pcr on the cap/ctm analyser for all hiv-exposed infants in public health facilities in kwazulu-natal province. during the study period from january 2014 to december 2014, the south african antiretroviral treatment guidelines11 recommended that all hiv-exposed infants have an hiv-1 pcr test at age six weeks, at six weeks post-cessation of breastfeeding and at any age less than 18 months that the child may be symptomatic (after age 18 months, serology was used for hiv diagnosis). paediatric guidelines recommend testing infants at the six-week immunisation visit and, for those who test positive, referral for confirmation of hiv infection and initiation of art. infants testing negative should be followed up and retested at age 18 months and/or after cessation of breastfeeding.11,12 laboratory database search we searched our laboratory database for reports on routine hiv-1 pcr test requests that were rejected during 2014. the database is managed with trakcare lab software (intersystems corporation, cambridge, massachusetts, united states) and the data were downloaded and stored in microsoft excel 2010 files (microsoft corporation, redmond, washington, united states). the data extracted included specimen numbers, reasons for specimen rejections and patient demographics, including the health facility where the dbs was collected (each facility was assigned a two-letter code). we also extracted the results for all hiv-1 cap/ctm pcr routine diagnostic tests conducted during 2014. laboratory experiment to validate the two-spot method specimen preparation residual edta blood specimens from previous successful, routine diagnostic hiv-1 pcr tests were selected and anonymised using a number allocated for this experiment. specimens with insufficient volume and specimens that were collected more than one day prior to testing were excluded. blood samples from 20 hiv-negative and 20 hiv-positive specimens were selected for the validation study.13 blood was pipetted onto pre-labelled ss906 filter paper cards by filling one circle completely (so that it contained 70 µl of blood), then filling two circles incompletely (so that one contained 30 µl and the other 40 µl of blood). examples of sufficient and insufficient specimens are shown in figure 1. the original specimen tubes were discarded as per routine biohazard waste disposal procedures. the blood spots were allowed to air-dry at room temperature. the sizes of the dbs were measured using calipers and recorded, then manually cut for hiv-1 pcr testing. figure 1: example filter paper card with sufficient and insufficient blood spots. validation experiment the complete (70 µl) dbs were processed using the standard (one-spot) method: each blood spot was inserted into a specimen preparation tube with lysis buffer, which had 1000 µl of specimen pre-extraction reagent (30 mm sodium citrate dehydrate, 42.5% guanidine thiocyanate, 4% polydocanol and 2% dithiothreitol) added to each tube. the tube was then subjected to thermomixing in a thermomixer comfort (eppendorf, hamburg, germany) at 56 °c for 10 minutes at 1000 rpm in order to elute the dbs. the two incomplete (30 µl and 40 µl) dbs were then combined in a single specimen preparation tube and processed using the above method. for both sample types, the dbs were pushed to one side when inserted into the specimen preparation tube, as per the standard procedure. both types of dbs were then tested in parallel over a period of five days for hiv-1 nucleic acids using the cap/ctm as per manufacturer’s instructions. briefly, the specimen preparation tubes were transferred to specimen racks, then loaded into the cap/ctm analyser, which is an automated closed system with combined extraction, real-time pcr amplification and detection allowing for reduced contamination. known low-positive and negative controls were included with every batch. the analyser software automatically determined the validity of the run, then determined the presence of hiv-1 nucleic acids based on the ct value, namely, the pcr cycle at which the signal indicated amplification. an hiv standard (world health organization 3rd hiv-1 international standard national institute for biological standards and control code: 10/152) was used to perform a dilution series and establish a lower limit of detection for the hiv-1 pcr test when using the two-spot method. replicates of the hiv standard were tested to establish precision.13 statistical methods statistical analyses were performed using ibm spss statistics for windows, version 22.0 (ibm corp., armonk, new york, united states). for the analysis of the laboratory database search, the frequency of each reason for specimen rejection was determined and for each health facility, a percentage of tests rejected as a result of pre-analytical problems was determined out of the total number of hiv-1 pcr tests requested. the hiv-1 pcr results of specimens processed pre-analytically using the standard method were then compared with specimens processed using the two-spot method. the chi-squared test was used to compare categorical variables. a p-value of < 0.05 was regarded as statistically significant. for the analysis of the laboratory experiment to validate the two-spot method, measures of sensitivity and specificity were used to describe the accuracy of the method. a scatter plot of ct values was used to compare the two methods. precision was determined by calculating the standard deviation and coefficient of variance of replicates. a probit regression analysis was used to determine the lower limit of detection of the assay. results laboratory database search the analysis of database records for 2014 revealed that 88 481 specimens were sent to dov for hiv-1 pcr testing, of which 3.8% were rejected or had no results. ninety-one (0.1%) test requests had no results as a result of analytical problems and 3276 (3.7%) were rejected because of pre-analytical problems (table 1). table 1: dried blood spot specimens received in 2014 at the department of virology laboratory at inkosi albert luthuli central hospital, kwazulu-natal province, south africa. insufficient specimen volume comprised 48.9% of the total number of rejections resulting from pre-analytical problems (table 2). other reasons for rejections included: inadequate information supplied on the test request form; incorrect clinical indication for the test; and poor specimen quality, such as incorrect specimen type, specimens that were too old for analysis and specimen cards that were expired. table 2: dried blood spots rejected because of pre-analytical problems in 2014 at the department of virology laboratory at inkosi albert luthuli central hospital, kwazulu-natal province, south africa. the data on the percentage of requests that were rejected per health facility were normally distributed (figure 2). the mean of the percentages of tests rejected for all health facilities in kwazulu-natal was 3.6%. two facilities (eh and gf, names blinded for publication purposes) were more than two standard deviations away from the mean, with rejection rates of 9.7% for eh and 7.6% for gf. figure 2: percentage of hiv-1 pcr requests rejected because of pre-analytical factors by health facilities in kwazulu-natal province, south africa, in 2014. each health facility is represented by a two-letter code. the analysis included data from 88 481 dried blood spot specimens. the horizontal line at 3.57% indicates the mean percentage of rejected requests for all facilities. the horizontal lines at 7.43% and -0.29% indicate two standard deviations from the mean. for all hiv-1 pcr test results in 2014 using the cap/ctm assay, there was an overall indeterminate rate of 1.7% (table 3). there were 10 307 valid positive and negative test results using the two-spot method. the two-spot method had an indeterminate rate of 1.9%. table 3: results of specimens tested by standard method and two-spot method in 2014 at the department of virology laboratory at inkosi albert luthuli central hospital, kwazulu-natal province, south africa. validation of the two-spot method the average diameter of the 30 µl dbs was 8.99 mm (95% confidence interval: 8.89–9.12 mm) and for the 40 µl dbs was 10.08 mm (95% confidence interval: 9.95–10.21 mm). a comparison of the qualitative results of patient specimens showed 100% agreement in accuracy between the two methods. the comparison of the ct values for the standard method versus the two-spot method showed that the pearson correlation coefficient was 0.997 (figure 3). in the precision study, 19 replicates of a specimen with a known concentration yielded a coefficient of variance of 0.0078. the probit regression analysis of the dilution series showed that 95% of replicates were detected at 3.6 log iu/ml (95% confidence interval: 2.9–4.925 log iu/ml). figure 3: validation experiment correlations between ct values for the standard (one-spot) and two-spot specimen processing methods. twenty positive and 20 negative specimens were processed by both the two-spot and the standard one-spot methods of pre-analytical specimen processing. the scatterplot shows the ct values obtained during real-time pcr amplification of the positive specimens. an overlaid line of linearity and pearson’s correlation coefficient of 0.997 shows the correlation between the ct values for each of the specimen processing methods. discussion this study is the first to describe the reasons for laboratory rejections of dbs specimens being submitted for hiv-1 pcr testing in kwazulu-natal province. almost 4% of dbs specimens that were submitted to the laboratory were rejected because of pre-analytical problems. our analysis showed that 48.9% of pre-analytical problems resulted from insufficient specimen volume and we identified two health facilities with high specimen rejection rates. insufficient dbs volume has been shown to affect the yield of hiv-1 nucleic acids14 and these specimens are therefore rejected by laboratories. others have also described challenges in the field with specimen collection and processing, as a result of specimen quality factors such as mislabeling or insufficient volume. rates of specimen rejection vary considerably and there is a paucity of data describing the frequency of each specimen quality issue.15,16 data such as specimen quality should be analysed in order to review the performance of hiv-1 pcr testing in the field and to standardise dbs specimen collection methods, storage and testing.14 this study has shown that analysing specimen quality can identify health facilities which require targeted interventions, such as retraining of healthcare workers. the prevention of mother-to-child transmission program in kwazulu-natal has shown significant success, largely because of the implementation of testing pregnant women and starting them on art,7 but problems have been described in the follow-up of mothers and infants.8,17 it has been shown that delaying hiv diagnosis in hiv-exposed infants results in a failure to link infants to hiv care, significant loss to follow up and mortality.18 at immunisation clinics in kwazulu-natal, nurses perform the specimen collection, whereas in hospital wards and clinics, some specimens are submitted by attending doctors. it has been shown that intense training and mentoring of nurses significantly improves the uptake of infant hiv testing and the overall quality of care.19 however, this needs to be expanded and implemented on a much larger scale.20,21 the need to expand nurse training will increase with the planned implementation of hiv-1 dna pcr testing of exposed neonates at birth.22 as this is being newly implemented, healthcare workers in labour wards and nurseries may not be as experienced with dbs specimen collection as those in immunisation clinics. we have described an innovative two-spot method for pre-analytical specimen processing that can reduce the number of dbs specimen rejections resulting from insufficient specimen volume. this method has been implemented in dov for approximately three years and is used as an adjunct to ongoing telephonic and electronic communication and training of nurses in the public healthcare sector. personal communications with other laboratories indicate that the two-spot method is being used in other hiv-1 pcr testing laboratories in south africa. however, to our knowledge the two-spot method has not yet been described in the scientific literature. the specimens processed using the two-spot method in 2014 yielded over 10 000 additional results from specimens that would otherwise have been rejected. furthermore, the proportion of specimens processed using the two-spot method with indeterminate results was comparable with that of all specimens processed in 2014. the validation study found that the two-spot method correlated well with the standard method and had good precision. the lower limit of detection of the hiv-1 pcr test when using the two-spot method was 3.6 log iu/ml, which is comparable to the 2.7 log iu/ml stated on the package insert for the standard method. future studies comparing the detection limits between the two methods may be required. limitations a major limitation with respect to analytical sensitivity is that the test is being used in infants within a prevention of mother to child transmission programme, most of whom have been exposed to art. the presence of these drugs has been stated as a limitation by the manufacturer on the hiv-1 pcr package insert. therefore, field evaluation of detection limits of the test may be required in a significantly art-exposed patient population such as south africa.23 another limitation of our study is that the volumes of 30 µl and 40 µl used in the validation study were selected in an attempt to simulate the real-world situation. however, a range of volumes could be present in dbs specimens in the field, for example, 20 µl and 50 µl. a third limitation is that the proportion of positive results using the two-spot method was significantly lower compared with the standard one-spot method. we speculate that this difference may be because of inconsistent selection of specimens for two-spot processing. practical application of the two-spot method would require an objective assessment of the dbs specimen to reduce the subjectivity of specimen processing in the laboratory. mean circle diameters for 30 µl and 40 µl spots can be provided to laboratory assistants on a template in order to demonstrate acceptable specimen volumes and aid the selection of patient specimens which are suitable for the two-spot method. we are in the process of implementing the use of such a template based on the findings of this study. a follow-up database analysis will be required to assess whether this intervention is effective. laboratories that plan to use or are currently using the two-spot method should consider this cautionary measure. conclusion in conclusion, we describe real-world problems with dbs specimen quality, which may have major implications for the management of hiv-infected infants in a resource-limited setting. the laboratory database can be used to monitor specimen quality and identify specific health facilities for interventions such as retraining of healthcare workers. as an adjunct to training, the two-spot method of specimen processing may be used to help reduce the number of specimens rejected. this method can potentially be applied to other hiv-exposed infant populations in resource-limited settings to reduce the need for patients having to repeat specimen collection and improve linkage to care. acknowledgements the authors are grateful to the patients of kwazulu-natal whose data were used for this study and to the laboratory staff of dov who conduct the hiv-1 pcr testing for the infants in the province. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. sources of support this research was supported by the department of virology, national health laboratory service, kwazulu-natal, south africa. authors’ contributions k.g. (national health laboratory service and university of kwazulu-natal) was the project leader. k.g. and p.m. (national health laboratory service and university of kwazulu-natal) were responsible for the project design; k.g. and n.s. (national health laboratory service) performed the laboratory work; and k.g. and r.p. (national health laboratory service and university of kwazulu-natal) performed the statistical analyses. k.g., r.p. and p.m. prepared the manuscript; and all authors reviewed the manuscript. references cotton mf, violari a, otwombe k, et al. early time-limited antiretroviral therapy versus deferred therapy in south african infants infected with hiv: results from the children with hiv early antiretroviral (cher) randomised trial. lancet. 2013 nov;382(9904):1555–1563. pubmed pmid: 24209829. pubmed central pmcid: 4104982. http://dx.doi.org/10.1016/s0140-6736(13)61409-9 violari a, cotton mf, gibb dm, et al. early antiretroviral therapy and mortality among hiv-infected infants. nejm. 2008 nov;359(21):2233–2244. pubmed pmid: 19020325. pubmed central pmcid: 2950021. http://dx.doi.org/10.1056/nejmoa0800971 ciaranello al, park je, ramirez-avila l, et al. early infant hiv-1 diagnosis programs in resource-limited settings: opportunities for improved outcomes and more cost-effective interventions. bmc med. 2011;9:59. pubmed pmid: 21599888. pubmed central pmcid: 3129310. http://dx.doi.org/10.1186/1741-7015-9-59 sibanda el, weller iv, hakim jg, et al. the magnitude of loss to follow-up of hiv-exposed infants along the prevention of mother-to-child hiv transmission continuum of care: a systematic review and meta-analysis. aids. 2013 nov;27(17):2787–2797. pubmed pmid: 24056068. pubmed central pmcid: 3814628. http://dx.doi.org/10.1097/qad.0000000000000027 stevens w, erasmus l, moloi m, et al. performance of a novel human immunodeficiency virus (hiv) type 1 total nucleic acid-based real-time pcr assay using whole blood and dried blood spots for diagnosis of hiv in infants. j clin microbiol. 2008 dec;46(12):3941–3945. pubmed pmid: 18923017. pubmed central pmcid: 2593254. http://dx.doi.org/10.1128/jcm.00754-08 barron p, pillay y, doherty t, et al. eliminating mother-to-child hiv transmission in south africa. bull world health organ. 2013 jan 1;91(1):70–74. pubmed pmid: 23397353. pubmed central pmcid: 3537246. http://dx.doi.org/10.2471/blt.12.106807 moodley p, parboosing r, moodley d. reduction in perinatal hiv infections in kwazulu-natal, south africa, in the era of more effective prevention of mother to child transmission interventions (2004–2012). j acquir immune defic synr. 2013 jul;63(3):410–415. pubmed pmid: 23535294. http://dx.doi.org/10.1097/qai.0b013e3182926931 smith sj, nimmo c, fredlund v, et al. early infant diagnosis of hiv and fast initiation of anti-retroviral therapy in a rural african setting: how well are we doing? paediatr int child health. 2014 aug;34(3):203–207. http://dx.doi.org/10.1179/2046905514y.0000000119 karcher ds, lehman cm. clinical consequences of specimen rejection: a college of american pathologists q-probes analysis of 78 clinical laboratories. arch pathol lab med. 2014 aug;138(8):1003–1008. pubmed pmid: 25076290. http://dx.doi.org/10.5858/arpa.2013-0331-cp jacobsz la, zemlin ae, roos mj, et al. chemistry and haematology sample rejection and clinical impact in a tertiary laboratory in cape town. clin chem lab med. 2011 dec;49(12):2047–2050. pubmed pmid: 21995606. http://dx.doi.org/10.1515/cclm.2011.743 department of health. the south african antiretroviral treatment guidelines 2013 [document on the internet]. c2013 [cited 2015 jul 22]. available from: http://www.kznhealth.gov.za/medicine/2013_art_guidelines.pdf. mckerrow n, stephen c, purchase se, et al. step-by-step guide for the management of children on art [document on the internet]. c2010 [cited 2015 jul 27]. available from: http://www.kznhealth.gov.za/arv/childguide.pdf. burd em. validation of laboratory-developed molecular assays for infectious diseases. clin microbiol rev. 2010 jul;23(3):550-76. pubmed pmid: 20610823. pubmed central pmcid: 2901657. http://dx.doi.org/10.1128/cmr.00074-09 smit pw, sollis ka, fiscus s, et al. systematic review of the use of dried blood spots for monitoring hiv viral load and for early infant diagnosis. plos one. 2014;9(3):e86461. pubmed pmid: 24603442. pubmed central pmcid: 3945725. http://dx.doi.org/10.1371/journal.pone.0086461 kouakou j, nobah m-t, tanoh a, et al. preliminary results of the demonstration phase of routine early hiv testing of hiv-exposed infants identified through pmtct programs in côte d’ivoire. abstract from aids 2008 – xvii international aids conference, mexico city: abstract no. 2008_mope0217. nkenfou cn, lobé ee, ouwe-missi-oukem-boyer o, et al. implementation of hiv early infant diagnosis and hiv type 1 rna viral load determination on dried blood spots in cameroon: challenges and propositions. aids res hum retroviruses. 2012 feb;28(2):176–181. pubmed pmid: 21679107. http://dx.doi.org/10.1089/aid.2010.0371 horwood c, haskins l, vermaak k, et al. prevention of mother to child transmission of hiv (pmtct) programme in kwazulu-natal, south africa: an evaluation of pmtct implementation and integration into routine maternal, child and women’s health services. trop med int health. 2010 sep;15(9):992–999. pubmed pmid: 20561313. http://dx.doi.org/10.1111/j.1365-3156.2010.02576.x massavon w, barlow-mosha l, mugenyi l, et al. factors determining survival and retention among hiv-infected children and adolescents in a community home-based care and a facility-based family-centred approach in kampala, uganda: a cohort study. isrn aids. 2014;2014:852489. pubmed pmid: 25006529. pubmed central pmcid: 4003865. fatti g, rundare a, pududu b, et al. an innovative approach to improve the quality of prevention of mother-to-child transmission of hiv programs through nurse clinical mentoring in south africa. j acquir immune defic syndr. 2013 jun 1;63(2):e76–78. pubmed pmid: 23666140. http://dx.doi.org/10.1097/qai.0b013e31828f5a5c zuber a, mccarthy cf, verani ar, et al. a survey of nurse-initiated and -managed antiretroviral therapy (nimart) in practice, education, policy, and regulation in east, central, and southern africa. j assoc nurses aids care. 2014 nov–dec;25(6):520–531. pubmed pmid: 24739661. http://dx.doi.org/10.1016/j.jana.2014.02.003 dohrn j, nzama b, murrman m. the impact of hiv scale-up on the role of nurses in south africa: time for a new approach. j acquir immune defic syndr. 2009 nov;52 suppl 1:s27–29. pubmed pmid: 19858933. http://dx.doi.org/10.1097/qai.0b013e3181bbc9e4 national department of health. national consolidated guidelines for the prevention of mother-to-child transmission of hiv (pmtct) and the management of hiv in children, adolescents and adults; 2015. mitchell c, dross s, beck ia, et al. low concentrations of hiv-1 dna at birth delays diagnosis, complicating identification of infants for antiretroviral therapy to potentially prevent the establishment of viral reservoirs. clin infect dis. 2014 apr;58(8):1190–1193. pubmed pmid: 24501389. pubmed central pmcid: 3967830. http://dx.doi.org/10.1093/cid/ciu068 abstract introduction methods results discussion acknowledgements references about the author(s) jonathan gwasupika department of clinical sciences, tropical diseases research centre, ndola, zambia victor daka department of public health, school of medicine, copperbelt university, ndola, zambia justin chileshe department of biomedical sciences, tropical diseases research centre, ndola, zambia moses mukosha department of pharmacy, school of health sciences, university of zambia, lusaka, zambia steward mudenda department of pharmacy, school of health sciences, university of zambia, lusaka, zambia bright mukanga department of public health, school of medicine, copperbelt university, ndola, zambia ruth l. mfune department of public health, school of medicine, copperbelt university, ndola, zambia gershom chongwe tropical diseases research centre, ndola, zambia citation gwasupika j, daka v, chileshe j, et al. covid-19 positive cases among asymptomatic individuals during the second wave in ndola, zambia. afr j lab med. 2023;12(1), a2119. https://doi.org/10.4102/ajlm.v12i1.2119 original research covid-19 positive cases among asymptomatic individuals during the second wave in ndola, zambia jonathan gwasupika, victor daka, justin chileshe, moses mukosha, steward mudenda, bright mukanga, ruth l. mfune, gershom chongwe received: 11 nov. 2022; accepted: 18 apr. 2023; published: 31 may 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: coronavirus disease 2019 (covid-19) is a worldwide public health concern for healthcare workers. about 80% of cases appear to be asymptomatic, and about 3% may experience hospitalisation and later die. less than 20% of studies have looked at the positivity rate of asymptomatic individuals. objective: this study investigated the covid-19 positivity rates among asymptomatic individuals during the second covid-19 wave at one of zambia’s largest testing centre. methods: this was a retrospective cross-sectional study conducted on routine surveillance and laboratory data at the tropical diseases research centre covid-19 laboratory in ndola, zambia, from 01 december 2020 to 31 march 2021. the study population was made up of persons that had tested for severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection as a requirement for travel. microsoft excel was used to come up with an epidemiological curve of daily covid-19 positive cases; proportions for gender were described using frequencies and percentages. results: a total of 11 144 asymptomatic individuals tested for sars-cov-2 were sampled for the study and 1781 (16.0%) returned positive results. the median age among those tested was 36 years (interquartile range: 29–46). testing for covid-19 peaked in the month of january 2021 (37.4%) and declined in march 2021 (21.0%). the epidemiological curve showed a combination of continuous and propagated point-source transmission. conclusion: the positivity rate of 16.0% among asymptomatic individuals was high and could imply continued community transmission, especially during january 2021 and february 2021. we recommend heightened testing for sars-cov-2 among asymptomatic individuals. what this study adds: this study adds critical knowledge to the transmission of covid-19 among asymptomatic travellers who are usually a key population in driving community infection. this knowledge is critical in instituting evidence-based interventions in the screening and management of travellers, and its control. keywords: asymptomatic individuals; covid-19 disease; positivity rate; sars-cov-2; zambia. introduction coronavirus disease 2019 (covid-19), caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), is a worldwide public health concern for healthcare workers, including physicians, public health specialists and researchers.1 the world health organization declared the outbreak of covid-19, which was first reported in wuhan, china, as a public health emergency of international concern on 30 january 2020,2 as it was posing a high risk to countries with vulnerable health systems.3 almost all 55 countries in africa have been affected by the coronavirus pandemic, with sub-saharan africa being the most affected.4 zambia recorded its first case of covid-19 on 18 march 20205 and has since recorded a total of 333 555 covid-19 cases and 4017 deaths as of 04 october 2022.6 severe acute respiratory syndrome coronavirus causes a number of human respiratory disease conditions, ranging from mild cold to severe respiratory distress syndrome, and it largely spreads between persons by respiratory droplets and contact routes.7 on the other hand, sars-cov-2 has been seen to spread faster than sars-cov, which was first reported in 2003 and caused previous outbreaks. accumulating evidence showed that sars-cov-2, unlike sars-cov, is transmitted by persons without symptoms.8 about 80% of cases appear to be asymptomatic,4,9,10 and about 3.3% may experience hospitalisation and later die. based on global biological, epidemiological and modelling evidence, asymptomatic covid-19 may play a substantial role in the pandemic trajectory.11 despite public health preventive measures such as hand hygiene, social distancing, quarantine and travel restrictions which were instituted, transmission of covid-19 seemed to be ongoing.12 vaccination against covid-19 has been shown to be effective against contracting severe forms of the disease.13 however, vaccination does not protect an individual from transmitting or becoming infected with sars-cov-2.14 zambia has experienced four waves of covid-19.1 during the first wave to about the fourth wave of covid-19, it was a requirement to have a negative polymerase chain reaction covid-19 certificate by everyone intending to travel.15,16 this study aimed to assess the positivity rate among asymptomatic travellers during the second wave of the covid-19 pandemic, and its determinants. methods ethical considerations ethical approval to carry out the study was obtained from the tdrc research ethics committee (irb registration number: 00002911). permission to carry out the study and access to patient information was obtained from the tropical diseases research centre management. informed consent was not obtained from any individual as there was no active participation in the study. confidentiality of patient information was adhered to and data were de-identified prior to analysis. study design and site this was a retrospective cross-sectional study conducted on surveillance and laboratory data collected at the tropical diseases research centre (tdrc) covid-19 laboratory in ndola, zambia, from 01 december 2020 to 31 march 2021. the tdrc is a national health research institution specialising in both infectious and non-infectious diseases. the tdrc covid-19 laboratory is accredited for certification of travellers by the african society for laboratory medicine and conducts approximately 400 covid-19 tests per day. study population and eligibility criteria the study population was made up of persons who were tested for sars-cov-2 infection during the second wave of covid-19. complete enumeration of the data set comprising individuals tested for covid-19 was obtained for analysis. eligibility for testing was based on getting tested for covid-19 as a mandatory requirement for international travel, regardless of age. tests of individuals that were collected from outside the tdrc and those that were done outside the stipulated period of the second wave were not included in the analysis. additionally, tests that were done after vaccination had begun were excluded. data collection data were collected from an already-prepared microsoft excel spreadsheet (microsoft corporation, redmond, washington, united states) and a case investigation form comprising the following information: date the test was done, age, gender, and results. an extraction data tool was used for data collection. variables with no clear labels and missing data were removed from the data set. data analysis microsoft excel was used to come up with an epidemiological curve of daily covid-19-positive cases, whereas proportions for gender were described using frequencies and percentages. age was described as a continuous variable and the mean, median, mode and range were used. to test for differences on the covid-19 test result, the chi-square test was used once assumptions were met to analyse binary variables; otherwise fisher’s exact test was used. for continuous variables; the mann-whitney ranksum test was used for skewed data. after stratifying covid-19 positivity by months, the one-way analysis of variance test was used to analyse for differences in age among groups. to predict factors associated with a positive test for covid-19, logistic regression methods were used. stata® software, version 14 se (stata corp., college station, texas, united states) was used for analysis. a p-value less than 0.05 was considered statistically significant at a confidence interval of 95%. results a total of 11 144 asymptomatic travellers tested for covid-19 were sampled for the study and 1781 tested positive, resulting in a positivity rate of 16.0% (table 1). the study participants had a median age of 36 years (interquartile range: 29 to 36 years). the test for covid-19 was noted to be done mostly by travellers in the age group 19 to 50 years. the youngest participant was 1 year old while the oldest was 92 years old. a majority of those tested were male travellers (73.2%; 8152/11 144); female travellers accounted for the remaining 26.8% (2992/11 144). the highest number of tests were completed in january 2021 (4170/11 144). table 1: basic characteristics of participants, ndola, zambia, december 2020 – march 2021. the proportion of female travellers that tested positive for covid-19 (18.4%) was greater than the proportion of male travellers (15.1%) with a p-value of 0.027 (table 2). among individuals who tested for covid-19 prior to travelling, about 7303 (83.2%) tested negative and were ndola residents, whereas among individuals that tested positive, 304 (12.9%) were not ndola residents. there were no positive results among individuals whose samples were collected orally. of the monthly tests done, 30/1557 (1.9%) were positive in december 2020, 1060/4170 (25.4%) were positive in january 2021, 532/3079 (17.3%) positive in february 2021 and 159/2338 (6.8%) positive in march 2021. table 2: covid-19 positivity rate and socio-demographics of participants in ndola, zambia, december 2020 – march 2021. when stratified by month of visit to the testing centre, there were more ndola residents seeking testing services at the tdrc laboratory in january 2021 (n = 948) than any other month included in the study (table 3). an equal peak number of non-ndola residents (n = 111) was seen in january 2021 and february 2021. the lowest number of travellers was seen in december 2020. table 3: covid-19 positivity rate stratified by months in ndola, zambia december 2020 – march 2021. coronavirus disease 2019 cases began to rise on 05 january 2021 and reached a peak on 26 january 2021 (figure 1). the cases remained high until 23 february 2021, when there was a reduction of 150 in the number of positive cases reported. figure 1: daily covid-19-positive cases in asymptomatic travellers in ndola, zambia, december 2020 – march 2021. female travellers had a 16.0% (adjusted odds ratio: 1.16; 95% confidence interval: 1.03 – 1.30; p = 0.012) increased chance of testing positive for sars-cov-2 compared to male travellers, after adjusting for age, residence, and month in which the test was done (table 4). table 4: predictors of positive covid-19 during the second wave in ndola, zambia, december 2020 – march 2021. discussion this study found a positivity rate of 16.0% (1781/11 144), with 69.1% (1231/1781) of male travellers being affected. the months of january 2021 and february 2021 recorded the highest rate of positivity. the epidemiological curve showed that the second wave of covid-19 lasted from december 2020 to the end of march 2021. further, the chances of testing positive for sars-cov-2 if an individual was female increased by about 16% (95% confidence interval: 1.03–1.30) compared to being male, after controlling for other variables. the positivity rate found in this study was similar to the positivity rate at the national level in zambia during the same period.6 despite the similarity, the national level positivity rate comprised both symptomatic and asymptomatic cases. the positivity rate found could have been higher if control measures of isolation and quarantine of cases and testing of people before travel were not put in place and followed.11 on the other hand, the positivity rate in this study was higher than the national rate of 10.6% during the first wave.15 this could have been due to differences in the attack rate and rate of transmission of the strain of coronavirus.16 conversely, a study in nigeria reported a higher positivity rate of 20.8% in the second wave which lasted from 25 october 2020 to 03 april 2021, with asymptomatic cases being the majority.17 a study done by avadhanula et al. showed a positivity rate of 11.4% among asymptomatic patients during the second wave between 18 march 2020 and 15 august 2020 in houston, texas, united states, which was much lower than the rate found in this study.18 this could have been due to differences in region, rate of transmission, adherence to recommended guidelines and utilisation of covid-19 vaccine as it was introduced in some countries earlier than others.17 our study and a study by ghosh, sarkar and chouhan, done in india between march 2021 and may 2021, thus confirmed the presence of covid-19 among asymptomatic individuals.19 our study found that a rise in covid-19 cases during the second wave of the pandemic was observed from december 2020 and ended in march 2021. the epidemiological curve for the daily cases of covid-19 showed a peak on 26 january 2021. in italy, different findings were reported in which the second wave began in august 2020 and continued to february 2021.20 a study in spain demonstrated that the second wave started on 01 july 2020 and ended on 15 october 2020, indicating that this period was different to what was obtained in our study.21 in india, the peak of cases was observed around 01 march 2021.19 these differences could be a result of differences in geographical locations and climatic conditions across the globe.22,23,24 this study found a statistically significant difference in positivity rate between female travellers compared with male travellers, with female travellers more likely to test positive. these findings are consistent with those in nigeria, where more asymptomatic female individuals than male tested positive.17 other studies have also reported similar findings in which female individuals had higher odds of positivity than male individuals.25,26 this could be due to female patients having a higher health-seeking behaviour than male patients.25 in a study done in netherlands, on data collected from march 2020 to august 2020, there was no significant difference in positivity rates between female patients and male patients.27 the study also found the age between 19 to 50 years to have a higher positivity rate compared to those who were 18 years or younger and those older than 50 years. this finding was not different from the study done in wuhan, china, and bahrain, ireland, that reported a higher prevalence of covid-19 in individuals who were less than 45 years old in wuhan, and 20 to 49 years in bahrain.28,29 this could be because those aged 18 years and younger were less susceptible to covid-19 during the second wave.30 in addition, control measures such as closure of school, colleges and universities may have contributed to the age group 18 years and younger having a low positivity rate.31 on the other hand, most of the individuals older than 50 years were symptomatic and prone to hospitalisation as compared to younger individuals.32 limitations this study had some limitations, one of which was that there was no control on the variables as this was a retrospective analysis of previously collected data. findings in this study may not be generalisable, as the data were obtained from one testing site. however, the study had good power and the results are a true reflection of the country’s positivity rate. a prospective study with more variables is recommended. conclusion the positivity rate was found to be 16.0%, implying that there was continued community transmission despite the instituted public health guidelines. age was not a predictor of a testing positive for covid-19, whereas the month in which a test was done, the sex of the individual and their place of residence were good predictors. the positivity rate reported in this study suggests the need to heighten testing of sars-cov-2 among asymptomatic individuals. acknowledgements we would like to acknowledge the staff in the molecular laboratory at the tropical diseases research centre for availing the data that was used in this study. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions j.g. conceptualised the study, conducted the formal analysis and wrote the first draft. v.d. conducted the formal analysis and review and editing of the manuscript. j.c., s.m., b.m. and r.l.m. performed data curation and reviewed the manuscript. m.m. performed the formal analysis and reviewed the manuscript. g.c. performed the editing, review of the manuscript and supervised the conduct of the study. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the data supporting the findings of this study are available from the corresponding author, v.d., upon request. disclaimer the views and opinions expressed in this article are solely of the authors and do not reflect the official policy or position of any affiliated organisation of the authors. references sokolovska l, sultanova a, cistjakovs m, murovska m. covid-19: the third wave of coronavirus infection outbreak. j transl sci. 2021;7(1):e1–e5. https://doi.org/10.15761/jts.1000389 bwire gm, paulo ls. coronavirus disease-2019: is fever an adequate screening for the returning travelers? trop med health. 2020;48(1):1–3. https://doi.org/10.1186/s41182-020-00201-2 yanez nd, weiss ns, romand ja, treggiari mm. covid-19 mortality risk for older men and women. bmc public health. 2020;20(1):e0248281. https://doi.org/10.1186/s12889-020-09826-8 tessema sk, nkengasong jn. understanding covid-19 in africa. nat rev immunol. 2021;21(8):469–470. https://doi.org/10.1038/s41577-021-00579-y mulenga lb, hines jz, fwoloshi s, chirwa l, siwingwa m, yingst s. prevalence of sars-cov-2 in six districts in zambia in july, 2020: a cross-sectional cluster sample survey. artic lancet glob health. 2021;9(6):e773–e781. https://doi.org/10.1016/s2214-109x(21)00053-x worldometer. zambia covid – coronavirus statistics – worldometer [homepage on the internet]. 2022 [cited 2022 oct 06]. available from: https://www.worldometers.info/coronavirus/country/zambia/ fwoloshi s, hines jz, barradas dt, et al. prevalence of severe acute respiratory syndrome coronavirus 2 among healthcare workers – zambia, july 2020. clin infect dis. 2021;73(6):e1321–e1328. https://doi.org/10.1093/cid/ciab273 johansson ma, quandelacy tm, kada s, et al. sars-cov-2 transmission from people without covid-19 symptoms. jama netw open. 2021;4(1):e2035057–e2035057. https://doi.org/10.1001/jamanetworkopen.2020.35057 tindale lc, stockdale je, coombe m, et al. evidence for transmission of covid-19 prior to symptom onset. elife. 2020;9:1–34. https://doi.org/10.7554/elife.57149 wei we, li z, chiew cj, yong se, toh mp, lee vj. presymptomatic transmission of sars-cov-2 – singapore, january 23 – march 16, 2020. mmwr morb mortal wkly rep. 2020;69(14):411–415. https://doi.org/10.15585/mmwr.mm6914e1 paleker m, tembo ya, davies m-a, et al. asymptomatic covid-19 in south africa – implications for the control of transmission. public health action. 2021;11(2):58. https://doi.org/10.5588/pha.20.0069 güner r, hasanoğlu i̇, aktaş f. covid-19: prevention and control measures in community. turk j med sci. 2020;50(9):571–577. https://doi.org/10.3906/sag-2004-146 williams tc, burgers wa. sars-cov-2 evolution and vaccines: cause for concern? lancet respir med. 2021;9(4):333–335. https://doi.org/10.1016/s2213-2600(21)00075-8 palmer bs. covid-19 eradication: stopping transmission between countries. bmj. 2021;373.e1 https://doi.org/10.1136/bmj.n1425 mulenga lb, hines jz, fwoloshi s, et al. prevalence of sars-cov-2 in six districts in zambia in july, 2020: a cross-sectional cluster sample survey. lancet glob health. 2021;9(6):e773–e781. https://doi.org/10.1016/s2214-109x(21)00053-x soriano v, ganado-pinilla p, sanchez-santos m, et al. main differences between the first and second waves of covid-19 in madrid, spain. int j infect dis. 2021;105:374–376. https://doi.org/10.1016/j.ijid.2021.02.115 akande ow, elimian ko, igumbor e, et al. epidemiological comparison of the first and second waves of the covid-19 pandemic in nigeria, february 2020-april 2021. bmj glob health. 2021;6(11):e007076. https://doi.org/10.1136/bmjgh-2021-007076 avadhanula v, nicholson eg, ferlic-stark l, et al. viral load of sars-cov-2 in adults during the first and second wave of covid-19 pandemic in houston, tx: the potential of the super-spreader. j infect dis. 2021;223(9):1528–1537. https://doi.org/10.1093/infdis/jiab097 ghosh dd, sarkar a, chouhan dp. covid-19 second wave: district level study of concentration of confirmed cases and fatality in india. environ challenges. 2021;5:100221. https://doi.org/10.1016/j.envc.2021.100221 coccia m. the impact of first and second wave of the covid-19 pandemic in society: comparative analysis to support control measures to cope with negative effects of future infectious diseases. environ res. 2021;197:111099. https://doi.org/10.1016/j.envres.2021.111099 iftimie s, lopez-azcona af, vallverdu i, et al. first and second waves of coronavirus disease-19: a comparative study in hospitalized patients in reus, spain. plos one. 2021;16(3):e0248029. https://doi.org/10.1101/2020.12.10.20246959 mudenda s. the second wave of covid-19 and risk of the third wave: factors affecting the continuous transmission, spread of, and increased mortality associated with coronavirus disease 2019 (covid-19). eur j environ public health. 2021;5(2):em0081. https://doi.org/10.21601/ejeph/11056 de cos o, castillo v, cantarero d. facing a second wave from a regional view: spatial patterns of covid-19 as a key determinant for public health and geoprevention plans. int j environ res public health. 2020;17(22):8468. https://doi.org/10.3390/ijerph17228468 chen s, prettner k, kuhn m, et al. climate and the spread of covid-19. sci rep. 2021;11(1):9042. https://doi.org/10.1038/s41598-021-87692-z stall nm, wu w, lapointe-shaw l, et al. sexand age-specific differences in covid-19 testing, cases, and outcomes: a population-wide study in ontario, canada. j am geriatr soc. 2020;68:2188–2191. https://doi.org/10.1111/jgs.16761 lapointe-shaw l, rader b, astley cm, et al. web and phone-based covid-19 syndromic surveillance in canada: a cross-sectional study. plos one. 2020;15(10):e0239886. https://doi.org/10.1371/journal.pone.0239886 ballering av, oertelt-prigione s, olde hartman tc, et al. sex and gender-related differences in covid-19 diagnoses and sars-cov-2 testing practices during the first wave of the pandemic: the dutch lifelines covid-19 cohort study. j womens health. 2021;30(12):1686–1692. https://doi.org/10.1089/jwh.2021.0226 yu c, zhou m, liu y, et al. characteristics of asymptomatic covid-19 infection and progression: a multicenter, retrospective study. virulence. 2020;11(1):1006. https://doi.org/10.1080/21505594.2020.1802194 almadhi ma, abdulrahman a, sharaf sa, et al. the high prevalence of asymptomatic sars-cov-2 infection reveals the silent spread of covid-19. int j infect dis. 2021;105:656–661. https://doi.org/10.1016/j.ijid.2021.02.100 starke kr, reissig d, petereit-haack g, schmauder s, nienhaus a, seidler a. the isolated effect of age on the risk of covid-19 severe outcomes: a systematic review with meta-analysis. bmj glob health. 2021;6(12):e006434. https://doi.org/10.1136/bmjgh-2021-006434 rotevatn ta, nygård k, espenhain l, et al. when schools were open for in-person teaching during the covid-19 pandemic – the nordic experience on control measures and transmission in schools during the delta wave. bmc public health. 2023;23(1):1–12. https://doi.org/10.1186/s12889-022-14906-y crimmins em. age-related vulnerability to coronavirus disease 2019 (covid-19): biological, contextual, and policy-related factors. public policy aging rep. 2020;30(4):142–146. https://doi.org/10.1093/ppar/praa023 article information authors: michael a. noble1 robert martin2 jean-bosco ndihokubwayo3 affiliations: 1program office for laboratory quality management, university of british columbia, canada 2department of global health, university of washington, united states 3world health organization regional office for africa, republic of the congo how to cite this article: noble ma, martin r, ndihokubwayo j-b. making great strides in medical laboratory quality. afr j lab med. 2014;3(2), art. #256, 2 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.256 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. making great strides in medical laboratory quality in this editorial... open access • content • references content top ↑ whilst the observation of blood and urine as a commentary of illness and disease can be traced back to hippocrates as early as 300 bc, the true roots and foundations of the modern medical laboratory as a vital investigation process to better understand pathology and diagnosis were established in the late-19th and early-20th centuries.1 modern day laboratory tests have become the cornerstone for objective data collection to assist, affirm and document diagnoses rather than depending on anecdotal and subjective opinion. use of highly-crafted optical lenses made microscopic examination of urine, sputum, blood and spinal fluid achievable. microbiology techniques for blood and sputum culture made the diagnosis of tuberculosis, diphtheria and typhoid both possible and documentable. examination for bilirubinaemia and abnormal glucose levels also became feasible. the first hospital laboratories were established in london (guys hospital) and baltimore (johns hopkins hospital)1 and, by the early 20th century, laboratories began to become a permanent part of the infrastructure of hospitals.also in the early part of the 20th century, professional organisations were emerging as self-regulating groups that addressed the competencies of laboratory professionals. however, by the 1940s, fw sunderman and w belk were concerned because the evidence and experience in united states laboratories indicated that physicians could not trust laboratory test results. in their key study2 they demonstrated that interlaboratory testing of identical samples resulted in considerable variability of test performance and results. this was the origin of proficiency testing as a valuable quality measure that ultimately led to the development of quality control, laboratory inspections and accreditation bodies across the united states, canada, europe and australia. fortunately, the foundations of the quality movement had already been developed by giants such as walter shewhart, w. edwards deming and others, in their studies of statistical design. medical laboratory quality control and the use of the levey-jennings chart3,4 were based upon the studies of shewhart. over the next 60 years, many countries saw continued growth and improvement in the medical laboratory based upon the tenets of quality and improvements in accreditation processes. lessons were learned from a variety of sources. deming5 and juran6 promoted quality measures as they assisted the post-war rebirth of japanese industry. the military developed tools to ensure that suppliers would deliver high quality, consistent and reliable provisions.6 standards documents, in particular the international organization for standardization (iso) 9001 (quality management) and its seed documents (http://www.iso.org/iso/home/store/catalogue_tc/catalogue_detail.htm?csnumber=46486), led the way to internationalising quality as we know it today. these efforts led to further refinements for testing laboratories and medical laboratories alike. arguably the most significant foundational document for medical laboratory quality improvement in the modern era has been the publication of iso 15189:2003, 2007 and 2012: medical laboratories – requirements for quality and competence.7 errors and challenges continued to occur, with perhaps the greatest challenge of the modern era being the recognition that containment of hiv would require even greater strides in healthcare improvement and enhancement of medical laboratory quality.8 great efforts by the us president’s emergency plan for aids relief (pepfar)9 programme, the bill and melinda gates foundation, the clinton health access initiative and the world health organization (who) pointed the way for laboratory improvement in every part of the world. it is through those processes and the active energy of the who’s regional office for africa (afro) that we see the growth and successes of quality management system programmes such as stepwise laboratory quality improvement process towards accreditation (slipta) and strengthening laboratory management toward accreditation (slmta). time has demonstrated that the path to improvement is not always a straight line; there are periods of success and periods of fallback. laboratory improvement must be experienced as a slow and gradual process based upon building a culture of quality, increasing knowledge and a goal of continual improvement. the study of risk management teaches us that we cannot predict all errors; but through the active practice of quality processes we can detect errors earlier and reduce the repetition of the same error time and time again. it is through the persistence of personal interest and action that we sustain quality efforts and have confidence in successful outcomes for the overall process of laboratory investigation. the authors of the excellent manuscripts in this special issue of the african journal of laboratory medicine, entitled transforming the quality of laboratory medicine through the strengthening laboratory management toward accreditation program, attest to the great strides that are being made through the progressive stepwise approach to adopting and embracing quality measures. but to them we put forward this challenge: whilst it is a great achievement to reach a level of success where the requirements of accreditation are met, the true accomplishment is reaching the point where that level of quality is an everyday practice and expectation, and the laboratory is ‘accreditation-ready’ over and over. when the inevitable slips and mistakes occur in laboratories that are accreditation-ready, the processes of error detection, correction and improvement, and progress back to quality, must occur quickly, smoothly and sustainably.10 references top ↑ 1.1. berger d. a brief history of medical diagnosis and the birth of the clinical laboratory. part 1 – ancient times through the 19th century. mlo med lab obs. 1999;31(7):28–30, 32, 34–40.1.2. belk wp, sunderman fw. a survey of the accuracy of chemical analyses in clinical laboratories. am j clin pathol. 1947;17(11):853–861. 1.3. levey s, jennings er. the use of control charts in the clinical laboratory. am j clin pathol. 1950;20(11):1059–1066. 1.4. levey s, koenig s. quality control standards for analysis of sodium, potassium, and calcium in urine. am j clin pathol. 1958;30(5):404–406. 1.5. walton m. the deming management method. new york, ny: perigee books; 1986. 1.6. juran jm. a history of managing for quality: the evolution, trends, and future directions of managing for quality. milwaukee, wi: asqc quality press; 1995. 1.7. noble ma. making progress in implementing quality in canadian medical laboratories. clin biochem. 2013;46(13–14):1194. http://dx.doi.org/10.1016/j.clinbiochem.2013.04.025 1.8. mfinanga sg, kahwa a, kimaro g, et al. dissatisfaction with the laboratory services in conducting hiv related testing among public and private medical personnel in tanzania. bmc health serv res. 2008;8:171. http://dx.doi.org/10.1186/1472-6963-8-171 1.9. woodcock s, fine g, mcclure k, et al. the role of standards and training in preparing for accreditation. am j clin pathol. 2010;134(3):388–392. http://dx.doi.org/10.1309/ajcp03tfpbkeyynt 1.10. noble ma. quality management in the medical laboratory. keynote presentation. association for pathologists in eastern, central, and southern africa (apecsa) arusha tanzania; august 2006. article information authors: racheal w. kimani1 anne w.t. muigai2 willie sang3 john n. kiiru3 samuel kariuki3 affiliations: 1institute of tropical medicine and infectious disease, jomo kenyatta university of agriculture and technology, kenya 2faculty of science, jomo kenyatta university of agriculture and technology, kenya 3centre for microbiology research, kenya medical research institute, kenya correspondence to: racheal kimani postal address: po box 6056-00200, thika, kenya dates: received: 26 apr. 2012 accepted: 09 apr. 2013 published: 17 oct. 2014 how to cite this article: kimani rw, muigai awt, sang w, kiiru jn, kariuki s. virulence factors in environmental and clinical vibrio cholerae from endemic areas in kenya. afr j lab med. 2014;3(1), art. #41, 7 pages. http://dx.doi.org/10.4102/ ajlm.v3i1.41 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. virulence factors in environmental and clinical vibrio cholerae from endemic areas in kenya in this original research... open access • abstract • introduction • research method and design    • detection of virulence factors from environmental and clinical v. cholerae    • amplification • results    • isolation of v. cholerae from environmental sources    • detection of virulence genes from environmental and clinical v. cholerae isolates • ethical considerations    • potential benefits and hazards • trustworthiness    • reliability    • validity • discussion    • isolation of environmental v. cholerae from western and coastal regions of kenya    • prevalence of virulence factors in environmental and clinical isolates of v. cholerae • conclusion • acknowledgements    • conflicting interests    • authors’ contributions • references abstract top ↑ background: since 1971, kenya has had repeated cholera outbreaks. however, the cause of seasonal epidemics of cholera is not fully understood and neither are the factors that drive epidemics, both in kenya and globally.objectives: the objectives of the study were to determine the environmental reservoirs of v. cholerae during an interepidemic period in kenya and to characterise their virulence factors. methods: one hundred (50 clinical, 50 environmental) samples were tested for v. cholerae isolates using both simplex and multiplex polymerase chain reaction. results: both sediments and algae from fishing and landing bays yielded isolates of v. cholerae. clinical strains were characterised along with the environmental strains for comparison. all clinical strains harboured ctxa, tcpa (el tor), ompu, zot, ace, toxr, hyla (el tor) and tcpi genes. prevalence for virulence genes in environmental strains was hyla (el tor) (10%), toxr (24%), zot (22%), ctxa (12%), tcpi (8%), hyla (26%) and tcpa (12%). conclusion: the study sites, including landing bays and beaches, contained environmental v. cholerae, suggesting that these may be reservoirs for frequent epidemics. improved hygiene and fish-handling techniques will be important in reducing the persistence of reservoirs. introduction top ↑ since 1971, kenya has suffered repeated cholera outbreaks. from 1974 to 1989, outbreaks were reported every year with an average case fatality rate of 3.6%.1 more cases have been reported in kenya since 2005 and an outbreak in 2007 had a case fatality of up to 5.6%.2 in 2011, 60 cholera cases, including 10 laboratory-confirmed cases and one refugee death, were reported in the world’s largest refugee camp in dadaab, kenya.3significant advances have been made in understanding the molecular basis of vibrio cholerae pathogenicity, including the identification of environmental reservoirs for the microorganism.4 it has also been shown that a 7th cholera pandemic spread from the bay of bengal in at least three independent but overlapping waves with a common ancestor in the 1950s and several transcontinental transmission events have been identified.5 the main reservoirs of v. cholerae are humans and aquatic sources such as brackish water and estuaries.6 ahmed et al.7 showed that multiple genetic lineages of v. cholerae were simultaneously infecting persons in kenya. this finding is consistent with the simultaneous emergence of multiple distinct genetic lineages of v. cholerae from endemic environmental reservoirs rather than recent introduction and spread by travelers. however, the cause of seasonal epidemics of cholera is not fully understood and neither are the factors that drive epidemics, both in kenya and globally. the objectives of this study were to determine the environmental reservoirs of v. cholera during an interepidemic period in kenya and to characterise their virulence factors. research method and design top ↑ the study was conducted in the coastal and western regions of kenya from february 2010 to november 2010. the coastal region sampling points were distributed in the cholera-endemic districts of kwale, malindi, mombasa and kilifi (figure 1). at each sampling point, preference was given to watering points and where there was human activity, as recommended by local health officials. the western region sampling points included the districts of kisumu, siaya, rachuonyo, homa bay, nandi hills, nyando, bondo, vihiga and busia. one sample of water, zooplankton, phytoplankton, sediments and/or floating vegetation was collected at each site as available, using the method described by huq et al.8 figure 1: coastal and western regions of kenya where environmental samples were collected. isolation began on the first day after collection of the environmental samples. one ml each of the zooplankton and phytoplankton homogenates was enriched in 10 ml (1x) alkaline peptone water. ten ml of each plant homogenate and sediment sample were also enriched in 5 ml triple-strength alkaline peptone water. on the second day, subculturing was done from the previous day’s alkaline peptone water inoculates on plates of thiosulphate citrate bile salts (tcbs). on day three, the presence of yellow mucoid colonies was considered presumptive for v. cholerae. presumptive colonies were tested by measuring their reaction to an oxidase reagent (1% dimethyl-p-phenylene-diamine dihydrochloride), along with other biochemical tests (voges proskauer, indole and carbohydrate fermentation). the environmental strains agglutinated with polyvalent o antisera and were sucrose fermenters. they formed large yellow mucoid colonies on the tcbs. colonies were also gram stained to confirm the morphological characteristics of the isolates. detection of virulence factors from environmental and clinical v. cholerae archived clinical isolates were sampled from the kenya medical research institute (kemri) laboratory. these isolates were mostly from outbreaks which occurred in the years 2009–2010. systematic random sampling was carried out in order to choose clinical strains. from a list of whole population strain numbers, a random strain number was chosen as the start strain. thereafter, every third strain from the whole population was sampled until a total of 50 samples were chosen, which is equal to the number of environmental strains. amplification deoxyribonucleic acid (dna) was isolated from freshly-cultured environmental and clinical v. cholerae isolates. the protocol was as follows: on the first day, bacterial isolates were grown at 37 °c overnight in mueller-hinton plates. cells were harvested and suspended in distilled water on the second day. the suspension was heated in a water bath at 95 °c for five minutes to lyse the cells. this was then centrifuged at 14 000 x g for six minutes in order to remove the protein debris. a sample supernatant was then stored at -20 °c for further use. polymerase chain reaction (pcr) was performed in a total volume of 32 µl containing 5 µl (5 ng/µl) of dna template, 1.0 µl (0.1 mm) each of forward and reverse primers, healthcare mastermix (ge healthcare, uk) and 25 µl nuclease-free water.all strains were then screened for the presence of genes encoding virulence determinants in v. cholerae including cholera toxin (ctxa), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), haemolysin (hlya), and nag-specific heat-stable toxin (st). detection of the tcpa gene specific to the el tor and classical biotypes was performed using a common forward primer and biotype-specific reverse primers. similarly, two forward primers were used for the detection of the biotype-specific haemolysin gene (hyla). pcr conditions and primers used for the detection of tcpa, ompu, tcpi, toxr and hyla genes were similar to those described previously by rivera et al.9 (table 1 and table 2), whilst detection of the ctxa gene was done using primers and conditions described before by fields et al.10 all pcr assays were performed using an automated dynax thermal cycler. pcr products were analysed by electrophoresis on 1.5% agarose gels, stained with ethidium bromide, visualised under uv light and recorded with the aid of a gel-documentation system. table 1: primers used in the study. table 2: primers pooled for multiplex pcr analysis. results top ↑ isolation of v. cholerae from environmental sources the coastal region had a total of 23 positive isolates out of 207 collected environmental samples (11%) (table 3). kwale had the most positive isolates, accounting for 43% of positive isolates collected in this region, followed by kilifi (35%) and mombasa (22%). no samples collected in malindi had positive isolates. the western region had 27 positive isolates out of 199 collected samples (12%). homa bay had the most positive isolates, accounting for 30% of positive isolates collected in this region, followed by kisumu, siaya and rachuonyo (15% each); nandi hills and nyando (7% each) and bondo, vihiga and busia (~4% each). table 3: sources of v. cholerae samples collected in coastal and western kenya by region. detection of virulence genes from environmental and clinical v. cholerae isolates all clinical strains (50 of 50) were pcr positive for the gene representing the el tor fragment for haemolysin (hyla) (figure 2), whilst only 10% (5 of 50) of the environmental strains were positive (table 4). in addition, all clinical strains were pcr positive for the gene representing the classical fragment for haemolysin as compared with 13 out of 50 (26%) of the environmental strains. for environmental v. cholerae strains, the results of the multiplex pcr yielded positive results for tcpa (el tor) at 12% (6 of 50), ctxa at 8% (4 of 50), zot at 24% (12 of 50), tcpi at 8% (4 of 50), toxr at 22% (11 of 50) and ompu at 0% (0 of 50) in the environmental strains. all clinical and environmental v. cholerae isolates (100%) were negative for the st gene. figure 2: example amplification of ompu, toxr and hyla genes in clinical vibrio cholera. table 4: results of polymerase chain reaction. table 4 part 2: results of polymerase chain reaction. ethical considerations top ↑ ethical approval was provided by the kenya national ethics review committee (approval number kemri/res/7/3/1). potential benefits and hazards there were no potential benefits or hazards to human subjects as they were not involved in the study. trustworthiness top ↑ reliability reliability is associated with accuracy, stability, consistency and reproducibility of the research. this was ensured by peer examination and open discussion with all participants. validity validity entails ensuring that the datasets gathered or items used are pertinent or relevant to the research conducted. this was ensured by use of appropriate research design and methods. discussion top ↑ isolation of environmental v. cholerae from western and coastal regions of kenya knowledge regarding environmental reservoirs of v. cholerae is of critical epidemiological and public health importance. we identified environmental reservoirs of v. cholera in kenya in a disease climate consisting of few reported cases. it is therefore likely that the v. cholerae existing in the environment amplify during the off-season to cause epidemics. prevalence of virulence factors in environmental and clinical isolates of v. cholerae in this study, the occurrence and distribution of selected virulence-associated genes in environmental and clinical strains of v. cholerae collected in kenya were demonstrated.environmental studies of v. cholerae have been done with the expectation that the v. cholerae strains possessing the entire complement of virulence genes would be isolated. in this study, as in previous similar studies, this did not happen. this can be explained by the fact that virulence genes are dispersed amongst environmental strains of v. cholerae and may be ferried about in mobile genetic elements. these genotypes of environmental v. cholerae are said to act as reservoirs for the virulence factors and potential mixing and matching leads to the formation of new pathogenic strains.11 two multiplex pcr assays revealed that all of the clinical strains were positive for ctx, zot, tcpi, and ace, as well as hlya (both el tor and classical), ompu and the toxr gene, suggesting the presence of an intact core toxin region in all isolates. these genes are found together and represent the genome of filamentous bacteriophage ctxφ. this implies that they possessed a ctxφ and tcp pathogenicity island. the pilus colonisation factor tcp acts as a receptor for ctxφ, which can infect non-toxigenic v. cholerae, leading to the emergence of new toxigenic strains of v. cholerae. the genes that encode the cholera toxin subunits ctxa and ctxb are localised to a ctx genetic element which is made up of a 4.6 kbp central core region and a 2.4 kbp repetitive sequence known as rs2.12 classical strains of v. cholerae o1 contain two copies of the ctx element, one on each chromosome. waldor and mekalanos13 were able to show that ctx is a filamentous bacteriophage related to the m13 coliphage.12 faruque, alberts and mekalanos14 reported that an environmental v. cholerae and two v. mimicus strains, all of which lacked tcp, were capable of being infected by ctxф. the v. cholerae pathogenicity island (vpi) is 39.5 kb in size and contains genes associated with virulence (tcp-acf cluster), the regulation of virulence (toxt and tcpp/h), the regulation of chemotaxis (tcpi or acfb) and mobility (int and orfi).9 the genes encoding tcp have been suggested to be part of a larger genetic element consisting of a cluster of genes. the tcpa el tor gene was positive in all clinical v. cholerae isolates and in 12% of environmental isolates in the current study, and the tcpa classical gene was negative in all isolates. harkey et al.15 suggested that regulators such as tcpi that act downstream of toxr and toxt may function to fine tune the expression of the tcp virulence determinant throughout the pathogenic cycle of v. cholerae. the tcpi encoding gene present in 8% of our environmental strains may thus have some other physiological functions as well.16 the outer membrane protein, ompu, was reported to be a potential adherence factor for v. cholerae. in this study, the gene was present in all of the v. cholerae strains of clinical origin that were tested, and negative in v. cholerae strains isolated from the environment. the haemolysin gene has been employed to differentiate between the two biotypes of v. cholera o1. two primer sets for the classical and el tor biotypes were used in this study. the v. cholerae strains of clinical origin from both biotypes were positive for the hyla gene. environmental isolates were 26% positive for haemolysin of the classical biotype and 10% for the el tor biotype. this phenomenon of detecting both fragments encoding for hyla specific to both the classical and the el tor biotypes in one isolate was observed in a similar study.17 nag-st in v. cholera o1 strains that is closely related to the heat-stable toxins produced by enterotoxigenic e. coli and other enteric pathogens was defined.18 the heat-stable enterotoxin production by the st genes was tested and none of the v. cholerae strains (of either environmental or clinical origin) were positive. the regulation and expression of genes for growth and survival depend on the regulon toxr, which is regulated by a cascade mechanism involving three known components: toxr, toxs and toxt. toxr, a 32-kda transmembrane protein, is the master regulator and its expression is dependent upon environmental growth conditions (incubation temperature, ph, osmolarity, bile salts, oxygen tension, hydrostatic pressure and amino acid composition of the medium.19 the toxr gene encodes a transcriptional activator controlling ct gene expression (ctxa), tcp biogenesis (tcpa), outer membrane protein expression (ompu), and at least 17 distinct genes in the o139 and o1 strains.20 in this study, the presence of the toxr gene was verified in all v. cholerae isolates of clinical origin and in 24% of the environmental isolates. conclusion top ↑ the implications of finding v. cholerae in environmental sources with virulence genes also found in clinical isolates are significant. the possibility is that this may lead to an epidemic strain arising through gene transfer that links genes that facilitate transmission in human populations, whilst those that foster persistence are proliferating in nearby environmental niches. gene interactions are therefore important to understanding the epidemiology and persistence of virulence factors and subsequent epidemic outbreaks. proper hygiene and fish-handling techniques should be ensured to cut the transmission from environmental sources to humans. acknowledgements top ↑ the authors would wish to thank the disease surveillance unit of the ministry of public health and sanitation, kenya for providing information on past cholera outbreaks. we also thank kemri–cmr members of staff for their support in this work. conflicting interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions r.k. (institute of tropical medicine and infectious disease, jomo kenyatta university of agriculture and technology [jkuat]) analysed the data and drafted the manuscript. s.k. (cmr–kemri), w.s. (cmr-kemri) and a.w.t.m. (faculty of science, jkuat) supervised the carrying out of this work. j.n.k. (cmr–kemri) supervised the laboratory protocols and execution of experiments. references top ↑ 1.world health organization. weekly epidemiol rec. 2004;79:236–244.2.world health organization. global task force on cholera control. cholera country profile [document on the internet]. [cited april]. available from: http://www.who.int/cholera/countries/kenyacountryprofile2010.pdf 3.nehebay s. cholera breaks out in kenya's dadaab refugee camp: un [page on the internet]. c2011 [cited 2012 sep 12]. available from http://www.reuters.com/article/2011/11/15/ozatp-kenya-un-refugees-idafjoe7ae0dr20111115 4.colwell rr, tamlin ml, brayton pr, et al. environmental aspects of vibrio cholerae in transmission of cholera. in: sack rb, zinnaka y, editors. advances in research on cholera and related diarrhoea. 7th ed. tokyo: ktk scientific publishers, 1990; pp. 327–343. 5.mutreja a, kim dw, thomson nr, et al. evidence for several waves of global transmission in the seventh cholera pandemic. nature 2011;477(7365):462–465. http://dx.doi.org/10.1038/nature10392 6.world health organization. cholera annual report 2011. weekly epidemiol rec. 2012;87(31–32), 289–304. 7.mohamed aa, oundo j, kariuki sm, et al. molecular epidemiology of geographically dispersed vibrio cholerae, kenya, january 2009–may 2010. emerging infectious diseases. 2012;18(6):n.p.. 8.huq a, sack rb, nizam a, et al. critical factors influencing the occurrence of vibrio cholerae in the environment of bangladesh. appl environ microbiol. 2005;71(8):4645–4654. http://dx.doi.org/10.1128/aem.71.8.4645-4654.2005 9.rivera in, chun j, huq a, et al. genotypes associated with virulence in environmental isolates of vibrio cholerae. appl environ microbiol. 2001;67(6):2421–2429. http://dx.doi.org/10.1128/aem.67.6.2421-2429.2001 10.fields pi, popovic t, wachsmuth k, et al. use of polymerase chain reaction for detection of toxigenic vibrio cholerae o1 strains from the latin american cholera epidemic. j clin microbiol. 1992;30(8):2118–2121. 11.karaolis dkr. a vibrio cholerae pathogenicity island associated with epidemic and pandemic strains. proc natl acad sci u s a. 1998;95(6):3134–3139. http://dx.doi.org/10.1073/pnas.95.6.3134 12.garg p, nandy rk, chaudhury p, et al. emergence of vibrio cholerae o1 biotype el tor serotype inaba from the prevailing o1 ogawa serotype strains in india. j clin microbiol. 2000;38(11):4249–4253. 13.waldor mk, mekalanos jj. lysogenic conversion by a filamentous phage encoding cholera toxin. science. 1996;272:1910–1914. http://dx.doi.org/10.1126/science.272.5270.1910 14.faruque sm, albert mj and mekalanos jj. epidemiology, genetics and ecology of toxigenic vibrio cholerae. microbiol mol biol rev. 1998;62:1301–1314. 15.harkey cw, everiss kd, peterson km. the vibrio cholerae toxin-coregulated-pilus gene tcpi encodes a homolog of methyl-accepting chemotaxis proteins. infect immun. 1994;62(7):2669–2678. 16.chakraborty s, mukhopadhyay ak, bhadra rk, et al. virulence genes in environmental strains of vibrio cholerae. appl environ microbiol. 2000:66(9):4022–4028. http://dx.doi.org/10.1128/aem.66.9.4022-4028.2000 17.singh dv, matte mh, matte gr, et al. molecular analysis of vibrio cholerae o1, o139, non-o1, and non-o139 strains: clonal relationships between clinical and environmental isolates. appl environ microbiol. 2001;67(2):910–921. http://dx.doi.org/10.1128/aem.67.2.910-921.2001 18.arita mt, honda t, miwatani t, et al. purification and characterization of a heat-stable enterotoxin of vibrio mimicus. fems microbiol lett. 1991;63(1):105–110. http://dx.doi.org/10.1111/j.1574-6968.1991.tb04513.x 19.dirita vj. co-ordinate expression of virulence genes by toxr in vibrio cholerae. mol microbiol. 1992;6(4):451–458. http://dx.doi.org/10.1111/j.1365-2958.1992.tb01489.x 20.herrington da. toxin, toxin-coregulated pili, and the toxr regulon are essential for vibrio cholerae pathogenesis in humans. j exp med. 1998;168(4):1487–1492. http://dx.doi.org/10.1084/jem.168.4.1487 21.shi l, miyoshi s, hiura m, et al. 1998. detection of genes encoding cholera toxin (ct), zonula occludens toxin (zot), accessory cholera enterotoxin (ace) and heat-stable enterotoxin (st) in vibrio mimicus clinical strains. microbiol immunol. 1998;42(12):823–828. http://dx.doi.org/10.1111/j.1348-0421.1998.tb02357.x 22.vicente ac, coelho am, salles ca. detection of vibrio cholerae and v. mimicus heat-stable toxin gene sequence by pcr. j med microbiol. 1997;46(5):398–402. http://dx.doi.org/10.1099/00222615-46-5-398 23.bhanumathi r, sabeena f, isac sr, et al. molecular characterization of vibrio cholerae o139 bengal isolated from water and the aquatic plant eichhornia crassipes in the river ganga, varanasi, india. appl environ microbiol. 2003;69:2389–2394. http://dx.doi.org/10.1128/aem.69.4.2389-2394.2003 abstract introduction research method and design results discussion trustworthiness acknowledgements references about the author(s) feyisayo jegede family health international 360 (fhi360), department of laboratory services, abuja, nigeria henry a. mbah labtrail global, smyrna, delaware, united states ado dakata department of haematology, bayero university/aminu kano teaching hospital, kano, nigeria dalhatu h. gwarzo department of haematology, bayero university/aminu kano teaching hospital, kano, nigeria surajudeen a. abdulrahman family health international 360 (fhi360), department of laboratory services, abuja, nigeria aisha kuliya-gwarzo department of haematology, bayero university/aminu kano teaching hospital, kano, nigeria citation jegede f, mbah ha, dakata a, et al. evaluating laboratory request forms submitted to haematology and blood transfusion departments at a hospital in northwest nigeria. afr j lab med. 2016;5(1), a381. http://dx.doi.org/10.4102/ajlm.v5i1.381 original research evaluating laboratory request forms submitted to haematology and blood transfusion departments at a hospital in northwest nigeria feyisayo jegede, henry a. mbah, ado dakata, dalhatu h. gwarzo, surajudeen a. abdulrahman, aisha kuliya-gwarzo received: 20 oct. 2015; accepted: 04 feb. 2016; published: 12 may 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the laboratory request form (lrf) is a communication link between laboratories, requesting physicians and users of laboratory services. inadequate information or errors arising from the process of filling out lrfs can significantly impact the quality of laboratory results and, ultimately, patient outcomes. objective: we assessed routinely-submitted lrfs to determine the degree of correctness, completeness and consistency. methods: lrfs submitted to the department of haematology (dh) and blood transfusion services (bts) of aminu kano teaching hospital in kano, nigeria, between october 2014 and december 2014, were evaluated for completion of all items on the forms. performance in four quality indicator domains, including patient identifiers, test request details, laboratory details and physician details, was derived as a composite percentage. results: of the 2084 lrfs evaluated, 999 were from dh and 1085 from bts. overall, lrf completeness was 89.5% for dh and 81.2% for bts. information on patient name, patient location and laboratory number were 100% complete for dh, whereas only patient name was 100% complete for bts. incomplete information was mostly encountered on bts forms for physician’s signature (60.8%) and signature of laboratory receiver (63.5%). none of the dh and only 9.4% of bts lrfs met all quality indicator indices. conclusion: the level of completion of lrfs from these two departments was suboptimal. this underscores the need to review and redesign the lrf, improve on training and communication between laboratory and clinical staff and review specimen rejection practices. introduction efficient laboratory service remains a foundation of modern healthcare systems. laboratory testing is an essential part of the clinical decision-making process, because it provides the majority of critical information required for making timely and informed decisions for patient care.1 relationships with laboratories and utilisation of laboratory services by physicians and other stakeholders in the healthcare system occur mainly through the use of laboratory request forms (lrfs) for two-way communication. requesting physicians may not fully utilize this important communication medium.2 inadequate information or errors arising from the process of filling out lrfs can have a significant impact on the quality of laboratory outputs and, ultimately, on patient safety.3,4 the notion of the brain-to-brain loop for laboratory diagnostics was first introduced by george lundberg over 30 years ago.5 the first step in this loop model involves the selection of appropriate laboratory tests in the brain of the physician, which is then communicated through the lrf. this is followed by numerous intermediary steps, such as identification of the patient, specimen collection and specimen handling; and then by the actual specimen analysis in the laboratory. the last steps involve the release of test results, either manually or electronically, for the physician’s review and reaction to the laboratory information, the interpretation of the results and the implementation of appropriate clinical action.5 traditionally, laboratory practice is divided into three phases (pre-analytical, analytical and post-analytical).6 evidence shows that the majority of laboratory errors (50% – 70%) occur during the pre-analytical phase and involve the handling of the lrf.7 errors occurring during the analytical phase average less than 10%,8 whereas errors occurring during the the post-analytical phase average about 15%.3 the preand post-analytical phases lie outside of the control of the laboratory, but contribute approximately 93% of total laboratory errors across the entire testing process.9,10,11,12 the most frequent pre-analytical errors as compiled by lippi12 are: missing sample and/or test request; wrong/missing identification; in vitro haemolysis; undue clotting; wrong container; and contamination from infusion route. other errors include: insufficient sample; inappropriate blood-to-anticoagulant ratio; insufficient mixing of the sample; or inappropriate transport and storage conditions. insufficient information or omission of information on the lrf may lead to laboratory errors,13 as well as make result interpretation difficult and delay communications with the requesting physician, moreso in patients with life-threatening medical conditions. misidentification of either the patient or the requested test have also been encountered frequently.14 the lrf not only provides information about the laboratory test being requested, but is also used to communicate results back to physicians and patients. the standard lrf contains demographic data and other information, such as location of the patient, laboratory information, physician’s name and signature, telephone number of the requesting physician, amongst others. pre-analytical errors, such as the absence of important clinical information on lrf, can have serious effects on patient care by causing post-analytical errors, such as inappropriate interpretative comments.15 the majority of errors occurring during the pre-analytical phase are a result of individual or system design defects.16 the pre-analytical phase should be prioritised so as to reduce errors across the entire laboratory testing process.16 in australia, planned interventions and sustained improvements in compliance with standards resulted in an immediate reduction in the proportion of incomplete lrfs, from 43% to 2%.14 in developed countries, laboratory quality management systems have been institutionalised, with functional and robust monitoring systems in place to detect and minimise errors before they occur at any phase in the laboratory work flow. unfortunately, the converse is true in most laboratories in developing countries, such as nigeria.17,18 in these countries, the focus tends to be on the analytical phase of the work flow without consideration of other factors or variables beyond the control of the laboratory. in nigeria, there are few studies on the handling of lrfs or the impact of the lrf on the pre-analytical phase of laboratory process. the objective of this study was to assess routinely-submitted lrfs for correctness, completeness and consistency and to evaluate the contributions of the lrf to quality service delivery. research method and design ethical considerations the study protocol was reviewed and approved by the aminu kano teaching hospital ethical committee (reference number: akth/mac/sub/12a-3/vi/1330) in line with the international standard of research requirement. team members were trained to retain patient confidentiality and patient names were not collected as part of the data set. study design and setting this was a retrospective, cross-sectional, descriptive study. all lrfs submitted to the department of haematology (dh) and the blood transfusion services (bts) of aminu kano teaching hospital, kano, nigeria, from october 2014 to december 2014 were reviewed systematically and evaluated for completeness, correctness and consistency. selection of dh and bts lrfs was based on presumed availability of lrfs and the high workload of these departments. aminu kano teaching hospital is a tertiary health institution located in kano state in northwestern nigeria. it is a 600-bed hospital which serves as the referral centre to kano and other neighbouring states, including katsina, jigawa and bauchi. data collection and analysis hard copies of both inpatient and outpatient lrfs received for routine laboratory investigations were reviewed and evaluated for the purposes of this study. data from the dh and bts were extracted manually from the lrfs and entered into an excel file (microsoft, redmond, washington, united states), then collated, cleaned and reviewed before analysis using the statistical package for social scientists (spss; version 21/2012; ibm, armonk, new york, united states). a score of 1 was used to indicate complete and correctly-filled information, whereas a score of 0 was recorded when any item was missing. a frequency distribution table was created to summarise the data collected. data were analysed and categorised into groups of quality indicators (qi), based on international federation of clinical chemistry-working group (ifcc-wg) guidelines.19 the qis used were: (1) patient identifiers (name, age, sex and unit number); (2) appropriateness of test request (request date and specimen type); (3) availability and completeness of laboratory details (eg. laboratory number and reference range); and (4) availability and completeness of physician’s details (doctor’s name and signature, name of consultant, phone and fax number). in this setting, ‘consultants’ head a medical team and are the most experienced senior clinicians. results a total of 2084 lrfs were evaluated, comprising 999 from dh and 1085 from bts. dh forms requested a total of 12 data elements, whereas bts forms requested a total of 18 data elements. department of haematology overall, 89.5% of dh forms were filled out completely (table 1). of all the required information on the lrf, only patient name, location within the hospital (ward) and laboratory number were filled out both completely and correctly for all patients. patient age, sex, request date, unit number, specimen type and clinical information were both available and correctly filled out for over 98% of the forms. of the 244 (24.4%) lrfs with incomplete information for either the doctor’s name and signature or the consultant’s name, a greater number of forms (n = 145; 14.5%) were missing the consultant’s name as compared with those missing the physician’s name and signature (n = 99; 9.9%). the current dh lrf does not request the phone number of the requesting physician, the time of specimen collection, the signature of the laboratory supervisor/manager to validate the patient results or the reference range. table 1: completeness of laboratory request forms submitted to department of haematology of aminu kano teaching hospital, kano, nigeria, from october 2014 to december 2014 (n = 999). blood transfusion service overall, 81.2% of bts lrfs were filled out completely (table 2), which was lower than for the dh. of all the variables expected to be completed on the lrfs, only patient name was available and completed on all forms examined. conversely, none of the bts forms had time of request completed. as observed with the dh forms, patient age was completed on 1071 forms (98.8%) and sex was completed on 1056 forms (97.4%). for 62 forms (5.7%), hospital/unit number was not indicated. for test request information, clinical information/diagnosis was completed on 877 forms (80.9%), whereas 1000 forms (92.2%) had information on specimen type completed. similarly, date of request was completed on 1005 forms (92.7%). for other lrf details, number of units of blood requested was complete on 1013 forms (93.4%), type of product requested on 866 forms (79.8%), and degree of urgency on 811 forms (74.7%). the physician’s name was missing on 124 forms (11.4%) and the physician’s signature on 660 forms (60.8%), whereas 271 forms (25%) were missing the supervising consultant’s name. all but one (0.1%) of the bts forms had laboratory number completed. almost all forms (n = 1082; 99.7%) had the date of sample collection completed, whereas only 396 (36.5%) had the signature of the laboratory receptionist (receiver) completed. the current bts form does not request the phone number of the requesting physician or the time of the request. table 2: completeness of laboratory request forms submitted to blood transfusion services of aminu kano teaching hospital, kano, nigeria, from october 2014 to december 2014 (n = 1085). major quality indicators overall, the most frequently occurring data quality gap identified on dh forms was completion of laboratory details (n = 0), followed by physician’s details, which were complete on 762 of the forms examined (76.3%; table 3). patient identifiers were available and complete on 968 dh forms (96.9%), and 991 forms (99.2%) had relevant fields for test request completed. the most frequently observed quality gap on bts forms was completion of physician’s details (n = 349; 32.2%), followed by completion of laboratory details (n = 396; 36.5%) and test request details (n = 522; 48.1%). the least commonly occurring data quality gap on bts forms was completion of patient identifiers (n = 1043; 96.1%). none of the dh request forms and only 102 bts forms (9.4%) examined met all of the qis analysed in this study. the majority of bts forms (n = 983; 90.6%) met one or more qi requirements. table 3: completeness of laboratory request forms submitted to department of haematology (n = 999) and blood transfusion services (n = 1085) of aminu kano teaching hospital, kano, nigeria, from october 2014 to december 2014: analysis based on major quality indicators according to international federation of clinical chemistry working group.19 discussion the study revealed that of the 12 required pieces of information on lrfs from the dh, only three (patient’s name, location within the hospital [ward] and laboratory number) were filled out both completely and correctly for all patients. for lrfs from the bts, of 18 pieces of required information, only patient name was filled out both completely and correctly for all patients. the most commonly incomplete item on dh forms was the specimen receiver’s signature, whereas for bts forms, specimen receiver’s signature and doctor’s signature were commonly incomplete. this study demonstrated that a higher proportion of lrfs from the dh were completed compared with lrfs from the bts (89.5% for dh vs 81.2% for bts). these findings are in agreement with a proportion of 84% completion previously reported from a similar study conducted in ile-ife, nigeria.20 however, our finding is in sharp contrast with a proportion of 1.73% for lrf completion reported from a similar study conducted in lagos, nigeria.21 in our study, the name of the requesting physician was completed on most dh and bts forms (90.1% for dh; 88.6% for bts). unfortunately, because of the design of the lrf, the contact details of the requesting physician, which may be needed for follow-up, are not requested. various studies conducted in south africa have reported comparable (89.6%)22 or a lower (65.2%)15 proportions for missing physician details and contact information. consultant name was well documented, with complete information on 75% of bts forms and 85.5% of dh forms. however, these proportions are lower than an ile-ife study reporting 96.6% completion of consultant-in-charge information.20 an australian study reported that 43% of forms lacked complete information; missing items included physician’s name and pager number(s).14 one reason for this variation in our setting may be attributed to work pressure on junior physicians and improper orientation regarding the impact of incomplete lrfs on the quality of patient care. this training is done by senior physicians without collaboration with laboratory professionals. in nigeria, it is not uncommon for physicians to be reluctant to follow guidance from medical laboratory professionals because of the prevailing power differential between physicians and other health care providers.23 healthcare workers’ attitudes18,24 toward the completion of lrfs cannot be overlooked, following reports of poor documentation of laboratory processes. in nigeria, it is common for staff to consider such documentation as unnecessary paperwork and an extra burden.18,24 our study reported that 99.8% of dh forms and 80.9% of bts forms had clinical diagnosis details completed. this is consistent with a similar study conducted in ile-ife, nigeria, which reported 93.2% completion of clinical diagnosis details.20 however, our findings contrast with the 65.9% completion of clinical details reported in lagos, nigeria,21 77% completion reported at nepal university teaching hospital25 and 22.7% completion reported at ghana tertiary hospital.13 furthermore, a study of lrfs conducted in cape town, south africa, reported that 20.8% had no diagnosis information and 25.3% had diagnosis information given in an abbreviated form.15 in an indian study, diagnosis was not indicated on 61.20% of forms.26 unfortunately in these cases, critical results found by the laboratory for 17.30% of the patients could not be communicated to them by the physicians because of incomplete forms.26 in our study, about 98% of dh and bts forms had complete information for patient age and sex, which is comparable to 86.4% completion for age and 99.8% completion for sex reported from the ile-ife, nigeria study.20 our findings are higher than the lagos, nigeria study, which reported 68% completion for patient age,21 as well as the much lower completion reported for patient age (25.6%) and sex (32.7%) in a ghanaian study.13 both our report and the ile-ife study20 found that the only well-completed parameter on the lrfs was patient information. the design of the request form may itself be a contributing factor to eliciting completion of some desired information, as patient demographic characteristics are displayed prominently at the beginning of each form. however, in addition to patient information, the lagos study found that the referring physician’s name was the most completed information (99%),21 which is better than our findings of 90.1% for dh forms and 88.6% for bts forms. we found that documentation of specimen type was better for dh (99.7%) compared with bts (92.2%). this is close to the 89.9% reported in the ile-ife, nigeria study.20 however, our finding contrasts with the much lower ~12% reported at the north indian neuropsychiatry institute.26 it is worth noting that only 4.3% of dh forms appropriately captured the signature of the laboratory receptionist. reception of samples from inpatients is usually in bulk; as such, the receptionist may be overwhelmed with work and therefore not able to individually sign all lrfs. other contributing factors may be: lack of proper training; and commitment to utilise standard operating procedures and guidelines in the laboratory. more importantly, information on result verification by the laboratory supervisor/manager before release was unavailable, as the lrfs examined in this study did not request this information. in general, none of the dh or bts forms examined in this study met all of the ifcc-wg qis.19 considering the frequency of omission of very vital information on both departments’ lrfs, including physician contact details, laboratory details, and test request, we suggest that both lrfs be redesigned to meet international standards. limitations one of the major limitation of this study is that the opinion of the healthcare workers involved in completing the lrfs was not sought. this would have given more insight into the reasons for the incomplete items. another limitation of this study is its design and the differences in the two lrfs. the dh form has a total of 12 items, whereas the bts form has 18 items. hence, comparing the quality and completion of the two forms for the different sections should be interpreted with caution in the light of these design variations. in addition, we did not classify the lrfs in terms of inpatient or outpatient, routine or emergency service. hence, the impact of the missing information on care of critical patients could not be assessed. recommendations this study demonstrates that the currently-used lrf for both dh and bts should be reviewed and redesigned. the redesign should include: the physician’s phone number, time of the sample collection, time of the request, signature of the laboratory supervisor (to validate results) and the biological reference range interval in line with iso 15189 requirements and standards.27 biological reference ranges serve as guidance for the proper interpretation of laboratory test results. there is a need to develop a laboratory quality manual, guidelines and standard operating procedures, especially for sample rejection practices, as well as details on utilisation and completion of lrfs. basic components of laboratory processing with an emphasis on the pre-analytical stage of laboratory work flow should be prominent in the orientation training of all new house officers, residents and other users of laboratory services. there is a need to revive and sustain joint physician-laboratory conferences and review meetings to share knowledge, strengthen communication and foster feedback for quality improvement. periodic comprehensive laboratory audits with an emphasis on lrf evaluation could be beneficial in comparing baseline information with post-evaluation data for continuous quality improvement efforts. extension of similar assessment of the lrf currently being used in other departments at aminu kano teaching hospital (microbiology, chemical pathology and histopathology) could also create an opportunity for improvement in the quality of laboratory outputs and, ultimately, on patient care. conclusion overall completion of lrfs submitted to dh was higher compared with those submitted to bts; however, completion of bts lrfs was higher when assessed according to qis. this study highlights the level of incompleteness of routinely-submitted lrfs and points out certain expected and vital pieces of information that were completely missing. this underscores the need to redesign the lrf, provide capacity building, strengthen communication between laboratory staff and physicians and enforce specimen rejection practices. trustworthiness the findings reported in this article reflect the outcome of work done on lrfs by our research team members who participated in the research design and excusion, collection, analysis of data and report writing. reliability the findings of the research presented in this report were based on a review of lrfs submitted routinely to the dh and bts of aminu kano teaching hospital in kano, nigeria. based on the study design, the findings are specific and limited to amino kano teaching hospital, nigeria. validity the findings, outcomes and recommendations from this study may be of benefit to developing countries, such as nigeria. in addition to the percentage performance reported, data were also subjected to four composite qis to evaluate the most important qis expected on lrfs. importantly, the findings and outcomes of this study will form a baseline for comparison in future and the practical recommendations offered will help aminu kano teaching hospital to make informed decisions about re-designing the assessed lrfs and to stimulate review of other lrfs in other sections of the hospital. acknowledgements the authors sincerely appreciate the aminu kano teaching hospital chief medical director, the ethical committee and staff of dh and bts, especially the laboratory staff, for the support and conducive atmosphere provided during data collection for the successful conduct of this study. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. the views expressed here are those of the authors and do not necessarily reflect those of aminu kano teaching hospital, kano or fhi-360. authors’ contributions f.j. conceptualised the study, performed the literature search and was involved in the writing of the manuscript. s.a.a. contributed to study conceptualisation and writing of the manuscript and performed statistical analysis. h.a.m. was the research team lead, co-conceptualised the study and critically reviewed the manuscript. a.k.-g. contributed to the literature search, conceptual contributions and writing of the manuscript. d.h.g. performed some of the experiments and contributed to the writing of the manuscript. a.d. performed the experiments, as well as being involved in data collection and writing of the manuscript. references harrison jp, mcdowell gm. the role of laboratory information systems in healthcare quality improvement. int j health care qual assur. 2008;21(7):679–691. levinson w, lesser cs, epstein rm. developing physician communication skills for patient-centered care. health affairs. 2010;29(7):1310–1318. http://dx.doi.org/10.1377/hlthaff.2009.0450 goswami b, singh b, chawla r, et al. evaluation of errors in a clinical laboratory: a one-year experience. clin chem lab med. 2010;48(1):63–66. http://dx.doi.org/10.1515/cclm.2010.006 ogbaini-emovon e, ojide ck, mordi rm, et al. inadequate information in laboratory test requisition in a tertiary hospital in benin city, nigeria. ann biomed sci. 2013;12(2):6–13. plebani m, favaloro ej, lippi g. patient safety and quality in laboratory and hemostasis testing: a renewed loop? semin thromb hemost. 2012;38(6):553–558. http://dx.doi.org/10.1055/s-0032-1315960 plebani m. errors in laboratory medicine and patient safety: the road ahead. clin chem lab med. 2007;45(6):700–707. http://dx.doi.org/10.1515/cclm.2007.170 plebani m, sciacovelli l, aita a, et al. harmonization of pre-analytical quality indicators. biochemia medica. 2014;24(1):105–113. http://dx.doi.org/10.11613/bm.2014.012 sonntag o. analytical interferences and analytical quality. clin chim acta. 2009;404(1):37–40. http://dx.doi.org/10.1016/j.cca.2009.03.031 singla p, parkash aa, bhattacharjee j. preanalytical error occurrence rate in clinical chemistry laboratory of a public hospital in india. clin lab. 2011;57(9–10):749–752. plebani m. the detection and prevention of errors in laboratory medicine. ann clin biochem. 2010;47(2):101–110. http://dx.doi.org/10.1258/acb.2009.009222 simundic am, lippi g. preanalytical phase—a continuous challenge for laboratory professionals. biochem medica (zagreb). 2012;22(2):145–149. lippi g, chance jj, church s, et al. preanalytical quality improvement: from dream to reality. clin chem lab med. 2011;49(7):1113–1126. http://dx.doi.org/10.1515/cclm.2011.600 olayemi e, asiamah-broni r. evaluation of request forms submitted to the haematology laboratory in a ghanaian tertiary hospital. pan afr med j. 2011;8(1):33. http://dx.doi.org/10.4314/pamj.v8i1.71148 burnett l, chesher d, mudaliar y. improving the quality of information on pathology request forms. ann clin biochem. 2004;41(1):53–56. http://dx.doi.org/10.1258/000456304322664708 zemlin ae, nutt l, burgess lj, et al. potential for medical error: incorrectly completed request forms for thyroid function tests limit pathologists’ advice to clinicians. s afr med j. 2009;99(9):668–671. da rin g. pre-analytical workstations: a tool for reducing laboratory errors. clin chim acta. 2009;404(1):68–74. http://dx.doi.org/10.1016/j.cca.2009.03.024 mbah h, ojo e, ameh j, et al. piloting laboratory quality system management in six health facilities in nigeria. plos one. 2014;9(12):e116185. http://dx.doi.org/10.1371/journal.pone.0116185 jegede fe, mbah ha, yakubu tn, et al. laboratory quality audit in 25 anti-retroviral therapy facilities in north west of nigeria. open j clin diagnostics. 2014;4:193–204. http://dx.doi.org/10.4236/ojcd.2014.44028[pdf] solberg he. the ifcc recommendation on estimation of reference intervals. the refval program. clin chem lab med. 2004;42(7):710–714. http://dx.doi.org/10.1515/cclm.2004.121 adegoke oa, idowu aa, jeje oa. incomplete laboratory request forms as a contributory factor to preanalytical errors in a nigerian teaching hospital. ajbr. 2011;5(3):82–85. oyedeji oa, ogbenna aa, iwuala so. an audit of request forms submitted in a multidisciplinary diagnostic center in lagos. pan afr med j. 2015;20:423. nutt l, zemlin ae, erasmus rt. incomplete laboratory request forms: the extent and impact on critical results at a tertiary hospital in south africa. ann clin biochem. 2008;45(5):463–466. http://dx.doi.org/10.1258/acb.2008.007252 aiyedun ji, chukwu ln, musa rh. interpersonal relationship between health care providers; a challenge to quality health care in university of abuja teaching hospital gwagwalada, f.c.t. abuja, nigeria. adv med biol sci res. 2014;2(4):101–108. jegede fe, mbah ha, aminu m, et al. evaluation of laboratory performance with quality indicators in infectious disease hospital, kano, nigeria. open j clin diagnostics. 2015;5:1–9. http://dx.doi.org/10.4236/ojcd.2015.51001 gyawali p, shrestha rk, bhattarai p, et al. evaluation of pre-analytical errors: inadequacies in the completion of laboratory requisition forms. j nepal assoc med lab sci. 2012;11(1):43–49. chhillar n, khurana s, agarwal r, et al. effect of pre-analytical errors on quality of laboratory medicine at a neuropsychiatry institute in north india. indian j clin biochem. 2011;26(1):46–49. http://dx.doi.org/10.1007/s12291-010-0082-2 international organization for standardization. iso 15189:2012: medical laboratories. requirements for quality and competence. geneva: international organization for standardization; 2012. abstract introduction methods results discussion acknowledgements references about the author(s) reuben n. abednego microbiology laboratory, national public health laboratory, dar es salaam, united republic of tanzania vitus silago department of microbiology and immunology, weill bugando school of medicine, catholic university of health and allied sciences, mwanza, united republic of tanzania citation abednego rn, silago v. antibacterial activity of soil-isolated bacillus altitudinis/pumilus complex against methicillin-resistant staphylococcus aureus from mwanza, tanzania. afr j lab med. 2023;12(1), a2167. https://doi.org/10.4102/ajlm.v12i1.2167 brief report antibacterial activity of soil-isolated bacillus altitudinis/pumilus complex against methicillin-resistant staphylococcus aureus from mwanza, tanzania reuben n. abednego, vitus silago received: 25 jan. 2023; accepted: 15 may 2023; published: 18 july 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract antimicrobial resistance in methicillin-resistant staphylococcus aureus and beta-lactamase-producing gram-negative bacteria is a global health concern necessitating research and the development of effective antimicrobial agents. this study, conducted in may 2020 in mwanza, tanzania, aimed to determine the antibacterial activity of metabolites from soil-isolated bacillus species against clinical bacterial pathogens. one soil-isolated bacillus species, identified as bacillus altitudinis/pumilus complex, showed antibacterial activity against gram-positive cocci, including a methicillin-resistant s. aureus strain with inducible clindamycin resistance, previously isolated from a patient with osteomyelitis. bacillus altitudinis/pumilus complex metabolites may be a potential source of antimicrobial agents against multidrug-resistant bacteria. what this study adds: the study supports existing research on the discovery and development of new antimicrobial agents against multi-drug-resistant bacteria. we report the antimicrobial activity of metabolites extracted from soil-isolated bacillus altitudinis/pumilus complex strains against gram-positive bacteria, including a methicillin-resistant staphylococcus aureus strain with inducible clindamycin resistance. keywords: bacillus altitudinis/pumilus complex; gram-positive cocci; methicillin-resistant staphylococcus aureus; discovery of new antibiotic agent; soil bacteria. introduction the upsurge of multidrug-resistant (mdr) infections has led to an increase in healthcare costs and mortalities. other negative impacts of mdr infections include prolonged days of hospitalisation and lack of prophylactic protection.1,2 extended-spectrum beta-lactamase-producing gram-negative bacteria, methicillin-resistant staphylococcus aureus (mrsa) and inducible clindamycin-resistant s. aureus are common mdr bacteria reported globally.3,4,5 according to estimates, by 2050, mdr infections could cost up to $100 trillion united states dollars annually and cause approximately 10 million deaths per year.6,7 moreover, a predictive statistical modelling study published in 2022 estimated that 1.27 million deaths were directly attributable to antimicrobial resistance (amr), and 4.95 million deaths were associated with amr globally in 2019.8 these deaths were largely due to mdr strains of escherichia coli, s. aureus, and klebsiella pneumoniae.8 these projections have led to urgent calls by the world health organization to guide and promote research and development of new effective antimicrobials against mdr strains.9 since 1987 to date, no new class of antibiotics has been discovered and successfully introduced for clinical use, particularly for the treatment of infections caused by mdr bacteria such as extended-spectrum beta-lactamase-producing gram-negative bacteria, mrsa and inducible clindamycin-resistant s. aureus strains.10 currently, scientists are struggling to discover potential sources of antibiotics with activity against mdr bacteria. various potential sources have been explored, including reptile blood,11 soil microorganisms,12 marine microorganisms,13 and plants.14 certain microorganisms produce antimicrobial compounds such as antibiotics, antifungals, and antivirals as a means of gaining a competitive advantage in their environment where resources such as food are limited.15 the majority of antibiotics in clinical use today were sourced from microorganisms.16 antimicrobials such as penicillin g (sourced from penicillium notatum), vancomycin (amycolatopsis orientalis), gentamicin (micromonospora purpurea), and lincosamides (streptomyces lincolnensis) are some examples that have been derived from microorganisms.16 this study aimed to test the antibacterial activity of bacillus species isolated from soil samples in mwanza, tanzania, against medically important pathogenic bacteria, including mdr strains such as extended-spectrum beta-lactamase-producing gram-negative bacteria, mrsa, and inducible clindamycin-resistant s. aureus isolated from clinical samples. methods ethical considerations this study was approved by the joint catholic university of health and allied sciences and bugando medical centre research ethics and review committee with research clearance certificate number: crec/298/2023. the current study used bacteria species previously isolated from clinical samples at bugando medical centre and stored at –80 °c in the microbiology laboratory at catholic university of health and allied sciences in mwanza, tanzania. participants’ informed consent forms were not applicable because archived bacteria were used. moreover, patient-related information such as socio-demographic data were not used in the current study, to ensure data confidentiality. study design, duration, sampling and setting this was a laboratory-based study conducted in may 2020 in mwanza, tanzania. surface soil samples were collected in sterile falcon™ 50 ml conical tubes (corning, glendale, arizona, united states) from 20 different locations of the same site at bugando hill in mwanza, tanzania. soil samples were sent to the microbiology laboratory at the catholic university of health and allied sciences and processed within 1 h of collection. isolation of bacillus species from soil samples soil samples were serially diluted in 0.85% sterile saline as described previously17 before being inoculated onto 5% sheep blood agar (ba; himedia, mumbai, india) plates. briefly, 1 g of soil sample was suspended in 5 ml of sterile saline, which was then vortexed for 15 s and allowed to settle for 5 min. one millilitre of the resulting supernatant was mixed with 4 ml of sterile saline, vortexed for 15 s, and then allowed to settle for 5 min. this step was repeated three times to obtain five-fold serial dilutions. from the fifth dilution, 1 ml of supernatant was inoculated on a ba plate using the pour plate technique. plates were incubated aerobically at 37 °c for 20 h. presumptive bacillus species colonies (large-sized, dry, flat, greyish to whitish, and beta-haemolytic colonies) were selected and sub-cultured on fresh ba plates to obtain pure colonies. these plates were incubated aerobically at 37 °c for 20 h. gram staining was used for the preliminary identification of bacillus species before further analysis. extraction of bacterial metabolites and antibacterial activity testing a loopful (10 µl) of each presumptive bacillus isolate was suspended in 1 ml of sterile nuclease-free water in an eppendorf tube (1.5 ml safe lock microcentrifuge tube; sigma-aldrich chemie gmbh, taufkirchen, germany). the tubes were incubated at 50 °c in a heating block for 30 min with intermittent vortex-mixing for 15 s every 10 min. we selected 50 °c for the extraction of bacterial metabolites because this temperature is insufficient for the destruction of metabolites. after incubation, the tubes were centrifuged at 12 000 revolutions per minute for 10 min, after which 0.8 ml of the supernatant from each tube was transferred into sterile eppendorf tubes. the supernatants containing metabolites from soil-isolated presumptive bacillus species were used immediately for antibacterial activity determination. the well diffusion method, as described by yilmaz and colleagues,18 was used to test the antibacterial activity of the extracted metabolites against one isolate each of extended-spectrum beta-lactamase-producing and non-producing e. coli and k. pneumoniae, mrsa and methicillin-susceptible s. aureus, inducible clindamycin-resistant s. aureus, coagulase-negative staphylococci, and streptococcus pyogenes. these bacterial pathogens were isolated from clinical samples of previous studies conducted in the same setting19,20 and archived at –80 °c in 20% glycerol stocks. the recovery of these bacterial strains was performed by subculture on ba plates which were incubated at 37 °c for 24 h. bacterial suspensions of these test strains were prepared using sterile saline and adjusted to 0.5 mcfarland standard solution (remel, lenexa, kansas, united states). the bacterial suspensions were then inoculated onto the surface of mueller hinton agar (mha; himedia, mumbai, india) plates. within 15 min of inoculation, wells of 6 mm in diameter were created in the inoculated plates using a cork borer (sigma aldrich, darmstadt, germany), and 100 µl of the suspension containing the extracted metabolites was then pipetted into the bored wells. the inoculated mha plates were incubated in an upright position in ambient air at 37 °c for 20 h. the diameters of zones of inhibitions were measured in millimetres from the edge of the inhibition zone to the margin of bacillus species metabolites. the complete absence of inhibition was referred to as ‘negative antibacterial activity’ and the presence of a clear zone of inhibition was referred to as ‘positive antibacterial activity’. this experiment was performed in duplicate to ensure the validity of our results. in cases of positive antibacterial activity, an average zone of inhibition was calculated and recorded as the final result. identification of bacillus species with positive antibacterial activity only one soil-isolated presumptive bacillus species with a clear zone of inhibition (‘positive antibacterial activity’) was further identified at the species level. we first conducted biochemical identification tests, including catalase production, coagulase production, oxidase production, urease production, dnase production, indole production, citrate utilisation, lactose fermentation, hydrolysis of bile aesculin, and motility tests. we also tested the capacity of the isolates to grow on ba supplemented with 7% nacl, as well as at higher incubation temperatures of 50 °c and 70 °c. the biochemical identification tests were not conclusive. for further species identification, we also used the vitek ms (biomérieux, baden, germany) system, which is an automated microbial identification system that uses the matrix assisted laser desorption ionization time-of-flight technology. the latter was performed at the microbiology laboratory, national public health laboratory in dar es salaam, tanzania. data analysis laboratory data were documented and analysed using microsoft excel (microsoft corporation, redmond, washington, united states). categorical data were expressed as percentages. results a total of 20 soil samples were collected and processed for the isolation of bacillus species. out of the 20 soil samples processed, 16 (80.0%) showed growth of presumptive bacillus species. of the 16 presumptive bacillus species isolated, only one (6.3%) isolate – s9d – showed antibacterial activity against gram-positive bacteria only, including the mrsa strain, which was also inducible clindamycin-resistant (table 1 and figure 1). figure 1: inhibition of methicillin-resistant staphylococcus aureus by bacillus altitudinis/pumilus complex isolated from soil in mwanza, tanzania, may 2020. the zone of growth inhibition is indicated with black arrows. table 1: antibacterial activity of bacillus species isolates from soil samples against clinical bacterial isolates in mwanza, tanzania, may 2020. the presumptive bacillus species isolate with positive antibacterial activity was gram-positive and rod-shaped with central spores on microscopic examination after gram staining. the isolate was also β-haemolytic on 5% sheep ba and demonstrated catalase production, sugar fermentation, growth on ba supplemented with 7% nacl, and growth at 50 °c (table 2). using vitek ms, the isolate was identified as bacillus altitudinis/pumilus complex with a confidence level of 99.9%. table 2: biochemical characteristics of a soil-isolated bacillus altitudinis/pumilus complex isolate with positive antibacterial activity against gram-positive bacteria isolated from clinical samples, mwanza, tanzania, may 2020. discussion in this study, we report a bacillus altitudinis/pumilus complex isolate with antimicrobial activity against clinical gram-positive bacterial strains, including an mrsa strain that was also inducible clindamycin-resistant. due to the presence of several closely related species in the bacillus genus, the vitek ms system used in this study could not identify the bacillus altitudinis/pumilus complex isolate at the species level.21 the study finding is similar to a previous study in brazil in 2020,22 where marine-isolated bacillus altitudinis/pumilus was shown to possess antimicrobial activity against mdr bacterial strains. similarly, another study in thailand in 2007 also reported that a soil-isolated bacillus pumilus showed antibacterial activity against gram-positive bacteria, including mrsa and vancomycin-resistant enterococcus faecalis.23 the potential antimicrobial activity of bacillus altitudinis/pumilus complex isolates has been linked to the production of bacilysins and bacteriocins.22 the report from thailand in 2007 documented that pumilicin 4 is a bacteriocin produced by a bacillus pumilus strain and had bactericidal activity against mrsa strains.23 we observed no antibacterial activity against gram-negative bacteria, indicating that bacillus altitudinis/pumilus complex may only be effective against gram-positive bacteria. similar findings were reported in brazil in 2021 where a bacillus altitudinis isolate from wetland sediment showed no antibacterial activity against gram-negative bacteria.21 the presence of the outer membrane in gram-negative bacteria limits cellular permeability,24 and may thus result in poor diffusion of antimicrobial metabolites into the cytoplasmic membrane for potential inhibition of bacterial growth.25 limitations due to limited resources and funds, we were not able to identify the presumptive bacillus species with negative antibacterial activity beyond the genus level. we also could not isolate, purify, or characterise the active metabolites from the bacillus altitudinis/pumilus complex isolate with positive antibacterial activity. conclusion our findings suggest that metabolites from soil-isolated bacillus altitudinis/pumilus complex can be a potential source of antimicrobial agents with activity against gram-positive bacteria, including mrsa and inducible clindamycin-resistant strains. acknowledgements authors would like to express their sincerely appreciation to the microbiology laboratory at catholic university of health and allied sciences in mwanza, tanzania, and microbiology laboratory at national public health laboratory in dar es salaam, tanzania. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions v.s. conceptualised the article. both v.s. and r.n.a. performed sample collection, laboratory procedures, and prepared the article. sources of support this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability all data presented in this article are available at microbiology laboratory at catholic university of health and allied sciences and can be accessed upon an official request to the corresponding author, v.s. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references friedman nd, temkin e, carmeli y. the negative impact of antibiotic resistance. clin microbiol infect. 2016;22(5):416–422. https://doi.org/10.1016/j.cmi.2015.12.002 wolf j. antibiotic resistance threatens the efficacy of prophylaxis. lancet infect dis. 2015;15(12):1368–1369. https://doi.org/10.1016/s1473-3099(15)00317-5 navidinia m. detection of inducible clindamycin resistance (mlsbi) among methicillin-resistant staphylococcus aureus (mrsa) isolated from health care providers. arch adv biosci. 2015;6(1):91–96. yehouenou cl, kpangon aa, affolabi d, et al. antimicrobial resistance in hospitalized surgical patients: a silently emerging public health concern in benin. ann clin microbiol antimicrob. 2020;19:1–10. https://doi.org/10.1186/s12941-020-00398-4 giufrè m, ricchizzi e, accogli m, et al. colonization by multidrug-resistant organisms in long-term care facilities in italy: a point-prevalence study. clin microbiol infect. 2017;23(12):961–967. https://doi.org/10.1016/j.cmi.2017.04.006 o’neill j. review on antimicrobial resistance: tackling a crisis for the health and wealth of nations. 2014. london: hm government; 2016. the world bank. by 2050, drug-resistant infections could cause global economic damage on par with 2008 financial crisis. washington, dc: world bank; 2016. murray cj, ikuta ks, sharara f, et al. global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. lancet. 2022;399(10325):629–655. https://doi.org/10.1016/s0140-6736(21)02724-0 world health organization. who publishes list of bacteria for which new antibiotics are urgently needed. geneva: who; 2017. silver ll. challenges of antibacterial discovery. clini microbiol rev. 2011;24(1):71–109. https://doi.org/10.1128/cmr.00030-10 bishop bm, juba ml, russo ps, et al. discovery of novel antimicrobial peptides from varanus komodoensis (komodo dragon) by large-scale analyses and de-novo-assisted sequencing using electron-transfer dissociation mass spectrometry. j proteome res. 2017;16(4):1470–1482. https://doi.org/10.1021/acs.jproteome.6b00857 begum k, mannan j, rahman m, mitchel-antoine a, opoku r. identification of antibiotic producing bacteria from soil samples of dhaka. j microbiol exp. 2017;4:00134. https://doi.org/10.15406/jmen.2017.04.00134 sosovele em, lyimo tj, hosea km. in vitro antimicrobial activity of extracts from marine streptomyces isolated from mangrove sediments of tanzania. j biochem technol. 2012;3(4):431–435. mwambete k. the in vitro antimicrobial activity of fruit and leaf crude extracts of momordica charantia: a tanzania medicinal plant. afri health sci. 2009;9(1):34–39. hibbing me, fuqua c, parsek mr, peterson sb. bacterial competition: surviving and thriving in the microbial jungle. nat rev microbiol. 2010;8(1):15–25. https://doi.org/10.1038/nrmicro2259 rajeev l. antibiotic discovery. mat meth. 2016;8:2671. https://doi.org/10.13070/mm.en.8.2671 chalasani ag, dhanarajan g, nema s, sen r, roy u. an antimicrobial metabolite from bacillus sp.: significant activity against pathogenic bacteria including multidrug-resistant clinical strains. frontiers in microbiol. 2015;6:1335. https://doi.org/10.3389/fmicb.2015.01335 yilmaz m, soran h, beyatli y. antimicrobial activities of some bacillus spp. strains isolated from the soil. microbiol res. 2006;161(2):127–131. https://doi.org/10.1016/j.micres.2005.07.001 silago v, kovacs d, msanga dr, et al. bacteremia in critical care units at bugando medical centre, mwanza, tanzania: the role of colonization and contaminated cots and mothers’ hands in cross-transmission of multidrug resistant gram-negative bacteria. antimicrob resist infect control. 2020;9(1):1–14. https://doi.org/10.1186/s13756-020-00721-w silago v, mushi mf, remi ba, et al. methicillin resistant staphylococcus aureus causing osteomyelitis in a tertiary hospital, mwanza, tanzania. j orthop surg res. 2020;15(1):1–6. https://doi.org/10.1186/s13018-020-01618-5 cavalini l, jankoski p, correa apf, brandelli a, motta as. characterization of the antimicrobial activity produced by bacillus sp. isolated from wetland sediment. an acade bras ciênc. 2021;93(suppl 4):e20201820. https://doi.org/10.1590/0001-3765202120201820 freitas-silva j, silva-oliveira t, muricy g, laport ms. bacillus strains associated to homoscleromorpha sponges are highly active against multidrug resistant bacteria. curr microbiol. 2020;77(5):807–815. https://doi.org/10.1007/s00284-019-01870-x aunpad r, na-bangchang k. pumilicin 4, a novel bacteriocin with anti-mrsa and anti-vre activity produced by newly isolated bacteria bacillus pumilus strain wapb4. curr microbiol. 2007;55(4):308–313. https://doi.org/10.1007/s00284-006-0632-2 delcour ah. outer membrane permeability and antibiotic resistance. biochim biophys acta. 2009;1794(5):808–816. https://doi.org/10.1016/j.bbapap.2008.11.005 garcia-gutierrez e, mayer mj, cotter pd, narbad a. gut microbiota as a source of novel antimicrobials. gut microbes. 2019;10(1):1–21. https://doi.org/10.1080/19490976.2018.1455790 abstract introduction methods results discussion acknowledgements references about the author(s) sam l. nsobya department of pathology, school of biomedical science, makerere university, kampala, uganda paul c. hewett population council, washington, district of columbia, united states sam kalibala population council, washington, district of columbia, united states barbara s. mensch population council, new york, new york, united statesphewett@popcouncil.org citation nsobya sl, hewett pc, kalibala s, mensch bs. performance of kalon herpes simplex virus 2 assay using dried blood spots among young women in uganda. afr j lab med. 2016;5(1), a429. http://dx.doi.org/10.4102/ajlm.v5i1.429 brief report performance of kalon herpes simplex virus 2 assay using dried blood spots among young women in uganda sam l. nsobya, paul c. hewett, sam kalibala, barbara s. mensch received: 24 feb. 2016; accepted: 25 may 2016; published: 16 aug. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract this study evaluated the performance of the kalon biological hsv2 igg enzyme-linked immunosorbent assay (kalon biological ltd, surrey, united kingdom) on dried blood spots (dbs) of various dilutions compared with plasma from young women aged 18–24 years in uganda. we estimated the sensitivity and specificity of three dbs dilutions using plasma as the reference. all three evaluated dbs dilutions yielded low sensitivities and specificities, with dbs 1:2 yielding the highest concurrence. other herpes simplex virus type 2 assays should be examined with regard to their utility for testing dbs. introduction herpes simplex virus type 2 (hsv-2) causes lifelong infections in exposed individuals and is a useful cumulative marker for unprotected sex. hsv-2 infection has also been shown to be an important co-infection for hiv, facilitating hiv acquisition and transmission, and accelerating disease progression.1 in uganda, it is estimated that about half of adults aged 15–59 years are infected with hsv-2.2 numerous studies have outlined the usefulness of dried blood spots (dbs) for the serologic diagnosis of infectious diseases, as well as for large-scale seroprevalence studies.3,4,5,6,7 since dbs do not require immediate refrigeration, occupy little space and are easily transported, they are an attractive means for biomarker-based studies, particularly in geographic settings with limited laboratory resources. recently-developed type-specific hsv antibody tests are based on the detection of antibodies to glycoprotein g1 (gg1), a marker for hsv-1 infection, and glycoprotein g2 (gg2), a marker for hsv-2 infection.8,9 validation of dbs for immunoglobulin g (igg)-based tests has been conducted using hsv-1-positive antibody samples from 22 healthy volunteers.10 hogrefe, ernst and su reported that testing data using a single dilution of dbs eluates (1:4) with the hsv type-specific enzyme-linked immunosorbent assay (elisa) method were similar to those of sera diluted to 1:101 using the standard herpeselect® elisa (focus diagnostics, california, united states).10 the efficiency of using igg eluted from dbs samples was found to be consistent with measurements of igg concentrations in most corresponding serum samples. however, results have been inconsistent when using the kalon biological hsv2 igg elisa assay (kalon biological ltd, surrey, united kingdom; hereafter kalon elisa hsv2 assay) when measuring antibodies to gg2 with the same testing protocol.10 in a household survey conducted in 2010, sexual behavior data and dbs specimens were collected from young women aged 18–24 years in kampala.11 when a 1:4 dilution was applied to our first 277 dbs specimens, the estimated hsv-2 prevalence was 2%. this prevalence was significantly lower than that reported previously in the 2004–2005 uganda hiv/aids sero-behavioural survey (uhsbs), which found a prevalence of 21% among women aged 15–19 years and 38% among women aged 20–24 years.2 when an additional 10 dbs specimens from our sample of young women were further analysed using a dilution of 1:2, the prevalence of hsv-2 increased three-fold.11 in the study reported here, we examined the performance of the kalon elisa hsv2 assay using dbs and plasma samples from stored specimens previously collected during the 2004–2005 uhsbs. the dbs laboratory assessment was conducted in late 2010 and early 2011. the study goal was to examine whether hsv-2 testing results based on dbs at various dilution levels were concordant with plasma-based results obtained from the same participants. methods ethical considerations participants who provided specimens to the 2004–2005 uhsbs consented to the long-term storage and future testing of their delinked blood specimens for which they would not receive results. we obtained additional permission for this study from the uganda ministry of health. the study was also reviewed and approved by the uganda virus research institute’s institutional review board (gc/127/11/10/15) and the population council institutional review board (protocol 433). study population this study was conducted using existing stored dbs and plasma specimens from the 2004–2005 uhsbs. the uhsbs was a nationally-representative household-based survey that sampled 19 656 adult respondents. the main objective of the uhsbs was to obtain national and sub-national estimates of hiv prevalence and selected indicators of hiv-related risk behaviours, programme coverage and hiv knowledge and attitudes. one of its specific objectives was to determine the magnitude and distribution of hiv and other sexually-transmitted infections, such as hsv-2, in uganda.2 the uhsbs estimated the national adult prevalence of hiv at 6.4% and that of hsv-2 at 44%.2 survey specimens and testing survey participants had venous blood samples drawn into 4.5 ml edta vacutainer tubes, from which dbs were produced using whatman ss903 specimen collection paper, air-dried overnight in plastic boxes and stored in lots of 20 separated by glassine paper in ziploc bags containing desiccants. in the field, blood was centrifuged and the plasma was transferred to microvials. plasma and dbs were transported periodically to a central laboratory in entebbe for processing and storage at -80 °c (plasma) and -20 °c (dbs). the plasma and dbs samples for the uhsbs were tested for hsv-2 antibodies using the kalon elisa hsv2 assay within several weeks of collection. results were classified as positive or negative using cutoffs as specified by the manufacturer. dbs validation study design for the purposes of this study, we randomly selected 110 stored dbs specimens from women aged 18–24 years whose plasma-based equivalents tested positive and 110 stored dbs specimens whose plasma-based equivalents tested negative using the kalon elisa hsv2 assay. laboratory testing and analysis was conducted at the molecular research laboratory in kampala as part of the makerere university-university of california, san francisco research collaboration on 2 may 2011. from each of these 220 dbs, a 6 mm-diameter (28 mm2) disk, containing approximately 50–75 µl of blood per spot, was punched out from the filter paper and soaked overnight at 4 °c in 150 µl of phosphate-buffered saline (ph 7.4). after the overnight elution step, the eluates were diluted at three different levels, 1:4, 1:3 and 1:2. each specimen was tested in triplicate with the kalon elisa hsv2 assay, as directed by the manufacturer’s instructions. data analysis frequencies of reactivity, non-reactivity, sensitivity and specificity with 95% confidence intervals were generated using sas® software, version 9.3 (sas institute inc., cary, north carolina, united states, 2011). dbs-based estimates of sensitivity and specificity were obtained using the plasma-based results as the reference. we determined the dilution level for dbs-based hsv-2 testing that yielded the highest (relative) sensitivity and specificity. results the final sample size included 210 individuals due to missing test results for 10 dbs. of the 210 plasma specimens, 104 (49.5%) were reactive and 106 (50.5%) were non-reactive for hsv-2 antibodies (table 1). the dbs 1:2 dilution yielded 116 (55.2%) reactive and 94 (44.8%) non-reactive results. dbs 1:3 produced 124 (59.0%) reactive and 86 (41.0%) non-reactive results, whereas dbs 1:4 yielded 110 (52.4%) reactive and 100 (47.6%) non-reactive results. table 1: hsv-2 seropositivity for plasma-based and dbs dilution-based assays using the kalon elisa hsv2 assay among young women in uganda, 2004–2005.† the 1:2 ratio of buffer and eluate yielded the highest sensitivity (84.6%) and specificity (73.6%) (table 2). the 1:3 dilution had the next-highest sensitivity (82.7%), but had the lowest specificity (64.2%). the 1:4 dilution showed the lowest sensitivity (73.1%) and a specificity of 67.9%. overall, 127 (60.5%) of the 210 specimens had concordant results between plasma and all three dbs dilutions (not shown in the tables). of the 127 concordant results, 69 (54.3%) were concordant positive and 58 (45.7%) were concordant negative. a total of 83 (39.5%) cases had a discordant result between plasma and at least one of the dbs dilutions. of the 83 discordant results, 35 (42.2%) were discordant positive and 48 (57.8%) were discordant negative. table 2: relative sensitivity, specificity and 95% confidence intervals of dried bloodspot dilutions tested with the kalon elisa hsv2 assay compared with plasma among young women in uganda, 2004–2005.† discussion the kalon elisa hsv2 assay has been shown to be sensitive and specific for hsv-2 diagnosis using plasma.2,12 in our study, a dilution of 1:2 showed the highest sensitivity and specificity compared with the 1:3 and 1:4 dilution levels. this estimated sensitivity and specificity is low relative to the plasma-based reference results. specifically, our findings differ from the estimated higher sensitivity and specificity found in a south african population which used the herpeselect elisa serological assay.13 however, earlier studies evaluating the plasma-based herpeselect elisa test in sub-saharan populations suggested a lower specificity than the plasma-based kalon elisa hsv2 assay.14 in addition, patterns of reactivity varied by dilution level. although not the optimal dilution based on sensitivity or specificity, dbs 1:3 had the highest proportional reactivity to hsv-2 antibodies (59.0%). all three dilutions yielded higher frequencies of reactivity than plasma (49.5%), specifically dbs 1:2 (55.2%), dbs 1:3 (59.0%) and dbs 1:4 (52.4%). we found low concordance between plasma-based and dbs-based results, which contrasts with the high concordance reported by hogrefe et al. using the herpeselect assay.10 limitations there are several limitations in our study. the sampling frame was limited to young women aged 18–24 years in uganda; thus, our results are not generalisable to men or older adults. additionally, the sample size was relatively small. further, we did not compare the quantity of igg in the dbs punch specimens to that in plasma specimens. finally, this study only used the kalon elisa hsv2 assay to evaluate dbs for hsv-2 diagnosis. other hsv-2 assays, such as the herpeselect 2 elisa igg and biokit hsv2 rapid assay are used in african countries and need to be further evaluated to compare their utility with dbs. conclusion in summary, our study examined the performance of the kalon elisa hsv2 assay, using dbs in a population of young women in uganda. dbs testing with the kalon elisa hsv2 assay revealed relatively low sensitivity and specificity compared with plasma-based results on the same individuals. while dbs would be an appealing and relatively simple means for biomarker-based studies, particularly in resource-constrained settings, the accuracy of this testing format would need to be substantially improved before its use could be recommended for epidemiological studies. acknowledgements the authors would like to thank angele marandet and wolfgang hladik at the united states centers for disease control for their support and encouragement in the development of the manuscript. we also thank joshua musinguzi of the ministry of health, aids control programme in uganda for his assistance in design and execution of the study. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. sources of support the study was funded by the national institutes of health grant number r01 hd 047764 s1, barbara s. mensch principal investigator, paul c. hewett, co-principal investigator. author contributions s.l.n. drafted the manuscript, which was reviewed and contributed to by p.c.h., s.k. and b.s.m. s.l.n. oversaw the testing of specimens in the laboratory and the reporting of the test results. s.l.n. and p.c.h. contributed to the analysis of the results. b.s.m. and p.c.h. conceived of the original study, with s.l.n. and s.k. assisting with its design and execution. references laeyendecker o, henson c, gray rh, et al. performance of a commercial, type-specific enzyme-linked immunosorbent assay for detection of herpes simplex virus type 2-specific antibodies in ugandans. j clin microbiol. 2004;42(4): 1794–1796. http://dx.doi.org/10.1128/jcm.42.4.1794-1796.2004 macro o. uganda hiv/aids sero-behavioural survey: 2004–2005. ministry of health; 2006. bailey nm, cunningham mp, kimber cd. the indirect fluorescent antibody technique applied to dried blood, for use as a screening test in the diagnosis of human trypanosomiasis in africa. trans r soc trop med hyg. 1967;61(5):696–700. http://dx.doi.org/10.1016/0035-9203(67)90135-6 beebe jl, briggs lc. evaluation of enzyme-linked immunoassay systems for detection of human immunodeficiency virus type 1 antibody from filter paper disks impregnated with whole blood. j clin microbiol. 1990;28(4):808–810. brody ja, mcalister r, haseley r, lee p. use of dried whole blood collected on filter paper disks in adenovirus complement fixation and measles hemagglutination inhibition tests. j immunol. 1964;92(6):854–857. jafri hs, torrico f, noh jc, et al. application of the enzyme-linked immunoelectrotransfer blot to filter paper blood spots to estimate seroprevalence of cysticercosis in bolivia. am j trop med hyg. 1998;58(3):313–315. takkouche b, iglesias j, alonso-fernandez jr, fernandez-gonzalez c, gestal-otero jj. detection of brucella antibodies in eluted dried blood: a validation study. immunol lett. 1995;45(1–2):107–108. http://dx.doi.org/10.1016/0165-2478(94)00247-o ashley rl, wald a. genital herpes: review of the epidemic and potential use of type-specific serology. clin microbiol rev. 1999;12(1):1–8. ashley r. type-specific antibodies to hsv-1 and -2: review of methodology. herpes. 1998;5:33–38. hogrefe wr, ernst c, su x. efficiency of reconstitution of immunoglobulin g from blood specimens dried on filter paper and utility in herpes simplex virus type-specific serology screening. clin diagn lab immunol. 2002;9(6):1338–1342. http://dx.doi.org/10.1128/cdli.9.6.1338-1342.2002 kelly ca, hewett pc, mensch bs, et al. using biomarkers to assess the validity of sexual behavior reporting across interview modes among young women in kampala, uganda. stud fam plann. 2014;45(1):43–58. http://dx.doi.org/10.1111/j.1728-4465.2014.00375.x lingappa j, nakku-joloba e, magaret a, et al. sensitivity and specificity of herpes simplex virus-2 serological assays among hiv-infected and uninfected urban ugandans. int j std aids. 2010;21(9):611–616. http://dx.doi.org/10.1258/ijsa.2009.008477 delany-moretlwe s, jentsch u, weiss h, et al. comparison of focus herpesselect and kalon hsv-2 gg2 elisa serological assays to detect herpes simplex virus type 2 antibodies in a south african population. sex transm infect. 2010;86(1):46–50. http://dx.doi.org/10.1136/sti.2009.036541 smith js, bailey rc, westreich dj, et al. herpes simplex virus type 2 antibody detection performance in kisumu, kenya, using the herpeselect elisa, kalon elisa, western blot and inhibition testing. sex transm infect. 2009;85(2):92–96. http://dx.doi.org/10.1136/sti.2008.031815 abstract introduction research method and design results discussion conclusion acknowledgements references about the author(s) anneta naidoo department of virology, nelson r mandela school of medicine, school of laboratory medicine and medical sciences, university of kwazulu-natal, durban, kwazulu-natal, south africa national health laboratory services, inkosi albert luthuli central hospital, durban, kwazulu-natal, south africa raveen parboosing department of virology, nelson r mandela school of medicine, school of laboratory medicine and medical sciences, university of kwazulu-natal, durban, kwazulu-natal, south africa national health laboratory services, inkosi albert luthuli central hospital, durban, kwazulu-natal, south africa pravi moodley department of virology, nelson r mandela school of medicine, school of laboratory medicine and medical sciences, university of kwazulu-natal, durban, kwazulu-natal, south africa national health laboratory services, inkosi albert luthuli central hospital, durban, kwazulu-natal, south africa citation naidoo a, parboosing r, moodley p. real-time polymerase chain reaction optimised for hepatitis c virus detection in dried blood spots from hiv-exposed infants, kwazulu-natal, south africa. afr j lab med. 2016;5(1), art. #269, 6 pages. http://dx.doi.org/10.4102/ajlm.v5i1.269 original research real-time polymerase chain reaction optimised for hepatitis c virus detection in dried blood spots from hiv-exposed infants, kwazulu-natal, south africa anneta naidoo, raveen parboosing, pravi moodley received: 30 sept. 2014; accepted: 08 jan. 2016; published: 18 mar. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: there is a paucity of data on the prevalence of hepatitis c virus (hcv) in children, particularly in sub-saharan africa. a major obstacle in resource-limited settings for polymerase chain reaction (pcr) testing is the necessity for specimen transportation and storage at low temperatures. there are numerous recent studies of using real-time hcv pcr for diagnosis and screening of plasma and serum, but few have looked at using dried blood spot (dbs) specimens. objectives: the aim of this study was to optimise a real-time hcv pcr method to detect hcv rna from infant dbs specimens for use as a tool for hcv surveillance in kwazulu-natal, south africa. method: the lightcycler® 2.0 instrument was used for the hcv pcr using the lightcycler® rna master sybr green i kit. template volume, primer concentration and primer annealing temperatures were optimised and the method was used on 179 dbs specimens from hiv-exposed infants in kwazulu-natal. results: primer concentrations adjusted to 0.25 µm and a template volume of 10 µl improved the pcr amplification. primer annealing temperatures lowered from 65 °c to 58 °c resulted in higher quantities of amplified pcr product. the limit of detection of the optimised hcv pcr assay was between 1200 iu/ml and 3580 iu/ml of hcv rna. hcv was not detected in any of the 179 dbs specimens. conclusion: the optimised real-time hcv pcr on infant dbs specimens performed well, but hcv was not found in this surveillance study. hiv infection may have little impact on the vertical transmission of hcv in this region. introduction hepatitis c virus (hcv) infects 3% of the global population,1 but there is a paucity of data on hcv prevalence in children, particularly children in sub-saharan africa.2 hcv is usually transmitted to infants vertically, with a rate that varies from 3% to 7%, although it may increase twoto five-fold with hiv co-infection.3 the prevalence of hcv is higher amongst hiv-positive patients because of shared routes of transmission and common risk factors.4 the estimated prevalence of hcv in sub-saharan africa was 3% amongst hiv-negative individuals in 2002, but prevalence increased to 7% amongst individuals with hiv co-infection by 2014.5 the prevalence of hcv/hiv co-infection in south africa was 0.1% in 20026 compared to 3.5% amongst individuals infected only with hcv.7 there are reasons to suspect that the prevalence of hcv may be high in kwazulu-natal, south africa. firstly, kwazulu-natal has a high antenatal hiv prevalence of 40.1%.8 secondly, kwazulu-natal has a high overall adult hcv prevalence of 6.4% and a much higher prevalence of hcv/hiv co-infection (13.4%),9 which may imply a high prevalence of hcv amongst pregnant woman. the world health organization suggests that screening for hcv in regions with a high prevalence of hiv should be conducted, but depends on hcv prevalence and resources.10 in a region with a high prevalence of both hiv and hcv, especially amongst pregnant women,9 it is important to determine the prevalence of hcv amongst infants. although most children with chronic hcv infection remain asymptomatic, some may have mild abnormalities in liver function and chronic hcv infection may progress to severe liver disease, end-stage liver disease and death.2 liver disease resulting from hcv infection is a major cause of mortality and morbidity amongst adults co-infected with hiv and hcv, which further compounds the hepatotoxic effects of antiretroviral therapy.4 infant diagnosis of hcv by molecular-based technologies is superior to serological techniques for detecting hcv infection by means of maternal antibodies circulating in infants.2 a major obstacle in resource-limited settings for polymerase chain reaction (pcr) testing is the necessity of transporting and storing specimens at low temperatures. however, using dried blood spot (dbs) specimens, which do not require refrigeration, significantly reduces pre-analytical problems and is widely used in molecular virology for the detection of viral nucleic acids.11 numerous studies have reported on the use of real-time pcr for hcv diagnosis using plasma and serum,12,13,14,15,16,17,18,19,20 although few have looked at dbs specimens.21,22,23,24,25 however, a few recent studies have found good correlations for adult serum and plasma compared with dbs specimens analysed using commercial hcv pcr assays.26,27,28,29 the prevalence of hcv amongst hiv-exposed infants in kwazulu-natal, south africa is unknown. therefore, the aim of this study was to optimise a real-time pcr assay to detect hcv rna from hiv-exposed infant dbs specimens for use as a tool for hcv surveillance in kwazulu-natal, south africa. research method and design ethical considerations ethical clearance was obtained from the university of kwazulu-natal biomedical research ethics committee (be273/090). informed consent was not required since this was a retrospective study in which we used discarded specimens from routine testing. all data were recorded and then anonymised for the purposes of the study. specifically, hiv pcr results, hcv pcr results, age and clinical diagnosis were recorded anonymously in a spreadsheet. no patient identifiers were recorded. study design this study was conducted retrospectively in the laboratory at the department of virology, national health laboratory service/university of kwazulu-natal, at the inkosi albert luthuli central hospital in durban, south africa. this is a reference laboratory which receives all dbs specimens from public sector facilities in kwazulu-natal for pcr testing of dna from hiv-exposed infants for early diagnosis of hiv infection. the number of dbs specimens tested by the laboratory has steadily increased each year from 13 699 in 2005 to 73 033 in 2012. the uptake of hiv pcr testing amongst hiv-exposed infants in kwazulu-natal has also increased from 18% in 2005 to 97.4% in 2011.30 a sample size of 179 was calculated using epi-info™ 6 (centre for disease control, atlanta, georgia, united states, 1998) using the number of live births in kwazulu-natal,30 the prevalence of hiv amongst antenatal attendees (which approximates the percentage of hiv-exposed infants)8 and the approximate hcv seropositivity in infants, based on data from the laboratory information system. the sample size was representative of hiv-exposed infants who attended public health facilities in kwazulu-natal and who required hiv pcr testing because of suspected hiv exposure or clinical suspicion of hiv infection. specimen collection as part of routine early infant diagnosis of hiv, infants’ blood is collected by heel-prick and spotted onto whatman filter paper in four circles. in the laboratory, two spots are cut out and used for routine hiv pcr testing within four to five days of dbs specimen collection. after releasing the results, the card (with the two unused spots) is routinely stored at room temperature for at least three months and then discarded. these discarded dbs specimens were tested for hcv after specimens had been stored for at least three months after initial collection. specimens with inadequate volume (where the entire circle was not filled with blood) were excluded from the study. acceptable dbs specimens were sequentially selected from august to december 2010 until 179 specimens were obtained. external quality control specimens were purchased from quality control for molecular diagnostics (qcmd; glasgow, scotland) and internal quality control specimens were prepared in-house. laboratory methods before automated extraction, using the nuclisens® easymag® extraction (biomérieux, marcy i’etoile, france), two dbs (approximately 50 µl blood per spot) were cut with sterilised scissors into a 1.5 ml eppendorf tube and 2 ml nuclisens® easymag® lysis buffer containing 5 m guanidinium thiocyanate was added to the tube. the lysis buffer was also added to the quality control specimens. the specimens were agitated for 30 minutes on an orbital shaker and centrifuged at 1500 x g for two minutes. supernatant was pipetted into transfer wells, which were then loaded onto the automated extraction system. briefly, cell-bound nucleic acids were released with the chaotropic agent − guanidinium thiocyanate − and then bound to silica particles, which were immobilised on a filter. impurities were removed by several washes in buffers containing guanidinium thiocyanate, 70% ethanol and acetone, after which the purified nucleic acids were eluted in molecular grade water. the lightcycler® 2.0 instrument (roche diagnostics, mannheim, germany) was used to perform reverse transcription and amplification in a one-step assay using the lightcycler® rna master sybr green i kit (roche diagnostics, mannheim, germany), which contained the enzymes thermus thermophilus dna polymerase, dntps, reaction buffer and sybr green i fluorescent dye. the primers used in the reverse transcription pcr were ky80 (sense) 5’-gca gaa agc gtc tag cca tgg cgt-3’ and ky78 (antisense) 5’-ctc gca agc acc cta tca ggc agt-3’. these primers target the highly-conserved 5’-non-coding region of the hcv genome to produce a pcr product of 244 bp and are known to detect all hcv genotypes.31 the reverse transcription pcr master mix consisted of pcr-grade water, mn(oac)2, both primers and the lightcycler® rna master sybr green i mix. the reverse transcription and pcr parameters were set according to the lightcycler® rna master sybr green i package insert. reverse transcription of the template rna was performed at 61 °c for 20 minutes. complementary dna (cdna) was amplified over 40 cycles of denaturation, annealing and extension. denaturation and extension were performed at the temperatures on the package insert. the annealing temperature was calculated using the melting temperatures of the primers, namely, 65 °c (4 °c subtracted from 69 °c), using standard pcr optimisation guidelines, whereby a range of annealing temperatures (55 °c to 65 °c) was used to determine the optimal annealing temperature, where efficiency of pcr amplification was maximal.32 melting curve analysis to identify pcr products was performed as described on the package insert. the pcr products were detected with a sybr green i dye, which intercalates with double-stranded dna and is detected at a wavelength of 530 nm. primer concentrations and template volumes were optimised using the methods described on the lightcycler® rna master sybr green i package insert. primer concentrations ranging from 0.1 µm to 0.5 µm, using two-fold dilutions, were used to determine the optimal primer concentration capable of detecting the lowest concentration of hcv rna. template volumes of 1 µl, 5 µl and 10 µl were used to determine the optimal starting concentration of the template rna. the hcv quantitative pcr proficiency panel (qcmd, glasgow, scotland) was prepared in five-fold dilutions as an external quality control standard to calculate the lower limit of detection of the optimised hcv rna pcr assay. for the preparation of the in-house internal quality control specimens, hcv-negative whole blood was provided by the south african national blood services (durban, south africa). these dbs specimens were prepared by spiking the hcv-negative whole blood with hcv-positive plasma (3 580 000 iu/ml) obtained from the national institute of communicable diseases (sandringham, johannesburg, south africa). this plasma was tested for hcv using the cobas ampliprep/cobas taqman hepatitis c virus assay on the cobas ampliprep and cobas taqman 48 analyser (roche diagnostics, mannheim, germany). ten-fold serial dilutions were made, with a starting concentration of 3 580 000 iu/ml of hcv rna, resulting in values of 3 580 000 iu/ml, 358 000 iu/ml, 35 800 iu/ml and 3580 iu/ml. unspiked whole blood was used as a negative control. fifty microliters (50 µl) of the positive and negative internal control specimens were spotted onto whatman filter paper to mimic infant dbs specimens. results primer concentrations adjusted to 0.25 µm and a template volume of 10 µl improved pcr amplification. an annealing temperature of 58 °c resulted in higher levels of amplified pcr product compared with an annealing temperature of 65 °c. quality control specimens with hcv rna values of < 10 000 iu/ml were detectable at an annealing temperature of 58 °c. detection at a melting temperature of 86 °c was indicative of an hcv-positive result (figure 1) for quality control specimens with viral loads of 13 646 iu/ml (high), 7063 iu/ml (medium) and 3972 iu/ml (low). the external quality control specimens with known hcv viral loads of 30 000 iu/ml, 6000 iu/ml and 1200 iu/ml were detected by the pcr, whereas external quality control specimens with an hcv viral load of 240 iu/ml were undetectable (figure 2). the in-house hcv quality control specimens with viral loads of 3 580 000 iu/ml, 358 000 iu/ml, 35 800 iu/ml and 3580 iu/ml were all detected by the hcv pcr (figure 3). figure 1: melting curves at a melting temperature of 86 °c represent hcv-positive controls. sixteen specimens that tested negative for hcv by serology were used as hcv-negative controls. figure 2: external quality controls. the melting curves show results of five-fold dilutions of hcv-positive external quality control specimens. these specimens were used to determine the lowest hcv rna-positive concentrations that the optimised pcr assay could detect. hcv rna at concentrations of 30 000 iu/ml, 6000 iu/ml and 1200 iu/ml were detected by the assay, whereas concentrations of 240 iu/ml of hcv rna and lower were undetectable. figure 3: internal quality controls. internal quality control dbs specimens were prepared by spiking hcv-negative whole blood with hcv-positive plasma. ten-fold dilutions were prepared with a starting concentration of 3580 000 iu/ml of hcv rna and added onto whatman filter paper. hcv-negative whole blood was used as a negative control. an hcv rna concentration of 3580 iu/ml was the lowest concentration of rna detected for dbs using the optimised pcr assay. the lowest concentration detected by the optimised hcv pcr was 1200 iu/ml (figure 2) when using the qcmd quality control specimens. the in-house internal quality control dbs specimens were detected with viral loads as low as 3580 iu/ml of hcv rna (figure 3). of the 179 infant dbs specimens, 13 tested hiv-positive and 166 tested hiv-negative via pcr. all 179 dbs specimens tested negative for hcv using the optimised real-time pcr assay. discussion we optimised a real-time pcr test for hcv using dbs specimens from infants as a simple method for qualitative hcv detection. although our method was qualitative, it was able to detect at least 3580 iu/ml and 1200 iu/ml in various types of quality control specimens. this could be useful for detecting acute hcv infections, which generally have viral loads less than 105 iu/ml.33 the detection of amplified nucleic acid using sybr green i is inexpensive and is an easier technical approach in comparison to other real-time pcr formats such as taqman and molecular beacon detection methods.14 it is possible that hcv rna viral load in our infant dbs specimens was below the level of detection of the assay, since hcv rna viral loads amongst infants in the first year of infection may be too low for detection.3 dbs specimens have improved the cost-effectiveness, coverage and availability of molecular-based testing in resource-limited settings.11,24,34,35 this has resulted in the development of several optimised pcr techniques to detect hcv using dbs.21,22,24,25 as with our method, several of these techniques are based on real-time pcr and, like de crignis,22 we used lightcycler® technology with sybr green i as the fluorophore.21,24,25 probe-based detection methods are known to improve the sensitivity of target detection, but increase the cost of the testing.21 the limit of detection varies amongst assays. our assay had a limit of detection of 3580 iu/ml, which is higher than previously reported limits of detection of 2500 iu/ml22 and 250 iu/ml.21 this difference may be attributed to differences in storage conditions of the dbs specimens before testing and differences in methods of rna extraction, which may result in variations in the number of copies of starting rna.11,25,34 additionally, dbs specimen volumes are lower when compared with plasma and serum specimens, which reduces the amount of rna extracted.34 thus, the correlation between hcv viral load from dbs and hcv viral load from plasma or serum may be imprecise, suggesting that dbs specimens are better suited to diagnosis, whereas plasma or serum are better suited to monitoring of hcv antiviral treatment.34 these methods for hcv testing using real-time pcr have been applied to hcv treatment monitoring,24 screening for hiv/hcv co-infection22 and surveillance for hcv amongst intravenous drug users.36 our real-time hcv pcr was used on dbs specimens from an infant population, which may be useful for hcv surveillance amongst infants. the use of dbs specimens for hcv pcr has several advantages.11,34,35 firstly, they are easier to collect than whole blood and present a lower infectious hazard. secondly, specimen transportation and storage are cheaper, since refrigeration is not required. lastly, the long-term stability of nucleic acids in dbs has been proven for molecular analysis. therefore, real-time hcv pcr using dbs specimens for hcv surveillance has been used to understand the molecular epidemiology of hcv.21,22,23,24,25,36 limitations our hcv pcr optimisation has several limitations. firstly, the results are not representative of all infants, since only specimens from hiv-exposed infants who require an hiv pcr were tested. secondly, our optimised hcv pcr may not be optimal for routine clinical diagnostic purposes, although it may be convenient for surveillance work. thirdly, we did not compare our dbs specimen method to those using whole blood. however, we did use alternative methods to evaluate our pcr assay, which included testing external quality control specimens with known hcv viral loads ranging from 1081 iu/ml to 34 594 iu/ml and clinical hcv-positive and hcv-negative specimens from a reference laboratory. further, positive and negative controls were included in each run. lastly, we chose not to follow previously described methods but to optimise our own method, because it was more cost-effective than other real-time techniques reported. in addition, we used existing equipment and in-house pcr consumables that the technical staff in our laboratory were already proficient in using. given that our dbs specimens were from hiv-exposed infants in kwazulu-natal, we expected to find a moderate prevalence of hcv amongst these infants as reported by other african countries with populations co-infected with hiv and hcv.37,38,39 infection with hiv is a risk factor for hcv transmission in co-infected pregnant women and is known to increase vertical transmission.3,4 the fact that we did not detect hcv rna in our study specimens may be because of the prolonged storage (≥ 3 months) of our dbs specimens at room temperature, which may have resulted in degradation of hcv rna.25 furthermore, perinatal transmission of hcv requires a high maternal hcv viral load.3 detection of hcv rna at birth is usually indicative of intrauterine infection, whereas infection during delivery is associated with hcv rna being detected only after infants are aged six months; in addition, delivery by caesarean section is less frequently associated with hcv transmission than vaginal delivery.3 however, clinical information, such as maternal hcv viral load, mode of delivery of infants, infant liver function markers, risk factors (such as hbv co-infection) and laboratory results (such as cd4 cell counts and hiv viral loads) were not available for our study. conclusion we have demonstrated that using an optimised real-time hcv pcr assay on dbs specimens from hiv-exposed infants is a good technique for hcv detection and may be well suited for surveillance purposes in resource-limited settings. to our knowledge, this is the first surveillance in south africa using an optimised real-time hcv pcr to report the prevalence of hcv amongst hiv-exposed infants. considering that hcv was not detected in our dbs specimens, hcv prevalence in this population of infants in kwazulu-natal, south africa may be low and may have little impact on hiv-exposed infants. therefore, further surveillance for hcv amongst hiv-exposed infants in our setting may not be necessary. however, hcv surveillance may still be prudent for hiv-exposed infants in countries with moderate to high hiv and hcv prevalence and should include collection of clinical information and data about confounding factors. acknowledgements competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. sources of support we would like to thank the national institute of communicable diseases, gauteng for providing us with serum samples positive for hcv. we also appreciate the south african national blood services, durban providing us with hcv-negative blood. authors’ contributions r.p. (university of kwazulu-natal/national health laboratory services) designed the project and analysed the data. a.n. (university of kwazulu-natal/national health laboratory services) performed the laboratory work and was responsible for writing the manuscript. p.m. (university of kwazulu-natal/national health laboratory services) assisted in writing and preparing the article references lavanchy d. evolving epidemiology of hepatitis c virus. clin microbiol infect. 2011;17(2):107–115. http://dx.doi.org/10.1111/j.1469-0691.2010.03432.x nel e, sokol rj, comparcola d, et al. viral hepatitis in children. j paediatr gastroenterol nutr. 2012;55(5):500–505. http://dx.doi.org/10.1097/mpg.0b013e318272aee7 yeung c-y, lee h-c, chan w-t, et al. vertical transmission of hepatitis c virus: current knowledge and perspectives. world j hepatol. 2014;6(9):643–651. http://dx.doi.org/10.4254/wjh.v6.i9.643 operskalski ea, kovacs a. hiv/hcv co-infection: pathogenesis, clinical complications, treatment, and new therapeutic technologies. curr hiv/aids rep. 2011;8(1):12–22. http://dx.doi.org/10.1007/s11904-010-0071-3 barth re, huijgen q, taljaard j, et al. hepatitis b/c and hiv in sub-saharan africa: an association between highly prevalent infectious diseases. a systematic review and meta-analysis. int j infect dis. 2010;14(12):e1024–e1031. http://dx.doi.org/10.1016/j.ijid.2010.06.013 hoffmann cj, dayal d, cheyip m, et al. prevalence and associations with hepatitis b and hepatitis c infection among hiv-infected adults in south africa. int j std aids. 2012;23(10):e10–e13. http://dx.doi.org/10.1258/ijsa.2009.009340 madhava v, burgess c, drucker e. epidemiology of chronic hepatitis c virus infection in sub-saharan africa. lancet infect dis. 2002;2(5):293–302. http://dx.doi.org/10.1016/s1473-3099(02)00264-5 national department of health. the 2013 national antenatal sentinel hiv prevalence survey, south africa. pretoria: national department of health; 2013. parboosing r, paruk i, lalloo ug. hepatitis c virus seropositivity in a south african cohort of hiv co-infected, arv naïve patients is associated with renal insufficiency and increased mortality. j med virol. 2008;80(9):1530–1536. http://dx.doi.org/10.1002/jmv.21262 world health organization. guidelines for the screening, care and treatment of persons with hepatitis c infection. geneva: world health organization; 2014. smit pw, elliott i, peeling rw, et al. an overview of the clinical use of filter paper in the diagnosis of tropical diseases. am j trop med hyg. 2014;90(2):195–210. http://dx.doi.org/10.4269/ajtmh.13-0463 albertoni g, castelo girão m, schor n. mini review: current molecular methods for the detection and quantification of hepatitis b virus, hepatitis c virus, and human immunodeficiency virus type 1. int j infect dis. 2014;25:145–149. http://dx.doi.org/10.1016/j.ijid.2014.04.007 chevaliez s, bouvier-alias m, rodriguez c, et al. the cobas ampliprep/cobas taqman hcv test, version 2.0, real-time pcr assay accurately quantifies hepatitis c virus genotype 4 rna. j clin microbiol. 2013;51(4):1078–1082. http://dx.doi.org/10.1128/jcm.02004-12 laughlin ts, nuccie b, rothberg pg. genotyping of hepatitis c virus by sequence analysis of the amplicon from the roche cobas ampliprep/cobas taqman viral load assay. j clin microbiol. 2010;48(2):671–672. http://dx.doi.org/10.1128/jcm.01519-09 mousavi-fard sh, merat s, shahzamani k, et al. development of a sybr green real time multiplex rt-pcr technique for simultaneous detection of hcv and gbv-c co-infection in plasma samples. mbd. 2014;1(1):41-49. paryan m, forouzandeh mm, kia v, et al. design and development of an in-house multiplex rt-pcr assay for simultaneous detection of hiv-1 and hcv in plasma samples. iran j microbiol. 2012;4(1):8–14. rozales fd, de-paris f, machado abmp, et al. optimization of one-step real-time pcr for the x-tail target of hcv as a diagnostic test. clin biomed res. 2014; 34(2):164–168. shahzamani k, sabahi f, merat s, et al. rapid low-cost detection of hepatitis c virus rna in hcv-infected patients by real-time rt-pcr using sybr green i. arch iran med. 2011;14(6):396–400. zhang ez, bartels dj, frantz jd, et al. development of a sensitive rt-pcr method for amplifying and sequencing near full-length hcv genotype 1 rna from patient samples. virol j. 2013:10:53. http://dx.doi.org/10.1186/1743-422x-10-53 zaghloul mhe, rizk em, elsayed ar, et al. detection of hcv-rna by real time pcr using sybrgreen dye i. int j adv res. 2015;3(6):1450–1458. bennett s, gunson rn, mcallister ge, et al. detection of hepatitis c virus rna in dried blood spots. j clin virol. 2012;54(2):106–109. http://dx.doi.org/10.1016/j.jcv.2012.02.004 de crignis e, re mc, cimatti l, et al. hiv and hcv detection in dbs by sybr green multiplex real time rt-pcr. j virol methods. 2010;165(1):51–56. http://dx.doi.org/10.1016/j.jviromet.2009.12.017 hope vd, hickman m, ngui sl, et al. measuring the incidence, prevalence and genetic relatedness of hepatitis c infections among a community recruited sample of injecting drug users, using dried blood spots. j viral hepat. 2011;18(4):262–270. http://dx.doi.org/10.1111/j.1365-2893.2010.01297.x santos c, reis a, dos santos cv, et al. the use of real-time pcr to detect hepatitis c virus rna in dried blood spots from brazilian patients infected chronically. j virol methods. 2011;179(1):17–20. http://dx.doi.org/10.1016/j.jviromet.2011.06.012 tuaillon e, mondain a, meroueh f, et al. dried blood spot for hepatitis c virus serology and molecular testing. hepatology. 2010;51(3):752–758. soulier a, poiteau l, rosa i, et al. dried blood spots: a tool to ensure broad access to hepatitis c screening, diagnosis, and treatment monitoring. j infect dis. 2015 sep 2. pii:jiv423. epub ahead of print. http://dx.doi.org/10.1093/infdis/jiv423 mutagi a, atherton c, wilson s, et al. development of qualitative hcv rna testing on dried blood spots as an adjunct screening tool to identify active hepatitis c infection. open forum infect dis. 2014;1(suppl 1):s376–s377. fouad na, mahedy aw, el-taher sm. application of dried blood spot testing for hepatitis c virus rna amplification. egypt j med microbiol. 2013;22(11):1–8. http://dx.doi.org/10.12816/0004921 ross rs, stambouli o, grüner n, et al. detection of infections with hepatitis b virus, hepatitis c virus, and human immunodeficiency virus by analyses of dried blood spots – performance characteristics of the architect system and two commercial assays for nucleic acid amplification. virol j. 2013;10:72. http://dx.doi.org/10.1186/1743-422x-10-72 moodley p, parboosing r, moodley d. reduction in perinatal hiv infections in kwazulu-natal, south africa, in the era of more effective prevention of mother to child transmission interventions (2004–2012). j acquir immune defic syndr. 2013;63(3):410–415. http://dx.doi.org/10.1097/qai.0b013e3182926931 young ky, resnick rm, myers tw. detection of hepatitis c virus rna by a combined reverse transcription-polymerase chain reaction assay. j clin micro. 1993;31(4):882–886. rapley r, whitehouse d, editors. molecular biology and biotechnology. 6th ed. cambridge, united kingdom: royal society of chemistry; 2014. mcgovern bh, birch ce, bowen mj, et al. improving the diagnosis of acute hepatitis c virus infection with expanded viral load criteria. clin infect dis. 2009;49(7):1051–1060. http://dx.doi.org/10.1086/605561 greenman j, roberts t, cohn j, et al. dried blood spot in the genotyping, quantification and storage of hcv rna: a systematic literature review. j viral hepat. 2015;22(4):353–361. http://dx.doi.org/10.1111/jvh.12345 snijdewind ijm, van kampen jja, fraaij pla, et al. current and future applications of dried blood spots in viral disease management. antiviral res. 2012;93(3):309–321. http://dx.doi.org/10.1016/j.antiviral.2011.12.011 hickman m, mcdonald t, judd a, et al. increasing the uptake of hepatitis c virus testing among injecting drug users in specialist drug treatment and prison settings by using dried blood spots for diagnostic testing: a cluster randomized control trial. j viral hepat. 2008;15(4):250–254. http://dx.doi.org/10.1111/j.1365-2893.2007.00937.x njouom r, pasquier c, ayouba a, et al. low risk of mother-to-child-transmission of hepatitis c virus in yaounde, cameroon: the anrs 1262 study. am j trop med hyg. 2005;73(2):460–466. ogboghodo bc, aigbirior ma, bazuaye gn, et al. hepatitis c virus and human immunodeficiency virus-i (hiv) co-infection in children in benin city, nigeria. afr. j. biomed. res. 2009;12(1):1–6. sadoh ae, sadoh we, iduoriyekemwen nj. hiv co-infection with hepatitis b and c viruses among nigerian children in an antiretroviral treatment programme. s. afr. j. child health. 2011;5(1):7–10.  article information authors: katy yao1 talkmore maruta2 elizabeth t. luman1 john n. nkengasong1 affiliations: 1international laboratory branch, division of global hiv/aids, us centers for disease control and prevention (cdc), atlanta, united states 2african society for laboratory medicine (aslm), addis ababa, ethiopia correspondence to: katy yao postal address: 1600 clifton road, ms: g45, atlanta, ga 30329-4018, united states dates: received: 14 may 2014 accepted: 03 july 2014 published: 16 sept. 2014 republished: 07 nov. 2014 how to cite this article: yao k, maruta t, luman et, nkengasong jn. the slmta programme: transforming the laboratory landscape in developing countries. afr j lab med. 2014;3(1), art. #194, 8 pages. http://dx.doi.org/10.4102/ ajlm.v3i2.194 note: article republished with updated references relating the special issue. copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the slmta programme: transforming the laboratory landscape in developing countries in this lessons from the field... open access • abstract • introduction • key components • variations from the basic implementation model    • cameroon    • lesotho • capacity building for programme scale-up • additional considerations    • country commitment    • site selection    • human resources • experience from africa    • mozambique – country ownership and sustainability    • rwanda – data-driven advocacy    • cameroon – expanding quality past the laboratory    • zimbabwe – overcoming contextual challenges • slmta’s global reach and influence outside africa • lessons learned • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: efficient and reliable laboratory services are essential to effective and well-functioning health systems. laboratory managers play a critical role in ensuring the quality and timeliness of these services. however, few laboratory management programmes focus on the competencies required for the daily operations of a laboratory in resource-limited settings. this report provides a detailed description of an innovative laboratory management training tool called strengthening laboratory management toward accreditation (slmta) and highlights some challenges, achievements and lessons learned during the first five years of implementation (2009–2013) in developing countries. programme: slmta is a competency-based programme that uses a series of short courses and work-based learning projects to effect immediate and measurable laboratory improvement, while empowering laboratory managers to implement practical quality management systems to ensure better patient care. a slmta training programme spans from 12 to 18 months; after each workshop, participants implement improvement projects supported by regular supervisory visits or on-site mentoring. in order to assess strengths, weaknesses and progress made by the laboratory, audits are conducted using the world health organization’s regional office for africa (who afro) stepwise laboratory quality improvement process towards accreditation (slipta) checklist, which is based on international organization for standardization (iso) 15189 requirements. these internal audits are conducted at the beginning and end of the slmta training programme. conclusion: within five years, slmta had been implemented in 617 laboratories in 47 countries, transforming the laboratory landscape in developing countries. to our knowledge, slmta is the first programme that makes an explicit connection between the performance of specific management behaviours and routines and iso 15189 requirements. because of this close relationship, slmta is uniquely positioned to help laboratories seek accreditation to iso 15189. introduction top ↑ efficient and reliable laboratory services are essential to a functioning health system as high-quality laboratory testing plays a key role in patient care, surveillance and outbreak investigation.1 poor laboratory quality and its negative impact on healthcare systems have been documented for resource-limited settings, including sub-saharan africa (ssa).2,3,4,5 using the number of accredited laboratories as a quality metric, a 2013 survey showed that 37 out of the 49 countries in ssa had no medical laboratories accredited to any internationally-recognised standards. of the 380 accredited laboratories in that region, 91% were in south africa and only 17% were public health laboratories.6 in recent years, however, several landmark events have drawn attention to the poor state of public health laboratories and have pushed for strengthening of laboratory systems and networks.1,7 one of these events was the issuance of the world health organization (who)–lyon statement in 2008,8 which called for countries with limited resources to pursue practical quality management systems and to adopt a stepwise approach to quality improvement and accreditation.4,7 another was the 2009 launch of a laboratory management training programme called ‘strengthening laboratory management toward accreditation’ (slmta).1 effective management and leadership are critical to strengthening health systems and the scaling up of health service delivery.9 recently, many countries and partners have initiated efforts to enhance management of health programmes and service delivery in developing countries, with measurable success.10,11,12,13,14,15,16,17,18 most of these management capacity-building efforts focused on managers from hospitals, primary healthcare centers (such as family planning, mother–child health, etc.), or vertical public health programmes (such as tuberculosis [tb] and hiv). existing laboratory management capacity-building efforts have primarily targeted senior laboratory officials where the focus is on laboratory policy, system and network development, 19,20,21,22,23 as opposed to daily operations of individual laboratories. training programmes are needed to enable laboratory managers to use available resources (staff, budgets, supplies, equipment, buildings and information) efficiently for planning, implementation and evaluation of service delivery in order to meet patients’ and clinicians’ expectations and public health needs.24 the slmta programme was created in response to the observed need for structured laboratory management training and quality improvement by the us centers for disease control and prevention (cdc), in collaboration with the american society for clinical pathology, the clinton health access initiative, and the world health organization’s regional office for africa (who afro). slmta is a competency-based management training programme which uses a series of short didactic courses and work-based applied learning projects with the goal of achieving immediate and measurable laboratory improvements. it provides a practical approach to addressing everyday challenges using available resources. the slmta training curriculum and implementation method were pilot-tested in 15 laboratories in uganda from august 2008 to march 2009, yielding promising results.24 slmta was then officially launched in 2009, with implementation beginning in 2010. as of the end of 2013, slmta had been rolled out in 47 countries and 617 laboratories, and had improved enrolled laboratories an average of 23 percentage points after one round of slmta training in a pre/post study using the who afro stepwise laboratory quality improvement process towards accreditation (slipta) checklist.25 this report provides a detailed description of the slmta programme and highlights some challenges, achievements and lessons learned during its first five years of implementation (2009–2013) in developing countries. key components top ↑ the design of the slmta curriculum and its implementation exemplify what is known as ‘good practice’ in management competencies development.19,26 the slmta curriculum covers the 10 key competencies of a laboratory manager: productivity; work area; inventory; procurement; equipment maintenance; quality assurance; specimens; laboratory testing; test result reporting; and document and records control. a total of 66 tasks and job routines define effective laboratory management and constitute the learning objectives of the curriculum.24 a typical slmta training programme spans from 12 to 18 months (figure 1). training is conducted in a series of three workshops, each lasting three to four days, utilising 44 instructional activities27 and more than 100 job aids. each activity provides hands-on, practice-based learning experience for specific management tasks. the total training time is approximately 60 hours to teach all 44 activities. figure 1: standard slmta implementation process. after each workshop, participants implement improvement projects in their home laboratories. there are two types of improvement projects: complicated projects that require extensive planning and data collection before and after the change; and simpler ‘just do it’ types of projects that can be implemented immediately with minimal time and resources (box 1). implementation of improvement projects requires teamwork involving the entire laboratory staff, thus ensuring that the projects become part of the laboratory’s continuous improvement processes. participants are encouraged to implement locally-appropriate solutions using existing resources. during the home-based learning period after each workshop, participants are supported by periodic supervisory visits or on-site mentoring guided by standardised tools. this structured supervision and support component is critical to the success of the slmta programme. box 1: examples of improvement projects. the formal laboratory evaluation component is designed to identify weaknesses and areas that require improvement, measure success of the programme and indicate future goals for the laboratory. evaluations are based on who afro’s five-stage accreditation-preparedness scheme, called slipta, which recognises laboratories according to their level of compliance with the international organisation for standardization (iso) 15189 standard.1 under the slipta scheme, laboratories are audited using the slipta checklist, which includes 111 items divided into 12 sections (table 1) based on the 12 quality system essentials from the clinical and laboratory standards institute (clsi).28 after an audit, a laboratory receives a score out of 258 points in order to determine its star rating – from ‘0’ (0–141 points, < 55%) to ‘5’ (244–258 points, ≥ 95%).29 not all laboratories will pursue accreditation; regardless, the slipta scheme provides the roadmap and motivation for laboratories to make steady improvement in service delivery and patient care. slmta and slipta are closely linked. the slipta checklist provides the slmta programme with a means to identify gaps and benchmark progress. slmta, on the other hand, equips laboratory management with the ability to implement quality management systems in order to improve their performance on the slipta scale and eventually achieve formal accreditation status. to support this link, individual slipta checklist items are mapped to each of the 44 instructional activities in the slmta curriculum so that participants know exactly which management action will fulfill the requirements of any given checklist item. because of this close linkage between the slmta curriculum and the slipta checklist, in june 2012, after modification of the slipta checklist, the slmta curriculum underwent revisions to remap the revised checklist items to slmta instructional activities. each laboratory participating in slmta conducts an internal audit at the beginning (baseline) and the end (exit) of the programme using the slipta checklist. the difference between baseline and exit scores, as well as their respective star ratings, is calculated in order to quantify the effects of the programme on laboratory function and quality (figure 1). in addition to the slipta scores, laboratories demonstrate their progress through improvement project data such as turn-around time, sample rejection rate, stock out rate, customer satisfaction survey results and before-and-after photographs of physical changes. table 1: sections of the who afro slipta checklist and star ratings. variations from the basic implementation model top ↑ some countries have customised slmta delivery to fit their local context. two notable variations are cameroon and lesotho, which adapted their programmes to address local challenges and to enhance existing laboratory capacity-building efforts. despite the variations, both adaptations adhere to the critical requirement of implementing slmta as a process (a series of workshops with improvement projects and mentoring) rather than a single training event. cameroon most countries conduct the slmta training in a central location. this centralised model provides logistical convenience, particularly when many laboratories are enrolled in the same round, allowing the programme to train many laboratories at one time. it also enables personnel from various laboratories to interact and learn from each other. however, there are drawbacks, including, (1) high costs associated with renting a venue and travelling participants; (2) staff must be absent from their laboratories for prolonged periods because of travel between home and training locations; and (3) a limited number of staff can attend the course, creating a potential divide between those who are trained and those who are not. working with a very limited budget, cameroon decentralised the workshops and conducted facility-based training, with teams traveling to the laboratories in the programme to provide training on site. whilst this model required more time from the trainers, it enabled hospital management and clinicians to be involved in the training alongside laboratory management, facilitating advocacy. in addition, it allowed the course to be better tailored to the needs of the individual laboratories, with all discussions related to site-specific challenges and solutions.30 lesotho the schedule and frequency of trainings for the initial slmta round in lesotho were modified in order to match existing mentorship timetables.31,32 at the time that slmta was adopted, the country had already begun a structured mentorship programme with an embedded mentor. this mentor soon became certified as a slmta trainer so that he could enhance on-going mentoring efforts with the slmta programme. these laboratories received slmta training one day per week over two blocks of six weeks each, spaced six months apart. the total training time was the same as the standard three-workshop model. because of the availability of a full-time mentor, these laboratories received more intensive and frequent monitoring visits – a total of 12 visits versus the standard six – and were able to implement numerous improvement projects. capacity building for programme scale-up top ↑ in order to facilitate programme scale-up, a training-of-trainers approach was used to develop indigenous trainers, who in turn implement the slmta programme in-country.27 because the quality and integrity of the programme relies heavily on these local trainers, it is critical that they are competent and well qualified. to achieve that goal, the programme has established strict screening criteria in order to ensure that potential trainers have the necessary availability, motivation and commitment, along with a technical background. a formal training-of-trainers course was developed in which slmta master trainers teach both the curriculum content and also facilitation skills. this two-week course provides a demanding but supportive environment where participants conduct teach-back of assigned activities from the curriculum and immediately receive constructive feedback from master trainers in order to improve their facilitation skills and understanding of the content. to graduate, participants must fulfill several requirements: (1) 100% daily attendance, including group work sessions; (2) equal responsibility in the preparation and facilitation of teach-back assignments; (3) 100% completion of homework; and (4) endorsement by a master trainer. participants and their organisations also receive reports providing performance reviews and recommendations on specific roles that they are competent to play in programme implementation. timely, specific, behaviour-focused feedback is the cornerstone of training-of-trainers. as such, the master trainers’ ability to mentor the participants and provide constructive feedback determines the quality of trainers produced. the rapid expansion of the slmta programme has resulted in the demand for more master trainers who can train trainers. given the crucial role that master trainers play in developing competent trainers, they must be highly motivated and effective, their qualifications must be impeccable and their development and selection process rigorous. to be considered as a master trainer candidate, he or she must: (1) be a certified slmta trainer; (2) have conducted the entire slmta process; (3) have the availability and commitment needed to be a strong asset to the programme; and (4) be nominated by an existing master trainer. eligible candidates are invited to a training-of-trainers course, where they apprentice under existing master trainers whilst sharing the course workload equally .27 throughout the course, these candidates receive coaching and feedback on their performance from master trainers and their competence and commitment are assessed constantly. additional considerations top ↑ country commitment countries adopting the slmta programme are advised to fulfill certain pre-requisites to ensure success. firstly, they must have a national laboratory policy and strategic plan, along with a laboratory technical working group in order to drive the initiative forward. secondly, countries must ensure financial and political support for slmta and a commitment to improving laboratory quality at all levels: ministry of health, hospital management, laboratory management and laboratory staff. it is critical that slmta sites have dedicated quality assurance and safety officers. it is also important for participants to remain in the same job or organisation throughout the duration of the programme and to be allowed the time needed to participate in the programme. site selection site selection should be based on several factors, including facility infrastructure, staffing levels, impact on coverage of patient care, geographic considerations and demonstration of site commitment. the number of laboratories enrolled for each round of slmta (i.e., cohort) has varied by country – ranging from one each in angola and swaziland to 27 in malawi.25 countries have been advised to start small and scale up progressively. however, political pressure for broader impact and the desire for more laboratories to benefit from slmta may have resulted in some countries enrolling large numbers of laboratories. four countries (ethiopia, malawi, nigeria and uganda) have enrolled > 20 laboratories in the first or subsequent slmta cohorts.25 enrolling a large number of laboratories requires more human and logistical resources for the provision of sufficient site monitoring and support. in addition, it is essential that there is good communication and coordination amongst trainers and mentors so as to ensure consistency throughout the group. most countries have continued to enroll new laboratories in subsequent slmta cohorts.25 kenya to date has initiated six cohorts of slmta, enrolling a total of 50 laboratories and seven blood banks. lesotho, a small country with only 19 laboratories, has reached a high coverage of 18 (95%) laboratories over three cohorts of slmta. human resources countries vary in their capacity to rollout the slmta programme. implementation requires three primary cadres: trainers to teach the curriculum; auditors to perform the internal audits; and mentors to facilitate the improvement projects. regional and in-country slmta training-of-trainer workshops conducted during the past five years have steadily produced more local trainers.27 although the demand for slmta trainers still exceeds the supply, the deficiency is less severe than that of qualified auditors and mentors. using unqualified auditors may lead to inaccurate audit findings and missed non-conformities. this gap is being addressed slowly as many countries are seeking partners’ help with regard to scaling up auditor training. mentorship and site visits may be the most challenging aspect of implementation and are often overlooked in the initial programme planning. site visits require personnel time, transportation resources (fuel, vehicle, driver) and lodging and per diem if overnight stays are necessary. if this component is not scheduled and budgeted properly from the beginning, countries often struggle to provide the onsite support and supervision that are critical to the programme’s success. site visits are necessary in order to check the progress of the improvement projects, assess effectiveness of the previous workshops, troubleshoot site-specific issues and provide motivation and encouragement. site visits often involve meetings with top facility management to advocate support for the laboratory. the length of site visits has varied greatly between countries and even amongst laboratories within the same slmta cohort, ranging from half a day to three or more days at each site. the frequency and length of site visits should be considered carefully and planned according to the size and scope of testing activities in the laboratory. in addition, the level of quality at baseline and progress thereafter, as well as site staff’s experience with regard to implementing quality systems, should be considered. laboratories needing more support should receive longer or more frequent visits to enable them to make measurable improvements and sustain their motivation. the need for extensive but affordable site support has led countries such as cameroon,30 mozambique,33 swaziland and zimbabwe34 to establish structured mentorship programmes with full-time facility-based local mentors – a model spearheaded by lesotho.32,35 this model has well-defined goals for each mentoring engagement, extended contact time on site, defined periods when mentors are absent, consistent approaches across laboratories and measurement of progress using standardised tools. mentors may come from the laboratories they are assigned to mentor, from a local partner, or from outside the country. mentors receive training in slmta implementation, mentorship and auditing. because of their extended participation in the laboratories they are mentoring, they are able to gain knowledge of the rhythms, practices and personalities of the laboratory, enabling them to facilitate the necessary changes in attitudes and behaviours. other strategies have been used to provide the needed support for the slmta laboratories. in kenya, for example, select slmta hospital laboratories were paired, or ‘twinned’, with internationally-accredited research laboratories. the accredited laboratories mentored the slmta laboratories in quality management system implementation.36 experience from africa top ↑ slmta was launched in africa in 2009. by the end of 2013, it had been implemented in 23 countries on the continent with a total of 503 participating laboratories, which constituted 87% of all the slmta-enrolled laboratories in the world.25 as the continent that launched slmta, africa has demonstrated to the world that with ingenuity, innovation and determination, implementing quality management systems is possible, despite resource limitations. to date, four slmta-enrolled laboratories in africa have been accredited to iso 15189, whilst many more are making great progress in continuous quality improvement.25 in the sections below, we highlight the experiences of four african countries. mozambique – country ownership and sustainability to develop a self-sufficient quality programme, mozambique integrated slmta within the existing structure of the ministry of health laboratory system. a national laboratory quality technical working group was established and a dedicated coordinator hired. the ministry of health provided the vision and leadership in implementation and advocacy, coordinated and financed the programme with partner support and pressed for slmta activities to be included in provincial and hospital annual plans and budgets. decentralising programme management to the provincial level has enabled them to increase programme coverage and lower the costs.33 rwanda – data-driven advocacy as with many other countries, rwanda’s laboratories suffered from chronic service disruptions as a result of reagent stock-out and equipment breakdowns from lack of maintenance. an improvement project was assigned to the slmta-enrolled laboratories, which tracked the number of tests not performed because of stock-out and equipment breakdowns over a three-month period. they then calculated the funds required to purchase needed reagents and maintain equipment, along with the revenue that would have been generated from these tests, finding that the missed income was far greater than the cost of preventing stock-out and equipment breakdowns. this return on investment analysis persuaded hospital management to prioritise reagent supplies and to contract with manufacturers to provide regular maintenance services for the laboratory equipment.37 cameroon – expanding quality past the laboratory in cameroon, management at one hospital witnessed the transformation of its laboratory after slmta and undertook to extend the quality into other units of the hospital. they formed their own quality improvement teams, which have reported improved hospital cleanliness, reduced patient waiting times, greater patient satisfaction, development of new treatment protocols and increased recognition of the importance of patient safety. additionally, a reduction in infection rates and stillbirths, as well as an increase in the number of patients served and hospital revenue, have been observed.38 zimbabwe – overcoming contextual challenges zimbabwe has suffered economic crises in the past few decades, resulting in deterioration of the healthcare system and a shortage of human resources. participants in its two slmta cohorts have identified creative solutions to overcome the extensive logistic and resource challenges. for example, standard operating procedures were hand-written in exercise books, levy-jennings charts were plotted manually and a paper-based system was used where computerised laboratory information systems were not available. hospitals recognised the value of accreditation and prioritised budgets for equipment calibration, service contracts and staff vaccinations. funding from the us president’s emergency plan for aids relief (pepfar) supported the establishment of a training and mentorship department at the zimbabwe national quality assurance program trust in order to develop local capacity to support slmta programme rollout and continued quality improvement for laboratory services.39 slmta’s global reach and influence outside africa top ↑ the slmta-driven laboratory quality improvement achieved in africa has inspired countries in other regions to follow suit, even in the absence of a regional or national accreditation preparedness scheme such as who afro’s slipta. outside the continent of africa, 24 countries from the caribbean region, central and south america and southeast asia have adopted the slmta programme and have used the slipta checklist to measure gaps and the progress of enrolled laboratories. the caribbean region, comprising many island countries with diverse geography, people, size and economy, has implemented slmta in 12 countries.25 after completing the slmta programme, bahama’s national hiv reference laboratory was accredited and two other enrolled laboratories in the region are also seeking international accreditation.40 in southeast asia, impressive results have also been observed in cambodia and vietnam, where one provincial laboratory that tests clinical as well as food and environmental samples was accredited to iso 17025 in 2013.25 a desire to automate data collection, analyse and manage slipta audit data more efficiently and to enable real-time graphical display of actionable results at audited facilities led to the development of a multi-lingual electronic tool in vietnam.41 this tool has been shared with the global slmta community. in latin america, a partnership was forged where 14 military laboratories from eight countries in the region were enrolled in promela (programa de mejoramiento de laboratorios de las fuerzas armadas de latinoamérica), an overarching laboratory improvement programme using slmta as its principle training tool in addition to other practical laboratory training and biosafety and/or infection control training. the fact that two africa-based master trainers (one anglophone, one lusophone) came to assist in the first spanish-speaking training-of-trainers in latin america underscores the benefits of standardised training and highlights slmta’s true global nature and its far-reaching network across borders and continents. lessons learned top ↑ throughout the slmta rollout, countries have overcome many challenges such as attrition of slmta-trained staff, encouraging the entire laboratory to work as a team, engaging hospital management, and insufficient mentorship capacity. table 2 summarises the most common challenges and offers corresponding recommendations to help guide future implementation. despite the challenges, slmta has worked successfully by demonstrating that with resolve, commitment and ingenuity, laboratory teams in developing countries can improve their service delivery using existing limited resources. it also demonstrates that starting with small tangible improvements (‘low-hanging fruit’) and gradually building upon early successes can boost laboratory teams’ confidence and motivate them to tackle the harder issues. this strategy is similar to the ‘little steps’ approach42 that has been shown to be effective in sustaining healthcare quality improvement efforts in developing countries. table 2: common challenges and recommendations for slmta implementation. within a few years, slmta has demonstrated its transformative power, emerging as a flagship programme for laboratory system strengthening in pepfar-supported countries. a recent 2013 institute of medicine report43 recognised that improvement of laboratories under pepfar support and guidance has been a signature achievement. in addition, it states that: pepfar’s laboratory efforts have had a fundamental and substantial impact on laboratory capacity in countries. this laboratory infrastructure has been, and continues to be, leveraged to improve the functioning of countries’ entire health systems.43 as laboratories do not exist in a vacuum, there have been calls38,44 for the slmta model to be adapted for the clinical settings in developing countries, with a goal toward overall hospital accreditation. this will ensure the sustainability of laboratory improvements and accreditation, and boost the centrality of quality management systems in hospital facilities, resulting in better patient care. slmta implementation has been supported primarily with pepfar resources. to ensure its longevity and viability beyond pepfar, countries must work hard to integrate the slmta components into normal laboratory operations, decentralise programme planning and budgeting to the provincial or lower level, look for ways to be financially self-sufficient (such as charging enrollment fees for privately-owned laboratories) and incorporate the curriculum into pre-service education. conclusion top ↑ after five years of implementation, slmta has proven to be an effective programme for the strengthening of laboratory health systems, with a focus on building management capacity in order to achieve quality services for improved patient care. evidence to date has indicated widespread success of the programme in its ability to facilitate continuous quality improvement in the enrolled laboratories. slmta has the unique potential to help laboratories make progress through the slipta process, improve quality of services and subsequently achieve accreditation to iso 15189. acknowledgements top ↑ the authors would like to thank dr. barbara mckinney, anna murphy and philip rotz for their significant contribution in the development of the slmta toolkit. the authors also extend their gratitude to all the slmta implementers for their tireless effort in improving the quality of laboratory systems and patient care in resource-limited settings. this research has been supported by pepfar through the cdc. the findings and conclusions in this article are those of the authors and do not necessarily represent the views of the cdc. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions k.y. (cdc) led the development of the slmta programme, oversaw its global implementation and wrote the manuscript. t.m. (aslm) played a key role in programme expansion and implementation and provided input to the manuscript. e.l. (cdc) provided substantial input to the writing of the manuscript. j.n. (cdc) provided high-level strategic direction for programme development, implementation and manuscript writing. references top ↑ 1.gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. pubmed pmid: 20716795 2.petti ca, polage cr, quinn tc, et al. laboratory medicine in africa: a barrier to effective health care. clin infect dis. 2006;42(3):377–382. 3.peter tf, rotz pd, blair dh, et al. impact of laboratory accreditation on patient care and the health system. am j clin pathol. 2010;134(4):550–555. 4.nkengasong jn. a shifting paradigm in strengthening laboratory health systems for global health: acting now, acting collectively, but acting differently. am j clin pathol. 2010;134(3):359–360. pubmed pmid: 20716789. 5.nkengasong jn, nsubuga p, nwanyanwu o, et al. laboratory systems and services are critical in global health: time to end the neglect? am j clin pathol. 2010;134(3):368–373. pubmed pmid: 20716791. 6.schroeder lf, amukele t. medical laboratories in sub-saharan africa that meet international quality standards. am j clin pathol. 2014;141(6):791–795. 7.alemnji ga, zeh c, yao k, et al. strengthening national health laboratories in sub-saharan africa: a decade of remarkable progress. trop med int health. 2014;19(4):450–458. 8.world health organization. joint who–cdc conference on health laboratory quality systems [document on the internet]. c2008 [cited 2014 aug 11]. available from: http://www.who.int/ihr/lyon/report20080409.pdf 9.waddington c, egger d, travis p, et al. towards better leadership and management in health: report on an international consultation on strengthening health leadership and management in low-income countries, 29 january – 01 february 2007, accra, ghana. making health systems work: working paper no. 10. who/hss/healthsystems/2007.3 [document on the internet]. c2007 [cited 2014 aug 11]. available from: http://www.who.int/management/working_paper_10_en_opt.pdf 10.matovu jkb, wanyenze rk, mawemuko s, et al. building capacity for hiv/aids program leadership and management in uganda through mentored fellowships. glob health action. 2011;4:10.3402/gha.v4i0.5815. pubmed pmid: 21364774. pubmed central pmcid: 3046003. 11.matovu jkb, wanyenze rk, mawemuko s, et al. strengthening health workforce capacity through work-based training. bmc int health hum rights. 2013;13:8. pubmed pmid: 23347473. pubmed central pmcid: 3565877. 12.kebede s, abebe y, wolde m, et al. educating leaders in hospital management: a new model in sub-saharan africa. int j qual health care. 2010;22(1):39–43. pubmed pmid: 19951963. pubmed central pmcid: 2803009. 13.bradley e, hartwig ka, rowe la, et al. hospital quality improvement in ethiopia: a partnership-mentoring model. int j qual health care. 2008;20(6):392–399. pubmed pmid: 18784268. 14.hartwig k, pashman j, cherlin e, et al. hospital management in the context of health sector reform: a planning model in ethiopia. int j health plann manage. 2008;23(3):203–218. pubmed pmid: 18157912. 15.kebede s, mantopoulos j, ramanadhan s, et al. educating leaders in hospital management: a pre-post study in ethiopian hospitals. glob public health. 2012;7(2):164–174. pubmed pmid: 21259143. 16.umble ke, brooks j, lowman a, et al. management training in vietnam’s national tuberculosis program: an impact evaluation. int j tuberc lung dis. 2009;13(2):238–246. pubmed pmid: 19146754. 17.rowe la, brillant sb, cleveland e, et al. building capacity in health facility management: guiding principles for skills transfer in liberia. hum resour health. 2010;8:5. pubmed pmid: 20298565. pubmed central pmcid: 2850875. 18.mansour m, mansour jb, el swesy ah. scaling up proven public health interventions through a locally owned and sustained leadership development programme in rural upper egypt. hum resour health. 2010;8(1):1. pubmed pmid: 20205749. pubmed central pmcid: 2822741. 19.egger d, travis p, dovlo d, et al. strengthening management in low income countries. making health systems work: working paper no. 1 [document on the internet]. c2007 [cited 2014 aug 11]. available from: http://www.who.int/management/working_paper_1_en_opt.pdf 20.gwu-aphl international institute for public health laboratory management, fall 2012 seminar: october 22 – november 2, 2012. dar es salaam, tanzania [page on the internet]. c2012 [cited 2013 may 31]. available from: http://www.aphl.org/aphlprograms/global/initiatives/documents/gh_2012_gwu-aphl-international-laboratory-management-institute-brochure.pdf 21.adams cl, mcclure k, bond k, et al. quality management systems for laboratory leadership: a novel international approach. poster presented at aabb annual meeting, 06 – 09 october 2012 boston, ma. 22.kariuki njenga m, traicoff d, tetteh c, et al. laboratory epidemiologist: skilled partner in field epidemiology and disease surveillance in kenya. j public health policy. 2008;29(2):149–164. pubmed pmid: 18523470. 23.nsubuga p, johnson k, tetteh c, et al. field epidemiology and laboratory training programs in sub-saharan africa from 2004 to 2010: need, the process, and prospects. pan afr med j. 2011;10:24. pubmed pmid: 22187606. pubmed central pmcid: 3224071. 24.yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3):401–409. pubmed pmid: 20716796. 25.yao k, luman e, slmta collaborating authors. evidence from 47 countries for slmta-driven improvement in laboratory quality management systems. afr j lab med. 2014. in press. 26.egger d, ollier e. managing the health millennium development goals – the challenge of management strengthening: lessons from three countries. making health systems work: working paper no. 8. who/hss/healthsystems/2007.1 [document on the internet]. c2007 [cited 2014 aug 11]. available from: http://www.who.int/management/working_paper_8_en_opt.pdf 27.maruta t, yao k, ndlovu n, et al. training-of-trainers: a strategy to build country capacity for slmta expansion and programme sustainability. afr j lab med. 2014. in press. 28.berte lm, boone dj, cooper g, et al. application of a quality management system model for laboratory services; approved guideline – third edition. clsi document gp26-a3 [isbn 1-56238-553-4]. vol 23 no 36. wayne, pa: clinical and laboratory standards institute; 2004. 29.world health organization regional office for africa. who guide for the stepwise laboratory improvement process towards accreditation in the african region (with checklist) [document on the internet]. c2011 [cited 2013 may 31]. available from: http://www.afro.who.int/index.php?option=com_docman&task=doc_download&gid=8642&itemid=2593 30.ndasi j, dimite l, mbome v, et al. decentralised, facility-based training as an alternative model for slmta implementation: the cameroon experience. afr j lab med. 2014. in press. 31.mothabeng d, maruta t, lebina m, et al. strengthening laboratory management towards accreditation: the lesotho experience. afr j lab med. 2012;1(1), art. #9, 7 pages. http://dx.doi.org/10.4102/ajlm.v1i1.9 32.maruta t, motebang d, wanyoike j, et al. impact of mentorship on who-afro strengthening laboratory quality improvement process towards accreditation (slipta). afr j lab med. 2012;1(1), art. #6, 8 pages. http://dx.doi.org/10.4102/ajlm.v1i1.6 33.masamha g, skaggs b, pinto i, et al. working towards a sustainable laboratory quality improvement programme: mozambique�s slmta story. afr j lab med. 2014. in press. 34.nzombe p, luman et, shumba e, et al. maximising mentorship: variations in laboratory mentorship models implemented in zimbabwe. afr j lab med. 2014. in press. 35.maruta t, rotz p, trevor p. setting up a structured laboratory mentoring programme. afr j lab med. 2013;2(1), art. #77, 7 pages. http://dx.doi.org/10.4102/ajlm.v2i1.77 36.makokha e, mwalili s, basiye f, et al. using institutional mentorship to roll out slmta in kenya. afr j lab med. 2014. in press. 37.nzabahimana i, sebasirimu s, gatabazi jb, et. al. innovative strategies for a successful slmta country program: the rwanda story. afr j lab med 2014. in press. 38.eno l, asong t, ngale e, et al. adapting the slmta programme for improved quality services in a regional hospital in cameroon. afr j lab med. 2014. in press. 39.simbi r. doing more with less: the zimbabwean story. paper presented at the first symposium on strengthening laboratory management toward accreditation (slmta) at the first international conference of the african society for laboratory medicine. cape town, south africa; 2012. 40. guevara g, gordon f, irving y, et al. the impact of slmta in improving laboratory quality systems in the caribbean region. afr j lab med. 2014. in press. 41.nguyen thi t, mckinney b, pierson a, et al. slipta e-tool improves laboratory audits in vietnam and cambodia. afr j lab med. 2014. in press. 42.umar n, litaker d, terris dd. toward more sustainable health care quality improvement in developing countries: the ‘little steps’ approach. qual manag health care. 2009 oct-dec;18(4):295-304. pubmed pmid: 19851237. 43.committee on the outcome and impact evaluation of global hiv/aids programs implemented under the lantos-hyde act of 2008; board on global health (bgh); board on children, youth, and families; institute of medicine. evaluation of pepfar. washington, dc: the national academies press; 2013. 44.mataranyika mn, beukes lk. view from the top: involvement of namibia�s health ministry in laboratory quality improvement and the building of a strong laboratory network. afr j lab med. 2014. in press. article information authors: bruce h. noden1,2 vincent nowaseb1,3 cornelia de waal-miller1 berta e. van der colf1 affiliations: 1department of health sciences, school of health and applied science, polytechnic of namibia, namibia 2department of entomology and plant pathology, oklahoma state university, united states 3national commission on research, science and technology, namibia correspondence to: bruce noden email: bruce.noden@okstate.edu postal address: 127 noble research center, oklahoma state university, ok 74078, united states dates: received: 10 sept. 2014 accepted: 30 june 2015 published: 20 aug. 2015 how to cite this article: noden bh, nowaseb v, de waal-miller c, van der colf be. profile, perceptions and future expectations of medical laboratory scientists in namibia. afr j lab med. 2015;4(1), art. #246, 7 pages. http://dx.doi.org/10.4102/ajlm.v4i1.246 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. profile, perceptions and future expectations of medical laboratory scientists in namibia in this original research... open access • abstract • introduction • research method and design    • materials and setting    • design and procedure    • analyses       • ethical considerations • results    • study population profile    • perceptions and experiences of the study population • future expectations and perceptions • discussion    • limitations • conclusions • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: public healthcare systems in sub-saharan africa are challenged by healthcare worker shortages, loss of trained staff and attrition to the private sector. studies have historically focused on medical doctors, nurses and pharmacists, with limited focus on medical laboratory scientists. objectives: this study addresses the professional perspectives and expectations of the first two classes of biomedical science students, who graduated from the polytechnic of namibia in 2012 and 2013. methods: a questionnaire was developed to capture qualitative and quantitative data from fourth-year students completing their final semester. data collected included: demographic information; students’ experience; professional expectations; and perceptions about the future of biomedical science education in namibia. results: amongst the 42 of 45 enrolled students who completed the questionnaire, nearly two-thirds anticipated working in government hospitals (29%) or industry (35%), with fewer planning careers in private hospitals (12%) or academia (14%). most expressed an interest in working abroad (64%) and/or in the capital (64%), with fewer interested in small urban areas (48%). only 7% expressed interest in working in a rural area. regarding their view of the future of biomedical science in namibia, 38% responded that it was encouraging, whereas the rest responded that it was uncertain (52%), negative (2%) or unknown (7%). conclusion: members of the first graduating classes of namibia’s nascent biomedical science degree programme reported a perceived lack of opportunity for professional advancement in the field if they remained in namibia. continued thought needs to be given to develop sustainable strategies and opportunities to retain namibian biomedical laboratory scientists in namibia. introduction top ↑ countries in sub-saharan africa are challenged by a lack of healthcare workers.1 on average, healthcare systems in african countries field less than 22.8 skilled healthcare workers per 10 000 population; far below the 59.4 per 10 000 population average documented in more developed countries.2 the already inadequate numbers of healthcare workers being trained are often further reduced by ’out‘-migration to other countries; it has been reported that 8000 nurses leave sub-saharan africa every year.3 this migration, commonly referred to as the “brain drain”, presents a difficult challenge for developing countries already facing substantial barriers to provide a minimal standard of healthcare services.4,5,6 for many healthcare workers, migration to affluent countries is driven by inadequate salaries, lack of training opportunities, limited chances for promotion, safety concerns and poor living conditions at home.4,5,7 it is also driven by healthcare worker shortages in developed countries.1 most evaluations of the “brain drain” phenomenon have focused on medical doctors,8,9,10 nurses5 and pharmacists.11 despite the important role of medical laboratory scientists in the medical diagnostic field, there is almost nothing in the current literature about the impact of this cadre’s out-migration on the health systems in developing countries.12 medical laboratory scientists perform the tests, generate the preliminary reports and, together with other qualified laboratory professionals, monitor the quality of clinical laboratories. altogether, this teamwork culminates in generating reliable results so that evidence-based diagnoses and effective patient care are delivered. the persistent shortage of medical professionals, including medical laboratory scientists, is well reported in countries such as the united kingdom and united states.13,14,15 there is, therefore, an increasing possibility that the “brain drain” phenomenon documented amongst doctors and nurses could also be extended to young african laboratory technicians and technologists and medical laboratory scientists. the resulting loss of these professionals would not only undercut the quality of healthcare delivery in africa, but will also drain the few resources available to the nascent laboratory science training programmes on the continent and may, eventually, cause them to be eliminated.16 namibia has historically relied on south african universities to train its medical personnel. the country also has a long history of reliance on foreign healthcare workers in all major health-related cadres (doctors, nurses, pharmacists and medical laboratory scientists). starting in 2008, the university of namibia (unam) has offered degree programmes in medicine and pharmacy. in the same year, the polytechnic of namibia (pon) introduced degree programmes in biomedical science and environmental health. the biomedical science programme at the pon was hosted by the school of health and applied sciences. prior to 2008, the technical staff of public and private laboratories comprised either foreigners or namibians who left the country to complete a three-year diploma in south african institutions. in 2005, the pon was approached by local stakeholders, as well as the ministry of health and social services, to create a training programme in-country. an advisory committee developed a curriculum for a four-year professional degree programme, which would allow graduates to move into a master’s programme and, ultimately, a doctorate in laboratory sciences. the four-year professional bachelor’s degree incorporated clinical chemistry, haematology, microbiology and molecular diagnostics with other important subjects, as well as a research project. after four years at the pon, the students complete a one-year internship in industry prior to registering as medical laboratory scientists. this programme, which partnered with cape peninsula university of technology (cput) in cape town, south africa, was the first such programme in southern africa. the curriculum and course were approved in 2007 and the first intake of students occurred in january 2008. as the programme developed, external assistance was provided by a president’s emergency plan for aids relief (pepfar) development grant through the local centers for disease control and prevention office and a twinning arrangement with university of arkansas for medical sciences. the new programme began with three faculty members teaching the first cohort of students. the programme grew each year, adding new faculty members, most recruited from industry. through time, the training laboratories were equipped for practical modules. the ministry of education offered study loans to students and the ministry of health and social services offered several bursaries. whilst starting off with excitement, there were considerable challenges in the first years of the programme. finding teaching venues in a dynamically-changing educational institution, in addition to challenges in finding faculty members, meant that practicals were not presented concurrently with theory sessions and international instructors were brought in to provide intense short practical modules. whilst all of these have now been addressed since the programme moved into its own building in 2014, the first two cohorts of students worked through the challenges of the early years. the pon graduated its first class of medical laboratory scientists in 2012. medical education is an important national investment, but the returns obtained are not always what are hoped for, or even expected.5,7 in namibia, where a trained, dedicated cadre of non-expatriate healthcare workers is desperately needed, it is important for nascent healthcare professional training degree programmes to understand what students expect from the education programme in which they have enrolled; and, more importantly, what they expect from their home county’s healthcare system in terms of career opportunities, remuneration and professional advancement after graduation. studies focused on the students, particularly in the early years of these valuable training programmes, provide namibian educators and policy makers with valuable information about how national investments in professional healthcare education may (or may not) benefit the national healthcare system.5,8,9,10,11 as an initial component of this research, the objective of this study was to describe and analyse the profile of the first two classes, which graduated in 2012 and 2013, in regard to personal and educational backgrounds, experiences whilst at the pon and future career expectations. research method and design top ↑ materials and setting consecutive fourth-year, final-semester students (study size: n = 22, 2011; n = 23, 2012) were enlisted from the first two classes graduating from the pon biomedical science programme in 2012 and 2013. only fourth-year students in the programme attending a tutorial during the preparation of their honours thesis research projects were provided an opportunity to complete the questionnaire. those who did not attend the tutorial (n = 3) were not given another opportunity. design and procedure a questionnaire was adapted from modipa and kambisya11 and nguyen et al.5 prior to filling out the questionnaire, the research team went through the questions with the students to ensure understanding. all students were provided as much time as necessary for full analysis of each question and a moderator was on hand to answer any questions. the questionnaire contained closedand open-ended questions concerning: the demographics and backgrounds of the students; their experiences and perceptions during the four years of training; and their expectations and final perceptions of the programme. analyses all data were entered into a microsoft excel™ spreadsheet and analysed using ibm spss statistics for windows, version 21.0 (ibm corp., armonk, ny 2012). for categorical data, pearson’s χ2 tests or fisher’s exact tests were used. binary logistic regression analysis was used to assess associations between population characteristics and desire to work abroad. p-values of less than 0.05 were considered to be statistically significant. ethical considerations the study and questionnaire were approved by the institutional research and publications committee of the pon, the ethical committee of the university, as per policy [irpc-poly/2011/7377/539]. the respondents voluntarily completed the questionnaire and all answers were recorded anonymously. completed questionnaires were securely organised and stored. results top ↑ study population profile of the 45 students enrolled in the programme, all 42 students who were present in the tutorial completed the questionnaire. three international students (angola, n = 1; botswana, n = 2) were not present the day the questionnaire was given, because they were still completing their in-service training commitments. amongst those who responded, the majority were either women (n = 32; 76%) or were between the ages of 21 and 25 years (n = 41; 98%) (table 1). two respondents (5%) were married. almost half of the namibian students were from northern regions (n = 20; 47%) and a third (n = 14; 33%) from central regions. the majority of the students had lived in an urban area before the age of 17 (n = 30; 71%), but most had rural experience (n = 27; 64%). in addition, almost two-thirds had attended government schools in urban areas (n = 27; 64%) and six (14%) had private education. the proportions of students with fathers (n = 14; 40%) or mothers (n = 12; 31%) with a tertiary educational experience were similar. table 1: characteristics of fourth-year students in the biomedical science programme at the polytechnic of namibia in 2011 and 2012. perceptions and experiences of the study population slightly more than half of the students learned of the programme through the media (newspaper or internet) or personal contacts (table 2). the choice to apply for the programme was predominantly self(n = 19; 42%) or family-influenced (n = 18; 40%). just over half (n = 24; 59%) chose biomedical science as their first choice. other students were interested in other medically-related programmes (24%), including medicine, forensics, pharmacy, dentistry and psychology, which were not yet offered in the country when the programme was initiated, or engineering (12%) and environmental health (5%), both of which were already being offered at the pon (data not included in table 2). table 2: perceptions and experiences of fourth-year students in the biomedical science programme at the polytechnic of namibia in 2011 and 2012. when asked why they chose biomedical science, the majority (n = 31; 78%) responded that it was because it was medically-oriented: ‘always wanted to do something medically related and stay in namibia and didn’t want pre-med at unam.’ (female, 22) ‘interested in medical field but didn’t want contact with ­patients.’ (female, 22) another six (15%) chose the programme because it sounded interesting: ‘i got amazed by the name. i never knew what it really was. went to try my luck at the pre-selection and just got in.’ ­(female, 23) ‘[g]ood in biology so pursued related courses. being a scientist sounded good.’ (male, 21) three of the students (8%) chose the programme because they wanted to help others: ‘[h]elp patients in order to save lives.’ (female, 25) ‘thought i would be more helpful and productive to work in a background career instead of an upfront one.’ (female, 22) with regard to living situations and financing of education, most students (n = 22; 49%) lived with family during their four years of study, whilst others rented accommodations off-campus (n = 18; 38%) or used the university housing (n = 7; 15%). forty per cent (n = 21) financed their education through student loans, whilst others were covered through bursaries (n = 15; 29%) or family support (n = 13; 25%). the studies of one student were financed through an agreement between unam and the pon, whilst another was financed through a trust fund. the main difficulties experienced were financial, with related mentions of transport (taking taxis to and from place of residence), supplies (textbooks, computers) and housing. future expectations and perceptions top ↑ most students indicated that they anticipated working in a government hospital setting (n = 15; 29%) or industry (n = 18; 35%) after completion of their training (table 3). a smaller group anticipated working in private hospitals n = 6; 12%) or in academia (n = 7; 14%). one student was focused on working in a forensic laboratory. when asked why they chose their particular settings for anticipated employment, responses could be summarised into four main areas. the first area centred on exposure to new things or new diseases, as can be seen from the following responses: table 3: future expectations and perceptions of fourth-year students in the biomedical science programme at the polytechnic of namibia in 2011 and 2012. ‘[e]xposure to all types of diseases as government [clinics] deal with patients with all backgrounds.’ (male, 24) ‘[e]xposure to wider range of more severe illnesses more common in low-income populations.’ (female, 23) the second area was focused on financial reasons or bursary repayment. the third area centred on the desire to improve one’s qualifications or personal growth and development, as shown by the following responses: ‘[i] believe these are the areas where my skills and qualifications will be utilised best and will also allow me to grow in my profession.’ (female, 22) ‘i have already worked in several med labs so for me to go into a different environment would be interesting and build my knowledge.’ (female, 23) finally, the fourth area was focused on personal professional choice (n = 2), such as a desire to improve the system: ‘so i can provide the best service possible which is not always done at government hospitals.’ (female, 22) ‘[t]o improve industry.’ (male, 22); personal choices (n = 3): ‘i don’t like hospitals and academia is not my field either.’ (female, 22) ‘i love to test water, to make sure it is sterile as this is the ­biggest need in all living beings.’ (male, 22) ‘i want to be a lecturer or training officer at pon one day so that i can train students very well and hopefully to [sic] make biomed the best course at pon.’ (female, 23); or altruism (n = 1): ‘[i] would like to help underprivileged people in society.’ (male, 25) with respect to location of practice, 55% of respondents provided more than one answer. sixty-four per cent (n = 27) wanted to work in the capital (windhoek), 48% (n = 20) in a small urban area 64% (n = 27); wanted to work abroad and 7% (n = 3) wanted to work in a rural area (table 3). of those considering working abroad, 48% (n = 13) preferred europe, 48% (n = 13) north america, 30% (n = 8) were considering working in another african country and 7% (n = 2) wanted to work in australia or new zealand. reasons given for wanting to work abroad included: (1) experiencing new ways of doing things; (2) exposure to new techniques; and (3) attraction to research possibilities not currently available in namibia. equal proportions of male (60%) and female (66%) students desired to work abroad. although not statistically significant, fewer students who started the programme in 2008 (52%) desired to work abroad compared with 2009 (76%). there were no regional differences amongst those desiring to work abroad. once more, though not statistically significant, more students with fathers (70%) or mothers (69%) with secondaryor tertiary-level education desired to work abroad compared with fathers (44%; p < 0.242) or mothers (43%; p < 0.225) with primary or unknown education (data not included in table 3). when asked whether they would have chosen to study biomedical science again, 24% (n = 10) answered in the affirmative; only half said they would recommend the programme to someone else (table 3). concerning their perception of the future of biomedical science in namibia, 38% (n = 16) responded that that it was bright and encouraging, whereas the rest were uncertain (n = 22; 52%), negative (n = 1; 2%) or unknown (n = 3; 7%). reasons given for being positive included: ‘[b]ecause new learning methods and training techniques are continuously developing.’ (male, 27) ‘we are needed, nip [namibia institute of pathology] is accredited. things [are] looking better. quality management is improving in labs.’ (female, 22) ‘[w]e are pioneers, so we can make changes as they need to be made.’ (female, 22) ‘i have faith in [the] country’s potential to develop.’ (female, 23) ‘[m]ore students in this field will make a difference.’ ­(female, 23) ‘[m]ore technologists = more development.’ (female, 24) most of those who responded with uncertainty focused on the lack of development in the industry and lack of opportunities arising from saturation of the market. of the 22 uncertain respondents, nine mentioned the limited job market and three mentioned a lack of alternatives for personal growth in the industry: ‘no means of profession ladder, small market, foreigners saturate the market, not many alternatives.’ (female, 23) ‘slow pace in namibia and there is very little interest by current professionals to develop the profession.’ (female, 22) ‘[the] current situation in the lab does not provide a very bright future for a young developing biomed scientists, but change is possible.’ (female, 22) one even mentioned a fear that positions would be replaced by automation in the near future. those who were ’unknown‘ in their responses cited lack of experience: ‘i can’t predict the future.’ (female, 23) ‘[n]o comment, as [i] am not in the industry yet.’ (female, 22) when asked what they anticipated doing in the future, the majority (n = 29; 58%) wished to further their education, whilst 28% (n = 14) wanted to explore other career fields and 14% (n = 7) planned to continue working in the laboratory. discussion top ↑ this study is the first of its kind to assess the characteristics, experiences and perceptions of medical laboratory science students in the biomedical science training programme in namibia. the implementation of a new health-related training programme in biomedical science in 2008 was unique for namibia as well the region. as such, a number of foreign students, albeit a small percentage, as well as students from all over namibia, were attracted to the programme. this novelty provided a diverse environment in which students from different cultures and socio-economic levels could interact professionally and support one another in their development. the characteristics of the first cohort of students were similar to other studies of medically-oriented students in other african countries.8,9,10,17,18,19 in a country where less than 1% of the adult population has a tertiary degree, one interesting component was the relatively high proportion of students whose parents had a tertiary degree (between 31% and 40%). whilst nguyen et al.5 found an association between desire to emigrate and mothers having a tertiary education, our study did not replicate this finding. this, possibly, speaks not only to their desire to work in a medically-focused profession but it could also be related to the pioneer spirit that these first two cohorts had as they worked through the challenges of a developing programme. having parents with tertiary degrees may have provided a stabilising factor which kept them pursuing their degrees. the results from the survey also highlighted some of the important challenges faced by students in those initial years. in fact, it was quite striking that 76% of the respondents indicated that they would not have chosen to study biomedical science again. there are a number of possible reasons for this result, some of which could be gleaned from their responses. first of all, biomedical science was not the first choice for over 40% of the respondents. respondents seemed to be attracted to medically-oriented careers but, for almost 25%, only after they were not able to get into other health-related programmes. they may have settled, instead, for a course that they did not know much about and were now looking at a career in something they were not sure about after having invested four years. secondly, financial difficulties, which were cited by almost one-third of the respondents, could have affected responses whilst reflecting on the questions. whilst some explicitly cited ’financial difficulties‘, the influence of finances was observed in the other challenges mentioned, such as transport, supplies and housing. for example, transport was critical, because 38% of the respondents lived off-campus and needed to take a taxi (us $2–3/day) to get to and from campus. the cost incurred by that alone meant that some did not have funds for a midday meal but had to wait to eat until they returned home in the evening. the housing conditions in which many students lived were also difficult. those living with relatives often lived in less-than-ideal situations, because the family members were distantly related or the students were an added burden to an already challenging family situation. this meant the students had to move often between supporting family members or put up with basic conditions that lacked running water or electricity. the challenges away from the campus often meant that classes were missed or stressful conditions were endured in order to achieve this degree. finally, the two-year period (2012–2013) during which the respondents were finishing the programme was filled with uncertainty as the namibian professional stakeholders, which first supported the programme, began to realise the implications of 23–24 graduates per year entering the workforce. this produced public questions concerning whether there would be enough positions for graduates. it is notable that shortly after these two groups of students graduated, the programme’s intake size was reduced from 30 to 15 to accommodate these industry concerns. taken together, it is still intriguing that 50% of the respondents would encourage someone else to study biomedical science at the pon. this may indicate that, whilst the programme may not have been what they had hoped for, they saw qualities that would benefit others. this aspect needs to be developed further in future studies focused on the alumni of the programme. in addition to the challenges faced by students, this study provides insights into the motivations and future expectations of this valuable cadre of health professionals and provides data suggesting the necessity of keeping them actively engaged in their home country. of the 42 students surveyed, 64% expressed interest in going abroad, even for a while, if given the chance. the main theme that resonated throughout their responses was that of perceived opportunity for advancement, either in experience or training. with respect to the preferred work location, respondents had a strong desire to work in urban areas or abroad. the reasons given were motivated primarily by a strong desire for opportunity and access to self-development. at that point in their training, the majority of respondents felt that the future of their profession was uncertain, mainly because they could only see job saturation in the near future as the industry is not growing fast enough to meet their needs. this desire for self-development opportunities resounds across sub-saharan africa as countries grapple with the out-movement of trained medical professionals to more affluent, opportunity-providing countries. according to our results, the responses of these namibian students to emigrate to more innovative environments in which to learn new technologies is similar to those of other african medical students,8,9,10,17,18,19 nurses,5,20 pharmacy students11 and medical laboratory professionals.6,16,21 this study provides a glimpse into yet another cadre of medical professionals in africa who are looking for ways to better themselves and move into positions where they can earn and achieve more from their training.5 the fact that a majority chose the programme themselves meant they saw potential to be satisfied in this profession. to keep them in it, however, careful consideration and innovative thinking is needed, if namibia wants to achieve its stated health development goals in the next 10 years.20,22 it was not surprising to find that only 7% of respondents were willing to work in rural areas. this is most likely because the majority grew up and attended schools in urban areas. less than 30% lived in rural areas and attended rural high schools before the age of 17. similar studies report that experience in urban areas creates a natural desire to stay in those areas.5 the same applies for rural experience and/or rural placement.11,23 often, this motivation is fuelled by more than just the higher earning potential.11 in the case of these students, the majority had lived and attended school in urban areas and, as a result, desired to stay connected, bringing in the theme of opportunity and future plans once again. the fact that most covered their education via loans means they have obligations to repay those debts. bursary arrangements often mean there is an expectation to pay back the sponsor by working for the sponsoring company for a certain number of years. those whose parents supplemented their tuition and living expenses may also have some kind of payback expectations, which keeps the student closer to the urban context where such opportunities are available. the equal desire of these namibian students to work either in europe or north america was notable. studies have reported that students often emigrate to their colonising countries because of shared national language and education systems.4 germany and south africa both have important roles in the historical development of namibia with both german and afrikaans being spoken by large portions of the population. now that namibia has developed medical training facilities in a number of disciplines, it will be important to monitor student emigration patterns.24 limitations one of the limitations of the study was the sample size. students in other years could have been included in the study group but the designers of the study felt it was important to capture the responses of the first two years of pioneering students in order to identify ways to improve the program. this served as a form of assessment, not only for the programme but also for the industry as a whole, as these students represented a cohort of highly-qualified namibians who did not leave the country to study abroad but rather persevered through challenges and difficulties. another limitation is the use of a cross-sectional approach to capture the perceptions and intentions of these students. as a questionnaire only monitors a student’s opinion on a particular day, it can only be interpreted as an idea of how they perceive their future at that point in time. conclusions top ↑ as the first study to assess the characteristics, experiences and perceptions of students in the nascent medical training programmes in namibia, these results provide a glimpse into yet another cadre of medical professionals in africa who are looking for ways to better themselves and move into positions where they can earn and achieve more from their training. the diversity of their backgrounds, challenges faced as they pursued training in a developing programme and concerns for the future have common threads with other health-related cadres in developing countries. the desire for meaningful engagement and opportunity and the uncertain view of how their training will integrate with the national programme means that merely having new medical training programmes in namibia is not enough. continued thought needs to be given by the stakeholders and government entities that originally envisioned the programmes in order to develop strategies and opportunities which will keep these professionals meaningfully engaged in the country. acknowledgements top ↑ the researchers would like to thank mr john pitman for editorial assistance. b.h.n. was partially supported by the oklahoma agricultural experiment station (okl-02909). competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions b.h.n. (polytechnic of namibia; oklahoma state university) conceptualised the study; developed the original proposal; obtained the ethical permissions for the study; developed the questionnaire and administered it; input the data; completed the analysis; and wrote the initial draft of the manuscript. c.dw.-m. (polytechnic of namibia), b.e.vdc. (polytechnic of namibia) and v.n. (polytechnic of namibia; national commission on research, science and technology) developed and administered the questionnaire. all authors modified and approved the final manuscript. references top ↑ hagopian a, thompson mj, fordyce m, et al. the migration of physicians from sub-saharan africa to the united states of america: measures of the african brain drain. hum resour health. 2004;2:17. http://dx.doi.org/10.1186/1478-4491-2-17 campbell j, dussault g, buchan j, et al. a universal truth: no health without a workforce. third global forum on human resources for health report [document on the internet]. c2013 [cited 2015 july 15]. global health workforce alliance and world health organization. available from: http://www.who.int/workforcealliance/knowledge/resources/hrhreport2013/en/ lazarus jv, wallace sa, liljestrand j. improving african health research capacity. scand j public health. 2010; 38(6):670–671. http://dx.doi.org/10.1177/1403494810372265 connell j, zurn p, stilwell b, et al. sub-saharan africa: beyond the health worker migration crisis? soc sci med. 2007;64(9):1876–1891. http://dx.doi.org/10.1016/j.socscimed.2006.12.013 nguyen l, ropers s, nderitu e, et al. intent to migrate among nursing students in uganda: measures of the brain drain in the next generation of health professionals. hum resour health. 2008;6:5. http://dx.doi.org/10.1186/1478-4491-6-5 sherr k, mussa a, chilundo b, et al. brain drain and health workforce distortions in mozambique. plos one. 2012;7:e35840. http://dx.doi.org/10.1371/journal.pone.0035840 saravia ng, miranda jf. plumbing the brain drain. bull world health organ. 2004;82(8):608–615. dambisya ym. career intentions of unitra medical students and their perceptions about the future. educ health. 2003;16(3):286–297. http://dx.doi.org/10.1080/13576280310001607442 ferrinho p, fronteira i, sidat m, et al. profile and professional expectations of medical students in mozambique: a longitudinal study. hum resour health. 2010;8:21. http://dx.doi.org/10.1186/1478-4491-8-21 ferrinho p, sidat m, fresta mj, et al. the training and professional expectations of medical students in angola, guinea-bissau and mozambique. hum resourc health. 2011;9:9. http://dx.doi.org/10.1186/1478-4491-9-9 modipa si, dambisya ym. profile and career preferences of pharmacy students at the university of limpopo, turfloop campus, south africa. educ health. 2008;21(3):164. reuter ml. the future of the medical technologist. lab med. 2000;31(11):598–599. http://dx.doi.org/10.1309/3cgx-c4qy-nln1-6a17 davis k. responding to the medical laboratory staffing shortage: the canadian perspective. clin leadersh manag rev. 2002;16(6):399–407. ward-cook k. medical laboratory workforce trends and projections: what is past is prologue. clin leadersh manag rev. 2002;16(6):364–369. beck s, doig, k. laboratory managers’ views on attrition and retention of laboratory personnel. clin lab sci. 2005;18(4):238–247. marinucci f, majigo m, wattleworth m, et al. factors affecting job satisfaction and retention of medical laboratory professionals in seven countries of sub-saharan africa. hum resour health. 2013;11:38. http://dx.doi.org/10.1186/1478-4491-11-38 fronteira i, rodrigues a, pereira c, et al. realities and professional expectations of medical students attending guinea bissau’s medical school in 2007 school year. acta med port. 2011;24(2):265–270. bailey n, mandeville kl, rhodes t, et al. postgraduate career intentions of medical students and recent graduates in malawi: a qualitative interview study. bmc med educ. 2012;12:87. http://dx.doi.org/10.1186/1472-6920-12-87 mandeville kl, bartley t, mipando m. future career plans of malawian medical students: a cross-sectional survey. hum resour health. 2012;10:29. http://dx.doi.org/10.1186/1478-4491-10-29 mutale w, ayles h, bond v, et al. measuring health workers’ motivation in rural health facilities: baseline results from three study districts in zambia. hum resour health. 2013;11:8. http://dx.doi.org/10.1186/1478-4491-11-8 mcclure k. student perceptions of the clinical laboratory science profession. clin lab sci. 2009;22(1):16–21. witt j. addressing the migration of health professionals: the role of working conditions and educational placements. bmc public health. 2009;9(suppl 1):s7.http://dx.doi.org/10.1186/1471-2458-9-s1-s7 kaye dk, mwanika a, sekimpi p, et al. perceptions of newly admitted undergraduate medical students on experiential training on community placements and working in rural areas of uganda. bmc med educ. 2010;10:47. http://dx.doi.org/10.1186/1472-6920-10-47 dambisya ym. the fate and career destinations of doctors who qualified at uganda’s makerere medical school in 1984: retrospective cohort study. bmj. 2004;329(7466):600–601. http://dx.doi.org/10.1136/bmj.38134.524387.ae article information authors: tjeerd a.m. datema1 linda oskam1 paul r. klatser1,2,3 affiliations: 1kit biomedical research, royal tropical institute, amsterdam, the netherlands2athena institute, vu university amsterdam, amsterdam, the netherlands 3amsterdam institute for global health and development, academic medical centre of the university of amsterdam, amsterdam, the netherlands correspondence to: linda oskam postal address: meibergdreef 39, 1105 az amsterdam, the netherlands dates: received: 26 may 2011 accepted: 11 nov. 2011 published: 13 dec. 2011 how to cite this article: datema tam, oskam l, klatser pr. review and comparison of quality standards, guidelines and regulations for laboratories. afr j lab med. 2011;1(1), art. #3, 7 pages. http://dx.doi.org/10.4102/ ajlm.v1i1.3 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. review and comparison of quality standards, guidelines and regulations for laboratories in this original research... open access • abstract • introduction • methodology    • selection of laboratory quality documents    • analysis of quality documents • results    • clinical quality documents       • iso 15189       • clsi gp26       • the clinical laboratory improvement amendments       • jci clinical laboratory standard    • non-clinical quality documents • discussion    • clinical laboratory quality documents    • non-clinical laboratory quality documents    • national versus international laboratory quality documents    • study limitations    • recommendations • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ background: the variety and number of laboratory quality standards, guidelines and regulations (hereafter: quality documents) makes it difficult to choose the most suitable one for establishing and maintaining a laboratory quality management system. objectives: there is a need to compare the characteristics, suitability and applicability of quality documents in view of the increasing efforts to introduce quality management in laboratories, especially in clinical diagnostic laboratories in low income and middle income countries. this may provide valuable insights for policy makers developing national laboratory policies, and for laboratory managers and quality officers in choosing the most appropriate quality document for upgrading their laboratories. method: we reviewed the history of quality document development and then selected a subset based on their current use. we analysed these documents following a framework for comparison of quality documents that was adapted from the clinical laboratory standards institute guideline gp26 quality management system model for clinical laboratory services. results: differences were identified between national and international, and non-clinical and clinical quality documents. the most salient findings were the absence of provisions on occurrence management and customer service in almost all non-clinical quality documents, a low number of safety requirements aimed at protecting laboratory personnel in international quality documents and no requirements regarding ethical behaviour in almost all quality documents. conclusion: each laboratory needs to investigate whether national regulatory standards are present. these are preferred as they most closely suit the needs of laboratories in the country. a laboratory should always use both a standard and a guideline: a standard sums up the requirements to a quality management system, a guideline describes how quality management can be integrated in the laboratory processes. introduction top ↑ after the development of the plan-do-check-act cycle by deming (based on the work of shewart) in the 1920s,1,2 the principles of quality management (qm) spread and became especially important in private industry. qm has many advantages, including protecting the product quality, customer safety and the company’s reputation. requirements for regulation of processes are recorded in documents called ‘standards’. national and international quality standards were developed by several organisations. simultaneously, governments forced the introduction of qm by means of laws and regulatory standards.in laboratory practice the necessity of qm also became apparent, although later than in industry. laboratory quality standards first focused only on testing laboratories and calibration laboratories, with the first international standard (iso guide 25) launched in 1978 by the international organization for standardization (iso). nowadays, many national and international quality standards for laboratory practice exist and quality laboratory practice has become the norm. in the case of full compliance with standard requirements a laboratory may be accredited: a formal recognition of competence and compliance to a quality standard. documents other than standards are important as well. quality standards are lists of requirements that need to be met in order to ensure quality practice. these standards do not contain explanations on how to implement the requirements or comply with them. in addition, standards are mostly not measurable. therefore, a laboratory needs guidelines describing how to implement the standard requirements and a checklist to visualise the degree to which it has met the standard. since 2008 the initiatives to strengthen laboratories in low and middle income countries (lmic) have evolved rapidly, especially in the clinical diagnostic laboratory sector.3 whilst efforts to increase health in lmic initially focused on improving treatment and care, the focus has now broadened to also improving laboratory diagnosis. stakeholders encourage countries to develop national standards, guidelines and regulations (hereafter called ‘quality documents’) to improve the quality of laboratory testing, contributing to meeting the millennium development goals (mdg) to achieve better health.4,5,6 in 2009 the world health organization (who) regional office for africa (who-afro) launched an accreditation checklist based on international quality documents5,7,8,9,10,11 and in 2005 the who regional office for south east asia (who-searo) recommended the expansion of the national accreditation scheme of thailand to member countries.2,12 the number of international laboratory quality documents is large and their scopes are diverse. as a result, deciding which document to use for implementing qm may be difficult: the authors have observed clinical laboratories implementing quality standards meant for non-clinical laboratories. this points to a lack of knowledge of decision makers regarding the existence of different types of quality documents for different types of laboratories. no review of widely used laboratory quality documents has been published to describe their characteristics and differences. we believe that there is a need for such information in the context of the rapidly increasing efforts to introduce qm in clinical laboratories. here we review documents that are widely used in qm implementation in laboratories, followed by an analysis of selected documents. the target audience of this study is the clinical laboratory sector. however, non-clinical laboratory quality documents were also included to provide perspective by showing their differences when compared with clinical laboratory quality documents (explaining why they should not be used by clinical laboratories). this analysis is intended for policy makers developing national laboratory quality policies, and for laboratory managers and staff with the ambition to implement a quality management system (qms). methodology top ↑ selection of laboratory quality documents laboratory qm development was reviewed using an unstructured internet search to identify quality documents that are internationally important and widely used. of these, seven quality documents were selected for further study – five international standards or guidelines and two national us regulations.iso 17025 – general requirements for test and calibration laboratories was included because it was the first internationally published standard (in 1978) on laboratory qm, originally named iso guide 25.13 this standard is presently widely used for testing and calibration laboratories. in 1979 in the usa, the food and drug administration (fda) enacted a national regulation called good laboratory practice for nonclinical laboratory studies (21cfr58) (hereafter called fda-glp).14,15 in 1981 the organization for economic cooperation and development (oecd) translated the fda-glp into international requirements for testing and calibration laboratories in its member states, titled principles on good laboratory practice (oecd-glp), consisting of a series of documents focusing on the different aspects of accreditation.16 the oecd-glp could be regarded as the second quality document to ever be developed specifically for international use. in 1988 a national regulation made expressly for clinical laboratories was enacted in the usa. this regulation is known as clinical laboratory improvement amendments (clia), coded 42cfr493.17 the clia is a national regulation, but the college of american pathologists (cap) uses it as the basis for its accreditation18 that is provided to clinical laboratories worldwide. therefore, the clia is also of international significance. iso 15189 – medical laboratories – particular requirements for quality and competence is the most widely used clinical laboratory standard. it was published for the first time in 2003.19 the version used in our analysis is from 2007. in 1999 the clinical and laboratory standards institute (clsi) developed gp26-a1, a guideline for establishing a qms in clinical laboratories. the development of the guideline started in 1997 by combining all the requirements of six quality documents (current at that time) into one document.20 in 2006 the iso 15189 requirements were incorporated into the third edition of this guideline (gp26-a3) titled: application of a quality management system model for laboratory services; approved guideline.21 in the spring of 2011 the fourth edition was published (gp26-a4). this guideline is used internationally to implement the requirements of iso 15189 in clinical laboratories. the joint commission international (jci) accreditation standard for clinical laboratories (second edition published in 2010) has a rather different background from the other quality standards included in this study. the jci provides accreditation to hospitals. as part of this accreditation, the jci developed the standard to include qm practices in hospital laboratories.22 because jci hospital accreditation is widespread, the significance of jci laboratory accreditation may also increase (table 1 provides an overview of the selected quality documents for analysis, including background information). table 1: overview of analysed quality documents with their most important characteristics. analysis of quality documents to guide the analysis, a framework was constructed consisting of three steps to analyse the selected quality documents. in step 1 the nature of the quality document was determined. we used the following definitions: • international standard: a consensus document developed for international use, summing up all the requirements for a qms. • international guideline: a more descriptive document than a standard, developed for international use, defining the intent of standard requirements and how these should be integrated in the laboratory processes. • national regulation: regulatory standard written by a national government. in step 2 a framework of 12 quality system essentials (qses) was adapted from the clsi guideline for implementing qm in clinical laboratories: gp26-a3.21 these 12 qses together cover all aspects of a qms (figure 1). when analysing the selected quality documents, their articles or sub-parts were allocated to one of the 12 qses to yield an indication of the level of coverage of total quality by each quality document (table 2). finally, in step 3 additional characteristics of each quality document were recorded. figure 1: the framework with the 12 quality system essentials used to analyse to which extent the content of quality documents covers all aspects of total quality management. table 2a: analysis of contents of national quality documents using the quality system essentials framework. table 2b: analysis of contents of international quality documents using the quality system essentials framework. results top ↑ the results of the analysis of the nature of the quality documents (analysis step 1) are provided (table 1). the fda-glp, developed for non-clinical research laboratories, and the clia, developed for clinical diagnostic laboratories, are both national regulatory standards. the quality documents developed for international use included the oecd-glp, iso 17025:2005, iso 15189:2007, clsi gp26-a3 and the jci clinical laboratory standard, 2nd edition. the oecd-glp and iso 17025 were developed for use in non-clinical laboratories and the iso 15189, clsi gp26 and jci clinical laboratory standard were developed for use in clinical laboratories.the results of determining the level of coverage of total quality by each quality document (analysis step 2) are provided (table 2). clinical quality documents in general, customers play an important role in clinical laboratory quality documents. whereas the non-clinical quality documents are generally written from the perspective of protecting the process and its product, the clinical quality documents are written to protect the customer from flawed results.the attention given to continuous improvement is much higher in clinical quality documents than in non-clinical quality documents. also, occurrence management (correct handling of nonconformities/accidents, followed by improvement measures to prevent similar occurrences in the future) receives much more attention in clinical quality documents than in non-clinical quality documents. iso 15189 of all clinical laboratory quality documents studied, iso 15189 is probably the most widely used standard worldwide. regional and international accreditation organisations such as the international laboratory accreditation cooperation (ilac), the interamerican accreditation cooperation (iaac), the asia pacific laboratory accreditation cooperation (aplac) and the european cooperation for accreditation (ea) recommend accreditation of clinical laboratories to iso 15189.23a notable characteristic of the iso 15189 standard is, besides the requirements to a qms, the inclusion of two annexes with recommendations: one contains all recommendations for a laboratory information system and another is completely dedicated to different aspects of ethics.24 clsi gp26 as a standard, iso 15189 is a document that only contains requirements for a qms, but no further explanation on why and how these requirements should be complied with. the clsi gp26 guideline contains much more information for laboratories on what qm is, how it is integrated in the laboratory work, and why certain activities should be performed. the clsi gp26 approaches every laboratory activity from a process workflow perspective. it contains a detailed description of this workflow by discussing each phase of the process using flow charts and process tables. the remainder of the document describes the requirements per qse, which are highly focused on continuous improvement (when compared to other clinical laboratory quality documents). the clinical laboratory improvement amendments the clia is a us federal document that applies to all clinical laboratory testing performed on humans except for clinical trials and fundamental research. this regulation is comprehensive and contains many discipline specific requirements (i.e. special requirements for histopathology, genetic testing, molecular techniques, etc.). remarkably, the nature of the clia is more similar to non-clinical quality documents as it is primarily focused on protecting the analysis process rather than customers and personnel. however, the clia, in contrast to most non-clinical standards, covers all 12 qses, which indicates that it is specifically designed for clinical laboratory practice. jci clinical laboratory standard the jci accreditation standard is slightly different from the aforementioned standards as it is a highly elaborate document that combines a standard with a guideline, describing the intent of each standard requirement and, uniquely, the measurable elements of each requirement. it is most elaborate on safety requirements, but it is the only clinical laboratory document which does not cover all 12 qses: information management is not covered as a topic in itself, although some requirements related to information management are present as part of different sections in the document. this standard is, similarly to the clia, highly elaborate on sub disciplines within laboratory practice providing quality assurance and quality control standards for each specific discipline. non-clinical quality documents the most salient findings are that the non-clinical quality documents are less customer focused and, instead, written from the perspective of protecting the process and its product.non-clinical quality documents are also less focused on continuous improvement; both the fda-glp and oecd-glp lack requirement on this qse. in addition, the fda-glp also lacks requirements on the purchasing and inventory element of the qms. another salient characteristic of most non-clinical quality documents (fda-glp, oecd-glp, and iso 17025) is the requirement to have a study plan or validated protocol in place besides standard operating procedures (sops). discussion top ↑ there are a number of quality documents specific for clinical laboratories and for non-clinical laboratories. we compared the characteristics, suitability and applicability of these quality documents in view of the increasing efforts to introduce qm in laboratories. clinical quality documents focus on both protecting the process, and the safety of the customer and the laboratory staff, whereas non-clinical documents aim primarily at protecting the safety and integrity of the analyses performed. moreover, non-clinical quality documents are generally not focused on continuous improvement, an aspect that could be regarded as one of the major goals of a qms. clinical laboratory quality documents the fact that iso 15189 includes a complete annex on ethics is exceptional when compared to all other quality documents. in clinical laboratory practice patient confidentiality and proper behaviour towards the patient are obvious ethical requirements. however, regulations regarding financial arrangements of the laboratory staff with external organisations or persons, or protection of the environment through correct waste-management are also ethical requirements. therefore, we recommend including a paragraph containing specific ethical norms with regard to laboratory practice in every quality document.it was observed that the clia is highly comprehensive when compared to other clinical quality documents. this may be related to the fact that the clia is adapted to a national situation (with laboratories in the usa generally having abundant resources to facilitate good quality practice), whilst iso 15189, as an international standard, has to maintain a certain level of generality to make it applicable in multiple countries that may have different standards of practice that are often determined by resource availability. the clia is more focused on protecting the process rather than the customers and personnel, making it different compared to other clinical quality documents. this may be a typical characteristic of a regulation that can have a narrower focus because other national regulations cover, for example, occupational safety (e.g. the usa 29 cfr part 1910 25): clinical laboratories in the usa following the clia need to comply with multiple other national regulations, whereas international standards and guidelines have to provide complete sets of requirements covering all aspects of laboratory practice. non-clinical laboratory quality documents most non-clinical laboratory quality documents are primarily process-focused. this may be the reason for the total absence of personnel safety requirements: the paragraphs of non-clinical quality documents shown in table 2 in the qse facilities and safety are all related to proper facilities and actions enabling safety of the process, not directed at the safety of personnel. this is illustrated by the requirement of the fda-glp in sub-part b, 58.29 (d): ‘personnel shall take necessary personal sanitation and health precautions designed to avoid contamination of test and control articles and test systems’.a characteristic found to be specific for non-clinical quality documents is the requirement to have a study plan or validated protocol in place besides sops. often, non-clinical laboratories perform research in addition to routine procedures. this research cannot be standardised in sops. notable, and probably related to the nature of test and calibration laboratories, is the low number of requirements aiming at the qse process improvement. in clinical laboratories, processes consist mainly of routinely performed procedures. continuous improvement is necessary to keep the performance of these procedures as efficient and effective as possible. in calibration laboratories, techniques should be performed with as little variance as possible, whereas variation in activities is inherent to the work of testing laboratories. incorporating continuous improvement is therefore complicated, if not impossible. qses that are left uncovered in all but one of the non-clinical quality documents are occurrence management and customer service. establishing procedures on customer service and occurrence management may be advisable in any environment. only iso 17025 contains requirements related to both qses, probably due to the incorporation of the highly customer-focused iso 9001 standard during its development.13, 26 national versus international laboratory quality documents in many countries, international quality documents serve as the basis for national quality documents. such adaption leads to documents that vary by country with regard to comprehensiveness. for example, the national guideline for clinical laboratories in the netherlands is more extensive than the iso 15189 standard.27 the same applies to the usa clia and fda-glp regulations. in contrast, the chinese standard is less extensive than the iso 15189, which makes it more feasible for laboratories in china to attain the standard.28 the required level of iso 15189 was considered to be too high in relation to the resources available, with the consequence that few laboratories in china tried to become accredited. by making the national standards easier to achieve, chinese laboratories were encouraged to implement qm leading to a substantial increase in accredited laboratories.28 on one hand some efforts on qm are better than no efforts at all, the concern however is the question in how far such simplified national standards can maintain adequate quality. this should be a topic of further research.nevertheless, it is recommended to use national quality regulations, if available, as they are often more detailed and optimally adapted to the national situation, and take into account the available resources. the problem is that national standards are currently almost exclusively available in high-income countries. in the last decade, several middle-income countries, for example thailand, mexico and argentina, have developed simplified national accreditation schemes based on international standards.29,30,31,32 recently, who-afro together with, among others, the usa centers for disease control and prevention (cdc) has launched an accreditation checklist based on clsi gp26 and iso 15189, which is tailor-made for implementation in clinical laboratories in lmic, and has started the roll-out in sub-saharan africa.7 a strong point is that the absence of national regulations is taken into account in this initiative: the who-afro accreditation checklist contains lots of questions regarding laboratory safety that would otherwise need to be covered by national regulations on occupational safety.11 iso 15189 only refers to national or regional regulations for safety requirements in such instances; these are absent in many countries. study limitations a potential weakness of this study is that the framework for analysis was adapted from a clinical laboratory quality document. this means that this framework was tailor-made for clinical laboratory quality documents and thus biased towards a clinical laboratory qms. although we therefore were able to correctly identify gaps in non-clinical quality documents compared to clinical quality documents, we were not able to identify gaps in clinical laboratory quality documents as compared to non-clinical quality documents. recommendations the type of laboratory and the resources available determine which document suits the practice of the laboratory best. national regulations are generally more detailed and tailor-made to the national laboratory system. international guidelines may be less detailed in order to remain applicable in multiple countries.laboratories planning to establish a qms need both a standard and a guideline. a standard provides no information on the reasons for implementation of the requirements, and standards are generally not measurable. the jci document illustrates the need for more than a standard: in this document the requirements (standard) are supplemented with a description of the intent of the requirements to prevent misinterpretation (guideline). in addition, measurable elements help the laboratory to determine whether each requirement is complied with. it is important that documents are chosen which suit the practice of a laboratory best. hence, clinical laboratories should choose a clinical laboratory standard, not the fda-glp, oecd-glp, iso 17025 or any other non-clinical laboratory document. several suggestions and recommendations remain to developers of quality documents. it was observed that non-clinical quality documents generally lack provisions on occurrence management, customer service and process improvement; we recommend including requirements on these aspects. ethics is also an element that applies to all types of organisations. increasing attention to ethical behaviour is highly recommended. acknowledgements top ↑ we would like to thank mirjam engelberts for useful suggestions. competing interests the authors declare that they have no financial or personal relationship(s), which may have inappropriately influenced them in writing this article. authors’ contributions t.a.m.d. (royal tropical institute) datema was involved in designing the study, analysing the documents and writing of the article. l.o. (royal tropical institute) was involved in designing and supervising the study and writing of the article. p.r.k. (royal tropical institute) was involved in designing the study and writing of the article. references top ↑ 1. the w. edwards deming institute [homepage on the internet]. c2011 [cited 2011 nov 11]. available from: http://deming.org/index.cfm?content=61 2. western electric history [homepage on the internet]. c2011 [cited 2011 nov 11]. available from: http://www.porticus.org/bell/westernelectric_history.html#western%20electric%20-%20a%20brief%20history 3. world health organization. joint who–cdc conference on health laboratory quality systems; 2008 apr 09–11; lyon, france. report no.: who/hse/ihr/lyo/2008.3. available from: http://www.who.int/ihr/lyon/report20080409.pdf 4. united nations general assembly. united nations millennium declaration; 2000 sept 08; new york city, usa. resolution no.: a/res/55/2. available from: http://www.un.org/millennium/declaration/ares552e.pdf 5. world health organization regional office for africa. the maputo declaration on strengthening of laboratory systems; 2008 jan 24; maputo, mozambique. available from: http://www.who.int/diagnostics_laboratory/maputo-declaration_2008.pdf 6. united nations. millennium development goals [homepage on the internet]. c2010 [cited 2011 nov 11]. available from: http://www.un.org/millenniumgoals 7. gershy-damet gm, rotz p, cross d, et al. the world health organization african region laboratory accreditation process: improving the quality of laboratory systems in the african region. am j clin pathol. 2010;134(3):393–400. http://dx.doi.org/10.1309/ajcptuuc2v1wjqbm, pmid:20716795 8. nkengasong jn, nsubuga p, nwanyanwu o, et al. laboratory systems and services are critical in global health: time to end the neglect? am j clin pathol. 2010;134(3):368–373. http://dx.doi.org/10.1309/ajcpmpsinq9brmu6, pmid:20716791 9. yao k, mckinney b, murphy a, et al. improving quality management systems of laboratories in developing countries: an innovative training approach to accelerate laboratory accreditation. am j clin pathol. 2010;134(3):401–409. http://dx.doi.org/10.1309/ajcpnbbl53fwuiqj, pmid:20716796 10. regional committee. strengthening public health laboratories in the who african region: a critical need for disease control; 2008 sept 02; yaounde, harmonization, implementation and improvement. trop med int health. cameroon. resolution no.: afr/rc58/r2. 11. datema tam, oskam l, van beers sm, klatser pk. critical review of the stepwise laboratory improvement process towards accreditation: suggestions for http://dx.doi.org/10.1111/j.1365-3156.2011.02917.x, pmid:22093245 12. kusum m, silva p. quality standards in health laboratories – implementation in thailand: a novel approach. world health organization regional office for south-east asia; 2005. report no.: sea-hlm-386 ed. 13. international organization for standardization. iso 17025 general requirements for test and calibration laboratories. geneva: iso; 2005. 14. food and drug administration. 21 cfr58 good laboratory practice for non-clinical laboratory studies. usa: food and drug administration; 1978. 15. food and drug administration. history of the fda [homepage on the internet]. c2011 [updated 2010 jul 29; cited 2011 nov 11]. available from: http://www.fda.gov/aboutfda/whatwedo/history/default.htm 16. organization for economic co-operation and development. principles on good laboratory practice. paris, france: organization for economic co-operation and development; 1998. 17. center for medicare and medicaid services (us). clinical laboratory improvement amendments [homepage on the internet]. c2011 [cited 2011 nov 11]. available from: http://www.access.gpo.gov/nara/cfr/waisidx_04/42cfr493_04.html 18. scully ta. continuance of the approval of the college of american pathologists as a clia accreditation organization. federal register, vol 66, no 177, 2001. 19. international organization for standardization. iso 15189 medical laboratories – particular requirements for quality and competence. geneva: iso; 2003. 20. berte lm. laboratory quality management: a roadmap. clin lab med. 2007;27(4):771–790. http://dx.doi.org/10.1016/j.cll.2007.07.008, pmid:17950897 21. clinical laboratory standards institute. application of a quality management system model for laboratory services, approved guideline gp26-a3. 3rd ed. wayne, pennsylvania: nccls; 2004. 22. joint commission international. accreditation standards for clinical laboratories. 2nd ed. oakbrook terrace illinois; joint commision international: 2010. 23. ilac – members (by category). international laboratory accreditation cooperation [homepage on the internet]. c2011 [cited 2011 nov 03]. available from: http://www.ilac.org/membersbycategory.html 24. international organization for standardization. iso 15189 medical laboratories – particular requirements for quality and competence. geneva: iso; 2007. 25. occupational safety and health administration. united states department of labor [homepage on the internet]. c2011 [cited 2011 nov 11]. available from: http://www.osha.gov/index.html 26. international organization for standardization. iso 9001 quality management systems – requirements. geneva: iso; 2008. 27. [foundation to promote the quality of the laboratory and the accreditation of laboratories in health care (cckl). fourth practical guideline for a quality system for a laboratory in the health care system]. 4th ed. bilthoven, the netherlands: cckl; 2005. dutch. 28. yang zh. the laboratory accreditation and regulations of clinical laboratory in china. clin biochem. 2009;42:310. http://dx.doi.org/10.1016/j.clinbiochem.2008.09.027, pmid:19863937 29. mazziotta d. accreditation of clinical laboratories in the latin-american region. clin biochem. 2009;42:309. http://dx.doi.org/10.1016/j.clinbiochem.2008.09.026, pmid:19863936 30. sierra-amor ri, lópez-martinez m. medical laboratory accreditation according to iso 15189:2003. the mexican experience. biochem med. 2007;17(2):1 88–192. 31. sierra-amor ri. mexican experience on laboratory accreditation according to iso 15189:2003. clin biochem. 2009;42:318. http://dx.doi.org/10.1016/j.clinbiochem.2008.09.095, pmid:19863944 32. wattanasri n, manoroma w, viriyayudhagorn s. laboratory accreditation in thailand: a systemic approach. am j clin pathol. 2010;134(4):534–540. http://dx.doi.org/10.1309/ajcpzyy19wmkmazt, pmid:20855633 hiv situation in senegal laboratory infrastructure in senegal quality assurance framework lessons learned and challenges quality assurance for poc acknowledgements references about the author(s) mouhamed a.s. mbengue laboratory of bacteriology and virology, cheikh anta diop university, chu aristide le dantec, dakar, senegal moussa sarr laboratory of bacteriology and virology, cheikh anta diop university, chu aristide le dantec, dakar, senegal westat, rockville, maryland, united states papa a. diaw laboratory of bacteriology and virology, cheikh anta diop university, chu aristide le dantec, dakar, senegal papa a.n. diall national committee for the control of aids, dakar, senegal maimouna d. toure laboratory of bacteriology and virology, cheikh anta diop university, chu aristide le dantec, dakar, senegal ndeye f.f.n. faye division for the control of aids and stds, ministry of health, dakar, senegal bousso gueye laboratory of bacteriology and virology, cheikh anta diop university, chu aristide le dantec, dakar, senegal ndeye c.t. kane laboratory of bacteriology and virology, cheikh anta diop university, chu aristide le dantec, dakar, senegal souleymane mboup laboratory of bacteriology and virology, cheikh anta diop university, chu aristide le dantec, dakar, senegal citation mbengue mas, sarr m, diaw pa, et al. establishing a national laboratory quality system for hiv diagnosis and monitoring in resource-limited settings: experience from senegal. afr j lab med. 2016;5(2), a440. http://dx.doi.org/10.4102/ajlm.v5i2.440 country profile establishing a national laboratory quality system for hiv diagnosis and monitoring in resource-limited settings: experience from senegal mouhamed a.s. mbengue, moussa sarr, papa a. diaw, papa a.n. diall, maimouna d. toure, ndeye f.f.n. faye, bousso gueye, ndeye c.t. kane, souleymane mboup received: 19 mar. 2016; accepted: 12 aug. 2016; published: 17 oct. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. hiv situation in senegal senegal is a western african country, with a population of around 14 million people and 14 administrative regions. based on the 2013 census, the total population of the country is estimated at 14 799 859 inhabitants, with a median population age of 18 years old.1 senegal, has a broad-based, pyramid-shaped age structure, which is characteristic of a population with a very high proportion of children and young people (table 1). in 2013, the crude birth rate was 37.2 per thousand, with a sex ratio at birth of around 105 men per 100 women. additionally, approximately 43% of the population is under 15 years of age, with an infant mortality rate of 47 deaths per 1000 births, and an overall risk of dying between birth and five years estimated at 72 deaths per 1000. the life expectancy at birth was 64 years in 2013.1 table 1: key hiv and health statistics in senegal. senegal has a concentrated hiv epidemic with high prevalence rates in most-at-risk groups, including commercial sex workers, but low prevalence rates in the general population in most regions (figure 1). based on the 2010–2011 senegal demographic and health survey, the global prevalence of hiv amongst women and men aged 15–49 years is 0.7%.2 more women are infected with hiv than men; the sex ratio is typically around 60 men per 100 women infected. however, the hiv seroprevalence is higher among vulnerable populations, with rates at 18.5% among sex workers, 17.8% among men having sex with men, and 9.4% among injection drug users.3,4 senegal has adopted the world health organization–joint united nations programme on hiv/aids recommended 90-90-90 targets.5 the adoption of this strategy means that the country is expected, by 2020, to have 90% of its population living with hiv diagnosed, 90% of all those diagnosed receiving sustained hiv treatment, and 90% of those receiving antiretroviral therapy having suppressed viral load measures.5 to achieve these outcomes, having good clinical laboratory services for diagnosis and follow-up will be critical.6 more specifically, investments will be needed to improve laboratory infrastructure, and to facilitate the access and availability of routine viral load and early infant diagnosis (eid) measures through the implementation of point-of-care (poc) diagnostic platforms along with an efficient and sustainable quality assurance programme. figure 1: hiv prevalence rate per region in senegal. laboratory infrastructure in senegal diagnostic laboratories in senegal operate within a three-tiered laboratory system: (1) district and peripheral health centre level laboratories; (2) regional level laboratories; and (3) central and hospital level laboratories (table 2). the organisation of the laboratory system is part of the overall health system structure and is placed under the leadership of the ministry of health. there is a bureau of laboratories within the ministry of health that is responsible for the implementation of policies defined by the government concerning the functioning and organisation of clinical laboratories. this office also promotes good clinical laboratory practice, not only for public medical laboratories, but also for private laboratories within the country. table 2: existing technologies or platforms hiv diagnosis, cd4 count, and viral load monitoring at each level of the tiered health system in senegal. the laboratory of bacteriology and virology (lbv) at cheikh anta diop university (cadu), which is located at le dantec hospital in the capital city, has been recognised by health authorities as the hiv national reference laboratory since 1986. in partnership with the ministry of health and the national aids programme, lbv/cadu is responsible for evaluating and validating diagnostic testing technologies, and technologies for cd4 count, viral load and eid. additionally, lbv/cadu ensures the supervision of other laboratories through a national external quality assessment (eqa) programme, through distribution of regular proficiency testing panels, collection of data for analysis, and follow-up and corrective actions in partnership with the ministry of health, the national committee for the control of aids and the division for the control of aids and sexually transmitted diseases. at the central and reference laboratory level, the hiv viral load, eid, cd4-count monitoring and hiv-diagnosis capabilities of lbv/cadu are described in table 2. at the regional and hospital laboratories level, these infrastructures are capable of performing hiv rapid diagnostic test confirmation, cd4-count measures and, at some of them, viral load and eid testing. however, the vast majority of the laboratory activities are conducted at the district level and peripheral health centre level. the capabilities for the district/peripheral health centre-level laboratories include: hiv rapid testing, cd4 count and collection of dried-blood specimens for eid. there are also few private laboratories operating at different levels of the health system. quality assurance framework quality assurance is the backbone of quality laboratory performance7 and proficiency testing is one of the major components of a quality assurance programme. in senegal, laboratory infrastructure is relatively well developed at the national and regional levels. however, issues such as inadequate infrastructure, shortage of qualified staff, lack of equipment, limited quality assurance and control procedures are frequently seen among district and peripheral health centre laboratories. the maintenance of a quality management system, including quality assurance, is crucial for having and providing good and reliable laboratory services.7,8,9 lbv/cadu has been working recently with the us centers for disease control and prevention to strengthen laboratory systems in africa and senegal though the implementation of quality management systems. the lbv/cadu-hosted afriqualab has been created for that purpose. afriqualab aims to improve laboratory quality in africa through regular organisation and distribution of proficiency testing panels across the african continent, including in francophone countries. afriqualab works in partnership with the us centers for disease control and prevention, westat and one world accuracy, a private canadian-based organisation specialising in eqa. during 2015, there were 174 participating laboratories from 28 countries, of which eight laboratories were from senegal. the proficiency testing organized by afriqualab is mostly free and focused on hiv-related proficiency testing panels, including hiv serology and flow cytometry. through technology transfer from the us centers for disease control and prevention, the programme is also offering free eqa for eid using dried-blood specimens and for hiv viral load using dried-tube specimens. currently, more than 60 laboratories are participating in this eqa sub-programme for eid using dried-blood specimens and for hiv viral load using dried-tube specimens in africa, including eight laboratories in senegal. additionally, afriqualab is also involved in an eqa programme focusing only on cd4 count technologies within senegal, with the support of the national aids programme and the public health agency of canada, an international programme for quality assessment and standardisation for immunological measures relevant to hiv. afriqualab also offers other panels, such as haematology, biochemistry, hepatitis b and c and mycobacteria, to participating labs using a fee-based service structure. lbv/cadu has made major improvements to its own quality management system and has recently achieved international organization for standardization 15189 accreditation of its medical laboratories. additionally, within senegal and at the national level, the ministry of health has recently released a national strategic plan that will guide the quality management system across the medical laboratory services and system in the country. lessons learned and challenges laboratories participating in the eqa programme for hiv rapid testing and cd4 (including poc) received feedback and, based on their results, have implemented corrective actions, as needed, to improve the quality of services, and were encouraged to participate in a laboratory network for continuous improvement. these outcomes are positive steps toward the implementation of quality management systems. the feedback received from the eqa programme has been used as an opportunity for participating laboratories to be more vigilant in many aspects of their work; for example, with respect to the expiration date for reagents, implementation of corrective actions, and on-site training for staff. the quality assurance programme and the external quality control activities must be followed up by systems-strengthening activities, such as staff training at all levels. we found that there is a need for more communication between the national reference laboratories and the bureau of laboratories, located at the ministry of health, regarding the type of corrective actions and support to be provided from the central/national level to regional and peripheral laboratories. there is also a need to provide incentives such as certificates of participation or achievement to successfully performing labs. quality assurance for poc currently, all 24 sites in-country with poc diagnostic technologies, including poc for cd4, are participating in an eqa programme organised by lbv. since 2004, lbv, with the support of the national aids programme, has provided voluntary, free-of-charge eqa of hiv rapid testing to assess the performance of laboratories conducting hiv diagnostics in dakar and other regions. currently, the eqa programme for hiv rapid testing uses dried-tube specimens. the proficiency testing panels consist of four specimens (two negatives, one hiv-1, one hiv-2) distributed at ambient temperature to participants. the results from the participants are sent to the national reference laboratory via email or cell-phone text messaging. costing and cost-effectiveness of hiv poc testing with regard to the 90-90-90 goals, the affordability of viral load testing and cd4 count measures is a key factor in plans to expand hiv laboratory services and scaling antiretroviral therapy across senegal. the country will need to put in place an affordable and sustainable strategy to reinforce the capacity of the public laboratories as well as to advocate for a strong political commitment from the ministry of health and sponsors. in partnership with the national aids programme, lbv/cadu is planning to estimate the costing and to undertake a cost-effectiveness study of implementing a nationwide hiv poc testing system, for cd4 count and viral load testing. the costing for, and cost-effectiveness of, a related quality assurance programme will be also taken into account. a model of costing for hiv poc testing, including quality assurance activities is currently under review with the international diagnostics centre of the london school of hygiene and tropical medicine. acknowledgements the authors are grateful to the staff of all sites participating in related eqa programmes. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. sources of support none. references agence nationale de la statistique et de la démographie. recensement général de la population et de l’habitat, de l’agriculture et de l’elevage. senegal: ansd; 2014. agence national de la statistique et de la démographie. enquête démographique et de santé et à indicateurs multiples 2010–2012. senegal: ansd; 2012. comité national de lutte contre le sida. rapport national de la riposte vih/sida 2014. yaoundé, cameroun: cnls; 2015. leprêtre a, ba i, lacombe k, et al. prevalence and behavioural risks for hiv and hcv infections in a population of drug users of dakar, senegal: the anrs 12243 udsen study. j int aids soc. 2015;18:19888. http://dx.doi.org/10.7448/ias.18.1.19888. ecollection 2015. joint united nations programme on hiv/aids. global aids response progress reporting 2015. geneva, switzerland: who press; 2015. nguyen s, ramos a, chang j, et al. monitoring the quality of hiv-1 viral load testing through a proficiency testing program using dried tube specimens in resource-limited settings. j clin microbiol. 2015;53(4):1129–1136. http://dx.doi.org/10.1128/jcm.02780-14. epub 2015 jan 21. garcia a, subbarao s, zhang g, et al. impact of proficiency testing program for laboratories conducting early diagnosis of hiv-1 infection in infants in lowto middle-income countries. j clin microbiol. 2014;52(3):773–780. http://dx.doi.org/10.1128/jcm.03097-13. epub 2013 dec 18. pai np, vadnais c, denkinger c, et al. point-of-care testing for infectious diseases: diversity, complexity, and barriers in lowand middle-income countries. plos one. 2012;9(9):e1001306. http://dx.doi.org/10.1371/journal.pmed.1001306. epub 2012 sep 4. miller wg, jones grd, horowitz gl, et al. proficiency testing/external quality assessment: current challenges and future directions. clin chem. 2011;57(12):1670–1680. http://dx.doi.org/10.1373/clinchem.2011.168641. epub 2011 sep 30. hiv situation in uganda uganda’s laboratory infrastructure and hiv-related testing services quality assurance in uganda laboratory and point-of-care quality assurance policy and framework in uganda acknowledgements references about the author(s) steven aisu central public health laboratory, ministry of health, kampala, uganda wilson nyegenye central public health laboratory, ministry of health, kampala, uganda victor bigira clinton health access initiative, kampala, uganda charles kiyaga central public health laboratory, ministry of health, kampala, uganda michael dfendu central public health laboratory, ministry of health, kampala, uganda sam acellam clinton health access initiative, kampala, uganda richard walwema infectious disease institute, kampala, uganda lydia nakiyingi infectious disease institute, kampala, uganda isaac sewanyana central public health laboratory, ministry of health, kampala, uganda aida namakula central public health laboratory, ministry of health, kampala, uganda richard batamwita central public health laboratory, ministry of health, kampala, uganda william lali world health organization, kampala, uganda citation aisu s, nyegenye w, bigira v, et al. quality assurance as an integral component of diagnostic testing in clinical laboratories and point-of-care testing: the uganda experience. afr j lab med. 2016;5(2), a447. http://dx.doi.org/10.4102/ajlm.v5i2.447 country profile quality assurance as an integral component of diagnostic testing in clinical laboratories and point-of-care testing: the uganda experience steven aisu, wilson nyegenye, victor bigira, charles kiyaga, michael dfendu, sam acellam, richard walwema, lydia nakiyingi, isaac sewanyana, aida namakula, richard batamwita, william lali received: 24 mar. 2016; accepted: 11 aug. 2016; published: 17 oct. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. hiv situation in uganda uganda, with an estimated population of 35 million people, is a landlocked country that forms part of the east african union member states. the country is divided into nine political regions (figure 1), in which there are 112 administrative districts. in order to make the administration and coordination of health services more efficient, the country is further divided into 14 health regions. figure 1: key hiv statistics in uganda, 2014, showing hiv prevalence by region. uganda is classified as a high burden hiv country with an estimated 1.6 million people living with hiv/aids and 140 000 new cases reported annually (table 1; figure 1).1 being one of only two countries where incidence was on the rise in 2013, hiv remains a major public health problem in uganda.2 hiv prevalence varies across the country, being highest in the central region and near urban centres and lowest in the mid-eastern and west nile regions. table 1: key hiv statistics in uganda, 2014. in 2013, the world health organization changed the recommendation for initiation of antiretroviral therapy from a cd4 count of 350 cells/mm3 to 500 cells/mm3.3 uganda was one of the first countries to adopt these recommendations and began implementing the revised treatment guidelines in april 2014.4 under the revised antiretroviral therapy guidelines, cd4 enumeration remains critical to staging the need for antiretroviral therapy among hiv-positive patients aged 15 years and above.5 in addition, the world health organization recommended the use of viral load testing as the preferred antiretroviral therapy monitoring approach. the ministry of health in uganda subsequently adopted viral load monitoring for antiretroviral therapy patients into the national guidelines. uganda’s viral load programme has since scaled up rapidly, but universal access has not yet been achieved. for patients who do not have access to viral load services, cd4 testing remains essential for monitoring antiretroviral therapy response. as uganda scales up the new world health organization/unaids 90-90-90 strategy,6 there has been a steep rise in the number of people in need of hiv testing and viral load monitoring across the country. it is estimated that over 850 000 people will be on antiretroviral therapy by the end of 2016. meeting these targets will require innovative ways of developing laboratory systems, including scaling up of point-of-care (poc) tests for hiv testing, diagnosis of opportunistic infections and monitoring response to treatment or disease progression. uganda’s laboratory infrastructure and hiv-related testing services the laboratory network in uganda consists of a five-tiered system covering the following levels: national, regional, general hospital (district), health centre iv and health centre iii. laboratories at national, regional and district levels are relatively well developed, whereas laboratories at lower health centre iv and iii level generally have inadequate space, no running water, frequent power interruptions and inadequate working space. in order to overcome some of these challenges, the country has established a national sample and results referral network based on hubs. a hub is a high volume laboratory that functions as a referral laboratory for 30–40 lower level laboratories. complex tests that cannot be handled at the hub, such as early infant diagnosis (eid), viral load testing, culture for tuberculosis and outbreak investigation samples, among others, are centralised at the central public health laboratory (cphl) and other reference laboratories.7,8,9 although testing capacity has been increased through deployment of modern high-throughput equipment, national testing targets for eid and viral load have not yet been met. it can be argued that lack of, or limited access to, laboratory services may be overcome through centralisation of testing services and building of new laboratories. however, this course of action requires substantial investment and could result in a significant delay. moreover, there is a nagging fear that if donor funding is significantly reduced or entirely stopped, such projects may be rendered ‘white elephants’. cognisant of such possibilities, over the last few years, uganda has adopted a combination of conventional testing platforms at higher level health facilities and poc testing at lower level health facilities. thus, in uganda, eid and viral load testing use roche molecular diagnostics and abbott molecular laboratory methods, while cd4 and hiv testing use a mix of conventional analysers and poc devices, including bd facscalibur®, bd facscount®, partec cyflow® counter and alere pima™ for cd4 counts, and elisa and rapid diagnostics tests for hiv. in addition, bd mgit is used for tuberculosis cultures and cepheid genexpert® for pcr. poc tests are deployed in remote rural areas or where test volumes are low, while conventional analysers are used mainly at hubs and in urban areas. quality assurance in uganda current quality assurance situation the quality assurance programme in uganda is still in its infancy. like many african countries, uganda has adopted the strengthening laboratory management towards accreditation and stepwise laboratory quality improvement process towards accreditation approaches to improving quality systems in laboratories. however, the supporting components to this programme, such as internal quality control procedures and external quality assessment (eqa), are quite weak. although cphl has put in place some measures in collaboration with the uganda virus research institute, the national tuberculosis and leprosy control program, the infectious diseases institute and the national drug authority to carry out some eqa functions, major gaps still exist. there is poor eqa coverage, particularly for poc testing; low response rates from participating facilities and lack of a comprehensive corrective action plans to support poorly performing laboratories. only panels for hiv testing and tuberculosis smear microscopy are produced locally, whereas panels for clinical chemistry, haematology, bacteriology, cd4 counts and viral load are supplied by external agencies. the eqa schemes and suppliers are not centrally coordinated. because uganda anticipates a rapid scale up of poc testing, cphl has developed plans to review the poc quality assurance policies and framework. laboratory and point-of-care quality assurance policy and framework in uganda current status of point-of-care quality assurance policy and framework uganda has a national health laboratory policy and strategic plan that guides the implementation of laboratory quality management systems across the health laboratory services. however, the strategies do not explicitly address poc testing deployment or poc testing quality issues. therefore, there is a need to review the national laboratory policy and strategic plan so as to address quality systems for poc testing. table 2 shows the results of a rapid assessment of the poc testing quality assurance policy and framework. while there is top-level leadership engagement (estimated score 80%), major gaps still exist for coordination at the national level (60%), definition of roles and responsibilities (60%), policies and strategic plans (40%), and availability of national laboratory standards (30%). table 2: status of point-of-care quality assurance policy and framework in uganda external quality assurance reforms for point-of-care testing a functional eqa scheme consists of six key components (figure 2), specifically: plan, define, implement, monitor, improve and evaluate. these components are arranged in a cyclical manner to demonstrate the concept of continual improvement. figure 2: components of a functional eqa scheme. in uganda, implementation of the eqa scheme will be done in three phases, as outlined below. phase 1: plan an hiv-related point-of-care testing quality system planning and developing an hiv-related poc quality system requires the review of the current guidelines and standards, and therefore requires the engagement of relevant leaders to execute the activities. with leadership engagement, establishment of the national quality assurance coordination team is easier and the roles and responsibilities of the selected teams can be defined (figure 3). before defining the expected quality of poc tests, there is a related need to conduct a situational analysis that may include mapping disease prevalence, as well as selecting and assessing sites. with the abovementioned background activity, the implementation plan will be developed with the further incorporation of quality assurance and policies. finally, a financial plan will be drafted with an emphasis on human resources and cost–benefit analysis. figure 3: proposed national external quality assessment coordination structure. phase 2: implement quality assurance for point-of-care testing the implementation strategy for quality assurance of poc testing requires quality assurance training and certification. this will further involve activities such as the process control, audits, site support supervision and mentorship, all of which constitute the fundamental components of capacity building. establishment of eqa (active, intermediate, passive) production, packaging/shipping through hubs, programme management and outsourcing will be implemented when the need arises (figure 4). the quality assurance supply chain and logistics programme can continuously be strengthened for improvement. figure 4: proposed courier system for the flow of national external quality assessment materials, forms, results and feedback. phase 3: sustain the quality assurance programme for the sustainability of the quality assurance programme planning, allocation of resources and continuous encouragement of socio-economic entrepreneurship are required. sustainability implementation will require focused monitoring and evaluation initiatives, country ownership and involvement, a high level of advocacy, and mobilisation of the community. feedback, corrective action and inter-lab comparisons are vital for quality assurance programmes, as these are the basis for improvement. cost of activities and resources for quality assurance improvement estimating the cost of eqa activities will include understanding the country’s needs based on identified interventions to inform key policy decisions and reducing high costs. external support will be sought to price the final model of service provision and to establish a cost-effective poc testing eqa scheme that is integrated in a comprehensive national eqa program. in cases where it will be necessary to produce eqa panels locally, the activity will be undertaken by the uganda national health laboratory and respective reference laboratories. eqa materials will be distributed using the hub system. technical assistance and other resources will be mobilised both internally and externally to support the programme. conclusion and way forward uganda is actively working to implement a robust quality assurance programme that will address poc testing in the country. although the quality assurance programme is still in its infancy, uganda has already adopted strengthening laboratory management towards accreditation and stepwise laboratory quality improvement process towards accreditation to improve overall laboratory quality systems and is focusing on how to improve internal quality control procedures and eqa. uganda has always taken great strides to meet challenges with innovative approaches. the country established a national sample and results referral network based on hubs to improve eid services, which also resulted in cost savings for the programme and in turn improved other health services. more recently, uganda performed a rapid assessment of the poc testing quality assurance policy and framework to identify major gaps that should be addressed. uganda understands the strength of working in a collaborative manner with all institutions to implement a sustainable country-owned eqa programme. cphl is working with the uganda virus research institute, the national tuberculosis and leprosy control program, the infectious diseases institute and the national drug authority to plan and carry out eqa functions necessary to support quality cd4, eid, and viral load services. acknowledgements the authors thank the ministry of health, the development partners and all the implementing partners within the health system of the country. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. sources of support none. references joint united nations programme on hiv/aids (unaids). the gap report. geneva, switzerland: unaids; 2014. ministry of health, macro i. uganda aids indicator survey (ais) 2011. kampala, uganda; ministry of health; 2011. world health organization. who issues new hiv recommendations calling for earlier treatment [page on the internet]. c2013 [cited 2016 sep 22]. available from: http://www.who.int/mediacentre/news/releases/2013/new_hiv_recommendations_20130630/en/ ministry of health, uganda. addendum to the national antiretroviral treatment guidelines [document on the internet]. c2013 [cited 2016 sep 27]. available from: http://preventcrypto.org/wp-content/uploads/2012/07/uganda-national-art-guidelines_2014.pdf ministry of health, uganda. uganda national policy guidelines for hiv counselling and testing [document on the internet]. c2012 [cited 2016 sep 22]. available from: http://www.who.int/hiv/pub/guidelines/uganda_art.pdf joint united nations programme on hiv/aids (unaids). 90-90-90: an ambitious treatment target to help end the aids epidemic. geneva, switzerland: unaids; 2014. pepfar, usaid. increasing viral load monitoring of people living with hiv on art in northern uganda in line with the 90-90-90 global targets [document on the internet]. c2016 [cited 2016 sep 27]. available from: https://www.usaidassist.org/sites/assist/files/improving_vl_testing_in_northern_uganda_june2016_a4_ada.pdf kiyaga c, sendagire h, joseph e, et al. uganda’s new national laboratory sample transport system: a successful model for improving access to diagnostic services for early infant hiv diagnosis and other programs. plos one. 2013;8(11):e78609. http://dx.doi.org/10.1371/journal.pone.0078609. ecollection 2013. stevens ws, gelman r, glencross dk, et al. evaluating new cd4 enumeration technologies for resource-constrained countries. evaluating diagnostics: the cd4 guide. nature rev microbiol. 2008;6(11 suppl):s29–s38. http://dx.doi.org/10.1038/nrmicro2000 reviewer acknowledgement open accesshttp://www.ajlmonline.org page 1 of 1 we would like to take this opportunity to thank all of those who provided scientific and logistical support for this special issue of the african journal of laboratory medicine: should names have inadvertently been excluded from this list, the publisher apologises 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professional term as reviewer to the african journal of laboratory medicine. please do not hesitate to contact me if you require assistance in performing this task. chantal parkins submissions@ajlmonline. org tel: +27 (0)21 975 2602 fax: +27 (0)21 975 4635 182 heidi albert george alemnji timothy amukele linda andiric rosemary audu ronald ballard debi boeras rachel crane karidia diallo dennis ellenberger paula fernandes glen fine peter fonjungo thomas gachuki emily griswold eduardo gudo emmanuel idigbe zachary katz luciana kohatsu paul lee kitty linde talkmore maruta martin matu barbara mckinney fausta mosha sikhulile moyo christina mwangi jane mwangi nqobile ndlovu clement ndongmo john nkengasong michael noble alaine nyaruhirira bryan nyary phoebe nzombe chin yi ou elde mel paladar chantal parkins linda parsons bethanie rammer philip rotz lee schroeder connie sexton judith shang ritu shrivastava edwin shumba beth skaggs penny smorenburg suzanne taylor hamilton tilley ralph timperi corey white shanita williams isatta wurie tun ye this research has been supported by the us president’s emergency plan for aids relief (pepfar) through the us centers for disease control and prevention. the findings and conclusions in this article are those of the authors and do not necessarily represent the official position of the us centers for disease control and prevention. use of trade names is for identification purposes only and does not constitute endorsement by the us centers for disease control and prevention. slmta steering committee: eduardo gudo emmanuel idigbe ismae whyms jean-bosco ndihokubwayo john n. nkengasong (chair) michael wanga nguyen thi thuong trevor peter tsehaynesh messele slmta technical advisory group: ana malla de abreu anthony emeribe fausta mosha george alemnji judith shang katy yao kelebeletse mokobela luciana kohatsu nqobile ndlovu sibongile zimuto talkmore maruta (chair) vireak voeurng hiv epidemiology in ethiopia hiv-related laboratory infrastructure in ethiopia for cd4, viral load and early infant diagnosis poc and quality assurance framework in ethiopia ethiopia’s experiences with implementing poc testing for cd4 and tuberculosis existing national quality assurance programmes challenges with poc testing acknowledgements references about the author(s) adisu kebede ethiopian public health institute, ethiopia yenew kebede centers for disease control and prevention, ethiopia laboratory branch, ethiopia adino desale ethiopian public health institute, ethiopia achamyeleh mulugeta ethiopian public health institute, ethiopia zelalem yaregal ethiopian public health institute, ethiopia atsbeha gebreegziabxier ethiopian public health institute, ethiopia yared tedla centers for disease control and prevention, ethiopia laboratory branch, ethiopia clement zeh centers for disease control and prevention, ethiopia laboratory branch, ethiopia gonfa ayana ethiopian public health institute, ethiopia citation kebede a, kebede y, desale a, et al. quality assurance for point-of-care testing: ethiopia’s experience. afr j lab med. 2016;5(2), a452. http://dx.doi.org/10.4102/ajlm.v5i2.452 country profile quality assurance for point-of-care testing: ethiopia’s experience adisu kebede, yenew kebede, adino desale, achamyeleh mulugeta, zelalem yaregal, atsbeha gebreegziabxier, yared tedla, clement zeh, gonfa ayana received: 30 mar. 2016; accepted: 11 aug. 2016; published: 17 oct. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. hiv epidemiology in ethiopia in 2015, there were an estimated 729 517 people living with hiv in ethiopia, including 95 094 children (aged 0–14 years), according to the 2015 estimation and projection package/spectrum modelling. hiv prevalence at the national level is 4.2% among urban populations and 0.6% rural popula