From 1994 to date we have been using the internal transcribed spacers (ITSs) nested polymerase chain reaction (PCR) to investigate the possibility of diagnosing Pneumocystis carinii pneumonia on non-invasive samples collected from HIV-positive patients with pulmonary involvement. The objectives were: (1) to test the sensitivity, specificity and prognostic value of PCR in diagnosis and follow up of PCP; (2) to investigate the eventual occurrence and role of asymptomatic carriers of P. carinii; (3) to evaluate the prognostic significance of blood PCR positivity versus respiratory samples; (4) to verify the occurrence of exogenous infections or endogenous reactivations in cases of recurrent P. carinii pneumonia; and (5) to study the possible correlation between P. carinii genotype identified and capability of blood dissemination, prior prophylactic treatments, clinical parameters and outcome of the patients.