Panfungal PCR assays using fresh frozen paraffin embedded tissue specimens for fungal species identification and the detection of azole-resistance mutations in the A. fumigatus cyp51A gene at a South Korean hospital

Abstract

Background: With rising concerns about changing fungal epidemiology and azole resistance in Aspergillus species, identifying fungal species and susceptibility patterns of mucorales and aspergillosis are crucial in the management of these diseases. The objectives of this study were to evaluate performance of panfungal PCR assays on formalin-fixed paraffin embedded (FFPE) samples for fungal species identification, and the detection of azole-resistance mutations in the Aspergillus fumigatus ( A.fumigatus ) cyp51A gene at a South Korean hospital. Methods: A total of 75 FFPE specimens with a histopathological diagnosis of aspergillosis or mucormycosis were identified during the 10-year study period (2006-2015). After deparaffinization and DNA extraction, panfungal PCR assays were conducted on FFPE samples for fungal species identification. The identified fungal species were compared with histopathological diagnosis. On samples identified as A.fumigatus , sequencings to identify frequent mutations in the cyp51A gene (tandem repeat 46 [TR46], L98H, and M220 alterations) that confer azole resistance were performed. Results: Specific fungal DNA was identified in 31 (41.3%) FFPE samples, and of these, 16 samples of specific fungal DNA were in accord with histopathological diagnosis of aspergillosis or mucormycosis. 15 samples had discordant histopathology and PCR results. No azole-mediating cyp51A gene mutation was revealed among nine cases of A. fumigatus . Moreover, no cyp51A mutations were identified among three cases with history of prior azole use. Conclusion: The pan-fungal PCR assay with FFPE sample may provide additional information on fungal species identification. No azole-resistance mediating mutations in the A. fumigatus cyp51A gene were identified among FFPE samples during study period.

Evaluating a semi‐nested PCR to support histopathology reports of fungal rhinosinusitis in formalin‐fixed paraffin‐embedded tissue samples

“…Although the site of the infection may affect the results, to set up a method independent of histopathological tests, in this study, a part of the sample was randomly taken from a PEB. While Jung et al reported a 41.3% positive rate for panfungal PCR due to long amplicon length and perhaps an ineffective DNA extraction kit 37 . This work found a concordance rate of 76% between semi‐nested PCR of the ITS region and histopathology tests, while others reported higher 21 or lower 38,39 concordance rates between these two methods.…”

“…While Jung et al reported a 41.3% positive rate for panfungal PCR due to long amplicon length and perhaps an ineffective DNA extraction kit. 37 This work found a concordance rate of 76% between semi‐nested PCR of the ITS region and histopathology tests, while others reported higher 21 or lower 38 , 39 concordance rates between these two methods.…”

“…In this work, we used a panfungal semi‐nested PCR to identify fungal elements in FFPE tissue samples by amplifying the ITS1‐5.8s‐ITS2 followed by ITS1 regions, which is consistent with a prior result. 47 Several researchers amplified other targets for detecting fungal DNA in FFPE samples, including 28S rDNA, 19 ITS2, 16 18S rDNA, 36 , 37 and mitochondrial tRNA. 36 Even though Cabaret et al claim that targeting mitochondrial DNA is preferable to ITS, 34 they got a lower positive rate by conventional PCR (10/16, 62.5%) than we did (56/64, 87.5%).…”