Detection and identification of fungi in bronchoalveolar lavage fluid from immunocompromised patients using panfungal PCR

Rapid diagnostics of fungal pneumonia and initiation of appropriate therapy are still challenging. In this study, we used two panfungal assays to test bronchoalveolar lavage fluid (BALF) samples to prove their ability to confirm invasive fungal disease diagnosis and identify causative agents. Two methods targeting different fungal rDNA regions were used, and the obtained PCR products were sequenced directly or after cloning. In total, 106 BALF samples from 104 patients were tested. After sequencing, we obtained 578 sequences. Four hundred thirty-seven sequences were excluded from further analysis due to duplication (n = 335) or similarity with sequences detected in the extraction control sample (n = 102); 141 unique sequences were analyzed. Altogether, 23/141 (16%) of the fungi detected belonged to pathogenic species, and 63/141 (45%) were identified as various yeasts; a variety of environmental or very rare fungal human pathogens represented 29/141 (21%) of the total and 26/141 (18%) were described as uncultured fungus. Panfungal PCR detected fungal species that would be missed by specific methods in only one case (probable cryptococcosis). Panfungal PCR followed by sequencing has limited use for testing BALF samples due to frequent commensal or environmental fungal species pickup.