Utilização de técnicas moleculares para o acompanhamento de pacientes cardiopatas chagásicos transplantados, como ferramenta para a identificação precoce de reativação da infecção

Abstract

Chagas disease is considered the third most common parasitic infection in the world, after malaria and schistosomiasis (WHO 2005). It is estimated that 7 to 8 million people are infected causing 12 thousand deaths each year (WHO, 2014). About 20-30% of infected people manifest the severe form of the disease developing cardiac complications that can lead to heart failure or sudden death (Britto et al., 2009). The factors determining the different clinical manifestations and leading to mild or severe forms of the disease are not yet fully understood. Moreover, it is unknown why some chagasic cardiac patients underwent to heart transplantation present disease reactivation while others do not. Significant contribution, however, has been attributed to genetic aspects of the parasite. Heart transplantation is a valid therapeutic option for patients with chronic Chagas disease in most advanced stage. However, after transplantation differential diagnosis of hostversus-graft disease and Chagas reactivation has been considered difficult impeding the correct treatment. In the present study, we evaluated the possibility of using methodologies based on PCRs targeted to nuclear (rDNA 24S) and mitochondrial (kDNA) markers for early diagnosis of the presence of Trypanosoma cruzi in heart of transplanted patients with chagasic cardiomiopathy. In addition, we characterized the DTUs (T. cruzi I to VI) of the parasites present in positive samples using sequential PCR tests, based on three polymorphic markers: the COII gene, the intergenic spacer miniexon and rDNA 24S gene to evaluate whether there was any DTU more associated with the reactivations. From 2008 to 2014, we analyzed 497 endomyocardial biopsies derived from 58 chagasic cardiac patients who underwent heart transplantation at the Hospital das Clínicas of UFMG. T. cruzi DNA was detected in 111 samples and 62% of patients have shown at least one positive result for the molecular diagnosis tests performed. Genotyping analyes using these 111 positive samples revealed that most of patients were infected by T. cruzi II supporting the idea that this is the main strain associated with the cardiac form and Chagas disease reactivation, at least in this geographical region. Clinical follow-up of 36 patients, positive for at least one our molecular tests, revealed that 29 of them presented clinical reactivation of Chagas disease in diferent time after the transplantation. Thus, there is a good association (80.55%) between the results generated by PCR analyses of both kDNA and rDNA 24S markers and reactivation of clinical disease. In addition, positive results were also obtained in the first six performed endomyocardial biopsies anticipating 1-32 months clinical reactivation, indicating that PCR may contribute to the early diagnosis of reactivation of Chagas disease with potential to support in treatment decisions.