The considerable impact of the SARS-CoV-2 pandemic and COVID-19 on the UK National Mycology Reference Laboratory activities and workload.

Starting late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a devastating global pandemic of coronavirus-19 disease (COVID-19) with ∼179 million cases and ∼3.9 million deaths to date. COVID-19 ranges from asymptomatic infection to severe illness with acute respiratory distress requiring critical care in up to 40% of hospitalized patients. Numerous reports have identified COVID-19-associated pulmonary aspergillosis (CAPA) as an important infective complication of COVID-19. In the UK, the pandemic has had unprecedented impacts on the National Health Service (NHS'): each wave of infections required hospitals to reconfigure for large surges in patients requiring intensive care, to the detriment of most aspects of non-COVID care including planned operations, outpatient appointments, general practitioner consultations and referrals. The UK National Mycology Reference Laboratory (MRL) offers a comprehensive service for the diagnosis and management of fungal disease nationwide, with a test portfolio that includes: diagnosis of allergies to fungal and other respiratory allergens; diagnosis of superficial and invasive/systemic fungal infections using traditional mycological, serological and molecular approaches; identification and susceptibility testing of the causative fungi; therapeutic drug monitoring of patients receiving antifungal therapy. Here, we describe the impact of the first 14 months of the COVID-19 pandemic on MRL activities. Changes to MRL workload closely mirrored many of the NHS-wide challenges, with marked reductions in 'elective' mycological activities unrelated to the pandemic and dramatic surges in tests that contributed to the diagnosis and management of COVID-19-related secondary fungal infections, in particular CAPA and candidemia in COVID-19 patients in intensive care. LAY SUMMARY: The COVID-19 pandemic has had an unprecedented impact on the UK National Health Service, with hospitals forced to repeatedly reconfigure to prepare for large surges in COVID-19 patients. Here we describe the impact of the first 14 months of the UK pandemic on the workload of the National Mycology Reference Laboratory.

COVID-19-Associated Invasive Aspergillosis: Data from the UK National Mycology Reference Laboratory.

COVID-19-associated pulmonary aspergillosis (CAPA) was recently reported as a potential infective complication affecting critically ill patients with acute respiratory distress syndrome following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, with incidence rates varying from 8 to 33% depending on the study. However, definitive diagnosis of CAPA is challenging. Standardized diagnostic algorithms and definitions are lacking, clinicians are reticent to perform aerosol-generating bronchoalveolar lavages for galactomannan testing and microscopic and cultural examination, and questions surround the diagnostic sensitivity of different serum biomarkers. Between 11 March and 14 July 2020, the UK National Mycology Reference Laboratory received 1,267 serum and respiratory samples from 719 critically ill UK patients with COVID-19 and suspected pulmonary aspergillosis. The laboratory also received 46 isolates of Aspergillus fumigatus from COVID-19 patients (including three that exhibited environmental triazole resistance). Diagnostic tests performed included 1,000 (1-3)-ß-d-glucan and 516 galactomannan tests on serum samples. The results of this extensive testing are presented here. For a subset of 61 patients, respiratory specimens (bronchoalveolar lavage specimens, tracheal aspirates, and sputum samples) in addition to serum samples were submitted and subjected to galactomannan testing, Aspergillus-specific PCR, and microscopy and culture. The incidence of probable/proven and possible CAPA in this subset of patients was approximately 5% and 15%, respectively. Overall, our results highlight the challenges in biomarker-driven diagnosis of CAPA, especially when only limited clinical samples are available for testing, and the importance of a multimodal diagnostic approach involving regular and repeat testing of both serum and respiratory samples.

MIC distributions for amphotericin B, fluconazole, itraconazole, voriconazole, flucytosine and anidulafungin and 35 uncommon pathogenic yeast species from the UK determined using the CLSI broth microdilution method.

BACKGROUND: Epidemiological cut-off values and clinical interpretive breakpoints have been developed for a number of antifungal agents with the most common Candida species that account for the majority of infections due to pathogenic yeasts species. However, less-common species, for which susceptibility data are limited, are increasingly reported in high-risk patients and breakthrough infections. METHODS: The UK National Mycology Reference Laboratory performs routine antifungal susceptibility testing of clinical yeast isolates submitted from across the UK. Between 2002 and 2016, >32 000 isolates representing 94 different yeast species were referred to the laboratory. Here we present antifungal susceptibility profiles generated over this period for amphotericin B, fluconazole, voriconazole, itraconazole, anidulafungin and flucytosine against 35 species of uncommon yeast using CLSI methodologies. MIC data were interpreted against epidemiological cut-off values and clinical breakpoints developed with Candida albicans, in order to identify species with unusually skewed MIC distributions that potentially indicate resistance. RESULTS: Potential resistance to at least one antifungal agent (>10% of isolates with MICs greater than the epidemiological cut-off or clinical breakpoint) was evidenced for 29/35 species examined here. Four species exhibited elevated MICs with all of the triazole antifungal drugs against which they were tested, and 21 species exhibited antifungal resistance to agents from at least two different classes of antifungal agent. CONCLUSIONS: This study highlights a number of yeast species with unusual MIC distributions and provides data to aid clinicians in deciding which antifungal regimens may be appropriate when confronted with infections with rarer yeasts.

Fluconazole Resistance in Isolates of Uncommon Pathogenic Yeast Species from the United Kingdom.

The triazole drug fluconazole remains one of the most commonly prescribed antifungal drugs, both for prophylaxis in high-risk patients and also as a second-line treatment option for invasive Candida infections. Established susceptibility profiles and clinical interpretive breakpoints are available for fluconazole with Candida albicans, Candida glabrata, Candida tropicalis, and Candida parapsilosis, which account for the majority of infections due to pathogenic yeast species. However, less common species for which only limited susceptibility data are available are increasingly reported in high-risk patients and from breakthrough infections. The UK National Mycology Reference Laboratory performs routine antifungal susceptibility testing of clinical isolates of pathogenic yeast submitted from across the United Kingdom. Between 2002 and 2016, ∼32,000 isolates were referred, encompassing 94 different yeast species. Here, we present fluconazole antifungal susceptibility data generated using a CLSI methodology over this 15-year period for 82 species (2,004 isolates) of less common yeast and yeast-like fungi, and amphotericin B, fluconazole, itraconazole, voriconazole, posaconazole, and anidulafungin, with members of the Nakaseomyces clade (C. glabrata, Candida nivariensis, and Candida bracarensis). At least 22 different teleomorph genera, comprising 45 species, exhibited high MICs when tested with fluconazole (>20% of isolates with MICs higher than the clinical breakpoint [≥8 mg/liter] proposed for C. albicans). Since several of these species have been reported anecdotally from breakthrough infections and therapeutic failures in patients receiving fluconazole, the current study underscores the importance of rapid and accurate yeast identification and may aid clinicians dealing with infections with rarer yeasts to decide whether fluconazole would be appropriate.

Fungal Infections of Implantation: More Than Five Years of Cases of Subcutaneous Fungal Infections Seen at the UK Mycology Reference Laboratory.

Subcutaneous fungal infections, which typically result from traumatic introduction (implantation) of fungal elements into the skin or underlying tissues, can present as a range of different clinical entities including phaeohyphomycosis, chromoblastomycosis, subcutaneous nodules or masses, and genuine eumycetoma. Here, we mined our laboratory information management system for such infections in humans and domestic animals for the period 2016-2022, including (i) fungal isolates referred for identification and/or susceptibility testing; (ii) infections diagnosed at our laboratory using panfungal PCR approaches on infected tissue; and (iii) organisms cultured in our laboratory from biopsies. In total, 106 cases were retrieved, involving 39 fungal species comprising 26 distinct genera. Subcutaneous infections with Alternaria species were the most frequent (36 cases), which possibly reflects the ubiquitous nature of this common plant pathogen. A substantial proportion of Alternaria spp. isolates exhibited reduced in vitro susceptibility to voriconazole. Notably, a significant number of subcutaneous infections were diagnosed in renal and other solid organ transplant recipients post transplantation, suggesting that humans may harbour "inert" subcutaneous fungal elements from historical minor injuries that present as clinical infections upon later immunosuppression. The current study underscores the diversity of fungi that can cause subcutaneous infections. While most organisms catalogued here were responsible for occasional infections, several genera (Alternaria, Exophiala, Phaeoacremonuim, Scedosporium) were more frequently recovered in our searches, suggesting that they possess virulence factors that facilitate subcutaneous infections and/or inhabit natural niches that make them more likely to be traumatically inoculated.

In Vitro Antifungal Drug Resistance Profiles of Clinically Relevant Members of the Mucorales (Mucoromycota) Especially with the Newer Triazoles.

Mucoromycoses (infections caused by members of the order Mucorales, phylum Mucoromycota [ex-Zygomycota]) are highly destructive, rapidly progressive infections, with dire prognoses especially when they occur in immunocompromised hosts. Current treatment guidelines recommend liposomal formulations of amphotericin B with adjunctive surgery as first line therapy, with the newer triazoles posaconazole or isavuconazole as alternative treatments, or as salvage therapy. Among the many organisms belonging to this order, a limited number of species in the genera Rhizopus, Mucor, Lichtheimia and Rhizomucor are responsible for most cases of human infection. Here, we present the minimum inhibitory concentration data (MICs) for amphotericin B, posaconazole, isavuconazole, itraconazole and voriconazole with a panel of over 300 isolates of the five most common agents of human infection (Lichtheimia corymbifera, Rhizopus arrhizus, R. microsporus, Rhizomucor pusillus and Mucor spp.) determined using the CLSI broth microdilution method. In agreement with previous studies, the most active antifungal drug for all Mucorales was amphotericin B, with MICs within the range that would predict susceptibility with Aspergillus fumigatus. Conversely, MICs for voriconazole against all species tested were high, and above the range associated with clinical efficacy with A. fumigatus. Interestingly, whilst isavuconazole and posaconazole MIC distributions indicated in vitro activity against some members of the Mucorales, activity was species-dependent for both agents. These data underscore the importance of accurate identification of the causative agents of mucoromycosis, coupled with antifungal susceptibility testing of individual isolates, in determining the optimal treatment of infections caused by these aggressive opportunistic human fungal pathogens.

MIC Distributions and Evaluation of Fungicidal Activity for Amphotericin B, Itraconazole, Voriconazole, Posaconazole and Caspofungin and 20 Species of Pathogenic Filamentous Fungi Determined Using the CLSI Broth Microdilution Method.

For filamentous fungi (moulds), species-specific interpretive breakpoints and epidemiological cut-off values (ECVs) have only been proposed for a limited number of fungal species-antifungal agent combinations, with the result that clinical breakpoints are lacking for most emerging mould pathogens. In the current study, we have compiled minimum inhibitory concentration (MIC) data for 4869 clinical mould isolates and present full MIC distributions for amphotericin B, itraconazole, voriconazole, posaconazole, and caspofungin with these isolates which comprise 20 species/genera. In addition, we present the results of an assessment of the fungicidal activity of these same five antifungal agents against a panel of 123 mould isolates comprising 16 of the same species.