Trypanosoma cruzi: polymerase chain reaction-based detection in dried feces of Triatoma infestans.

PCR was employed to detect Trypanosoma cruzi DNA in Triatoma infestans dry fecal spots collected on filter papers. Both insects fed on experimentally infected monkeys and insects collected in a Paraguayan endemic area for Chagas' disease were examined. When the insects fed on a chronically infected monkey with low parasitemia as revealed by direct microscopic observation (DMO), T. cruzi was detected in the insect feces by PCR as soon as 2 days postfeeding. When the same experiment was performed on monkeys with parasitemia levels below the limit of detection by DMO, the degree of positivity found through PCR-Southern hybridization, applied on Day 8 postfeeding, was superior to that obtained through xenoculture. These results suggest that PCR can be used to speed the xenodiagnosis results with great sensitivity. On the other hand, when applied to the feces of triatomines collected in the field, 84% were positive by PCR-Southern hybridization, whereas only 26% were positive by DMO. Therefore, PCR could also be applied to the monitoring of the infection status of triatomines which infest rural dwellings by examining only the feces left on paper sensors hung on the walls of the houses.

Russomando G ，Rojas de Arias A ，Almiron M ，Figueredo A ，Ferreira ME ，Morita K ... -  《EXPERIMENTAL PARASITOLOGY》

Suitability of a rapid DNA isolation and amplification for detection of Trypanosoma cruzi in Triatoma infestans dry fecal spots collected on filter paper.

A rapid DNA extraction was used for T. cruzi detection in triatomines dry fecal spots collected on filter paper and analyzed by PCR. Fifty T. infestans were fed on experimentally infected Balb/C mice with high T. cruzi parasitemia and divided into five groups of ten triatomines, and 100 triatomines were infected with lower parasitemia and divided into five groups of 20 triatomines. One dry fecal spot was analyzed per group on days 1, 2, 3, 4 and 5 post feeding. Amplification targeted T. cruzi TCZ sequence and resulted positive from day 4 after bugs feeding in the two models (high and lower parasitemial. The rapid DNA isolation and PCR proposed are suitable for detection of T. cruzi DNA in filter paper and should be considered in field research.

Braz LM ，Raiz-Júnior R ，Alárcon RS ，Gakiya E ，Amato-Neto V ，Okay TS ... -  《Parasite》

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

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. 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. 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. 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.

Pizarro JC ，Lucero DE ，Stevens L 《BMC INFECTIOUS DISEASES》

Trypanosoma cruzi detection in blood by xenodiagnosis and polymerase chain reaction in the wild rodent Octodon degus.

We detected Trypanosoma cruzi in blood samples of the wild rodent Octodon degus by xenodiagnosis and a polymerase chain reaction (PCR) using the domestic and wild vectors of Chagas disease, Triatoma infestans and Mepraia spinolai, respectively. We captured 35 rodents and extracted DNA from blood samples and intestinal contents of vectors fed on O. degus. Our results indicate that the percentage of rodents naturally infected with T. cruzi depends on the biologic sample used for PCR and on the vector species for xenodiagnosis. The PCR with blood samples did not detect T. cruzi DNA, but the PCR with intestinal contents showed that both vectors were positive for T. cruzi. The PCR performed with M. spinolai intestinal contents detected four times more T. cruzi-positive O. degus than the PCR with Triatoma infestans intestinal contents (22.9% and 5.7%, respectively). We report the improvement of T. cruzi detection in sylvatic animals by a combination of PCR and xenodiagnosis using sylvatic vectors, especially in disease-endemic areas with low parasitemias in mammals.

Campos R ，Botto-Mahan C ，Ortiz S ，Acuña M ，Cattan PE ，Solari A ... -  《AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE》

Trypanosoma cruzi: specific detection of parasites by PCR in infected humans and vectors using a set of primers (BP1/BP2) targeted to a nuclear DNA sequence.

In the present work we evaluate Trypanosoma cruzi DNA detection by PCR using the nuclear oligonucleotides BP1/BP2 as primers. These primers are targeted to the 5' and 3' ends of the coding region for the flagellar protein F29. An amplification product of BP1/BP2 is a DNA band 692 bp long. Titration assays were performed to evaluate the minimum amount of parasite DNA that can be detected by this assay, resulting in 10 fg (equivalent to about 1/20 of the genome). The assay was also performed using T. cruzi DNA from different strains, clones, and human-derived isolates obtaining, in all cases, amplification products. No DNA amplification was observed when the PCR was performed using DNA from Leishmania braziliensis, but when T. rangeli DNA was used, a 615-bp-long fragment was amplified. Under appropriate gel conditions T. cruzi and T. rangeli DNA amplicons could be differentiated. When both conventional xenodiagnosis and PCR detection of parasite DNA in the feces of insect vectors fed with blood from infected patients were compared, 10 of 20 samples were positive by both techniques. However, 2 other samples with positive serology were also positive by PCR. When PCR was performed on blood samples from infected and uninfected individuals, 62 of 65 serologically positive human samples amplified the BP1/BP2 692-bp T. cruzi DNA fragment (sensitivity >95%). The 3 negative samples were positive when Southern blot hybridization was performed using the radiolabeled PCR amplification product as probe (sensitivity 100%).

Silber AM ，Búa J ，Porcel BM ，Segura EL ，Ruiz AM ... -  《EXPERIMENTAL PARASITOLOGY》