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

Evolution of Chagas’ disease in Brazil. Epidemiological perspective and challenges for the future: a critical review

Aims: This paper aimed to provide a critical review of the evolution of Chagas’ disease in Brazil, its magnitude, historical development and management, and challenges for the future. Methods: A literature search was performed using PubMed, SciELO and Google Scholar and throughout collected articles’ references. Narrative analysis was structured around five main themes identified: vector transmission, control program, and transfusion, oral and congenital transmission. Results: In Brazil, the Chagas’ disease Control Program was fully implemented in the 1980s, when it reached practically all the endemic areas, and in 1991, the Southern Cone Initiative was created, aiming to eliminate the disease transmission through eliminating the Triatoma infestans and controlling blood banks. As a result, the prevalence of chagasic donors in blood banks reduced from 4.4% in the 80s to 0.2% in 2005. In 2006, PAHO certified the interruption of transmission of Chagas’ disease through this vector in Brazil. However, there are still challenges, such as the domiciliation of new vector species, the need for medical care of the infected individuals, the prevention of alternative mechanisms of transmission, the loss of political concern regarding the disease and, the weakening of the control program. Conclusion: Despite the progress towards control, there are still many challenges ahead to maintain and expand such control and minimise the risk of re-emergence