RESUMO
BACKGROUND: A question of epidemiological relevance in Chagas disease studies is to understand Trypanosoma cruzi transmission cycles and trace the origins of (re)emerging cases in areas under vector or disease surveillance. Conventional parasitological methods lack sensitivity whereas molecular approaches can fill in this gap, provided that an adequate sample can be collected and processed and a nucleic acid amplification method can be developed and standardized. We developed a duplex qPCR assay for accurate detection and quantification of T. cruzi satellite DNA (satDNA) sequence in samples from domestic and sylvatic mammalian reservoirs. The method incorporates amplification of the gene encoding for the interphotoreceptor retinoid-binding protein (IRBP), highly conserved among mammalian species, as endogenous internal amplification control (eIAC), allowing distinction of false negative PCR findings due to inadequate sample conditions, DNA degradation and/or PCR interfering substances. RESULTS: The novel TaqMan probe and corresponding primers employed in this study improved the analytical sensitivity of the assay to 0.01 par.eq/ml, greater than that attained by previous assays for Tc I and Tc IV strains. The assay was tested in 152 specimens, 35 from 15 different wild reservoir species and 117 from 7 domestic reservoir species, captured in endemic regions of Argentina, Colombia and Mexico and thus potentially infected with different parasite discrete typing units. The eIACs amplified in all samples from domestic reservoirs from Argentina and Mexico, such as Canis familiaris, Felis catus, Sus scrofa, Ovis aries, Equus caballus, Bos taurus and Capra hircus with quantification cycles (Cq's) between 23 and 25. Additionally, the eIACs amplified from samples obtained from wild mammals, such as small rodents Akodon toba, Galea leucoblephara, Rattus rattus, the opossums Didelphis virginiana, D. marsupialis and Marmosa murina, the bats Tadarida brasiliensis, Promops nasutus and Desmodus rotundus, as well as in Conepatus chinga, Lagostomus maximus, Leopardus geoffroyi, Lepus europaeus, Mazama gouazoubira and Lycalopex gymnocercus, rendering Cq's between 24 and 33. CONCLUSIONS: This duplex qPCR assay provides an accurate laboratory tool for screening and quantification of T. cruzi infection in a vast repertoire of domestic and wild mammalian reservoir species, contributing to improve molecular epidemiology studies of T. cruzi transmission cycles.
Assuntos
Doença de Chagas/veterinária , Reservatórios de Doenças/parasitologia , Mamíferos/parasitologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Animais Domésticos/parasitologia , Animais Selvagens/parasitologia , Doença de Chagas/diagnóstico , Primers do DNA/genética , Sondas de DNA/genética , DNA de Protozoário/isolamento & purificação , DNA Satélite/isolamento & purificação , Proteínas do Olho/genética , Proteínas de Ligação ao Retinol/genética , Sensibilidade e Especificidade , Trypanosoma cruziRESUMO
Chagas disease, caused by the protozoan Trypanosoma cruzi, is an important public health concern in areas extending from South America northward into the southern United States of America. Although this hemoflagellate has many wild and domestic mammalians reported as reservoir hosts, studies on this subject are scarce in Nuevo León state, a region located in northeastern Mexico. This cross-sectional study showed that the general prevalence of T. cruzi infection in Nuevo León state was 14.5% (35/241), this percentage matching the ones determined by PCR and traditional diagnostics. Localities and infected mammals did not significantly differ (χ2 = 6.098, p = 0.192); however the number of infected animals was highly correlated with mammalian species (p = 0.009). Striped skunks (Mephitis mephitis) were found to be the most infected overall (11/34, 32.3%), while dogs (Canis familiaris) had the lowest prevalence. In conclusion, although the prevalence of T. cruzi infection in small mammals was lower in Nuevo León than in other states of Mexico, our results provide new locality records, including striped skunks, opossums (Didelphis marsupialis) and dogs, and extend the recorded area to woodrats (Neotoma micropus).
La enfermedad de Chagas, causada por el protozoario Trypanosoma cruzi, es un problema importante para la salud pública en una vasta región que se extiende en dirección norte desde Sudamérica hasta el sur de los Estados Unidos de América. Aunque este hemoflage-lado tiene muchos mamíferos silvestres y domésticos reportados como reservorios, los estudios sobre este tema son escasos en el estado de Nuevo León, localizado en el noreste de México. Se efectuó un relevamiento de la prevalencia de T. cruzi en pequeños mamíferos cubriendo 9 municipios del estado de Nuevo León y 3 tipos de asentamiento (rural, suburbano y urbano). Se observó una prevalencia general de la infección del 14,5% (35/241) usando PCR y diagnóstico tradicional para detectarla. No se determinó una asociación estadísticamente significativa entre las localidades relevadas y las especies de mamíferos infectados (χ² = 6,098, p = 0,192); sin embargo, el número de animales infectados se correlacionó con la especie de mamífero (p = 0,009). La mayor prevalencia de T. cruzi se detectó en los zorrillos (Mephitis mephitis) (11/34; 32,3%); la menor (13/136; 9,5%), en los perros (Canis familiaris). La prevalencia de la infección por T. cruzi en pequenos mamíferos fue más baja en Nuevo León que en otros estados de México. Estos resultados proveen nuevos registros de localidad incluyendo zorrillos, zarigüeyas o tlacuaches (Didelphis marsupialis) y perros, y también amplían el área registrada para las ratas de campo (Neotoma micropus).
Assuntos
Animais , Cães , Reservatórios de Doenças , Doença de Chagas , Doenças do Cão , América do Sul , Trypanosoma cruzi/isolamento & purificação , Prevalência , Estudos Transversais , Doença de Chagas/veterinária , Doenças do Cão/parasitologia , Doenças do Cão/epidemiologia , Mamíferos , México/epidemiologiaRESUMO
Chagas disease, caused by the protozoan Trypanosoma cruzi, is an important public health concern in areas extending from South America northward into the southern United States of America. Although this hemoflagellate has many wild and domestic mammalians reported as reservoir hosts, studies on this subject are scarce in Nuevo León state, a region located in northeastern Mexico. This cross-sectional study showed that the general prevalence of T. cruzi infection in Nuevo León state was 14.5% (35/241), this percentage matching the ones determined by PCR and traditional diagnostics. Localities and infected mammals did not significantly differ (χ2=6.098, p=0.192); however the number of infected animals was highly correlated with mammalian species (p=0.009). Striped skunks (Mephitis mephitis) were found to be the most infected overall (11/34, 32.3%), while dogs (Canis familiaris) had the lowest prevalence. In conclusion, although the prevalence of T. cruzi infection in small mammals was lower in Nuevo León than in other states of Mexico, our results provide new locality records, including striped skunks, opossums (Didelphis marsupialis) and dogs, and extend the recorded area to woodrats (Neotoma micropus).