Molecular identification of T. cruzi lineages and populations in end-stage Chagas heart disease patients undergoing heart transplantation: predominance of lineage I

BURGOS JM; DIEZ M; VIGLIANO C; BISIO M; DUFFY T; FAVALORO L; FAVALORO R; LEVIN MJ; SCHIJMAN AG

Buenos Aires, Argentina

Congreso; XVI Congreso Mundial de Cardiología; 2008

The aim of this study was to identify T. cruzi lineages directly from target tissues and bloodstream of endstage Chagas heart disease (ESChHD) patients who underwent heart trasplantation (HT) and developed Chagas reactivation (RA). Patients´ groups ESChHD: seven argentinean patients submitted to HT due to end-stage chronic Chagas heart disease. non-ChHD: three patients seropositive for T. cruzi, who underwent heart transplantation because of concomitant disorders (fibrosis, myocardial infarct and valvular heart diseases). Indeterminate Chagas disease patients (IChD): forty six T. cruzi seropositive individuals without signs and symptoms of cardiac manifestations. Molecular identification of parasite lineages (Lg) Attempts to identify the parasite lineages were carried out directly in peripheral blood and tissue biopsy samples. Lg-PCR-based lineage identification was assessed by: i) Amplification of the intergenic spacer of the spliced-leader genes ii) Hot-start heminested amplification of the dimorphic D7 domain of the 24S-ribosomal RNA genes, and iii) Real Time Hemi-nested PCR targeted to the A10 DNA fragment. Analysis of minicircle signatures The 330-bp minicircle variable region of the kinetoplastid genome was amplified by PCR and the products were digested (RFLP-PCR) with MspI + RsaI restriction enzymes. Results ESChHD: Between 1 and 6 weeks after transplantation, Tc I DNA was detected in peripheral blood samples from 5/7 ESChHD patients, Tc IId DNA in one case and Tc IIe in the remaining one. Lg-PCR positivity occurred earlier in Tc II reactivated patients than in those infected with Tc I, suggesting that Tc II displayed higher parasitemia levels. Consecutive post-transplant blood samples, characterized by Lg-PCR and RFLP-PCR, revealed that the same bloodstream populations persisted during RA, which was also observed in a patient who suffered two RA episodes. Among 5 ESChHD-RA patients, 4 presented lesions (epidermic chagomas and/or endomyocardial biopsy samples) with the same parasite lineages that were detected in bloodstream. In each patient, the minicircle signatures from blood and tissue samples were nearly identical, indicating that the parasites causing the lesions were those found in blood. The fifth patient had skin chagomas and endomyocardial biopsy samples positive for Tc IId/e DNA despite only lineage I was detected in blood. Minicircle signatures of the parasite populations characterized from blood, skin and EMB were all different. Interestingly, T. cruzi II was linked to all our 3 ESChD patients with myocarditis reactivation whereas Tc I was only detected in skin chagomas. Non-ChHD group: Between 4 and 6 weeks after transplantation, T. cruzi IId populations were identified in 2 patients. The other one was PCR negative. IChD group: Out of the 19 characterized patients, 17 were infected by Tc IId parasites and 2 by Tc I. The 27 remaining patients were PCR negative. Finally, our study showed a differential distribution of bloodstream lineages I and II between ESChHD patients and the other patients groups (Fisher exact test, p = 0.005), in contrast of the original assumption of innocuity of T. cruzi I in southern endemic regions of America.