PCR reveals significantly higher rates of Trypanosoma cruzi infection than microscopy in the Chagas vector, Triatoma infestans: High rates found in Chuquisaca, Bolivia

<p>Abstract</p> <p>Background</p> <p>The Andean valleys of Bolivia are the only reported location of sylvatic <it>Triatoma infestans</it>, the main vector of Chagas disease in this country, and the high human prevalence of <it>Trypanosoma cruzi </it>infection in this region is hypothesized to result from the ability of vectors to persist in domestic, peri-domestic, and sylvatic environments. Determination of the rate of <it>Trypanosoma </it>infection in its triatomine vectors is an important element in programs directed at reducing human infections. Traditionally, <it>T. cruzi </it>has been detected in insect vectors by direct microscopic examination of extruded feces, or dissection and analysis of the entire bug. Although this technique has proven to be useful, several drawbacks related to its sensitivity especially in the case of small instars and applicability to large numbers of insects and dead specimens have motivated researchers to look for a molecular assay based on the polymerase chain reaction (PCR) as an alternative for parasitic detection of <it>T. cruzi </it>infection in vectors. In the work presented here, we have compared a PCR assay and direct microscopic observation for diagnosis of <it>T. cruzi </it>infection in <it>T. infestans </it>collected in the field from five localities and four habitats in Chuquisaca, Bolivia. The efficacy of the methods was compared across nymphal stages, localities and habitats.</p> <p>Methods</p> <p>We examined 152 nymph and adult <it>T. infestans </it>collected from rural areas in the department of Chuquisaca, Bolivia. For microscopic observation, a few drops of rectal content obtained by abdominal extrusion were diluted with saline solution and compressed between a slide and a cover slip. The presence of motile parasites in 50 microscopic fields was registered using 400× magnification. For the molecular analysis, dissection of the posterior part of the abdomen of each insect followed by DNA extraction and PCR amplification was performed using the TCZ1 (5' – CGA GCT CTT GCC CAC ACG GGT GCT – 3') and TCZ2 (5' – CCT CCA AGC AGC GGA TAG TTC AGG – 3') primers. Amplicons were chromatographed on a 2% agarose gel with a 100 bp size standard, stained with ethidium bromide and viewed with UV fluorescence.</p> <p>For both the microscopy and PCR assays, we calculated sensitivity (number of positives by a method divided by the number of positives by either method) and discrepancy (one method was negative and the other was positive) at the locality, life stage and habitat level. The degree of agreement between PCR and microscopy was determined by calculating Kappa (<it>k</it>) values with 95% confidence intervals.</p> <p>Results</p> <p>We observed a high prevalence of <it>T. cruzi </it>infection in <it>T. infestans </it>(81.16% by PCR and 56.52% by microscopy) and discovered that PCR is significantly more sensitive than microscopic observation. The overall degree of agreement between the two methods was moderate (Kappa = 0.43 ± 0.07). The level of infection is significantly different among communities; however, prevalence was similar among habitats and life stages.</p> <p>Conclusion</p> <p>PCR was significantly more sensitive than microscopy in all habitats, developmental stages and localities in Chuquisaca, Bolivia. Overall we observed a high prevalence of <it>T. cruzi </it>infection in <it>T. infestans </it>in this area of Bolivia; however, microscopy underestimated infection at all levels examined.</p

Microsatellites Reveal a High Population Structure in Triatoma infestans from Chuquisaca, Bolivia

Chagas disease is a protozoan infection caused by the parasite Trypanosoma cruzi. Chagas is prevalent throughout Central and South America, and it remains a chief concern in Bolivia. A movement that began in 1991 called the Southern Cone Initiative has been successful in reducing the incidence of Chagas disease in the Southern Cone countries of Argentina, Brazil, Chile, and Uruguay; but due to socio-economic and other factors, incidence remains high in Bolivia. The most important mode of transmission of T. cruzi to humans and other mammals is through feces of triatomine bugs. Thus, disease control and transmission prevention focus on elimination of triatomine vectors, and more specifically in Bolivia, it focuses on the elimination of Triatoma infestans. This study focuses on T. infestans in the Department of Chuquisaca, Bolivia. Ten highly variable microsatellite markers were used to analyze the population structure of insects collected in different towns. Statistical analyses show that T. infestans are highly structured, which means that they colonize on a small geographic scale. The results also suggest little active dispersal. These findings should be implemented during control efforts so that insecticide spraying focuses on geographic areas of colonization and re-colonization

The improbable transmission of Trypanosoma cruzi to human: the missing link in the dynamics and control of Chagas disease

Chagas disease has a major impact on human health in Latin America and is becoming of global concern due to international migrations. Trypanosoma cruzi, the etiological agent of the disease, is one of the rare human parasites transmitted by the feces of its vector, as it is unable to reach the salivary gland of the insect. This stercorarian transmission is notoriously poorly understood, despite its crucial role in the ecology and evolution of the pathogen and the disease. The objective of this study was to quantify the probability of T. cruzi vectorial transmission to humans, and to use such an estimate to predict human prevalence from entomological data. We developed several models of T. cruzi transmission to estimate the probability of transmission from vector to host. Using datasets from the literature, we estimated the probability of transmission per contact with an infected triatomine to be 5.8x10(-4) (95%CI: [2.6; 11.0] x 10(-4)). This estimate was consistent across triatomine species, robust to variations in other parameters, and corresponded to 900-4,000 contacts per case. Our models subsequently allowed predicting human prevalence from vector abundance and infection rate in 7/10 independent datasets covering various triatomine species and epidemiological situations. This low probability of T. cruzi transmission reflected well the complex and unlikely mechanism of transmission via insect feces, and allowed predicting human prevalence from basic entomological data. Although a proof of principle study would now be valuable to validate our models' predictive ability in an even broader range of entomological and ecological settings, our quantitative estimate could allow switching the evaluation of disease risk and vector control program from purely entomological indexes to parasitological measures, as commonly done for other major vector borne diseases. This might lead to different quantitative perspectives as these indexes are well known not to be proportional one to another