Rapid simple and nested polymerase chain reaction for the diagnosis of Pneumocystis carinii pneumonia.

Abstract

We have developed a rapid and easy extraction procedure for polymerase chain reaction (PCR) protocols. Using this simplified step, we evaluated the sensitivity and the specificity of a simple PCR using the primers of Wakefield et al, and of a nested PCR, using new internal primers selected by us, in a total of 89 bronochoalveolar lavage (BAL) fluid samples from 43 immunosuppressed patients. In 13 patients, Pneumocystis carinii pneumonia (PCP) was diagnosed by immunofluorescent antibody (IFA) staining performed on BAL cells cytospun on microscope slides. In seven of these patients we attempted to estimate the post-treatment persistence of P. carinii in BAL, by PCR. After a rapid 2-h extraction procedure, simple and nested PCR were positive in all cases of PCP. SImple and nested PCR both had a 100% sensitivity and a 98 and 84% specificity respectively, compared to IFA. After completion of treatment, BAL liquids from asymptomatic patients were no longer positive by both PCR techniques, whereas the BAL fluid of a patient who was still symptomatic was positive by simple and nested PCR. In follow-up BAL fluids of patients with proven PCP, persistence of P. carinii was detected for a longer period by nested PCR than by simple PCR. Simple PCR is a very rapid and sensitive assay for the diagnosis of PCP in BAL fluid and gives clear-cut results in the case of doubtful IFA staining results. Nested PCR seems to improve the sensitivity of the detection of P. carinii in BAL fluid, but the clinical relevance of a positive result remains to be investigated..