Evaluation of a polymerase chain reaction reverse hybridization line probe assay for the detection and identification of medically important fungi in bronchoalveolar lavage fluids.

Abstract

An assay system in which polymerase chain reaction (PCR) amplification of the ITS-1 region of ribosomal DNA (rDNA) is combined with a reverse-hybridization line probe assay (LiPA) was used for the identification of six Candida species and four Aspergillus species in pure cultures of clinical isolates, as well as in bronchoalveolar lavage (BAL) fluid samples from 42 patients with various underlying diseases. The results were compared with the results obtained with conventional routine identification methods as well as with a commercial enzyme-linked immunosorbent assay (ELISA) galactomannan detection assay and an Aspergillus-specific PCR. No discrepancies between the PCR-LiPA system and routine methods were found for pure cultures of Candida and Aspergillus species except in the case of Aspergillus versicolor. In BAL fluid samples in which Candida species were cultured, the PCR-LiPA system identified more species than did the routine methods. When routine analyses of patient samples were supplemented by adding data obtained by repurifying and re-identifying cultures and by taking isolates obtained from other body sites into account, the results agreed with PCR-LiPA system results in 81% of the cases (34/42). Most of the remaining discrepancies (6/8) involved cases in which such supplementary data were not available. In BAL fluid samples from which A. fumigatus was cultured, the agreement between the PCR-LiPA system and the routine methods was low. Only 2 of 11 BAL samples shown to contain A. fumigatus in ELISA and genus-specific PCR assays were positive in PCR-LiPA system. The PCR-LiPA system enables the simultaneous detection and identification of different fungal species present in pure or mixed populations within 6 h in a single assay. Optimization is required, however, before it is useful as a diagnostic tool in the clinical microbiology laboratory.