Invasive Fungal Infection Diagnostics: Overcoming Challenges of Deploying Molecular Methods on Formalin Fixed Paraffin-Embedded Specimens

Abstract

Background In immunocompromised patients, rapid diagnosis of fungal infections is vital to improve clinical outcomes. Tissue biopsies are a valuable source of diagnostic information, allowing for information to be gathered from the site of infection. Formalin-fixed paraffin-embedded (FFPE) tissues are often still available after histopathology and microbiology fail to identify the etiology. That, combined with their stability make FFPE samples often the best and only specimen remaining from biopsy. The fixative and embedding wax present challenges for molecular biology that must be taken into consideration. We aimed to develop methods for generating positive controls and extracting fungal DNA from FFPE tissues for three fungal diagnostic assays (Aspergillus quantitative polymerase chain reaction (qPCR) panel, Mucorales qPCR, and fungal next-generation sequencing (NGS)). Methods Positive controls were generated by injecting human lung tissue specimens with 1.2e4 conidia/mL of fungal pathogens of interest (Aspergillus terreus, A. fumigatus, A. niger, Rhizopus microsporus, and Candida albicans) then culturing in yeast extract-peptone-dextrose broth for 15 hours in a shaking incubator at 37°C, 300 rpm. Tissues were prepared as FFPE blocks. For each nucleic acid extraction, two 10µm scrolls were processed using the Takara DEXPAT kit, with the addition of a lyticase step for fungal lysis and nucleic acid cleanup on the Biomerieux EasyMag platform. Triplicate extractions were performed for each block, and the eluates analyzed by specific (Aspergillus and Mucorales) qPCR. To ensure the resulting DNA is useful for sequencing, primers specific to ITS1 and ITS2 were used to generate amplicons and quantified. Results A. fumigatus was detected at an average threshold cycle (Ct) of 29 with a CV% of 2.7% for the pan-Aspergillus assay and 29 Ct, CV of 1.9% for the A. fumigatus assay. A. terreus was detected at 31 Ct, CV% of 0.7%. A. niger was detected at 34 Ct, CV% of 2.8% for the pan-Aspergillus assay. The Mucorales qPCR successfully detected R. microsporus at Ct 27, CV% of 1.3%. Neither the Aspergillus nor the Mucorales qPCR assays detected eluates for non-cognate targets. In addition, the ITS1 / ITS2 amplifications were successful in yielding an average of 11/3, 12/24, 7/21, 19/35 and 25/37, ng/uL for A. terreus, A. fumigatus, A. niger, R. microsporus, and C. albicans, respectively. Conclusion Reliable FFPE-tissue scroll was generated as a positive control for each of the fungal assays tested. The extraction methods generated precise and accurate results. These results support the use of conidia injection of tissues followed by incubation in rich media to create FFPE controls for fungal qPCR and NGS. These will be essential to control laboratory molecular microbiology assays performed on FFPE tissue specimens submitted for informing a fungal etiology of an invasive infection.