Multilocus Polymerase Chain Reaction Restriction Fragment—Length Polymorphism Genotyping of Trypanosoma cruzi (Chagas Disease): Taxonomic and Clinical Applications

Marlene Rozas 1 2 Simonne De Doncker 1 Vanessa Adaui 0 1 Ximena Coronado 2 Christian Barnabe 3 Michel Tibyarenc 3 Aldo Solari 2 Jean-Claude Dujardin 1 0 Instituto de Medicina Tropical Alexander von Humboldt, Laboratorio de Parasitolog a Molecular , Lima , Peru 1 Institute of Tropical Medicine , Molecular Parasitology, Antwerp , Belgium 2 Programa de Biolog a Celular y Molecular, Institute de Ciencias Biomedicas, Facultad de Medicina, Universidad de Chile , Santiago 3 Ge ne tique et Evolution des Maladies Infectieuses, Unite Mixte de Recherche Centre National de Recherche Scientifique/Institut de Recherche pour le Developpement 2724 , Montpellier , France 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 electrophoresisand 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. Trypanosoma cruzi is the etiologic agent of Chagas disease, which affects 20 million people in Latin America [1]. T. cruzi has a broad host range and infects wild and domestic mammals, thus giving rise to a large reservoir of parasites. The parasites are transmitted by several species of bloodsucking reduviid bugs as well as through blood transfusion, passage by infected women to offspring, and even organ transplantation. - Chagas disease presents a short acute phase followed by a lifelong chronic phase characterized by clinical pleomorphism [2]. Natural populations of T. cruzi are characterized by a great genetic diversity, and this led to a series of classification attempts [35]. The most recent and commonly accepted classification is based on random amplified polymorphic DNA (RAPD) and multilocus enzyme electrophoresis (MLEE) and distinguishes 2 major lineages (called TCI and TCII, the second lineage being further divided into 5 phylogenetic subdivisions: IIa, IIb, IIc, IId, and IIe). The term discrete typing unit (DTU) was adopted to designate a set of stocks that are genetically more similar to each other than to any other stock; the existence of 6 DTUs in total was proposed [57]. T. cruzi presents a clonal replication mode [8], but it has been hypothesized that the evolutionary emergence of some DTUs could have been the result of recombination: (i) with DTU IIa and IIc likely originating from a fusion between ancestral DTU I and IIb strains [9] and (ii) with IId and IIe probably resulting from the hybridization of strains similar to DTU IIb with DTU IIc [7, 9]. Identification of the different DTUs is highly relevant for clinical and epidemiological studies. On the one hand, it allows exploration of the possible link between the genetic diversity of T. cruzi and the pleomorphism in severity and symptoms of the disease that is observed in different geographic regions [10 12] and/or chemotherapeutical failure [13]. On the other hand, considering the association of specific DTUs to sylvatic or domestic cycles [1, 6], it permits the molecular tracking of sylvatic T. cruzi populations, which might constitute a potential reservoir of reinfestation in the domestic transmission cycle (which is specifically targeted by the currently running control programs). In this context, there is a need for simple, sensitive, and discriminatory molecular methods allowing a direct application in host samples. In Leishmania, another Trypanosomatid, we recently showed that polymerase chain reaction multilocus restriction fragmentlength polymorphism (PCR-RFLP) analysis (MLP analysis) constituted an adequate answer to this need [14, 15]. The objective of the present study was to apply MLP analysis to 12 gene loci in a sample representative of the diversity of T. cruzi. We particularly focused on genes involved in host-parasite relationships (antigens and others) because these may be prone to polymorphism as an adaptive answer to the immune selective pressure [16]. Results were compared with those obtained by RAPD and MLEE, respectively, for taxonomic validation and practical applications for molecular epidemiological studies of Chagas disease were considered. The proof-of-evidence for the direct applicability of our method on host samples was demonstrated. MATERIALS AND METHODS Parasite strains and host samples. Twenty parasite strains of T. cruzi representative of the 6 major DTUs and 1 strain of T. cruzi marinkellei (table 1), all previously typed by MLEE were selected for this study. DNA was isolated from epimastigote cultures as described elsewhere [6]. Blood samples from 10 humans and 21 other mammals (4 Euneomys chinchilloides, 4 Abrothrix olivaceus, 3 Thylamys elegans, 5 Phyllotis darwini, and 5 Capra hircus) and intestinal contents from 19 of the vector Mepraia spinolai were previously obtained in the fourth region of Chile, an area where Chagas disease is hyperendemic. All samples were shown by kDNA amplification [13, 17, 18] to contain T. cruzi. Informed consent was obtained from patients or their parents or guardians. Human experimentation guidelines of the Instituut voor Tropische Geneeskunde were followed. Ethics clearance was obtained from the ethics committee of the Facultad de Medicina, Universidad de Chile, Santiago, Chile. PCR assays. Twelve loci (2 complete [coding and noncod Gainesville ing] sequences of kmp11 [19] and 1f8 [20], 9 coding sequences of TcCRT [21], gp72 [22], Tcgp63-II [23], SAPA [24], fl-160 [25], hsp70, hsp60 [26], cruzipain [27], sa85-1 [28], and 1 intergenic sequence of hsp70) were selected for the analysis. The target sequences differed in copy number and chromosome location [1928]. The corresponding sequences were retrieved from GenBank and were aligned using ClustalW (version 1.8; availa (...truncated)