Development of conventional and real-time multiplex PCR-based assays for estimation of natural infection rates and Trypanosoma cruzi load in triatomine vectors

Abstract

Abstract Background Chagas disease is a complex anthropozoonosis with distinct domestic and sylvatic mammal species acting as potential reservoirs. The diversity of vector species and their habitats are among the factors that hinder the control of the disease. Control programs periodically monitor the prevalence of T. cruzi infection in insect bugs through microscopical observation of diluted feces. However, microscopy presents limited sensitivity in samples with low parasite numbers, difficulties in examining all evolutionary stages of the insect and may in turn be limited to differentiate T. cruzi from other morphologically similar trypanosomatids. Here, we report two highly sensitive and accurate methodologies to infer T. cruzi infection rates and to quantify parasite load in the gut of field-collected triatomines. Methods Triatomines were manually collected in the period 2011–2012 and 2014–2015, in domestic, peridomestic or sylvatic habitats in rural areas of 26 municipalities, encompassing three distinct Brazilian biomes: Caatinga, Cerrado and Atlantic Rainforest. Following morphological and taxonomical identification, the search for flagellated protozoa was performed by optical microscopy. A conventional PCR targeting T. cruzi kDNA and a TaqMan qPCR directed to the parasite nuclear satellite DNA (SAT) were developed, both in multiplex, with the triatomine 12S subunit ribosomal RNA gene, used as internal amplification control. Both methods were used for detection (kDNA-PCR) and parasite load quantification (SAT-DNA-qPCR), to investigate T. cruzi infection in captured triatomines. Results The combined methods were assayed on a panel of 205 field-collected triatomine samples. Diagnostic analysis revealed 21% positivity for the kDNA-PCR, whereas microscopic examination enabled identification of T. cruzi in only 7.0% of the PCR-positive samples. Negative PCR results were confirmed by the absence of T. cruzi flagellates using microscopy. Caatinga biome yielded the highest T. cruzi infection rate (60%), followed by the Atlantic Rainforest and Cerrado with 7.1 and 6.1%, respectively. In addition, a wide range distribution of parasite load, varying from 8.05 × 10-2 to 6.31 × 1010 was observed with a median of 2.29 × 103 T. cruzi/intestine units. When parasite load was analyzed by triatomine species, a significantly higher median was found for Panstrongylus lutzi in comparison with Triatoma brasiliensis. Conclusions Our results demonstrate highly sensitive PCR-based methodologies to monitor T. cruzi infection in triatomines. In addition, the qPCR assay offers the possibility of further evaluation parasite load, as a promising biomarker of the vectorial capacity of triatomines in Chagas disease endemic areas.

Keywords

• Trypanosoma cruzi
• Multiplex PCR
• Real-time qPCR
• Triatomines
• Natural infection
• Parasite load