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.

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.

Rapid molecular identification of pathogenic yeasts by pyrosequencing analysis of 35 nucleotides of internal transcribed spacer 2.

Rapid identification of yeast species isolates from clinical samples is particularly important given their innately variable antifungal susceptibility profiles. Here, we have evaluated the utility of pyrosequencing analysis of a portion of the internal transcribed spacer 2 region (ITS2) for identification of pathogenic yeasts. A total of 477 clinical isolates encompassing 43 different fungal species were subjected to pyrosequencing analysis in a strictly blinded study. The molecular identifications produced by pyrosequencing were compared with those obtained using conventional biochemical tests (AUXACOLOR2) and following PCR amplification and sequencing of the D1-D2 portion of the nuclear 28S large rRNA gene. More than 98% (469/477) of isolates encompassing 40 of the 43 fungal species tested were correctly identified by pyrosequencing of only 35 bp of ITS2. Moreover, BLAST searches of the public synchronized databases with the ITS2 pyrosequencing signature sequences revealed that there was only minimal sequence redundancy in the ITS2 under analysis. In all cases, the pyrosequencing signature sequences were unique to the yeast species (or species complex) under investigation. Finally, when pyrosequencing was combined with the Whatman FTA paper technology for the rapid extraction of fungal genomic DNA, molecular identification could be accomplished within 6 h from the time of starting from pure cultures.

An improved protocol for the preparation of total genomic DNA from isolates of yeast and mould using Whatman FTA filter papers.

Here, we present a significantly improved version of our previously published method for the extraction of fungal genomic DNA from pure cultures using Whatman FTA filter paper matrix technology. This modified protocol is extremely rapid, significantly more cost effective than our original method, and importantly, substantially reduces the problem of potential cross-contamination between sequential filters when employing FTA technology.

Exophiala campbellii causing a subcutaneous palmar cyst in an otherwise healthy UK resident.

Exophiala is a ubiquitous genus encompassing more than forty species, a number of which have been associated with superficial or systemic infections in humans, and other hot- or cold-blooded animals. Here we report a human case of subcutaneous mycotic cyst caused by Exophiala campbellii. To our knowledge, this is only the third reported human infection caused by E. campbellii, all three of which involved subcutaneous nodules in patients who had resided in the United Kingdom.

Pyrosequencing analysis of 20 nucleotides of internal transcribed spacer 2 discriminates Candida parapsilosis, Candida metapsilosis, and Candida orthopsilosis.

Two new cryptic sister species, Candida orthopsilosis and Candida metapsilosis, were recently identified by consistent DNA sequence differences among several genes within the genetically heterogeneous Candida parapsilosis complex. Here, we present data demonstrating that Pyrosequencing analysis of 20 nucleotides of internal transcribed spacer region 2 rapidly and robustly distinguishes between these three closely related Candida species.

Adiaspiromycosis due to Emmonsia crescens is widespread in native British mammals.

Adiaspiromycosis caused by Emmonsia crescens is primarily a respiratory disease affecting small mammals, especially members of the Families Rodentia, Carnivora and Mustelidae. Although isolated reports exist of adiaspiromycosis in free-living British wildlife, the extent of infection in wild animals in the UK, and the significance of any associated pathology are unclear. Here, we report the results of histopathological examination of lungs of free-living wild mammals from the south-west UK coupled with digestion of lung material in potassium hydroxide followed by centrifugation and microscopic examination for the presence of adiaspores. The combined results showed that almost one-third (27/94, 28.7%) of animals examined had evidence of infection with E. crescens. Attempts to culture E. crescens from infected lungs were unsuccessful. However, E. crescens could be confirmed as the causative agent by PCR amplification and sequencing of DNA from adiaspores micro-dissected from animal lungs. The prevalence of adiaspiromycosis was largely independent of animal species or precise geography. Adiaspore burdens in most animals were low, consistent with transient exposure to E. crescens. However, burdens in several animals suggested heavy or repeated exposures to E. crescens, and were considered sufficient to have significantly impaired respiratory function. Finally, since E. crescens is apparently widespread in UK mammals and the first UK human case of adiaspiromycosis was reported recently, we present data obtained using a previous isolate of E. crescens demonstrating that both the mycelial and adiaspore phases of the organism are susceptible to amphotericin B, voriconazole, itraconazole and caspofungin.

Candida nivariensis, an emerging pathogenic fungus with multidrug resistance to antifungal agents.

In 2005, Candida nivariensis, a yeast species genetically related to Candida glabrata, was described following its isolation from three patients in a single Spanish hospital. Between 2005 and 2006, 16 fungal isolates with phenotypic similarities to C. nivariensis were submitted to the United Kingdom Mycology Reference Laboratory for identification. The strains originated from various clinical specimens, including deep, usually sterile sites, from patients at 12 different hospitals in the United Kingdom. PCR amplification and sequencing of the D1D2 and internal transcribed spacer 1 (ITS1) regions of the nuclear ribosomal gene cassette confirmed that these isolates from the United Kingdom are genetically identical to C. nivariensis. Biochemically, C. glabrata and C. nivariensis are distinguished by their differential abilities to assimilate trehalose. However, in contrast to the original published findings, we found that C. glabrata isolates, but not C. nivariensis isolates, are capable of assimilating this substrate. Antifungal susceptibility tests revealed that C. nivariensis isolates are less susceptible than C. glabrata isolates to itraconazole, fluconazole, and voriconazole and to have significantly higher flucytosine MICs than C. glabrata strains. Finally, C. nivariensis could be rapidly distinguished from the other common pathogenic fungus species by pyrosequencing of the ITS2 region. In the light of these data, we believe that C. nivariensis should be regarded as a clinically important emerging pathogenic fungus.

Molecular identification of pathogenic fungi.

Systemic fungal infections represent a major cause of morbidity and mortality in immunocompromised patients. The ever-increasing number of yeast species associated with human infections that are not covered by conventional identification kits, and the fact that moulds isolated from deep infections are frequently impossible to identify using classical methods due to lack of sporulation, has driven the need for rapid, robust molecular identification techniques. We recently developed a rapid method of preparing fungal genomic DNAs using Whatman FTA filters, which has greatly facilitated molecular identification. Mould isolates cultured from dark grain mycetomas (destructive infections of skin/subcutaneous tissues that progress to involve muscle and bone) invariably fail to produce features by which they can be identified and were taxonomic mysteries. PCR amplification and sequencing of 250 bp of the internal transcribed spacer region 1 (ITS1) allowed us to distinguish between the known agents of mycetoma, to describe three new species associated with this disease and to define phylogenetic relationships. For yeasts, 153 isolates encompassing 47 species that had failed to be identified using classical methods were unambiguously identified by conventional sequencing of 350 bp of the 26S rRNA D1D2 region. These represented 5% of the isolates examined and included common species with atypical biochemical and phenotypic profiles, and rarer species infrequently associated with infection. Our recent studies indicate that FTA extraction coupled with pyrosequencing of 25 bp of ITS2 could potentially identify most common yeast species from pure culture in half a day. Together, these data underscore the importance of molecular techniques for fungal identification.

The evolution and evaluation of a whole blood polymerase chain reaction assay for the detection of invasive aspergillosis in hematology patients in a routine clinical setting.

BACKGROUND: Invasive aspergillosis (IA) is associated with high mortality. Successful outcome with treatment is linked to early diagnosis. The utility of classic diagnostic methods, however, is limited. METHODS: To aid in the diagnosis of IA, we retrospectively assessed our diagnostic service, using real-time polymerase chain reaction (PCR) and galactomannan sandwich enzyme-linked immunosorbent assay (ELISA). RESULTS: A total of 203 patients at risk of invasive fungal infection were screened by PCR, and 116 of the patients were also tested by ELISA. The patient group comprised 176 patients with hematological malignancy and 28 control patients with evidence of invasive candidal infection. Consensus European Organisation for Research and Treatment of Cancer and Mycoses Study Group criteria were used to classify fungal infection, which, by definition, excluded the PCR result. The PCR method was sensitive (up to 92.3% sensitivity) and specific (up to 94.6% specificity) and had good agreement with the galactomannan ELISA (76.7%) and high-resolution computed tomography scan results. CONCLUSIONS: A negative PCR result can be used to rule out IA and to limit the need for empirical antifungal therapy; thus, it has a role in diagnosing IA infections, especially in combination with antigen testing. PCR-positive cases classified as "false positives" regularly reflect the limitations of classic microbiological procedures or restricted use of consensus clinical methods employed to classify infection.

A consensus on fungal polymerase chain reaction diagnosis?: a United Kingdom-Ireland evaluation of polymerase chain reaction methods for detection of systemic fungal infections.

The limitations of classical diagnostic methods for invasive fungal infections (IFIs) have led to the development of molecular techniques to aid in the detection of IFIs. Despite good published performance, interlaboratory reproduction of these assays is variable, and no consensus has been reached for an optimal method. This publication describes the first multicenter study of polymerase chain reaction methods, for the detection of Aspergillus and Candida species, currently used in the UK and Ireland by distribution and analysis of multiple specimen control panels. All three Candida methods were comparable, achieving a satisfactory level of detection (10 cfu), and the method of preference was dependent on the requirements of the particular laboratory. The results for the five Aspergillus assays were more variable, but two methods (2Asp and 4Asp) were superior (10(1) conidia). Formally, the overall performances of the two Aspergillus assays were comparable (kappa statistic = 0.77). However, on the Roche LightCycler, there was a clear sample-type effect that greatly reduced the detection limit of the 4Asp method when testing whole blood samples. Therefore, the preferred Aspergillus method relied on the amplification platform available to the user. This study represents the initial process to achieve a consensus method for the diagnosis of IFIs.

Molecular identification of unusual pathogenic yeast isolates by large ribosomal subunit gene sequencing: 2 years of experience at the United kingdom mycology reference laboratory.

Rapid identification of yeast isolates from clinical samples is particularly important given their innately variable antifungal susceptibility profiles. We present here an analysis of the utility of PCR amplification and sequence analysis of the hypervariable D1/D2 region of the 26S rRNA gene for the identification of yeast species submitted to the United Kingdom Mycology Reference Laboratory over a 2-year period. A total of 3,033 clinical isolates were received from 2004 to 2006 encompassing 50 different yeast species. While more than 90% of the isolates, corresponding to the most common Candida species, could be identified by using the AUXACOLOR2 yeast identification kit, 153 isolates (5%), comprised of 47 species, could not be identified by using this system and were subjected to molecular identification via 26S rRNA gene sequencing. These isolates included some common species that exhibited atypical biochemical and phenotypic profiles and also many rarer yeast species that are infrequently encountered in the clinical setting. All 47 species requiring molecular identification were unambiguously identified on the basis of D1/D2 sequences, and the molecular identities correlated well with the observed biochemical profiles of the various organisms. Together, our data underscore the utility of molecular techniques as a reference adjunct to conventional methods of yeast identification. Further, we show that PCR amplification and sequencing of the D1/D2 region reliably identifies more than 45 species of clinically significant yeasts and can also potentially identify new pathogenic yeast species.

Ultra-rapid preparation of total genomic DNA from isolates of yeast and mould using Whatman FTA filter paper technology - a reusable DNA archiving system.

Conventional methods for purifying PCR-grade fungal genomic DNA typically require cell disruption (either physical or enzymatic) coupled with laborious organic extraction and precipitation stages, or expensive column-based technologies. Here we present an easy and extremely rapid method of preparing yeast and mould genomic DNAs from living cultures using Whatman FTA filter matrix technology. Aqueous suspensions of yeast cells or hyphal fragments and conidia (in the case of moulds) are applied directly (or after freeze-thawing) to dry FTA filters. Inoculated filters are then subjected to brief microwave treatment, to dry the filters and inactivate the organisms. Filter punches are removed, washed rapidly, dried and placed directly into PCR reactions. We show that this procedure inactivated all of the 38 yeast and 75 mould species tested, and generated PCR-grade DNA preparations in around 15 minutes. A total of 218 out of 226 fungal isolates tested liberated amplifiable DNA after application to FTA filters. Detection limits with yeast cultures were approximately 10 colony-forming units per punch. Moreover, we demonstrate that filter punches can be recovered after PCR, washed and used in fresh PCR reactions without detectable cross-contamination. Whatman FTA technology thus represents a cheap, ultra-rapid method of fungal genomic DNA preparation, and also potentially represents a powerful fungal DNA archiving and storage system.

Arthroderma olidum, sp. nov. A new addition to the Trichophyton terrestre complex.

In 1981, four fungal isolates from hair of the European badger (Meles meles) were examined by Dr Phyllis Stockdale at the Commonwealth Mycological Institute, Kew, and deposited in the UK National Collection of Pathogenic Fungi as an undescribed member of the Trichophyton terrestre complex. The present paper formalizes the complete description of a new ascomycete taxon, Arthroderma olidum following successful recent attempts to re-isolate the same fungus from the soil of Badger holes in South West England. Furthermore, using ribosomal RNA gene sequencing, we show that the asexual form of A. olidum is conspecific with the recently described Trichophyton eboreum1 isolated from a human skin specimen in Germany.

Polycytella hominis is a mutated form of Scedosporium apiospermum.

PCR amplification and sequencing of two separate regions of the nuclear ribosomal repeat region revealed that Polycytella hominis, a hyphomycete isolated from a human case of mycetoma, was genetically indistinguishable from Scedosporium apiospermum (the anamorph of Pseudallescheria boydii). These organisms also exhibited remarkably similar susceptibility profiles to common antifungal agents. P. hominis is thus likely to be a mutant of S. apiospermum showing abnormalities of sporulation, for which a possible mechanism is discussed. Polycytella hominis should thus be regarded as a synonym of Scedosporium apiospermum.