Evaluation of adult chronic Chagas' heart disease diagnosis by molecular and serological methods

Abstract

Chagas' disease caused by Trypanosoma cruzi is endemic in Latin America. T. cruzi presents heterogeneous populations and comprises two main genetic lineages, named T. cruzi I and T. cruzi II. Diagnosis in the chronic phase is based on conventional serological tests, including indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (ELISA), and diagnosis in the acute phase based on parasitological methods, including hemoculture. The objective of this study was to evaluate the diagnostic procedures of Chagas' disease in adult patients in the chronic phase by using a PCR assay and conventional serological tests, including TESA-blot as the gold standard. Samples were obtained from 240 clinical chronic chagasic patients. The sensitivities, compared to that of TESA-blot, were 70% for PCR using the kinetoplast region, 75% for PCR using the nuclear repetitive region, 99% for IIF, and 95% for ELISA. According to the serological tests results, we recommend that researchers assess the reliability and sensitivity of the commercial kit Chagatest ELISA recombinant, version 3.0 (Chagatest Rec v3.0; Wiener Lab, Rosario, Argentina), due to the lack of sensitivity. Based on our analysis, we concluded that PCR cannot be validated as a conventional diagnostic technique for Chagas' disease. These data have been corroborated by low levels of concordance with serology test results. It is recommended that PCR be used only for alternative diagnostic support. Using the nuclear repetitive region of T. cruzi, PCR could also be applicable for monitoring patients receiving etiologic treatment.

FIG. 1.

Results comparing the phenol-chloroform DNA isolation method and Bio-Rad AquaPure Genomics for blood tissue based on 100 positive samples. The differences between the results obtained with stDNA and kDNA were statistically significant (P =0.040 and P = 0.047, respectively).

FIG. 2.

Results comparing the phenol-chloroform DNA isolation method and Bio-Rad AquaPure Genomics for blood tissue based on DNA concentration measurement by spectrophotometry. The difference between the results obtained was statistically significant (P =0.017).

FIG. 3.

(A) Results of PCR amplification of a 166-bp fragment with T. cruzi nuclear repetitive region-specific primers cruzi1 and cruzi2 extracted from DNA. Lane 1, patient sample 1; lane 2, patient sample 2; lane 3, patient sample 123; lane 4, patient sample 220; lane 5, patient sample 72; lane 6, positive control T. cruzi I strain Dm11; lane 7, positive control T. cruzi II strain VS; lane 8, molecular weight marker. (B) Results of PCR amplification of a 330-bp fragment with T. cruzi minicircle-specific primers 121 and 122 extracted from DNA. Lane 1, molecular weight marker; lane 2, patient sample 1; lane 3, patient sample 2; lane 4, patient sample 123; lane 5, positive control T. cruzi II strain VS; lane 6, positive control T. cruzi I strain Dm11; lane 7, negative control 1; lane 8, negative control 2. (C) Results of PCR amplification of a 110-bp fragment of the human β-globin region as an internal control. Lane 1, patient sample 1; lane 2, patient sample 2; lane 3, patient sample 123; lane 4, patient sample 200; lane 5, patient sample 127; lane 6, patient sample 122; lane 7, patient sample 256; lane 8, molecular weight marker.