Field Application of Polymerase Chain Reaction Diagnosis and Strain Typing of Trypanosoma cruzi in Bolivian Triatomines

Abstract

A new approach for direct identification and characterization of Tiypaiiosoiiia cruzi stocks in biological samples was tested for field applicability on an extensive sample of feces collected from triatomine vectors from four different species found in Bolivia. The first step of the technique is polymerase chain reaction (PCR) amplification of the hypervnriable region of kinetoplast DNA minicircles of T. cruzí parasites. In this report, 345 fecal samples were analyzed and the PCR results were compared with microscopic examination. For Triatoina iilfesraizs, the principal Bolivian vector, both techniques were in concordance 85.3% of the time. For the three other species, RIzodrzius pictipes, Erciyi-us m m " u s , and Triaroiiia sordida, the fecal samples were all negative by microscopic examination whereas PCR results showed several T. crirzi-infected insects in each species. The second step of the procedure is the characterization of the T. ci-uzi clones by means of hybridization of the PCR products with clone-specific probes generated by the PCR. We used two probes corresponding to major clones circulating in high frequency in Bolivia (as shown by previous population genetic studies using isoenzyme characterization). We obtained four primary results: 1) we confirm the importance of two major clones in Bolívia in two distinct regions; 2) we report high rates of mixed infections (multiple clones in a single vector) in Triatoina iilfestaizs, up to 22% and 35% in Cochabamba and La Paz departments, respectively; 3) the results favor the absence of interaction between different clones; and 4) we find, for the first time, evidence of the major clones circulating in three species of triatomines that are known as mainly sylvatic species. The origin of these clones, sylvatic or domestic, is also discussed.

Suitability of a rapid DNA isolation and amplification for detection ofTrypanosoma cruziinTriatoma infestansdry fecal spots collected on filter paper

“…These results, together with those run with DNA from Leishmania amazonensis, L. braziliensis and L. chagasii, corroborated the specificity of our amplification system. Nevertheless, all negative filter paper samples revealed the 144 bp DNA fragment after addition of T. cruzi DNA thus confirming the inexistence of amplification inhibitor factors what was crucial to rule out the presence of inhibitors, that have already been reported by Breniere et al (1995) who used the same boiling procedure of the present study, and Araújo et al (2002) who used a more complex extraction procedure with phenol-chloroform extractions. Dorn et al (1999;2001) have associated amplification inhibition to the type of biological sample, suggesting that digestive tract samples, especially stomach contents are more prone to inhibit PCR.…”

“…Cirurgicos-00426) and eluted in 200 µl of sterile distilled water, in a 1.5 ml polypropylene microtube for the DNA extraction. Then, samples were boiled at 100°C for 20 min., re-centrifuged and supernatants containing DNA were stored at -20°C until amplification (Breniere et al, 1995). After thawing, samples were spun in a refrigerated micro centrifuge (Centrifuge 5417R, Eppendorf-2233, Hamburg, Germany) at 15,300 xg, for 5 min.…”

Immunization with recombinant paraflagellar rod protein induces protective immunity against Trypanosoma cruzi infection

“…This divergence has resulted from the clonal evolution of T. cruzi, characterized by a structured population where several discrete genotypes persist with little or no genetic exchange between groups [35], although hybrid genotypes have been identified suggesting that genetic exchange can occur in rare instances [27]. Of particular relevance to vaccine design, mixed infections with different clones are frequently observed in both the insect vector and humans [36,37]. Thus, it is imperative that any vaccine designed to protect against T. cruzi be composed of antigens that are highly conserved among the diverse strains of T. cruzi known to infect man.…”

A simplified method for sample collection and DNA isolation for polymerase chain reaction detection of Trypanosoma rangeli and Trypanosoma cruzi in triatomine vectors

“…However, none of these methods can specifically detect these trypanosomes. Recently, some authors have used molecular assays in order to specifically detect T. rangeli and T. cruzi in vertebrate and invertebrate hosts using distinct DNA extraction methods (Moser et al 1989, Brenière et al 1995, Russomando et al 1996, Shikanai-Yasuda et al 1996, Souto et al 1999, Vallejo et al 1999.…”