PLOS NEGLECTED TROPICAL DISEASES RESEARCH ARTICLE Rapid diagnostic tests and ELISA for diagnosing chronic Chagas disease: Systematic revision and meta-analysis Sandra Helena Suescún-Carrero ID1*, Philippe Tadger1,2, Carolina Sandoval Cuellar1, Lluis Armadans-Gil3, Laura Ximena Ramı́rez López1 1 Universidad de Boyacá, Tunja, Colombia, 2 Real World Solutions, IQVIA, Zaventem, Belgium, 3 Epidemiology and Preventive Medicine Service, Hospital Universitari Vall d’Hebron—Universitat Autónoma de Barcelona, Barcelona, Spain a1111111111 a1111111111 * ssuescun27@uniboyaca.edu.co a1111111111 a1111111111 a1111111111 Abstract Objective OPEN ACCESS To determine the diagnostic validity of the enzyme-linked immunosorbent assay (ELISA) Citation: Suescún-Carrero SH, Tadger P, Sandoval and Rapid Diagnostic Tests (RDT) among individuals with suspected chronic Chagas Dis- Cuellar C, Armadans-Gil L, Ramı́rez López LX ease (CD). (2022) Rapid diagnostic tests and ELISA for diagnosing chronic Chagas disease: Systematic revision and meta-analysis. PLoS Negl Trop Dis Methodology 16(10): e0010860. https://doi.org/10.1371/journal. pntd.0010860 A search was made for studies with ELISA and RDT assays validity estimates as eligibility criteria, published between 2010 and 2020 on PubMed, Web of Science, Scopus, and Editor: Eva Clark, Baylor College of Medicine, UNITED STATES LILACS. This way, we extracted the data and assessed the risk of bias and applicability of the studies using the QUADAS-2 tool. The bivariate random effects model was also used to Received: May 13, 2022 estimate the overall sensitivity and specificity through forest-plots, ROC space, and we visu- Accepted: October 3, 2022 ally assessed the heterogeneity between studies. Meta-regressions were made using sub- Published: October 18, 2022 group analysis. We used Deeks’ test to assess the risk of publication bias. Peer Review History: PLOS recognizes the benefits of transparency in the peer review Results process; therefore, we enable the publication of all of the content of peer review and author 43 studies were included; 27 assessed ELISA tests; 14 assessed RDTs; and 2 assessed responses alongside final, published articles. The ELISA and RDTs, against different reference standards. 51.2 % of them used a non-com- editorial history of this article is available here: parative observational design, and 46.5 % a comparative clinical design (“case-control” https://doi.org/10.1371/journal.pntd.0010860 type). High risk of bias was detected for patient screening and reference standard. The Copyright: © 2022 Suescún-Carrero et al. This is ELISA tests had a sensitivity of 99% (95% CI: 98–99) and a specificity of 98% (95% CI: 97– an open access article distributed under the terms of the Creative Commons Attribution License, 99); whereas the Rapid Diagnostic Tests (RDT) had values of 95% (95% CI: 94–97) and which permits unrestricted use, distribution, and 97% (95% CI: 96–98), respectively. Deeks’ test showed asymmetry on the ELISA assays. reproduction in any medium, provided the original author and source are credited. Conclusions Data Availability Statement: All relevant data are within the manuscript and its Supporting ELISA and RDT tests have high validity for diagnosing chronic Chagas disease. The analy- Information files. sis of these two types of evidence in this systematic review and meta-analysis constitutes PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0010860 October 18, 2022 1 / 26 PLOS NEGLECTED TROPICAL DISEASES Chagas disease diagnosis: Systematic revision and meta-analysis Funding: SHSC, LXRL, CSC, PT were funded by an input for their use. The limitations included the difficulty in extracting data due to the lack the Universidad de Boyacá, Colombia. LAG was of information in the articles, and the comparative clinical-type design of some studies. funded by the Universitat Autónoma de Barcelona, Barcelona, Spain. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared Author summary that no competing interests exist. Chagas disease (CD), an infection caused by the Trypanosoma cruzi parasite, affects between 8 and 10 million people worldwide. It is considered one of the main problems of public health in Latin America, and the international migration has caused infected sub- jects to scatter through the rest of the world, making CD a global health problem. There- fore, it is important to diagnose this infection using laboratory tests, which sometimes becomes a problem due to lack of reference tests and the existence of different types of tests with different sensitivity and specificity values, added to the difficulty in detecting the parasite in its chronic phase. This systematic review and meta-analysis determined the diagnostic validity of the enzyme-linked immunosorbent assay (ELISA) and rapid diag- nostic tests (RDT) among individuals with suspected chronic CD. It included 43 studies and concluded that both ELISA and RDTs had adequate diagnostic performance. It is nec- essary to better understand these two types of diagnostic tests to facilitate clinical decision making about the disease and improve access to treatment for the population at risk. Introduction American Trypanosomiasis or Chagas disease (CD), caused by the protozoan Trypanosoma cruzi, continues to be an important cause of illness, disability and death [1]. In recent years, CD has positioned itself as the main parasitic disease in Latin America and as one of the 13 most neglected tropical diseases [2]. It is estimated that about 100 million people are at risk of being infected with T. cruzi, in the region, and that there are about 8 to 10 million already infected; with 30,000 new cases per year due to all forms of transmission, which leads to 12,000 annual deaths [3]. In addition, the international migration has caused infected individuals from Latin America to migrate all over the world, which now makes the disease a problem for the global health systems [4]. CD has two forms: acute and chronic. The acute phase is usually asymptomatic or can pres- ent as a nonspecific, self-limited febrile syndrome that resolves in approximately 90 % of untreated infected individuals [5]. On the other hand, in its chronic phase, around 60% to 70% of patients do not present any apparent symptoms; 30% of the subjects develop cardiomyopa- thies with a clinical variety, including arrhythmias, aneurysms, dilated cardiomyopathy, and sudden death [6]. It is essential and important to diagnose T. cruzi infection using laboratory tests in order to prescribe the best treatment and, this way, stop the progression of the disease and prevent its transmission [7,8]. However, one limitation is the complexity of the diagnostic process, which is sometimes hampered by the lack of a reference standards, by the availability of multiple types of assays with different sensitivity and specificity values, and by the great difficulty of detecting the parasite in the chronic phase of the disease [9]. The World Health Organization (WHO) recommends using two conventional tests for diagnosing chronic CD, based on differ- ent principles and the detection of different antigens. Furthermore, in the case of ambiguous or inconclusive results, a third technique should be used [10]. Thus, serological tests, such as indirect immunofluorescence, indirect hemagglutination, enzyme-linked immunosorbent PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0010860 October 18, 2022 2 / 26 PLOS NEGLECTED TROPICAL DISEASES Chagas disease diagnosis: Systematic revision and meta-analysis assay (ELISA), and immunochromatographic tests or rapid diagnostic tests (RDT) are used [11]. They can be qualitative or semi-quantitative, based on different antigens; some use a multi-epitope antigen and others use a combination of recombinant proteins [12]. The Pan American Health Organization states that the evidence on the validity of tests for diagnosing CD has been considered high in the case of ELISA tests and chemiluminescence analysis, and moderate for RDTs [13]. Each technique has different features in relation to the antigenic tar- gets used, the population evaluated, the cut-off points and the equipment used; therefore, a direct comparison of test performance is more difficult [14]. Taking into account the afore- mentioned, the purpose of this study was to summarize the evidence available on the diagnos- tic validity of ELISA and immunochromatographic tests (RDT) in individuals with suspected diagnose of chronic CD. Methods Protocol and registration This systematic review and meta-analysis was carried out according to the PRISMA-DTA guidelines (Preferred Reporting Items for Systematic Reviews and Meta-analysis of Diagnostic Test Accuracy Studies -The PRISMA-DTA Statement) [15] for the abstract and the body of the manuscript (S1 and S2 Checklists). The protocol was registered in the PROSPERO data- base (International Prospective Register of Systematic Reviews) with number CRD42020186588. Eligibility criteria The search included studies that estimated sensitivity and specificity of ELISA or RDT index tests for chronic CD, with participants over five years old, patients with chronic CD, and patients without this disease; studies conducted in endemic and non-endemic areas for CD, that described the reference standards used, studies with a cross-sectional design and a case- control type; written in English, Spanish and Portuguese, published between 2010 and 2020; with research done with volunteers and with samples that included humans. Studies indicating that patients were receiving treatment for CD, those that were related exclusively to acute infection or in newborns, and those with mixed data on patients with acute and chronic infec- tion were excluded. Data sources The databases used for the search, which was carried out from May to August 2020, were: Pubmed/Medline, Scopus; ISIWeb/Web of Science, and LILACS. The corresponding authors of articles included were contacted by email to inquire about missing data or request clarifica- tion on studies. Study search and selection The standard search strategy described in The Joanna Briggs Institute Reviewers’ Manual 2015 [16] was used. Thus, there was an initial limited search to identify relevant keywords and indexing terms, followed by a comprehensive search in the databases included with strategies for each of the search engines (S1 Database). Two reviewers (SHSC-LXRL) assessed article titles and abstracts in an independent and blinded manner. Disagreements in the inclusion of studies were resolved by consensus, taking into account that the abstracts should meet the pro- posed eligibility criteria. Subsequently, the articles were reviewed in full text. PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0010860 October 18, 2022 3 / 26 PLOS NEGLECTED TROPICAL DISEASES Chagas disease diagnosis: Systematic revision and meta-analysis Data collection process Two authors (SHSC-LXRL) extracted the following data independently: author(s), year of pub- lication, type of participants, study area, index test, reference test, study period, country of implementation, number of patients and healthy subjects, total number of participants, sensi- tivity and specificity, risk of bias and applicability. Definitions for data extraction The subjects included in the different studies were classified into: patients who had lived or resided in an endemic area for CD and patients who reside in a non-endemic area. The study area was considered endemic if CD occurred in this geographic area; and as a non-endemic area, otherwise. The index tests were considered commercial when they were part of a brand of laboratory diagnostic reagents, validated by medical device regulatory agen- cies and those available on the market; and considered in-house tests when studies indicated that immunoadsorption assays had been designed with different peptides or proteins with the application of non-standard “internal” methods. RDTs are those immunochromatographic assays that throw qualitative results and can be read at first sight. Reference tests met the standard if they included a combination of serological tests with dif- ferent antigens detecting antibodies against T. cruzi, and an additional test to reach a definitive diagnosis if the results were inconclusive. The study design was considered clinical-comparative or case-control type if a group of par- ticipants diagnosed with chronic CD and a group without this diagnosis had been included; and it was considered non-comparative if a consecutive and representative series of patients with suspected CD had taken the test to be evaluated, as well as the reference test. Risk of bias and applicability Three authors (SHSC-LXLR-CSC) assessed the methodological quality and risk of bias of the studies included, in a blinded and independent manner, using the Quality Assessment of Diag- nostic Accuracy Studies-2 (QUADAS-2) tool, which comprises four domains: patient screen- ing, index test, reference test, and flow and time [17]. Each domain was assessed for risk of bias, and the first three domains were also assessed for applicability. The QUADAS-2 tool was adjusted to the needs of this review, as follows: the risk of bias in patient screening was considered high if a consecutive or random sample of patients had not been used; and unclear if patient recruitment was not specified. The risk of bias related to the index test was considered unclear if there was no specification that the results of the index tests were interpreted without knowing the results of the reference test. The risk of bias related to reference tests was considered high if these tests were interpreted knowing the results of the index test, or if a single reference test had been used (taking into account that the WHO estab- lishes that serological diagnosis in the chronic phase of CD should be based on positive results in two tests that are based on different immunological principles and, in case of inconsistency, on a third test). Diagnostic accuracy measures The reported measures were sensitivity and specificity for each of the index tests assessed for diagnosing chronic CD. When the studies did not have these two measures, they were calcu- lated based on the number of true positives and negatives, as well as on the number of false positives and negatives and the total number of patients. PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0010860 October 18, 2022 4 / 26 PLOS NEGLECTED TROPICAL DISEASES Chagas disease diagnosis: Systematic revision and meta-analysis Summary of results Sensitivity and specificity were modeled bivariately with binomial-normal random effects, with a gold standard (GS) assumption, but also with an imperfect gold standard (IGS) model. The GS models were fitted with a Bayesian and classical approach; and the IGS model with a Bayesian approach only. Models were selected with the deviance information criterion (DIC) for the Bayesian models, and with the likelihood ratio test for the classical models. Six possible models for the GS were evaluated according to the type of distribution that followed the ran- dom effects (normal or mixed normal) and the type of connection (logit, cloglog and probit), and the best model was selected according to the smallest DIC with at least two points differ- ence. The specification of the model with the best fit (in bamdit metadiag) was reproduced in the rest of the packages (meta4diag: Binomial-normal with probit, and metandi and IGS: Bino- mial-normal with logit) to facilitate comparisons. The bivariate random effects model was used to estimate the overall sensitivity and specific- ity and their respective 95 % confidence intervals (CI). The results were plotted in forest-plots and ROC space (R DTAplots program), and heterogeneity between studies was assessed visu- ally. R 1.3 software (DTAplots, bamdit::plotcompare and meta4diag::meta-regression) [18], Stata 15 (metandi) [19], midas and JAGS were used to conduct the meta-analysis. Additional analyses Meta-regressions were carried out with potential modifiers of diagnostic validity (bamdit plot- compare and meta4diag meta-regression). The variables of interest were study design (clinical comparative or non-comparative), study area (endemic or non-endemic), study risk (low or high risk of bias), sample type (serum, whole blood or not applicable) for the RDTs, and type of test (commercial or in-house) for the ELISA tests but not for the RDTs because of the low number of studies, which made it impossible to estimate them. All variables were categorized at two levels in both the ELISA and RDT assays to facilitate the comparison of predictive regions and validity estimates. A QUADAS-2 assessment was applied in each study in order to analyze by subgroups. The three levels of the QUADAS-2 became two: low risk and high risk (which included the high risk and unclear categories). Of the 7 items of the tool, item 1 (patient screening) and item 3 (reference standard) were consid- ered since they were the only ones with a sufficient number of studies with a high risk of bias. In the rest of items, most studies were low risk. A sensitivity analysis was carried out excluding influential outliers. Influential studies were reviewed based on the assumption that the subsequent interval distribution of study weight should include one. The publication bias was assessed using Deeks’ asymmetry test, which was considered statistically significant with a value of p < 0.1 [20]. Results Study selection As shown in Fig 1, 897 publications were initially identified, of which 739 were eliminated due to duplication in the databases. Of the remaining 158 publications, 75 did not meet the selec- tion criteria in the review by title and abstract. Of the remaining 83 articles, 40 were excluded for the following reasons: 17 due to inadequate study design, 9 did not meet the diagnostic ref- erence test criteria suggested by the WHO, 8 due to non-concordance between the index test and the tests that were to analyze the present investigation (ELISA or RDT), 4 used a popula- tion that was in the acute phase of the disease or were studies that analyzed subjects in the PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0010860 October 18, 2022 5 / 26 PLOS NEGLECTED TROPICAL DISEASES Chagas disease diagnosis: Systematic revision and meta-analysis Fig 1. Study selection. https://doi.org/10.1371/journal.pntd.0010860.g001 acute and chronic phase and 2 included patients with previous treatment for CD. Finally, 43 full-text articles were used for qualitative and quantitative analysis [9,10,21–61]. Study features The 43 selected studies were published between 2010 and 2019, with between 3 and 6 studies being published per year, except for 2015, a year in which there were no publications. 27 arti- cles [9,21,22,24,25,27–35,38,41,42,44–46,48,51,53,55,57–59] evaluated ELISA tests; 14 used RDT [10,23,26,32,37,39,40,43,47,50,52,54,60,61] and 2 [36,56] evaluated both tests (Table 1). In 33 articles (76 for ELISA tests and 39 for RDT), several substudies included aspects such as tests with different peptides, populations from different countries, participation of several reference laboratories or evaluation of different index tests. This data is presented and analyzed by separated in the present investigation. Six articles did not present sensitivity and specificity data [22,24,25,37,38,51] but were included considering that they indicated true and false posi- tive and negative data. A total of 30,356 participants were reported in the 43 articles (from 56 to 10,284 subjects per study). According to the nature of the sample, 76.7 % of the investigations were carried out with serum [9,21,22,24–30–36,39,41,42,44,45,48–51,53–60], 7% with venous blood [40,46,61], 7% with venous blood and serum [23,43,52], 4.7% with capillary blood [10,37], 2.3% with serum, plasma, venous blood and capillary [47]; and 2.3 % did not report the type of sample used [39]. Of those studies where ELISA tests were assessed, 17 were carried out with in-house tests [21,24,26,28,31,35,38,40,44,46,48,52,53,55,60–62]; 12 were commercial trials [9,22,24,25,30,34,36,42,45,48,55,56], and one article studied an in-house assay and a commer- cial assay [26]. The brands assessed that were present in more than one study were Architect PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0010860 October 18, 2022 6 / 26 Table 1. Qualitative analysis of studies selected (ELISA and RDT assays). Ref Author / Type of Type of participants Study area Index test Reference test Study period Country Number of Number of Total Sensit. Specif. year test sick healthy patients patients 1 Briceño 2010 ELISA Healthy patients and participants with Endemic AgA-ELISA Bioschile Ingenieria Genetica S.A Not stated Venezuela 89 477 566 98.8% 97%� (19) chronic CD and lymphoblastogenesis area Kit, BioKIT and Pharmatest and Chagasic cardiomyopathy or (Laboratorios Pharmatest) asymptomatic Immunofluorescence of epimastigotes Indirect hemagglutination and ELISA) 2 Aria 2016 _ ELISA Blood donors Endemic ELISA Chagas test IICS V.1 Chagatest ELISA recombinant v 3.0 Not stated Paraguay 33 23 56 97% 91%� (30) a Participants who were positive for area from Wiener. syphilis, Hepatitis B and C, HIV, HTLV. Aria 2016 _ ELISA Chagas test IICS V.1 BiosChile Test ELISA 97% 95%� b 4 Llano 2014 ELISA Participants with leishmaniasis and heart Endemic Chagas (Trypanosoma cruzi) ELISA and IFI Not stated Colombia 78 21 99 96% 98%� (52) disease. Symptomatic and asymptomatic and Non- IgG-ELISA (NovaTec No brand reported endemic Immunodiagnostica GmbH) area 5 Bergmann ELISA Blood donors from Brazil Endemic Chagatest (Wiener Lab, Immunotransference with TESA Not stated Brazil 122 39 161 99% 99% / (56) 2013 _a area Argentina). antigen (T. cruzi excretor-secretor 18%� antigen) Bergmann CHAGATEK (Biolab-Mérieux, 98% 75%� No brand reported 2013 _b Rio de Janeiro, Brazil). Bergmann EIAgen T. cruzi IgG + IgM 99% 53%� PLOS NEGLECTED TROPICAL DISEASES 2013 _c (Adaltis, Bologna, Italy) 7 da Silva ELISA Participants with Chagas disease, Endemic ELISA home test IgG whole and ELISA and indirect 2006 Brazil 60 54 114 100% 100% (58) 2012_a indeterminate form, cardiac disorders, area subclasses hemagglutination assay IHA digestive disorders with or without Ig G whole: dilution 1:40 No brand reported cardiac disorders da Silva ELISA home test IgG 100% 90.7% Healthy participants, without Chagas 2012_b Ig G 1: dilution 1:10 disease, with other diseases such as HIV, da Silva hepatitis C, syphilis, visceral ELISA home test IgG 100% 88,9% 2012_c leishmaniasis, tegumentary leishmaniasis Ig G 2: dilution 1:10 da Silva ELISA home test IgG 95% 98.1% 2012_d Ig G 3: dilution 1:20 8 Pimenta ELISA Individuals in the rural endemic area Endemic ELISA IBMP-8.1 ELISA and indirect Not stated Argentina, 215 122 337 95.3% 100% (59) 2019 _a area hemagglutination Bolivia, Paraguay No brand reported Pimenta ELISA IBMP-8.4 100% 100% 2019 _b 9 Pierimarchi ELISA Migrants and blood donors Endemic ELISA automated using 2 In house ELISA, HAI Not stated Venezuela 55 77 132 98.1% 100%� (60) 2013_a and Non- different antigens of T. cruzi. (a No brand reported Italy PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0010860 October 18, 2022 endemic recombinant protein and a BioELISA Chagas, Biokit, Spain area complete extract of T. cruzi) TcF (recombinant antigen) Commercial immunomatographic Pierimarchi ELISA automated using 2 96.3% 100%� 2013_b different antigens of T. cruzi. (a rapid test (Chagas Quick Test, Cypress Diagnostics, Belgium) recombinant protein and a complete extract of T. cruzi) IMT 10 Praast 2011 ELISA Blood donors from Germany and Endemic Abbott Architect Chagas BioMérieux ELISA cruzi. Not stated Germany 655 9629 10284 99.8% 99.9% (20) patients from Brazil and Guatemala, and Non- Wiener Lab test Chagas ELISA Bolivia, Argentina and USA endemic recombinant 3.0. area Abbott Chagas confirmatory immunoblot assay (Continued ) 7 / 26 Chagas disease diagnosis: Systematic revision and meta-analysis Table 1. (Continued) Ref Author / Type of Type of participants Study area Index test Reference test Study period Country Number of Number of Total Sensit. Specif. year test sick healthy patients patients 12 Caicedo ELISA Sera from Instituto Nacional de Salud, Endemic Test ELISA Chagas III BIOS ELISA and IFI home test 2014 to 2016 Colombia 256 245 501 99.2% 97.9% (8) 2019_a public health and department area Indirect hemagglutination (Wiener laboratories, blood donors Chagatest HAI) Caicedo Nonconventional methods 92.5% 97.5% (T. cruzi excretor-secretor antigen) 2019_b Synthetic peptides Umelisa (TESA, bioMérieux Immunoblot) Chagas SUMA Caicedo Recombinant antigens Architect 98.4% 97.9% 2019_c System Chagas ARCHI Caicedo BioELISA Chagas BIOKIT 98% 94.6% 2019_d Caicedo Chagatest ELISA recombinant v. 98.8% 97.9% 2019_e 4 WIENER Caicedo T. cruzi AB DIAPRO 95.7% 97.1% 2019_f Caicedo 7.Chagas ELISA IgM + IgG 99.6% 97.5% 2019_g VIRCEL 13 Caballero ELISA Blood donors Endemic ELISA home test with Burunga TESA-blot Western blot Not stated Panama and 40 113 153 100% 82.3% (22) 2019_a Participants with visceral leishmaniasis, area genotype Chagatest Rec v3.0 Wiener kit Brazil cutaneous and mucocutaneous Caballero ELISA home test with MM1 100% 77.8% leishmaniasis, T. rangely, rheumatic 2019_b genotype fever, toxoplasma, and P. falciparum PLOS NEGLECTED TROPICAL DISEASES Caballero ELISA home test with Jose-IMT 100% 84.9% 2019_c genotype Caballero ELISA home test with Y 100% 88.4% 2019_d genotype Caballero ELISA home test with FCI 100% 81.4% 2019_e genotype Caballero ELISA home test with JJ 100% 78.7% 2019_f genotype 15 Tonelli ELISA Participants with indeterminate form, Endemic ELISA home test ELISA (Gold Analisa, Brazil), IHA Not stated Brazil 53 70 123 60.3% 72.8%� (24) 2018_a chagasic myocarditis, tegumentary area Epitope 1 of B cells derived from (Wiener Lab., Argentina) and IFA leishmaniasis, visceral leishmaniasis the family of Mucin Associated (Bio-Manguinhos, Brazil) Surface Proteins (MASP) Tonelli ELISA home test 100% 97.1%� 2018_b Epitope 2 of B cells derived from the family of Mucin Associated Surface Proteins (MASP) PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0010860 October 18, 2022 Tonelli ELISA home test 100% 67.1%� 2018_c Epitope 3 of B cells derived from the family of Mucin Associated Surface Proteins (MASP) Tonelli ELISA home test 100% 82.8%� 2018_d Combination of epitopes 1, 2 and 3 of B cells derived from the family of Mucin Associated Surface Proteins (MASP) Tonelli ELISA home test 100% 100%� 2018_e Combination of epitopes 2 and 3 of B cells derived from the family of Mucin Associated Surface Proteins (MASP) 16 Pérez 2018 ELISA Migrants from Bolivia Non- Architect Chagas Immunochromatogra-phy tests January Spain 307 3844 4151 92.5% 100% (25) endemic (ICT) and/or indirect 2014-August area immunofluorescence (IFI) 2017 (Continued ) 8 / 26 Chagas disease diagnosis: Systematic revision and meta-analysis Table 1. (Continued) Ref Author / Type of Type of participants Study area Index test Reference test Study period Country Number of Number of Total Sensit. Specif. year test sick healthy patients patients 17 Peverengo ELISA Blood donors Endemic ELISA ELISA (Chagatest ELISA) e IHA Not stated Argentina 67 67 134 90.2% 100% (26) 2018_ a area CP1 [antigens FRA and SAPA (Chagatest IHA) from Wiener Lab (Ags)] (Argentina) IFI Peverengo CP3, composed of antigenic 100% 92.5% 2018_ b determinants MAP, TcD y TSSAII / V / VI Peverengo CP1+CP3: 100% 100% 2018_ c 20 Mucci 2017 ELISA Healthy patients, patients infected with Endemic ELISA with synthesized peptides ELISA whole, HAI Not stated Argentina 62 16 78 97% 97% (29) T.cruzi, in the asymptomatic chronic area No brand reported stage of the disease without heart or gastrointestinal compromise, participants with tegumentary leishmaniasis 23 Neves ELISA Serum samples from individuals from Endemic ELISA of recombinant proteins ELISA recombinant Immuno-ELISA Not stated Brazil 280 20 300 98.9% 100% (33) 2016_a� CD endemic areas in Pernambuco area IBMP8.1 Chagas (Wama Diagnóstica, São (Brazil) of the reference laboratory for Palo, Brazil, batch 14D061), Neves IBMP8.2 98.2% 90% CD (RLCD, Oswaldo). Cruz Foundation ELISA whole Chagas III (BIOSChile, 2016_b� / PE, Brazil) Ingenierı́a Genética S.A., Santiago, Neves IBMP8.3 Chile, batch 1F130525) 95.4% 95% 2016_c� PLOS NEGLECTED TROPICAL DISEASES Neves IBMP8.4 99.6% 100% 2016_d� 24 Neves ELISA Sera obtained from the serum bank of a Endemic Immuno-ELISA Chagas (batch IFI IFA (Immunocruzi; Biomérieux) Not stated Brazil 186 499 685 97.3% 100% (34) 2016_a reference laboratory of chagasic area 14D061; Wama Diagnóstica, São Western blot (TESA blot; participants with cardiac disorders. Paulo, Brazil) and Pathozyme Biomérieux, Rio de Janeiro, Brazil) Non-chagasic participants, blood donors Neves Chagas (batch 7042779; Omega 99.5% 99.2% with dengue, filariasis, hepatitis B and C, 2016_b Diagnostics, Scotland, UK), HIV, HTLV, leishmaniasis, leptospirosis, recombinant rubella, measles, schistosomiasis and Neves syphilis Chagas III (batch 1F130525; 100% 100% 2016_c BIOSChile, Ingenierı́a Genética S.A., Santiago, Chile) Neves Gold ELISA Chagas (batch 99.5% 97% 2016_d CHA132A; Rem, São Paulo 26 Reis 2014_a ELISA Brazilian chagasic and non-chagasic Endemic 26_a: ELISA rTc_11623.20 ELISA recombinant ELISA v. 3.0 kit, Not stated Brazil 58 45 103 94.8% 98.2% (36) participants, patients with chagasic heart area Chagatest Reis 2014_b 26_b: ELISA rTc_N_10421.310 89.6% 94.6% disease, cutaneous and visceral HAI Wiener Laboratorio Rosario, PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0010860 October 18, 2022 Reis 2014_c leishmaniasis 26_c: combination of a and b Argentina 95.5% 98.1% IFI Sigma Chemical Company, Missouri, USA Western blot (TESAcruzi, bioMerieux Brazil) 27 Izquierdo ELISA Migrants and blood donors Non- ELISA chemiluminescent ELISA whole ELISA ID-PaGIA Not stated Spain 92 58 150 100% 98.3% (37) 2013 Participants with visceral leishmaniasis endemic (DiaMed, Cressier sur Morat, area Switzerland) and Chagas Bioelisa Assay (Biokit, Lliçà d’Amunt, Spain) RDT 28 Cervantes ELISA Participants with leishmaniasis, Endemic Dot-ELISA ELISA whole, western blot Not stated Mexico 96 153 360 97% 89% (38) 2013 tuberculosis, neurocysticercosis, taeniasis area No brand reported and toxoplasmosis 30 Iborra 2012 ELISA Migrants from endemic countries in Non- Chemiluminescent ELISA whole T. cruzi ELISA test Not stated Spain 76 89 165 100% 96.6% (40) Latin America endemic immunoassay of microparticles system; Ortho Clinical Diagnostic, area (ARCHITECT Chagas Abbott) USA) IFI Immunofluor CHAGAS kit; Biocientı́fica S.A., Buenos Aires, Argentina Discordance: immunochromatogra- phy Onsite Chagas Ab Combo- Cassete (CTK Biotech, Inc. USA). (Continued ) 9 / 26 Chagas disease diagnosis: Systematic revision and meta-analysis Table 1. (Continued) Ref Author / Type of Type of participants Study area Index test Reference test Study period Country Number of Number of Total Sensit. Specif. year test sick healthy patients patients 31 Longhi 2012 ELISA Participants with Kalaazar disease, Endemic ELISA of T. cruzi and ELISA whole, IFI, HAI Not stated Brazil 228 108 336 100% 95.2% (42) leishmaniasis, lupus erythematosus, non- area recombinant antigens JL7 No brand reported chagasic cardiomyopathies, schistosomiasis, juvenile diabetes, idiopathic megaesophagus, and South American blastomycosis 33 Hernández ELISA Patients from La Paz, Bolivia who Endemic ELISA of fusion polypeptides ELISA Wiener Chagatest-ELISA Not stated Bolivia, Brazil, 76 54 130 98% 97%� (44) 2010_a attended a parasitology laboratory and Non- TcBCDE Recombinant version 3.0 (Wiener Spain and other Healthy participants from non-endemic endemic Laboratorios, Santa Fé, Argentina) European areas such as Germany and Mongolia, area countries and patients with visceral or cutaneous leishmaniasis, syphilis and brucellosis. Hernández Patients from Santa Cruz, Bolivia who ELISA of fusion polypeptides ELISA Wiener Chagatest-ELISA 64 21 85 98% 94% 2010_b attended a hospital TcBCDE Recombinant version 3.0 (Wiener Laboratorios, Santa Fé, Argentina) Bioelisa Chagas (Biokit, Barcelona, Spain) ELISA recombinant Chagas Stat-Pak (Chembio Diagnostic Systems, Medford, NY), Hernández Patients from Brazil who attended a ELISA of fusion polypeptides IFI 165 216 381 99% 99% 2010_c university hospital TcBCDE HAI Wiener PLOS NEGLECTED TROPICAL DISEASES T. cruzi crude extract ELISA (EIE Biomanguinhos; Fiocruz, Rio de Janeiro, Brazil) 35 Dopico ELISA Serum samples from Latin American Non- ELISA IBMP 8.1 ELISA whole ORTHO T. cruzi Not stated Spain 347 331 678 99.4% 100% (46) 2019_a pregnant women with toxoplasmosis and endemic ELISA Test System (Ortho Clinical Zika. area Diagnostics Inc., Raritan, USA) Dopico ELISA IBMP 8.4 99.1% 99.7% ELISA recombinant Bioelisa 2019_b CHAGAS (Biokit S.A., Barcelona, Spain) or BIO-FLASH Chagas (automated chemiluminescent assay; Biokit S.A., Barcelona, Spain) 37 Abras 2016 ELISA Participants with leishmaniasis, Non- ELISA Chemiluminescent ELISA whole, recombinant, western January 2009 Spain 114 200 314 100% 97.6% (48) toxoplasmosis, amoebic liver abscess, endemic Architect blot to December malaria, strongyloidiasis, visceral larva area No brand reported 2012 migrans, cytomegalovirus, HIV, parvovirus, Epstein Barr, hepatitis B and C, syphilis, and Lyme disease PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0010860 October 18, 2022 39 Berrizbeitia ELISA Participants with leishmaniasis, Endemic ELISA of epimastigotes ELISA whole, IFI, HAI Not stated Venezuela 50 70 120 100% 74% (50) 2012_a ascariasis, strongyloidiasis and area secretion/excretion proteins No brand reported trichuriasis Optic density 0,400 Berrizbeitia ELISA of epimastigotes 98% 80% 2012_b secretion/excretion proteins Optic density 0,500 Berrizbeitia ELISA of epimastigotes 98% 88% 2012_c secretion/excretion proteins Optic density 0,600 40 Neves ELISA Healthy and chronic CD patients, Endemic ELISA of chimeric proteins ELISA whole and recombinant Not stated Brazil, other Latin 595 526 1121 96.4% 99.6% (51) 2018_a participants with leishmaniasis area IBMP 8.1 ELISA Chagas III (batch 1F130525; American BIOSChile, Ingenierı́a Genética S.A., countries and the Neves ELISA of chimeric proteins 8.2 93.5% 99.6% Santiago, Chile) USA 2018_b Immuno-ELISA Chagas (batch Neves ELISA of chimeric proteins 8.3 14D061; Wama Diagnostica, São 96.8% 100% 2018_c Paulo, Brazil) Neves ELISA of chimeric proteins 8.4 Pathozyme Chagas (Omega 99.4% 100% 2018_d Diagnostics, Scotland, United Kingdom) Gold ELISA Chagas (Rem, São Paulo, Brazil) (Continued ) 10 / 26 Chagas disease diagnosis: Systematic revision and meta-analysis Table 1. (Continued) Ref Author / Type of Type of participants Study area Index test Reference test Study period Country Number of Number of Total Sensit. Specif. year test sick healthy patients patients 41 Neves ELISA Participants positive for dengue, Endemic ELISA of chimeric proteins ELISA whole Not stated Brazil, USA, 825 630 1455 97.4% 99.4% (53) 2017_a filariasis, Hepatitis B and C, HIV, HTLV, area IBMP 8.1 Immuno-ELISA Chagas (Wama Mexico, leishmaniasis, leptospirosis, measles, Diagnóstica-SP, Brazil, batch Nicaragua, Neves ELISA of chimeric proteins 94.3% 99.6% rubella, schistosomiasis and syphilis 14D061) Guatemala, 2017_b IBMP 8.2 ELISA Chagas III (BIOSChile, Honduras and Neves ELISA of chimeric proteins Ingenierı́a Genética S.A., Santiago, Argentina 97.9% 99.9% 2017_c IBMP 8.3 Chile, batch 1F130525) Neves ELISA of chimeric proteins 99.3% 100% 2017_d IBMP 8.4 3 Mendicino RDT Study conducted in whole bloods to Endemic WL Check Chagas (Wiener Lab Chagatest ELISA, Wiener Lab SAIC Not stated Argentina 64 177 241 87.3% 98.8%� (41) 2014_ a patients that go to primary care clinics in area SAIC, Argentina) IHA (IHA Chagas Poly- chaco, the rural areas of a province of Argentina Lemos Laboratory SRL, Argentina) with clinical suspicion of CD IFI in case of discordance. Mendicino Study conducted in serum 67 171 238 95.7% 100%� 2014_ b 6 Acosta RDT Participants who were positive for Endemic Immunochromatogra-phy test ELISA Chagas test IICS-UNA Not stated Paraguay 97 105 202 97% 95%� (57) 2013_a toxoplasmosis, syphilis, tuberculosis, area for qualitative detection of IgG rheumatoid factor and hepatitis anti Trypanosoma cruzi Acosta Immunochromatogra-phy test 51 43 94 94% 100%� 2013_b SD Bioline–Korea 11 Lozano 2019 RDT Inhabitants of the cities Yacuiba and Endemic Chagas Stat-Pak (CSP; Chembio Lysate antigen ELISA from Wiener April to Bolivia 304 381 685 97.7% 97.4%� PLOS NEGLECTED TROPICAL DISEASES (21) _a Villa Montes (province of Gran Chaco, area Inc., Medford, USA) and recombinant from Wiener August 2018 department of Tarija) Discordance: ELISA (Chagatek, Lozano 2019 Chagas Detect Plus (CDP; 98.4% 87.1%� Laboratorio Lemos, Buenos Aires, _b InBIOS International Inc., Seattle, USA) Argentina) 14 Mendicino RDT Inhabitants of the northern province of Endemic RDT A: WL Check Chagas IHA (IHA Chagas Polychaco, Lemos Not stated Argentina 42 64 106 90.5% 100%� (23) 2018_ a Santa Fe, located in Gran Chaco area (Wiener Lab SAIC, Argentina) Laboratory SRL) ELISA (Chagatest ELISA, Wiener Mendicino RDT B: SD BiolineChagasAb 97.6% 93.8%� 2018_ b Rapid (Standard Diagnostics Lab SAIC) When results were discordant, IFI Inc., Korea) was performed with commercial conjugates and smear prepared with epimastigotes of T. cruzi of the strain Tulahuen 0.10 19 Navarro RDT Migrants in Spain from Bolivia, Ecuador, Non- Rapid immunochromato- IFI, ELISA May 2008 to Spain 57 219 276 88% 94%� (28) 2011 Peru endemic graphy test (ICT) (Simple December area Chagas WB, Operon) 2009 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0010860 October 18, 2022 21 Angheben RDT Migrants and travelers from endemic Non- Rapid immunochromato- ELISA whole ELISA for Chagas April 2009 to Italy 256 384 640 82.8% 98.7% (31) 2017 countries of Latin America endemic graphy test Chagas Quick Test III1, BioChile, Chile (Lys ELISA) June 2015 area (CQT), Cypress diagnostics of ELISA recombinant Bio-Elisa Belgium Chagas, Biokit, Spain (Ric-ELISA). 22 Egüez RDT People who went to the reference Endemic Chagas Stat-Pak (CST; Chembio Elisa whole Wiener Lab (Rosario, March to May Bolivia 209 133 342 87% 93.2% (32) 2017_a laboratory Laboratorio de Referencia area Inc., Medford, USA) Argentina), ELISA Wiener v2.0 2014 Departamental Chuquisaca in Sucre ELISA recombinant ELISA Wiener Egüez Chagas Detect Plus (CDP; 93.4% 95.2% v3.0 2017_b InBios Inc., Seattle, USA) HAI IHA test, Chagas Polychaco kit from laboratorio Lemos (Buenos Aires, Argentina) 25 Shah 2014_a RDT Participants with early and advanced Endemic Chagas Detect Plus (CDP) ELISA recombinant Wiener April-May Bolivia 292 293 585 96.2% 98.8% (35) heart disease area (InBios International Inc, recombinant v3.0 ELISA 2013 Seattle) IFI does not report any brand Serum HAI IHA (Chagas Polychaco kit; Lemos Laboratories, Buenos Aires, Shah 2014_b Chagas Detect Plus (CDP) 99.3% 96.9% Argentina (InBios International Inc, Seatle) Blood (Continued ) 11 / 26 Chagas disease diagnosis: Systematic revision and meta-analysis Table 1. (Continued) Ref Author / Type of Type of participants Study area Index test Reference test Study period Country Number of Number of Total Sensit. Specif. year test sick healthy patients patients 29 Flores RDT Migrants and travelers to endemic Non- Operon Immunochromatogra- ELISA whole, IFI, PCR Not stated Spain 63 188 251 100% 92.6% (39) 2012_a countries of Latin America, natives and endemic phy test (ICT-Operon; Simple Does not report any brand people born in Spain, with epidemiologic area Stick Chagas serum and plasma risk factors. Flores Simple ChagasWB Operon S.A., 91.8% 93.7% Participants with visceral leishmaniasis 2012_b Spain) and malaria. Peripheral blood Flores Simple ChagasWB Operon S.A., 86.1% 94.8% 2012_c Spain) Capillary blood 32 Barfied RDT No patient characteristics reported Endemic Chagas STATPAK from ELISA whole BioMerieux ChagaTek Not stated Argentina 190 185 375 95.3% 99.5% (43) 2011_a area Laboratorio Lemos, Argentina ELISA 15 minutes ELISA recombinant Laboratorio Lemos Biozima Chagas recombinant Barfied Chagas STATPAK from 95.8% 99.5% 2011_b Laboratorio Lemos, Argentina 20 minutes Barfied PATH Rapid Test from Lemos 97.9% 96.2% 2011_c 15 minutes Barfied PATH Rapid Test from Lemos 99.5% 96.8% 2011_d 20 minutes Barfied PATH Rapid Test from Lemos 98.9% 94% PLOS NEGLECTED TROPICAL DISEASES 2011_e 25 minutes 34 Chapouis RDT Latin American migrants from several Non- Stat-Pak in blood ELISA whole and recombinant June to Switzerland 125 874 999 95.2% 99.9% (45) 2010_a countries where the disease is endemic. endemic Stat-Pak assay: the bioMerieux Elisa November Chapouis area Stat-Pak in serum cruzi 2008 96% 99.8% Biokit bioelisa Chagas 2010_b 36 Reithinger RDT Patients from Argentina Endemic Trypanosoma Detect MRA ELISA whole, IFI, HAI 2000 to 2007 United Kingdom 40 61 101 82.5% 96.7% (47) 2010_a area rapid test; Inbios, Seattle, WA in Does not report any brand Reithinger Patients from Ecuador Trypanosoma Detect MRA Chagas III 51 49 100 84.3% 95.9% 2010_b rapid test; Inbios, Seattle, WA in (BiosChile Ingenierı́a Genética S.A., Santiago, Chile) Chagatek ELISA (bioMérieux, Buenos Aires, Argentina) IFI, HAI does not report any brand Reithinger Patients from México Trypanosoma Detect MRA Kit Chagas III (BiosChile Ingenieria 40 60 100 77.5% 100% 2010_c rapid test; Inbios, Seattle, WA in Genétca S.A., Santiago, Chile) IFI, HAI does not report any brand PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0010860 October 18, 2022 Reithinger Patients from Venezuela Trypanosoma Detect MRA ELISA home test 40 25 65 95% 100% 2010_d rapid test; Inbios, Seattle, WA in IFI, HAI does not report any brand 42 Sánchez RDT Existing samples from the serum banks Endemic Rapid test from different ELISA whole. IFI, HAI Not stated Argentina, Brazil, 237 237 474 90.1% 91% (54) 2014_a in each national reference laboratory area commercial brands: OnSite Does not report any brand Colombia, Costa Chagas Ab Rapid test Rica and Mexico Sánchez WL Check Chagas 88.7% 97% 2014_b Sánchez Trypanosoma Detect Rapid Test 92.9% 94% 2014_c Sánchez Chagas Quick Test 92.9% 93.2% 2014_d Sánchez Chagas Stat-Pak assay 87.2% 93.2% 2014_e Sánchez SD Chagas Ab Rapid 90.7% 94% 2014_f Sánchez Serodia Chagas 94.2% 94.7% 2014_g Sánchez ImmunoComb II Chagas Ab 97.2% 94% 2014_h (Continued ) 12 / 26 Chagas disease diagnosis: Systematic revision and meta-analysis Table 1. (Continued) Ref Author / Type of Type of participants Study area Index test Reference test Study period Country Number of Number of Total Sensit. Specif. year test sick healthy patients patients 43 López 2010 RDT Adult patients from Central and South Non- Simple CHAGASWB (Operon S. ELISA whole (ELISAc) home test Not stated Spain 49 92 148 92.5% 96.8% (55) America who went to the Primary Care endemic A, Spain) ELISA recombinant Bioelisa Chagas, Center Clot in Barcelona for a CD area BiokitS.A., Spain screening. Western blot 18 Antinori ELISA Migrants from Latin America (Brazil, Non- (ARCHITECT Chagas, Abbott, Unknown reference standard July 30, 2013 Italy 48 453 501 100% 99.7% (27) 2018_ a and Bolivia, Ecuador; Salvador and Peru) endemic Chicago, IL, USA.) to July 30, RDT who participated in health promotion area 2014 Antinori (BioELISA Chagas III, 95.9% 99.7% 2018_ b programs and screening BiosChile, Santiago, Chile) Antinori (Trypanosoma cruzi IgG Rapid 89.2% 92.5% 2018_ c Test, ImmunoSpark, SD, Rome, Italy) 38 Whittman ELISA Participants who are blood donors at the Non- Ortho ELISA ELISA whole ELISA Ortho ELISA September USA 500 300 800 92.4% 100% (49) 2019_a and American Red Cross–migrants and endemic ELISA recombinant Abbott PRISM 2006 to June RDT native American area (Abbott Laboratories, Abbott Park, 2018 Whittman Hemagen ELISA 88% 100% IL) 2019_b Radioimmunoprecipi-tation RIPA, Whittman Wiener ELISA Quest Diagnostics (Chantilly, VA) 94% 99.3% 2019_c Immunochromatogra-phy Abbott Whittman InBios rapid test enzyme strip assay (ESA) 97.4% 92.3% 2019_d PLOS NEGLECTED TROPICAL DISEASES � The authors of this paper calculated the sensitivity and specificity Sensit: Sensitivity Specif: Specificity https://doi.org/10.1371/journal.pntd.0010860.t001 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0010860 October 18, 2022 13 / 26 Chagas disease diagnosis: Systematic revision and meta-analysis PLOS NEGLECTED TROPICAL DISEASES Chagas disease diagnosis: Systematic revision and meta-analysis Chagas Abbott [9,30,34,36,48,55], ELISA Chagas III BIOSChile [9,36,41], and Chagatest Wie- ner [9,25]. 11 different RDTs were assessed; the most used commercial brands were Chagas Detect Plus InBios [10,40,43,54,56], Chagas Stat-Pak assay ChemBio [10,40,50,52,60], and WL Check Chagas Wiener [23,32,60]. Of the 16 studies that analyzed RDTs, 8 did not report who read the results [23,26,36,37,40,54,60,61], 4 did not specify whether or not they had training in reading this type of test [10,47,50 and 56] and 4 studies indicated that the reading was done by qualified personnel [32,39,43,52]. 62.7 % of the studies were carried out in countries with endemic regions and native popula- tion such as Brazil, Argentina, Bolivia, Venezuela, Paraguay, Mexico, Colombia, Guatemala, Panama, Ecuador, Peru, Nicaragua, Honduras and Costa Rica. 27.9 % of the studies were car- ried out in non-endemic regions, with a migrant population from countries such as Spain, the United States, Italy, Switzerland and the United Kingdom. 51.2 % of the studies had a non-comparative observational design (cohort or cross-sec- tional) [10,23,27,32,36–41,43,44,46,48–53,57–59]; 46.5 % were comparative clinical design studies (case-control) [9,21,22,24–26,28–31,33–35,42,45,47,54–56,60] and 2.3 % corresponded to mixed studies (comparative and non-comparative) [61]. Of the 43 studies analyzed, 18 of them [21,22,27,31,33,38,42,44–47,49,51,53,55,57–59] included individuals with other diseases to assess cross-reaction. These diseases were infectious parasitic diseases such as cutaneous, mucocutaneous, visceral and tegumentary leishmaniasis, malaria, toxoplasmosis, schistosomiasis, strongyloidiasis, filariasis, neurocysticercosis, taenia- sis, ascariasis, trichuriasis, amoebic liver abscess, visceral larva migrans, and patients positive for Trypanosoma rangeli; bacterial, such as syphilis, rheumatic fever, leptospirosis, tuberculo- sis, brucellosis, and Lyme disease; viral infections such as hepatitis B and C, HIV, rubella, mea- sles, dengue, Zika, parvovirus, HTLV, Epstein-Barr, or cytomegalovirus; fungal infections, such as South American blastomycosis; and other non-infectious diseases, such as lymphoblas- togenesis, juvenile diabetes, lupus erythematosus, and idiopathic megaesophagus. However, only 13 of these [21,26,27,29,31,38,42,45–47,55,58,59] reported cross-reaction percentages, which ranged between 0% and 62.5%, and whose main reaction was related to Leishmania spp. Risk of bias and applicability The quality assessment of the studies included in the analysis of ELISA tests is shown in S1 and S2 Figs; and those of RDTs are shown in S3 and S4 Figs. The risk of bias was assessed in the four domains: 1. Patient selection was assessed at high risk of bias in 19 articles for ELISA tests and in 6 for RDT because a consecutive or random sample of patients was not used, since it was unclear in 5 articles for ELISA tests, and because it did not specify patient recruitment; and it was assessed as low risk in 9 studies for RDTs. 2. The risk of bias related to the index test was assessed as unclear in 19 studies for ELISA and in 7 for RDTs because they did not state clearly whether the index test results were inter- preted without knowledge of the reference standard results. 3. The bias related to the reference test had 19 studies for ELISA tests and 7 for RDTs, which were classified as high risk because the result of the reference test was interpreted knowing the results of the index test, or a single diagnostic test was used as a reference standard ([62] [63],). 4. Flow and time were assessed as high risk of bias in four studies for ELISA tests, as not all patients received the same reference standard; while all included studies for RDTs were found to be at low risk of bias on this dimension. PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0010860 October 18, 2022 14 / 26 PLOS NEGLECTED TROPICAL DISEASES Chagas disease diagnosis: Systematic revision and meta-analysis Regarding the applicability in the first three domains, 100% of the articles that assessed both ELISA and RDTs tests were classified as low risk because they coincided with the review question. Synthesis of results Selecting the model for Elisa and Rapid Diagnostic Tests. The Bayesian model (GS) that best fit the analysis of ELISA tests was the binomial-normal with the logit or the probit as the link function (DIC = 630 and DIC = 631, which can be considered equivalent [S1 Table]). Sim- ilarly, for the RDT analysis, with the binomial-normal probit model (DIC = 416 [S2 Table]); thus, a probit link was used for the two tests analyzed. Sensitivity and specificity of ELISA tests. The ELISA tests had an overall sensitivity of 99% (95% CI: 98–99) and an overall specificity of 98% (95% CI: 97–99) (Fig 2). Some studies presented outliers in sensitivity [33] and specificity [25]. In the predictive region, greater vari- ability is observed for specificity than for sensitivity (Fig 3), therefore, it is observed that there is greater heterogeneity in specificity than in sensitivity. Sensitivity and specificity of Rapid Diagnostic Tests. The RDTs had an overall sensitiv- ity of 95% (95% CI: 94–97), and an overall specificity of 97% (95% CI: 96–98). There were studies with atypical values or outliers [10,54] (Those that did not follow the patron of most of Fig 2. Sensitivity and specificity of ELISA tests in the studies included in the meta-analysis. TP: true positives; FP: false positives; FN: false negatives; TN: true negatives. https://doi.org/10.1371/journal.pntd.0010860.g002 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0010860 October 18, 2022 15 / 26 PLOS NEGLECTED TROPICAL DISEASES Chagas disease diagnosis: Systematic revision and meta-analysis Fig 3. Predictive region in the ROC space, with all the studies for ELISA tests. https://doi.org/10.1371/journal.pntd.0010860.g003 the studies, which means that they were strayed of the tendency) (Fig 4). The predictive region (Fig 5) is more symmetric, and a slightly higher variability is observed for sensitivity. There- fore, it is identified that there is a slightly greater heterogeneity for sensitivity. Subgroup analysis. For the ELISA tests, sensitivity estimates were similar by subgroups. The punctual estimate ranged from 98% to 99%, and CIs ranged from 97% to 99%. As for speci- ficity, the sensitivity value ranged between 95% and 100%; the subgroup with low risk of bias was the one that showed the lowest specificity, with 95% (95% CI: 91–97), and the non-endemic area was the one that reported the highest specificity, 100% (95% CI: 99–100) (S5 Fig). Regarding the ELISA tests, the moderators that showed the greatest difference in the predic- tive region were study design (clinical comparative or non-comparative), where the compara- tive studies presented low heterogeneity, while the non-comparative studies had greater heterogeneity; and the study risk subgroup (low or high risk of bias), because the studies are similar when they present risk, showing a lower precision (S6 Fig). For the RDTs, the sensitivity estimates by subgroups vary more than the specificity. The punctual sensitivity estimates ranged from 91% to 97%, and CIs ranged from 86% to 98%, with the most notable difference (with no overlapping CIs) also observed between the non-compar- ative design subgroup, with 97 % (95 % CI: 96–98), and the comparative clinical design, with 93 % (95 % CI: 90–95). The punctual specificity estimate ranges from 95% to 98%, and CIs range from 93% to 99%. The most notable difference (without overlapping CIs) is between the non-comparative design subgroup, with 98% (95% CI: 97–99), and the clinical comparative design, with 95% (95% CI: 93–99) (S7 Fig). Regarding the RDTs, the moderators that show the greatest difference in the predictive region are the subgroups study area (endemic or non-endemic), sample type (serum, whole blood or not applicable), and study risk (low or high risk of bias) (S8 Fig). PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0010860 October 18, 2022 16 / 26 PLOS NEGLECTED TROPICAL DISEASES Chagas disease diagnosis: Systematic revision and meta-analysis Fig 4. Sensitivity and specificity of Rapid Diagnostic Tests in the studies included in the meta-analysis. TP: true positives; FP: false positives; FN: false negatives; TN: true negatives. https://doi.org/10.1371/journal.pntd.0010860.g004 Analysis of influential observations. Four [9,25,33,34] and five [10,23,40,52,54] were the most influential for the ELISA test studies and the RDT respectively. For each test, two models were fitted (one with all studies and one for all studies except the influential ones) in order to observe the effect of excluding influential studies on the accuracy and predictive region. Both models showed a similar predictive region. The influential studies of ELISA tests were always studies with a clinical-comparative design; whereas the ones for RDTs were non-comparative studies (S9 Fig). Publication bias. Asymmetry was observed in the funnel plots for ELISA tests; whereas no asymmetry was observed in the RDTs. The result of the Deeks’ asymmetry test was statistically significant for ELISA tests (p < 0.01), but not for RDTs (p = 0.64). Discussion Summary of evidence The combined sensitivity and specificity of ELISA tests were 99% (95% CI: 98–99) and 98% (95% CI: 97–99); whereas the ones for RDTs were 95% (95% CI: 94–97) and 97% (95% CI: 96– 98). The overall sensitivity of RDTs was lower than that of ELISA tests. According to the results PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0010860 October 18, 2022 17 / 26 PLOS NEGLECTED TROPICAL DISEASES Chagas disease diagnosis: Systematic revision and meta-analysis Fig 5. Predictive region in the ROC space, with all the studies for RDTs. https://doi.org/10.1371/journal.pntd.0010860.g005 obtained in this meta-analysis, the sensitivity and specificity of ELISA tests were higher than those reported in another meta-analysis, in which ELISA tests were compared with RDTs [64] and in which the sensitivity was 97.7. % (96.7%-98.5%) and specificity 96.3% (94.6%-97.6%). The sensitivity of ELISA tests was also higher than in another meta-analysis, where different types of tests were analyzed for diagnosing CD and in which they obtained a sensitivity result of 90% (89%-91%) and a specificity of 98%. % (98 %-98 %) [65], as in this study. Regarding RDTs, the sensitivity and specificity data obtained in this study were lower than those docu- mented in another meta-analysis, where they included clinical trials with recruitment of cohorts of individuals at risk of exposure to T. cruzi, which were 96.6 % (CI95%: 91.3–98.7) and 99.3 % (CI95 %: 98.4–99.7), respectively [66]. Regarding heterogeneity, in the ELISA tests it was identified that this measure was higher in specificity than in sensitivity, which is similar to that reported by Brazil [64], while in the RTDs, the heterogeneity was limited, being slightly higher for sensitivity, coinciding with what was described in the systematic review carried out by Angheben [39], results that differ from those reported by Afonso [65]. This meta-analysis showed greater specificity for ELISA tests in non-endemic areas, and greater sensitivity for endemic areas. The diagnostic performance of RDTs was the same for endemic and non-endemic areas, unlike what Angheben et al. [66] reported, where perfor- mance was higher in endemic than non-endemic areas. No differences in sensitivity were observed between the in-house and commercial ELISA tests. The same showed to be true for specificity in the two types of tests. This differs from what was stated in the meta-analysis by Afonso et al. [65], where the commercial tests were more sensitive than the in-house ones; however, the specificity was similar between the two, as it was observed in this meta-analysis. PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0010860 October 18, 2022 18 / 26 PLOS NEGLECTED TROPICAL DISEASES Chagas disease diagnosis: Systematic revision and meta-analysis All ELISA test assays were made on serum, with the exception of the study by Cervantes- Landı́n et al. [46], who used venous blood impregnated on filter paper before its coagulation. However, RDTs were made on all types of samples, mostly serum [23,26,32,36,50,54,56,60], but also venous blood and serum [23,43,52]; venous blood [40,61]; capillary blood [10,37]; serum, venous blood, and capillary blood [47], and one study did not specify the sample type [39]. These RDTs are frequently used in Point-of-Care tests (immediate diagnostic analyses), which are performed outside the laboratory, closer to the patient, with easily transportable material and equipment, and which results are available in minutes or in less than one hour. Its application is greater in developing countries [67] since the use of samples such as capillary blood -easy to collect without requiring collection tubes or centrifugation-, would be the best choice for RDTs. The results obtained in this study, depending on the type of sample, allow us to infer that the diagnostic performance of the RDTs was good, regardless of the type of sample. The trypanosomatids that affect men in America belong to the Leishmania and Trypano- soma genera. ELISA tests have been valuable for diagnosing these two agents, but their speci- ficity may be low due to the cross-reactivity between the two species of parasites; thus, it is important to take this into account, especially when you want to know the prevalence of these two diseases in endemic areas [68]. Furthermore, most of the areas where T. cruzi is found are co-endemic for Leishmania sp. and T. rangeli, which complicates the diagnosis of CD [69]. This coincides with what was documented in 13 of the studies analyzed, according to which the percentage of cross-reaction is between 0% and 62.5% with various diseases, among which leishmaniasis stands out. Limitations The items in which a risk of bias was detected in more articles were patient screening and ref- erence standard for the two types of tests assessed; furthermore, the risk of bias associated with the index test was unclear in more than 50% of the studies for ELISA tests. On the other hand, in the applicability items, the studies selected did not present a risk, similar to that described in the meta-analysis carried out by Angheben et al. [66], in relation to the quality of the RDT tests. For the ELISA tests, the sensitivity in the studies with low or high risk of bias was very similar, different from that described by Afonso et al. [65], who reported higher sensitivity in low-risk studies than in high-risk ones. As for specificity, it was higher in the articles that pre- sented a high risk of bias, different from what was reported in this same meta-analysis, where the studies with low risk showed greater specificity. 46.5% of the studies were found to be comparative, and 2.3% mixed studies (comparative and non-comparative). This influences the quality of the studies included because the best design to assess the validity of diagnostic tests is the non-comparative one (cohort or cross-sec- tional), where a consecutive and representative series of patients with suspected disease are given the test to be evaluated and the reference test in a blind and independent manner and interpreted in the absence of any additional clinical information, which will not be available when the test is used in the practice, either. In the literature found on this topic, most studies evaluating diagnostic tests take into account the blinding of the reference test results, but few of them have used this non-compara- tive design [70]. In this meta-analysis, lower sensitivity and specificity values have been found in RDT studies with a comparative clinical design, where a group of patients diagnosed with the disease and a group without this diagnosis were included. This is an unexpected result since studies with a clinical comparative or case-control design tend to overestimate sensitivity and specificity; however, it is possible that this effect only occurs when severe cases are PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0010860 October 18, 2022 19 / 26 PLOS NEGLECTED TROPICAL DISEASES Chagas disease diagnosis: Systematic revision and meta-analysis included in the case group [71]. Additionally, a high risk of publication bias was identified for ELISA tests, which coincides with the study by Afonso et al. [65]. The meta-analysis could have been affected by the search made in only four databases because not all studies related to the topic might have been included. Regarding the studies included, ELISA and RDT tests were not compared because only one study [36] had done so; the same was true for the in-house and commercial RDT subgroups which could not be assessed since only one study [26] included in-house RDTs. The quality of the systematic review was also influenced by the results of the selected articles and their design (some of which were clinical-comparative); that is, some variables could not be explored in the subgroup analysis because they were not reported in many studies, for instance, study period, cross reactions, distribution by rural and urban area, generation or ver- sion of diagnostic tests, estimation of cut-off points, geographic areas of the strains used as sources of antigens, and the type of antigen (multiepitope or combination of recombinant proteins). Subsequent studies should follow the instructions given by the WHO and carry out two tests in parallel using different antigens due to the immunogenic diversity of the different strains of the parasite, the immune response of the patients, and the existence of cross-reac- tions with other trypanosomatids that coexist in endemic areas [72]. Likewise, a sufficient number of samples should be included to evaluate the cross-reactions between chronic CD and Leishmania infection, considering that patients with either of the two infections, or with mixed infection, may be misdiagnosed given the crossed serological reactions when combina- tions of uncharacterized antigens are used [22]. Regarding the current status of the implementation of RDTs for diagnosing chronic CD in endemic areas of Latin America, these are used as tests of choice in screening programs for its detection, early treatment and control, and they represent a first approach at point of care for the rapid diagnosis of CD in endemic countries [73]. In addition, since RDTs are easier to use than ELISA, it would be feasible to use them more often in screening programs, which would facilitate the detection of CD cases without ignoring the current recommendations to confirm positive results through conventional methods. Conclusions According to this systematic review, ELISA and Rapid Detection Tests (RDTs) have a high validity for diagnosing chronic CD; however, the overall sensitivity of the second test was lower than that of the first one, so it is important to better study the variables that influence the validity of the RDTs, some of which had not been taken into account in some of the studies included. The usefulness of RDTs for screening CD in epidemiological contexts, such as endemic regions that are difficult to access or non-endemic regions with a high prevalence of chronic CD, should also be assessed, as well as the inclusion of RDTs in the diagnostic algorithms used for its detection, in order to improve access to treatment since the first level of primary health care. Supporting information S1 Checklist. PRISMA-DTA for abstracts checklist. (DOCX) S2 Checklist. PRISMA-DTA checklist. Checklist for reporting of systematic reviews and meta-analysis of diagnostic test accuracy studies. (DOCX) PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0010860 October 18, 2022 20 / 26 PLOS NEGLECTED TROPICAL DISEASES Chagas disease diagnosis: Systematic revision and meta-analysis S1 Database. Database search strategy. (DOCX) S1 Table. ELISA test. Bivariate model goodness. (DOCX) S2 Table. RDTs Bivariate model goodness. (DOCX) S1 Fig. Risk of bias and applicability of ELISA tests. (TIFF) S2 Fig. Summary of risk of bias and applicability of ELISA tests. (TIFF) S3 Fig. Risk of bias and applicability of RDTs. (TIFF) S4 Fig. Summary of risk of bias and applicability of RDTs. (TIFF) S5 Fig. Estimated sensitivity and specificity of ELISA tests by subgroups. (TIFF) S6 Fig. Predictive region of ELISA tests by subgroups. (TIFF) S7 Fig. Estimated sensitivity and specificity of RDTs by subgroups. (TIFF) S8 Fig. Predictive region of RDTs by subgroups. (TIFF) S9 Fig. Predictive region in the global ROC space and after excluding the most influential studies for ELISA (A) and RDTs (B). (TIFF) Author Contributions Conceptualization: Sandra Helena Suescún-Carrero. Data curation: Sandra Helena Suescún-Carrero, Philippe Tadger, Carolina Sandoval Cuellar, Laura Ximena Ramı́rez López. Formal analysis: Sandra Helena Suescún-Carrero, Lluis Armadans-Gil, Laura Ximena Ramı́rez López. Funding acquisition: Sandra Helena Suescún-Carrero, Carolina Sandoval Cuellar, Laura Ximena Ramı́rez López. Investigation: Sandra Helena Suescún-Carrero, Philippe Tadger, Carolina Sandoval Cuellar, Lluis Armadans-Gil, Laura Ximena Ramı́rez López. Methodology: Sandra Helena Suescún-Carrero, Lluis Armadans-Gil. Project administration: Sandra Helena Suescún-Carrero, Laura Ximena Ramı́rez López. Resources: Sandra Helena Suescún-Carrero, Carolina Sandoval Cuellar, Laura Ximena Ramı́rez López. 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