Chagas disease is still a major public health issue in many Latin American countries. One of the current major challenges is to find an association between Trypanosoma cruzi discrete typing units (DTUs) and clinical manifestations of the disease. In this study, we used a multilocus conventional PCR and quantitative real time PCR (qPCR) approaches to perform the molecular typing and parasite load quantification directly from blood specimens of 65 chronic Chagas disease patients. All patients were recruited at the same health center, but their place of birth were widely distributed in different geographic regions of Brazil. Of the 65 patients, 35 (53.8%) presented positive amplification by real time qPCR, being 20 (30.7%) with the clinical indeterminate form and 15 (23.1%) with the cardiac form of the disease. The parasite load median for all positive patients was 2.54 [1.43–11.14] parasite equivalents/mL (par. Eq./mL), with the load ranging from 0.12 to 153.66 par. Eq./mL. Noteworthy, the parasite load was significantly higher in patients over 70 years old (median 20.05 [18.29–86.86] par. Eq./mL). Using guanidine-EDTA blood samples spiked with reference T. cruzi strains, belonging to the six DTUs, it was possible to genotype the parasite up to 0.5 par. Eq./mL, with high specificity. Of the patients with positive qPCR, it was possible to identify the T. cruzi DTU in 28 patients (80%). For the remaining patients (20%), at least a partial result was obtained. Analysis of specimens showed prevalences of TcVI, TcII and mixed infection TcVI+TcII equal to 40%, 17.1% and 14.3%, respectively. In addition, two patients were infected by TcV, and one patient was coinfected by TcIII+TcVI, These last three patients were in stage A of chronic chagasic cardiomyopathy (CCC), and they were born at the Bahia State (northeast region of Brazil). When T. cruzi genotypes were compared with the parasite load, more elevated parasite loads were observed in patients infected by TcII in general (parasite load median of 7.56 par. Eq./mL) in comparison to patients infected by TcVI (median of 2.35 par. Eq./mL). However, while the frequency of CCC was 50% in patients infected by TcVI and TcV, only 16.7% of patients infected by TcII evolved to CCC. Taking together, our results contribute to update the epidemiological knowledge of T. cruzi DTUs in Brazil, and highlight the age of patient and infection by TcII as important features that lead to the observation of higher parasitemia levels.

BackgroundMolecular tests can allow the rapid detection of tuberculosis (TB) and multidrug-resistant TB (MDR-TB). TB-SPRINT 59-Plex Beamedex® is a microbead-based assay developed for the simultaneous spoligotyping and detection of MDR-TB. The accuracy and cost evaluation of new assays and technologies are of great importance for their routine use in clinics and in research laboratories. The aim of this study was to evaluate the performance of TB-SPRINT at three laboratory research centers in Brazil and calculate its mean cost (MC) and activity-based costing (ABC).MethodsTB-SPRINT data were compared with the phenotypic and genotypic profiles obtained using Bactec™ MGIT™ 960 system and Genotype® MTBDRplus, respectively.ResultsCompared with MGIT, the accuracies of TB-SPRINT for the detection of rifampicin and isoniazid resistance ranged from 81 to 92% and 91.3 to 93.9%, respectively. Compared with MTBDRplus, the accuracies of TB-SPRINT for rifampicin and isoniazid were 99 and 94.2%, respectively. Moreover, the MC and ABC of TB-SPRINT were USD 127.78 and USD 109.94, respectively.ConclusionTB-SPRINT showed good results for isoniazid and rifampicin resistance detection, but still needs improvement to achieve In Vitro Diagnostics standards.

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