Trypanosoma cruzi discrete typing units in patients of Chagas disease and Triatoma infestans after insecticide spraying in Chile

Chagas disease is widespread in Chile, distributed in rural and peri-urban areas in the seven endemic regions of the country. Human Chagas disease presents two distinct phases: the acute phase, which appears just after infection, and the chronic phase, which may last several years. After a long asymptomatic phase, around 30% of infected individuals develop chronic disease with severe damage to the heart and digestive system [Arribada et al., 1986]. Microscopic examinations of fresh or stained blood smears, xenodiagnosis and hemoculture are methods used for detected T. cruzi during the acute phase. In contrast, the detection is by circulating antibodies during the chronic phase. Triatoma infestans (Hemiptera, R eduviidae), a strictly hematophagous and almost exclusively domestic species, is the main vector of Trypanosoma cruzi , the causative agent of Chagas disease, in the southern cone of South America [Lent and Wygodzinsky 1979, Zeledón 1983]. Domestic animals are excellent hosts for these insects. The peri-domestic area around human houses in rural villages is very important, because it usually includes heavily infested goat corrals, chicken coops, rabbit cages and storerooms, which in many cases are just a few meters from the house [Gürtler 1993, 1997, Cecere et al., 1997, López et al., 1999, OMS 2002, Catalá et al., 2004]. In Chile, it attempted disruption of the domestic cycle of transmission of T. cruzi by means such as health education, improvement of housing and vector elimination by applying insecticides to human dwellings. Along northern areas where the domiciliary vectors were present systematic insecticide spraying with the support of health authorities it was applied during the last 20 years. Goat corrals are the main refuge for the peri-domestic populations of T. infestans and one of the ecotopes in which pyrethroid insecticides show low efficiency against these insects [Cecere etal., 1997, Gürtler etal., 2004]. Chicken coops are also very frequent a nd maintain abundant vector populations. Infested peri-domestic places could act as sources of housere-infestation after insecticide application [Gürtler etal., 2004], because of the movement of insects between habitats [Schofield 1985]. Although T. cruz i infection in peri-domestic T. infestans is not as frequent as in intra-domestic insects, the peri-domestic structures may be important sources of vector specimens. Chagas disease represents a major public health problem in America, with an estimated 16-18 million people infected by Trypanosoma cruzi [Moncayo, 2003]. In the most endemic areas of this disease of Chile, the Norte Grande and Norte Chico, there are an estimated 150,000 infected people, even though the only domiciliary vector Triatoma infestans has been controlled since 2000 [Lorca et al., 2001]. However, there are still geographic areas with infection rates of 0.55% and very low T. infestans infestation rates [ Jercic etal., 2011]. The clinical symptoms of chagasic patients are cardiological or/and digestive dysfunction in about 1/3 of the chronic infected subjects. In Chile most cases are reported as diseases in other organs (50.6%), followed by cardiological dysfunction (44 .6%) and digestive dysfunction (4.7%) [Moncayo, 2003]. The etiologic agent T. cruzi is composed of six DTUs (TcI-TcVI). Efforts to find the association between the infective T. cruzi DTUs with clinical manifestations have been made all over Latin America [ Zingales etal. , 2012]. In Chile there are reports indicating that TcI, TcII, TcV and TcVI, studied with different molecular markers, are prevalent (including mixed infection) [Sanchez etal., 1993; Rozas etal., 2005; Coronado etal., 2006; Arenas etal., 2012]. T. cruzi belongs to the order Kinetoplastida, a group of parasitic organisms with an organelle called a kinetoplast, which contains DNA in concatenated minicircles and maxicircles. The minicircles are very abundant (10,000 - 20,000 copies/cell), and therefore represent a perfect DNA target for diagnosis by means of PCR-DNA amplification. These amplicons are also useful for genotyping the T. cruzi DTUs, since minicircles are composed of different classes which may be used to characterize T. cruzi lineages by DNA-DNA hybridization methods [ Veas etal. , 1991; Brenière etal., 1998; Torres et al., 2004]. Each minicircle (1400bp) contains four constant regions intercalated with four hypervariable regions, which vary among the different minicircle classes present in each T. cruzi DTU [Arenas et al., 2012]. The medical entomology laboratory of the Institute of Public Health, analyzed samples of T. infestans . The Regional Ministerial Secretariats (SEREMIS) of Atacama, Valparaíso and Metropolitana provided triatomines captured in the period 2005-2010 of which 28.5% were infected. Parallel to the studies with the vector, there were a series of studies of serological screenings in children under 5 years old and their families between the regions of Arica and Parinacota and O’Higgins. Five thousand one hundred eleven screenings were preformed, with 28 positive cases (0.55%), a value close to the 0.7% prevalence obtained by the National Health Survey (NHS) 2009-2010 (Jercic et al., 2011). Our study, framed by the recommendations of the Pan american Health Organization Initiative of Countries of the Southern Cone, sought to evaluate the effectiveness of the programs of vector elimination carried out in Chile [OPS, 2002]. The Chilean Health Ministry and the Public Health Institute (PHI) con firmed each chagasic patient by means of serological methods, as well as the infected or non-infected status of the triatomines, by means of PCR diagnosis. This epidemiological information is relevant to direct the necessary efforts to spray human dwellings with insecticides and try to eradicate T. infestans. In this study, we evaluate the T. cruzi DTUs present in random representative samples of these patients and T. infestans collected in all endemic areas of the country under the entomological surveillance program of the Ministry of Health. Our aim is to identify T. cruzi DTUs currently circulating in T. infestans and non-treated chronic adult patients to analyze the T. cruzi DTUs circulating in the survivors in both hosts in an extensive area of Chile . We pretend to analyze the interactions between T. cruzi parasites and its hosts in order to evaluate their impact on the environmental health. To accomplish this goal we used a direct method to genotype T. cruzi , to avoid selection of T. cruzi clones fromm ixed infections during the parasite isolation and amplification processes.