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》

A new method for forensic DNA analysis of the blood meal in chagas disease vectors demonstrated using Triatoma infestans from Chuquisaca, Bolivia.

Feeding patterns of the vector are important in the epidemiology of Chagas disease, the leading cause of heart disease in Latin America. Chagas disease is caused by the parasite, Trypanasoma cruzi, which is transmitted by blood feeding insects. Historically, feeding behaviours of haematophagous insects have been investigated using serological reactions, which have detection limits in terms of both taxonomic resolution, and quantity and quality of the blood meal. They are labor intensive, require technical expertise, need fresh or frozen samples and antibodies often are either not available commercially or the resources for synthesis and purification are not available. We describe an assay to identify vertebrate blood meal sources, and the parasite T. cruzi using species-specific PCR assays from insect vectors and use the method to provide information regarding three questions: (1) Do domestic and peri-domestic (chicken coop and animal corral) habitats vary in the blood meals detected in the vectors? (2) What is the pattern of multiple blood meals? (3) Does the rate of T. cruzi infection vary among habitats and is it associated with specific blood meal types? Assays based on the polymerase chain reaction were evaluated for identification of the blood meal source in the heamatophagous Chagas disease vector Triatoma infestans. We evaluate a technique to identify 11 potential vertebrate food sources from the complex mixture extracted from the vector's abdomen. We tested the assay on 81 T. infestans specimens collected from the Andean highlands in the department of Chuquisaca, located in central Bolivia, one of the regions in South America where sylvatic T. infestans have been reported. This area is suggested to be the geographic origin of T. infestans and has very high human infection rates that may be related to sylvatic vector populations. The results of the assays revealed that a high percentage of insects collected in human dwellings had fed on peri-domestic animals. In contrast, one insect from a chicken coop but no bugs from corrals tested positive for human blood. Forty-eight percent of insects tested positive for more than one vertebrate species. T. cruzi infection was detected in 42% of the specimens. From the epidemiological point of view, the results reveal an overall pattern of movement from peri-domestic structures to human habitations for T. infestans in this region of Bolivia as well as the important role of pigs, dogs, chickens and guinea pigs in the dynamics of T. cruzi infection.

Pizarro JC ，Stevens L 《PLoS One》

Sylvatic Triatoma infestans (Reduviidae, Triatominae) in the Andean valleys of Bolivia.

Triatoma infestans is the main vector of Chagas disease in the Southern Cone countries. Wild populations of T. infestans appear widespread throughout the Andean valleys of Bolivia. In Cotapachi (2750 m asl), all sorts of rocky outcrops, regardless of their size, provided good refuges for T. infestans. Of the 1120 ecotopes investigated, 330 (29.5%) contained triatomines and 92% of the collected insects were nymphal instars. In the cold season, triatomine densities were similar in small and large outcrops. During the hot season, bug densities were higher in the larger outcrops, particularly in those located in peridomestic sites. T. infestans populations apparently produced one generation per year. Over half the sampled bugs were positive for T. cruzi infection. At Mataral (1750 m asl), a site located in the inter-Andean Chaco, a new morph of T. infestans was detected in a sylvatic environment.

Cortez MR ，Emperaire L ，Piccinali RV ，Gürtler RE ，Torrico F ，Jansen AM ，Noireau F ... -  《ACTA TROPICA》

Development of conventional and real-time multiplex PCR-based assays for estimation of natural infection rates and Trypanosoma cruzi load in triatomine vectors.

Moreira OC ，Verly T ，Finamore-Araujo P ，Gomes SAO ，Lopes CM ，de Sousa DM ，Azevedo LR ，da Mota FF ，d'Avila-Levy CM ，Santos-Mallet JR ，Britto C ... -  《Parasites & Vectors》

Field application of polymerase chain reaction diagnosis and strain typing of Trypanosoma cruzi in Bolivian triatomines.

Breniere SF ，Bosseno MF ，Telleria J ，Carrasco R ，Vargas F ，Yaksic N ，Noireau F ... -  《AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE》