RESUMO
Molecular techniques have revealed a high prevalence of Pneumocystis colonization in wild mammals. Accurate quantification of Pneumocystis sp. is essential for the correct interpretation of many research experiments investigating this organism. The objectives of this study were to detect the presence of Pneumocystis sp. in bats by qPCR, and to distinguish colonization from infection. Probes and primers for real time PCR (qPCR) were designed based on the gene of major surface glycoprotein (MSG) of Pneumocystis sp., in order to analyze 195 lung tissue samples from bats captured (2007-2009). All samples were also analyzed by nested PCR, using oligonucleotide primers designed for the gene encoding the mitochondrial small subunit rRNA (mtSSU rRNA) to confirm the results. The qPCR assay was standardized using a standard curve made with the DNA extracted from bronchoalveolar lavage positive for Pneumocystis jirovecii. The average Ct was found to be between 13 and 14 (calibration curve) for the detection of infection with Pneumocystis sp. and above these values for colonization. It was considered as negative samples the ones that had Ct values equal to 50. Out of the total 195 samples, 47 (24.1%) bat lung DNA samples were positive for Pneumocystis sp. by qPCR. The most common bat species found were: Tadarida brasiliensis (23.4%), Histiotus velatus (17.0%), Desmodus rotundus (14.9%) and Molossus molossus (8.5%). The average cycle threshold of the positive samples (bats) was 25.8 and standard deviation was 1.7. The DNA samples with Ct values greater than 14 suggest that these animals might be colonized by Pneumocystis sp. Results obtained in this study demonstrated the usefulness of the qPCR procedure for identification of Pneumocystis sp. and for distinction between its colonizing or infectious status in bats.
Assuntos
Portador Sadio/veterinária , Quirópteros/microbiologia , Reservatórios de Doenças/microbiologia , Infecções por Pneumocystis/transmissão , Pneumocystis/isolamento & purificação , Animais , Brasil , Líquido da Lavagem Broncoalveolar/microbiologia , Portador Sadio/epidemiologia , Portador Sadio/microbiologia , Quirópteros/classificação , DNA Fúngico/análise , DNA Fúngico/genética , Proteínas Fúngicas/genética , Especificidade de Hospedeiro , Pulmão/microbiologia , Glicoproteínas de Membrana/genética , Pneumocystis/genética , Pneumocystis carinii/genética , Pneumocystis carinii/isolamento & purificação , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase em Tempo Real , Ribotipagem , Especificidade da EspécieRESUMO
Pneumocystis pneumonia (PcP) is a serious fungal infection among immunocompromised patients. In developed countries, the epidemiology and clinical spectrum of PcP have been clearly defined and well documented. However, in most developing countries, relatively little is known about the prevalence of pneumocystosis. Several articles covering African, Asian and American countries were reviewed in the present study. PcP was identified as a frequent opportunistic infection in AIDS patients from different geographic regions. A trend to an increasing rate of PcP was apparent in developing countries from 2002 to 2010.
