Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples

Background: Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI–TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR).Methods/Principal Findings: The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm.Conclusions/Significance: Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production.This work received financial support from the Ministry of Science and Technology of Argentina [PICT 2011-0207 to AGS] and the National Scientific and Technical Research Council in Argentina (CONICET) [PIP 112 2011-010-0974 to AGS]. Work related to evaluation of biological samples was partially sponsored by the Pan-American Health Organization (PAHO) [Small Grants Program PAHO-TDR]; the Drugs and Neglected Diseases Initiative (DNDi, Geneva, Switzerland), Wellcome Trust (London, United Kingdom), SANOFI-AVENTIS (Buenos Aires, Argentina) and the National Council for Science and Technology in Mexico (CONACYT) [FONSEC 161405 to JMR]

Influence of Triatoma dimidiata in Modulating the Virulence of Trypanosoma cruzi Mexican Strains

The epidemiology of Chagas disease is complex. There are different vectors and reservoirs and different clinical manifestations. In order to assess whether the biological behavior of three strains isolated in southeastern Mexico (H4 isolated from human, Z17 isolated from Didelphis sp., and V isolated from T. dimidiata) could be modified during passage through the vector T. dimidiata, the parasitemia curve, the amount of amastigote nests, and mortality of BALB/c infected with blood trypomastigotes of T. cruzi were evaluated. Strains were maintained in continuous passage from mouse to mouse and in animals infected with metacyclic trypomastigotes. The parasitemia curves were significantly different () between mice to mice and triatoma to mice groups in strains H4 and Z17, and was also observed fewer amastigote nests in cardiac tissue ( strain H4 with higher number versus all groups and Z17 between mice to mice and triatoma to mice) 45 days after inoculation. It is concluded that T. dimidiata influences in modulating the virulence of strains of T. cruzi in the region. Further studies of the intestinal tract of the insect in search for some protein molecules involved in regulating may clarify the virulence of the parasite