Publications by authors named "Matthew C Fisher"

124 Publications

Genomic epidemiology of a case cluster in Glasgow, Scotland, 2018.

Microb Genom 2021 Mar 23;7(3). Epub 2021 Feb 23.

Broad Institute of MIT and Harvard, Cambridge, MA, USA.

In 2018, a cluster of two cases of cryptococcosis occurred at the Queen Elizabeth University Hospital (QEUH) in Glasgow, Scotland (UK). It was postulated that these cases may have been linked to pigeon droppings found on the hospital site, given there have been previous reports of associated with pigeon guano. Although some samples of pigeon guano taken from the site yielded culturable yeast from genera related to , they have since been classified as or spp., and no isolates of were recovered from either the guano or subsequent widespread air sampling. In an attempt to further elucidate any possible shared source of the clinical isolates, we used whole-genome sequencing and phylogenetic analysis to examine the relationship of the two isolates from the QEUH cases, along with two isolates from sporadic cases treated at a different Glasgow hospital earlier in 2018. Our work demonstrated that these four clinical isolates were not clonally related; while all isolates were from the VNI global lineage and of the same mating type (MATα), the genotypes of the two QEUH isolates were separated by 1885 base changes and belonged to different sub-lineages, recently described as the intercontinental sub-clades VNIa-93 and VNIa-5. In contrast, one of the two sporadic 2018 clinical isolates was determined to belong to the VNIb sub-lineage and the other classified as a VNIV/VNI hybrid. Our work demonstrated that the two 2018 QEUH isolates and the two prior clinical isolates were all genetically distinct. It was not possible to determine whether the QEUH genotypes stemmed from independent sources or from the same source, i.e. pigeons carrying different genotypes, but it should be noted that whilst members of allied genera within the were isolated from the hospital environment, there were no environmental isolations of .
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http://dx.doi.org/10.1099/mgen.0.000537DOI Listing
March 2021

Microbial Grazers May Aid in Controlling Infections Caused by the Aquatic Zoosporic Fungus .

Front Microbiol 2020 21;11:592286. Epub 2021 Jan 21.

Department of Evolution, Ecology and Behaviour, Biosciences Building, University of Liverpool, Liverpool, United Kingdom.

Free-living eukaryotic microbes may reduce animal diseases. We evaluated the dynamics by which micrograzers (primarily protozoa) apply top-down control on the chytrid () a devastating, panzootic pathogen of amphibians. Although micrograzers consumed zoospores (∼3 μm), the dispersal stage of chytrids, not all species grew monoxenically on zoospores. However, the ubiquitous ciliate , which likely co-occurs with , grew at near its maximum rate ( = 1.7 d). A functional response (ingestion vs. prey abundance) for , measured using spore-surrogates (microspheres) revealed maximum ingestion ( ) of 1.63 × 10 zoospores d, with a half saturation constant () of 5.75 × 10 zoospores ml. Using these growth and grazing data we developed and assessed a population model that incorporated chytrid-host and micrograzer dynamics. Simulations using our data and realistic parameters obtained from the literature suggested that micrograzers could control and potentially prevent chytridiomycosis (defined as 10 sporangia host). However, simulated inferior micrograzers (0.7 × and 1.5 × ) did not prevent chytridiomycosis, although they ultimately reduced pathogen abundance to below levels resulting in disease. These findings indicate how micrograzer responses can be applied when modeling disease dynamics for and other zoosporic fungi.
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http://dx.doi.org/10.3389/fmicb.2020.592286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7858660PMC
January 2021

Emerging infections and the integrative environment-health sciences: the road ahead.

Nat Rev Microbiol 2021 03;19(3):133-135

MRC Centre for Global Infectious Disease Analysis, Imperial College School of Public Health, Imperial College London, London, UK.

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http://dx.doi.org/10.1038/s41579-021-00510-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7797887PMC
March 2021

Post-epizootic microbiome associations across communities of neotropical amphibians.

Mol Ecol 2021 03 20;30(5):1322-1335. Epub 2021 Jan 20.

MRC Centre for Global Infectious Disease Analysis, School of Public Health, Imperial College, London, UK.

Microbiome-pathogen interactions are increasingly recognized as an important element of host immunity. While these host-level interactions will have consequences for community disease dynamics, the factors which influence host microbiomes at larger scales are poorly understood. We here describe landscape-scale pathogen-microbiome associations within the context of post-epizootic amphibian chytridiomycosis, a disease caused by the panzootic chytrid fungus Batrachochytrium dendrobatidis. We undertook a survey of Neotropical amphibians across altitudinal gradients in Ecuador ~30 years following the observed amphibian declines and collected skin swab-samples which were metabarcoded using both fungal (ITS-2) and bacterial (r16S) amplicons. The data revealed marked variation in patterns of both B. dendrobatidis infection and microbiome structure that are associated with host life history. Stream breeding amphibians were most likely to be infected with B. dendrobatidis. This increased probability of infection was further associated with increased abundance and diversity of non-Batrachochytrium chytrid fungi in the skin and environmental microbiome. We also show that increased alpha diversity and the relative abundance of fungi are lower in the skin microbiome of adult stream amphibians compared to adult pond-breeding amphibians, an association not seen for bacteria. Finally, stream tadpoles exhibit lower proportions of predicted protective microbial taxa than pond tadpoles, suggesting reduced biotic resistance. Our analyses show that host breeding ecology strongly shapes pathogen-microbiome associations at a landscape scale, a trait that may influence resilience in the face of emerging infectious diseases.
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http://dx.doi.org/10.1111/mec.15789DOI Listing
March 2021

Cross-Disciplinary Genomics Approaches to Studying Emerging Fungal Infections.

Life (Basel) 2020 Nov 28;10(12). Epub 2020 Nov 28.

Department of Infectious Disease Epidemiology, MRC Centre for Global Infectious Disease Analysis, St Mary's Campus, Imperial College London, London W2 1PG, UK.

Emerging fungal pathogens pose a serious, global and growing threat to food supply systems, wild ecosystems, and human health. However, historic chronic underinvestment in their research has resulted in a limited understanding of their epidemiology relative to bacterial and viral pathogens. Therefore, the untargeted nature of genomics and, more widely, -omics approaches is particularly attractive in addressing the threats posed by and illuminating the biology of these pathogens. Typically, research into plant, human and wildlife mycoses have been largely separated, with limited dialogue between disciplines. However, many serious mycoses facing the world today have common traits irrespective of host species, such as plastic genomes; wide host ranges; large population sizes and an ability to persist outside the host. These commonalities mean that -omics approaches that have been productively applied in one sphere and may also provide important insights in others, where these approaches may have historically been underutilised. In this review, we consider the advances made with genomics approaches in the fields of plant pathology, human medicine and wildlife health and the progress made in linking genomes to other -omics datatypes and sets; we identify the current barriers to linking -omics approaches and how these are being underutilised in each field; and we consider how and which -omics methodologies it is most crucial to build capacity for in the near future.
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http://dx.doi.org/10.3390/life10120315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761180PMC
November 2020

Discriminating lineages of Batrachochytrium dendrobatidis using quantitative PCR.

Mol Ecol Resour 2020 Nov 24. Epub 2020 Nov 24.

MRC Centre for Global Infectious Disease Analysis, Department of Infectious Disease Epidemiology, Imperial College London, London, UK.

The ability to detect and monitor infectious disease in a phylogenetically informative manner is critical for their management. Phylogenetically informative diagnostic tests enable patterns of pathogen introduction or changes in the distribution of genotypes to be measured, enabling research into the ecology of the pathogen. Batrachochytrium dendrobatidis (Bd), a causative agent of chytridiomycosis in amphibian populations, emerged worldwide in the 21st century and is composed of six lineages which are display varying levels of virulence in their hosts. Research into the distribution, ecology and pathogenicity of these lineages has been hampered by an inability to type lineage efficiently. Here, we describe a lineage-specific TaqMan qPCR assay that differentiates the two lineages of Bd most commonly associated with chytridiomycosis: BdGPL and BdCAPE. We demonstrate how this assay can be used for the surveillance of wild populations of amphibians in Southern Africa using skin swabs, tissue samples and cultured isolates.
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http://dx.doi.org/10.1111/1755-0998.13299DOI Listing
November 2020

Rapid Detection of Azole-Resistant Aspergillus fumigatus in Clinical and Environmental Isolates by Use of a Lab-on-a-Chip Diagnostic System.

J Clin Microbiol 2020 10 21;58(11). Epub 2020 Oct 21.

Centre for Bio-Inspired Technology, Department of Electrical and Electronic Engineering, Faculty of Engineering, Imperial College London, London, United Kingdom.

has widely evolved resistance to the most commonly used class of antifungal chemicals, the azoles. Current methods for identifying azole resistance are time-consuming and depend on specialized laboratories. There is an urgent need for rapid detection of these emerging pathogens at point-of-care to provide the appropriate treatment in the clinic and to improve management of environmental reservoirs to mitigate the spread of antifungal resistance. Our study demonstrates the rapid and portable detection of the two most relevant genetic markers linked to azole resistance, the mutations TR34 and TR46, found in the promoter region of the gene encoding the azole target 51A. We developed a lab-on-a-chip platform consisting of: (i) tandem-repeat loop-mediated isothermal amplification; (ii) state-of-the-art complementary metal-oxide-semiconductor microchip technology for nucleic acid amplification detection; and (iii) a smartphone application for data acquisition, visualization, and cloud connectivity. Specific and sensitive detection was validated with isolates from clinical and environmental samples from 6 countries across 5 continents, showing a lower limit of detection of 10 genomic copies per reaction in less than 30 min. When fully integrated with a sample preparation module, this diagnostic system will enable the detection of this ubiquitous fungus at the point-of-care, and could help to improve clinical decision making, infection control, and epidemiological surveillance.
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http://dx.doi.org/10.1128/JCM.00843-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587087PMC
October 2020

A Low-Cost Tebuconazole-Based Screening Test for Azole-Resistant Aspergillus fumigatus.

Curr Protoc Microbiol 2020 09;58(1):e112

MRC Centre for Global Infectious Disease Analysis, Department of Infectious Disease Epidemiology, Imperial College London, London, United Kingdom.

The global emergence of azole resistance in Aspergillus fumigatus is resulting in health and food security concerns. Rapid diagnostics and environmental surveillance methods are key to understanding the distribution and prevalence of azole resistance. However, such methods are often associated with high costs and are not always applicable to laboratories based in the least-developed countries. Here, we present and validate a low-cost screening protocol that can be used to differentiate between azole-susceptible "wild-type" and azole-resistant A. fumigatus isolates. © 2020 The Authors. Basic Protocol 1: Preparation of Tebucheck multi-well plates Basic Protocol 2: Inoculation of Tebucheck multi-well plates.
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http://dx.doi.org/10.1002/cpmc.112DOI Listing
September 2020

Genome-wide mapping using new AFLP markers to explore intraspecific variation among pathogenic Sporothrix species.

PLoS Negl Trop Dis 2020 07 1;14(7):e0008330. Epub 2020 Jul 1.

Laboratory of Emerging Fungal Pathogens, Department of Microbiology, Immunology, and Parasitology, Discipline of Cellular Biology, Federal University of São Paulo (UNIFESP), São Paulo, Brazil.

Sporotrichosis is a chronic subcutaneous mycosis caused by Sporothrix species, of which the main aetiological agents are S. brasiliensis, S. schenckii, and S. globosa. Infection occurs after a traumatic inoculation of Sporothrix propagules in mammals' skin and can follow either a classic route through traumatic inoculation by plant debris (e.g., S. schenckii and S. globosa) or an alternative route through zoonotic transmission from animals (e.g., S. brasiliensis). Epizootics followed by a zoonotic route occur in Brazil, with Rio de Janeiro as the epicenter of a recent cat-transmitted epidemic. DNA-based markers are needed to explore the epidemiology of these Sporothrix expansions using molecular methods. This paper reports the use of amplified-fragment-length polymorphisms (AFLP) to assess the degree of intraspecific variability among Sporothrix species. We used whole-genome sequences from Sporothrix species to generate 2,304 virtual AFLP fingerprints. In silico screening highlighted 6 primer pair combinations to be tested in vitro. The protocol was used to genotype 27 medically relevant Sporothrix. Based on the overall scored AFLP markers (97-137 fragments), the values of polymorphism information content (PIC = 0.2552-0.3113), marker index (MI = 0.002-0.0039), effective multiplex ratio (E = 17.8519-35.2222), resolving power (Rp = 33.6296-63.1852), discriminating power (D = 0.9291-0.9662), expected heterozygosity (H = 0.3003-0.3857), and mean heterozygosity (Havp = 0.0001) demonstrated the utility of these primer combinations for discriminating Sporothrix. AFLP markers revealed cryptic diversity in species previously thought to be the most prevalent clonal type, such as S. brasiliensis, responsible for cat-transmitted sporotrichosis, and S. globosa responsible for large sapronosis outbreaks in Asia. Three combinations (#3 EcoRI-FAM-GA/MseI-TT, #5 EcoRI-FAM-GA/MseI-AG, and #6 EcoRI-FAM-TA/MseI-AA) provide the best diversity indices and lowest error rates. These methods make it easier to track routes of disease transmission during epizooties and zoonosis, and our DNA fingerprint assay can be further transferred between laboratories to give insights into the ecology and evolution of pathogenic Sporothrix species and to inform management and mitigation strategies to tackle the advance of sporotrichosis.
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http://dx.doi.org/10.1371/journal.pntd.0008330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7329091PMC
July 2020

Threats Posed by the Fungal Kingdom to Humans, Wildlife, and Agriculture.

mBio 2020 05 5;11(3). Epub 2020 May 5.

Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada

The fungal kingdom includes at least 6 million eukaryotic species and is remarkable with respect to its profound impact on global health, biodiversity, ecology, agriculture, manufacturing, and biomedical research. Approximately 625 fungal species have been reported to infect vertebrates, 200 of which can be human associated, either as commensals and members of our microbiome or as pathogens that cause infectious diseases. These organisms pose a growing threat to human health with the global increase in the incidence of invasive fungal infections, prevalence of fungal allergy, and the evolution of fungal pathogens resistant to some or all current classes of antifungals. More broadly, there has been an unprecedented and worldwide emergence of fungal pathogens affecting animal and plant biodiversity. Approximately 8,000 species of fungi and Oomycetes are associated with plant disease. Indeed, across agriculture, such fungal diseases of plants include new devastating epidemics of trees and jeopardize food security worldwide by causing epidemics in staple and commodity crops that feed billions. Further, ingestion of mycotoxins contributes to ill health and causes cancer. Coordinated international research efforts, enhanced technology translation, and greater policy outreach by scientists are needed to more fully understand the biology and drivers that underlie the emergence of fungal diseases and to mitigate against their impacts. Here, we focus on poignant examples of emerging fungal threats in each of three areas: human health, wildlife biodiversity, and food security.
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http://dx.doi.org/10.1128/mBio.00449-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7403777PMC
May 2020

Response to Comment on "Amphibian fungal panzootic causes catastrophic and ongoing loss of biodiversity".

Science 2020 03;367(6484)

Wildlife Health Ghent, Department of Pathology, Bacteriology, and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium.

Lambert question our retrospective and holistic epidemiological assessment of the role of chytridiomycosis in amphibian declines. Their alternative assessment is narrow and provides an incomplete evaluation of evidence. Adopting this approach limits understanding of infectious disease impacts and hampers conservation efforts. We reaffirm that our study provides unambiguous evidence that chytridiomycosis has affected at least 501 amphibian species.
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http://dx.doi.org/10.1126/science.aay2905DOI Listing
March 2020

Chytrid fungi and global amphibian declines.

Nat Rev Microbiol 2020 06 25;18(6):332-343. Epub 2020 Feb 25.

Institute of Zoology, Zoological Society of London, London, UK.

Discovering that chytrid fungi cause chytridiomycosis in amphibians represented a paradigm shift in our understanding of how emerging infectious diseases contribute to global patterns of biodiversity loss. In this Review we describe how the use of multidisciplinary biological approaches has been essential to pinpointing the origins of amphibian-parasitizing chytrid fungi, including Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans, as well as to timing their emergence, tracking their cycles of expansion and identifying the core mechanisms that underpin their pathogenicity. We discuss the development of the experimental methods and bioinformatics toolkits that have provided a fuller understanding of batrachochytrid biology and informed policy and control measures.
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http://dx.doi.org/10.1038/s41579-020-0335-xDOI Listing
June 2020

A New Lineage of Cryptococcus gattii (VGV) Discovered in the Central Zambezian Miombo Woodlands.

mBio 2019 11 12;10(6). Epub 2019 Nov 12.

Molecular Microbiology Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

We discovered a new lineage of the globally important fungal pathogen on the basis of analysis of six isolates collected from three locations spanning the Central Miombo Woodlands of Zambia, Africa. All isolates were from environments (middens and tree holes) that are associated with a small mammal, the African hyrax. Phylogenetic and population genetic analyses confirmed that these isolates form a distinct, deeply divergent lineage, which we name VGV. VGV comprises two subclades (A and B) that are capable of causing mild lung infection with negligible neurotropism in mice. Comparing the VGV genome to previously identified lineages of revealed a unique suite of genes together with gene loss and inversion events. However, standard restriction fragment length polymorphism (RFLP) analysis could not distinguish between VGV and VGIV isolates. We therefore developed a new RFLP method that can reliably identify the newly described lineage. Our work highlights how sampling understudied ecological regions alongside genomic and functional characterization can broaden our understanding of the evolution and ecology of major global pathogens. is an environmental pathogen that causes severe systemic infection in immunocompetent individuals more often than in immunocompromised humans. Over the past 2 decades, researchers have shown that falls within four genetically distinct major lineages. By combining field work from an understudied ecological region (the Central Miombo Woodlands of Zambia, Africa), genome sequencing and assemblies, phylogenetic and population genetic analyses, and phenotypic characterization (morphology, histopathological, drug-sensitivity, survival experiments), we discovered a hitherto unknown lineage, which we name VGV (variety five). The discovery of a new lineage from an understudied ecological region has far-reaching implications for the study and understanding of fungal pathogens and diseases they cause.
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http://dx.doi.org/10.1128/mBio.02306-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6851281PMC
November 2019

Captivity and Infection by the Fungal Pathogen Perturb the Amphibian Skin Microbiome.

Front Microbiol 2019 23;10:1834. Epub 2019 Aug 23.

Department of Infectious Disease Epidemiology, MRC Centre for Global Infectious Disease Analysis, School of Public Health, Imperial College London, London, United Kingdom.

The emerging fungal pathogen, () is responsible for the catastrophic decline of European salamanders and poses a threat to amphibians globally. The amphibian skin microbiome can influence disease outcome for several host-pathogen systems, yet little is known of its role in infection. In addition, many experimental amphibian disease studies to date have relied on specimens that have been kept in captivity for long periods without considering the influence of environment on the microbiome and how this may impact the host response to pathogen exposure. We characterized the impact of captivity and exposure to on the skin bacterial and fungal communities of two co-occurring European newt species, the smooth newt, and the great-crested newt, . We show that captivity led to significant losses in bacterial and fungal diversity of amphibian skin, which may be indicative of a decline in microbe-mediated protection. We further demonstrate that in both and infection was associated with changes in the composition of skin bacterial communities with possible negative consequences to host health. Our findings advance current understanding of the role of host-associated microbiota in infection and highlight important considerations for amphibian conservation programmes.
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http://dx.doi.org/10.3389/fmicb.2019.01834DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6716147PMC
August 2019

Global epidemiology of emerging Candida auris.

Curr Opin Microbiol 2019 12 3;52:84-89. Epub 2019 Jul 3.

MRC Centre for Global Infectious Disease Analysis, Imperial College London, London, United Kingdom. Electronic address:

The discovery in 2009 of a new species of yeast, Candida auris, heralded the arrival of a novel emerging human infectious disease. This review highlights the unique characteristics of C. auris that have lled to it being of public health concern worldwide, namely public health concern, namely its global emergence, its ability to cause nosocomial outbreaks in healthcare settings, its innate and emerging resistance to multiple antifungal drugs and its resilience in the face of hygiene and infection control measures. Genomic epidemiology has identified four emergences of C. auris marked by four clades of the pathogen. These clades of C. auris are genetically dissimilar and are associated with differential resistance to antifungal drugs, suggesting that they will continue to phenotypically diverge into the future. The global emergence of C. auris testifies to the unmapped nature of Kingdom Fungi, and represents a new nosocomial threat that will require enhanced infection control across diverse healthcare and community settings.
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http://dx.doi.org/10.1016/j.mib.2019.05.008DOI Listing
December 2019

Elevated Prevalence of Azole-Resistant Aspergillus fumigatus in Urban versus Rural Environments in the United Kingdom.

Antimicrob Agents Chemother 2019 09 23;63(9). Epub 2019 Aug 23.

MRC Centre for Global Infectious Disease Analysis, Department of Infectious Disease Epidemiology, Imperial College London, London, United Kingdom.

Azole resistance in the opportunistic pathogen is increasing, dominated primarily by the following two environmentally associated resistance alleles: TR/L98H and TR/Y121F/T289A. By sampling soils across the South of England, we assess the prevalence of azole-resistant (AR) in samples collected in both urban and rural locations. We characterize the susceptibility profiles of the resistant isolates to three medical azoles, identify the underlying genetic basis of resistance, and investigate their genetic relationships. AR was detected in 6.7% of the soil samples, with a higher prevalence in urban (13.8%) than rural (1.1%) locations. Twenty isolates were confirmed to exhibit clinical breakpoints for resistance to at least one of three medical azoles, with 18 isolates exhibiting resistance to itraconazole, 6 to voriconazole, and 2 showing elevated minimum inhibitory concentrations to posaconazole. Thirteen of the resistant isolates harbored the TR/L98H resistance allele, and six isolates carried the TR/Y121F/T289A allele. The 20 azole-resistant isolates were spread across five genetic subtypes, t01, t02, t04B, t09, and t18 with t02 being the predominant subtype. Our study demonstrates that AR can be easily isolated in the South of England, especially in urban city centers, which appear to play an important role in the epidemiology of environmentally linked drug-resistant .
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http://dx.doi.org/10.1128/AAC.00548-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6709452PMC
September 2019

Nonrandom Distribution of Azole Resistance across the Global Population of Aspergillus fumigatus.

mBio 2019 05 21;10(3). Epub 2019 May 21.

MRC Centre for Global Infectious Disease Analysis, Department of Infectious Disease Epidemiology, Imperial College London, London, United Kingdom.

The emergence of azole resistance in the pathogenic fungus has continued to increase, with the dominant resistance mechanisms, consisting of a 34-nucleotide tandem repeat (TR)/L98H and TR/Y121F/T289A, now showing a structured global distribution. Using hierarchical clustering and multivariate analysis of 4,049 isolates collected worldwide and genotyped at nine microsatellite loci using analysis of short tandem repeats of (STR), we show that can be subdivided into two broad clades and that alleles TR/L98H and TR/Y121F/T289A are unevenly distributed across these two populations. Diversity indices show that azole-resistant isolates are genetically depauperate compared to their wild-type counterparts, compatible with selective sweeps accompanying the selection of beneficial mutations. Strikingly, we found that azole-resistant clones with identical microsatellite profiles were globally distributed and sourced from both clinical and environmental locations, confirming that azole resistance is an international public health concern. Our work provides a framework for the analysis of isolates based on their microsatellite profile, which we have incorporated into a freely available, user-friendly R Shiny application (AfumID) that provides clinicians and researchers with a method for the fast, automated characterization of genetic relatedness. Our study highlights the effect that azole drug resistance is having on the genetic diversity of and emphasizes its global importance upon this medically important pathogenic fungus. Azole drug resistance in the human-pathogenic fungus continues to emerge, potentially leading to untreatable aspergillosis in immunosuppressed hosts. Two dominant, environmentally associated resistance mechanisms, which are thought to have evolved through selection by the agricultural application of azole fungicides, are now distributed globally. Understanding the effect that azole resistance is having on the genetic diversity and global population of will help mitigate drug-resistant aspergillosis and maintain the azole class of fungicides for future use in both medicine and crop protection.
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http://dx.doi.org/10.1128/mBio.00392-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6529631PMC
May 2019

Amphibian fungal panzootic causes catastrophic and ongoing loss of biodiversity.

Science 2019 03;363(6434):1459-1463

Wildlife Health Ghent, Department of Pathology, Bacteriology, and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium.

Anthropogenic trade and development have broken down dispersal barriers, facilitating the spread of diseases that threaten Earth's biodiversity. We present a global, quantitative assessment of the amphibian chytridiomycosis panzootic, one of the most impactful examples of disease spread, and demonstrate its role in the decline of at least 501 amphibian species over the past half-century, including 90 presumed extinctions. The effects of chytridiomycosis have been greatest in large-bodied, range-restricted anurans in wet climates in the Americas and Australia. Declines peaked in the 1980s, and only 12% of declined species show signs of recovery, whereas 39% are experiencing ongoing decline. There is risk of further chytridiomycosis outbreaks in new areas. The chytridiomycosis panzootic represents the greatest recorded loss of biodiversity attributable to a disease.
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http://dx.doi.org/10.1126/science.aav0379DOI Listing
March 2019

Dynamic ploidy changes drive fluconazole resistance in human cryptococcal meningitis.

J Clin Invest 2019 03 28;129(3):999-1014. Epub 2019 Jan 28.

Centre for Global Health, Institute for Infection and Immunity, St. George's, University of London, United Kingdom.

Background: Cryptococcal meningitis (CM) causes an estimated 180,000 deaths annually, predominantly in sub-Saharan Africa, where most patients receive fluconazole (FLC) monotherapy. While relapse after FLC monotherapy with resistant strains is frequently observed, the mechanisms and impact of emergence of FLC resistance in human CM are poorly understood. Heteroresistance (HetR) - a resistant subpopulation within a susceptible strain - is a recently described phenomenon in Cryptococcus neoformans (Cn) and Cryptococcus gattii (Cg), the significance of which has not previously been studied in humans.

Methods: A cohort of 20 patients with HIV-associated CM in Tanzania was prospectively observed during therapy with either FLC monotherapy or in combination with flucytosine (5FC). Total and resistant subpopulations of Cryptococcus spp. were quantified directly from patient cerebrospinal fluid (CSF). Stored isolates underwent whole genome sequencing and phenotypic characterization.

Results: Heteroresistance was detectable in Cryptococcus spp. in the CSF of all patients at baseline (i.e., prior to initiation of therapy). During FLC monotherapy, the proportion of resistant colonies in the CSF increased during the first 2 weeks of treatment. In contrast, no resistant subpopulation was detectable in CSF by day 14 in those receiving a combination of FLC and 5FC. Genomic analysis revealed high rates of aneuploidy in heteroresistant colonies as well as in relapse isolates, with chromosome 1 (Chr1) disomy predominating. This is apparently due to the presence on Chr1 of ERG11, which is the FLC drug target, and AFR1, which encodes a drug efflux pump. In vitro efflux levels positively correlated with the level of heteroresistance.

Conclusion: Our findings demonstrate for what we believe is the first time the presence and emergence of aneuploidy-driven FLC heteroresistance in human CM, association of efflux levels with heteroresistance, and the successful suppression of heteroresistance with 5FC/FLC combination therapy.

Funding: This work was supported by the Wellcome Trust Strategic Award for Medical Mycology and Fungal Immunology 097377/Z/11/Z and the Daniel Turnberg Travel Fellowship.
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http://dx.doi.org/10.1172/JCI124516DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391087PMC
March 2019

Rapid and Sensitive Detection of Azole-Resistant Aspergillus fumigatus by Tandem Repeat Loop-Mediated Isothermal Amplification.

J Mol Diagn 2019 03 4;21(2):286-295. Epub 2018 Dec 4.

Centre for Bio-Inspired Technology, Institute of Biomedical Engineering, Department of Electrical and Electronic Engineering.

Invasive fungal infections caused by multiazole-resistant Aspergillus fumigatus are associated with increasing rates of mortality in susceptible patients. Current methods of diagnosing infections caused by multiazole-resistant A. fumigatus are, however, not well suited for use in clinical point-of-care testing or in the field. Loop-mediated isothermal amplification (LAMP) is a widely used method of nucleic acid amplification with rapid and easy-to-use features, making it suitable for use in different resource settings. Here, we developed a LAMP assay to detect a 34 bp tandem repeat, named TR34-LAMP. TR34 is a high-prevalence allele that, in conjunction with the L98H single-nucleotide polymorphism, is associated with the occurrence of multiazole resistance in A. fumigatus in the environment and in patients. This process was validated with both synthetic double-stranded DNA and genomic DNA prepared from azole-resistant isolates of A. fumigatus. Use of our assay resulted in rapid and specific identification of the TR34 allele with high sensitivity, detecting down to 10 genomic copies per reaction within 25 minutes. Fluorescent and colorimetric detections were used for the analysis of 11 clinical isolates as cross validation. These results show that the TR34-LAMP assay has the potential to accelerate the screening of clinical and environmental A. fumigatus to provide a rapid and accurate diagnosis of azole resistance, which current methods struggle to achieve.
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http://dx.doi.org/10.1016/j.jmoldx.2018.10.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6419584PMC
March 2019

Transcriptional Heterogeneity of VGII Compared with Non-VGII Lineages Underpins Key Pathogenicity Pathways.

mSphere 2018 10 24;3(5). Epub 2018 Oct 24.

Infectious Disease and Microbiome Program, The Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.

is a pathogenic yeast of humans and other animals which causes disease predominantly in immunocompetent hosts. Infection begins when aerosolized yeast or spores enter the body, triggering an immune response, including engulfment by macrophages. To understand the early transcriptional signals in both the yeast and its mammalian host, we performed a time-course dual-transcriptome sequencing (RNA-seq) experiment for four lineages of (lineages VGI to IV) interacting with mouse macrophages at 1, 3, and 6 h postinfection. Comparisons of to gene expression levels indicated that lineage VGII is transcriptionally divergent from non-VGII lineages, including differential expression of genes involved in capsule synthesis, capsule attachment, and ergosterol production. Several paralogous genes demonstrated subfunctionalization between lineages, including upregulation of capsule biosynthesis-related gene and downregulation of in VGIII. Isolates also compensate for lineage-specific gene losses by overexpression of genetically similar paralogs, including overexpression of capsule gene in VGIV, which have lost the gene. Differential expression of one in five genes was detected following coincubation with mouse macrophages; all isolates showed high induction of oxidative-reduction functions and downregulation of capsule attachment genes. We also found that VGII switches expression of two laccase paralogs (from to ) during coincubation of macrophages. Finally, we found that mouse macrophages respond to all four lineages of by upregulating FosB/Jun/Egr1 regulatory proteins at early time points. This report highlights the evolutionary breadth of expression profiles among the lineages of and the diversity of transcriptional responses at this host-pathogen interface. The transcriptional profiles of related pathogens and their responses to host-induced stresses underpin their pathogenicity. Expression differences between related pathogens during host interaction can indicate when and how these genes contribute to virulence, ultimately informing new and improved treatment strategies for those diseases. In this paper, we compare the transcriptional profiles of five isolates representing four lineages of in rich media. Our analyses identified key processes, including those involving cell capsule, ergosterol production, and melanin, that are differentially expressed between lineages, and we found that VGII has the most distinct profile in terms of numbers of differentially expressed genes. All lineages have also undergone subfunctionalization for several paralogs, including capsule biosynthesis and attachment genes. Most genes appeared downregulated during coincubation with macrophages, with the largest decrease observed for capsule attachment genes, which appeared to be coordinated with a stress response, as all lineages also upregulated oxidative stress response genes. Furthermore, VGII upregulated many genes that are linked to ergosterol biosynthesis and switched from expression of the laccase to expression of Finally, we saw a pronounced increase in the FosB/Jun/Egr1 regulatory proteins at early time points in bone marrow-derived macrophages, marking a role in the host response to This work highlights the dynamic roles of key virulence genes in response to macrophages.
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http://dx.doi.org/10.1128/mSphere.00445-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6200987PMC
October 2018

Surveillance for Azole-Resistant in a Centralized Diagnostic Mycology Service, London, United Kingdom, 1998-2017.

Front Microbiol 2018 20;9:2234. Epub 2018 Sep 20.

Fungal Pathogens Laboratory, National Heart and Lung Institute, Imperial College London, London, United Kingdom.

is the leading cause of invasive aspergillosis. Treatment is hindered by the emergence of resistance to triazole antimycotic agents. Here, we present the prevalence of triazole resistance among clinical isolates at a major centralized medical mycology laboratory in London, United Kingdom, in the period 1998-2017. A large number ( = 1469) of clinical isolates from unselected clinical specimens were identified and their susceptibility against three triazoles, amphotericin B and three echinocandin agents was carried out. All isolates were identified phenotypically and antifungal susceptibility testing was carried out by using a standard broth microdilution method. Retrospective surveillance (1998-2011) shows 5/1151 (0.43%) isolates were resistant to at least one of the clinically used triazole antifungal agents. Prospective surveillance (2015-2017) shows 7/356 (2.2%) isolates were resistant to at least one triazole antifungals demonstrating an increase in incidence of triazole-resistant in our laboratory. Among five isolates collected from 2015 to 2017 and available for molecular testing, three harbored TR/L98H alteration in the gene that are associated with the acquisition of resistance in the non-patient environment. These data show that historically low prevalence of azole resistance may be increasing, warranting further surveillance of susceptible patients.
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http://dx.doi.org/10.3389/fmicb.2018.02234DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6158360PMC
September 2018

Diagnosing Emerging Fungal Threats: A One Health Perspective.

Front Genet 2018 11;9:376. Epub 2018 Sep 11.

Department of Infectious Disease Epidemiology, Imperial College London, London, United Kingdom.

Emerging fungal pathogens are a growing threat to global health, ecosystems, food security, and the world economy. Over the last century, environmental change and globalized transport, twinned with the increasing application of antifungal chemical drugs have led to increases in outbreaks of fungal diseases with sometimes catastrophic effects. In order to tackle contemporary epidemics and predemic threats, there is a pressing need for a unified approach in identification and monitoring of fungal pathogens. In this paper, we discuss current high throughput technologies, as well as new platforms capable of combining diverse data types to inform practical epidemiological strategies with a focus on emerging fungal pathogens of wildlife.
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http://dx.doi.org/10.3389/fgene.2018.00376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6141620PMC
September 2018

Climate structuring of infection in the threatened amphibians of the northern Western Ghats, India.

R Soc Open Sci 2018 Jun 13;5(6):180211. Epub 2018 Jun 13.

Ecology, Behaviour and Evolution Research Group, School of Biological and Marine Sciences, University of Plymouth, Drake Circus, Plymouth, Devon PL4 8AA, UK.

() is a pathogen killing amphibians worldwide. Its impact across much of Asia is poorly characterized. This study systematically surveyed amphibians for across rocky plateaus in the northern section of the Western Ghats biodiversity hotspot, India, including the first surveys of the plateaus in the coastal region. These ecosystems offer an epidemiological model system since they are characterized by differing levels of connectivity, edaphic and climatic conditions, and anthropogenic stressors. One hundred and eighteen individuals of 21 species of Anura and Apoda on 13 plateaus ranging from 67 to 1179 m above sea level and 15.89 to 17.92° North latitude were sampled. Using qPCR protocols, 79% of species and 27% of individuals tested were positive for . This is the first record of in caecilians in India, the Critically Endangered and Endangered . Mean site prevalence was 28.15%. Prevalence below the escarpment was 31.2% and 25.4% above. The intensity of infection (GE) showed the reverse pattern. Infection may be related to elevational temperature changes, thermal exclusion, inter-site connectivity and anthropogenic disturbance. Coastal plateaus may be thermal refuges from . Infected amphibians represented a wide range of ecological traits posing interesting questions about transmission routes.
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http://dx.doi.org/10.1098/rsos.180211DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6030269PMC
June 2018

High prevalence of triazole resistance in clinical Aspergillus fumigatus isolates in a specialist cardiothoracic centre.

Int J Antimicrob Agents 2018 Nov 10;52(5):637-642. Epub 2018 Aug 10.

Fungal Pathogens Laboratory, National Heart and Lung Institute, Imperial College London, UK; Department of Microbiology, Royal Brompton and Harefield NHS Foundation Trust, London, UK.

Objectives: To evaluate the prevalence of triazole-resistant Aspergillus fumigatus and common molecular cyp51A polymorphisms amongst clinical isolates in a specialised cardiothoracic centre in London, UK.

Methods: All A. fumigatus isolates were prospectively analysed from April 2014 to March 2016. Isolates were screened with a four-well VIPcheck™ plate to assess triazole susceptibility. Resistance was confirmed with a standard microbroth dilution method according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. Triazole-resistant A. fumigatus isolates were subjected to a mixed-format real time polymerase chain reaction (RT-PCR) assay (AsperGenius) to detect common cyp51A alterations.

Results: We identified 167 clinical A. fumigatus isolates from 135 patients. Resistance to at least one azole antifungal drug was confirmed in 22/167 (13.2%) of isolates from 18/135 (13.3%) patients, including 12/74 (16.2%) patients with cystic fibrosis (CF). The highest detection rate of azole-resistant A. fumigatus was among the 11- to 20-y age group. All triazole-resistant isolates (n = 22) were resistant to itraconazole, 18 showed cross-resistance to posaconazole and 10 displayed reduced susceptibility to voriconazole. No pan-azole-resistant A. fumigatus was identified. TR/L98H was identified in 6/22 (27.3%) of azole-resistant isolates and detectable in 5/12 (42%) patients with CF.

Conclusions: In our specialist cardiothoracic centre, the prevalence of triazole-resistant A. fumigatus is alarmingly high (13.2%). The majority of azole-resistant isolates were from patients with CF. We found a higher prevalence of the environmentally driven mutation TR/L98H in our A. fumigatus isolates than in published UK data from other specialist respiratory centres, which may reflect differing patient populations managed at these institutions.
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http://dx.doi.org/10.1016/j.ijantimicag.2018.08.004DOI Listing
November 2018

MARDy: Mycology Antifungal Resistance Database.

Bioinformatics 2018 09;34(18):3233-3234

MRC Centre for Outbreak Analysis and Modelling, Imperial College London, London, UK.

Summary: The increase of antifungal drug resistance is a major global human health concern and threatens agriculture and food security; in order to tackle these concerns, it is important to understand the mechanisms that cause antifungal resistance. The curated Mycology Antifungal Resistance Database (MARDy) is a web-service of antifungal drug resistance mechanisms, including amino acid substitutions, tandem repeat sequences and genome ploidy. MARDy is implemented on a Linux, Apache, MySQL and PHP web development platform and includes a local installation of BLASTn of the database of curated genes.

Availability And Implementation: MARDy can be accessed at http://www.mardy.net and is free to use. The complete database can be retrieved, ordered by organism, gene and drug. Missing or new mycological antifungal resistance data can be relayed to the development team through a contribute entry form. Updates and news will be publicized via a dedicated Twitter feed: @MARDYfungi.
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http://dx.doi.org/10.1093/bioinformatics/bty321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6137992PMC
September 2018

Author Correction: Genomic epidemiology of the UK outbreak of the emerging human fungal pathogen Candida auris.

Emerg Microbes Infect 2018 05 31;7(1):104. Epub 2018 May 31.

National Heart and Lung Institute, Imperial College London, London, SW3 6LR, UK.

Correction to: Emerging Microbes & Infections (2018) 7,43 https://doi.org/10.1038/s41426-018-0045-x ; published online 29 March 2018.
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http://dx.doi.org/10.1038/s41426-018-0098-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5981204PMC
May 2018

Breaching Pathogeographic Barriers by the Bat White-Nose Fungus.

mBio 2018 05 22;9(3). Epub 2018 May 22.

MRC Centre for Global Infectious Disease Analysis, Imperial College London, London, United Kingdom

Bat white-nose syndrome has become associated with unparalleled mortality in bat species across the United States since 2006. In a recent article, Drees and colleagues (mBio 8:e01941-17, 2017, https://doi.org/10.1128/mBio.01941-17) utilized both whole-genome sequencing and microsatellite data to explore the origin and spread of the causative agent of bat white-nose syndrome, The research by Drees et al. supports the hypothesis that was introduced into North America from Europe, with molecular dating suggesting a divergence from European isolates approximately 100 years ago. The approaches described in this study are an important contribution toward pinpointing the origins of this infection and underscore the need for more rigorous international biosecurity in order to stem the tide of emerging fungal pathogens.
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http://dx.doi.org/10.1128/mBio.00897-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5964353PMC
May 2018

The Cryptococcus neoformans Titan cell is an inducible and regulated morphotype underlying pathogenesis.

PLoS Pathog 2018 05 18;14(5):e1006978. Epub 2018 May 18.

Medical Research Council Centre for Medical Mycology at the University of Aberdeen, Aberdeen Fungal Group, Institute of Medical Sciences, Foresterhill, Aberdeen, United Kingdom.

Fungal cells change shape in response to environmental stimuli, and these morphogenic transitions drive pathogenesis and niche adaptation. For example, dimorphic fungi switch between yeast and hyphae in response to changing temperature. The basidiomycete Cryptococcus neoformans undergoes an unusual morphogenetic transition in the host lung from haploid yeast to large, highly polyploid cells termed Titan cells. Titan cells influence fungal interaction with host cells, including through increased drug resistance, altered cell size, and altered Pathogen Associated Molecular Pattern exposure. Despite the important role these cells play in pathogenesis, understanding the environmental stimuli that drive the morphological transition, and the molecular mechanisms underlying their unique biology, has been hampered by the lack of a reproducible in vitro induction system. Here we demonstrate reproducible in vitro Titan cell induction in response to environmental stimuli consistent with the host lung. In vitro Titan cells exhibit all the properties of in vivo generated Titan cells, the current gold standard, including altered capsule, cell wall, size, high mother cell ploidy, and aneuploid progeny. We identify the bacterial peptidoglycan subunit Muramyl Dipeptide as a serum compound associated with shift in cell size and ploidy, and demonstrate the capacity of bronchial lavage fluid and bacterial co-culture to induce Titanisation. Additionally, we demonstrate the capacity of our assay to identify established (cAMP/PKA) and previously undescribed (USV101) regulators of Titanisation in vitro. Finally, we investigate the Titanisation capacity of clinical isolates and their impact on disease outcome. Together, these findings provide new insight into the environmental stimuli and molecular mechanisms underlying the yeast-to-Titan transition and establish an essential in vitro model for the future characterization of this important morphotype.
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http://dx.doi.org/10.1371/journal.ppat.1006978DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5959070PMC
May 2018