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The detection of Trypanosoma cruzi in Triatoma infestans: comparison of a PCR-based assay with microscopical examination

The laboratory diagnosis of human American trypanosomiasis (Chagas disease) is currently usually based on serology or the detection of a Trypanosoma cruzi parasitaemia (Maldonado et al., 2004). Unfortunately, most of the serological methods perform poorly when used to detect the re-activation of infections, and are therefore not recommended for determining post-treatment cure (PortelaLindoso and Shikanai-Yasuda, 2003). Parasitaemia can be detected by the direct microscopical examination of samples of blood or buffy coats, haemoculture or xenodiagnosis. Although xenodiagnosis remains the ‘gold standard’, this technique requires expertise and is both cumbersome and time-consuming, often requiring 30–60 days to yield a final result. Despite these drawbacks, xenodiagnosis is indicated in the acute phase of disease or during possible reactivation, whenever the other techniques give negative results (Luquetti and Rassi, 2000). Romaña and Briones (1954) found that infections could sometimes be detected in triatomine bugs fed on acute cases of Chagas disease as early as 6 days post-feed. In the last decade, the use of PCR to detect the DNA of Try. cruzi in faecal samples collected from bugs used in xenodiagnosis has not only increased the sensitivity of the diagnosis but also shortened the time needed to obtain a useful result (Russomando et al., 1996; ShikanaiYasuda et al., 1996). The aim of the present study was to explore and compare the potential usefulness of PCR and direct microscopical examination for detecting Try. cruzi infection in bugs used for xenodiagnosis. Most (220) of the 275 bugs used — thirdand fourth-instar, laboratoryreared Triatoma infestans — were fed on BALB/c mice that had been experimentally infected with the Y strain of Try. cruzi, while the mice were in the acute phase of disease and had low parasitaemias (of approximately 10 parasites/ml blood). The aim of using mammalian hosts with low parasitaemias was to mimic the situation found, during re-activation of Chagas disease, in immunosuppressed heart-transplant patients. The other 55 bugs were fed on uninfected mice, as negative controls. Digestive-tract samples (faeces or abdominal contents) were collected from 25 bugs (20 that had fed on infected mice and five of the negative controls) 1, 2, 3, 4, 5, 10, 15, 20, 25, 30 and 60 days post-feed, with different bugs being used at each timepoint. Each sample was split into two aliquots. A subsample (2–10 ml) of one aliquot was mixed with one to five volumes of 0.9% NaCl solution and then checked by microscopy (at 6400, as a wet smear), while the same volume of the other aliquot was checked by PCR (see below). The primers used in the PCR — F2 (59TGC ACT CGG CTG ATC GTT TTC GAG-39) and B3 (59-AGG GTT GTT TGG TGT CCA GTG TGTG-39), both produced by Integrated DNA Technologies (Belo Horizonte, Brazil) — were designed to produce a 144-bp amplicon from the TCZ repetitive sequence of Try. cruzi (Moser et al., 1989). Genomic DNA was extracted, from a mixture of a digestive-tract aliquot Annals of Tropical Medicine & Parasitology, Vol. 101, No. 5, 461–465 (2007)