Background: Trypanosoma cruzi, the causative agent of Chagas disease, and T. rangeli are kinetoplastid parasites endemic to Latin America. Although closely related to T. cruzi and capable of infecting humans, T. rangeli is non-pathogenic. Both parasite species are transmitted by triatomine bugs, and the presence of T. rangeli constitutes a confounding factor in the study of Chagas disease prevalence and transmission dynamics. Trypanosoma cruzi possesses high molecular heterogeneity: seven discrete typing units (DTUs) are currently recognized. In Ecuador, T. cruzi TcI and T. rangeli KP1(-) predominate, while other genetic lineages are seldom reported. Methods: Infection by T. cruzi and/or T. rangeli in different developmental stages of triatomine bugs from two communities of southern Ecuador was evaluated via polymerase chain reaction product size polymorphism of kinetoplast minicircle sequences and the non-transcribed spacer region of the mini-exon gene (n = 48). Forty-three mini-exon amplicons were also deep sequenced to analyze single-nucleotide polymorphisms within single and mixed infections. Mini-exon products from ten monoclonal reference strains were included as controls. Results: Trypanosoma cruzi genetic richness and diversity was not significantly greater in adult vectors than in nymphal stages III and V. In contrast, instar V individuals showed significantly higher T. rangeli richness when compared with other developmental stages. Among infected triatomines, deep sequencing revealed one T. rangeli infection (3%), 8 T. cruzi infections (23.5%) and 25 T. cruzi + T. rangeli co-infections (73.5%), suggesting that T. rangeli prevalence has been largely underestimated in the region. Furthermore, deep sequencing detected TcIV sequences in nine samples; this DTU had not previously been reported in Loja Province. Conclusions: Our data indicate that deep sequencing allows for better parasite identification/typing than amplicon size analysis alone for mixed infections containing both T. cruzi and T. rangeli, or when multiple T. cruzi DTUs are present. Additionally, our analysis showed extensive overlap among the parasite populations present in the two studied

Analysis of genetic polymorphism is a powerful tool for epidemiological surveillance and research. Powerful inference from pathogen genetic variation, however, is often restrained by limited access to representative target DNA, especially in the study of obligate parasitic species for which ex vivo culture is resource-intensive or bias-prone. Modern sequence capture methods enable pathogen genetic variation to be analyzed directly from host/vector material but are often too complex and expensive for resource-poor settings where infectious diseases prevail. This study proposes a simple, cost-effective ‘genome-wide locus sequence typing’ (GLST) tool based on massive parallel amplification of information hotspots throughout the target pathogen genome. The multiplexed polymerase chain reaction amplifies hundreds of different, user-defined genetic targets in a single reaction tube, and subsequent agarose gel-based clean-up and barcoding completes library preparation at under 4 USD per sample. Our study generates a flexible GLST primer panel design workflow for Trypanosoma cruzi, the parasitic agent of Chagas disease. We successfully apply our 203-target GLST panel to direct, culture-free metagenomic extracts from triatomine vectors containing a minimum of 3.69 pg/μl T. cruzi DNA and further elaborate on method performance by sequencing GLST libraries from T. cruzi reference clones representing discrete typing units (DTUs) TcI, TcIII, TcIV, TcV and TcVI. The 780 SNP sites we identify in the sample set repeatably distinguish parasites infecting sympatric vectors and detect correlations between genetic and geographic distances at regional (< 150 km) as well as continental scales. The markers also clearly separate TcI, TcIII, TcIV and TcV + TcVI and appear to distinguish multiclonal infections within TcI. We discuss the advantages, limitations and prospects of our method across a spectrum of epidemiological research.

Trypanosoma cruzi, causative agent of Chagas disease, and T. rangeli are kinetoplastid parasites endemic to Latin America. Although closely-related to T. cruzi and capable of infecting humans, T. rangeli is non-pathogenic. Both parasite species are transmitted by triatomine bugs, and the presence of T. rangeli constitutes a confounding factor in the study of Chagas prevalence and transmission dynamics. T. cruzi possesses high molecular heterogeneity: six discrete typing units (DTUs) are currently recognized. In Ecuador, TcI predominates while other DTUs are seldom reported. Here, infection by T. cruzi and/or T. rangeli in triatomine bugs from two communities of southern Ecuador was evaluated via PCR product size polymorphism of kinetoplast-minicircle sequences and the non-transcribed spacer region of the mini-exon gene. Additionally, mini-exon amplicons were deep-sequenced to analyze single nucleotide polymorphisms and mixed infections. As a control, mini-exon products from several monoclonal reference strains were included. T. cruzi genetic diversity was significantly greater in adult vectors than in nymphal stage V vectors. Among infected triatomines, deep sequencing revealed one T. rangeli infection (3%), 9 T. cruzi infections (27.3%) and 23 T. cruzi / T. rangeli mixed infections (69.7%), suggesting that T. rangeli prevalence has been largely underestimated in the region. Furthermore, deep-sequencing detected TcIV sequences in six samples (first TcIV record in southern Ecuador). Our data indicate that amplicon size analysis alone is not reliable for parasite identification/typing in mixed infections containing both T. cruzi and T. rangeli, or when multiple T. cruzi DTUs are present. Additionally, our analysis showed extensive overlap among the parasite populations present in the two studied localities (ca. 28 km apart); suggesting active parasite dispersal over the study area. Our results highlight the value of amplicon sequencing methodologies to clarify the population dynamics of kinetoplastid parasites in endemic regions and inform control campaigns in southern Ecuador.