Development and Clinical Application of a Panfungal PCR Assay To Detect and Identify Fungal DNA in Tissue Specimens

Abstract

Given the rise in the incidence of invasive fungal infections (IFIs) and the expanding spectrum of fungal pathogens, early and accurate identification of the causative pathogen is essential. We developed a panfungal PCR assay that targets the internal transcribed spacer 1 (ITS1) region of the ribosomal DNA gene cluster to detect fungal DNA in fresh and formalin-fixed, paraffin-embedded (PE) tissue specimens from patients with culture-proven (n ‫؍‬ 38) or solely histologically proven (n ‫؍‬ 24) IFIs. PCR products were sequenced and compared with sequences in the GenBank database to identify the causal pathogen. The molecular identification was correlated with results from histological examination and culture. The assay successfully detected and identified the fungal pathogen in 93.6% and 64.3% of culture-proven and solely histologically proven cases of IFI, respectively. A diverse range of fungal genera were identified, including species of Candida, Cryptococcus, Trichosporon, Aspergillus, Fusarium, Scedosporium, Exophiala, Exserohilum, Apophysomyces, Actinomucor, and Rhizopus. For five specimens, molecular analysis identified a pathogen closely related to that identified by culture. All PCR-negative specimens (n ‫؍‬ 10) were PE tissues in which fungal hyphae were visualized. The results support the use of the panfungal PCR assay in combination with conventional laboratory tests for accurate identification of fungi in tissue specimens.

High-Throughput Identification and Quantification of Candida Species Using High Resolution Derivative Melt Analysis of Panfungal Amplicons

“…[1][2][3][4][5][6] Culture-based diagnosis of fungal infections is inadequate in that many species do not culture efficiently or require unacceptably long incubations. 7 Antigen-based tests for galactomannan or ␤-glucan are improvements over culture, but are either too specific, too insensitive, plagued by false positives, or not yet validated by widespread testing.…”

Systemic infection by Spencermartinsiella sp. in a Nile crocodile (Crocodylus niloticus)

“…are the main fungal pathogens causing dermatitis and pneumonia (SCHUMACHER, 2003). This report describes the pathological findings and diagnosis of systemic HENDOLIN et al, 2000;CHEN et al, 2001;SCUPHAM et al, 2006;LAU et al, 2007;MANTER;VIVANCO, 2007). The ITS region sequences, especially the ITS2 region, are of great importance to differentiate clinically relevant yeast species (LEAW et al, 2006).…”

Microsphaeropsis arundinis: an emerging cause of phaeohyphomycosis in cats and people

“…In 2010, our group again reported disease caused by M. arundinis infection affecting the distal extremity of a cat (Figure 1), although this animal also had a lesion on its face 4 . Halliday molecularly characterised the internal transcribed spacer (ITS1), 5.8S and ITS2 regions and the D1/D2 region of the 28S rDNA gene cluster of the available human (four) and feline (two) isolates 4,5 . These included a case (contributed by Tom Gottlieb) of refractory dermal plaques in a renal transplant recipient ( Figure 2; Table 1).…”

“…This could be done using not only colonial material or fresh biopsy specimens from representative lesions but also paraffin-embedded formalin-fixed tissues 5 . Unlike the situation in people where most patients appear to be immunosuppressed by comorbid disease In veterinary practice, frustratingly, repeat samples for culture and…”