Validation of a Real-Time PCR Assay for Detection of Pneumocystis jiroveci in Respiratory Specimens
Komal Arora, Kumari Vadlamudi, Leon W Razai, Jonathan R Lindner, Wieslaw Furmaga, Hongxin Fan. University of Texas Health Science Center, San Antonio, TX
Background: The diagnosis of Pneumocystis pneumonia(PCP) relies on microscopic identification of the stained organism. Due to its instability outside human body, the microscopic identification of Pneumocystis Jiroveci(PJ) results in significant number of false negatives. The organism cannot be cultured in-vitro which further complicates the diagnosis. Early diagnosis of PCP affects prognosis. Compared to microscopy, diagnosis by PCR is more rapid & sensitive. The quantitation of fungal load helps to differentiate active pneumonia from colonization. We validated a real-time PCR(RTPCR) that detects & quantifies the PJ load in respiratory specimens.
Design: Thirty-six specimens (34 BAL, 2 sputum) were collected from patients with clinical suspicion of PCP. DNA was extracted by QIAGEN EZ1 Tissue Kit and analyzed for presence of PJ using real-time PCR assay on ABI Prism 7900HT Sequence Detection System. The assay utilized primers and TaqMan probe targeting the PJ dihydrofolate reductase (DHFR2) gene region. Each sample was tested in duplicate. Human albumin gene was co-amplified and used as a normalizer to control for the number of cells tested. Quantitative load was reported as PJ copies/100,000 cells. A positive control plasmid containing partial PJ DHFR2 gene sequence was established from a PJ positive patient sample.
Results: Among 36 specimens, PJ positivity was in agreement between PCR and microscopy in 4 specimens (PJ load 4.4x102 - 3.5x106 copies/100,000 cells) confirming assay's accuracy. The PCR was more sensitive to detect 1 additional low positive PJ (5 copies/100,000 cells) in a sputum sample. The positivity of this sample was also confirmed by 2 other PCR assays targeting different PJ gene regions (KEX1 & beta-tubulin). The specificity of the assay was tested on selected viral, bacterial & human DNA samples with no cross reactions being observed. The DHFR2 PCR was linear across 8 orders of magnitude and was sensitive enough to detect 10 copies of PJ on serially diluted positive plasmid sample.
Assay's precision & reproducibility were assessed by testing 2 specimens in different dilutions on 4 runs. The results were in agreement among replicates.
Conclusions: The RTPCR for detection of PJ is rapid, more sensitive, accurate, specific and reproducible.
Wednesday, March 6, 2013 9:30 AM
Poster Session V # 246, Wednesday Morning