Evaluation of Molecular Methods to Identify Chagas Disease and Leishmaniasis in Blood Donation Candidates in Two Brazilian Centers.

In Brazil, blood donation is regulated by the Brazilian Ministry of Health, and all States follow the same protocol for clinical and laboratory screening. Brazil is an endemic country for Chagas disease (CD), caused by Trypanosoma cruzi, and for leishmaniasis, caused by a species of Leishmania spp. Screening for leishmaniosis is not routinely performed by blood banks. Given the antigenic similarity between T. cruzi and Leishmania spp., cross-reactions in serological tests can occur, and inconclusive results for CD have been found. The objective of this study was to apply molecular techniques, e.g., nPCR, PCR, and qPCR, to clarify cases of blood donation candidates with non-negative serology for CD and to analyze the difference between the melting temperature during real-time PCR using SYBR Green. Thirty-seven cases that showed non-negative results for CD using chemiluminescent microparticle immunoassay (CMIA) tests from blood banks in Campo Grande, MS, and Campinas, SP, were analyzed. In the serum samples, 35 samples were evaluated by ELISA, and 24.3% (9/35) showed positive results for CD. nPCR was able to detect 12 positive results in 35 samples (34.28%). qPCR for T. cruzi was quantifiable in the samples that showed a value ≥0.002 par eq/mL (parasite equivalents per milliliter), and in 35 samples, 11 (31.42%) were positive. Of all evaluated samples using the described tests (CMIA, ELISA, nPCR, and qPCR), 18 (48.6%) were positive for CD. For MCA by qPCR, the melting temperature was 82.06 °C ± 0.46 for T. cruzi and 81.9 °C ± 0.24 for Leishmania infantum. The Mann-Whitney test showed a significant value of p < 0.0001. However, the differentiation between T. cruzi and L. infantum could not be considered due to temperature overlap. For leishmaniasis, of the 35 samples with non-negative serology for CD tested by the indirect fluorescent antibody test (IFAT), only one sample (2.85%) was positive (1:80). The PCR for Leishmania spp. was performed on 36 blood samples from donation candidates, and all were negative. qPCR for L. infantum showed 37 negative results for the 37 analyzed samples. The data presented here show the importance of performing two different tests in CD screening at blood banks. Molecular tests should be used for confirmation, thereby improving the blood donation system.

Biological characterization of Trypanosoma cruzi epimastigotes derived from trypomastigotes isolated from Brazilian chagasic patients.

Chagas disease (CD), caused by Trypanosoma cruzi, occurs in several countries in Latin America and non-endemic countries. Heterogeneity among T. cruzi population has been the Achilles' heel to find a better treatment for CD. In this study, we characterized the biochemical parameters and mitochondrial bioenergetics of epimastigotes differentiated from eight T. cruzi isolates (I1-I8) obtained from Brazilian CD patients. Molecular analysis of parasites DTUs grouped all of them as TcII. The profile of the growth curves in axenic cultures was distinct among them, except for I1 and I3 and I2 and I4. Doubling times, growth rates, cell body length, and resistance to benznidazole were also significantly different among them. All the isolates were more glucose-dependent than other T. cruzi strains adapted to grow in axenic culture. Mitochondrial bioenergetics analysis showed that each isolate behaved differently regarding oxygen consumption rates in non-permeabilized and in digitonin-permeabilized cells in the presence of a complex II-linked substrate. When complex IV-linked respiratory chain substrate was used to provide electrons to the mitochondrial respiratory chain (MRC), similarity among the isolates was higher. Our findings show that TcII epimastigotes derived from patients' trypomastigotes displayed their own characteristics in vitro, highlighting the intra-TcII diversity, especially regarding the functionality of mitochondrial respiratory complexes II and IV. Understanding T. cruzi intraspecific biological features help us to move a step further on our comprehension regarding parasite's survival and adaptability offering clues to improve the development of new therapies for CD.

Parasite load evaluation by qPCR and blood culture in Chagas disease and HIV co-infected patients under antiretroviral therapy.

Chagas disease also known as American trypanosomiasis, is caused by Trypanosoma cruzi and transmitted by triatominae-contaminated feces. It is considered a neglected tropical disease that affects 6 to 7 million people worldwide. The reactivation of Chagas disease occurs when the chronically infected hosts are not able to control T. cruzi infection, generating recurrence of the acute phase. HIV is the main immunosuppressive infection that can lead to the reactivation of chronic Chagas disease in AIDS conditions. In co-infected patients, the reactivation of Chagas disease is related to their high parasite load, high HIV viral load, and CD4 T-cell counting less than 200/mm3, which may evolve to meningoencephalitis and myocarditis. Eight T. cruzi/HIV co-infected patients under antiretroviral therapy (ART) and ten Chagas disease patients without HIV infection that attended at Study Group of Chagas Disease, Hospital de Clínicas, University of Campinas (GEdoCh/HC/UNICAMP-SP) and Pontifical Catholic University of Campinas SP (PUCC/SP) were evaluated. Tests for Chagas disease were performed, such as qPCR and T. cruzi blood culture. The patient's medical records were analyzed to verify clinical and epidemiological data, viral load, and CD4 T-cell counting since the outset of ART. For both groups, we found no statically significant differences between parasite load via blood culture and qPCR. In T. cruzi/HIV co-infected subjects, we observed a significant increase of CD4 T-cells counting and viral load decrease, which became undetectable over the years after ART. Parasites isolated from the patient's blood culture were genotyped, being the majority of them infected with TcII and one case of mixed infection (TcII and TcV/TcVI). These results were expected according to the region of origin of the patients. We suggest that the parasite load be monitored through qPCR in T.cruzi/HIV co-infected patients. We conclude that ART in people living with HIV improves infection and immunosuppression control, enabling the natural evolution of the American trypanosomiasis.

Using a Chagas disease hospital database: a clinical and epidemiological patient profile.

INTRODUCTION: Chagas disease (CD) prevention and control rely on studies of its distribution, characteristics of individuals affected and mode of transmission. CD data in Brazil are scarce; a retrospective analysis of the clinical characteristics of 80 patients treated at the Clinical Hospital of UNICAMP, Campinas, Brazil, was performed. METHODS: Patient data records were analyzed. RESULTS: Thirty percent of the patients probably got infected through vector-borne transmission, 65% came from endemic areas, a predominance of cardiac and cardiodigestive forms was found among males, and the cardiac form prevailed (51%). CONCLUSIONS: The results update the view on the epidemiology of CD in Campinas, Brazil.

Serological and molecular inquiry of Chagas disease in an Afro-descendant settlement in Mato Grosso do Sul State, Brazil.

Furnas do Dionísio is a Brazilian Afro-descendant settlement in the city of Jaraguari, 21.4 miles from Campo Grande, Mato Grosso do Sul, Brazil. Approximately 96 families live in this quilombola (Maroon) settlement, also known in Brazil as a remnant community of descendants of African slaves. Recent studies found 20% of households were infested by triatomines, 18% of insects captured in the community were infected by Trypanosoma cruzi, and 22.7% of dogs presented T. cruzi antibodies. The low prevalence of Chagas disease observed in humans in Mato Grosso do Sul State is attributed to its arrival via colonist migration and subsequent transplacental transmission. In order to gain a better understanding of the T. cruzi cycle in residents of the study community, serological and molecular tests were carried out to diagnose Chagas disease. In the present study, 175 residents between 2 and 80 years old were included. A total of 175 participants were interviewed and 170 provided blood samples, which were tested for T. cruzi antibodies with serological tests. Molecular diagnosis was performed in 167 participants by PCR (KDNA) and NPCR (satellite DNA) tests. One of the 170 samples tested positive for all serological tests performed. The overall frequency of Chagas disease in the community was low (0.6%). Interview responses revealed that 66.3% knew of triatomine insects and 65.7% reported having had no contact with them. Physical improvements to residences, together with vector surveillance and control by the State and municipal governments and local ecological conservation contribute to the low frequency of the Chagas disease in this quilombola community.

The detection of Trypanosoma cruzi by nested-PCR in elderly patients: relationship to the clinical and epidemiological profile.

Chagas disease, which is caused by Trypanosoma cruzi, is transmitted primarily by triatomine bugs, although the incidence of new cases has decreased as a result of vector control. In Brazil, most of those affected have the chronic form of the disease and are generally elderly individuals who require appropriate clinical follow-up. In this work, we undertook a descriptive study in which 85 patients were interviewed and blood samples were collected for molecular analyses based on the amplification of parasite satellite DNA. The cardiac form of the disease was the most prevalent among the patients and hypertension was the most frequent comorbidity; polypharmacy was detected in 34% of the cases. Serological tests were positive in 95% of cases while 36% were positive in nested-polymerase chain reaction. These findings indicate an increased use of medications and a larger number of age-related diseases in elderly patients with Chagas disease, even in patients with low parasitemia. We conclude that elderly patients with Chagas disease require special attention and that further studies should be done with elderly individuals who carry this disease.

Using a Chagas disease hospital database: a clinical and epidemiological patient profile

Abstract INTRODUCTION: Chagas disease (CD) prevention and control rely on studies of its distribution, characteristics of individuals affected and mode of transmission. CD data in Brazil are scarce; a retrospective analysis of the clinical characteristics of 80 patients treated at the Clinical Hospital of UNICAMP, Campinas, Brazil, was performed. METHODS: Patient data records were analyzed. RESULTS: Thirty percent of the patients probably got infected through vector-borne transmission, 65% came from endemic areas, a predominance of cardiac and cardiodigestive forms was found among males, and the cardiac form prevailed (51%). CONCLUSIONS: The results update the view on the epidemiology of CD in Campinas, Brazil.