Quantitative PCR on nasopharyngeal aspirate (NPA) can achieve high sensitivity and specificity in diagnosing Pneumocystis pneumonia (PCP) compared to microscopic examination of bronchoscopic specimens in a population with low HIV prevalence. Since NPA is a minimally invasive procedure, it is ideal as a screening test for PCP. C urrently, microbiological confirmation of Pneumocystis pneumonia (PCP) requires the identification of Pneumocystis cysts or trophozoites using microscopy. Bronchoscopic specimens are preferred because Pneumocystis resides mainly in the alveolar space (1). However, both transbronchoscopic bronchoalveolar lavage and sputum induction pose significant risk to the patient (2-4). Upper respiratory tract specimens are not recommended because of low sensitivity when traditional microscopic examination is used (5). PCR has higher sensitivity than microscopic examination in the detection of Pneumocystis (6) and may overcome the problem of low sensitivity associated with microscopic examination of upper respiratory tract specimens. In this study, we sought to determine whether nasopharyngeal aspirate (NPA) specimens could be used for the diagnosis of PCP.Patients of the Hong Kong West Cluster Hospitals with bronchoscopic specimens (bronchoalveolar lavage fluid [BALF] or bronchial aspirate) submitted to our laboratory for Pneumocystis examination from 1 January 2010 to 31 March 2012 were identified using the laboratory information system. In our laboratory, Pneumocystis cysts were visualized with methenamine silver stain by using standard procedures (7). With this method, Pneumocystis trophozoites or sporozoites could not be detected. Archived NPA specimens from these patients collected during the same hospitalization were retrieved. Only the earliest NPA specimen was tested if multiple specimens were available from the same patient. NPA specimens were collected as described previously (8). Clinical and laboratory data were obtained using the clinical management system. Patients without archived NPA specimens were excluded. A patient was considered to have definite PCP if Pneumocystis was identified in the bronchoscopic specimen by microscopic examination (9). This study has been approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster.DNA was extracted from 200 l of NPA specimens and from other fungi using the QIAamp DNA kit (Qiagen, Hilden, Germany) and DNeasy plant minikit (Qiagen, Hilden, Germany), respectively, according to the manufacturer's instructions (10). Real-time quantitative PCR (qPCR) targeting a 124-bp fragment of the mitochondrial large subunit rRNA (mt LSU rRNA) gene of Pneumocystis jirovecii was performed as described previously (11). Plasmid suspensions were used as standards for quantification and positive controls (Fig. 1).We verified the positive results from the mitochondrial (mt) large-subunit (LSU) rRNA qPCR (LSU-qPCR) by a conventional PCR targeting the mitochondrial small-subunit rRNA (mt SSU rRNA) gene of P. jiro...