An update on current and novel molecular diagnostics for the diagnosis of invasive fungal infections

ABSTRACTBackground Invasive fungal infections cause millions of infections annually, but diagnosis remains challenging. There is an increased need for low-cost, easy to use, highly sensitive and specific molecular assays that can differentiate between colonized and pathogenic organisms from different clinical specimens.Areas covered We reviewed the literature evaluating the current state of molecular diagnostics for invasive fungal infections, focusing on current and novel molecular tests such as polymerase chain reaction (PCR), digital PCR, high-resolution melt (HRM), and metagenomics/next generation sequencing (mNGS).Expert opinion PCR is highly sensitive and specific, although performance can be impacted by prior/concurrent antifungal use. PCR assays can identify mutations associated with antifungal resistance, non-Aspergillus mold infections, and infections from endemic fungi. HRM is a rapid and highly sensitive diagnostic modality that can identify a wide range of fungal pathogens, including down to the species level, but multiplex assays are limited and HRM is currently unavailable in most healthcare settings, although universal HRM is working to overcome this limitation. mNGS offers a promising approach for rapid and hypothesis-free diagnosis of a wide range of fungal pathogens, although some drawbacks include limited access, variable performance across platforms, the expertise and costs associated with this method, and long turnaround times in real-world settings.KEYWORDS: Molecular diagnosticspolymerase chain reactionPCRdigital PCRmetagenomicsnext-generation sequencinghigh resolution meltinvasive fungal disease Article highlights Polymerase chain reaction (PCR) is a highly sensitive and specific molecular assay and can identify mutations associated with antifungal resistance, although performance can be impacted by prior/concurrent antifungal use.Droplet digital PCR (dPCR) can be multiplexed to detect individual pathogens and to screen for mutations potentially associated with antifungal resistance, has the potential for enhanced detection of low DNA concentrations, particularly relevant to fungal infection and has increased tolerance to inhibition compared to quantitative PCR, although current options are still limited.High resolution melt (HRM) is a rapid and highly sensitive diagnostic modality that can identify a wide range of fungal pathogens, including down to the species level, but multiplex assays are limited and HRM is currently unavailable in most healthcare settings.Metagenomics next-generation sequencing (mNGS) offers a promising approach for rapid and hypothesis-free diagnosis of a wide range of fungal pathogens, although some drawbacks include limited access, variable performance across platforms, the expertise and costs associated with this method, and long turnaround times in real-world settings.True point-of-care molecular diagnostics assays are still needed that are available in lower resource settings and that can complement culture and conventional fungal biomarkers.Declaration of interestsJDJ received research funding from Astellas, F2G, and Pfizer – all outside of the submitted work. PLW performed diagnostic evaluations and received meeting sponsorship from Bruker, Dynamiker, and Launch Diagnostics; Speaker’s fees, expert advice fees and meeting sponsorship from Gilead; Speaker and expert advice fees from F2G and speaker fees MSD and Pfizer: Speaker’s fees and performed diagnostic evaluations for Associates of Cape Cod and IMMY. Speakers’ fees from Qiagen. Expert advice fees from Mundipharma – all outside of the submitted work. SEK performed diagnostic evaluations and received meeting sponsorship from AusDiagnostics and received speaker fees and conference travel funding from Pfizer. SIF is a scientific cofounder, director, and advisor of MelioLabs, Inc., and has an equity interest in the company. NIAID award number R01AI134982 has been identified for conflict of interest management based on the overall scope of the project and its potential benefit to MelioLabs, Inc.; however, the research findings included in this particular publication may not necessarily relate to the interests of MelioLabs, Inc. The terms of this arrangement have been reviewed and approved by the University of California, San Diego, in accordance with its conflict of interest policies. MH received research funding from Gilead, Astellas, MSD, IMMY, Mundipharma, Scynexis, F2G and Pfizer – all outside of the submitted work. GRT received research and consulting fees from Astellas, Amplyx, Cidara, F2G, Mayne, Melinta, Mundipharma, Scynexis, and served on the DRC for Pfizer – all outside of the submitted work. All other authors declare no conflict of interest.Reviewer disclosuresPeer reviewers on this manuscript have no relevant financial or other relationships to disclose.Author contributionsJDJ, MH, and GRT conceived and designed the study. JDJ, PLW, SEK, TG, SF, MH, and GRT wrote the initial draft. JDJ, PLW, SEK, TG, SF, MH, and GRT provided critical comments. All authors read and approved the final manuscript.Additional informationFundingThis work was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health [award number R01AI134982] to SIF.