DTI Drug Target Insights 2023; 17: 78-89ISSN 1177-3928 | DOI: 10.33393/dti.2023.2596REVIEW Drug Target Insights - ISSN 1177-3928 - www.aboutscience.eu/dti © 2023 The Authors. This article is published by AboutScience and licensed under Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0). Commercial use is not permitted and is subject to Publisher’s permissions. Full information is available at www.aboutscience.eu Efficacy of LAMP assay for Mycobacterial spp. detection to prevent treatment delays and onset of drug resistance: a systematic review and meta-analysis Gurvinder Singh Bumbrah1, Sarika Jain2, Zeeshan Fatima3,4 and Saif Hameed3 1Department of Forensic Sciences, Amity School of Applied Sciences, Amity University Haryana, Gurugram, Manesar - India 2Department of Mathematics, Amity School of Applied Sciences, Amity University Haryana, Gurugram, Manesar - India 3Amity Institute of Biotechnology, Amity University Haryana, Gurugram, Manesar - India 4Department of Medical Laboratory Sciences, College of Applied Medical Sciences, University of Bisha, Bisha - Saudi Arabia ABSTRACT Background: Tuberculosis (TB) remains a deadly disease affecting one-third population globally. Long turnaround time and poor sensitivity of the conventional diagnostics are the major impediments for faster diagnosis of Myco- bacterial spp to prevent drug resistance. To overcome these issues, molecular diagnostics have been developed. They offer enhanced sensitivity but require sophisticated infrastructure, skilled manpower and remain expensive. Methods: In that context, loop-mediated isothermal amplification (LAMP) assay, recommended by the WHO in 2016 for TB diagnosis, sounds as a promising alternative that facilitates visual read outs. Therefore, the aim of the present study is to conduct a meta-analysis to assess the diagnostic efficiency of LAMP for the detection of a panel of Mycobacterium spp. following PRISMA guidelines using scientific databases. From 1600 studies reported on the diagnosis of Mycobacterium spp., a selection of 30 articles were identified as eligible to meet the criteria of LAMP based diagnosis. Results: It was found that most of the studies were conducted in high disease burden nations such as India, Thai- land, and Japan with sputum as the most common specimen to be used for LAMP assay. Furthermore, IS6110 gene and fluorescence-based detections ranked as the most used target and method respectively. The accuracy and precision rates mostly varied between 79.2% to 99.3% and 73.9% to 100%, respectively. Lastly, a quality assessment based on QUADAS-2 of bias and applicability was conducted. Conclusion: LAMP technology could be considered as a feasible alternative to current diagnostics considering high burden for rapid testing in low resource regions. Keywords: Diagnosis, LAMP, Meta-analysis, Mycobacteria, Therapeutics, Tuberculosis Received: May 1, 2023 Accepted: May 12, 2023 Published online: June 7, 2023 Corresponding authors: Drs. Saif Hameed and Zeeshan Fatima Amity Institute of Biotechnology Amity University Haryana Gurugram, Manesar-122413 - India saifhameed@yahoo.co.in; drzeeshanfatima@gmail.com spine, or brain (1). Worldwide, TB is the 13th leading cause of death and the second raging infectious killer after human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) (2). In 2020, an estimated 10 million people got ill with TB worldwide, the infection being divided as 5.6 million men, 3.3 million women, and 1.1 million children. TB affects most of the countries among all age groups and can be fatal if not treated properly. Moreover, the emergence of drug-resistant strains has further complicated the problem and has become a rising obstacle against efficient therapeu- tics (3). Therapeutics are available but the effective control of the disease is impeded due to the lack of rapid and accurate diagnostics. Under such significant circumstances, there is an urgent need for rapid, accurate, and cost-effective diagnos- tic test for TB to identify new cases and reduce the time-to- treatment and prevent its further transmission. The current available methods are primarily based on smear microscopy (acid-fast staining), culture, and nucleic Introduction Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) remains a deadly disease affecting millions of people worldwide. It is estimated to affect approximately one-third of the global population and is becoming one of the most fatal infectious diseases. MTB usually attacks the lungs, but TB bacteria can infect any part of the body such as kidney, https://doi.org/10.33393/dti.2023.2596 https://creativecommons.org/licenses/by-nc/4.0/legalcode mailto:saifhameed@yahoo.co.in mailto:drzeeshanfatima@gmail.com Bumbrah et al Drug Target Insights 2023; 17: 79 © 2023 The Authors. Published by AboutScience - www.aboutscience.eu acid amplification. Although methods based on acid-fast staining are sensitive, they pose problems in low-resource places and are time-consuming (4). The solid culture method requires around 4-8 weeks, while liquid-based culture meth- ods also require around 10-14 days (4). Nucleic acid amplifica- tion techniques are based on polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP). Although hemi-nested PCR based on GeneXpert for MTB detection is rapid, sensitive, and specific, it also poses challenges of high cost and high end equipment dependency, which limits its implementation in low-resource regions (5). LAMP is an iso- thermal DNA amplification method that relies on four or six pairs of primers to amplify minute quantities of DNA within a shorter period with simple operation, making it more suit- able for low-resource regions (6). Thus, research in TB diag- nostics aims to find an efficient, reproducible, cost-effective tool with minimal infrastructure requirements. LAMP is a popularly adopted new age technology for rapid nucleic acid amplification which is widely used for pathogen (virus, bac- teria, and malaria) detection including severe acute respira- tory syndrome coronavirus 2 (SARS CoV-2) (7-9). LAMP-based detection methods have been proved to be more sensitive than GeneXpert assay. In fact, the World Health Organization (WHO) has endorsed LAMP for TB as a replacement for smear microscopy for peripheral settings (10). In pursuit of developing better diagnostics, which are cru- cial for achieving global elimination of TB, we performed a systematic review and meta-analysis to access the diagnostic accuracy of LAMP to detect mycobacteria. Even if couple of studies have depicted the efficacy of LAMP during the last decade, an updated version is missing. Moreover, most of these studies were specific to either pulmonary or extrapul- monary TB. Therefore, the present study not only offers an up-to-date diagnostic performance of LAMP for TB detection but also covers other Mycobacterium spp. The pooled sen- sitivity and specificity of LAMP were analyzed against differ- ent references. Further, diagnostic efficiency was determined based on reference methods, target genes, and detection methods of LAMP. Taken together, we aimed to evaluate the diagnostic potency of LAMP as a tool for detection of myco- bacteria to address the current TB diagnosis burden in low- resource places. Methods The Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) guidelines (11) were followed for identification of eligible studies in the present systematic review and meta-analysis. Search strategy Diverse scientific databases, for example, PubMed, Google Scholar, Science Direct, Scopus, BioRxiv, and MedRxiv, were searched to screen for studies performed using LAMP for TB diagnostics from the year 2000 till March 2022. The terms such as LAMP, Tuberculosis, Mycobacterium and mycobacte- ria were used in various combinations during our research without any limitations: “LAMP + Tuberculosis” or “LAMP + Mycobacterium” or “LAMP + mycobacteria” or “LAMP + TB” or “LAMP + Tuberculosis + Mycobacterium” or “LAMP + Tuberculosis + mycobacteria” for PubMed, Science Direct, and Google Scholar without using any language restriction. The retrieved results were screened for duplication and con- formity with the prespecified eligibility criteria. Study eligibility criteria Inclusion criteria This systematic review and meta-analysis included: (1) both peer-reviewed and preprint original articles on LAMP technology used for detection of any mycobacterial species such as MTB, M. bovis, and M. africanum; (2) only full-text articles written in English language; and (3) articles that contain data on true-positive (TP), false-positive (FP), false- negative (FN), and true-negative (TN) values for the assay or have sufficient data so that the number of TP, FP, FN, and TN (performed on clinical samples) could be determined. Exclusion criteria Exclusion was made for: (1) studies based on non-isother- mal amplification; (2) studies where data are irretrievable; (3) review articles, editorials, commentaries, proceedings, etc.; (4) foreign language articles (other than English) based on LAMP-mediated detection of mycobacteria. Data extraction Potential articles after reviewing titles and abstracts fol- lowed by full text for inclusion were extracted by two authors (G.S.B. and Z.H.). Consultation from two independent authors (S.J. and S.H.) was made to eliminate the doubt about any discrepancy. The extracted information from included stud- ies had authors, year of publication, location of study, sample size, types of specimens, target genes, detection method, and standard reference method. The data extracted for eval- uation of diagnostic accuracy for LAMP were performed by using either respiratory or non-respiratory specimens with any of the reference methods such as smear microscopy, culture, and GeneXpert. The important parameters in this meta-analysis such as TP, TN, FP, and FN of all studies were either extracted or calculated to provide their sensitivity and specificity values. The included studies (n = 30) were then assessed for their methodological quality to reduce system- atic biases and inferential errors from the collected data. Statistical analysis The quantitative analysis of the included studies (n = 30) from the data extracted such as the values of TP, FP, TN, FN and sample size was performed. Furthermore, the values of sensitivity and sensitivity were mined or calculated from the available data. Moreover, pooled sensitivity and speci- ficity of LAMP associated with 95% confidence interval (CI) were estimated. To maintain the accuracy and precision, the formulas: Accuracy = [TP + TN/TP + TN + FP + FN]* 100 and Precision = [TP/TP+FP]* 100 (12,13) were used. Accuracy and precision are important characteristics of any measurement. Mycobacterial diagnostic efficiency of LAMP assay80 © 2023 The Authors. Drug Target Insights - ISSN 1177-3928 - www.aboutscience.eu/dti Accuracy is the degree of closeness of measured value to a standard value. However, precision provides the informa- tion regarding the closeness of multiple measured values to each other. Accuracy and precision are independent of each other. Forest plot for sensitivity and specificity were plotted using R-software along with summary receiver operating characteristic (SROC) for the given study. Quality assessment Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool was used to assess the methodological quality of the eligible studies. The risk of bias in the included studies (n = 30) was assessed from four areas of bias, for example, patient selection, index test, reference standards, and flow timing (14,15). For each QUADAS-2 domain specific yes/no questions were tailored. Following these criteria, the eligible studies were then refereed for low, unclear, or high risks of bias. Furthermore, we also judged to generate low, unclear, or high-risk applicability. Results Literature survey We followed the PRISMA guidelines (11) to search the literature for the present study (Fig. 1). The major scientific databases viz. PubMed, Science Direct, Scopus, BioRxiv, and MedRxiv have been extensively searched applying the above inclusion criteria and around 1,600 articles were extracted. From the 1,600 articles, we included the ones that were published after the year 2000 since the inception of LAMP technology (6) and thus excluded 22 articles. Further, only articles written in English language were considered and thus excluded 44 articles. Reading the titles and abstracts of these studies allowed to exclude further 1,029 articles comprising the review articles, editorials, proceedings etc. Following this exclusion, we removed the duplicated articles and further excluded 390 articles. Additional 73 articles were irrelevant as they didn’t use LAMP technology for the diagnosis of any mycobacterial species and were excluded, leaving a panel composed of 42 eligible studies. Lastly, from the 42 included articles, further 12 articles were also eliminated because their TP, FP, TN, and TN values were either not specified in these articles or the sensitivity and specificity values could not be calculated. Altogether, we observed that only 30 arti- cles were eligible for detailed meta-analysis (Fig. 1) consider- ing all the exclusion criteria. Study characteristics and meta-analysis Table I shows the data extracted from the eligible stud- ies mentioning the details of authors, year of publication, country of study, types of specimens, target genes, detection method, and reference methods. Figure 2 shows the coun- try-wise distribution of 30 identified articles included in the present study. Most of the studies (43.3%; n = 13) were con- ducted in the high TB burden nations such as India followed Fig. 1 - Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) flowchart depicts search of the literature and screening strategy for meta-analysis. Bumbrah et al Drug Target Insights 2023; 17: 81 © 2023 The Authors. Published by AboutScience - www.aboutscience.eu Ta bl e I - C ha ra ct er is tic s an d ou tc om es o f t he in cl ud ed s tu di es (n  =  3 0) S. N o. A ut ho r Jo ur na l Co un tr y Re fe re nc e M et ho d Sp ec im en Ta rg et G en e D et ec ti on M et ho d TP TN FP FN Si ze Se ns iti vi ty Sp ec ifi ci ty 1 Bo eh m e et a l ( 20 07 ) J C lin M ic ro bi ol Sw itz er la nd Cu ltu re , s m ea r m ic ro sc op y Sp ut um gy rB Fl uo re sc en ce , tu rb id it y 17 3 50 0  4  5  6 82 97 .7 0% 99 % 2 Pa nd ey et a l ( 20 08 ) J M ed M ic ro bi ol Ja pa n A ci d- fa st s ta in in g, ba ct er ia l c ul tu re , ra di ol og y Sp ut um 16 S rR N A Fl uo re sc en ce  9 0  9 8  6  6  2 00 94 % 94 .2 0% 3 Po ud el et a l ( 20 09 ) Ka th m an du U ni v M ed J (K U M J) N ep al Sm ea r m ic ro sc op y, cu ltu re , r ad io lo gy Sp ut um 16 S rR N A Fl uo re sc en ce  9 7  9 6  6  3  2 02 97 % 94 .1 2% 4 G eo jit h et a l ( 20 11 ) J M ic ro bi ol M et ho ds In di a Cu ltu re , P CR r ev er se - hy br id iz ati on li ne p ro be as sa y, g en ot yp e M TB E as sa y Sp ut um ri m M Co lo ri m et ry , g el el ec tr op ho re si s  1 7  1 7  1 21    56 44 .7 0% 94 .4 0% 5 G eo rg e et a l ( 20 11 ) PL oS O ne In di a Fl uo re sc en ce s m ea r m ic ro sc op y, c ul tu re Sp ut um ri m M Co lo ri m et ry , g el el ec tr op ho re si s  3 1  3 6  2  2    71 93 .9 0% 94 .7 0% 6 M it ar ai et a l ( 20 11 ) In t J T ub er c Lu ng D is Ja pa n Cu ltu re , s m ea r m ic ro sc op y, n uc le ic a ci d am pl ifi ca tio n (N A A) Sp ut um gy rB Fl uo re sc en ce , tu rb id it y 19 6  8 8  9 27  3 20 87 .9 0% 90 .7 0% 7 N ag de v et a l ( 20 11 ) J C lin M ic ro bi ol In di a PC R Ce re br os pi na l flu id IS 61 10 Tu rb id it y  1 5    8  2  2    27 88 .2 3% 80 % 8 Se th i et a l ( 20 13 ) J C lin L ab A na l In di a Sm ea r m ic ro sc op y, cu ltu re , P CR Sp ut um 16 S rR N A , IS 61 10 Co lo ri m et ry , g el el ec tr op ho re si s  8 7  3 0  0 16  1 33 84 .5 0% 10 0% 9 Ca o et a l ( 20 15 ) J M ic ro bi ol M et ho ds Ch in a Sm ea r m ic ro sc op y, cu ltu re , P CR Sp ut um IS 61 10 Fl uo re sc en ce  9 8  1 8  5  2  1 23 98 .0 0% 78 .3 0% 10 Jo on et a l ( 20 15 ) In t J T ub er c Lu ng D is In di a Sm ea r m ic ro sc op y, cu ltu re , P CR En do m et ria l fl ui d, ur in e, b lo od , s em en , ce re br os pi na l fl ui d, pl eu ra l fl ui d, p us , pe ric ar di al fl ui d, pe rit on ea l fl ui d, in te sti na l a nd ly m ph no de b io ps y tis su e IS 61 10 , M PB 64 , sd aA Co lo ri m et ry  2 8 26 2 23  2  3 15 93 .3 0% 91 .9 0% 11 M oo n et a l ( 20 15 ) J M ed M ic ro bi ol Ko re a Cu ltu re , s m ea r m ic ro sc op y Sp ut um hs pX Co lo ri m et ry , tu rb id it y, g el el ec tr op ho re si s  3 2 25 5  3 13  3 03 71 .1 0% 98 .8 0% 12 Bo ja ng et a l ( 20 16 ) J I nf ec t G am bi a Sm ea r m ic ro sc op y, cu ltu re , G en eX pe rt M TB /R IF Sp ut um 16 S rR N A Fl uo re sc en ce  9 8 15 7 10  1  2 66 99 .0 0% 94 .0 0% 13 G ra y et a l ( 20 16 ) J C lin M ic ro bi ol Sw itz er la nd Cu ltu re , s m ea r m ic ro sc op y Sp ut um gy rB Fl uo re sc en ce 33 1 ## # 52 61 17 77 84 .4 0% 96 .6 0% 14 Ka ku et a l ( 20 16 ) Jp n J I nf ec t D is Ja pa n Sm ea r m ic ro sc op y, cu ltu re Sp ut um gy rB , IS 61 10 Fl uo re sc en ce 13 4 31 2  5  2 1 47 2 86 .5 0% 98 .4 0% 15 M od i et a l ( 20 16 ) In t J T ub er c Lu ng D is In di a Cu ltu re , r ad io lo gy , st ai ni ng , P CR Ce re br os pi na l flu id IS 61 10 , M PB 64 Fl uo re sc en ce , g el el ec tr op ho re si s, tu rb id it y 14 4 10 0  0    6 25 0 96 .0 0% 10 0. 00 % (C on tin ue d) Mycobacterial diagnostic efficiency of LAMP assay82 © 2023 The Authors. Drug Target Insights - ISSN 1177-3928 - www.aboutscience.eu/dti 16 Sh ar m a et a l ( 20 16 ) Tu be rc ul os is (E di nb ) In di a PC R, c ul tu re , s m ea r m ic ro sc op y N ee dl e as pi ra te IS 61 10 , M PB 64 Fl uo re sc en ce , g el el ec tr op ho re si s, tu rb id it y 10 8  5 0  0  1 2 17 0 90 .0 0% 10 0. 00 % 17 Sh ar m a et al (2 01 6) J O rt ho p Re s In di a Cu ltu re , s ta in in g, P CR Sy no vi al fl ui d, pu s IS 61 10 , M PB 64 Fl uo re sc en ce , g el el ec tr op ho re si s, tu rb id ity  8 1  5 0  0    9 14 0 90 .0 0% 10 0. 00 % 18 Jo on e t a l (2 01 7) J M ic ro bi ol M et ho ds In di a PC R, c ul tu re , s m ea r m ic ro sc op y Sp ut um IS 61 10 , M PB 64 Co lo ri m et ry , g el el ec tr op ho re si s  1 7 21 2  6    1 23 6 94 .4 0% 97 .2 0% 19 Re dd y et a l (2 01 7) In t J T ub er c Lu ng D is So ut h Af ri ca Cu ltu re , s m ea r m ic ro sc op y, X pe rt Sp ut um gy rB Fl uo re sc en ce 11 9 51 4 17  4 5 69 5 72 .6 0% 96 .8 0% 20 Ya da v et a l (2 01 7) In t J T ub er c Lu ng D is In di a Cu ltu re , s m ea r m ic ro sc op y, G en eX pe rt Sp ut um gy rB , IS 61 10 Fl uo re sc en ce  8 2 36 8  3    0 45 3 10 0. 00 % 99 .2 0% 21 Ki m e t a l (2 01 8) A nn L ab M ed Ko re a Cu ltu re , s m ea r m ic ro sc op y, P CR Sp ut um gy rB , IS 61 10 Fl uo re sc en ce , tu rb id it y  8 7 18 6  0  1 7 29 0 83 .6 0% 10 0. 00 % 22 N gu ye n et al (2 01 8) D ia gn M ic ro bi ol In fe ct D is V ie tn am Sm ea r m ic ro sc op y, cu ltu re , X pe rt M TB /R IF Sp ut um gy rB , IS 61 10 Co lo ri m et ry , flu or es ce nc e  1 5 44 5 23  1 8 50 1 45 .5 0% 95 .1 0% 23 Pe re ra e t a l (2 01 8) Ce yl on M ed J Sr i L an ka Sm ea r m ic ro sc op y, cu ltu re Cu ltu re is ol at es ri m M Co lo ri m et ry  3 1  1 0  5    0  4 6 10 0. 00 % 66 .6 7% 24 Jo on e t a l (2 01 9) J M ic ro bi ol M et ho ds In di a Cu ltu re , s m ea r m ic ro sc op y, G en eX pe rt M TB /R IF a ss ay , P CR , LA M P- LF D a ss ay Sp ut um s da A Co lo ri m et ry  1 3  9 2  2    0 10 7 10 0. 00 % 97 .8 7% 25 Ph et su ks ir i et a l ( 20 19 ) Jp n J I nf ec t D is Th ai la nd Cu ltu re , i m m un o- ch ro m at og ra ph ic te st Sp ut um m pt 64 Co lo ri m et ry 14 4    5  1    1 15 1 99 .3 1% 83 .3 3% 26 Pu na ti et a l (2 01 9) Br az J M ic ro bi ol In di a Cu ltu re , P CR Fe ca l s am pl es IS 90 0 Tu rb id it y, g el el ec tr op ho re si s, co lo ri m et ry , la te ra l fl ow de vi ce  8 6 29 4  9    0 38 9 10 0. 00 % 97 .0 2% 27 Ra jp ut e t a l (2 01 9) J M ic ro bi ol M et ho ds In di a Cu ltu re , s m ea r m ic ro sc op y, P CR Fl ui ds , u ri ne , pu s IS 61 10 , Pa b, M PB 64 Co lo ri m et ry , flu or es ce nc e  9 0  3 2  9  2 3 15 4 79 .6 5% 78 .0 5% 28 H an e t a l (2 02 0) BM C In fe ct D is Ch in a Xp er t M TB /R IF , S AT -T B as sa y Pl eu ra l fl ui ds gy rB , IS 61 10 Fl uo re sc en ce  5 9  4 1  1 16 4 26 5 26 .5 0% 97 .6 0% 29 Ph et su ks ir i et a l ( 20 20 ) Jp n J I nf ec t D is Th ai la nd M ic ro sc op y, c ul tu re , PC R, r ad io lo gy Sp ut um 16 S rR N A Co lo ri m et ry , flu or es ce nc e, g el el ec tr op ho re si s, im m un o- ch ro m at og ra ph y 11 9 10 2 24 7 25 2 94 .4 4% 80 .9 5% 30 Ph et su ks ir i et a l ( 20 20 ) Re v In st M ed T ro p Sa o Pa ul o Th ai la nd Xp er t M TB /R IF , c ul tu re , sm ea r m ic ro sc op y Sp ut um 16 S rR N A Co lo ri m et ry , flu or es ce nc e, g el el ec tr op ho re si s 12 6  7 1  0 7 20 4 94 .7 4% 10 0. 00 % LA M P = lo op -m ed ia te d is ot he rm al a m pl ifi ca tio n; L FD = la te ra l fl ow d ip sti ck ; P CR = p ol ym er as e ch ai n re ac tio n. Ta bl e I - (C on tin ue d) Bumbrah et al Drug Target Insights 2023; 17: 83 © 2023 The Authors. Published by AboutScience - www.aboutscience.eu Fig. 2 - Country-wise distribution of included studies (n = 30) repor- ted in the present investigation. Table II - Accuracy and precision of the included studies (n = 30) S. No. Study Accuracy Precision 1 Boehme et al (2007) 98.68 97.74 2 Pandey et al (2008) 94.00 93.75 3 Poudel et al (2009) 95.54 94.17 4 Geojith et al (2011) 60.71 94.44 5 George et al (2011) 94.36 93.93 6 Mitarai et al (2011) 88.75 95.60 7 Nagdev et al (2011) 85.18 88.23 8 Sethi et al (2013) 87.96 100.00 9 Cao et al (2015) 94.30 95.14 10 Joon et al (2015) 92.06 54.90 11 Moon et al (2015) 94.71 91.42 12 Bojang et al (2016) 95.86 90.74 13 Gray et al (2016) 93.64 86.42 14 Kaku et al (2016) 94.49 96.40 15 Modi et al (2016) 97.60 100.00 16 Sharma et al (2016) 92.94 100.00 17 Joon et al (2017) 97.03 73.91 18 Reddy et al (2017) 91.07 87.50 19 Sharma et al (2016) 93.57 100.00 20 Yadav et al (2017) 99.33 96.47 21 Kim et al (2018) 94.13 100.00 22 Nguyen et al (2018) 91.81 39.47 23 Perera et al (2018) 89.13 86.11 24 Joon et al (2019) 98.13 86.66 25 Phetsuksiri et al (2019) 98.67 99.31 26 Punati et al (2019) 97.68 90.52 27 Rajput et al (2019) 79.22 90.90 28 Han et al (2020) 37.73 98.33 29 Phetsuksiri et al (2020) 87.69 83.21 30 Phetsuksiri et al (2020) 96.56 100.00 values ranged from 0.67 to 1.00 (Fig. 6). A total of 27 out of the 30 included studies showed pooled sensitivity greater than 70%. Only three studies reported sensitivity values of 26% and 45% each (19-21). In terms of FP rate (1-specificity), 27 included studies showed a pooled FP rate higher than 80% (Fig. 7). Additionally, the accuracy and precision rates of included studies were calculated and varied between 37.73% and 99.33%. The analysis proved that 22 studies displayed more than 90% accuracy with only 4 studies depicting less than 80% accuracy (Tab. II). Likewise, the precision rates var- ied between 39.47% and 100%. The analysis showed that 21 studies exhibited more than 90% precision rate with only 3 studies depicting less than 80%. Of note, we observed that six studies displayed 100% precision rate. by Thailand and Japan (each 10%; n = 3). Two studies each were also conducted in countries such as China, Korea, and Switzerland (6.3%; n = 2). Apart from this, one study each, that is, 3.3%, was from countries included such as Gambia, Nepal, South Africa, Sri Lanka, and Vietnam. Although most of the included articles do not mention about the patient details, the type of specimen (Fig. 3) used in most of the studies was sputum (42.8%; n = 21). In addition, some studies have been tested on other specimens such as cerebrospinal fluid (n = 4), fecal samples (n = 1), urine (n = 3), blood (n = 2), and pleural fluid (n = 3) for the detection of mycobacteria by using LAMP. Furthermore, the standard smear microscopy, culture assay, and PCR-based methods were used as references either alone or in combination (n = 30). Of note, radiology was also used (n = 4) to validate LAMP results as a reference standard (Tab. I), with one study using immunochromatography (16). Next, we examined the various target genes used for the eligible stud- ies. Ten different types of target genes including hspX, IS900, mpt64, Pab, sdaA, rimM, 16SrRNA, MPB64, gyrB, and IS6110 were used in the included studies (n = 30). IS6110 gene was most frequently used in the included studies (n = 14; 31.18%) followed by gyrB (n = 9, 20.45%), 16SrRNA (n = 6, 13.63%), and MPB64 (n = 6, 13.63%) genes (Fig. 4). Furthermore, while analyzing detection methods used for these 30 studies, fluo- rescent method (n = 19, 32.39%) was the most frequently performed followed by colorimetry (n = 14, 25.35%), gel electrophoresis (n = 11, 20.00%) and turbidity (n = 9, 16.36%) methods (Fig. 5). In 53.33% (n = 16) of studies, more than one detection method was used. In 16.66% (n = 5) of studies, com- bination of three methods was used while in only two studies (6.66%), combination of four different methods was reported (17,18). Among all the eligible studies, 4 studies showed 100% sensitivity, while for 16 studies this parameter was higher than 90%. Similarly, 6 studies exhibited 100% specificity while 90% or more specificity was observed in 24 studies (Tab. I). Furthermore, upon analysis of sensitivity and speci- ficity using forest plot at 95% CI, we found that the sensitiv- ity values varied between 0.26 and 1.00 and the specificity Quality assessment of the study Almost two-thirds of the included studies (22 out of 30 studies) have a high risk of patient selection bias due to Mycobacterial diagnostic efficiency of LAMP assay84 © 2023 The Authors. Drug Target Insights - ISSN 1177-3928 - www.aboutscience.eu/dti Fig. 3 - Distribution of type of spe- cimen for detection of mycobacte- ria in the included studies (n = 30). Fig. 4 - Distribution of target ge- nes reported in the included stu- dies (n = 30). non-random patient selection and case-control study design (Fig. 8, Tab. I). Around 26% (8 out of 30) of the included stud- ies have low risk of patient selection bias because these studies provided sufficient details about patient inclusion/ exclusion criteria; 86% of included articles (26 out of 30 studies) present low risk of index test bias because these tests clearly stated the quantitative detection read-outs with reported thresholds. Moreover, these studies explicitly declared that their index and reference tests were done simultaneously in parallel to each other or that testing was blinded from each other. Two studies (19,22) were reported without defined detection thresholds. One study (23) had unclear risk of index test bias as the quantitative detection thresholds were not explained. It was either unclear whether index test results were interpreted with knowledge of refer- ence test results or if only qualitative read-out was used for Bumbrah et al Drug Target Insights 2023; 17: 85 © 2023 The Authors. Published by AboutScience - www.aboutscience.eu Fig. 5 - Distribution of type of de- tection method for mycobacteria in the included studies (n = 30). Fig. 6 - The Forest plot of sensi- tivity and specificity of included studies (n = 30) on the diagnostic performance of loop-mediated isothermal amplification (LAMP) technique. Mycobacterial diagnostic efficiency of LAMP assay86 © 2023 The Authors. Drug Target Insights - ISSN 1177-3928 - www.aboutscience.eu/dti at high risk as it did not provide any information on whether the samples for a reference test and the index test were taken at the same time. Our review question did not focus on any patient demographics. None of the included studies attempted to exclude patients based on demographics and thus had no “concern of patient selection applicability” (Fig. 8, Tab. I). Index tests of all studies have generally been used for Point-of-care test (POCTs) and thus have low concern of index test applicability. Reference standard tests of nearly all studies were culture, smear microscopy, Xpert test, PCR, or combina- tions of them. Thus, we graded these studies as having low concern of standard test applicability. Discussion Early and correct diagnosis of all the TB forms is pertinent for effective treatment of the disease and prevention of the spread of infection, particularly in nations which have high burden. The currently available diagnostics rely mostly on smear microscopy, culture, and PCR-based methods which are not only time-consuming and low sensitive but cumber- some and costly (25,26). LAMP assay provides a faster and innovative point-of-care diagnostic alternative as it is cost- effective, sensitive, and gives results in less than 1 hour due to amplification under isothermal condition by strand dis- placement activity of Bst DNA polymerase and visual read- outs (27-30). In fact, the efficiency of LAMP in diagnosis of pulmonary TB is evident from wide ranges of studies (31-35). Additionally, LAMP has been successfully deployed for diag- nosis of other forms of TB such as tuberculous meningitis (36,37), osteoarticular TB (38), and tubercular lymphadenitis (39). Although a few studies have evaluated the diagnostic validity of LAMP by meta-analysis for diagnosis of MTB (40), Fig. 7 - Summary receiver operating characteristic (SROC) depicts loop-mediated isothermal amplification (LAMP) diagnostic perfor- mance in mycobacteria diagnosis. Fig. 8 - Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2) summary of items for risk of bias and applicability in included studies (n = 30). Green color depicts the low risk of biasedness, yellow color depicts the unclear risk of bia- sedness, and red color depicts the high risk of biasedness. reading the results. Hence, index test bias of these studies was unclear. For the rest of the included studies, almost all (n = 30) have low risk of reference standard bias because they provided enough information about the standard reference test used in the study. Half of the studies (15 out of 30) have an unclear risk of flow and timing bias as there is not enough information, whether reference standard results were interpreted with the knowledge of the results of the index test. One study (24) was Bumbrah et al Drug Target Insights 2023; 17: 87 © 2023 The Authors. Published by AboutScience - www.aboutscience.eu pulmonary TB (41), and extrapulmonary TB (42), an updated meta-analysis covering all forms of mycobacteria was still missing. Hence, the aim of the present study was to system- atically review and perform the meta-analysis to assess the diagnostic accuracy of the LAMP assay for detection of all forms of mycobacteria. This meta-analysis revealed that most of the studies were conducted in high TB burden countries such as India, Thailand, and Japan (Fig. 2). We observed that for the detec- tion of mycobacteria sputum could be considered as the most chosen sample (Fig. 3). When considering the target genes, we found a variety of genes that were used in the included studies. However, IS6110 ranked first among all evaluated genes in the included studies (Fig. 4). This occurrence could be due to the presence of multiple copies of IS6110 present in the MTB genome (43). However, other target genes such as 16s rRNA and gyrB were also prominent. Next, we consid- ered the detection method that was used for assessing the LAMP results. Most of the studies used fluorescence-based methods followed by colorimetry, gel electrophoresis, and turbidity, with no justification of their choices (Fig. 5). The prominence of fluorescence methods could be due to their increased sensitivity for the detection. Exceptionally, only one study mentioned lateral flow-based detection method despite market applicability. Forest plot was used to calculate the sensitivity and speci- ficity. The pooled sensitivity values of meta-analysis ranged between 0.26 and 1.0 (Fig. 6) and forest plot and SROC curve revealed a pooled specificity value between 0.67 and 1.0 (Fig. 7) with 95% CI. The accuracy and precision were calcu- lated for the included studies and for 16 studies we found that the accuracy rate was higher than their corresponding precision rates and vice versa for 14 articles upon intra-com- parison of accuracy with precision (Tab. II). The current study also exhibited few limitations. Firstly, we observed high risk of patient selection bias or index test bias in almost two-thirds of the eligible studies (Fig. 8). Therefore, the use of unbiased patient cohorts and double-blinded index test may be recommended for future studies. Secondly, few stud- ies showed the highest performance with 100% sensitivity and specificity, respectively, hence displaying the lowest QUADAS risk and concerns in all the domains. Furthermore, lack of subgroup analysis and the use of solely peer-reviewed English language articles were also additional limitations. Hence, although the current meta-analysis should be interpreted with caution, however, we believe that it will not impact the robust- ness of the analysis leading to further improved studies and reviews. Particularly considering the growing significance of LAMP-based detection for TB comparable to other methods, such studies may be encouraged (43-45). Conclusion Despite suffering from few disadvantages, like false positiv- ity due to heavy reliance on indirect detection methods such as turbidity and nonspecific dyes and not providing any addi- tional benefits like information on mutations, drug resistance etc., the LAMP technique could be a promising molecular test to enhance case detection before conventional time-consum- ing culture. Its simplicity, less turnaround time, and cost-effec- tiveness are major attractions for clinical laboratories. Also, it will be unjust to rely on single point-of-care test for TB suc- cessfully in various kinds of populations and resource avail- ability. Although the unit cost is higher than smear microscopy and culture-based methods, it is likely to offer good value for money relative to conventional methods. In a nutshell, the present study endorses the use of LAMP assay as a promis- ing alternative for detection of mycobacteria, particularly in regions which are financially compromised, where drug-resis- tant strains are not prevalent and PCR-based tests cannot be done so frequently. The faster diagnosis through LAMP could provide an alternative solution for failed medications to cur- rent therapeutics due to delayed diagnosis and subsequent development of drug resistance, thereby providing an oppor- tunity to employ this new information in improving treatment strategies. However, the LAMP assay still must be improved to turn to a strong and competitive alternative to other molecu- lar diagnostic methods. Acknowledgments We are grateful to Akansha Bhatt and Reva Gautam for their assistance in reviewing the literature and to Muriel Billamboz for assistance in English language editing. Author contributions G.S.B.: search, data extraction, validation. G.S.B., S.J.: data analysis. 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