Serological tests are preferentially used for the diagnosis of Chagas' disease (CD) during the chronic phase because of the low parasitemia and high anti-Trypanosoma cruzi antibody titers. However, the current methods showed several disadvantages, as contradictory or inconclusive results, mainly related to the characteristics of the antigens used, in general, crude or whole parasites, but also due to antigen production protocol and the experimental conditions used in serological tests. Thus, better-quality serological assays are urgently needed. Here, we performed a wide immunogenomic screen strategy to identify conserved linear B-cell epitopes in the predicted proteome based on genome sequence from T. cruzi strains to will be applied as synthetic peptides in the serodiagnosis of the chronic CD. Three B-cell epitopes derived from mucin-associated surface protein (MASP) family, expressed in both infective parasite stages, trypomastigote and amastigotes, conserved in T. cruzi strains, and highly divergent as compared with Leishmania spp. proteome, were selected for this study. The results demonstrated that synthetic peptide 2 and a mixture of peptides (Mix II: peptides 2 and 3) were able to identify all chronic CD cases, indeterminate or Chagas cardiomyopathy clinical presentation, and simultaneously able to discriminate infections caused by Leishmania parasites, with high accuracy (98.37 and 100.00%, respectively) and agreement (kappa index = 0.967 and 1.000, respectively) with direct methods as compared to current diagnostic pipeline performed by reference laboratories in Brazil. This study represents an interesting strategy for the discovery of new antigens applied to serologic diagnosis of infectious diseases and for the technological development of platforms for large-scale production of diagnostic tests.
A kDNA PCR enzyme-linked immunosorbent assay (kDNA PCR-ELISA) for the diagnosis of human visceral leishmaniasis (HVL) was developed. The detection limit of the reaction, precision measurements, and cut-off of the kDNA PCR-ELISA were defined in a proof-of-concept phase. A reference strain of Leishmania (Leishmania) infantum and a bank of 14 peripheral blood samples from immunocompetent patients with VL were characterized using techniques considered gold standards, and 11 blood samples obtained from healthy individuals of an endemic area were also assessed. Phase II evaluation determined the performance of the assay in peripheral blood samples from 105 patients with VL (adults and children), 25 patients with Leishmania/HIV coinfection, 40 healthy individuals, and 33 asymptomatic individuals living in endemic areas. The kDNA PCR-ELISA exhibited satisfactory precision, with a detection limit of 0.07 fg of DNA from L. (L.) infantum and 1 parasite/mL blood. The overall sensitivity of the assay for all groups studied was 100% (95% confidence interval [CI]: 97.1–100%), and the specificity was 95% (95% CI: 83.5–98.6%). The kDNA PCR-ELISA was shown to be a useful tool for VL symptomatic and asymptomatic individuals diagnosis and its use in endemic countries may help monitor control interventions.
BACKGROUND Visceral Leishmaniasis (VL) is an infectious disease that is a significant
cause of death among infants aged under 1 year and the elderly in Brazil.
Serodiagnosis is a mainstay of VL elimination programs; however, it has
significant limitations due to low accuracy.OBJECTIVE This study aimed to evaluate three recombinant Leishmania
infantum proteins (rFc, rC9, and rA2) selected from previous
proteomics and genomics analyses to develop enzyme-linked immunosorbent
assay (ELISA) and immunochromatographic tests (ICT) for the serodiagnosis of
human VL (HVL) and canine VL (CVL).METHODS A total of 186 human (70 L. infantum-infected symptomatic,
20 other disease-infected, and 96 healthy) and 185 canine (82 L.
infantum-infected symptomatic, 27 L.
infantum-infected asymptomatic, and 76 healthy) sera samples were
used for antibody detection.FINDINGS Of the three proteins, rA2 (91.5% sensitivity and 87% specificity) and rC9
(95.7% sensitivity and 87.5% specificity) displayed the best performance in
ELISA-HVL and ELISA-CVL, respectively. ICT-rA2 also displayed the best
performance for HVL diagnosis (92.3% sensitivity and 88.0% specificity) and
had high concordance with immunofluorescence antibody tests (IFAT),
ELISA-rK39, IT-LEISH®, and ELISAEXT. ICT-rFc, ICT-rC9,
and ICT-rA2 had sensitivities of 88.6%, 86.5%, and 87.0%, respectively, with
specificity values of 84.0%, 92.0%, and 100%, respectively for CVL
diagnosis.MAIN CONCLUSIONS The three antigens selected by us are promising candidates for VL diagnosis
regardless of the test format, although the antigen combinations and test
parameters may warrant further optimisation.
Recognising the importance of Chagas disease in Brazil, Bambuí set up epidemiological
surveillance for Chagas disease in 1974 and was the first municipality to do so. To
ascertain the current epidemiology of Chagas disease in this municipality, 1.782
blood samples from the general population were analysed; 7.7% of samples were found
to be seropositive for Chagas disease. A strong positive correlation between
increasing age and Chagas disease was evident in both genders, with the highest
prevalence in individuals aged over 60 years. Clinically, the cardiodigestive form of
Chagas disease was the most common in these samples. These data confirm the
interruption of Trypanosoma cruzi transmission, in parallel with a
still important residual morbidity of Chagas disease in the county, thus supporting
political decisions that will prioritise epidemiological surveillance and medical
treatment of Chagas disease in the coming years.
Diagnosis of Trypanosoma cruzi (T. cruzi) infection in the chronic phase of Chagas disease (CD) is performed by serologic testing. Conventional tests are currently used with very good results but require time, laboratory infrastructure, and expertise. Rapid diagnostic tests (RDTs) are an alternative as the results are immediate and do not require specialized knowledge, making them suitable for epidemiologic studies and promising as a screening tool. Nevertheless, few studies conducted comparative evaluations of RDTs to validate the results and assess their performance. In this study, we analyzed four trades of rapid tests (OnSite Chagas Ab Combo Rapid Test-United States, SD Bioline Chagas AB-United States, WL Check Chagas-Argentina, and TR Chagas Bio-Manguinhos-Brazil) using a panel of 190 samples, including sera from 111 infected individuals, most of whom had low T. cruzi antibody levels. An additional 59 samples from uninfected individuals and 20 sera from individuals with other diseases, mainly visceral leishmaniasis, were included. All tests were performed by three independent laboratories in a blinded manner. Results showed differences in sensitivity from 92.8 to 100%, specificity from 78.5 to 92.4%, and accuracy from 90.5 to 95.3% among the four assays. The results presented here show that all four RDTs have high overall diagnostic ability. However, WL Check Chagas and TR Chagas Bio-Manguinhos were considered most suitable for use in screening studies due to their high sensitivity combined with good performance. Although these two RDTs have high sensitivity, a positive result should be confirmed with other tests to confirm or rule out reactivity/positivity, especially considering possible cross-reactivity with individuals with leishmaniasis or toxoplasmosis.
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