Diagnostic use of PCR for detection of Pneumocystis carinii in oral wash samples

Abstract

There is a need to develop noninvasive methods for the diagnosis of Pneumocystis carinii pneumonia in patients unable to undergo bronchoscopy or induction sputum. Oral wash specimens are easily obtained, and P. carinii nucleic acid can be amplified and demonstrated by PCR. In routine clinical use, easy sample processing and single-round PCR are needed to ensure rapid analysis and to reduce the risk of contamination. We developed a single-round Touchdown PCR (TD-PCR) protocol with the ability to detect PCR inhibition in the specimen. The TD-PCR was evaluated in a routine diagnostic laboratory and was compared to a previously described PCR protocol (mitochondrial RNA) run in a research laboratory. Both PCR methods amplified a sequence of the mitochondrial rRNA gene of P. carinii. Paired bronchoalveolar lavage (BAL) and oral wash specimens from 76 consecutive human immunodeficiency virus type 1-infected persons undergoing a diagnostic bronchoscopy were included. The TD-PCR procedure was quicker than the mitochondrial PCR procedure (<24 versus 48 h) and, compared to microscopy, had sensitivity, specificity, and positive and negative predictive values of 89, 94, 93, and 91%, respectively, for oral wash specimens and 100, 91, 90, and 100%, respectively, for BAL specimens. Our results suggest that oral wash specimens are a potential noninvasive method to obtain a diagnostic specimen during P. carinii pneumonia infection and that it can be applied in a routine diagnostic laboratory.

FIG. 1

PCR results after DNA extraction by Chelex 100. PCR products were subjected to agarose gel electrophoresis and stained with ethidium bromide. MW, molecular weight marker. Lanes: 1 and 6, true-negative samples amplifying the internal process control at 656 bp; 2, 4, and 5, P. carinii-positive samples amplifying both the internal process control and P. carinii DNA at 346 bp; 3, false-negative sample, since no internal process control was present due to inhibition of the PCR.