Molecular ID of cinically important fungal pathogens from formalin fixed paraffin embedded tissue
December 06 2016
The frequency and diversity of lethal invasive fungal pathogens has moved beyond Aspergillus fumigatus to include a plethora of pathogens including the non-fumigatus species of Aspergillus, the Mucormycetes, Fusarium and Scedosporium species and a variety of melanised fungi. These fungi have different antifungal susceptibilities, demanding genus and species identification in complex immunocompromised patients where early, reliable detection and appropriate management is crucial to improving survival.
While current histopathology can prove invasive fungal infections by the demonstration of fungal elements and host reactions in formain fixed paraffin embedded (FFPE) tissue specimens, identification to genus or species level based on morphological characteristics is limited. In addition fungal cultures from tissue biopsy specimens, when performed, remain negative in a substantial number of cases. Molecular species-level identification is possible but has been technically challenging.
Salehi et al 2016, evaluated a real-time quantitative PCR (qPCR) assay which targets the internal spacer (ITS) region of ribosomal DNA (rDNA) to detect and identify genus and species of clinically relevant Aspergillus, the Mucormycetes, Fusarium and Scedosporium from FFPE tissue specimens obtained from patients with histologically proven IFDs. This retrospective multicentre study correlated the molecular and histopathological mould identification results of 102 formalin-fixed parafin-embedded (FFPE) tissue specimens obtained between 2008 and 2014, from Iran.
A key analytical finding was the high sensitivity (94%) of the DNA extraction method utilised: automated EZ1 extraction instrument (Qiagen, Venlo, The Netherlands) in combination with a DNA tissue kit according to the manufacturer’s recommendations. DNA extraction from FFPE tissue is difficult because of the small amounts of DNA suitable for amplification and DNA degradation. The method used in this study allowed for excellent yield compared to the 60-80% amplification success rates of other current methods.
The qPCR assay evaluated showed an overall sensitivity of 64% for the identification of fungi from FFPE compared to histopathological analysis, as shown in Table below. (Table 2, Saleh et al 2016).
Of importance were the findings below that demonstrate that histopathological features of moulds may be easily confused in tissue sections:
- Fusarium oxysporum and Fusarium solani DNA was detected in five specimens but diagnosed as Aspergillus by histopathology
- Aspergillus flavus, Scedosporium apiospermum and Syncephalastrum were detected from samples classified as mucormycosis
- Molecular results (R. oryzae and A flavus) correlated with only one of four tissue samples histologically suspected of having concomitant aspergillosis and mucormycosis; two with A. flavus only and one Mucor isolate
PCR-based techniques are not without their own challenges and limitations; DNA degradation, PCR inhibitors present in samples, lack of standardisation of optimal sample type, primer selection and PCR formats.
However, direct identification by qPCR assays such as those evaluated in this study demonstrate promise for a rapid, reliable adjunctive tool for the accurate identification of clinically relevant fungal pathogens. Sequence based analysis should also be possible, using a similar protocol.
Salehi et al 2016: J Clin Microbiol 54:2798–2803. doi:10.1128/JCM.01185-16.