Abstract

A systematic review with meta-analysis was performed to estimate the accuracy of IgM ELISA for Leptospirosis diagnosis. A search of Medline, Lilacs, Embase, Cochrane Central Register of Controlled Trials and Grey literature (Google Scholar and British Library) was conducted. The medical subject headings (MeSHs) and the words “leptospirosis”, “human leptospirosis” and “IgM ELISA” were used. Fifty-two studies were analyzed, which included 10,775 samples. The pooled sensitivity of all the studies was 86% (CI 95%, 85%-87%) and specificity was 90% (CI 95%, 89%-91%). In studies of the acute phase, the sensitivity and specificity were 84% (CI 95%, 82%-85%) and 91% (CI 95%, 90%-91%), respectively. In conclusion, IgM ELISA is sensitive for use as an initial screen for leptospiral infections.

Key words
Human leptospirosis; IgM ELISA; Leptospirosis diagnosis; Meta-analysis

Resumo

O objetivo desta revisão sistemática e meta-análise foi avaliar a acurácia do ELISA IgM para o diagnóstico precoce da leptospirose em humanos. A busca foi realizada nas seguintes bases de dados: Medline, PubMed, LILACS, Embase e Cochrane Central Register of Controlled Trials e Grey literature (Google Scholar and British Library). As palavras-chaves usadas foram: “leptospirosis”, “human leptospirosis” e “IgM ELI-SA”. Foram analisados 52 estudos, que incluíram 10.775 amostras. A sensibilidade e especificidade combinada de todos os estudos foram 86% (CI 95%, 85%-87%) e 90% (CI 95%, 89%-91%), respectivamente. Nos estudos de fase aguda, a sensibilidade e especificidade foram, respectivamente, 84% (CI 95%, 82%-85%) e 91% (CI 95%, 90%-91%). Conclui-se que o ELISA IgM é um teste sensível para rastreamento inicial da leptospirose.

Palavras-chave
Leptospirose humana; ELISA IgM; Diagnóstico; Meta-análise

Introduction

Leptospirosis is a neglected infectious disease caused by spirochetes from the genus Leptospira. It constitutes the most widespread zoonosis and is emerging as a major public health problem with outcomes ranging from subclinical infections to fatal pulmonary hemorrhage and Weil's syndrome¹1. Pappas G, Papadimitriou P, Siozopoulou V, Christou L, Akritidis N. The globalization of leptospirosis: worldwide incidence trends. Int J Infect Dis 2008;12(4):351-357..

Leptospirosis has a broad geographical distribution, occurring in both rural and urban areas of tropical, subtropical and temperate regions. The disease outbreaks in developed countries are usually associated with occupational exposure, tourism or sporting events¹1. Pappas G, Papadimitriou P, Siozopoulou V, Christou L, Akritidis N. The globalization of leptospirosis: worldwide incidence trends. Int J Infect Dis 2008;12(4):351-357..

Leptospirosis is transmitted by contact of abraded skin or mucous membranes with water or soil contaminated with urine from reservoir animals, such as rodents²2. Bharti AR, Nally JE, Ricaldi JN, Matthias MA, Diaz MM, Lovett MA, Levett PN, Gilman RH, Willig MR, Gotuzzo E, Vinetz JM; Peru-United States Leptospirosis Consortium. Leptospirosis: a zoonotic disease of global importance. Lancet Infect Dis 2003;3(12):757-771.. More than 500.000 cases of severe leptospirosis are reported each year, with mortality rates exceeding 10%³3. World Health Organization (WHO). Report of the Second Meeting of the Leptospirosis Burden Epidemiology Reference Group (LERG). WHO: Geneva; 2011.. A new global estimate estimates that the overall annual incidence is 1 million cases and 60,000 deaths⁴4. Hagan JE, Costa J, Calcagno M, Kane M, Torgerson P, Martinez-Silveira MS, Stein C, Abela-Ridder B, Ko AI. Global morbidity and mortality of leptospirosis: a systematic review. PLoS Negl Trop Dis 2015; 9(9):e0003898.

The microscopic agglutination test (MAT) is most often used as a reference test⁵5. Limmathurotsakul D, Turner EL, Wuthiekanun V, Thaipadungpanit J, Suputtamongkol Y, Chierakul W, Smythe LD, Day NP, Cooper B, Peacock SJ. Fool's gold: why imperfect reference tests are undermining the evaluation of novel diagnostics: a re-evaluation of 5 diagnostic tests for leptospirosis. Clin Infect Dis 2012;55(3):322-331.. Standard tests are tedious, laborious and require well-equipped laboratories with experienced staff and are therefore restricted to a few centers. Because the initial presentation of leptospirosis may be difficult to discern from other infectious diseases, rapid and accurate diagnosis is essential to prevent the progression of the more severe form of the disease, particularly in developing countries²2. Bharti AR, Nally JE, Ricaldi JN, Matthias MA, Diaz MM, Lovett MA, Levett PN, Gilman RH, Willig MR, Gotuzzo E, Vinetz JM; Peru-United States Leptospirosis Consortium. Leptospirosis: a zoonotic disease of global importance. Lancet Infect Dis 2003;3(12):757-771..

Traditional serological methods, such as the ELISA, are widely used to diagnose leptospirosis. Antileptospires IgM may be detected 4 to 5 days after the onset of symptoms, before detection of IgG and agglutinating antibodies, and persist at least 5 months in patients⁶6. Silva MV, Camargo ED, Batista L, Vaz AJ, Brandão AP, Nakamura PM, Negrão JM. Behaviour of specific IgM, IgG and IgA class antibodies in human leptospirosis during the acute phase of the disease and during convalescence. J Trop Med Hyg 1995;98(4):268-272.. ELISA can be performed with minimal training and typically provides results in 2–4 hours. The aim of this study was to perform a systematic review and meta-analysis of the literature to verify the accuracy of the IgM ELISA for leptospirosis diagnosis.

Methods

All methods for analysis, inclusion/exclusion criteria, data extraction and quality assessment were specified in advance. It was performed a systematic review according to a prospective protocol using PRISMA–statement guidelines⁷7. Liberati A, Altman DG, Tetzlaff J, Mulrow C, Gøtzsche PC, Ioannidis JP, Clarke M, Devereaux PJ, Kleijnen J, Moher D. The PRISMA statement for reporting systematic reviews and meta-analysis of studies that evaluate healthcare interventions: explanation and elaboration. BMJ 2009;339:b2700.^,88. Medeiros LR, Rosa MI, Silva BR, Reis ME, Simon CS, Dondossola ER, Cunha Filho JS. Accuracy of magnetic resonance in deeply infiltrating endometriosis: a systematic review and meta-analysis. Arch Gynecol Obstet 2015;291(3):611-621.. The review protocol is registered at PROSPERO (International prospective register of systemic reviews, http://www.crd.york.ac.uk/prospero; CRD42014 009784).

The electronic databases Medline via Pubmed, Lilacs (through Scielo interface), Cochrane Central Register of Controlled Trials, Embase and Grey literature (Google Scholar and British Library) were searched for papers published from January 1969 to July 2014. The following terms were used, both as text words and, as appropriate, Medical Subjects Heading (MeSH), or equivalent subject heading/thesaurus terms: Leptospirosis, Human Leptospirosis and IgM ELISA

This sensitive filter was created by combining three filters to identify diagnostic studies via the Boolean operators “OR” and “AND”. The search was limited to human studies and had no language restrictions. Reference lists of all available primary studies were reviewed to identify additional relevant citations. The complete search strategy is available on request.

Abstracts/titles identified from the search were screened by two reviewers. Disagreements about study inclusion or exclusion were initially solved by consensus, and if agreement was not possible, they were arbitrarily resolved by a third reviewer.

Cross-sectional and cohort studies, prospective and retrospective, which evaluated IgM enzyme-linked immunosorbent assay (Elisa) in Leptospirosis diagnosis were included. Studies that used the index test IgM Elisa to diagnose leptospirosis in patients were analyzed. The diagnostic reference standard was the result of the MAT with confirmation based on the result on the same serum sample as used for the index test. Therefore, the primary outcome analyzed was the presence of Leptospirosis.

It was extracted data on the studies, patients and test characteristics using a standardized form. Data were abstracted as 2 x 2 tables regarding IgM Elisa vs MAT in leptospirosis diagnosis (positive vs negative by cut-off). It was also calculated the sensitivities, specificities, and Odds Ratio diagnostic (DOR). Studies that lacked the data needed to construct 2 x 2 contingency tables were excluded. The assessment of non–English–language articles was performed independently following translation (if necessary). Any disagreement was resolved by consensus for studies published in all languages. Final inclusion or exclusion was made with reference to a selection criteria checklist.

Disagreements about study inclusion or exclusion were initially solved by consensus, and if agreement was not possible, they were arbitrarily resolved by another reviewer. The agreement statistics among reviewers were computed.

The methodological quality assessment for diagnostic accuracy was performed according to criteria from the Quality Assessment of Diagnostic Accuracy Studies (QUADAS 2)⁹9. Whiting PF, Rutjes AW, Westwood ME, Mallett S, Deeks JJ, Reitsma JB, Leeflang MM, Sterne JA, Bossuyt PM; QUADAS-2 Group. QUADAS-2: a revised tool for the quality assessment of diagnostic accuracy studies. Ann Intern Med 2011;155(8):529-536.. QUADAS-2 is designed to assess the quality of primary diagnostic accuracy studies, and it consists of four domains: patient selection, index test, reference standard, and flow of patients through the study and timing of the index tests(s) and reference standard “flow and timing”. Signaling questions are included to help judge the risk of bias⁸8. Medeiros LR, Rosa MI, Silva BR, Reis ME, Simon CS, Dondossola ER, Cunha Filho JS. Accuracy of magnetic resonance in deeply infiltrating endometriosis: a systematic review and meta-analysis. Arch Gynecol Obstet 2015;291(3):611-621.. The Quality assessment of studies was independently performed using the Review Manager 5.2 software¹⁰10. Review Manager (RevMan) [computer program]. Version 5.2. 2012 Copenhagen: The Nordic Cochrane Centre, The Cochrane Collaboration. Altman DG. Some common problems in medical research. In: Altman DG, editor. Practical statistics for medical research. 9ª ed. London: Chapman; 1999. p. 396-439..

The rates were calculated as true positive (TPR, sensitivity), false positive (FPR, 1 – specificity), true negative (TN) and false negative (FN)¹¹11. Altman DG. Some common problems in medical research. In: Altman DG (eds). Practical statistics for medical research, 9 ed. London: Chapman; 1999. p. 396-439.. If any cell containing “0” was present in the contingency table, 0,5 was added to each cell to facilitate the calculations; if the study contained two cells with “0”, the study was excluded from the analysis¹²12. Irwig L, Tosteson AN, Gatsonis C, Lau J, Colditz G, Chalmers TC, Mosteller F. Guidelines for meta-analyses evaluating diagnostic tests. Ann Intern Med 1994; 120(8):667-676..

Bivariate analysis was used to calculate pooled estimates of sensitivity, specificity, and DOR in addition to 95% confidence intervals (CIs) for the summary estimates¹³13. Reitsma JB, Glas AS, Rutjes AWS, Scholten RJ, Bossuyt PM, Zwinderman AH. Bivariate analysis of sensitivity and specificity produce informative summary measures in diagnostic reviews. J Clin Epidemiol 2005; 58(10):982-990.. The bivariate model preserves the 2-dimensional nature of diagnostic data by analyzing the logit transformed sensitivity and specificity of each study in a single model and considers both within-study and be-tween-study variability, in contrast to the Littenberg and Moses method that departs from a fixed effects model¹⁴14. Gatsonis C, Paliwal P. Meta-analysis of diagnostic and screening test accuracy evaluations: methodologic primer. AJR Am J Roentgenol 2006; 187(2):271-281.. To detect cut-off threshold effects, the relationship between sensitivity and specificity was evaluated by the Spearman's correlation coefficient. Pooled estimates were only calculated for studies showing sufficient clinical and statistical homogeneity. I²2. Bharti AR, Nally JE, Ricaldi JN, Matthias MA, Diaz MM, Lovett MA, Levett PN, Gilman RH, Willig MR, Gotuzzo E, Vinetz JM; Peru-United States Leptospirosis Consortium. Leptospirosis: a zoonotic disease of global importance. Lancet Infect Dis 2003;3(12):757-771. or Q tests (commonly used in meta-analysis) are not recommended for assessing statistical homogeneity in diagnostic reviews because they do not consider the association between sensitivity and specificity¹⁵15. Deeks JJ, Macaskill P, Irwig L. The performance of tests of publication bias and other sample size effects in systematic reviews of diagnostic test accuracy was assessed. J Clin Epidemiol 2005; 58(9):882-893.. The DOR can relate to different combinations of sensitivities and specificities and describes the odds of the positive test resulting in participants with the disease compared with the odds of a positive test resulting in those without disease. A single diagnostic odds ratio corresponds to a set of sensitivities and specificities depicted by the SROC. It can change according to the threshold and to the ROC curve used to define an abnormal examination resulted in the expected trade-off between sensitivity and specificity.

A summary receiver operating characteristic curve was generated using data from all thresholds using the Littenberg and Moses method. Additionally, the area under the curve (AUC) can summarize the inherent capacity of a test for discriminating a diseased from a non-diseased subject. Accurate tests usually have AUCs close to 1, and poor tests usually have AUCs close to 0.5¹⁶16. Stata Corporation. Stata Statistical Software version 11: College Station, TX/Stata Corporation; 2009.. Sensitivity analyses were performed to assess excluding studies with a high risk of verification bias according to QUADAS 2. To analyze publication bias, inverted funnel plots of the logarithmic odds ratio (OR) of individual studies were plotted against sample size¹⁵15. Deeks JJ, Macaskill P, Irwig L. The performance of tests of publication bias and other sample size effects in systematic reviews of diagnostic test accuracy was assessed. J Clin Epidemiol 2005; 58(9):882-893..

The statistical analysis was performed with the software Stata 11¹⁷17. Zamora J, Abraira V, Muriel A, Khan K, Coomarasamy A. Meta-DiSc: a software for meta-analysis of test accuracy data. BMC Med Res Methodol 2006; 6:31., Meta-DiSc^®1818. Terpstra WJ, Ligthart GS, Schoone GJ. Serodiagnosis of human leptospirosis by enzyme-linked-immuno-sorrbent-assay (ELISA). Zentralbl Bakteriol A 1980; 247(3):400-405. (version 1.4), and Review Manager 5.2¹⁰10. Review Manager (RevMan) [computer program]. Version 5.2. 2012 Copenhagen: The Nordic Cochrane Centre, The Cochrane Collaboration. Altman DG. Some common problems in medical research. In: Altman DG, editor. Practical statistics for medical research. 9ª ed. London: Chapman; 1999. p. 396-439..

Results

A total of 545 studies were identified: 510 studies were identified using the database search and 35 additional records were identified through other sources. Seventy-nine full-text articles were retrieved; 27 were excluded after further scrutiny. Fifty-two primary studies, including 10,775 serum samples, met the criteria for inclusion and were included in the meta-analysis 19-69 (Figure 1).

Details of the participants and interventions are summarized in Table 1 ¹⁸18. Terpstra WJ, Ligthart GS, Schoone GJ. Serodiagnosis of human leptospirosis by enzyme-linked-immuno-sorrbent-assay (ELISA). Zentralbl Bakteriol A 1980; 247(3):400-405.^–6969. Vedhagiri K, Velineni S, Timoney JF, Shanmughapriya S, 1 Vijayachari P, Narayanan R, Natarajaseenivasan K. Detection of LipL32-specific IgM by ELISA in sera of patients with a clinical diagnosis of leptospirosis. Pathog Glob Health 2013;107(3):130-135.. Most studies were prospective, except for two⁴¹41. Bajani MD, Ashford DA, Bragg SL, Woods CW, Aye T, Spiegel RA, Plikaytis BD, Perkins BA, Phelan M, Levett PN, Weyant RS. Evaluation of four commercially available rapid serologic tests for diagnosis of leptospirosis. J Clin Microbiol 2003;41(2):803-809.^,4444. Obregón AM, Martínez G, Martínez R, Llop A, Rodríguez I, Rodríguez J, Fernández C. Serological response by ELISA and MAT in Cuban volunteers vaccinated with vax SPIRAL. Rev Cubana Med Trop 2004;56(2):148-1451..

The quality assessment results are presented in Figure 2 ¹⁹19. Pappas MG, Ballou WR, Gray MR, Takafuji ET, Miller RN, Hockmeyer WT. Rapid serodiagnosis of leptospirosis using the IgM-specific Dot-ELISA: comparison with the microscopic agglutination test. Am J Trop Med Hyg 1985; 34(2):346-354.^–6969. Vedhagiri K, Velineni S, Timoney JF, Shanmughapriya S, 1 Vijayachari P, Narayanan R, Natarajaseenivasan K. Detection of LipL32-specific IgM by ELISA in sera of patients with a clinical diagnosis of leptospirosis. Pathog Glob Health 2013;107(3):130-135.. Thirteen studies fulfilled all criteria of QUADAS 2¹⁹19. Pappas MG, Ballou WR, Gray MR, Takafuji ET, Miller RN, Hockmeyer WT. Rapid serodiagnosis of leptospirosis using the IgM-specific Dot-ELISA: comparison with the microscopic agglutination test. Am J Trop Med Hyg 1985; 34(2):346-354.^,2020. Da Silva MV, Camargo ED, Vaz AJ, Souza, AMC, Ueda M, Sakata, EE. Teste imunoenzimático (ELISA) para detecção de anticorpos circulantes da classe IgM na leptospirose humana. Rev Inst Med Trop São Paulo [online]1988; [acessado 2017 Ago 25];30(2):95-100. Disponível em: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0036-46651988000200008&lng=pt.
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http://www.scielo.br/scielo.php?script=s... ^,4444. Obregón AM, Martínez G, Martínez R, Llop A, Rodríguez I, Rodríguez J, Fernández C. Serological response by ELISA and MAT in Cuban volunteers vaccinated with vax SPIRAL. Rev Cubana Med Trop 2004;56(2):148-1451. and two studies showed unclear risk of bias in the flow timing³⁹39. Effler PV, Bogard AK, Domen HY, Katz AR, Higa HY, Sasaki DM. Evaluation ofeight rapid screening tests for acute leptospirosis in Hawaii. J Clin Microbiol 2002;40(4):1464-1469.^,4545. Vitale G, La Russa C, Galioto A, Chifari N, Mocciaro C, Caruso R, Micalizzi A, Mansueto P, Di Rosa S, Mansueto S. Evaluation of an IgM-ELISA test for the diagnosis of human leptospirosis. New Microbiol 2004;27(2):149-154.. Two studies have indicated high risk of bias in the patient selection in the applicability criteria⁵⁰50. McBride AJ, Santos BL, Queiroz A, Santos AC, Hartskeerl RA, Reis MG, Ko AI. Evaluation of four whole-cell Leptospira-based serological tests for diagnosis of urban leptospirosis. Clin Vaccine Immunol 2007;14(9):1245-1248.^,5151. McBride AJ, Pereira FA, da Silva ED, Ferreira AG, Reis MG, Ko AI. Evaluation of the EIE-IgM-Leptospirose assay for the serodiagnosis of leptospirosis. Acta Trop 2007;102(3):206-211., and two studies demonstrated a high risk of bias in evaluating the index test⁴⁸48. de Abreu Fonseca C, Teixeira de Freitas VL, Caló Romero E, Spinosa C, Arroyo Sanches MC, da Silva MV, Shikanai-Yasuda MA. Polymerase chain reaction incomparison with serological tests for early diagnosis of human leptospirosis. Trop Med Int Health 2006;11(11):1699-1707.^,6565. Kumar A, Rajasekar V, Selvakumar G, Dhanalakshmi DP. Comparison study between in-house IgM DOT-ELISA and the microscopic agglutination test (MAT) for the diagnosis of human leptospirosis. Int Research J of Pharmacy 2012;3(4):314-317.. In the other studies, there were some unclear applicability criteria in the index test and reference standard¹⁹19. Pappas MG, Ballou WR, Gray MR, Takafuji ET, Miller RN, Hockmeyer WT. Rapid serodiagnosis of leptospirosis using the IgM-specific Dot-ELISA: comparison with the microscopic agglutination test. Am J Trop Med Hyg 1985; 34(2):346-354.^,2121. Da Silva MV, Camargo ED, Vaz AJ, Souza AMC, Chieffi PP, Sakata EE. Imunodiagnóstico da leptospirose humana através do teste ELISA-IgM, empregando-se diferentes preparações antigênicas a partir de sorotipos prevalentes de Leptospira interrogans. Rev Inst Med Trop São Paulo [online] 1990 [acessado 2017 Ago 25]; 32(4):233-239. Disponível em: http://www.scielo.br/scielo.php?pid=S0036-46651990000400001&script=sci_abstract&tlng=pt
http://www.scielo.br/scielo.php?pid=S003... ^,2323. Cinco M, Balanzin A, Banfi DAE. Evaluation of an immunoenzimatic test (ELISA) for the diagnosis of leptospirosis in Italy. Eur J of Epidemiology 1992;8(5):677-682.^–2626. Ribeiro MA, Brandão AP, Romero EC. Evaluation of diagnostic tests for human leptospirosis. Braz J Med Biol Res 1996;29(6):773-777.^,2929. Da Silva MV, Nakamura PM, Camargo ED, Batista L, Vaz AJ, Romero EC, Brandao AP. Immunodiagnosis of human leptospirosis by dot-ELISA for the detection of IgM, IgG, and IgA antibodies. Am J Trop Med Hyg 1997;56(6):650-655.^,3030. Brandão AP, Camargo ED, da Silva ED, Silva MV, Abrão RV. Macroscopic agglutination test for rapid diagnosis of human leptospirosis. J Clin Microbiol 1998;36(11):3138-3142.^,3232. Smits HL, van der Hoorn MA, Goris MG. Simple latex agglutination assay for rapid serodiagnosis of human leptospirosis. J Clin Microbiol 2000;38(3):1272-1275.^,3434. Levett PN, Branch SL, Whittington CU, Edwards CN, Paxton H. Two methods for rapid serological diagnosis of acute leptospirosis. Clin Diagn Lab Immunol 2001;8(2):349-351.^,3535. Smits HL, Chee HD, Eapen CK, Kuriakose M, Sugathan S, Gasem MH, Yersin C, Sakasi D, Lai-A-Fat RF, Hartskeerl RA, Liesdek B, Abdoel TH, Goris MG, Gussenhoven GC. Latex based, rapid and easy assay for human leptospirosis in a single test format. Trop Med Int Health 2001;6(2):114-118.^,3838. Céspedes Z M, Glenny A M, Felices AV, Balda J L, Suárez MV. Prueba de Elisa indirecta para la detección de anticuerpos IgM para el diagnóstico de Leptospirosis humana. Revista Peruana de Medicina Experimental y Salud Pública 2002;19(1):1924-1927.^,4040. Levett PN, Branch SL. Evaluation of two enzyme-linked immunosorbent assay methods for detection of immunoglobulin M antibodies in acute leptospirosis. Am J Trop Med Hyg 2002;66(6):745-748.^,4242. Sehgal SC, Vijayachari P, Sugunan AP, Umapathi T. Field application of Lepto lateral flow for rapid diagnosis of leptospirosis. J Med Microbiol 2003;52(Pt 10):897-901.^,4343. Nakarin J, Pradutkanchana S. Evaluation of enzyme-linked immunosorbent assay and indirect hemagglutination assay for detection of leptospiral antibody by using three different antigens. J Med Assoc Thai 2004;87(10):1218-1224.^,4646. Tansuphasiri U, Deepradit S, Phulsuksombati D, Tangkanakul W. Two simple immunoassays using endemic leptospiral antigens for serodiagnosis of human leptospirosis. Southeast Asian J Trop Med Public Healt. 2005;36(2):302-311.^,4747. Blacksell SD, Smythe L, Phetsouvanh R, Dohnt M, Hartskeerl R, Symonds M, Slack A, Vongsouvath M, Davong V, Lattana O, Phongmany S, Keolouangkot V, White NJ, Day NP, Newton PN. Limited diagnostic capacities of two commercial assays for the detection of Leptospira immunoglobulin M antibodies in Laos. Clin Vaccine Immunol 2006;13(10):1166-1169.^,4949. Ooteman MC, Vago AR, Koury MC. Evaluation of MAT, IgM ELISA and PCR methodsfor the diagnosis of human leptospirosis. J Microbiol Methods 2006;65(2):247-257.^,5353. Dey S, Mohan CM, Ramadass P, Nachimuthu K. Diagnosis of leptospirosis by recombinant antigen based single serum dilution ELISA. Indian J Med Res 2008;128(2):172-177.^,5454. Honarmand HR, Nezafat TM, Heydarzadeh A, Soltani B, Mirzajani E, Asmar M. Evaluation an in–house IgM-ELISA for the diagnosis of human leptospirosis. Journal of Semnan University of Medical Sciences 2008;9(4):309-313.^,5757. Pol S, Bharadwaj R. Evaluation of high performance liquid chromatography purified leptospiral antigen for the diagnosis of leptospirosis. Jpn J Infect Dis 2009;62(6):428-431.^,6464. Desakorn V, Wuthiekanun V, Thanachartwet V, Sahassananda D, Chierakul W, Apiwattanaporn A, Day NP, Limmathurotsakul D, Peacock SJ. Accuracy of a commercial IgM ELISA for the diagnosis of human leptospirosis in Thailand. Am J Trop Med Hyg 2012;86(3):524-527.^,6767. Bourhy P, Vray M, Picardeau M. Evaluation of an in-house ELISA using the intermediate species Leptospira fainei for diagnosis of leptospirosis. J Med Microbiol 2013;62(Pt 6):822-857.^–6969. Vedhagiri K, Velineni S, Timoney JF, Shanmughapriya S, 1 Vijayachari P, Narayanan R, Natarajaseenivasan K. Detection of LipL32-specific IgM by ELISA in sera of patients with a clinical diagnosis of leptospirosis. Pathog Glob Health 2013;107(3):130-135..

The robustness of the results was tested by repeating the analysis using a different statistical model (random effects model). Some studies were identified as outliers, and one re-analysis was performed without them. However, no significant difference was found in the sensitivity or specificity; therefore, those papers were not excluded from the meta-analysis.

All 52 studies selected were included in the meta-analysis. Statistical analyses were performed on both the acute and unspecific phase and only the acute phase. Analysis with excluding particular studies with high risk of bias ⁴⁸48. de Abreu Fonseca C, Teixeira de Freitas VL, Caló Romero E, Spinosa C, Arroyo Sanches MC, da Silva MV, Shikanai-Yasuda MA. Polymerase chain reaction incomparison with serological tests for early diagnosis of human leptospirosis. Trop Med Int Health 2006;11(11):1699-1707.^,6565. Kumar A, Rajasekar V, Selvakumar G, Dhanalakshmi DP. Comparison study between in-house IgM DOT-ELISA and the microscopic agglutination test (MAT) for the diagnosis of human leptospirosis. Int Research J of Pharmacy 2012;3(4):314-317. in relation to the index test were conducted, and because there was no significant change they were maintained the meta-analysis.

IgM ELISA for the diagnosis of human Leptospirosis had a pooled sensitivity in all studies of 0.86 (95% CI, 0.85 – 0.87). The pooled specificity in all studies was 0.90 (95% CI, 0.89 – 0.91). The estimates for heterogeneity were highly consistent across studies: sensitivity: QT = 914.77, P-value < 0.0001; inconsistency I² = 94.4%; and specificity: QT = 738.48, P-value < 0.0001; inconsistency I² = 93.1% (Figure 3).

IgM ELISA for the diagnosis of human leptospirosis had a pooled sensitivity in the acute phase of 0.84 (95% CI, 0.82 – 0.85), and the specificity of Leptospirosis in the acute phase was 0.91 (95% CI, 0.90 - 0.91). The estimates for heterogeneity were highly consistent across studies: sensitivity: QT = 764.77, P-value < 0.0001; I² = 95.3%; and specificity: QT = 435.55, P-value < 0.0001; I² = 91.7% (data not shown).

The DOR was 82.06 (95% CI, 45.77-147.12), QT=595.94, P-value = 0.001 in all studies and 67.11 (95% CI, 33.53-134.29), QT = 426.33, P-value = 0.001 in the acute phase (data not shown).

SROC curves were constructed due to heterogeneity in the DOR. The AUC for the ROC curve was estimated by a trapezoidal rule 95. The resulting summary ROC curves are shown with operating points for sensitivity and specificity. The AUC was 0.960 in all studies and 0.952 in the acute phase respectively (Figure 4).

Covariable-type studies were separated into prospective and retrospective design, and the meta-regression analysis indicated no association between type of studies and outcome (P = 0.32).

Begg's funnel plot and Egger's test were performed to assess the publication bias of the literature in all comparison models. The shape of the funnel plot reveals any evidence of obvious asymmetry. Then, the Egger's test was used to provide statistical evidence of funnel plot symmetry for total phase (P for bias = 0.001) and acute phase (P for bias = 0.008), indicating publication bias (data not shown).

Discussion

In summary, this systematic review showed that IgM ELISA in all phases had a sensitivity of 0.86 and specificity of 0.84, whereas the acute phase had a sensitivity of 0.90 and specificity of 0.91.

The results showed that IgM ELISA could be useful as a screening and a confirmatory test, especially in regions with small laboratories that have difficulty performing other techniques such as MAT.

A recent systematic review included 35 studies up to 2010 and analyzed ELISA (IgM, IgG and IgA). In the present study, 55 studies with IgM only were included and analyzed the accuracy of IgM in the acute phase of the disease. We found a higher sensitivity compared to IgM results Signorini et al.⁷¹71. Agampodi SB, Matthias MA, Moreno AC, Vinetz JM. Utility of quantitative polymerase chain reaction in leptospirosis diagnosis: association of level of leptospiremia and clinical manifestations in Sri Lanka. Clin Infect Dis 2012;54(9):1249-1255., 86 versus 80%, respectively.

It was found high heterogeneity between studies. It is expected in meta-analyses of diagnostic test accuracy because it comes from observational studies, study designs and different cutoff points. This high heterogeneity was also observed in the meta-analysis performed by Signorini et al.⁷¹71. Agampodi SB, Matthias MA, Moreno AC, Vinetz JM. Utility of quantitative polymerase chain reaction in leptospirosis diagnosis: association of level of leptospiremia and clinical manifestations in Sri Lanka. Clin Infect Dis 2012;54(9):1249-1255..

A rapid diagnostic test provides a quick test result but does not indicate an early test. The ideal rapid test should have high accuracy, be easy to perform, interpret, inexpensive, and stable and give the result within 2 hours⁷⁰70. Banoo S, Bell D, Bossuyt P, Herring A, Mabey D, Poole F, Smith PG, Sriram N, Wongsrichanalai C, Linke R, O'Brien R, Perkins M, Cunningham J, Matsoso P, Nathanson CM, Olliaro P, Peeling RW, Ramsay A. Evaluation of diagnostic tests for infectious diseases: general principles. Nat Rev Microbiol 2010;8(Suppl.):S17-29..

There are two phases of Leptospira infection: (1) between 3-7 days or acute septicemic phase with nonspecific symptoms such as myalgia and headache. The leptospires are detectable in the blood stream, decrease until 15 days⁷²72. Signorini ML, Lottersberger J, Tarabla HD, Vanasco NB. Enzyme-linked immunosorbent assay to diagnose human leptospirosis: a meta-analysis of the published literature. Epidemiol Infect. 2013;141(1):22-32 and (2) the start in the second week after the onset of symptoms, and the antibodies usually persist for several months⁶6. Silva MV, Camargo ED, Batista L, Vaz AJ, Brandão AP, Nakamura PM, Negrão JM. Behaviour of specific IgM, IgG and IgA class antibodies in human leptospirosis during the acute phase of the disease and during convalescence. J Trop Med Hyg 1995;98(4):268-272.. During this phase, leptospires are eliminated from the blood stream as IgM antibodies increase⁷³73. Levett PN. Leptospirosis. Clinical Microbiology Reviews 2001; 14(2):296-326..

The rapid test depends on the detectable presence of anti-Leptospira antibodies already presented during the acute phase of the disease⁷⁴74. Ahmed A, Anthony RM, Hartskeerl RA. A simple and rapid molecular method for Leptospira species identification. Infect Genet Evol 2010;10(7):955-962.. Molecular tests that detect the causative agent can be confirmed during the first 5 days after the onset of the disease⁷⁵75. Ahmed A, Klaassen HLMB, Van der Veen M, van der Linden H, Goris MGA, Hartskeerl RA. Evaluation of real-time PCR and culturing for the detection of leptospires in canine samples. Advances in Microbiology 2012;2(2):162-170.. It is very important that a test be rapid and sensitive, because the earlier the diagnosis the faster the treatment decision.

Whereas molecular tests, such as the polymerase chain reaction (PCR), that demonstrate the presence of the causative agent in a clinical sample mainly during the first 5 days after the onset of the disease (DPO), serological tests depend on the accumulation of detectable amounts of anti-Leptospira antibodies in the late acute to convalescent samples⁷⁴74. Ahmed A, Anthony RM, Hartskeerl RA. A simple and rapid molecular method for Leptospira species identification. Infect Genet Evol 2010;10(7):955-962.^–7676. Goris MGA, Leeflang MMG, Boer KR, Goeijenbier M, van Gorp ECM4, Wagenaar JFP, Hartskeerl RA. Establishment of valid laboratory case definition for human leptospirosis. J Bacteriol Parasitol [periódico na Internet]. 2012 [acessado 2017 Ago 25];3. Disponível em: 10.4172/2155-9597.1000132
https://doi.org/10.4172/2155-9597.100013... .

Rapid diagnostic tests should ideally be accurate, simple to use, relatively inexpensive, easy to interpret, stable under extreme conditions, require little or no processing, and give the results within 1–2 hours⁷⁰70. Banoo S, Bell D, Bossuyt P, Herring A, Mabey D, Poole F, Smith PG, Sriram N, Wongsrichanalai C, Linke R, O'Brien R, Perkins M, Cunningham J, Matsoso P, Nathanson CM, Olliaro P, Peeling RW, Ramsay A. Evaluation of diagnostic tests for infectious diseases: general principles. Nat Rev Microbiol 2010;8(Suppl.):S17-29.. Again, it is very important that a test be rapid and sensitive, because the earlier the diagnosis the faster the treatment decision.

Often, an early diagnosis or reference standard is employed in referral centers where confirmation is performed by experts. The rapid diagnosis is highly useful at the peripheral facilities and might be integral for early outbreak warning and useful for monitoring outbreaks if a rapid unusual accumulation of cases might provide an early alert, provided that specimens are collected, transported, and stored in an adequate manner⁷⁶76. Goris MGA, Leeflang MMG, Boer KR, Goeijenbier M, van Gorp ECM4, Wagenaar JFP, Hartskeerl RA. Establishment of valid laboratory case definition for human leptospirosis. J Bacteriol Parasitol [periódico na Internet]. 2012 [acessado 2017 Ago 25];3. Disponível em: 10.4172/2155-9597.1000132
https://doi.org/10.4172/2155-9597.100013... .

This review, which included retrospective and prospective studies, had the following limitations: i) high heterogeneity found between studies; ii) use of selected samples and the choice of case definition may be a source of bias; and iii) it is a misunderstanding that rapid tests are easy and therefore do not require experience; iv) it may reflect population-related differences, such as past exposure to leptospirosis, exposure to environmental leptospires, or infection with other infectious agents.

In conclusion, in the meta-analysis, the diagnosis of leptospirosis was ascertained by definite clinical criteria and standard MAT criteria. Also, IgM ELISA is sufficiently sensitive for use as an initial screen for leptospiral infections. The IgM ELISA showed higher sensitivity (84%) and specificity (91%) in the diagnosis of acute leptospiral infection and can be used as a rapid test for the detection of the disease, therefore improving the prognosis of patients and decreasing the lethality of leptospirosis.

Acknowledgments

The study was supported by the University of Extremo Sul Catarinense.

References

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