Smulian Ag

Source of Pneumocystis carinii in recurrent episodes of pneumonia in AIDS patients.

OBJECTIVE To investigate the hypothesis that P.carinii special form hominis (P.c. hominis) reinfections occur in AIDS patients. DESIGN Polymerase chain reaction (PCR) was used to identify patients who had different P.c. hominis mitochondrial DNA (mtrRNA) genotypes in the two disease episodes (genotype switching). P.c. hominis from these patients were analysed with an allele-specific PCR (ASP) assay to determine whether the genotype found in a second disease episode were present in the first disease episode. To assess the possible contributions of other factors to genotype switching, data on the sampling method and drugs used to treat each patient were compiled. METHODS Bronchoalveolar lavage fluid (BALF) was subjected to PCR using primers that amplified a 346 base-pair region of the mtrRNA locus known to be polymorphic at site 85 of the amplicon. Samples from patients in whom the P.c. hominis mtrRNA sequence had changed at site 85 in the two disease episodes were studied by ASP in which primers designed to prime synthesis from the allele of the mtrRNA sequence found in second episodes of disease were used in PCR of P.c. hominis DNA from first episodes of P. carinii pneumonia. RESULTS In four of five patients who produced P.c. hominis with different mtrRNA genotypes during first and second episodes, ASP did not detect the second-episode genotype in first-episode BALF. There was no evidence that either sampling methods or drug-resistance contributed to genotype switching. CONCLUSIONS P.c. hominis reinfections occur in AIDS patients.

Characterization of multiple unique cDNAs encoding the major surface glycoprotein of rat-derived Pneumocystis carinii.

The major surface glycoprotein (MSG) of rat-derivedPneumocystis carinii represents a group of related molecules that are encoded by multiple genes. We isolated seven unique MSG cDNAs from a library prepared from a single infected rat lung. The cDNAs displayed both conserved and variant regions to previously described cDNAs. These clones contained inserts that ranged in size from 0.4 to 1.8 kb and all contained a poly(A) tail. The largest clone, Pc1410, hybridized to all 15 chromosomes resolved by pulsed-field gel electrophoresis (PFGE). Protein produced by in vitro translation from Pc1410 was immunoprecipitated with affinity-purified MSG antibodies. The clones were characterized by DNA sequencing of their 3′ and 5′ ends. Analysis of the untranslated and coding regions demonstrated that the clones contained unique and conserved regions of sequence, but none of the clones were identical. Isolation of seven additional unique clones picked from a single screening of a cDNA library suggests that numerous MSG transcripts exist within a population ofP. carinii.

Analysis of Pneumocystis carinii organism burden, viability and antigens in bronchoalveolar lavage fluid in AIDS patients with pneumocystosis: correlation with disease severity.

ObjectivesWe examined 96 bronchoalveolar lavage fluid (BALF) specimens from AIDS patients with proven Pneumocystis carinii pneumonia (PCP) in order to compare the relationship of organism burden, viability and antigen expression with disease severity at the time of clinical presentation. MethodsTinctorial analysis of BALF specimens with proven PCP using Diff-Quik, cresyl echt violet and erythrosin B stains to evaluate organism burden and viability. P. carinii antigen examination was performed by Western blot analysis. ResultsP. carinii cluster ratios were more sensitive than cyst counts as an indicator of organism burden, and correlated well with the alveolar-arterial oxygen gradient as a measure of disease severity. Erythrosin B, the vital stain used to measure P. carinii viability, displayed a wide range of values and provided little useful information. Antigens of 35–45 and 95 kD, which were specific for P. carinii, were found by immunoblot analysis in BALF cellular fraction of most patients with pneumocystosis. By contrast, antigens of 52 and 66 kD, which were found in both BALF supernatant and cellular fractions of P. carinii patients and controls, most likely represented albumin and immunoglobulin G heavy chain, respectively, of host origin. The 35–45 kD antigen was found in 88% of the BALF specimens and appeared to represent an important marker of P. carinii infection. The 95 kD antigen was detected in 49% of the specimens. ConclusionsWe conclude that analysis of P. carinii characteristics in BALF specimens of patients with pneumocystis may provide additional information. These data will also be helpful in developing more sensitive assays and in targeting specific P. carinii factors for future investigation.