PCR-SSCP and automated DNA sequencing used to investigate a cluster of Pneumocystis carinii pneumonia (PCP)

During a two month period a cluster of four cases of PCP occurred in bone marrow transplant/leukemic patients at Princess Margaret Hospital (PMH). The literature on Pneumocystis carinii (PC) has argued against person-to- person transmission. Some case-reports have suggested outbreaks in immunocompromised patients, although typing techniques have not yet been developed to support this theory. Nucleotide sequence variation was recently described in a portion of the large-subunit mitochondrial rRNA gene of PC. We performed a two-step polymerase chain reaction (PCR) on 13 known IFA positive bronchoalveolar lavage specimens which had been frozen at -20 degrees C. Primer pairs were used to amplify a 346 bp fragment of the large-subunit mitochondrial rRNA gene. PCR was positive on three of the four cases, nine unrelated samples from HIV-positive patients, and one temporally unrelated PMH sample. Single- stranded conformation polymorphism (SSCP) was then performed using the 13 PCR products. Identical patterns were obtained for all 13, suggesting no variation between cases and positive controls for the portion of the gene studied. However, sequencing confirmed that one HIV-positive control and one case had a single base pair mutation at position 85, effectively ruling out the possibility of a point source outbreak in this case. PCR-SSCP may have application to PC typing were a more variable gene available for study, and with restriction of PC-PCR products into smaller fragments may facilitate detection of mutational changes. Such a typing system may ultimately provide more insight into the variability and mode of acquisition of this organism.