Reverse-transcription PCR increases sensitivity of broad-range fungal detection in bronchoalveolar lavage fluid. (22nd November 2021)

• Record Type: Journal Article
• Authors: Glasgow, Heather L; Cruz, Kurtis; Murphy, Sean C
• Is Part Of: Medical mycology. Volume 60:Number 1(2022)
• Publication Date: 2021-11-22
• Subjects: fungal infection -- reverse transcription PCR -- 28S rRNA -- broad range -- molecular diagnosis; Medical mycology -- Periodicals; Veterinary mycology -- Periodicals; Mycology -- Periodicals; Mycoses -- Periodicals; Pathogenic fungi -- Periodicals; 616.969005
• DOI: 10.1093/mmy/myab061
• Languages: English
• ISSNs: 1369-3786

Abstract

Abstract: Broad-range PCR targeting 28S D1-D2 ribosomal DNA (rDNA) identifies numerous fungi but has limited sensitivity in clinical specimens. Ribosomal RNA (rRNA) vastly outnumbers rDNA, suggesting reverse transcription (RT)-PCR could improve detection. Among contrived samples, RT-PCR decreased 28S PCR cycle threshold values by 10--12 cycles and lowered the limit of detection > 2000-fold. Among 32 bronchoalveolar lavage specimens, RT-PCR detected 12/15 (80%) fungal PCR- or culture-positive specimens, versus 6/12 (50%) by 28S PCR, 9/12 (75%) by any fungal PCR, and 13/15 (87%) by culture. RT-PCR newly identified fungi in 4/17 (24%) PCR- and culture-negative specimens. RT substantially increased 28S PCR sensitivity overall. Lay summary: Fungal infection remains difficult to diagnose in the laboratory. Here, we have shown that detecting ribosomal RNA and DNA, rather than only ribosomal DNA, in a broad range fungal assay results in a significant enhancement in the ability to detect and identify fungal pathogens in clinical samples.