Imperial College London

Professor Matthew Fisher

Faculty of MedicineSchool of Public Health

Professor of Fungal Disease Epidemiology
 
 
 
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Contact

 

+44 (0)20 7594 3787matthew.fisher Website

 
 
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Location

 

VC3Norfolk PlaceSt Mary's Campus

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Summary

 

Publications

Publication Type
Year
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177 results found

Sewell T, Zhu J, Rhodes J, Hagen F, Mels JF, Fisher M, Jombart Tet al., 2019, Non-random distribution of azole resistance across the global population of Aspergillus fumigatus, mBio, Vol: 10, ISSN: 2150-7511

The emergence of azole resistance in the pathogenic fungus Aspergillus fumigatus has continued to increase, with the dominant resistance mechanisms, consisting of a 34-nucleotide tandem repeat (TR34)/L98H and TR46/Y121F/T289A, now showing a structured global distribution. Using hierarchical clustering and multivariate analysis of 4,049 A. fumigatus isolates collected worldwide and genotyped at nine microsatellite loci using analysis of short tandem repeats of A. fumigatus (STRAf), we show that A. fumigatus can be subdivided into two broad clades and that cyp51A alleles TR34/L98H and TR46/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 A. fumigatus 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 A. fumigatus genetic relatedness. Our study highlights the effect that azole drug resistance is having on the genetic diversity of A. fumigatus and emphasizes its global importance upon this medically important pathogenic fungus.IMPORTANCE Azole drug resistance in the human-pathogenic fungus Aspergillus fumigatus 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 fungic

JOURNAL ARTICLE

Sewell TR, Zhang Y, Brackin AP, Shelton JMG, Rhodes J, Fisher MCet al., 2019, Elevated prevalence of azole resistant Aspergillus fumigatus in urban versus rural environments in the United Kingdom, Publisher: Cold Spring Harbor Laboratory

<jats:title>Abstract</jats:title><jats:p>Azole resistance in the opportunistic pathogen <jats:italic>Aspergillus fumigatus</jats:italic> is increasing, dominated primarily by two environmentally-associated resistance alleles: TR<jats:sub>34</jats:sub>/L98H and TR<jats:sub>46</jats:sub>/Y121F/T289A. Using an environmental sampling strategy across the South of England we assess the prevalence of azole resistant <jats:italic>A. fumigatus</jats:italic> (AR<jats:italic>Af</jats:italic>) in soil samples collected in both urban and rural locations. We characterise the susceptibility profiles of the resistant isolates to three medical azoles, identify the underlying genetic basis of resistance and investigate their genetic relationships. AR<jats:italic>Af</jats:italic> was detected in 6.7% of the soil samples, with a higher prevalence in urban (13.8%) compared to rural (1.1%) locations. Nineteen isolates were confirmed to exhibit clinical breakpoints for resistance to at least one of three medical azoles, with 18 isolates exhibiting resistance to itraconazole, four to voriconazole, with two also showing additional elevated minimum inhibitory concentration to posaconazole. Thirteen of the resistant isolates harboured the TR<jats:sub>34</jats:sub>/L98H resistance allele and six isolates carried TR<jats:sub>46</jats:sub>/Y121F/T289A allele. The 19 azole-resistant isolates were spread across five <jats:italic>csp1</jats:italic> genetic subtypes, t01, t02, t04B, t09 and t18 with t02 the predominant subtype. Our study demonstrates that AR<jats:italic>Af</jats:italic> can be easily isolated in the South of England, especially in urban city centres, which appear to play an important role in the epidemiology of environmentally-linked drug resistant <jats:italic>A. fumigatus</jats:italic>.</jats:p>

WORKING PAPER

Scheele BC, Pasmans F, Skerratt LF, Berger L, Martel A, Beukema W, Acevedo AA, Burrowes PA, Carvalho T, Catenazzi A, De la Riva I, Fisher MC, Flechas SV, Foster CN, Frías-Álvarez P, Garner TWJ, Gratwicke B, Guayasamin JM, Hirschfeld M, Kolby JE, Kosch TA, La Marca E, Lindenmayer DB, Lips KR, Longo AV, Maneyro R, McDonald CA, Mendelson J, Palacios-Rodriguez P, Parra-Olea G, Richards-Zawacki CL, Rödel M-O, Rovito SM, Soto-Azat C, Toledo LF, Voyles J, Weldon C, Whitfield SM, Wilkinson M, Zamudio KR, Canessa Set al., 2019, Amphibian fungal panzootic causes catastrophic and ongoing loss of biodiversity, Science, Vol: 363, Pages: 1459-1463, ISSN: 0036-8075

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.

JOURNAL ARTICLE

Stone NRH, Rhodes J, Fisher MC, Mfinanga S, Kivuyo S, Rugemalila J, Segal ES, Needleman L, Molloy SF, Kwon-Chung J, Harrison TS, Hope W, Berman J, Bicanic Tet al., 2019, Dynamic ploidy changes drive fluconazole resistance in human cryptococcal meningitis, Journal of Clinical Investigation, Vol: 129, Pages: 999-1014, ISSN: 0021-9738

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 hetero

JOURNAL ARTICLE

Yu L-S, Rodriguez-Manzano J, Malpartida-Cardenas K, Sewell T, Bader O, Armstrong-James D, Fisher MC, Georgiou Pet al., 2018, Rapid and sensitive detection of azole-resistant Aspergillus fumigatus by tandem-repeat loop-mediated isothermal amplification., Journal of Molecular Diagnostics, ISSN: 1525-1578

Invasive human fungal infections caused by multi-azole resistant Aspergillus fumigatus are associated with increasing rates of mortality in susceptible patients. Current methods of diagnosing infections caused by multi-azole 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 multi-azole 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.

JOURNAL ARTICLE

Abdolrasouli A, Scourfield A, Rhodes J, Shah A, Elborn JS, Fisher MC, Schelenz S, Armstrong-James Det al., 2018, High prevalence of triazole resistance in clinical Aspergillus fumigatus isolates in a specialist cardiothoracic centre, International Journal of Antimicrobial Agents, Vol: 52, Pages: 637-642, ISSN: 0924-8579

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. TR34/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 TR34/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.

JOURNAL ARTICLE

Farrer R, Ford C, Rhodes J, Delorey T, May R, Fisher M, Cloutman-Green E, Balloux F, Cuomo Cet al., 2018, Transcriptional heterogeneity of Cryptococcus gattii VGII compared with non-VGII lineages underpins key pathogenicity pathways, mSphere, Vol: 3, ISSN: 2379-5042

Cryptococcus gattii 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 RNA-seq experiment for four lineages of C. gattii (VGI-IV) interacting with mouse macrophages at 1hr, 3hr and 6hr post infection. Comparison of in vitro to ex vivo gene expression indicates lineage VGII is transcriptionally divergent to non-VGII lineages, including differential expression of genes involved in capsule synthesis, capsule attachment and ergosterol production. Various paralogs demonstrate sub-functionalisation between lineages including an upregulation of capsule biosynthesis-related gene CAP2, and downregulation of CAP1 in VGIII. Isolates also compensate for lineage-specific gene-losses by over-expression of genetically similar paralogs, including an over-expression of capsule gene CAS3 in VGIV having lost CAS31. Differential expression of one in five C. gattii genes was detected following co-incubation with mouse macrophages; all isolates showed high induction of oxidative-reduction functions and a downregulation of capsule attachment genes. We also show that VGII switches expression of two laccase paralogs (from LAC1 to LAC2) during co-incubation of macrophages. Finally, we found that mouse macrophages respond to all four lineages of C. gattii by upregulating FosB/Jun/Egr1 regulatory proteins at early time points. This study highlights the evolutionary breadth of expression profiles amongst the lineages of C. gattii and the diversity of transcriptional responses at this host-pathogen interface.

JOURNAL ARTICLE

Farrer RA, Ford CB, Rhodes J, Delorey T, May RC, Fisher MC, Cloutman-Green E, Balloux F, Cuomo CAet al., 2018, Transcriptional Heterogeneity of Cryptococcus gattii VGII Compared with Non-VGII Lineages Underpins Key Pathogenicity Pathways., mSphere, Vol: 3

Cryptococcus gattii 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 C. gattii (lineages VGI to IV) interacting with mouse macrophages at 1, 3, and 6 h postinfection. Comparisons of in vitro to ex vivo 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 CAP2 and downregulation of CAP1 in VGIII. Isolates also compensate for lineage-specific gene losses by overexpression of genetically similar paralogs, including overexpression of capsule gene CAS3 in VGIV, which have lost the CAS31 gene. Differential expression of one in five C. gattii 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 LAC1 to LAC2) during coincubation of macrophages. Finally, we found that mouse macrophages respond to all four lineages of C. gattii by upregulating FosB/Jun/Egr1 regulatory proteins at early time points. This report highlights the evolutionary breadth of expression profiles among the lineages of C. gattii and the diversity of transcriptional responses at this host-pathogen interface.IMPORTANCE The transcriptional profiles of related pathogens and their responses to host-induced stresses underp

JOURNAL ARTICLE

Abdolrasouli A, Petrou MA, Park H, Rhodes J, Rawson T, Moore L, Donaldson H, Holmes A, Fisher M, Armstrong-James Det al., 2018, Surveillance for azole-resistant Aspergillus fumigatus in a centralized diagnostic mycology service, London, United Kingdom, 1998-2017, Frontiers in Microbiology, Vol: 9, ISSN: 1664-302X

Background/Objectives: Aspergillus fumigatus 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.Methods: A large number (n = 1469) of clinical A. fumigatus 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.Results: 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 A. fumigatus in our laboratory. Among five isolates collected from 2015 to 2017 and available for molecular testing, three harbored TR34/L98H alteration in the cyp51A gene that are associated with the acquisition of resistance in the non-patient environment.Conclusion: These data show that historically low prevalence of azole resistance may be increasing, warranting further surveillance of susceptible patients.

JOURNAL ARTICLE

Nash A, Sewell T, Farrer R, Abdolrasouli A, Shelton J, Fisher M, Rhodes JLet al., 2018, MARDy: mycology antifungal resistance database, Bioinformatics, Vol: 34, Pages: 3233-3234, ISSN: 1367-4803

Summary:The increase of antifungal drug resistance is a major global human health concern andthreatens agriculture and food security; in order to tackle these concerns, it is important to understandthe 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 andPHP web development platform and includes a local installation of BLASTn of the database of curatedgenes.Availability and implementation:MARDy can be accessed at http://www.mardy.net and is free touse. The complete database can be retrieved, ordered by organism, gene and drug. Missing or newmycological antifungal resistance data can be relayed to the development team through a contribute entryform.

JOURNAL ARTICLE

Ghosh PN, Fisher MC, Bates KA, 2018, Diagnosing Emerging Fungal Threats: A One Health Perspective, FRONTIERS IN GENETICS, Vol: 9, ISSN: 1664-8021

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.

JOURNAL ARTICLE

Canessa S, Bozzuto C, Campbell Grant EH, Cruickshank SS, Fisher MC, Koella JC, Lötters S, Martel A, Pasmans F, Scheele BC, Spitzen-van der Sluijs A, Steinfartz S, Schmidt BRet al., 2018, Decision-making for mitigating wildlife diseases: From theory to practice for an emerging fungal pathogen of amphibians, Journal of Applied Ecology, Vol: 55, Pages: 1987-1996, ISSN: 0021-8901

Conservation science can be most effective in its decision-support role when seeking answers to clearly formulated questions of direct management relevance. Emerging wildlife diseases, a driver of global biodiversity loss, illustrate the challenges of performing this role: in spite of considerable research, successful disease mitigation is uncommon. Decision analysis is increasingly advocated to guide mitigation planning, but its application remains rare. Using an integral projection model, we explored potential mitigation actions for avoiding population declines and the ongoing spatial spread of the fungus Batrachochytrium salamandrivorans (Bsal). This fungus has recently caused severe amphibian declines in north-western Europe and currently threatens Palearctic salamander diversity. Available evidence suggests that a Bsal outbreak in a fire salamander (Salamandra salamandra) population will lead to its rapid extirpation. Treatments such as antifungals or probiotics would need to effectively interrupt transmission (reduce probability of infection by nearly 90%) in order to reduce the risk of host extirpation and successfully eradicate the pathogen. Improving the survival of infected hosts is most likely to be detrimental as it increases the potential for pathogen transmission and spread. Active removal of a large proportion of the host population has some potential to locally eradicate Bsal and interrupt its spread, depending on the presence of Bsal reservoirs and on the host's spatial dynamics, which should therefore represent research priorities. Synthesis and applications. Mitigation of Batrachochytrium salamandrivorans epidemics in susceptible host species is highly challenging, requiring effective interruption of transmission and radical removal of host individuals. More generally, our study illustrates the advantages of framing conservation science directly in the management decision context, rather than adapting to it a posteriori.

JOURNAL ARTICLE

Abdolrasouli A, Bercusson AC, Rhodes JL, Hagen F, Buil JB, Tang AYY, de Boer LL, Shah A, Milburn AJ, Elborn JS, Jones AL, Meis JF, Fisher MC, Schelenz S, Simmonds NJ, Armstrong-James Det al., Airway persistence by the emerging multi-azole-resistant Rasamsonia argillacea complex in cystic fibrosis, Mycoses, ISSN: 0933-7407

Infections caused by Rasamsonia argillacea complex have been reported in various clinical settings. Cystic fibrosis (CF) is one of the main underlying conditions. An observational cohort study of CF patients with Rasamsonia in respiratory samples was conducted. Eight isolates from six patients were identified as R. argillacea complex and tested for antifungal susceptibility. All isolates had high MICs to voriconazole and posaconazole and low MECs to echinocandins. Four patients experienced lung function decline in the year preceding first Rasamsonia isolation. This continued in the year following first isolation in three out of four cases. Antifungal therapy was initiated in two patients, to which only one exhibited a clinical response. Three out of six patients died within three years of isolating Rasamsonia. Genotyping suggests that similar genotypes of Rasamsonia can persist in CF airways. Consistent with other fungi in CF, the clinical impact of airway colonization by Rasamsonia is variable. In certain patients, Rasamsonia may be able to drive clinical decline. In others, though a clear impact on lung function may be difficult to determine, the appearance of Rasamsonia acts as a marker of disease severity. In others it does not appear to have an obvious clinical impact on disease progression. This article is protected by copyright. All rights reserved.

JOURNAL ARTICLE

Fisher MC, 2018, Epidemiological definitions, terminology and classifications with reference to fungal infections of animals, Emerging and Epizootic Fungal Infections in Animals, Pages: 17-27, ISBN: 9783319720913

© Springer International Publishing AG, part of Springer Nature 2018. Emerging infections caused by fungi have become a widely recognised global phenomenon in animal species and populations worldwide. This chapter details the vocabulary and grammar that is used to discuss such infections. Much of this terminology is specific to the field of mycology, and careful usage is required in the scientific literature and discussion in order to maintain clarity of expression.

BOOK CHAPTER

Martel A, Pasmans F, Fisher MC, Grogan LF, Skerratt LF, Berger Let al., 2018, Chytridiomycosis, Emerging and Epizootic Fungal Infections in Animals, Pages: 309-335, ISBN: 9783319720913

© Springer International Publishing AG, part of Springer Nature 2018. The amphibian fungal disease chytridiomycosis is considered one of the greatest threats to biodiversity. This lethal skin disease is caused by chytridiomycete fungi belonging to the genus Batrachochytrium. Although sudden amphibian population declines had occurred since the 1970s in the Americas and Australia, mass mortalities were not observed until the 1990s. The fungus Batrachochytrium dendrobatidis (Bd) was identified as the cause of these declines. It is estimated that Bd has caused the rapid decline or extinction of at least 200 amphibian species, which is probably an underestimation due to the cryptic behaviour of many amphibians such as many salamanders and also the lack of monitoring. A second chytrid species, B. salamandrivorans (Bsal), has recently emerged and caused mass mortality in salamanders in Belgium, the Netherlands and Germany, affecting most salamander and newt taxa in the amphibian community and is considered a major threat to the western Palearctic amphibian biodiversity. In this chapter we review the epidemiology, host pathogen interactions and mitigation strategies of both chytrid pathogens.

BOOK CHAPTER

Thorpe CJ, Lewis TR, Fisher MC, Wierzbicki CJ, Kulkarni S, Pryce D, Davies L, Watve A, Knight MEet al., 2018, Climate structuring of Batrachochytrium dendrobatidis infection in the threatened amphibians of the northern Western Ghats, India, Royal Society Open Science, Vol: 5, ISSN: 2054-5703

Batrachochytrium dendrobatidis (Bd) is a pathogen killing amphibians worldwide. Its impact across much of Asia is poorly characterized. This study systematically surveyed amphibians for Bd 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 Bd. This is the first record of Bd in caecilians in India, the Critically Endangered Xanthophryne tigerina and Endangered Fejervarya cf. sahyadris. 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 Bd. Infected amphibians represented a wide range of ecological traits posing interesting questions about transmission routes.

JOURNAL ARTICLE

Cremin Í, Watson O, Heffernan A, Imai N, Ahmed N, Bivegete S, Kimani T, Kyriacou D, Mahadevan P, Mustafa R, Pagoni P, Sophiea M, Whittaker C, Beacroft L, Riley S, Fisher Met al., 2018, An infectious way to teach students about outbreaks, Epidemics, Vol: 23, Pages: 42-48, ISSN: 1755-4365

The study of infectious disease outbreaks is required to train today’s epidemiologists. A typical way to introduce and explain key epidemiological concepts is through the analysis of a historical outbreak. There are, however, few training options that explicitly utilise real-time simulated stochastic outbreaks where the participants themselves comprise the dataset they subsequently analyse. In this paper, we present a teaching exercise in which an infectious disease outbreak is simulated over a five-day period and subsequently analysed. We iteratively developed the teaching exercise to offer additional insight into analysing an outbreak. An R package for visualisation, analysis and simulation of the outbreak data was developed to accompany the practical to reinforce learning outcomes. Computer simulations of the outbreak revealed deviations from observed dynamics, highlighting how simplifying assumptions conventionally made in mathematical models often differ from reality. Here we provide a pedagogical tool for others to use and adapt in their own settings.

JOURNAL ARTICLE

Rhodes J, Abdolrasouli A, Farrer RA, Cuomo CA, Aanensen DM, Armstrong-James D, Fisher MC, Schelenz Set al., 2018, Genomic epidemiology of the UK outbreak of the emerging human fungal pathogen Candida auris (vol 7, pg 43, 2018), EMERGING MICROBES & INFECTIONS, Vol: 7, ISSN: 2222-1751

JOURNAL ARTICLE

Rhodes J, Fisher MC, 2018, Breaching pathogeographic barriers by the bat white-nose fungus, mBio, Vol: 9, ISSN: 2150-7511

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, Pseudogymnoascus destructans The research by Drees et al. supports the hypothesis that P. destructans 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.

JOURNAL ARTICLE

Dambuza IM, Drake T, Chapuis A, Zhou X, Correia J, Taylor-Smith L, LeGrave N, Rasmussen T, Fisher MC, Bicanic T, Harrison TS, Jaspars M, May RC, Brown GD, Yuecel R, MacCallum DM, Ballou ERet al., 2018, The Cryptococcus neoformans Titan cell is an inducible and regulated morphotype underlying pathogenesis, PLoS Pathogens, Vol: 14, ISSN: 1553-7366

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.

JOURNAL ARTICLE

Fisher MC, Hawkins NJ, Sanglard D, Gurr SJet al., 2018, Worldwide emergence of resistance to antifungal drugs challenges human health and food security, Science, Vol: 360, Pages: 739-742, ISSN: 0036-8075

The recent rate of emergence of pathogenic fungi that are resistant to the limited number of commonly used antifungal agents is unprecedented. The azoles, for example, are used not only for human and animal health care and crop protection but also in antifouling coatings and timber preservation. The ubiquity and multiple uses of azoles have hastened the independent evolution of resistance in many environments. One consequence is an increasing risk in human health care from naturally occurring opportunistic fungal pathogens that have acquired resistance to this broad class of chemicals. To avoid a global collapse in our ability to control fungal infections and to avoid critical failures in medicine and food security, we must improve our stewardship of extant chemicals, promote new antifungal discovery, and leverage emerging technologies for alternative solutions.

JOURNAL ARTICLE

Fisher MC, Ghosh P, Shelton JMG, Bates K, Brookes L, Wierzbicki C, Rosa GM, Farrer RA, Aanensen DM, Alvarado-Rybak M, Bataille A, Berger L, Boell S, Bosch J, Clare FC, Courtois EA, Crottini A, Cunningham AA, Doherty-Bone TM, Gebresenbet F, Gower DJ, Hoglund J, James TY, Jenkinson TS, Kosch TA, Lambertini C, Laurila A, Lin C-F, Loyau A, Martel A, Meurling S, Miaud C, Minting P, Ndriantsoa S, O'Hanlon SJ, Pasmans F, Rakotonanahary T, Rabemananjara FCE, Ribeiro LP, Schmeller DS, Schmidt BR, Skerratt L, Smith F, Soto-Azat C, Tessa G, Toledo LF, Valenzuela-Sanchez A, Verster R, Voeroes J, Waldman B, Webb RJ, Weldon C, Wombwell E, Zamudio KR, Longcore JE, Garner TWJet al., 2018, Development and worldwide use of non-lethal, and minimal population-level impact, protocols for the isolation of amphibian chytrid fungi, Scientific Reports, Vol: 8, ISSN: 2045-2322

Parasitic chytrid fungi have emerged as a significant threat to amphibian species worldwide, necessitating the development of techniques to isolate these pathogens into culture for research purposes. However, early methods of isolating chytrids from their hosts relied on killing amphibians. We modified a pre-existing protocol for isolating chytrids from infected animals to use toe clips and biopsies from toe webbing rather than euthanizing hosts, and distributed the protocol to researchers as part of the BiodivERsA project RACE; here called the RML protocol. In tandem, we developed a lethal procedure for isolating chytrids from tadpole mouthparts. Reviewing a database of use a decade after their inception, we find that these methods have been applied across 5 continents, 23 countries and in 62 amphibian species. Isolation of chytrids by the non-lethal RML protocol occured in 18% of attempts with 207 fungal isolates and three species of chytrid being recovered. Isolation of chytrids from tadpoles occured in 43% of attempts with 334 fungal isolates of one species (Batrachochytrium dendrobatidis) being recovered. Together, these methods have resulted in Non-lethal isolation of chytrids from amphibiansa si gnificant reduction and refinement of our use of threatened amphibian species and have improved our ability to work with this group of emerging pathogens.

JOURNAL ARTICLE

O'Hanlon SJ, Rieux A, Farrer RA, Rosa GM, Waldman B, Bataille A, Kosch TA, Murray KA, Brankovics B, Fumagalli M, Martin MD, Wales N, Alvarado-Rybak M, Bates KA, Berger L, Boell S, Brookes L, Clare F, Courtois EA, Cunningham AA, Doherty-Bone TM, Ghosh P, Gower DJ, Hintz WE, Hoglund J, Jenkinson TS, Lin C-F, Laurila A, Loyau A, Martel A, Meurling S, Miaud C, Minting P, Pasmans F, Schmeller DS, Schmidt BR, Shelton JMG, Skerratt LF, Smith F, Soto-Azat C, Spagnoletti M, Tessa G, Toledo LF, Valenzuela-Sanchez A, Verster R, Voros J, Webb RJ, Wierzbicki C, Wombwell E, Zamudio KR, Aanensen DM, James TY, Gilbert MTP, Weldon C, Bosch J, Balloux F, Garner TWJ, Fisher MCet al., 2018, Recent Asian origin of chytrid fungi causing global amphibian declines, SCIENCE, Vol: 360, Pages: 621-+, ISSN: 0036-8075

JOURNAL ARTICLE

Colley T, Sehra G, Chowdhary A, Alanio A, Kelly SL, Kizawa Y, Armstrong-James D, Fisher MC, Warrilow AGS, Parker JE, Kelly DE, Kimura G, Nishimoto Y, Sunose M, Onions S, Crepin D, Lagasse F, Crittall M, Shannon J, McConville M, King-Underwood J, Naylor A, Bretagne S, Murray J, Ito K, Strong P, Rapeport Get al., 2018, In vitro and in vivo efficacy of a novel and long acting fungicidal azole, PC1244 on Aspergillus fumigatus infection, Antimicrobial Agents and Chemotherapy, Vol: 62, ISSN: 0066-4804

The antifungal effects of the novel triazole, PC1244, designed for topical or inhaled administration, againstA. fumigatushave been tested in a range ofin vitroandin vivostudies. PC1244 demonstrated potent antifungal activities against clinicalA. fumigatusisolates (N=96) with a MIC range of 0.016--0.25 μg/ml, whereas the MIC range for voriconazole was 0.25--0.5 μg/ml. PC1244 was a strong tight-binding inhibitor of recombinantA. fumigatusCYP51A and CYP51B (sterol 14α-demethylase) enzymes and strongly inhibited ergosterol synthesis inA. fumigatuswith an IC50of 8 nM. PC1244 was effective against a broad spectrum of pathogenic fungi (MIC ranged from <0.0078∼2 μg/ml), especially onAspergillus terreus,Trichophyton rubrum,Candida albicans,Candida glabrata,Candida krusei,Cryptococcus gattii,Cryptococcus neoformans and Rhizopus oryzaePC1244 also proved to be quickly absorbed into bothA. fumigatushyphae and bronchial epithelial cells, producing persistent antifungal effects. In addition, PC1244 showed fungicidal activity (MFC, 2 μg/ml), which was 8-fold more potent than voriconazole.In vivo, once daily intranasal administration of PC1244 (3.2 ∼ 80μg/mL) to temporarily neutropenic, immunocompromised mice 24h after inoculation with itraconazole-susceptibleA. fumigatussubstantially reduced fungal load in the lung, galactomannan in serum and circulating inflammatory cytokines. Furthermore, 7 days extended prophylaxis with PC1244 showed superiorin vivoeffects when compared against 1 day of prophylactic treatment, suggesting accumulation of the effects of PC1244. Thus, PC1244 has the potential to be a novel therapy for the treatment ofA. fumigatusinfection in the lungs of humans.

JOURNAL ARTICLE

Rhodes JL, Abdolrasouli A, Farrer R, Cuomo C, Aanensen D, Armstrong-James D, Fisher M, Schelenz Set al., 2018, Genomic epidemiology of the UK outbreak of the emerging human fungal pathogen Candida auris, Emerging Microbes and Infections, Vol: 7, ISSN: 2222-1751

Candida auris was first described in 2009, and has since caused nosocomial outbreaks, invasive infections and fungaemia across at least 19 countries in five continents. An outbreak of C. auris occurred in a specialised cardiothoracic London hospital between April 2015 and November 2016, which to date has been the largest outbreak within the UK, involving a total of 72 patients. To understand the genetic epidemiology of C. auris infection, both within this hospital and within a global context, we sequenced the outbreak isolate genomes using Oxford Nanopore Technologies and Illumina to detect antifungal resistance alleles and to reannotate the C. auris genome. Phylogenomic analysis placed the UK outbreak in the India/Pakistan clade, demonstrating an Asian origin: the outbreak showed similar genetic diversity to that of the entire clade and limited local spatiotemporal clustering was observed. One isolate displayed resistance to both echinocandins and 5-flucytosine; the former was associated with a serine to tyrosine amino acid substitution in the gene FKS1, and the latter was associated with a phenylalanine to isoleucine substitution in the gene FUR1. These mutations add to a growing body of research on multiple antifungal drug targets in this organism. Multiple differential episodic selection of antifungal resistant genotypes has occurred within a genetically heterogenous population across this outbreak, creating a resilient pathogen and making it difficult to define local-scale patterns of transmission as well as implementing outbreak control measures.

JOURNAL ARTICLE

Rhodes J, Abdolrasouli A, Farrer R, Cuomo C, Aanensen D, Armstrong-James D, Fisher M, Schelenz Set al., 2018, Rapid genome sequencing for outbreak analysis of the emerging human fungal pathogen Candida auris, Emerging Microbes and Infections, Vol: 7, ISSN: 2222-1751

Candida auris was first described in 2009, and it has since caused nosocomial outbreaks, invasive infections, and fungaemia across at least 19 countries on five continents. An outbreak of C. auris occurred in a specialized cardiothoracic London hospital between April 2015 and November 2016, which to date has been the largest outbreak in the UK, involving a total of 72 patients. To understand the genetic epidemiology of C. auris infection both within this hospital and within a global context, we sequenced the outbreak isolate genomes using Oxford Nanopore Technologies and Illumina platforms to detect antifungal resistance alleles and reannotate the C. auris genome. Phylogenomic analysis placed the UK outbreak in the India/Pakistan clade, demonstrating an Asian origin; the outbreak showed similar genetic diversity to that of the entire clade, and limited local spatiotemporal clustering was observed. One isolate displayed resistance to both echinocandins and 5-flucytosine; the former was associated with a serine to tyrosine amino acid substitution in the gene FKS1, and the latter was associated with a phenylalanine to isoleucine substitution in the gene FUR1. These mutations add to a growing body of research on multiple antifungal drug targets in this organism. Multiple differential episodic selection of antifungal resistant genotypes has occurred within a genetically heterogenous population across this outbreak, creating a resilient pathogen and making it difficult to define local-scale patterns of transmission and implement outbreak control measures.

JOURNAL ARTICLE

Bates K, Clare F, O'Hanlon S, Bosch J, Brookes L, McLaughlin E, Daniel O, Garner T, Fisher M, Harrison Xet al., 2018, Amphibian chytridiomycosis outbreak dynamics are linked with host skin bacterial community structure, Nature Communications, Vol: 9, ISSN: 2041-1723

Host-associated microbes are vital for combatting infections and maintaining health. In amphibians, certain skin-associated bacteria inhibit the fungal pathogen Batrachochytrium dendrobatidis (Bd), yet our understanding of host microbial ecology and its role in disease outbreaks is limited. We sampled skin-associated bacteria and Bd from Pyrenean midwife toad populations exhibiting enzootic or epizootic disease dynamics. We demonstrate that bacterial communities differ between life stages with few shared taxa, indicative of restructuring at metamorphosis. We detected a significant effect of infection history on metamorph skin microbiota, with reduced bacterial diversity in epizootic populations and differences in community structure and predicted function. Genome sequencing of Bd isolates supports a single introduction to the Pyrenees and reveals no association between pathogen genetics and epidemiological trends. Our findings provide an ecologically relevant insight into the microbial ecology of amphibian skin and highlight the relative importance of host microbiota and pathogen genetics in predicting disease outcome.

JOURNAL ARTICLE

Fisher M, Ghosh P, Shelton J, Bates K, Brookes L, Wierzbicki C, Rosa G, Farrer R, Aanensen D, Alvarado-Rybak M, Bataille A, Berger L, Boell S, Bosch J, Clare F, Courtois E, Crottini A, Cunningham A, Doherty-Bone T, Gebresenbet F, Gower D, Hoglund J, Jenkinson T, Kosch T, James T, Lambertini C, Laurila A, Lin C-F, Loyau A, Martel A, Meurling S, Miaud C, Minting P, Ndriantsoa S, Ribeiro L, Ribeiro L, Pasmans F, Rakotonanahary T, Rabemananjara F, Schmeller D, Schmidt B, Skerratt L, Smith F, Soto-Azat C, Tessa G, Toledo LF, Valenzuela-Sanchez A, Verster R, Voros J, Waldman B, Webb R, Weldon C, Wombwell E, Zamudio K, Longcore J, Garner Tet al., 2018, Development and worldwide use of a non-lethal and minimal population-level impact protocols for the isolation of chytrids from amphibians, Publisher: bioRxiv

Parasitic chytrid fungi have emerged as a significant threat to amphibian species worldwide, necessitating the development of techniques to isolate these pathogens into sterile culture for research purposes. However, early methods of isolating chytrids from their hosts relied on killing amphibians. We modified a pre-existing protocol for isolating chytrids from infected animals to use toe clips and biopsies from toe webbing rather than euthanizing hosts, and distributed the protocol to interested researchers worldwide as part of the BiodivERsA project RACE; here called the RML protocol. In tandem, we developed a lethal procedure for isolating chytrids from tadpole mouthparts. Reviewing a database of use a decade after their inception, we find that these methods have been widely applied across at least 5 continents, 23 countries and in 62 amphibian species, and have been successfully used to isolate chytrids in remote field locations. Isolation of chytrids by the non-lethal RML protocol occured in 18% of attempts with 207 fungal isolates and three species of chytrid being recovered. Isolation of chytrids from tadpoles occured in 43% of attempts with 334 fungal isolates of one species (Batrachochytrium dendrobatidis) being recovered. Together, these methods have resulted in a significant reduction and refinement of our use of threatened amphibian species and have improved our ability to work with this important group of emerging fungal pathogens.

WORKING PAPER

Valenzuela-Sánchez A, O'Hanlon SJ, Alvarado-Rybak M, Uribe-Rivera DE, Cunningham AA, Fisher MC, Soto-Azat Cet al., 2017, Genomic epidemiology of the emerging pathogen Batrachochytrium dendrobatidis from native and invasive amphibian species in Chile., Transboundary and Emerging Diseases, ISSN: 0931-184X

Emerging fungal diseases represent a threat to food security, animal and human health worldwide. Amphibian chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), has been associated with catastrophic and well-documented amphibian population declines and extinctions. For the first time, Bd was cultured from native and non-native wild amphibians in Chile. Phylogenomic analyses revealed that Chilean isolates AVS2, AVS4 and AVS7 group within the global panzootic lineage of Bd (BdGPL) in a single highly supported clade that includes a genotype previously isolated from the United Kingdom. Our results extend the known distribution of BdGPL in South America and suggest a single and relatively recent introduction of BdGPL into the country, providing additional support to the role of anthropogenic activity in the global spread of this panzootic lineage.

JOURNAL ARTICLE

Fernandez-Loras A, Fernandez-Beaskoetxea S, Arriero E, Fisher MC, Bosch Jet al., 2017, Early exposure to Batrachochytrium dendrobatidis causes profound immunosuppression in amphibians, EUROPEAN JOURNAL OF WILDLIFE RESEARCH, Vol: 63, ISSN: 1612-4642

Fungal pathogens have evolved a broad suite of strategies aiming at evading the host immune response. Amphibians are globally infected by the panzootic chytrid fungus Batrachochytrium dendrobatidis (Bd) and, while robust innate immune defences have been characterised, there is little evidence for the existence of effective adaptive immunity. We determine the immune response of the common midwife toad following challenge by Bd as larvae. Immune function was described for both the cell-mediated and antibody-mediated immune responses following infectious challenge as larval amphibians. While there were no significant differences in the ratio of neutrophils/lymphocytes between infected and uninfected individuals, early exposure of tadpoles to Bd significantly dampened the levels of circulating immunoglobulins (IgM and IgY) in the serum of juveniles after metamorphosis. Our results show that Bd immunosuppresses amphibians when infection occurs as larvae with potentially broad effects on the remodelling of immunity during metamorphosis.

JOURNAL ARTICLE

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