Emerging aspects related to the application of biomolecular techniques to the diagnosis of Pneumocystis carinii pneumonia: our experience with ITSs primers.

Abstract

From April 1995 to September 1997 we examined by different polymerase chain reactions (PCRs) several biological samples in order to assess the diagnostic value of biomolecular techniques to detect Pneumocystis carinii DNA in respiratory (BAL and oropharyngeal washes) and blood samples (serum and peripheral blood mononuclear cells (PBMC)) collected from AIDS patients with respiratory disease. We present our results obtained with the application of the Internal Transcribed Spacers (ITSs) nested PCR. One hundred and twenty-two BALs, 142 oropharyngeal washes, 200 sera, 126 PBMC and 50 plasma samples collected from 122 AIDS patients with acute respiratory disease were examined and processed as follows. Serum samples . 7-ml blood samples were withdrawn by sterile Vacutainer venipuncture into tubes containing clotting gelatin, aliquoted and stored at −20°C within 4 h from collection. For patients with BAL proven P. carinii pneumonia (PCP), in addition to the blood sample collected before bronchoscopy, other samples were drawn during follow up. PBMC . 7-ml blood samples were collected in Vacutainer heparinized tubes and cells separated by a Ficoll-Paque Plus (Pharmacia Biotech, Sweden) gradient in RPMI 1640 with 20 mM HEPES (HyClone Europe Ltd., The Netherlands). The cell pellet was washed twice in RPMI, resuspended in 1 ml PBS, aliquoted and stored at −80°C until used. Plasma . 70-ml blood samples were collected in Vacutainer heparinized tubes and plasma aliquoted and stored at −20°C until used. Oropharyngeal samples (gargling) . 10 ml sterile normal saline, gargled for about 2 min fast, was …