Multilocus polymerase chain reaction restriction fragment-length polymorphism genotyping of Trypanosoma cruzi (Chagas disease): Taxonomic and clinical applications

Background. Trypanosoma cruzi, the agent of Chagas disease, is subdivided into 6 discrete typing units (DTUs); their identification is important to understand clinical pleomorphism and track sylvatic DTUs that might (re-)invade domestic foci of the disease and jeopardize the running control programs. Methods. The genetic polymorphism of 12 loci was analyzed by multilocus polymerase chain reaction restriction fragment-length polymorphism (PCR-RFLP) analysis (MLP analysis) in a sample representative of the diversity within T. cruzi. We paid particular attention to genes involved in host-parasite relationships, because these may be prone to polymorphism as an adaptive answer to the immune selective pressure. Results. The results of MLP analysis were shown to agree with the current multilocus enzyme electrophoresis-and random amplified polymorphic DNA-based classification of T. cruzi in 6 DTUs, thereby providing a taxonomic validation of our method. Our data supported hypotheses of genetic recombination within T. cruzi. We demonstrated direct applicability of PCR-RFLP analysis to blood of mammal hosts and intestine content of vector insects. Domestic DTUs were encountered in wild animals, and, reciprocally, sylvatic DTUs were encountered in humans, raising questions about changes of transmission patterns. Conclusions. MLP analysis represents a new alternative to existing molecular methods for T. cruzi typing. It might offer an invaluable support to clinical and epidemiological studies and to control programs.