title: PCR reveals significantly higher rates of Trypanosoma cruzi infection than microscopy in the Chagas vector, Triatoma infestans: high rates found in Chuquisaca, Bolivia.
abstract: BACKGROUND
  The Andean valleys of Bolivia are the only reported location of sylvatic Triatoma infestans, the main vector of Chagas disease in this country, and the high human prevalence of Trypanosoma cruzi 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 Trypanosoma infection in its triatomine vectors is an important element in programs directed at reducing human infections. Traditionally, T. cruzi 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 T. cruzi infection in vectors. In the work presented here, we have compared a PCR assay and direct microscopic observation for diagnosis of T. cruzi infection in T. infestans 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.
  
  
  METHODS
  We examined 152 nymph and adult T. infestans 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 400x 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. 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 (k) values with 95% confidence intervals.
  
  
  RESULTS
  We observed a high prevalence of T. cruzi infection in T. infestans (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.
  
  
  CONCLUSIONS
  PCR was significantly more sensitive than microscopy in all habitats, developmental stages and localities in Chuquisaca, Bolivia. Overall we observed a high prevalence of T. cruzi infection in T. infestans in this area of Bolivia; however, microscopy underestimated infection at all levels examined.
authors.affiliation: Department of Biology, University of Vermont, Burlington, VT 04505, USA. jpizzaro@uvm.edu
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Pizarro
authors.forename: David E
authors.initials: DE
authors.lastname: Lucero
authors.forename: Lori
authors.initials: L
authors.lastname: Stevens
mesh_terms: D000818:Animals; D001838:Bolivia / Q000453:epidemiology; D014355:Chagas Disease / Q000453:epidemiology / Q000635:transmission / Q000662:veterinary; D019353:Endemic Diseases; D006801:Humans; D007303:Insect Vectors / Q000469:parasitology*; D008853:Microscopy; D016133:Polymerase Chain Reaction; D015995:Prevalence; D012680:Sensitivity and Specificity; D014224:Triatoma / Q000469:parasitology*; D014349:Trypanosoma cruzi / Q000235:genetics* / Q000302:isolation & purification*