Population genomics confirms acquisition of drug-resistant Aspergillus fumigatus infection by humans from the environment.

Infections caused by the fungal pathogen Aspergillus fumigatus are increasingly resistant to first-line azole antifungal drugs. However, despite its clinical importance, little is known about how susceptible patients acquire infection from drug-resistant genotypes in the environment. Here, we present a population genomic analysis of 218 A. fumigatus isolates from across the UK and Ireland (comprising 153 clinical isolates from 143 patients and 65 environmental isolates). First, phylogenomic analysis shows strong genetic structuring into two clades (A and B) with little interclade recombination and the majority of environmental azole resistance found within clade A. Second, we show occurrences where azole-resistant isolates of near-identical genotypes were obtained from both environmental and clinical sources, indicating with high confidence the infection of patients with resistant isolates transmitted from the environment. Third, genome-wide scans identified selective sweeps across multiple regions indicating a polygenic basis to the trait in some genetic backgrounds. These signatures of positive selection are seen for loci containing the canonical genes encoding fungicide resistance in the ergosterol biosynthetic pathway, while other regions under selection have no defined function. Lastly, pan-genome analysis identified genes linked to azole resistance and previously unknown resistance mechanisms. Understanding the environmental drivers and genetic basis of evolving fungal drug resistance needs urgent attention, especially in light of increasing numbers of patients with severe viral respiratory tract infections who are susceptible to opportunistic fungal superinfections.

Comparison of in vivo pathogenicity of four Candida auris clades in a neutropenic bloodstream infection murine model.

Candida auris is an emerging worldwide concern, but comparative data about the virulence of different C. auris lineages in mammalian hosts is lacking. Different isolates of the four prevalent C. auris clades (South Asian n = 5, East Asian n = 4, South African n = 5, and South American n = 5) were compared to assess their virulence in a neutropenic murine bloodstream infection model with C. albicans as reference. C. auris, regardless of clade, proved to be less virulent than C. albicans. Highest overall mortality at day 21 was observed for the South American clade (96%), followed by the South Asian (80%), South African (45%) and East Asian (44%) clades. Fungal burden results showed close correlation with lethality. Histopathological examination revealed large aggregates of blastoconidia and budding yeast cells in the hearts, kidneys and livers but not in the spleens. The myocardium of apparently healthy sacrificed mice as well as of mice found moribund showed contraction band necrosis in case of all lineages. Regardless of clade, the heart and kidneys were the most heavily affected organs. Isolates of the same clade showed differences in virulence in mice, but a markedly higher virulence of the South American clade was clearly demonstrated.

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.

Farnesol increases the activity of echinocandins against Candida auris biofilms.

Candida auris biofilms exhibit decreased susceptibility to echinocandins, which is associated with poorer clinical outcomes. Farnesol is a quorum-sensing molecule enhancing the activity of antifungals; therefore, we evaluated the in vitro effect of farnesol with anidulafungin, caspofungin, or micafungin against biofilms using fractional inhibitory concentration indexes (FICI), Bliss independence model, LIVE/DEAD-assay and scanning electron microscopy. Based on mathematical models, farnesol caused synergism in eleven out of twelve cases (FICIs range 0.133-0.507; Bliss synergy volume range 70.39-204.6 µM2%). This was confirmed by microscope images of combination-exposed biofilms. Our study showed the prominent effect of farnesol with echinocandins against C. auris biofilms.

In vitro activity of rezafungin against common and rare Candida species and Saccharomyces cerevisiae.

BACKGROUND: Rezafungin is a novel echinocandin with excellent activity against common Candida species; however, limited data are available regarding rare Candida species. METHODS: We determined the in vitro susceptibility of 689 clinical isolates of 5 common and 19 rare Candida species, as well as Saccharomyces cerevisiae. The activity of rezafungin was compared with that of anidulafungin, caspofungin, micafungin, amphotericin B and fluconazole, using CLSI broth microdilution methodology (Fourth Edition: M27). RESULTS: Rezafungin MIC90 values were 0.06 mg/L for Candida albicans (n=125), Candida tropicalis (n=51), Candida dubliniensis (n=22), Candida inconspicua (n=41), Candida sojae (n=10), Candida lipolytica (n=10) and Candida pulcherrima (n=10), 0.12 mg/L for Candida glabrata (n=81), Candida krusei (n=53), Candida kefyr (n=52) and Candida fabianii (n=15), 0.25 mg/L for Candida lusitaniae (n=46) and Candida auris (n=19), 0.5 mg/L for Candida metapsilosis (n=15) and S. cerevisiae (n=21), 1 mg/L for Candida orthopsilosis (n=15) and Candida guilliermondii (n=27) and 2 mg/L for Candida parapsilosis sensu stricto (n=59). Caspofungin MIC90 values were 0.25-2 mg/L for all species, while micafungin and anidulafungin MIC90 values were similar to those of rezafungin. Fluconazole resistance was found in C. albicans (5.6%) and C. glabrata (4.9%); rezafungin was effective against these isolates as well. Amphotericin B MIC values did not exceed 2 mg/L. CONCLUSIONS: Rezafungin showed excellent in vitro activity against both WT and azole-resistant Candida species, as well as against S. cerevisiae. Rezafungin had similar activity to other echinocandins (excluding caspofungin) against common Candida species and, notably, against clinically relevant uncommon Candida species.

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.

Candida auris Isolates of the Southern Asian and South African Lineages Exhibit Different Phenotypic and Antifungal Susceptibility Profiles In Vitro.

Candida auris is a serious nosocomial health risk, with widespread outbreaks occurring in hospitals worldwide. Sequence analyses of outbreak isolates revealed that C. auris has simultaneously emerged as four distinct continentally restricted clonal lineages. We previously reported multiple independent introductions of C. auris isolates from at least three of these lineages (the Southern Asia, South African, and Japanese/Korean lineages) into hospitals across the United Kingdom and that isolates circulating in the United Kingdom displayed two different cell phenotypes which correlated with differences in virulence in Galleria mellonella wax moths. Here, we compared the phenotypic characteristics and antifungal susceptibilities of isolates representative of the three geographic clades circulating in the United Kingdom. Isolates of the South African and Japanese/Korean lineages, but not those of the Southern Asian lineage, grew well on media containing actidione. However, unlike Southern Asian lineage isolates, they were unable to produce even rudimentary pseudohyphae in culture. Importantly, although all isolates were fluconazole resistant in vitro, fluconazole and voriconazole exhibited significantly higher MICs against isolates of the South African lineage than against isolates of the Southern Asian lineage. A similar trend was seen with minimum fungicidal concentrations (MFCs), with higher MFCs of the triazole antifungal agents being seen for the South African lineage isolates. Finally, the formation of large cellular aggregates was seen only with isolates of the South African and Japanese/Korean lineages, which correlates with the reduced virulence observed previously in Galleria wax moths inoculated with such isolates. Intriguingly, aggregation could be reversibly induced in isolates of the Southern Asian lineage by exposure to triazole and echinocandin antifungals but not by exposure to amphotericin B or flucytosine.

Rapid and robust identification of clinical isolates of Talaromyces marneffei based on MALDI-TOF mass spectrometry or dimorphism in Galleria mellonella.

Talaromyces marneffei is a thermally dimorphic fungal pathogen that causes serious infections particularly in patients with human immunodeficiency virus (HIV). Although the mould form typically produces a characteristic red-diffusing pigment, and conidia from penicillate heads, several nonpathogenic Talaromyces/Penicillium species are morphologically and phenotypically similar. While those other species do not exhibit thermal dimorphism, conversion of T. marneffei to the distinctive fission yeast form in vitro is arduous and frequently incomplete. Here we show that T. marneffei can be rapidly and unambiguously discriminated from related nonpathogenic Talaromyces/Penicillium spp., either by matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry or conversion to fission yeast after introduction into Galleria mellonella. Conversion of T. marneffei conidia to the fission yeast form in G. mellonella larvae occurred as early as 24 h post inoculation at 37oC. Identification by MALDI-TOF was possible after supplementation of the commercial Bruker database with in-house mass spectral profiles created from either the yeast or mycelial phase of T. marneffei. In addition, we show that in-house generated mass spectral profiles could be successfully used to identify T. marneffei with a recently published on-line MALDI-TOF database, circumventing the need to create extensive in-house additional databases for rarely encountered fungal pathogens.

A novel dermatophyte relative, Nannizzia perplicata sp. nov., isolated from a case of tinea corporis in the United Kingdom.

A novel dermatophyte was isolated from skin scales of a female patient presenting with tinea corporis of the wrist and arm. Her principal risk factor was long-term corticosteroid use for underlying Lupus autoimmune syndrome. Microscopic examination of skin scales from lesions revealed hyphae consistent with dermatophyte infection, and a morphologically identical fungus grew in pure culture on all cultures of skin scales. Repeat isolation of the same organism from persistent lesions five months later confirmed the novel species as the causative agent. Microscopic examination revealed predominantly smooth, thin-walled macroconidia, with large numbers of unicellular aleuriospores of varied shapes and sizes. Since the isolate exhibited considerable microscopic pleomorphism, sharing morphological features consistent with several dermatophyte genera, it was subjected to multi-locus phylogenetic analyses employing a total of six different loci. Sequence analyses of all loci revealed that the isolate clustered with species within Nannizzia but diverged from all known members of the genus by 2 to 13% depending on locus analyzed. The isolate exhibited high minimum inhibitory concentrations for terbinafine in vitro, which might explain why the infection had failed to respond to two cycles of oral treatment with this antifungal agent. Interestingly, sequences in GenBank of an unnamed "Microsporum sp" isolated from leg skin of a patient in the Czech Republic showed greater than 99% identity across all of the loci analysed in common, indicating that this novel organism, which we describe here as Nannizzia perplicata sp. nov., is likely not restricted to the UK.

Isolates of the emerging pathogen Candida auris present in the UK have several geographic origins.

Candida auris has recently emerged as a serious nosocomial health risk, with widespread outbreaks in numerous hospitals worldwide and the existence of geographic region-specific discrete clonal lineages. Here we have compared the rDNA sequences of 24 isolates of Candida auris from 14 different hospital centers in the United Kingdom with those of strains from different international origins present in the public sequence databases. Here we show that UK isolates of C. auris fall into three well-supported clades corresponding to lineages that have previously been reported from India, Malaysia and Kuwait, Japan and Korea, and South Africa, respectively.

Rapid Identification of Clinically Relevant Members of the Genus Exophiala by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry and Description of Two Novel Species, Exophiala campbellii and Exophiala lavatrina.

Exophiala is a ubiquitous pleomorphic genus comprising at least 40 species, many of which have been associated with superficial, visceral, or systemic infections in humans, other mammals, or cold-blooded animals. In this study, we investigated the potential of matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) for the identification of Exophiala species. A total of 89 isolates (including 50 human and 4 animal clinical isolates) stored in the National Collection of Pathogenic Fungi were identified by PCR amplification and sequencing of internal transcribed spacer region 1. Eighty-three of the isolates corresponded to 16 known species within Exophiala/Rhinocladiella The remaining six isolates are shown by phylogenetic analyses based on four loci to represent two novel Exophiala species. Four isolates from domestic bathrooms which form a sister species with Exophiala lecanii-corni are described here as Exophiala lavatrina sp. nov. The remaining two isolates, both from subcutaneous infections, are distantly related to Exophiala oligosperma and are described here as Exophiala campbellii sp. nov. The triazoles and terbinafine exhibited low MICs against all Exophiala isolates in vitro MALDI-TOF MS successfully distinguished all 18 species and identified all isolates after appropriate reference spectra were created and added to commercial databases. Intraspecific mean log scores ranged from 1.786 to 2.584 and were consistently significantly higher than interspecific scores (1.193 to 1.624), with the exception of E. lecanii-corni and E. lavatrina, for which there was considerable log score overlap. In summary, MALDI-TOF MS allows the rapid and accurate identification of a wide range of clinically relevant Exophiala species.

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.

Comparative Pathogenicity of United Kingdom Isolates of the Emerging Pathogen Candida auris and Other Key Pathogenic Candida Species.

Candida auris, first described in 2009, has since emerged as an important, multidrug-resistant, nosocomial agent of candidemia, with large outbreaks reported worldwide and high mortality rates associated with therapeutic failure. The current study employed C. auris isolates from a variety of centers in the United Kingdom to evaluate the pathogenicity of this emerging pathogen compared to that of other common pathogenic yeast species in the invertebrate Galleria mellonella infection model. We showed that C. auris isolates differ in their growth characteristics in vitro, with a proportion of isolates failing to release daughter cells after budding, resulting in the formation of large aggregates of cells that cannot be physically disrupted. Our results also demonstrate strain-specific differences in the behavior of C. auris in G. mellonella, with the aggregate-forming isolates exhibiting significantly less pathogenicity than their nonaggregating counterparts. Importantly, the nonaggregating isolates exhibited pathogenicity comparable to that of C. albicans, which is currently accepted as the most pathogenic member of the genus, despite the fact that C. auris isolates do not produce hyphae and produce only rudimentary pseudohyphae either in vitro or in G. mellonella. IMPORTANCE The incidence of invasive candidiasis, which includes candidemia and deep tissue infections, continues to rise and is associated with considerable mortality rates. Candida albicans remains the most common cause of invasive candidiasis, although the prevalence of non-albicans species has increased over recent years. Since its first description in 2009, Candida auris has emerged as a serious nosocomial health risk, with widespread outbreaks in numerous hospitals worldwide. However, despite receiving considerable attention, little is known concerning the pathogenicity of this emerging fungal pathogen. Here, using the Galleria mellonella insect systemic infection model, we show strain-specific differences in the virulence of C. auris, with the most virulent isolates exhibiting pathogenicity comparable to that of C. albicans, which is currently accepted as the most pathogenic member of the genus.

Epidemiology, antifungal susceptibility, and pathogenicity of Candida africana isolates from the United Kingdom.

Candida africana was previously proposed as a new species within the Candida albicans species complex, together with C. albicans and C. dubliniensis, although further phylogenetic analyses better support its status as an unusual variant within C. albicans. Here we show that C. africana can be distinguished from C. albicans and C. dubliniensis by pyrosequencing of a short region of ITS2, and we have evaluated its occurrence in clinical samples by pyrosequencing all presumptive isolates of C. albicans submitted to the Mycology Reference Laboratory over a 9-month period. The C. albicans complex constituted 826/1,839 (44.9%) of yeast isolates received over the study period and included 783 isolates of C. albicans, 28 isolates of C. dubliniensis, and 15 isolates of C. africana. In agreement with previous reports, C. africana was isolated exclusively from genital specimens, in women in the 18-to-35-year age group. Indeed, C. africana constituted 15/251 (6%) of "C. albicans" isolates from female genital specimens during the study period. C. africana isolates were germ tube positive, grew significantly more slowly than C. albicans and C. dubliniensis on conventional mycological media, could be distinguished from the other members of the C. albicans complex by appearance on chromogenic agar, and were incapable of forming chlamydospores. Here we present the detailed evaluation of epidemiological, phenotypic, and clinical features and antifungal susceptibility profiles of United Kingdom isolates of C. africana. Furthermore, we demonstrate that C. africana is significantly less pathogenic than C. albicans and C. dubliniensis in the Galleria mellonella insect systemic infection model.

Assessment of accuracy of identification of pathogenic yeasts in microbiology laboratories in the United kingdom.

Rapid, accurate identification of yeast isolates from clinical samples has always been important given their innately variable antifungal susceptibility profiles. Recently, this has become paramount with the proposed introduction of species-specific interpretive breakpoints for MICs obtained in yeast antifungal susceptibility tests (M. A. Pfaller, D. Andes, D. J. Diekema, A. Espinel-Ingroff, D. Sheehan, and CLSI Subcommittee for Antifungal Susceptibility Testing, Drug Resist. Updat. 13:180-195, 2010). Here, we present the results of a 12-month evaluation of the accuracy of identifications that accompany yeast isolates submitted to the Mycology Reference Laboratory (United Kingdom) for either confirmation of identity or susceptibility testing. In total, 1,781 yeast isolates were analyzed, and the robustness of prior identifications obtained in microbiology laboratories throughout the United Kingdom was assessed using a combination of culture on chromogenic agar, morphology on cornmeal agar, and molecular identification by pyrosequencing. Over 40% of isolates (755) were submitted without any suggested identification. Of those isolates with a prior identification, 100 (9.7%) were incorrectly identified. Error rates ranged from 5.2% (for organisms submitted for antifungal susceptibility testing) to 18.2% (for organisms requiring confirmation of identity) and varied in a strictly species-specific manner. At least 50% of identification errors would be likely to affect interpretation of MIC data, with a possible impact on patient management. In addition, 2.3% of submitted cultures were found to contain mixtures of at least two yeast species. The vast majority of mixtures had gone undetected in the referring laboratory and would have impacted the interpretation of antifungal susceptibility profiles and patient management. Some of the more common misidentifications are discussed according to the identification method employed, with suggestions for avoiding such misinterpretations.

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.

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.

Activity of voriconazole, itraconazole, fluconazole and amphotericin B in vitro against 1763 yeasts from 472 patients in the voriconazole phase III clinical studies.

The susceptibility of 1763 yeast isolates (from 22 species and seven genera) was tested using Clinical and Laboratory Standards Institute M27-A2 microdilution methodology. Candida spp. predominated (97.1%), mainly C. albicans (51.4%), C. glabrata (16.4%) and C. tropicalis (13.7%), followed by Trichosporon spp. (1.1%) and Cryptococcus neoformans (1.0%). Most isolates came from blood/catheters (72.0%) or the oesophagus/oropharynx (11.3%). The voriconazole, itraconazole, fluconazole and amphotericin B MIC90 values (minimum inhibitory concentration for 90% of the isolates) for all isolates were 1.0, 2.0, 64 and 1.0 microg/mL, respectively. Voriconazole MICs correlated with those for fluconazole (r = 0.91) and itraconazole (r = 0.90). Only 109 isolates (6.2%) had voriconazole MICs > or = 4.0 microg/mL; all were C. albicans, C. glabrata or C. tropicalis resistant to itraconazole (and most to fluconazole). Isolates from 22 patients with amphotericin MICs > or = 2.0 microg/mL (range 2.0-16.0 microg/mL) were also cross-resistant to one or more of the triazoles. Patients (n = 34) with voriconazole-resistant isolates showed a 56% response to voriconazole therapy, and those patients (n = 261) with susceptible isolates showed a 71% response. Twenty-three voriconazole-treated patients had baseline resistant isolates, in eight patients voriconazole resistance developed during therapy and in three patients a different resistant species arose during therapy. Thus, voriconazole MICs correlate with those of fluconazole and itraconazole and may predict clinical outcome.