Invasive fungal diseases (IFDs) present an increasing global burden in immunocompromised and other seriously ill populations, including those caused by pathogens which are inherently resistant or less susceptible to antifungal drugs. Early diagnosis encompassing accurate detection and identification of the causative agent and of antifungal resistance is critical for optimum patient outcomes. Many molecular-based diagnostic approaches have good clinical utility although interpretation of results should be according to clinical context. Where an IFD is in the differential diagnosis, panfungal PCR assays allow the rapid detection/identification of fungal species directly from clinical specimens with good specificity; sensitivity is also high when hyphae are seen in the specimen including in paraffin-embedded tissue. Aspergillus PCR assays on blood fractions have good utility in the screening of high risk hematology patients with high negative predictive value (NPV) and positive predictive value (PPV) of 94 and 70%, respectively, when two positive PCR results are obtained. The standardization, and commercialization of Aspergillus PCR assays has now enabled direct comparison of results between laboratories with commercial assays also offering the simultaneous detection of common azole resistance mutations. Candida PCR assays are not as well standardized with the only FDA-approved commercial system (T2Candida) detecting only the five most common species; while the T2Candida outperforms blood culture in patients with candidemia, its role in routine Candida diagnostics is not well defined. There is growing use of Mucorales-specific PCR assays to detect selected genera in blood fractions. Quantitative real-time Pneumocystis jirovecii PCRs have replaced microscopy and immunofluorescent stains in many diagnostic laboratories although distinguishing infection may be problematic in non-HIV-infected patients. For species identification of isolates, DNA barcoding with dual loci (ITS and TEF1α) offer optimal accuracy while next generation sequencing (NGS) technologies offer highly discriminatory analysis of genetic diversity including for outbreak investigation and for drug resistance characterization. Advances in molecular technologies will further enhance routine fungal diagnostics.

Pulmonary cryptococcosis describes an invasive lung mycosis caused by Cryptococcus neoformans or Cryptococcus gattii complex. It is often a high-consequence disease in both immunocompromised and immunocompetent populations, and may be misdiagnosed as pulmonary malignancy, leading to a delay in therapy. Epidemiology follows that of cryptococcal meningoencephalitis, with C. gattii infection more common in certain geographic regions. Diagnostic tools include histopathology, microscopy and culture, and the detection of cryptococcal polysaccharide antigen or Cryptococcus-derived nucleic acids. All patients with lung cryptococcosis should have a lumbar puncture and cerebral imaging to exclude central nervous system disease. Radiology is key, both as an adjunct to laboratory testing and as the initial means of detection in asymptomatic patients or those with non-specific symptoms. Pulmonary cryptococcomas (single or multiple) may also be associated with disseminated disease and/or cryptococcal meningitis, requiring prolonged treatment regimens. Optimal management for severe disease requires extended induction (amphotericin B and flucytosine) and consolidation therapy (fluconazole) with close clinical monitoring. Susceptibility testing is of value for epidemiology and in regions where relatively high minimum inhibitory concentrations to azoles (particularly fluconazole) have been noted. Novel diagnostic tools and therapeutic agents promise to improve the detection and treatment of cryptococcosis, particularly in low-income settings where the disease burden is high.

Scedosporium/Lomentospora fungi are increasingly recognized pathogens. As these fungi are resistant to many antifungal agents, early diagnosis is essential for initiating targeted drug therapy. Here, we review the microbiological tools for the detection and diagnosis of invasive scedosporiosis and lomentosporiosis. Of over 10 species, Lomentospora prolificans, Scedosporium apiospermum, S. boydii and S. aurantiacum cause the majority of infections. Definitive diagnosis relies on one or more of visualization, isolation or detection of the fungus from clinical specimens by microscopy techniques, culture and molecular methods such as panfungal PCR or genus-/species-specific multiplex PCR. For isolation from respiratory tract specimens, selective media have shown improved isolation rates. Species identification is achieved by macroscopic and microscopic examination of colonies, but species should be confirmed by ITS with or without β-tubulin gene sequencing or other molecular methods. Matrix-assisted laser desorption ionization-time of flight mass spectrometry databases are improving but may need supplementation by in-house spectra for species identification. Reference broth microdilution methods is preferred for antifungal susceptibility testing. Next-generation sequencing technologies have good potential for characterization of these pathogens. Diagnosis of Scedosporium/Lomentospora infections relies on multiple approaches encompassing both phenotypic- and molecular-based methods.

This systematic review and meta-analysis aimed to examine the performance of polymerase chain reaction (PCR) assays for diagnosing mucormycosis. A standardised search was conducted from conception to December 3rd 2024 using PubMed, Embase, Global Health, and Cochrane library. Original studies that used PCR-based methods on any human specimen to diagnose mucormycosis were analysed for eligibility. Using a bivariate meta-analysis, the diagnostic performance of PCR was examined against the European Organisation for Research and Treatment of Cancer-Mycoses Study Group Education and Research Consortium 2020 (EORTC-MSGERC) definitions of proven and probable invasive mould disease, which was modified to include all patients at risk of mucormycosis. The study protocol was registered on the PROSPERO database (CRD42023478667). Of 4855 articles, a total of 30 met inclusion criteria, including 5920 PCR reactions on 5147 non-duplicate specimens from 819 cases of proven/probable mucormycosis and 4266 patients who did not meet the EORTC-MSGERC 2020 criteria. According to specimen type, sensitivity of PCR varied (p < 0.001) whereas specificity was similar (p = 0.662). Bronchoalveolar lavage fluid offered the highest sensitivity of 97.5% (95% CI 83.7-99.7%), specificity of 95.8% (95% CI 89.6-98.4%), positive likelihood ratio (LR+) of 23.5, and negative likelihood ratio (LR-) of 0.03. Tissue provided sensitivity of 86.4% (95% CI 78.9-91.5%), specificity of 90.6% (95% CI 78.1-96.3%), LR+ of 9.2, and LR- of 0.15. Blood provided reduced sensitivity of 81.6% (95% CI 70.1-89.4%), specificity of 95.5% (95% CI 87.4-98.5%), DOR of 95, LR+ of 18.3, and LR- of 0.19. Formalin-fixed paraffin-embedded specimens yielded the lowest sensitivity of 73.0% (95% CI 61.0-82.3%), highest specificity of 96.4% (CI 95% 87.5-99.0%), LR+ of 20.2, and LR- of 0.28. The covariates best explaining heterogeneity of the overall analysis were specimen type, study design (cohort versus case-control) and disease prevalence while patient population (COVID-19 versus other) and PCR (conventional versus quantitative) had less impact on heterogeneity. This meta-analysis confirms the high performance of PCR for diagnosing mucormycosis and supports the instatement of PCR detection of free-DNA in blood, BALF and tissue into future updated definitions and diagnostic guidelines for mucormycosis. None.

Invasive fungal diseases (IFDs) comprise a growing healthcare burden, especially given the expanding population of immunocompromised hosts. Early diagnosis of IFDs is required to optimise therapy with antifungals, especially in the setting of rising rates of antifungal resistance. Molecular techniques including nucleic acid amplification tests and whole genome sequencing have potential to offer utility in overcoming limitations with traditional phenotypic testing. However, standardisation of methodology and interpretations of these assays is an ongoing undertaking. The utility of targeted Aspergillus detection has been well-defined, with progress in investigations into the role of targeted assays for Candida, Pneumocystis, Cryptococcus, the Mucorales and endemic mycoses. Likewise, whilst broad-range polymerase chain reaction assays have been in use for some time, pathology stewardship and optimising diagnostic yield is a continuing exercise. As costs decrease, there is also now increased access and experience with whole genome sequencing, including metagenomic sequencing, which offers unparalleled resolution especially in the investigations of potential outbreaks. However, their role in routine diagnostic use remains uncommon and standardisation of techniques and workflow are required for wider implementation.

Nosocomial clusters of fungal infections, whilst uncommon, cannot be predicted and are associated with significant morbidity and mortality. Here, we review reports of nosocomial outbreaks of invasive fungal disease to glean insight into their epidemiology, risks for infection, methods employed in outbreak detection including genomic testing to confirm the outbreak, and approaches to clinical and infection control management. Both yeasts and filamentous fungi cause outbreaks, with each having general and specific risks. The early detection and confirmation of the outbreak are essential for diagnosis, treatment of affected patients, and termination of the outbreak. Environmental sampling, including the air in mould outbreaks, for the pathogen may be indicated. The genetic analysis of epidemiologically linked isolates is strongly recommended through a sufficiently discriminatory approach such as whole genome sequencing or a method that is acceptably discriminatory for that pathogen. An analysis of both linked isolates and epidemiologically unrelated strains is required to enable genetic similarity comparisons. The management of the outbreak encompasses input from a multi-disciplinary team with epidemiological investigation and infection control measures, including screening for additional cases, patient cohorting, and strict hygiene and cleaning procedures. Automated methods for fungal infection surveillance would greatly aid earlier outbreak detection and should be a focus of research.