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Chagas' disease diagnosis: Comparative analysis of recombinant ELISA with conventional ELISA and the haemagglutination test
Abstract
Serological screening for Chagas' disease in the blood banks of South America is carried out by using two different assays that generally show a high number of inconclusive results. To establish a combination of two tests that can minimize the number of inconclusive results, we compared a recombinant enzyme-linked immunosorbent assay (ELISA) with two conventional tests.
Serum samples from chagasic patients (n = 112), from non-chagasic individuals (n = 143) and from patients with other diseases (n = 32) were tested using three assays: recombinant ELISA (Rec-ELISA); conventional ELISA (Con-ELISA); and the indirect haemagglutination (IHA) test.
When we evaluated the data by matching the Rec-ELISA and the IHA test, 52 inconclusive results were obtained. When Rec-ELISA and Con-ELISA were matched, only four inconclusive results were observed.
Our investigation indicates that the use of two ELISAs with different antigen preparations provides an effective test combination for blood bank screening of Chagas' disease.
... In the present study, the results of the ELISA were confirmed by blood culture and PCR in a high percentage of samples, affirming the use of ELISA as the sole test for screening prospective blood donors. Satisfactory results for different ELISA kits have been previously reported (Oelemann et al., 1998;Leiby et al., 2000;Blejer, Saguier, Salamone, 2001;Gadelha et al., 2003;Pirard et al., 2005). However, the results presented in this study must be interpreted cautiously by considering the samples analyzed, the serological tests and reagents, the technical procedures, quality control, and the protocols for extraction and amplification of the DNA, and taking into account that the study area has a low prevalence of Chagas' disease. ...
... Of the conventional serological tests used to diagnose Chagas' disease, the IHA has the lowest sensitivity (Oelemann et al., 1998;Leiby et al., 2000;Blejer, Saguier, Salamone, 2001;Gadelha et al., 2003;Pirard et al., 2005). However, in the present study, the IHA was able to confirm the diagnosis of one individual with an inconclusive ELISA and a negative IIF result. ...
... However, in the present study, the IHA was able to confirm the diagnosis of one individual with an inconclusive ELISA and a negative IIF result. This result corroborates the concerns of several investigators (Blejer, Saguier, Salamone, 2001;Gadelha et al., 2003;Pirard et al., 2005;Araújo, Vianna, Berne, 2008) who have emphasized the importance of combining several effective tests when screening for Chagas' disease at the blood banks, thereby reducing false-negative or inconclusive results and crossreactions (Saéz-Alquezar et al., 1998). ...
The objective of this study was to evaluate, through blood culture and PCR, the results of the ELISA for Chagas' disease in the screening of blood donors in the public blood-supply network of the state of Paraná, Brazil, and to map the epidemiological profile of the donors with respect to their risk of infection by Trypanosoma cruzi. The negative and positive results of the ELISA were confirmed by blood culture and PCR for 190/191 individuals (99.5%). For one individual (0.5%), the ELISA was inconclusive, blood culture and IIF were negative, and IHA and PCR positive. Three individuals (1.6%) were positive for T. cruzi on all the tests. Donors were predominantly female, and natives of Paraná, of rural origin, had observed or been informed of the presence of the vector in the municipalities where they resided, had never received a blood transfusion, had donated blood 1 to 4 times, and reported no cases of Chagas' disease in their families. We concluded that PCR and blood culturing have excellent potential for confirming the results of the ELISA, and that candidate blood donors with negative or positive tests have a similar risk of infection by T. cruzi, indicating that the ELISA test is sufficiently safe for screening blood prior to use.
... The analysis identified 24 published studies that used HmT as a diagnostic technique for CD. After analysis, only seven studies [63,66,76,[84][85][86][87] were selected. A total of 1450 subjects were studied. ...
... Sensitivity and specificity are reported with a mean (95% confidence limits). The Forest plot depicts the estimated sensitivity and specificity (black squares) and its 95% confidence limits (horizontal black line)[63,66,76,[84][85][86][87]. ...
The present systematic review and meta-analysis about the accuracy of diagnostic tests aim to describe the findings of literature over the last thirty years for the diagnosis of Chagas disease (CD). This work aimed to determine the accuracy of diagnostic techniques for CD in the disease's acute and chronic phases. The PubMed database was searched for studies published between 1990 and 2021 on CD diagnostics. Fifty-six published studies that met the criteria were analyzed and included in the meta-analysis, evaluating diagnostic accuracy through sensitivity and specificity. For Enzyme-Linked Immunosorbent Assay (ELISA), Fluorescent Antibody Technique (IFAT), Hemagglutination Test (HmT), Polymerase Chain Reaction (PCR), and Real-Time Polymerase Chain Reaction (qPCR) diagnosis methods, the sensitivity had a median of 99.0%, 78.0%, 75.0%, 76.0%, and 94.0%, respectively; while specificity presented a median of 99.0%, 99.0%, 99.0%, 98.0%, and 98.0%, respectively. This meta-analysis showed that ELISA and qPCR techniques had a higher performance compared to other methods of diagnosing CD in the chronic and acute phases, respectively. It was concluded utilizing the Area Under the Curve restricted to the false positive rates (AUCFPR), that the ELISA diagnostic test presents the highest performance in diagnosing acute and chronic CD, compared to serological and molecular tests. Future studies focusing on new CD diagnostics approaches should be targeted.
... The analysis identified 24 published studies that used HmT as a diagnostic technique for CD. After analysis, only seven studies [63,66,76,[84][85][86][87] were selected. A total of 1450 subjects were studied. ...
... Sensitivity and specificity are reported with a mean (95% confidence limits). The Forest plot depicts the estimated sensitivity and specificity (black squares) and its 95% confidence limits (horizontal black line)[63,66,76,[84][85][86][87]. ...
The present systematic review and meta-analysis about the accuracy of diagnostic tests aim to describe the findings of literature over the last thirty years for the diagnosis of Chagas disease (CD). This work aimed to determine the accuracy of diagnostic techniques for CD in the disease’s acute and chronic phases. The PubMed database was searched for studies published between 1990 and 2021 on CD diagnostics. Fifty-six published studies that met the criteria were analyzed and included in the meta-analysis, evaluating diagnostic accuracy through sensitivity and specificity. For Enzyme-Linked Immunosorbent Assay (ELISA), Fluorescent Antibody Technique (IFAT), Hemagglutination Test (HmT), Polymerase Chain Reaction (PCR), and Real-Time Polymerase Chain Reaction (qPCR) diagnosis methods, the sensitivity had a median of 99.0%, 78.0%, 75.0%, 76.0%, and 94.0%, respectively; while specificity presented a median of 99.0%, 99.0%, 99.0%, 98.0%, and 98.0%, respectively. This meta-analysis showed that ELISA and qPCR techniques had a higher performance compared to other methods of diagnosing CD in the chronic and acute phases, respectively. It was concluded utilizing the Area Under the Curve restricted to the false positive rates (AUCFPR), that the ELISA diagnostic test presents the highest performance in diagnosing acute and chronic CD, compared to serological and molecular tests. Future studies focusing on new CD diagnostics approaches should be targeted
... A third test is performed when discordant results are obtained. Serological discrepancies are not uncommon, especially when using crude antigen-based serologies and in countries where Leishmania spp. is endemic [11][12][13]. To date, none of the existing serological techniques have proved sufficiently specific and sensitive to be recommended as a single test for the screening of the disease. ...
... As expected, these tests lacked specificity due to frequent cross-reactions with related protozoa, such as Leishmania spp. and Trypanosoma rangeli [12,13]. The development of techniques using recombinant antigens [14,16] and, more recently, chimeric recombinant antigens [15], has helped to overcome the previous problem of specificity, as well as to improve the sensitivity of the serological tests. ...
Objectives
Imported Chagas disease (CD) is an emerging health problem in Europe due to immigration from endemic countries. Although WHO currently recommends two different serological methods to establish diagnosis, new tools like the Architect Chagas assay have potential for use as a single diagnostic test. Our objective was to determine an optimal signal‐to‐cutoff (S/CO) value for the Architect Chagas assay to diagnose CD with a single test.
Methods
A retrospective study conducted at the 12 de Octubre University Hospital (Madrid, Spain). All patients with requests for Chagas screening between January 2014 and August 2017 were consecutively included. All samples were routinely tested with the Architect assay. Negative samples (S/CO<0.8) required no further testing. Immunochromatographic testing (ICT) and/or indirect immunofluorescence (IFI) were used to confirm samples with S/CO≥0.8. Receiver operator characteristic (ROC) curve analysis determined the Architect S/CO value that yielded 100% specificity and positive predictive value. SPSS software, version 22.0 was used for data analysis.
Results
4153 samples were analysed; 361(8.69%) gave a reactive Architect Chagas result. 261/361(72.3%) were women; median age was 38 years old (2‐79). 92.8% were Bolivian. 307 (85.0%) were confirmed as cases of Chagas; 52 (14.4%) were not infected; 2 (0.6%) were not evaluable. Seroprevalence was 7.39%. An S/CO ≥3.80 yielded 100% specificity (95% confidence interval [CI], 0.93‐1.00) and 100% positive predictive value (95% CI, 0.99‐1.00).
Conclusions
Using S/CO ≥3.80, the Architect Chagas could be used as a single test for diagnosis of chronic CD in Bolivian immigrants. Patients with S/CO between 0.80 and 3.80 would require additional testing.
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... As some investigations had data regarding two or more tests, the number of reports, tests, and data to analyse will not match the number of reports included in the review. Data concerning commercial ELISA were extracted from 28 reports (Lorca et al. 1992, Pan et al. 1992, Carvalho et al. 1993, Teixeira et al. 1994, Hamerschlak et al. 1997, Oelemann et al. 1998, Houghton et al. 1999, Leiby et al. 2000, Gadelha et al. 2003, Arrieta et al. 2004, Enciso et al. 2004, Moretti et al. 2004, Pirard et al. 2005, Duarte et al. 2006, Malan et al. 2006, Caballero et al. 2007, Tobler et al. 2007, Gorlin et al. 2008, Otani et al. 2009, Remesar et al. 2009, Añez et al. 2010, Flores-Chávez et al. 2010, Barfield et al. 2011, De Marchi et al. 2011, Pereira et al. 2012, Araújo & Berne 2013 (Fig. 1) including 26 different combinations of trademarks and test names. Data from 12 commercial ELISA-rec reports were extracted (Pastini et al. 1994, Gomes et al. 2001, Gadelha et al. 2003, Pirard et al. 2005, Blejer 2006, Chang et al. 2006, Caballero et al. 2007, Ramírez et al. 2009, Remesar et al. 2009, Villagrán et al. 2009, Añez et al. 2010, Pereira et al. 2012 (Fig. 1), including eight different combinations of trademarks and test names. ...
... Data concerning commercial ELISA were extracted from 28 reports (Lorca et al. 1992, Pan et al. 1992, Carvalho et al. 1993, Teixeira et al. 1994, Hamerschlak et al. 1997, Oelemann et al. 1998, Houghton et al. 1999, Leiby et al. 2000, Gadelha et al. 2003, Arrieta et al. 2004, Enciso et al. 2004, Moretti et al. 2004, Pirard et al. 2005, Duarte et al. 2006, Malan et al. 2006, Caballero et al. 2007, Tobler et al. 2007, Gorlin et al. 2008, Otani et al. 2009, Remesar et al. 2009, Añez et al. 2010, Flores-Chávez et al. 2010, Barfield et al. 2011, De Marchi et al. 2011, Pereira et al. 2012, Araújo & Berne 2013 (Fig. 1) including 26 different combinations of trademarks and test names. Data from 12 commercial ELISA-rec reports were extracted (Pastini et al. 1994, Gomes et al. 2001, Gadelha et al. 2003, Pirard et al. 2005, Blejer 2006, Chang et al. 2006, Caballero et al. 2007, Ramírez et al. 2009, Remesar et al. 2009, Villagrán et al. 2009, Añez et al. 2010, Pereira et al. 2012 (Fig. 1), including eight different combinations of trademarks and test names. Finally, data concerning PCR were extracted from 24 reports (Avila et al. 1993, Wincker et al. 1994, Britto et al. 1995, Espinoza et al. 1996, Junqueira et al. 1996, Carriazo et al. 1998, Chiaramonte et al. 1999, Gomes et al. 1999, Ribeiro-dos-Santos et al. 1999, Castro et al. 2002, Gutierrez et al. 2004, Duarte et al. 2006, Gil et al. 2007, Piron et al. 2007, Fitzwater et al. 2008, Deborggraeve et al. 2009, Ferrer et al. 2009, Ramírez et al. 2009, Batista et al. 2010, Gilber et al. 2013, Sabino et al. 2013), but only one commercial test was found (Deborggraeve et al. 2009, De Winne et al. 2014). ...
Chronic Chagas disease diagnosis relies on laboratory tests due to its clinical characteristics. The aim of this research was to review commercial enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) diagnostic test performance. Performance of commercial ELISA or PCR for the diagnosis of chronic Chagas disease were systematically searched in PubMed, Scopus, Embase, ISI Web, and LILACS through the bibliography from 1980-2014 and by contact with the manufacturers. The risk of bias was assessed with QUADAS-2. Heterogeneity was estimated with the I2 statistic. Accuracies provided by the manufacturers usually overestimate the accuracy provided by academia. The risk of bias is high in most tests and in most QUADAS dimensions. Heterogeneity is high in either sensitivity, specificity, or both. The evidence regarding commercial ELISA and ELISA-rec sensitivity and specificity indicates that there is overestimation. The current recommendation to use two simultaneous serological tests can be supported by the risk of bias analysis and the amount of heterogeneity but not by the observed accuracies. The usefulness of PCR tests are debatable and health care providers should not order them on a routine basis. PCR may be used in selected cases due to its potential to detect seronegative subjects.
... Studies on screening tests for Chagas disease reported sensitivity rates for the ELISA kit of bioMérieux ranging from 98.38% to 98.5% and specificity rates from 94.30% to 99.93%. (20,21) Sensitivity rates ranging from 52.75% to 96.5% and specificity from 87% to 100% (22)(23)(24) have been described for IIF. Sensitivity rates from 98.5% to 100% and specificity between 99.69% and 100% have been reported for IHA. ...
... Sensitivity rates from 98.5% to 100% and specificity between 99.69% and 100% have been reported for IHA. (19,20) Regarding the Bio-Manguinhos-FIOCRUZ kits, Gadelha et al. (22) found 100% sensitivity and specificity for the c-ELISA kit and sensitivity of 98.2% for the rec-ELISA kit. ...
The frequent occurrence of inconclusive serology in blood banks and the absence of a gold standard test for Chagas'disease led us to examine the efficacy of the blood culture test and five commercial tests (ELISA, IIF, HAI, c-ELISA, rec-ELISA) used in screening blood donors for Chagas disease, as well as to investigate the prevalence of Trypanosoma cruzi infection among donors with inconclusive serology screening in respect to some epidemiological variables.
To obtain estimates of interest we considered a Bayesian latent class model with inclusion of covariates from the logit link.
A better performance was observed with some categories of epidemiological variables. In addition, all pairs of tests (excluding the blood culture test) presented as good alternatives for both screening (sensitivity > 99.96% in parallel testing) and for confirmation (specificity > 99.93% in serial testing) of Chagas disease. The prevalence of 13.30% observed in the stratum of donors with inconclusive serology, means that probably most of these are non-reactive serology. In addition, depending on the level of specific epidemiological variables, the absence of infection can be predicted with a probability of 100% in this group from the pairs of tests using parallel testing.
The epidemiological variables can lead to improved test results and thus assist in the clarification of inconclusive serology screening results. Moreover, all combinations of pairs using the five commercial tests are good alternatives to confirm results.
... The CS tests used in clinical laboratories vary within countries according to available reagents and protocols; the most widely used are listed as follows: (i) the complement fixation reaction (CFR), as first described by Guerreiro and Machado (1913) and later modified by Muniz and Freitas (1944a, b); (ii) the direct agglutination of killed parasites; (iii) the indirect agglutination of red blood cells pre-sensitized with parasite antigens and (iv) the indirect immunofluorescence tests (IIF) with fixed cultured parasites. The standardization of these various tests and protocols was made possible by a multicentric study involving several laboratories in different countries (Camargo et al. 1986) and other studies with commercially available tests (Leiby et al. 2000, Gadelha et al. 2003. ...
... Serum samples collected at different periods of time during this human T. cruzi infection were valuable for evaluating the success of the specific therapy and to establish a cure; all tests became negative after 300 days. Several other purified antigens from T. cruzi are commercially available for the diag-nosis of Chagas disease using ELISA, but only one has been evaluated in controlled clinical trials using treatedpresumably-cured patients (Silva et al. 2002, Gadelha et al. 2003, Lorena et al. 2008. The data are summarized in Table V and discussed below. ...
In previous work, we proposed alternative protocols for following patients with treated Chagas disease and these are reviewed herein. Evidence was provided to support the following: (i) functional anti-trypomastigote antibodies are indicative of ongoing chronic Trypanosoma cruzi infections; (ii) specific antibodies detected by conventional serology (CS) with epimastigote extracts, fixed trypomastigotes or other parasite antigens may circulate years after parasite elimination; (iii) functional antibodies are evidenced by complement-mediated lysis of freshly isolated trypomastigotes, a test which is 100% specific, highly sensitive, and the first to revert after T. cruzi elimination and (iv) the parasite target for the lytic antibodies is a glycoprotein of high molecular weight (gp160) anchored at the parasite surface. The complement regulatory protein has been cloned, sequenced and produced as a recombinant protein by other groups and is useful for identifying functional anti-T. cruzi antibodies in ELISA tests, thus dispensing with the need for live trypomastigotes to manage treated patients. If used instead of CS to define cures for Chagas patients, ELISA will avoid unnecessary delays in finding anti-T. cruzi drugs. Other highly sensitive techniques for parasite DNA detection, such as PCR, need to be standardized and included in future protocols for the management of patients with drug-treated Chagas disease.
... Nevertheless, due to its good specificity, HAI can be used with other serological tests for diagnosing chagasic infection. Gadelha et al. [36] compared the ELISA and HAI methods in a group of individuals from an endemic region who were suspected to carry the disease and showed 92% positivity for ELISA, without any inconclusive results. HAI, on the other hand, presented a positive result in 59% of the samples and an inconclusive one in 33%. ...
... HAI, on the other hand, presented a positive result in 59% of the samples and an inconclusive one in 33%. Nevertheless, the conjoint analysis of the 2 methods showed 52.6% positivity and 45.5% inconclusive results [36]. Therefore, when the diagnosis is based on 2 serological methods, inconclusive results tend to increase, but such practice presents false positives and cross reactions [37,38]. ...
Despite the existence of highly sensitive tests, inconclusive serological results are frequent in chronic chagasic infection. This study aimed to define a diagnostic conduct for 30 individuals with inconclusive serology (G3) for chagasic infection assisted at the Outpatient Unit for Infectious and Parasitic Diseases of the Botucatu School of Medicine. Twenty-one individuals with negative serology (G1) and 33 with positive serology (G2) were also studied. Serological methods ELISA, HAI, IFI and immunoblotting TESA-cruzi were used for G1, G2 and G3, and parasitological methods xenodiagnosis, hemoculture and PCR-LIT were used for G2 and G3 individuals. ELISA, HAI and IFI were performed in 5 different blood samples in G2 and G3. TESA-cruzi was carried out only once in G1, G2 and G3 and, since it is the most sensitive, it was utilized as standard. In G3, positivity for ELISA reached 86% in the fifth blood sample; the ELISA+HAI+IFI combination showed a maximum of 44.8% in the second sample; and TESA-cruzi, 76% in one single sample. Xenodiagnosis positivity was 9.4%; hemoculture showed 15.2%; and PCR-LIT exhibited 22% positivity in G2. Nevertheless, in G3, positivity percentage was 3.4% for xenodiagnosis, 6.7% for PCR-LIT, and no positive result was found for hemoculture. In G3, PCR-LIT resolved one case which was still inconclusive according to serology tests. In order to define inconclusive diagnoses, the results suggest the combined use of ELISA+HAI+IFI in 2 blood samples, decreasing the occurrence of false positive/negative results. If results remain inconclusive, the performance of TESA-cruzi and PCR-LIT, if necessary, is recommended.
... Regarding methods for diagnosing T. cruzi infection, with exception of the study that evaluated newborns, all other studies employed serological tests accompanied by a second different method to determine the clinical form of disease. The serological tests, such as indirect immunofluorescence, indirect hemagglutination, and enzyme-linked immunosorbent assays [54], are simple, accessible, and effective for detecting anti-T. cruzi antibodies in the chronic phase. ...
T cells recognize their ligand, the peptide major histocompatibility complex (MHC), via the T-cell receptor (TCR), which is composed of covalently linked α and β or γ and δ chains. This recognition is critical for T-cell ontogeny and controls the selection, activation, and function of T lymphocytes. Specific TCR αβ variable regions have been associated with immunopathogenesis of Chagas disease. Here, we present a systematic review that compiles experimental in vivo and human data regarding the preferential expression of variable alpha (Vα) and variable beta (Vβ) chain regions in Trypanosoma cruzi infection. The original studies indexed in PubMed/Medline, Scopus, and Web of Science databases were screened according to the PRISMA strategy. The analysis showed that expression of TCR Vα subfamilies were evaluated in one human study, and, unlike TCR Vβ, TCR Vα presented a more restricted usage. Despite the great variability in the usage of TCR Vβ regions in human Chagas disease, a down-regulation of TCR Vβ5 expression by T cells from patients in the acute phase of the disease was shown. Opposingly, this TCR region was found overly expressed in CD4+ T cells from chronic Chagas patients. It was also demonstrated that murine Vβ9+ T cells derived from nonlymphoid organs of T . cruzi -infected animals had a modulatory profile, while splenic Vβ9+ T cells produced inflammatory cytokines, indicating that although they display the same TCR Vβ region usage, these cells are functionally distinct. Despite the limitations of few papers and year of publication of the studies, compiling the data derived from them reveals that further investigation of TCR usage will point to their potential role in protective or pathogenic responses, as biomarkers of disease progression, and in the search for dominant peptides potentially useful for the development of vaccines or therapies.
... These tests have different principles of antibody recognition, improving the diagnosis of T. cruzi infection. Although their routine application, some problems are quite frequent in serological diagnosis, notably cross-reaction [10,11], which is more frequent using polyclonal antisera. A common cause of cross-reactivity is molecular mimicry between parasite-and self-epitopes. ...
Introduction: The diagnosis in Chagas disease is a challenge because most infections with Trypanosoma cruzi are asymptomatic and currently serological tests have limitations, such as cross-reactivity with other trypanosomatids. Real-time PCR (qPCR) is a useful procedure that allows T. cruzi detection even when the parasitic load is very low and seems interesting for monitoring the response to trypanocidal treatment and elucidating cases with doubtful serological results.
Areas covered: This systematic review aimed to investigate the applications and relevance of qPCR in human Chagas disease, and focus on the methodological aspects.
Expert opinion: The results showed that blood samples with the TaqMan procedure direct to nuclear DNA (nDNA) sequences are used the most. However, a high variability among laboratories concerning the qPCR methods make it difficult to compare between studies and the use in routine surveillance laboratories, even if some works had performed an analytical validation of T. cruzi qPCR to try to counteract this. Nevertheless, the detection of T. cruzi by qPCR has multiple advantages including fast results, reduction of carryover contamination compared to conventional PCR, and high sensitivity and specificity. This study has given an overview of assays using qPCR in human Chagas disease and has shown the relevance of this technique in diagnosis.
... The performance of ELISAs depends on the antigenic matrix employed to detect anti-T. cruzi antibodies (12,13). Conventional tests make use of either fractionated T. cruzi lysates or whole-cell epimastigote homogenates, resulting in a complex antigenic mixture of unknown and variable composition. ...
Chimeric T. cruzi -antigens have been proposed as a diagnostic tool for chronic Chagas disease (CD), both in endemic and non-endemic settings. Antibody response varies in accordance to each T. cruzi strain, presenting challenges to the use of antigens lacking demonstrated cross-reactivity with Leishmania spp. Our group expressed four chimeric proteins (IBMP-8.1, 8.2, 8.3 and 8.4) and previously assessed their diagnostic performance to determine cross-reactivity with Leishmania spp. Herein, we validated our findings using sera samples from different Brazilian endemic geographic areas reporting either Chagas Disease, visceral or American cutaneous leishmaniasis (ACL), or both. Overall, 829 serum samples were evaluated using commercial and IBMP enzyme-linked immunosorbent assays. Due to the absence of a reference assay to diagnosis CD, latent class analysis (LCA) was performed through the use of a statistical model. The incidence of cross-reactivity for ACL-positive samples varied from 0.35% (IBMP-8.3) to 0.70% (IBMP-8.1 and IBMP-8.2). Regarding visceral leishmaniasis (VL)-positive samples, the IBMP-8.2 and IBMP-8.3 antigens cross-reacted with six (3.49%) and only one sample (0.58%), respectively. No cross-reactivity with either ACL or VL was observed for the IBMP-8.4 antigen. Similarly, no cross-reactions were found when VL-positive samples were assayed with IBMP-8.1. The agreement among the results obtained using IBMP antigens ranged from 97.3% for IBMP-8.2, 99% for IBMP-8.1 and IBMP-8.3 to 100% for IBMP-8.4, demonstrating almost perfect agreement with LCA. Accordingly, in light of the negligible cross-reactivity with both ACL and VL, we suggest the use of IBMP antigens in regions with co-endemicity for T. cruzi and Leishmania spp.
... The use of a concurrent examination of samples with a technique based on recombinant antigens could help in confirming the infectious status of suspected cases. 44 One of the most significant problems in the serological diagnosis of T. cruzi infection is the absence of a gold standard test. Hence, institutions should opt for various tests using distinct technical principles to confirm the diagnosis in discrepant samples. ...
Introduction:
Infection by Trypanosoma cruzi is challenging to blood bank supplies in terms of accurate diagnosis, mostly due to its clinical complexity. Infected individuals may remain asymptomatic for years, albeit they may have circulating parasites potentially transferable to eventual receptors of a transfusion.
Objective:
Although risk donors are systematically excluded through a survey, an important residual risk for transmission remains, evidencing the need to implement additional actions for the detection of T. cruzi in blood banks.
Method:
A review of the scientific literature is presented with the objective of identifying relevant publications on this subject.
Results:
We discuss the diagnostic considerations of this chronic infection on transfusion medicine and some recent advances in the processing of blood and derivatives units.
Conclusion:
Finally, recommendations are made on how the transmission of T. cruzi can be avoided through the implementation of better diagnostic and pathogen control measures at blood banks.
... Existe otro kit, el ChagaTest® (Wiener, Argentina), que utiliza antígenos obtenidos por tecnología de ADN recombinante que ha mostrado excelentes resultados. Pero se ha señalado que la aplicación de un ELISA tradicional y un ELISA recombinante juntos mejora la sensibilidad y especificidad del diagnóstico serológico (Gadelha et al. 2003). ...
p style="text-align: justify;">LA ENFERMEDAD DE CHAGAS ES UN PROBLEMA DE SALUD pública en toda Latinoamérica; alrededor de 20 millones de personas están infectadas y 200 millones están en riesgo de contraer la enfermedad. En 2006, la prevalencia en Centroamérica era del 7%. Actualmente no existe vacuna contra el protozoo y el tratamiento disponible resulta, aparte de poco efectivo, muy tóxico para el paciente. Los programas de control de vectores han ayudado a reducir los índices de infestación en Latinoamérica, pero aún falta mucho por hacer. En Nicaragua, la enfermedad de Chagas está subvalorada y los trabajos publicados son muy pocos. Es necesario investigar sobre esta enfermedad en nuestro país con otro enfoque, uno que no subvalore la enfermedad y ayude a desarrollar métodos diagnósticos y posibles tratamientos. Este artículo recopila información sobre los trabajos realizados por los grupos más importantes de investigación en Chagas de Nicaragua en cuanto a epidemiología, control vectorial, diagnóstico y caracterización molecular.</p
... However, its performance depends on the antigen preparations used to detect the anti-T. cruzi antibodies [8,9]. Whole-cell homogenates or fractionated lysates of T. cruzi at the epimastigote stage have been used as complex mixtures of antigens for detecting T. cruzi infection. ...
Background:
The performance of current serologic tests for diagnosing chronic Chagas disease (CD) is highly variable. The search for new diagnostic markers has been a constant challenge for improving accuracy and reducing the number of inconclusive results.
Methodology/principal findings:
Here, four chimeric proteins (IBMP-8.1 to -8.4) comprising immunodominant regions of different Trypanosoma cruzi antigens were tested by enzyme-linked immunosorbent assay. The proteins were used to detect specific anti-T. cruzi antibodies in the sera of 857 chagasic and 689 non-chagasic individuals to evaluate their accuracy for chronic CD diagnosis. The antigens were recombinantly expressed in Escherichia coli and purified by chromatographic methods. The sensitivity and specificity values ranged from 94.3% to 99.3% and 99.4% to 100%, respectively. The diagnostic odds ratio (DOR) values were 6,462 for IBMP-8.1, 3,807 for IBMP-8.2, 32,095 for IBMP-8.3, and 283,714 for IBMP-8.4. These chimeric antigens presented DORs that were higher than the commercial test Pathozyme Chagas. The antigens IBMP-8.3 and -8.4 also showed DORs higher than the Gold ELISA Chagas test. Mixtures with equimolar concentrations were tested in order to improve the diagnosis accuracy of negative samples with high signal and positive samples with low signal. However, no gain in accuracy was observed relative to the individual antigens. A total of 1,079 additional sera were used to test cross-reactivity to unrelated diseases. The cross-reactivity rates ranged from 0.37% to 0.74% even for Leishmania spp., a pathogen showing relatively high genome sequence identity to T. cruzi. Imprecision analyses showed that IBMP chimeras are very stable and the results are highly reproducible.
Conclusions/significance:
Our findings indicate that the IBMP-8.4 antigen can be safely used in serological tests for T. cruzi screening in blood banks and for chronic CD laboratory diagnosis.
... However, the performance of these tests is dependent on the antigen preparations used to detect the anti-T. cruzi antibodies [9]. Earlier versions of serological tests used whole extracts of the non-invasive epimastigote forms of T. cruzi, because this is the safest parasite stage, which is also the most cost-efficient to culture. ...
The performance of serologic tests in chronic Chagas disease diagnosis largely depends on the type and quality of the antigen preparations that are used for detection of anti-Trypanosoma cruzi antibodies. Whole-cell T. cruzi extracts or recombinant proteins have shown variation in the performance and cross-reactivity. Synthetic chimeric proteins comprising fragments of repetitive amino acids of several different proteins have been shown to improve assay performances to detect Chagasic infections. Here, we describe the production of four chimeric T. cruzi proteins and the assessment of their performance for diagnostic purposes. Circular Dichroism spectra indicated the absence of well-defined secondary structures, while polydispersity evaluated by Dynamic Light Scattering revealed only minor aggregates in 50 mM carbonate-bicarbonate (pH 9.6), demonstrating that it is an appropriate buffering system for sensitizing microplates. Serum samples from T. cruzi-infected and non-infected individuals were used to assess the performance of these antigens for detecting antibodies against T. cruzi, using both enzyme-linked immunosorbent assay and a liquid bead array platform. Performance parameters (AUC, sensitivity, specificity, accuracy and J index) showed high diagnostic accuracy for all chimeric proteins for detection of specific anti-T. cruzi antibodies and differentiated seropositive individuals from those who were seronegative. Our data suggest that these four chimeric proteins are eligible for phase II studies.
... Among these, EIA is the most used because of its high automation level and flexibility to using different antigen preparations. 12 Antigens for the EIA assays are provided by total or semipurified homogenate of epimastigote forms of T. cruzi. As it could be expected, there is considerable variation in the reproducibility, feasibility, and reliability of such positive control samples. ...
There is a significant heterogeneity in reported performance of serological assays for Chagas disease diagnosis. The conventional serology testing in laboratory diagnosis and in blood banks is unsatisfactory because of a high number of inconclusive and misclassified results. We aimed to assess the quality of four commercially available enzyme-linked immunosorbent assay tests for their ability to detect Trypanosoma cruzi antibodies in 685 sera samples. Cross-reactivity was assessed by using 748 sera from patients with unrelated diseases. Initially, we found that the reactivity index against T. cruzi antigen was statistically higher in sera from Chagas disease patients compared with those from non-chagasic patients, supporting the notion that all evaluated tests have a good discriminatory ability toward the diagnosis of T. cruzi infection in patients in the chronic phase of the disease. Although all tests were similarly sensitive for diagnosing T. cruzi infection, there were significant variations in terms of specificity and cross-reactivity among them. Indeed, we obtained divergent results when testing sera from patient with unrelated diseases, particularly leishmaniasis, with the levels of cross-reactivity being higher in tests using whole T. cruzi extracts compared with those using recombinant proteins. Our data suggest that all four tests may be used for the laboratory diagnosis and routine blood screening diagnose for Chagas disease. We also emphasize that, despite their general good performance, caution is needed when analyzing the results when these tests are performed in areas where other diseases, particularly leishmaniasis, are endemic.
... This bimodal distribution of antibody levels in Chagas has not previously been described, probably because old studies in highly endemic regions were based on less sensitive assays. 9 Most data using contemporary EIAs, which were primarily developed for use in low-endemic BD populations, are based on donor screening in these populations. 2,10 In low-endemic regions, a similar number of low-reactive samples could only be obtained after screening a huge number of BDs. ...
Low-level seroreactive donor samples that are inconsistently detected by different Trypanosoma cruzi immunoassays are common, but the population distribution has not been reported in an endemic region. The objective was to understand the distribution of low-level reactive samples using highly sensitive immunoassays and the relationship with epidemiologic evidence of exposure to T. cruzi.
Blood donors (BDs) were recruited in two blood banks located in Chaco province, in northeastern Argentina, from June 2006 to March 2007. Donors completed a Chagas exposure questionnaire and provided blood samples. All samples were tested in parallel with five contemporary and commercially available enzyme immunoassays for T. cruzi and a subgroup by a chemiluminescent assay.
Of the 1423 enrolled donors, 304 (21.4%) tested positive on all assays while 93 (6.5%) were reactive on at least one assay (inconclusive). Epidemiologic evidence of exposure to T. cruzi was significantly higher among positive and inconclusive donors compared to seronegative BD (p values range from 0.01 to <0.001 depending on the exposure). Histograms of the signal-to-cutoff values from all positive samples showed clear bimodal distributions for the whole parasite lysate assays, but not for the one recombinant antigen-based assay. Low antibody level responses were present in 30% to 40% of the reactives, depending on the assay.
The population of individuals exposed to T. cruzi in highly endemic regions has a bimodal distribution of antibody response to the parasite. Although the clinical significance of low-level reactivity is not fully established, these results may reflect evolving seroreversions after spontaneously resolved infections.
© 2015 AABB.
... Note that the NCH 2 samples that showed falsepositive results in FC-ATE are from patients with VL and ACL. The false-positive results found may be due to shared antigens among the Trypanosomatideos, triggering frequently a cross reactivity between T. cruzi and Leishmania spp. in serological assays, a phenomenon previously described (Vexenat et al., 1996;Gadelha et al., 2003). Despite the high sensitivity of different serological tests for the diagnosis of Chagas disease, the cross-reactivity mainly with sera from the VL and ACL patients is still frequent Amato Neto et al., 2005;Berrizbeitia et al., 2006Berrizbeitia et al., , 2012. ...
... También se dispone de ensayos no convencionales, a excepción de los test de ELISA, en su mayoría son pruebas rápidas basadas en inmunocromatografía y aglutinación (tabla 1). Prácticamente, todos los ensayos de ELISA destacan por una sensibilidad > 97%, y son recomendados como técnicas de elección para realizar el cribado 12,17 . Menos estudiadas están las técnicas rápidas, aunque su utilidad es indiscutible por la sencillez de su ejecución y la rapidez (10 min) 18 . ...
... Los resultados de los ensayos con proteínas recombinantes no difieren estadísticamente respecto de los realizados con extractos de parásitos, al menos no en seis países de Latinoamérica. [85][86][87][88][89] Los ensayos para diagnóstico de la nueva generación parecen ser los microarreglos; estas plataformas tienen el potencial de realizar diagnósticos de enfermedades infecciosas diferenciales altamente específicos, por lo que el número de aplicaciones excede en mucho el disponible por cualquier otra metodología conocida. 90,91 En el caso de las enfermedades infecciosas, esta plataforma ha permitido por ejemplo el estudio poblacional y patrones de resistencia de la tuberculosis. ...
Chagas disease represents one of the more significant public health problems in the Americas. Information regarding the genome and proteome of vectors and parasite, as well as their interactions, will be essential to develop specific and effective diagnostic and preventive tools. Advances that have contributed to the design, implementation, and efficacy of disease surveillance and control activities are reviewed. Genomic and proteomic information has contributed to a better understanding of vector distributions and dispersion, diversity, population dynamics, and control targets (populations and species). In addition, genomic and proteomic studies have impacted parasite diagnostics, Trypanosoma cruzi population dynamics, pharmacological treatment and knowledge of parasite-host interactions. Discussion of these contributions includes expectations for future basic and applied research questions.
... Along with this, several commercial ELISA kits with recombinant protein mixtures display equivalent or even higher sensitivities and specificities than those produced by kits with total parasite homogenate. (Gadelha et al., 2003;Pirard et al., 2005;Remesar et al., 2009;Caballero et al., 2007) These works have studied kits using Ag1, Ag2, Ag30, Ag13 together with Ag36 recombinant antigens (Chagatest Rec from Wiener lab, Argentina), and FRA and CRA recombinant antigens (Biomanguinhos, Friocruz, Brazil). However, another study reported that Chagatest Rec v3.0 (Wiener) displayed a rather low 95% sensitivity. ...
... Evaluation of a mixture of recombinant antigens or synthetic peptides showed high performance (sensitivity and specificity 100%). The construction of quimeric proteins, (multi-epitope fused antigens), has shown high performance by ELISA ( Gadelha et al. 2003;Aguirre et al. 2006;Camussone et al. 2009). ...
The rTSSA-II (recombinant Trypomastigote Small Surface II) antigen was evaluated by ELISA to detect anti-Trypanosoma cruzi antibodies in sera from naturally infected dogs and humans. For this evaluation ELISA-rTSSA-II was standardized and groups were classified according to the results obtained through xenodiagnosis, ELISA and PCR. Sensitivity (Se), Specificity (Sp), Kappa index (KI) and area under curve (AUC) were determined. The Se was determined by using 14 sera from dogs infected with T. cruzi VI (TcVI) whereas Sp was determined by using 95 non-chagasic sera by xenodiagnosis, ELISA-Homogenate and PCR. The performance of ELISA-rTSSA-II in dog sera was high (AUC=0·93 and KI=0·91). The Se was 92·85% (1 false negative) and Sp was 100%. Two sera from dogs infected with TcI and 1 with TcIII were negative. For patients infected with T. cruzi, reactivity was 87·8% (36/41), there was only 1 indeterminate, and Sp was 100%. Fifty-four sera from non-chagasic and 68 sera from patients with cutaneous leishmaniasis did not react with rTSS-II. ELISA-rTSSA-II showed a high performance when studying sera from naturally infected dogs and it also presented 100% Sp. This assay could be an important tool to carry out sero-epidemiological surveys on the prevalence of T. cruzi circulating lineages in the region.
... (figure 1) After applying inclusion/exclusion criteria, 114 texts were excluded, 4 of them were discarded due duplicated data in different reports by the same author -leaving 103 texts for data extraction. Several reports had data from two or more tests simultaneously; therefore, there were 115 regular ELISA reports[22,25, (Additional file 1), 49 ELISA-rec reports [22,23,61,76, 79,80,85,90,92,97,98,103,106107108109110111112113114115116 (Additional file 2) and 21 PCR reports[26,100,117118119120121122123124125126127128129130 (Additional file 3), generating 185 tests results to analyze (figure 1). ...
Most current guidelines recommend two serological tests to diagnose chronic Chagas disease. When serological tests are persistently inconclusive, some guidelines recommend molecular tests. The aim of this investigation was to review chronic Chagas disease diagnosis literature and to summarize results of ELISA and PCR performance.
A systematic review was conducted searching remote databases (MEDLINE, LILACS, EMBASE, SCOPUS and ISIWeb) and full texts bibliography for relevant abstracts. In addition, manufacturers of commercial tests were contacted. Original investigations were eligible if they estimated sensitivity and specificity, or reliability -or if their calculation was possible - of ELISA or PCR tests, for chronic Chagas disease.
Heterogeneity was high within each test (ELISA and PCR) and threshold effect was detected only in a particular subgroup. Reference standard blinding partially explained heterogeneity in ELISA studies, and pooled sensitivity and specificity were 97.7% [96.7%-98.5%] and 96.3% [94.6%-97.6%] respectively. Commercial ELISA with recombinant antigens studied in phase three investigations partially explained heterogeneity, and pooled sensitivity and specificity were 99.3% [97.9%-99.9%] and 97.5% [88.5%-99.5%] respectively. ELISA's reliability was seldom studied but was considered acceptable. PCR heterogeneity was not explained, but a threshold effect was detected in three groups created by using guanidine and boiling the sample before DNA extraction. PCR sensitivity is likely to be between 50% and 90%, while its specificity is close to 100%. PCR reliability was never studied.
Both conventional and recombinant based ELISA give useful information, however there are commercial tests without technical reports and therefore were not included in this review. Physicians need to have access to technical reports to understand if these serological tests are similar to those included in this review and therefore correctly order and interpret test results. Currently, PCR should not be used in clinical practice for chronic Chagas disease diagnosis and there is no PCR test commercially available for this purpose. Tests limitations and directions for future research are discussed.
... 10,[98][99][100][101][102] Because techniques such as radioimmunoprecipitation assay and immunoblot are not yet standardised, conventional and recombinant ELISAs seem the best methods to fulfi l the criteria of sensitivity, reproducibility, and predictive values. [90][91][92][93][94][95][96][97] In the case of PCR, studies of methods devised in individual laboratories showed low sensitivity. 32,[103][104][105][106] Optimum indications for PCR, once validated and standardised, could be diagnosis and follow-up of the acute phase including the congenital form; 80 monitoring to detect reactivation before clinical onset in immunodefi cient patients (either qualitively by conversion of previous consistently negative PCR or quantitively by increasing parasitic load-eg, in graft recipients); early assessment of the response to treatment by quantitative ultrasensitive PCR; 70,106 and genotyping. ...
More than 100 years after the discovery of human American trypanosomiasis by Carlos Chagas, our knowledge and management of the disease are profoundly changing. Substantial progress made by disease control programmes in most endemic areas contrasts with persisting difficulties in the Gran Chaco region in South America and the recent emergence of the disease in non-endemic areas because of population movements. In terms of pathogenesis, major discoveries have been made about the life cycle and genomics of Trypanosoma cruzi, and the role of the parasite itself in the chronic phase of the disease. From a clinical perspective, a growing number of arguments have challenged the notion of an indeterminate phase, and suggest new approaches to manage patients. New methods such as standardised PCR will be necessary to ensure follow-up of this chronic infection. Although drugs for treatment of Chagas disease are limited, poorly tolerated, and not very effective, treatment indications are expanding. The results of the Benznidazole Evaluation For Interrupting Trypanosomiasis (BENEFIT) trial in 2012 will also help to inform treatment. Mobilisation of financial resources to fund research on diagnosis and randomised controlled trials of treatment are international health priorities.
... Los resultados de los ensayos con proteínas recombinantes no difieren estadísticamente respecto de los realizados con extractos de parásitos, al menos no en seis países de Latinoamérica. [85][86][87][88][89] Los ensayos para diagnóstico de la nueva generación parecen ser los microarreglos; estas plataformas tienen el potencial de realizar diagnósticos de enfermedades infecciosas diferenciales altamente específicos, por lo que el número de aplicaciones excede en mucho el disponible por cualquier otra metodología conocida. 90,91 En el caso de las enfermedades infecciosas, esta plataforma ha permitido por ejemplo el estudio poblacional y patrones de resistencia de la tuberculosis. ...
Chagas disease represents one of the more significant public health problems in the Americas. Information regarding the genome and proteome of vectors and parasite, as well as their interactions, will be essential to develop specific and effective diagnostic and preventive tools. Advances that have contributed to the design, implementation, and efficacy of disease surveillance and control activities are reviewed. Genomic and proteomic information has contributed to a better understanding of vector distributions and dispersion, diversity, population dynamics, and control targets (populations and species). In addition, genomic and proteomic studies have impacted parasite diagnostics, Trypanosoma cruzi population dynamics, pharmacological treatment and knowledge of parasite-host interactions. Discussion of these contributions includes expectations for future basic and applied research questions.
... When the data were evaluated by matching the Rec-ELI-SA and IHA test, 52 inconclusive results were obtained. When Rec-ELISA and Con-ELISA were matched, only four inconclusive results were observed, indicating that the use of two enzyme immunoassays with different antigen preparations provides a secure test combination for diagnosis of Chagas disease and blood-bank screening (Gadelha et al. 2003). The EIE-Rec kit presents a number of other advantages. ...
In the acute phase and in the chronic forms of Chagas disease, the etiological diagnosis may be performed by detection of the parasite using direct or indirect parasitological methods and by the presence of antibodies in the serum by way of serological tests. Several techniques are easily available, ranging from the simplest wet smear preparation to immuno-enzymatic assays with recombinant antigens that will meet most diagnostic needs. Other tests under evaluation include a molecular test using polymerase chain reaction, which has shown promising results and may be used as a confirmatory test both in the acute and chronic phases of the disease. Better rapid tests are needed for diagnosis, some of which are already under evaluation. Additionally, there is a need for tools that can identify patients cured shortly after specific treatment. Other needs include a marker for prognosis and early diagnosis of congenital transmission.
... A positive result in two ELISA tests with different antigenic compositions is considered to sufficiently corroborate a result and define whether or not a donor or patient is infected. In addition, it could help reduce false-negative results and crossreactions (Gadelha et al., 2003). ...
American Trypanosomiasis, or Chagas' disease, is a parasitic infection caused by the protozoan Trypanosoma cruzi. It is endemic in a large area of the American continent, extending from Mexico to Argentina. Imported Chagas' disease is now appearing as a new threat to non-endemic countries, mostly because of a steady increase of foreign residents from Latin America. Chagas' disease becomes chronic in the vast majority of infected individuals. This finding gives rise to problems for blood transfusion services. In non-endemic countries, transfusion is the most likely infection route. Strategies to reduce transmission by transfusion include blood donor selection and deferral, blood donation testing, leukoreduction, filters and pathogen inactivation systems. Policies in endemic and non-endemic areas are quite different: in endemic countries, universal screening of blood donations for T. cruzi antibody detection is mandatory. However, in non-endemic countries, there are two different approaches: one is the deferral of people at risk of Chagas' disease and the second approach is to accept the blood donation if specific laboratory assay results are negative. This second approach is being introduced in countries where there is a substantial Latin American population, such as United States, Spain and France. The assays used for the detection of T. cruzi are mostly immunologic, made with T. cruzi antigen homogenates or with recombinant antigens. Complementary assays, such as indirect immunofluorescence (IIF) or immunoblot, can help ascertain antibody specificity.
... A number of recombinant T. cruzi antigens have been tested for their use in diagnosis, but have not been incorporated into laboratory kits due to technical and financial constraints. A recombinant ELISA test that uses two recombinant antigens, cytoplasmic repetitive antigen and flagellar repetitive antigen, was designed to overcome some of the inconveniences in the preparation of antigenic parasite components [56]. These studies concluded that the use of two ELISAs with different antigen preparations provides an effective test combination for blood bank screening of Chagas' disease. ...
Trypanosoma cruzi is the etiologic agent of Chagas' disease, a chronic inflammatory condition that results in heart and digestive complications. The first draft of the parasite genome is now complete and it is expected that, along with the published genomic and proteomic analyses discussed herein, it will lead to the identification of crucial genes and proteins directly associated with disease. This article reviews the current research trends addressing host-parasite interaction, parasite genetic variability and diagnosis. These advances will certainly bring about major developments not only in our understanding of Trypanosoma cruzi biology, but also in the application of new technologies to disease prevention and control.
... In this context, two recombinant antigens of T. cruzi, cytoplasmic repetitive antigen (CRA) and flagellar repetitive antigen (FRA), displayed better results when used in combination than separately (Krieger et al., 1992). The sensitivity and specificity were 98.2-100% and 100%, respectively, and no cross-reactivity was observed with other parasitic diseases, such as leishmaniasis (Gomes et al., 2001;Gadelha et al., 2003). Since the sensitivity of serological tests is usually higher in multiple epitope formats, the genes encoding the relevant epitopes of K9, K26 and K39 were joined to produce a chimeric recombinant protein (Boarino et al., 2005). ...
Human visceral leishmaniasis (HVL) is endemic in the tropical and sub-tropical regions of Africa, Asia, the Mediterranean, Southern Europe and South and Central America, with approximately 500,000 new cases reported annually. As dogs are considered to be the major reservoirs for HVL, the accurate diagnosis of disease in these animals is important. Diagnosis of canine visceral leishmaniasis (CVL) is performed mainly by direct parasitological methods that can yield false-negative results, either because of the very low number of Leishmania spp. organisms in clinical samples (bone marrow and lymph nodes) or because morphological identification is difficult. In addition, these methods are invasive. Conventional serological techniques are limited by cross-reactivity with other parasitic diseases and because several technical procedures have not been standardised. The development of polymerase chain reaction based approaches and immunoassays based on the use of recombinant antigens aimed at improving the sensitivity and specificity of CVL diagnosis is discussed.
... Two antigens with these characteristics were used in this study: cytoplasmatic repetitive antigen (CRA), present in evolved forms of the epimastigote and amastigote of T. cruzi, and flagellar repetitive antigen (FRA), present in the epimastigote and trypomastigote forms of the parasite (9,10). The immune response to these antigens has already been studied in mice (11)(12)(13)(14), in their diagnostic potential and to monitor posttreatment cure (15)(16)(17). However, it is not known which isotypes of immunoglobulin G (IgG) are involved in the immune response generated in patients with Chagas' disease in the presence of these recombinant antigens (Ags-Recs). ...
The wide range of clinical Chagas' disease manifestations, of which heart involvement is the most significant, because of its characteristics, frequency and consequences, and lack of treatment and cure, justify research in this area. Specific immunoglobulin G (IgG) antibody subclasses have been associated with human Chagas' disease. Thus, in this study, the profile of IgG subclasses against cytoplasmic (CRA) and flagellar (FRA) recombinant repetitive T. cruzi-specific antigens was correlated with cardiac (CARD, n=33), cardiodigestive (CD, n=7), and indeterminate (IND, n=20) forms of Chagas' disease by indirect enzyme-linked immunosorbent assay (ELISA). IgG subclasses were detected in almost all Chagas patients studied. Nevertheless, only specific IgG2 isotype FRA was found with a significant statistical difference in CARD patients when compared to IND patients. This result suggests the potential use of this isotype for prognostic purposes, for monitoring the progression of chronic Chagas' disease, and for predicting the risk of CARD damage. This is important information, as it could help physicians to evaluate and manage the treatment of their patients. However, a follow-up study is necessary to confirm our result.
... Two recombinant antigens (Rec-Ags), cytoplasmatic repetitive antigen (CRA) and flagellar repetitive antigen (FRA), already studied in murine model (26)(27)(28)(29), have been successfully used in the immunodiagnosis of Chagas' disease (30)(31)(32)(33). These antigens were also used to evaluate the cure for Chagas' disease in patients from Minas Gerais (Brazil) treated in the acute phase of the infection (34). ...
We propose to analyze the relation between the cellular immune response of Chagas' disease patients after in vitro stimulation of peripheral blood mononuclear cells (PBMC) with recombinant antigens cytoplasmatic repetitive antigen (CRA) or flagellar repetitive antigen (FRA) of T. cruzi and the chronic clinical forms of disease. Cells were stimulated using phytohemagglutinin, CRA, FRA, or a soluble antigen of Epimastigota (Ag-Epi) for 24 hr, 72 hr, or 6 days. The proliferation of cells was evaluated after 6 days of culture by quantification of incorporated 3H-thymidine. Cytokines were measured in the supernatants obtained after 24 hr (tumor necrosis factor [TNF]-alpha and interleukin [IL]-4), 72 hr (IL-10), and 6 days (interferon [IFN]-gamma) using enzyme-linked immunosorbent assay (ELISA). Cells of the Chagas patients stimulated with the recombinant antigens exhibited higher proliferation responses compared with that of non-Chagas (NC) individuals. However, when proliferation was compared between patients with the cardiac form (CF) or indeterminate form (IF), it was not possible to establish a difference in the response. So far as the cytokines secreted in the culture supernatants after stimulation in vitro with T. cruzi antigens were concerned, the results showed that CRA, as well as Epi-Ag, were able to stimulate the production of TNF-alpha and IFN-gamma in Chagas patients as compared with NC individuals. However, the cytokine levels after stimulation with the T. cruzi antigens were not different between the patients with CF and IF. CRA was capable of inducing a T helper type 1 (Th1) immune response, with elevated production of TNF-alpha and IFN-gamma in Chagas patients that are carriers of CF and IF clinical forms.
Con el objetivo de incrementar la precisión diagnóstica, la Organización Mundial de la Salud recomienda la realización de dos o más pruebas in- munoserológicas para el diagnóstico de la enfermedad de Chagas en etapa crónica. El objetivo de este trabajo fue realizar una revisión sistemática rápida acerca del desempeño de las técnicas inmunoserológicas y méto- dos moleculares en la población general. Se identi caron 178 estudios de los cuales fueron incluidos nueve. Las técnicas de ELISA mostraron la mayor sensibilidad (82-98%) y especi cidad (96-100%). Los métodos rá- pidos mostraron valores de sensibilidad entre 88-93% y especi cidad 97- 100%, mientras que los métodos moleculares (PCR) presentaron niveles muy variables de sensibilidad (22-92%) y especi cidad (70-100%). Estos resultados muestran que las técnicas de ELISA cuentan con una sensibili- dad y especi cidad adecuadas. La PCR, al igual que los métodos rápidos, mostró una gran variabilidad en los resultados, debido principalmente a la heterogeneidad de la técnicas y profusión de métodos elaborados de manera in house.
PhD Thesis. This document in portuguese has two papers, one with a systematic review of PCR and ELISA tests for diagnosis of chronic Chagas disease, and a second one with clinical prediction model for chronic Chagas disease diagnosis.
Various infectious agents can be transmitted by blood exposure, which comprises of transfusion, of which hemoparasites that are commonly absent from European countries but that can have infected blood donor candidates born, raised or having been living in the Tropics. Among those hemoparasites is Trypanosoma cruzi, responsible for Chagas disease. T. cruzi is responsible for acute post-transfusion infections every year in endemic areas (South America) and also, more incidently, in North America. There are situations which expose European blood donors to this risk and the present essay discusses arguments which have now been taken into consideration by certain transfusion systems such as the French one.
We evaluated the recombinant antigen SAPA (Shed Acute Phase Antigen) for the detection of Trypanosoma cruzi antibodies in sera from naturally infected dogs. The technique used was ELISA and the antigens were a homogenate of parasite T. cruzi (ELISA-H) and the recombinant SAPA (ELISA-SAPA). We analyzed 93 sera from dogs by ELISA-H and ELISA-SAPA, which were grouped as follows: G1: 11 negative control sera from the city of Salta, G2: 11 positive control sera from dogs naturally infected with T. cruzi and G3: 71 samples of dogs belonging to a Chagas disease-endemic area. The sensitivity and specificity of ELISA-SAPA were 100 %. The kappa index between ELISA-H and ELISA-SAPA was 0,85. These results confirm the use of SAPA antigen in the diagnosis of infection with T. cruzi in dogs.
Unlabelled:
In this prospective study we evaluated the performance characteristics of a specific and sensitive antigen preparation (AgA) used in an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-Trypanosoma cruzi antibodies in serum samples, for Chagas' disease diagnosis. The antigen production was achieved by combination of nutritional stress and autoclaving the parasites. Specificity and sensitivity were evaluated in two separate tests, using 152 sera from healthy individuals and 175 sera from Chagas' patients (70 by xenodiagnosis). Cross-reactivity was tested using 289 sera from patients who had a parasitological diagnosis of a disease known to induce antigenic responses towards T. cruzi. All of these sera were tested with our AgA-ELISA and with 3 commercial diagnosis kits. To evaluate the agreement of results between our AgA-ELISA and a "gold standard" test for Chagas, we tested 566 sera from an endemic area.
Results:
sensitivity and specificity were 100%; cross-reactivity was the lowest compared with commercial kits. Overall agreement with the gold standard test was excellent (kappa = 0.92). AgA-ELISA exhibits levels of sensitivity, specificity and cross-reactivity comparable or superior to those shown, obtained with the commercial kits used in our country, while being at least 10 times less expensive. This balance between diagnostic accuracy and cost makes AgA-ELISA useful for blood bank screening in poor regions of the world suffering from Chagas' disease. Further validations of this antigenic formulation in other countries are necessary.
The absence of a gold standard test for Trypanosoma cruzi antibodies represents a problem not only for the evaluation of screening tests, but also for appropriate blood donor counseling. The aim of this study was to estimate the sensitivity and specificity of multiple blood donor screening tests for T. cruzi antibodies in Argentina.
From June 2006 to March 2007 a sample of 1455 blood donors was recruited from two blood banks in Chaco province, an area of Argentina with highly endemic T. cruzi infection. Samples were tested by three epimastigote lysate enzyme immunoassays (EIAs), one recombinant antigen EIA, two indirect hemagglutination assay (IHA) tests, a particle agglutination assay (PA), and a research trans-sialidase inhibition assay (TIA). Sensitivity and specificity were estimated using latent class analysis (LCA).
LCA estimated the consensus prevalence of T. cruzi infection at 24.5%. Interassay correlation was higher among the four EIA tests and TIA compared to IHA tests. Assay sensitivities varied from 96 to 99.7 for different EIAs, 91% for TIA, 84% for PA, and 66 to 74% for IHA tests. Relative to the LCA, assay specificities were from 96% to almost 100%.
Based on the comparison of several tests in a large population from an endemic area for T. cruzi infection, our data showed an adequate sensitivity for EIA tests in contrast to PA and IHA assays. The latter tests should no longer be used for blood donor screening.
The availability of the Trypanosoma cruzi genome is only a small piece of the
puzzle of this parasite’s biology. A high percentage of hypothetical proteins, few
genes characterized and limitations on heterologous protein expression are the
present scenario for this biological model. Thus, the need for development of a highthroughput
gene characterization platform becomes evident. Ideally, such platform
should allow efficient cloning and disponibility of different applications. In this context,
the goal of this study was to use the GatewayÒ technology (Invitrogen) to construct
plasmid vectors suitable for different uses. Our constructs contained 35.1 intergenic
regions, T. cruzi Dm28c 18S ribosome or the T7 bacteriophage promoters, antibiotic
resistance genes, and several tags for multiple purposes. Besides T. cruzi vectors, a
Crithidia fasciculata heterologous protein expression system was created based on
pNUS-H1 and modified for use of the bacteriophage T7 RNA polymerase. The
advantages of the C. fasciculata system are that it is related to T. cruzi but it is not
pathogenic for humans. Three T. cruzi genes were used to validate our strategy.
Antibodies against the products of these genes were used to detect the presence of
the recombinant proteins in T. cruzi and C. fasciculata extracts, and to compare the
localization of the native proteins with that of the recombinant proteins tagged with cmyc
epitope or fluorescent proteins GFP, CFP and YFP in T. cruzi. The results
obtained with these antibodies corroborated the results obtained with our system.
TAP tag protein complex purification was also used for strategy validation. Our
results show that this platform is a fast and efficient cloning system that allows the
characterization of Trypanosoma cruzi genes by different biological approaches. The
development of this high-throughput platform is a step further to large scale
applications such as the T. cruzi ORFeome.
Bioelectrodes to detect immunoglobulin G (IgG) antibodies occurring in sera of patients suffering from American trypanosomiasis were assembled. The device consisted of a gold electrode modified with a thiol sensitized with parasite proteins. The assemblage was accomplished by adsorbing IgG antibodies from confirmed infected patients followed by adsorption of anti-human IgG labeled with a redox enzyme. The appliance was used as a working electrode in a three-electrode cell containing a soluble charge-transfer mediator, also behaving as enzyme cosubstrate. The method is based on the measurement of the catalytic current after addition of the enzyme substrate, occurring when a positive serum is used to build up the biosensor. The discrimination efficiency between positive and negative sera was 100% for the samples studied. A 0.9525 correlation coefficient was obtained for results acquired by using this approach and one commercial diagnostic kit. The reproducibility, evaluated by the percentage coefficient of variation, varied between 7 and 20%. The sensitivity was 12.4 ng mL(-1) IgG, which is in the same order as that obtained with the commercial kit. Stability of the device was studied for a 7-day period and the results showed no significant change (p = 0.218). Leishmaniasic sera showed cross-reactivity when total parasite homogenate was used as antigen.
A kit based on an enzyme immunoassay, EIE-Recombinant-Chagas-Biomanguinhos, developed by the Oswaldo Cruz Foundation, was evaluated for the serodiagnosis of chronic Chagas disease. Evaluation was performed with 368 serum samples collected from individuals living in an endemic area for Chagas disease: 131 patients in the chronic phase with confirmed clinical, epidemiological, and serological diagnosis (indirect immunofluorescence, indirect hemagglutination or enzyme-linked immunosorbent assay) and 237 nonchagasic seronegative individuals were considered negative control. The EIE-Recombinant-Chagas-Biomanguinhos kit showed high sensitivity, 100% (CI 95%: 96.4-100%) and high specificity, 100% (CI 95%: 98-100%). The data obtained were in full agreement with clinical and conventional serology data. In addition, no cross-reaction was observed with sera from patients with cutaneous (n=14) and visceral (n=3) leishmaniasis. However, when these sera were tested by conventional serological assays for Chagas disease, cross-reactions were detected in 14.3% and 33.3% of the patients with cutaneous and visceral leishmaniasis, respectively. No cross-reactions were observed when sera from nonchagasic seronegative patients bearing other infectious disease (syphilis, n=8; HTLV, n=8; HCV, n=7 and HBV, n=12) were tested. In addition, sera of patients with inconclusive results for Chagas disease by conventional serology showed results in agreement with clinical evaluation, when tested by the kit. These results are relevant and indicate that the referred kit provides a safe immunodiagnosis of Chagas disease and could be used in blood bank screening.
Objetivos. Determinar la seroprevalencia de la infección por Trypanosoma cruzi en Ushuaia, la ciudad más austral del mundo. Métodos. Se analizaron muestras de suero de 2 991 personas, obtenidas entre enero de 1995 y diciembre de 1996. Las muestras fueron procesadas por hemaglutinación indirecta (HAI) e inmunoensayo enzimático (ELISA) o inmunofluorescencia indirecta (IFI). Resultados. La seroprevalencia general de la infección por T. cruzi fue de 6,8%. La prevalencia según el país de origen fue de 41,1% en los bolivianos, 5,0% en los argentinos y 0,9% en los chilenos; en embarazadas fue de 5,9%, en exámenes obligatorios de 6,3% y en consultas dirigidas de 30,8%. Conclusiones. Se destaca la magnitud de la infección por T. cruzi en una zona donde no existe el insecto vector. Debido al riesgo de la transmisión congénita y transfusional, es necesario mantener el control de la sangre a transfundir y reforzar el seguimiento de los hijos de mujeres infectadas para un diagnóstico precoz y tratamiento oportuno de la infección.
The commercially available diagnostic tests for Chagas’ disease employ whole extracts or semipurified fractions of Trypanosoma cruzi epimastigotes. Considerable variation in the reproducibility and reliability of these tests has been reported by different research laboratories, mainly due to cross-reactivity with other pathogens and standardization of the reagents. The use of recombinant antigens for the serodiagnosis of Chagas’ disease is recommended to increase the sensitivity and specificity of serological tests. Expressed in Escherichia coli, as fusion products with glutathione S-transferase, six T. cruzi recombinant antigens (H49, JL7, A13, B13, JL8, and 1F8) were evaluated in an enzyme-linked immunosorbent assay for Chagas’ disease. The study was carried out with a panel of 541 serum samples of chagasic and nonchagasic patients from nine countries of Latin America (Argentina, Bolivia, Brazil, Chile, Colombia, El Salvador, Guatemala, Honduras, and Venezuela). The optimal concentration of each recombinant antigen for coating of plates was determined with the help of 125I-labelled recombinant proteins. While the specificity of the epimastigote antigen was 84% because of false positives from leishmaniasis cases, for the recombinant antigens it varied from 96.2 to 99.6%. Recombinant antigens reacted with 79 to 100% of serum samples from chronic chagasic patients. In this way, it is proposed that a mixture of a few T. cruzi recombinant antigens should be employed in a diagnostic kit to minimize individual variation and promote high sensitivity in the diagnosis of Chagas’ disease.
A Trypanosoma cruzi antigen which is shed into the culture medium by the trypomastigote stage of the parasite and detected in blood of acutely infected mice was cloned and characterized. We designate this antigen shed acute phase antigen (SAPA). Five protein bands with apparent molecular masses ranging from 160 to 200 kDa were detected by immunoblotting of plasma from infected mice and in supernatants of cultured trypomastigotes upon reaction with antibodies against SAPA. A serum obtained from a patient acutely infected with Chagas' disease revealed a similar set of polypeptides in supernatants of cultured trypomastigotes when tested by immunoblotting. SAPA seems thus to be a major shed protein during the acute period of the disease. Twenty-six of 28 sera from human acute cases of Chagas' disease tested reacted with SAPA. Conversely, only 8-10% of sera from chronic cases of the disease contained detectable levels of antibody against SAPA. Sera from rabbits infected with six different parasite strains all contained antibodies against SAPA. Antibodies against SAPA are detectable 15 days after the manifestation of acute Chagas' disease symptoms in humans and 15 days post-infection in sera from mice and rabbits. The nucleotide sequence of a genomic clone encoding the 3' end of the SAPA gene revealed the presence of 14 tandemly arranged 12-amino acid-long repeats. A 39-amino acid-long region that is very hydrophobic precedes the stop codon. Due to its early appearance it might be possible to design diagnostic assays which are based on SAPA for identification of recently infected cases of Chagas' disease.
An enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibodies against Trypanosoma cruzi. Two synthetic T. cruzi peptides, TcD and PEP2, were used. The specificity and sensitivity of the peptide ELISA were determined with 260 serum samples from individuals living in an area in which Chagas' disease is endemic. ELISAs were performed with the peptides singly or in combination. The evaluation of these tests showed that 168 (93.8%) of 179 serum samples from T. cruzi-infected patients were positive when TcD peptide was used as antigen; 164 (91.6%) samples were positive with PEP2, and 178 (99.4%) samples were positive when the two peptides were combined. Thus, the sensitivity of the ELISA using the two peptides exceeded 99%. The specificity was evaluated by using a panel of 118 serum samples that included samples from 81 individuals living in an area of endemicity with negative serology for Chagas' disease and from 37 patients from areas in which T. cruzi was not endemic but with other pathologies, such as leishmaniasis, tuberculosis, and leprosy. Only two false-positive serum samples were found in this group of individuals, giving a test specificity of more than 98%. Because these peptides can be synthesized and are very stable at room temperature, the use of such reagents can improve the standardization and reproducibility of ELISAs for the serodiagnosis of T. cruzi infection.
The commercially available diagnostic tests for Chagas' disease employ whole extracts or semipurified fractions of Trypanosoma cruzi epimastigotes. Considerable variation in the reproducibility and reliability of these tests has been reported by different research laboratories, mainly due to cross-reactivity with other pathogens and standardization of the reagents. The use of recombinant antigens for the serodiagnosis of Chagas' disease is recommended to increase the sensitivity and specificity of serological tests. Expressed in Escherichia coli, as fusion products with glutathione S-transferase, six T. cruzi recombinant antigens (H49, JL7, A13, B13, JL8, and 1F8) were evaluated in an enzyme-linked immunosorbent assay for Chagas' disease. The study was carried out with a panel of 541 serum samples of chagasic and nonchagasic patients from nine countries of Latin America (Argentina, Bolivia, Brazil, Chile, Colombia, El Salvador, Guatemala, Honduras, and Venezuela). The optimal concentration of each recombinant antigen for coating of plates was determined with the help of 125I-labelled recombinant proteins. While the specificity of the epimastigote antigen was 84% because of false positives from leishmaniasis cases, for the recombinant antigens it varied from 96.2 to 99.6%. Recombinant antigens reacted with 79 to 100% of serum samples from chronic chagasic patients. In this way, it is proposed that a mixture of a few T. cruzi recombinant antigens should be employed in a diagnostic kit to minimize individual variation and promote high sensitivity in the diagnosis of Chagas' disease.
The polypeptides of 46 and 58kDa were recognized in different T. cruzi strains (Y, WSL and Colombiana) by serum of all chagasic patients studied. These polypeptides were isolated from T. cruzi Y strain and used in ELISA. The sensitivity and specificity were 97.6% [CI 95%: 86-100%] and 100% [CI 95%: 89.3-100%], respectively when Tc 46 was used. When Tc 58 was used the sensitivity and specificity were 100% [CI 95%: 89.6-100%] and 90.2% [CI 95%: 75.9-96.8%], respectively.Os polipeptídeos de 46 e 58kDa foram reconhecidos em diferentes cepas de T. cruzi (Y, WSL e Colombiana) pelo soro de todos os pacientes chagásicos estudados. Estes polipeptídeos foram isolados da cepa Y e usados em ELISA. A sensibilidade e especificidade foram 97,6% [CI 95%: 86-100%] e 100% [CI 95%: 89,3-100%], respectivamente quando Tc 46 foi usada. Quando Tc 58 foi utilizada a sensibilidade e especificidade foram de 100% [CI 95%: 89,6-100%] and 90,2% [CI 95%: 75,9-96,8%], respectivamente.
BACKGROUND: Chagas' disease is transmitted to man either by the bite of insects harboring Trypanosoma cruzi or by the transfusion of blood from infected donors. The conventional serologic testing as presently used in blood banks in South America is unsatisfactory, because of a high number of inconclusive and false-positive results. Other methods such as polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) with recombinant antigens have been proposed, but inherent difficulties have so far precluded their adoption in the large-scale screening required by blood banks. STUDY DESIGN AND METHODS: A highly sensitive and specific chemiluminescent ELISA using a purified trypomastigote glycoconjugate antigen and a complex epimastigote antigen was devised for the diagnosis of active T. cruzi infection. RESULTS: Chemiluminescent ELISA was 100-percent sensitive in the diagnosis of 100 cases of confirmed Chagas' disease. Inconclusive results and false-positive reactions were eliminated in a panel of 115 sera. The specificity of the chemiluminescent ELISA was 100 percent with a purified trypomastigote glycoconjugate antigen and 99.7 percent with a complex epimastigote antigen when applied to 1000 normal human sera and 288 heterologous sera from patients with other infections, including leishmaniasis, and vaccinated individuals. CONCLUSION: The chemiluminescent ELISAs provide a test that is highly sensitive (purified trypomastigote glycoconjugate and complex epimastigote antigens) and specific (purified trypomastigote glycoconjugate antigen) for Chagas' disease diagnosis. It can be used in blood bank screening and to monitor the treatment of patients undergoing chemotherapy.
Blood transfusion is one of the principal routes of transmission of Chagas' disease, a major endemic disease in Latin America. Methods for blood screening are not accurate and may yield false results that lead to high social and economic costs. This study compares two methods of diagnosing Chagas' disease (indirect immunofluorescence and hemagglutination) and several enzyme-linked immunosorbent assays (ELISAs) with regard to specificity and sensitivity, by using human sera with known serologic and parasitologic characteristics, as well as samples with discrepant results on conventional serologic tests. An ELISA using recombinant antigens showed no cross-reactivity with sera that were positive for other diseases. All evaluated ELISAs performed well, and their use may lead to a reduction of more than 50 percent in the number of discordant sera. Further improvements are needed in view of the complexity of the serologic diagnosis of Chagas' disease.
We tested two Trypanosoma cruzi recombinant antigens in a diagnostic test for Chagas' disease. These antigens were a cytoplasmic repetitive antigen (CRA) and a flagellar repetitive antigen (FRA). The results indicate that the recombinant antigens give better results when used in combination than when used separately, and that the removal of the beta-galactosidase portion of the recombinant fusion proteins increases the specificity of the diagnostic test for Chagas' disease. In addition, a direct enzyme-linked immunosorbent assay (ELISA), which involves the use of peroxidase-labeled antigens to detect the immune-complexes, was developed and compared with a conventional ELISA. The results indicate that the recombinant (CRA+FRA) ELISA is better than the conventional ELISA in the diagnosis of Chagas' disease, providing 100% specificity and sensitivity in all sera tested to date. The recombinant ELISA was compared with conventional serologic tests (hemagglutination and immunofluorescence) for Chagas' disease diagnosis, and the results show that the recombinant ELISA does not give rise to false-positive results that are observed with the two other tests. The use of the recombinant ELISA should be useful in the prevention of transmission of Chagas' disease by blood transfusions.
Here we describe the characterization of a Trypanosoma cruzi DNA sequence (clone A13) that codes for a polypeptide recognized by IgM and IgG antibodies from sera of acute and congenital chagasic patients. Antibodies to A13 antigen are also detected in the sera of chronic patients with different clinical forms of Chagas' disease, but not in sera of patients with leishmaniasis or other parasitic diseases. The antigenic determinants encoded by clone A13 are found in amastigotes and trypomastigotes of several T. cruzi strains, but not in the noninfective epimastigotes. The DNA sequence of the recombinant clone reveals one open reading frame encoding 251 amino acids without tandemly repeated sequences. Our data suggest that the A13 antigen may be useful for the development of serodiagnostic procedures.
Trypanosoma cruzi genes were cloned in lambda gt11 and screened with an anti-trypomastigote antiserum. Two out of twelve clones were selected in view of their reactivity with human chagasic sera. One clone encodes a flagellar antigen (FRA) of more than 300 kDa, whereas the other corresponds to a roughly 225-kDa cytoplasmic antigen (CRA). The flagellar antigen is present in both epimastigotes and trypomastigotes, but the cytoplasmic antigen is not found in trypomastigotes. The CRA clone is entirely composed of at least 23 copies of a 42-bp repeat and the FRA gene contains at least 14 copies of a 204-bp motif. The FRA gene hybridizes to a RNA of about 10 kb, while the CRA gene detects a transcript of 5.2 kb.
To identify Trypanosoma cruzi target antigens in overt Chagas' heart disease, a parasite lambda gt11 cDNA library was screened with the serum of a patient with a severe chagasic heart involvement (JL). Using a phage dot array immunoassay, 5 highly antigenic clones, JL1, JL5, JL7, JL8, and JL9, were probed with sera from clinically characterized T. cruzi infected subjects. The correlation of cloned T. cruzi antigen recognition with the clinical status of the subjects led to the identification of a recombinant antigen, JL5, that reacted predominantly with sera from patients with Chagas' heart disease. The antigenic determinant of the JL5 recombinant was a small 35 amino acid peptide. The nucleotide and the deduced amino acid sequence, together with other experimental data, allowed identification as the C-terminal portion of a T. cruzi P ribosomal protein. The C-terminal undecapeptide in JL5, EDDDMGFGLFD, was highly homologous to the same region of the human P protein SD(D/E)DMGFGLFD. The latter sequence has been identified as the P protein epitope in systemic lupus erythematosus (SLE). Positive SLE sera reacted with the JL5 recombinant phage, suggesting that the T. cruzi P protein might induce antibodies with a similar specificity to that of P antibodies in SLE.
In spite of being separated by more than 20 million years of evolution, the murine and human immune systems share extensive similarities. Thus, experimental results obtained with the murine model may have predictive value for human Chagas' disease. Challenge of the H-2 congenic mouse stains A.SW (H-2s) and A.CA (H-2f) with Trypanosoma cruzi yields different results. The A.CA animals die approximately 12 days postinfection, while A.SW mice survive indefinitely. A 45-kD protein (Tc45), an antigen differentially recognized by the A.SW strain, is present in cultured epimastigotes and blood trypomastigotes. We describe here its purification from epimastigotes. The presence of Tc45 was monitored and a single band was detected. Since the molecular weights of Tc45, cruzipain, cruzain, and a 46-kD parasite polypeptide are similar, it was important to determine if these molecules are related. A complete lack of homology was observed when the sequence of cruzain, cruzipain, and the 46-kD polypeptide were compared with the preliminary sequence of Tc45.
In the acute phase of Chagas' disease, when the parasitemia is high, diagnosis can be easily made using conventional parasitological methods. During the chronic phase, due to the low parasitemia, diagnosis is performed mainly by immunological methods. Conventional serological techniques are limited by cross-reactivity with other parasitic diseases, non-standardization of reagents, and the diversity of technical procedures. Methods are being developed to improve the sensitivity and specificity of diagnosis using molecular approaches. PCR-based detection systems and the use of recombinant antigens in ELISA are the most promising.
Chagas' disease is transmitted to man either by the bite of insects harboring Trypanosoma cruzi or by the transfusion of blood from infected donors. The conventional serologic testing as presently used in blood banks in South America is unsatisfactory, because of a high number of inconclusive and false-positive results. Other methods such as polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) with recombinant antigens have been proposed, but inherent difficulties have so far precluded their adoption in the large-scale screening required by blood banks.
A highly sensitive and specific chemiluminescent ELISA using a purified trypomastigote glycoconjugate antigen and a complex epimastigote antigen was devised for the diagnosis of active T. cruzi infection.
Chemiluminescent ELISA was 100-percent sensitive in the diagnosis of 100 cases of confirmed Chagas' disease. Inconclusive results and false-positive reactions were eliminated in a panel of 115 sera. The specificity of the chemiluminescent ELISA was 100 percent with a purified trypomastigote glycoconjugate antigen and 99.7 percent with a complex epimastigote antigen when applied to 1000 normal human sera and 288 heterologous sera from patients with other infections, including leishmaniasis, and vaccinated individuals.
The chemiluminescent ELISAs provide a test that is highly sensitive (purified trypomastigote glycoconjugate and complex epimastigote antigens) and specific (purified trypomastigote glycoconjugate antigen) for Chagas' disease diagnosis. It can be used in blood bank screening and to monitor the treatment of patients undergoing chemotherapy.
Determine the seroprevalence of Trypanosoma cruzi infection in Ushuaia, Argentina, which is the southernmost city in the world.
Serum samples were analyzed from 2,991 people, obtained between January 1995 and December 1996. The samples were processed using indirect hemagglutination and either enzyme-linked immunosorbent assay or indirect immunofluorescence.
The general seroprevalence of T. cruzi infection was 6.8%. According to the residents' country of origin, prevalence was 41.1% among Bolivians, 5.0% among Argentines, and 0.9% among Chileans. The prevalence found in pregnant women was 5.9%; in compulsory examinations (such as for a job or for immigrants settling permanently in Argentina), it was 6.3%; and in examinations done based on clinical or epidemiological reasons to suspect infection with Chagas' disease, it was 30.8%.
There is an alarming level of T. cruzi infection in this region, where the insect vector does not even exist. Given the risk of transmission congenitally and from blood transfusions, controls must be maintained on donated blood. In addition, follow-up of children of infected women should be strengthened so that these children receive early diagnosis and timely treatment of the infection.
Diagnosis of individuals infected by Trypanosoma cruzi is performed mainly by serological tests using crude antigens, which might crossreact with other infections. In the past ten years, many recombinant T. cruzi proteins and synthetic peptides have been described, and some are already on the market. Managers of laboratories and blood banks need to make decisions on a cost-benefit basis whether to include these new-generation tests. Here, we indicate antigens that are likely to prove most useful.
We used the EIE-Recombinant-Chagas-Biomanguinhos kit (EIE-Rec kit) developed by the Oswaldo Cruz Foundation, Brazil, to monitor cure of chagasic patients who were treated during the acute phase of T. cruzi infection. Treated patients were previously studied by parasitological and serological tests and classified as cured patients (CP) (n = 10), dissociated patients (DP) (n = 6), and noncured patients (NCP) (n = 6). When sera of these patients were assayed by EIE-Rec kit all sera from NCP and all sera from CP showed positive and negative reactions, respectively. These results were in full agreement with those obtained previously by the classical tests. Two DP showed a positive reaction; the remaining four displayed a negative reaction, similar to that observed in sera from nonchagasic (NCh) individuals, and could therefore be considered CP. Our results suggest that the EIE-Rec kit could be used to monitor the efficacy of Chagas' disease treatment.
PCR and sero-diagnosis of chronic Chagas' disease: biotecnological advances119 23 World Health Organization: Control of Chagas' disease
- Gomes
- Ym
Gomes YM: PCR and sero-diagnosis of chronic Chagas' disease: biotecnological advances. Appl Biochem Biotechnol 1997; 66:119 23 World Health Organization: Control of Chagas' disease. WHO Technical Report Series 1991; 811:38–47
An appraisal of Chagas' disease serodiagnosis Chagas' Disease (American Trypanosomiasis): its Impact on Transfusion and Clinical Medicine
- S Camargo
- Z Brener
- Camargo Me
Camargo ME: An appraisal of Chagas' disease serodiagnosis; in Wendel S, Brener Z, Camargo ME, Rassi A, eds. Chagas' Disease (American Trypanosomiasis): its Impact on Transfusion and Clinical Medicine. São Paulo, Brazil, ISBT, 1992: 165 –168 22
World Health Organization: The Southern Cone Initiative
World Health Organization: The Southern Cone Initiative. TDR
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