WORKSHOPS ON OPPORTUNISTIC PROTISTS 9s

Evaluation of PCR technique for Diagnosing Pneumocystk can'nii Pneumonia in HIV Positive Patients using Oropharyngeal washings.

BETI'INA LUNDGREN" , THOMAS BENFIELP AND E N S D. LLJNDGREV. 'Dept of Clin. Microbiol.,' Dept Infect. Dis., Hvidovre Hospital, Copenhagen, Denmark

Routine diagnosis of Pneumocystis carinii pneumonia (PCP) requires either bronchoscopy or induced sputum followed by microscopic evaluation of specimens obtained from the lung. The diagnostic yield of bronchoalveolar lavage specimens @&-fluid) in diagnosing PCP from patients with AIDS using conventional stains is more than 95%. Throughout the last five yean PCR has been evaluated as a diagnostic tool in diagnosing PCP. The technique increases the sensitivity compared to immunofluorescent staining technique in induced sputum [ 11 but not in BAL fluid [2]. Since PCR is still more time consuming than the routine diagnosic procedures for PCP, it will require good sensitivity on non-invasive specimens to use PCR as a diagnostic tool for PCP. Wakefield et all has previous described PCR in diagnosing PCP in oropharyngeal washings (OW) &om 3 1 HIV positive patients, of whom 18 had PCP based on clinical progression and response to treatment and visualization of P. carinii in BAL specimens. They found a sensitivity of 56% (10/18) by ethidium bromide stained gels and 78% (14/18) by visualization of the ampliGcaton product by oligoblotting [3]. The objective of this study was to evaluate the diagnostic value of identifying P. carinii in OW by PCR from consecutive HN-infected patients undergoing diagnostic bronchoscopy.

MATERTALS AND METHODS. Oropharyngeal washings and bronchoscopic alveolar lavage(BAL) were obtained from 49 HlV positive patients, who underwent bronchoscopy due to respiratory illness. Routine diagnosis of PCP was based on microscopy of BAL specimens stained with Giemsa and indirect immunoflourescent technique. Oropharyngeal washings were obtained by patients gargling and rinsing their mouth with 10 ml sterile saline, after overnight fast, and immediately prior to bronchoscopy. As controls OW was collected from 20 hospital staff with and 20 staff without exposure to patients with PCP. DNA was extracted from 2 ml of OW and BAL using simple Proteinase K digestion, followed by PCR as previous described [3]. The P. carinii specific amplification product (346 base pair product from mitochodrid ribosomal RNA) was visualised on ethidium bromide stained agarose gels as well as by Southern hybridization and oligoblotting.

RESULTS AND DISCUSSION. In 25 (5 1%) of the 49 patients$ carinii was identified by conventional staining technique in BAL fluid, and of those 24 (96%) had also amplifiable P. carinii DNA in BAL fluid (table 1). Of the 25 patients with verified PCP, 18 (72%) corresponding OW'S were FCR positive. All PCR positive OW except one were visualised on ethidium stained gels, whereas the last sample was o d y positive by oligoblotting. The remaining 24 of the 49 patients were negative by conventional staining. Twenty-tree (96%) of these 24 were also negative by PCR, whereas the last patient was PCR positive in both BAL and OW (table 1). This patient had received pentamidine profylaxis, had clinical PCP and responded to anti-PCP therapy. We did not find any correlation between relative number of P. carinii organisms in BAL-fluid detected by conventional stains and the result of PCR from BAL and OW. Both patients with a high and low number of P. carinii organisms in the BAL specimens

were PCR positive in OW.

Table 1. Detection of P. carinii DNA by PCR in BAL fluid and in oropharyngeal washings (OW) in 49 patients, of whom had 25 microscopically verified PCP by examination of BAL fluid.

PCP (n=25) No PCP (n=24)

Pos. PCR - BAL fluid

POS. PCR - OW None of the 20 HIV negative hospital staff with exposure to patients with PCP or the 20 staff without exposure to patients with PCP were had PCR positive OW. The sensitivity of diagnosing PCP in non invasive OW in HIV positive patients is 72% and the specificity is 96%. Using the PCR previous described by Wakefield et a1 [3], and a simple DNA extraction from specimens with protinase K, we were able to maintain a good sensitivity and specificity. This non- invasive method for diagnosing PCP may serve as an adjuvant to routine diagnostic procedures, especially in severely ill patients unable to undergo more invasive procedures. [Sponsored in part by ECA Biomed-1; "Pneumocystis and

Pneumocystosis" 1.

1. Lipschik GY, Gill VJ, Lundgren JD, Andrawis VA, Nelson NA, Nielsen JO, Ognibene IT, Kovacs JA Luncet (1992) 3 4 0 203. 2. Skot J, k c h e AX, Kolmos HJ, Nielsen JO, Mathiesen LR, Lundgren JD. ScandJinfectDis.(l995) 27: 363. 3. Wakefield AE, Miller RF, Guiver LA, Hopkin JM. Q. J Med(1993) 86: 401.