RAPID SIMPLE AND NESTED POLYMERASE CHAIN-REACTION FOR THE DIAGNOSIS OF PNEUMOCYSTIS-CARINII PNEUMONIA

We have developed a rapid and easy extraction procedure for polymerase chain reaction (PCR) protocols. Using this simplified step, we evalua ted the sensitivity and the specificity of a simple PCR using the prim ers of Wakefield et al, and of a nested PCR, using new internal primer s selected by us, in a total of 89 bronchoalveolar lavage (BAL) fluid samples from 43 immunosuppressed patients, In 13 patients, Pneumocysti s carinii pneumonia (PCP) was diagnosed by immunofluorescent antibody (IFA) staining performed on BAL cells cytospun on microscope slides, I n 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. S imple and nested PCR both had a 100% sensitivity and a 98 and 84% spec ificity 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 symp tomatic was positive by simple and nested PCR. In follow-up BAL fluids of patients with proven PCP, persistence of P. carinii was detected f or a longer period by nested PCR than by simple PCR, Simple PCR is a v ery rapid and sensitive assay for the diagnosis of PCP in BAL fluid an d gives clear-cut results in the case of doubtful IFA staining results . Nested PCR seems to improve the sensitivity of the detection of P. c arinii in BAL fluid, but the clinical relevance of a positive result r emains to be investigated.