Real-time PCR and Nested-PCR assays for detection of Pneumocystis sp. in Lung Tissues of Bats*

Background: Pneumocystis constitutes a highly diversifi ed biological group, with numerous species, which are strongly host-specifi c and well adapted to live inside the lungs of a diverse range of mammals. The detection of DNA from Pneumocystis in clinical specimens by PCR assays is leading to important advances in pneumonia caused by Pneumocystis and its epidemiology. The aim of this study was to analyze two different diagnostic methods, real-time PCR (qPCR) using primers based in the Major Surface Glycoprotein (MSG) of Pneumocystis sp. and conventional nested PCR using primers designed to the small subunit of mitochondrial rRNA (mtSSU rRNA) for detection of Pneumocystis DNA in lung tissue from bats. Materials, Methods & Results: Bats (195 samples) were captured (2007-2009) in caves, forests, and urban areas, were obtained from the Program of Rabies Control of two states in Brazil: Mato Grosso and Rio Grande do Sul, located respectively in the Mid-Western and Southern regions of the country approximately 2000 km apart. Lung tissue (250 mg) was fi nely minced, homogenized with crushing and DNA extraction was carried out with commercial kit. DNA samples the lung tissue of bats were analyzed by nested PCR, using oligonucleotide primers designed for the gene encoding the mitochondrial small subunit r RNA (mtSSU rRNA) and Taqman probe and primers for qPCR were selected based on the Major Surface Glycoprotein (MSG) of Pneumocystis sp. Chi-square (P < 0.001 was considered signifi cant) and the McNemar’s test was used to analyze nested PCR and qPCR as methods of detection of Pneumocystis sp. and the Kappa was calculated by Win Episcope 2.0. To assess the sensitivity and specifi city of the qPCR assay, a nested PCR assay was considered as the reference method. The positivity was 36.4% in the nested PCR and 24.1% using the qPCR. Concordance was obtained in 68.2% of the samples (133/195). It was demonstrated that there was no statistically signifi cant difference between the techniques used and, both tests proved to be specifi c for the detection of Pneumocystis species. Specifi city was 71% for the nested PCR and 84.6% for the qPCR. Pneumocystis was detected (71/195) by the nested PCR assay in 14 species:. Tadarida brasiliensis, Histiotus velatus, Desmodus rotundus, Molossus molossus, Glossophaga soricina, Nyctinomops laticaudatus, Promops nasutus, Artibeus sp., Eptesocus furinalus, Lasurus blossevillii, Molossus currentium, Molossus rufus, Myotis levis and Nyctinomops macrotis. Discussion: This study detected the DNA from Pneumocystis through the nested PCR and qPCR assays, and the frequency found is comparable to that obtained in a previous study, which used the nested PCR in Central American, South American and European countries. Pneumocystis sp. was observed in a high number of different bat species (14) in two Brazilian States (RS and MT). The qPCR showed a higher specifi city in comparison to the nested PCR. The literature has similar fi ndings to the results obtained by this research, employing the same tests and genes. The nested PCR and qPCR assays are indicated in the diagnosis of Pneumocystis sp. in bats and it is important to highlight that a better diagnostic precision is achieved with the association of both tests. Additionally, this study was the fi rst to detect Pneumocystis sp. in the lungs of bats using qPCR.