RESUMO
BACKGROUND: The detection of Trypanosoma cruzi genetic material in clinical samples is considered an important diagnostic tool for Chagas disease. We have previously demonstrated that PCR using clot samples yields greater sensitivity than either buffy coat or whole blood samples. However, phenol-chloroform DNA extraction from clot samples is difficult and toxic. The objective of the present study was to improve and develop a more sensitive method to recover parasite DNA from clot samples for the diagnosis of Chagas disease. METHODOLOGY/PRINCIPAL FINDINGS: A total of 265 match pair samples of whole blood-guanidine (GEB) and clot samples were analyzed; 150 were from Chagas seropositive subjects. DNA was extracted from both whole blood-guanidine samples, using a previously standardized methodology, and from clot samples, using a newly developed methodology based on a combination of the FastPrep technique and the standard method for GEB extraction. A qPCR targeting the nuclear satellite sequences was used to compare the sample source and the extraction method. Of the 150 samples from Chagas positive individuals by serology, 47 samples tested positive by qPCR with DNA extracted by both GEB and clot, but an additional 13 samples tested positive only in DNA extracted from clot. No serology-negative samples resulted positive when tested by qPCR. CONCLUSIONS: The new methodology for DNA extraction from clot samples improves the molecular diagnosis of Chagas disease.
Assuntos
Doença de Chagas/diagnóstico , DNA de Protozoário/sangue , Trypanosoma cruzi/genética , Doença de Chagas/parasitologia , DNA de Protozoário/genética , Testes Diagnósticos de Rotina/métodos , Humanos , Técnicas de Diagnóstico Molecular , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Testes Sorológicos/métodos , Trypanosoma cruzi/isolamento & purificaçãoRESUMO
About 20-30% of people infected with Chagas disease present with chronic Chagas cardiomyopathy (CCC), the most serious and frequent manifestation of the disease, while others remain asymptomatic and often do not experience Chagas-specific mortality. It is not currently well understood what causes these differential disease outcomes, but a genetic predisposition within the host could play an important role. This study examined variants in the NLRP1, CARD, and CASP1 inflammasome genes among 62 T. cruzi seropositive patients from Bolivia (38 cases with CCC and 24 asymptomatic controls) to uncover associations with CCC. All subjects underwent a complete medical examination including electrocardiogram (EKG) and echocardiogram. After genotype calling and quality control filtering with exclusion of 3 cases and 3 controls, association analysis was performed across 76 directly genotyped SNPs in NLRP1, CARD, and CASP1 genes, adjusting for age, sex, and population stratification. One SNP (rs11651270; Bonferroni-corrected p = 0.036) corresponding to a missense mutation in NLPR1 was found to be significant after adjustment for multiple testing, and a suggestive association was seen in CARD11 (rs6953573; Bonferroni-corrected p = 0.060). Although limited by sample size, the study results suggest variations in the inflammasome, particularly in NLRP1 and CARD11, may be associated with CCC.