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BACKGROUND: New antifungal agents are required to mitigate against azole-resistant Aspergillus and drug-resistant non-Aspergillus moulds. The novel orotomide, olorofim (F2G, Manchester, UK), has potent fungicidal activity against Aspergillus including azole-resistant Aspergillus fumigatus, Lomentospora prolificans and Scedosporium spp. Development of olorofim-specific clinical breakpoints/epidemiological cut-off values requires reliable MIC data. OBJECTIVES: Determine the in vitro activity of olorofim compared with standard antifungals against mould pathogens at an Australian hospital. MATERIALS AND METHODS: Olorofim MICs were determined for 507 clinical mould isolates using the CLSI M38-A3 standard. MICs of amphotericin B, anidulafungin, posaconazole, voriconazole and isavuconazole were obtained using Sensititre™ YeastOne YO10 and AUSNMRCI panels (Thermo-Fisher Scientific). RESULTS: A. fumigatus sensu stricto was the commonest species (33.3%) followed by L. prolificans (18.3%), Scedosporium (11.4%) and Fusarium (6%) species. Olorofim modal MICs were ≤0.25 mg/L (MIC90 0.25 mg/L) for all Aspergillus except Aspergillus Section Usti (1 mg/L); MICs for nine azole-resistant/non-wild-type A. fumigatus ranged from 0.008 to 0.125 mg/L. The MIC90 of olorofim for L. prolificans was 0.5 mg/L, 0.25-0.5 mg/L for Scedosporium spp. and 8 mg/L for the F. solani complex but with modal MICs of 0.25 and 0.008 mg/L for F. oxysporum and F. proliferatum complexes, respectively. For Verruconis gallopava (n = 10), the olorofim MIC90 was 0.06 mg/L (voriconazole MIC90 2 mg/L, isavuconazole MICs of 4->8 mg/L). Olorofim had little activity against other dematiaceous moulds including Exophiala species. CONCLUSIONS: Olorofim was highly active against Aspergillus spp. including azole-resistant A. fumigatus, L. prolificans, Scedosporium spp. and some Fusarium species with the new finding of potent activity against V. gallopava.

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Past analysis of laboratory methods used for mycology specimens revealed significant variation in practices, many of which fell short of recommended procedures. In 2016 these findings led to a set of recommendations for laboratories to consider modification of their methods where appropriate, to analyse current laboratory methods used by participants in the Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP) Mycology module, and to compare these to the 2016 recommendations. Seven test items, with 105-107 participants each, were analysed. Several laboratories (7-12%) did not handle specimens as recommended in an appropriate biological safety cabinet. Direct microscopy was not performed on tissue specimens 23-25% of the time. The most used staining method was potassium hydroxide with an optical brightener for fluorescent microscopy (49%) followed by Gram stain (33%). While 17-25% of laboratories used three or more media, use of four or more was uncommon (<3%). Between 9-13% of participants used only a single non-inhibitory medium for cultures. Urine specimens were incubated longer than recommended with 57% of laboratories incubating for >7days and 24% >21 days. Duration of incubation was shorter than recommended for several specimen types with 36% of skin specimens and 37-48% of tissue specimens being kept ≤21 days. For cultures kept >7 days, 13% were inspected daily but for those incubating >14 days only 3%. The methods of several laboratories remain outside recommended practice. An updated set of recommendations are made.

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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.

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Histoplasmosis, a significant mycosis primarily prevalent in Africa, North and South America, with emerging reports globally, poses notable health challenges, particularly in immunocompromised individuals such as people living with HIV/AIDS and organ transplant recipients. This systematic review, aimed at informing the World Health Organization's Fungal Priority Pathogens List, critically examines literature from 2011 to 2021 using PubMed and Web of Science, focusing on the incidence, mortality, morbidity, antifungal resistance, preventability, and distribution of Histoplasma. We also found a high prevalence (22%-44%) in people living with HIV, with mortality rates ranging from 21% to 53%. Despite limited data, the prevalence of histoplasmosis seems stable, with lower estimates in Europe. Complications such as central nervous system disease, pulmonary issues, and lymphoedema due to granuloma or sclerosis are noted, though their burden remains uncertain. Antifungal susceptibility varies, particularly against fluconazole (MIC: ≥32 mg/l) and caspofungin (MICs: 4-32 mg/l), while resistance to amphotericin B (MIC: 0.125-0.16 mg/l), itraconazole (MICs: 0.004-0.125 mg/l), and voriconazole (MICs: 0.004-0.125 mg/l) remains low. This review identifies critical knowledge gaps, underlining the need for robust, globally representative surveillance systems to better understand and combat this fungal threat.

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Recognizing the growing global burden of fungal infections, the World Health Organization established a process to develop a priority list of fungal pathogens (FPPL). In this systematic review, we aimed to evaluate the epidemiology and impact of infections caused by Fusarium spp., Scedosporium spp., and Lomentospora prolificans to inform the first FPPL. PubMed and Web of Sciences databases were searched to identify studies published between January 1, 2011 and February 23, 2021, reporting on mortality, complications and sequelae, antifungal susceptibility, preventability, annual incidence, and trends. Overall, 20, 11, and 9 articles were included for Fusarium spp., Scedosporium spp., and L. prolificans, respectively. Mortality rates were high in those with invasive fusariosis, scedosporiosis, and lomentosporiosis (42.9%-66.7%, 42.4%-46.9%, and 50.0%-71.4%, respectively). Antifungal susceptibility data, based on small isolate numbers, showed high minimum inhibitory concentrations (MIC)/minimum effective concentrations for most currently available antifungal agents. The median/mode MIC for itraconazole and isavuconazole were ≥16 mg/l for all three pathogens. Based on limited data, these fungi are emerging. Invasive fusariosis increased from 0.08 cases/100 000 admissions to 0.22 cases/100 000 admissions over the time periods of 2000-2009 and 2010-2015, respectively, and in lung transplant recipients, Scedosporium spp. and L. prolificans were only detected from 2014 onwards. Global surveillance to better delineate antifungal susceptibility, risk factors, sequelae, and outcomes is required.

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SUMMARYAlthough Scedosporium species and Lomentospora prolificans are uncommon causes of invasive fungal diseases (IFDs), these infections are associated with high mortality and are costly to treat with a limited armamentarium of antifungal drugs. In light of recent advances, including in the area of new antifungals, the present review provides a timely and updated overview of these IFDs, with a focus on the taxonomy, clinical epidemiology, pathogenesis and host immune response, disease manifestations, diagnosis, antifungal susceptibility, and treatment. An expansion of hosts at risk for these difficult-to-treat infections has emerged over the last two decades given the increased use of, and broader population treated with, immunomodulatory and targeted molecular agents as well as wider adoption of antifungal prophylaxis. Clinical presentations differ not only between genera but also across the different Scedosporium species. L. prolificans is intrinsically resistant to most currently available antifungal agents, and the prognosis of immunocompromised patients with lomentosporiosis is poor. Development of, and improved access to, diagnostic modalities for early detection of these rare mold infections is paramount for timely targeted antifungal therapy and surgery if indicated. New antifungal agents (e.g., olorofim, fosmanogepix) with novel mechanisms of action and less cross-resistance to existing classes, availability of formulations for oral administration, and fewer drug-drug interactions are now in late-stage clinical trials, and soon, could extend options to treat scedosporiosis/lomentosporiosis. Much work remains to increase our understanding of these infections, especially in the pediatric setting. Knowledge gaps for future research are highlighted in the review.

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Rose bengal (RB) solutions coupled with a green laser have proven to be efficient in clearing resilient nail infections caused by Trichophyton rubrum in a human pilot study and in extensive in vitro experiments. Nonetheless, the RB solution can become diluted or dispersed over the tissue and prevented from penetrating the nail plate to reach the subungual area where fungal infection proliferates. Nanoparticles carrying RB can mitigate the problem of dilution and are reported to effectively penetrate through the nail. For this reason, we have synthesized RB-encapsulated chitosan nanoparticles with a peak distribution size of ~200 nm and high reactive oxygen species (ROS) production. The RB-encapsulated chitosan nanoparticles aPDT were shown to kill more than 99% of T. rubrum, T. mentagrophytes, and T. interdigitale spores, which are the common clinically relevant pathogens in onychomycosis. These nanoparticles are not cytotoxic against human fibroblasts, which promotes their safe application in clinical translation.

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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.

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BACKGROUND: Global emergence of rapidly developing resistance to multiple antifungal drugs and high mortality pose challenges to the treatment of invasive Candida auris infections. New therapeutic approaches are needed, such as repurposing drugs including combination with antifungals. Statins have been reported to exert antifungal effects against various Candida species. OBJECTIVES: Our study investigated potential synergy between the statins (rosuvastatin and fluvastatin) and azoles (voriconazole, posaconazole and isavuconazole) on clinical isolates of C. auris. METHODS: Twenty-one clinical isolates of C. auris were obtained. Chequerboard assays based on the CLSI broth microdilution method were used to assess synergy based on FIC index (FICI) calculations of MICs of individual drugs and in combinations. RESULTS: Single drug geometric mean (GM) MICs of fluvastatin and rosuvastatin were ≥128 mg/L in all 21 isolates. GM (range) MICs of posaconazole, voriconazole and isavuconazole were 0.259 (0.016-1 mg/L), 0.469 (0.016-2 mg/L) and 0.085 (0.004-1 mg/L), respectively. Combination of azoles with fluvastatin showed synergy in 70%-90% of C. auris isolates. In particular, voriconazole/fluvastatin resulted in 16-fold reduction in voriconazole MIC and synergy in 14/21 (67%) isolates. Posaconazole/fluvastatin resulted in 8-fold reduction in posaconazole MIC and synergy in 19/21 (90%) isolates.Combining rosuvastatin with the azoles also showed synergy against C. auris in 40%-60% of the isolates and additive effect in 40%-50%. None of the combinations was antagonistic. CONCLUSIONS: Our results provide a rationale for pursuing in vivo synergy tests as well as clinical studies to explore tolerability, treatment outcomes, optimal dose and exposure targets.

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Background: New and emerging risks for invasive aspergillosis (IA) bring the need for contemporary analyses of the epidemiology and outcomes of IA, in order to improve clinical practice. Methods: The study was a retrospective, multicenter, cohort design of proven and probable IA in adults from 10 Australasian tertiary centres (January 2017-December 2020). Descriptive analyses were used to report patients' demographics, predisposing factors, mycological characteristics, diagnosis and management. Accelerated failure-time model was employed to determine factor(s) associated with 90-day all-cause mortality (ACM). Findings: Of 382 IA episodes, 221 (in 221 patients) fulfilled inclusion criteria - 53 proven and 168 probable IA. Median patient age was 61 years (IQR 51-69). Patients with haematologic malignancies (HM) comprised 49.8% of cases. Fifteen patients (6.8%) had no pre-specified immunosuppression and eleven patients (5.0%) had no documented comorbidity. Only 30% of patients had neutropenia. Of 170 isolates identified, 40 (23.5%) were identified as non-Aspergillus fumigatus species complex. Azole-resistance was present in 3/46 (6.5%) of A. fumigatus sensu stricto isolates. Ninety-day ACM was 30.3%. HM (HR 1.90; 95% CI 1.04-3.46, p = 0.036) and ICU admission (HR 4.89; 95% CI 2.93-8.17, p < 0.001) but not neutropenia (HR 1.45; 95% CI 0.88-2.39, p = 0.135) were associated with mortality. Chronic kidney disease was also a significant predictor of death in the HM subgroup (HR 3.94; 95% CI 1.15-13.44, p = 0.028). Interpretation: IA is identified in high number of patients with mild/no immunosuppression in our study. The relatively high proportion of non-A. fumigatus species complex isolates and 6.5% azole-resistance rate amongst A. fumigatus sensu stricto necessitates accurate species identification and susceptibility testing for optimal patient outcomes. Funding: This work is unfunded. All authors' financial disclosures are listed in detail at the end of the manuscript.

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Performance of panfungal PCR-DNA sequencing assays for diagnosis of invasive fungal disease on formalin-fixed, paraffin-embedded tissue (FFPE) is influenced by many variables. Interpretation of a positive result can be challenging due to the need to differentiate colonisers and contaminants from clinically significant pathogens. We conducted a retrospective audit on FFPE tissue specimens that underwent panfungal PCR from January 2021 to August 2022. Panfungal PCR results from samples where fungal elements were visualised on histopathology were compared with results from samples where no fungal elements were visualised. The cost per clinically significant positive sample in each group was calculated. Of the 248 FFPE tissues sampled, 18.1% (45/248) had fungal forms seen on histopathology. Panfungal PCR was positive in 22/45 samples (48.9%), with 16 (35.6%) results deemed clinically significant. For the remaining 203 specimens, panfungal PCR was positive in 19 (9.4%) samples with only six (3.0%) clinically significant. The average cost per clinically significant result was AUD 258.13 in the histopathology positive group and AUD 3,105.22 in the histopathology negative group. Our data suggest panfungal PCR has limited clinical utility in FFPE tissue when no fungal elements are seen. Restricting the assay to only those samples that are positive on histopathological examination aids interpretation of PCR positive results and conserves laboratory resources.

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Mucormycosis is an uncommon, yet deadly invasive fungal infection caused by the Mucorales moulds. These pathogens are a WHO-assigned high-priority pathogen group, as mucormycosis incidence is increasing, and there is unacceptably high mortality with current antifungal therapies. Current diagnostic methods have inadequate sensitivity and specificity and may have issues with accessibility or turnaround time. Patients with diabetes mellitus and immune compromise are predisposed to infection with these environmental fungi, but COVID-19 has established itself as a new risk factor. Mucorales also cause healthcare-associated outbreaks, and clusters associated with natural disasters have also been identified. Robust epidemiological surveillance into burden of disease, at-risk populations, and emerging pathogens is required. Emerging serological and molecular techniques may offer a faster route to diagnosis, while newly developed antifungal agents show promise in preliminary studies. Equitable access to these emerging diagnostic techniques and antifungal therapies will be key in identifying and treating mucormycosis, as delayed initiation of therapy is associated with higher mortality.

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Fungal infections are a recognized complication of immunosuppression in solid organ transplant recipients. Phaeohyphomycoses are fungal infections caused by a diverse group of dematiaceous fungi. Methods: We share the learning points from 2 Australian cases of phaeohyphomycosis secondary to Phaeacreomonium species (spp). A literature review was performed using Medline, Embase, and Google Scholar to identify this condition among kidney transplant recipients. Results: With the 2 cases reported in this article, a total of 17 cases were identified in the literature. Phaeacremonium spp is ubiquitous in humid and temperate flora, including Australia. Minor trauma is likely the source of inoculation in most cases and diagnosis is often delayed. Presently, no guidelines for management exist given the rarity of this condition. Most known cases have been treated with surgical debulking combined with long-course antifungal therapy. Conclusion: This paper describes 2 Australian cases of phaeohyphomycosis in kidney transplant recipients. A high index of suspicion, especially in the immunosuppressed, is essential for timely diagnosis in kidney transplant recipients. There are several diagnostic and therapeutic challenges that remain with this condition.

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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.

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Resistance to azoles in Candida tropicalis is increasing and may be mediated by genetic characteristics. Using whole genome sequencing (WGS), we examined the genetic diversity of 82 bloodstream C. tropicalis isolates from two countries and one ATCC strain in a global context. Multilocus sequence typing (MLST) and single nucleotide polymorphism (SNP)-based phylogenies were generated. Minimum inhibitory concentrations (MIC) for antifungal agents were determined using Sensititre YeastOne YO10. Eleven (13.2%) isolates were fluconazole-resistant and 17 (20.5%) were classified as fluconazole-non susceptible (FNS). Together with four Canadian isolates, the genomes of 12 fluconazole-resistant (18 FNS) and 69 fluconazole-susceptible strains were examined for gene mutations associated with drug resistance. Fluconazole-resistant isolates contained a mean of 56 non-synonymous SNPs per isolate in contrast to 36 SNPs in fluconazole-susceptible isolates (interquartile range [IQR] 46−59 vs. 31−48 respectively; p < 0.001). Ten of 18 FNS isolates contained missense ERG11 mutations (amino acid substitutions S154F, Y132F, Y257H). Two echinocandin-non susceptible isolates had homozygous FKS1 mutations (S30P). MLST identified high genetic diversity with 61 diploid sequence types (DSTs), including 53 new DSTs. All four isolates in DST 773 were fluconazole-resistant within clonal complex 2. WGS showed high genetic variation in invasive C. tropicalis; azole resistance was distributed across different lineages but with DST 773 associated with in vitro fluconazole resistance.

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This study aimed to validate the performance of the custom formulated Sensititre YeastOne One (SYO) microdilution plate which includes isavuconazole (AUSNMRC1) to perform susceptibility testing on clinically relevant yeast and mould species across three Australian reference laboratories. The minimum inhibitory concentration (MIC) results were compared with the IVD approved SYO YO10 microdilution plate and isavuconazole gradient strips. A total of 127 isolates were tested on both the YO10 and AUSNMRC1 plates. The overall essential agreement (EA) and categorical agreement (CA) for the eight common drugs was 99.9% and 98.8%, respectively. The EA was 96.9% for the isavuconazole MICs obtained using the AUSNMRC1 plate and gradient strip. The MIC results for all nine antifungals on the AUSNMRC1 panel were highly reproducible for all quality control and reference strains and the overall EA and CA for 45 clinical strains tested across all three participating laboratories were >93% and 94.1%, respectively. These findings demonstrate the SYO AUSNMRC1 plate provides a commercial means to determine isavuconazole MICs by broth microdilution testing.

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Cryptococcus neoformans and Cryptococcus gattii are the principle causative agents of cryptococcosis. Differences in epidemiological and clinical features, and also treatment, mean it is important for diagnostic laboratories to distinguish between the two species. Molecular methods are potentially more rapid than culture and cryptococcal antigen (CRAG) detection; however, commercial PCR-based assays that target Cryptococcus do not distinguish between species. Here, we developed a real-time PCR assay targeting the multicopy mitochondrial cytochrome b (cyt b) gene to detect C. neoformans and C. gattii in clinical specimens. Assay performance was compared with culture, histopathology, CRAG and panfungal PCR/DNA sequencing. The cyt b-directed assay accurately detected and identified all eight C. neoformans/gattii genotypes. High-resolution melt curve analysis unambiguously discriminated between the two species. Overall, assay sensitivity (96.4%) compared favorably with panfungal PCR (76.9%) and culture (14.5%); assay specificity was 100%. Of 25 fresh frozen paraffin embedded (FFPE) specimens, assay sensitivity was 96% (76% for panfungal PCR; 68% for histopathology). The Cryptococcus-specific PCR is a rapid (~4 h) sensitive method to diagnose (or exclude) cryptococcosis and differentiate between the two major species. It is suitable for use on diverse clinical specimens and may be the preferred molecular method for FFPE specimens where clinical suspicion of cryptococcosis is high.