One of the greatest concerns in Chagas' disease is the absence of reliable methods for the evaluation of chemotherapy efficacy in treated patients. The tests available to evaluate cure after the specific treatment are the complement-mediated lysis (CoML) and flow cytometry tests, but they are not feasible for routine clinical use. In this study, we evaluated an enzyme-linked immunosorbent assay (ELISA) based on the recombinant Trypanosoma cruzi complement regulatory protein (rCRP) as a method to determine parasite clearance in comparison to the CoML and other methods such as conventional serology, hemoculture, and PCR in serum samples of 31 patients collected before and after the treatment, monitored for an average of 27.7 months after chemotherapy. The results showed that the percentage of patient samples that were positive by rCRP ELISA was reduced from 100 to 70.3, 62.5, 71.4, and 33.4% in the first, second, third, and fourth years after treatment, respectively, while the samples positive by CoML were reduced to 85.2, 81.2, 71.4, and 33.4% during the same period, demonstrating the same significant tendency in the reduction of positive samples. On the other hand, the conventional serology (CS) tests did not present this reduction. The percentage of samples positive by PCR was initially 77.4% and decreased to 55.5, 68.7, 47.7, and 50.0% at the fourth year after treatment, confirming the drastic clearance of circulating parasites after treatment. Our results strongly suggest that the rCRP ELISA was capable of detecting the early therapeutic efficacy in treated patients and confirmed its superiority over the CS tests and parasitologic methods.

Currently, diagnosis of Chagas' disease is based on serological methods, but due to the high occurrence of inconclusive results, more reliable methods are needed. The use of recombinant antigens for serodiagnosis of Chagas' disease is recommended in order to increase the sensitivity and specificity of the serological tests. The Trypanosoma cruzi complement regulatory protein (CRP) is a surface glycoprotein present on the trypomastigote forms of the parasite, and the recombinant CRP (rCRP) was cloned in a mammalian expression system and purified by affinity chromatography. The purified recombinant protein was used as an antigen in an enzyme-linked immunosorbent assay (rCRP ELISA) in order to verify its sensitivity and specificity compared with other established methods. In this evaluation, a panel of 184 serum samples distributed among chronic chagasic patients (n ‫؍‬ 65), blood bank donors (n ‫؍‬ 100), and patients infected with Leishmania spp. (n ‫؍‬ 19) was used. The sensitivity and specificity of the rCRP ELISA were 100% when compared to conventional serology and complement-mediated lysis tests from these groups. When hemoculture and PCR tests were evaluated for diagnosis of chronic chagasic patients, using the rCRP ELISA as a reference test, the positivities were found to be 64.62 and 81.54%, respectively, showing a higher degree of sensitivity of the test. The data demonstrate that rCRP ELISA was able to discriminate between chronic chagasic patients and nonchagasic individuals, such as blood donors and patients with leishmaniasis. Thus, the rCRP is an excellent antigen for use in Chagas' disease diagnosis, due to the absence of false-negative or false-positive results.

HighlightsThis is the first meta-analysis of individual data in chronic Trypanosoma cruzi infection after treatment.The probability of seroreversion is variable along the course of follow-up.An interaction was found between age at treatment and country setting.The course of parasitological/molecular tests after treatment needs to be assessed.

Intraspecific variability among Cystoisospora belli isolates and its clinical implications in human cystoisosporosis have not been established. In this study, the restriction fragment length polymorphisms in a 1.8-kb amplicon of the small subunit ribosomal DNA (SSU rDNA) of the parasite was investigated in 20 C. belli-positive stool samples obtained from 15 HIV-infected patients. Diarrheic syndrome was observed in all patients with cystoisosporosis and the number of diarrheic episodes per patient during hospitalization ranged from 1 to 26 (mean of 9.64 ± 9.30), with a mean duration of 2 to 12 days (mean of 5.90 ± 3 days). Three restriction profiles (RF) were generated with MboII digestion, which were named RFI, RFII, and RFIII. Two isolates obtained from a patient with extraintestinal cystoisosporosis showed distinct restriction profiles with MboII. This study demonstrates that patients can be infected with different C. belli genotypes, and this information may be useful for identifying new C. belli genotypes infecting humans.

Trypanosoma cruzi trypomastigotes are able to resist lysis via the complement system, which involves many surface proteins including the complement regulatory protein (CRP). To examine the diversity in CRP recognition among strains of T. cruzi, the expression levels of the translated protein on trypomastigote surfaces were analyzed by flow cytometry, and associations between protein expression and the biological behavior of these strains, especially the ability to induce lytic antibodies in animal models, were assessed. The highly virulent T. cruzi strains Ninoa, INC-5, and Colombiana and the less virulent strains CL-Brener, LGB-231, and JG were used in the experiments. An expression profile analysis showed that the Colombiana and INC-5 strains have higher translated protein levels and induced higher production of antibodies in mice than the other strains. Our results indicated that there are differences in the surface expression of CRP between parasite strains, with a tendency for the most virulent strains to have higher expression levels. Combined, these results contribute to a better understanding of CRP functions and the complexity of host-parasite interactions, considering the large number of virulence factors involved in the process.

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The genetic variability of 61 Trypanosoma cruzi isolates from 47 chronic chagasic patients of Minas Gerais state was analyzed by random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) using M13-40, lambdagt11-F, and L15996 primers. Cluster analysis by unweighted pair group method analysis was applied to RAPD profiles, and cluster analysis used to verify a possible correlation among different clinical forms of the disease from these patients. The T. cruzi isolates showed distinct grouping on tree topology, with the isolates not being possible to establish a correlation to the clinical forms of Chagas' disease. These data showed that the T. cruzi isolates from these patients would compose a group of populations well correlated genetically.

Background The screening of Trypanosoma cruzi -infected blood donors using two serological techniques frequently leads to conflicting results. This fact prompted us to evaluate the diagnostic performance of four “in-house” immunodiagnostic tests and two commercially available enzyme-linked immunosorbent assays (ELISAs). Material and Methods One hundred and seventy-nine blood donors, whose screening for Chagas disease was doubtful, underwent three in-house ELISAs, one in-house immunoblotting test (TESA-blot), and two commercial ELISAs (bioMérieux and Wiener) in an attempt to define the presence or absence of infection. Simultaneously, 29 donors with previous positive results from three conventional serological tests and 30 donors with constant negative results were evaluated. Results The ELISA-Wiener showed the highest rate in sensitivity (98.92%) and the ELISA-bioMérieux, the highest specificity (99.45%), followed by the TESA-blot, which showed superior performance, with lower false-negative (2.18%) and false-positive (1.12%) rates. In series, the combination composed of the TESA-blot and ELISA-bioMérieux showed slightly superior performance, with trifunctional protein deficiency (TFP) = 0.01%. Conclusion Our study confirms the high sensitivity and specificity of commercial kits. To confirm the presence or absence of T. cruzi infection, the combination of TESA-blot and ELISA-bioMérieux may be suggested as the best alternative. Individually, the TESA-blot performed the closest to the gold standard; however, it is not commercially available.