Detection of Pneumocystis carinii and characterization of mutations associated with sulfa resistance in bronchoalveolar lavage samples from human immunodeficiency virus-infected subjects

Abstract

One hundred ninety-four bronchoalveolar specimens were evaluated by microscopic examination and by amplification of a sequence of a Pneumocystis carinii dihidropteroate synthase gene for identification of mutations linked to sulfa resistance. PCR sensitivity and specificity were 100 and 86.7%, respectively, compared to results of microscopic examination. However, 7 out of 19 microscopy-negative, PCR-positive samples were collected from subjects with a clinically high probability of P. carinii pneumonia, suggesting that PCR may be more sensitive than microscopic examination, although the absolute performance of PCR cannot be determined. Mutations were identified in 28 out of 70 (40%) PCR-positive specimens and were significantly more common in patients exposed to sulfa drugs (21 out of 29 [72.4%]) than in those not exposed to sulfa drugs (4 out of 35 [11.4%]).

FIG. 1.

DG-DGGE analysis. (A) Photograph of DG-DGGE patterns showing the electrophoretic mobility of PCR fragments from wild-type (lanes 1 and 6) and mutant (lanes 2 and 4) DHPS sequences. Heteroduplex patterns, obtained by mixing wild-type and mutant sequences, are showed in lanes 3 and 5. Amino acids that differ from those in the wild-type sequence are underlined. (B) Sensitivity of DG-DGGE analysis for detection of minority populations of the P. carinii DHPS gene. Plasmids containing wild-type or mutant (A-55) DHPS sequences were mixed in various concentrations and were analyzed by DG-DGGE. The percentage of mutant DNA is indicated above each lane. wt, wild type.