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Candida haemulonii complex species can be multidrug-resistant and cause infections such as candidemia. This study determined the genetic relationship between isolates from Brazil and the United States through whole-genome sequencing and performed antifungal susceptibility testing to investigate drug resistance. Contrary to what is widely described, most isolates were susceptible to azoles. However, an atypical susceptibility profile was found in 50% of Candida pseudohaemulonii strains, including resistance to the three echinocandins. Isolates from both countries formed distinct clusters with wide genetic diversity. Isolates from three hospitals in Brazil were clonal and involved in candidemia cases, pointing to the importance of improving hospital infection control measures and molecular identification.

Genomic analyses are widely applied to epidemiological, population genetic and experimental studies of pathogenic fungi. A wide range of methods are employed to carry out these analyses, typically without including controls that gauge the accuracy of variant prediction. The importance of tracking outbreaks at a global scale has raised the urgency of establishing high-accuracy pipelines that generate consistent results between research groups. To evaluate currently employed methods for whole-genome variant detection and elaborate best practices for fungal pathogens, we compared how 14 independent variant calling pipelines performed across 35 Candida auris isolates from 4 distinct clades and evaluated the performance of variant calling, single-nucleotide polymorphism (SNP) counts and phylogenetic inference results. Although these pipelines used different variant callers and filtering criteria, we found high overall agreement of SNPs from each pipeline. This concordance correlated with site quality, as SNPs discovered by a few pipelines tended to show lower mapping quality scores and depth of coverage than those recovered by all pipelines. We observed that the major differences between pipelines were due to variation in read trimming strategies, SNP calling methods and parameters, and downstream filtration criteria. We calculated specificity and sensitivity for each pipeline by aligning three isolates with chromosomal level assemblies and found that the GATK-based pipelines were well balanced between these metrics. Selection of trimming methods had a greater impact on SAMtools-based pipelines than those using GATK. Phylogenetic trees inferred by each pipeline showed high consistency at the clade level, but there was more variability between isolates from a single outbreak, with pipelines that used more stringent cutoffs having lower resolution. This project generated two truth datasets useful for routine benchmarking of C. auris variant calling, a consensus VCF of genotypes discovered by 10 or more pipelines across these 35 diverse isolates and variants for 2 samples identified from whole-genome alignments. This study provides a foundation for evaluating SNP calling pipelines and developing best practices for future fungal genomic studies.

Aspergillus fumigatus, an environmental mold, causes life-threatening infections. Studies on the phylogenetic structure of human clinical A. fumigatus isolates are limited. Here, we performed whole genome sequencing of 24 A. fumigatus isolates collected from 18 patients in U.S. healthcare facilities in California. Single-nucleotide polymorphism (SNP) differences between patient isolates ranged from 187 to 70 829 SNPs. For five patients with multiple isolates, we calculated the within-host diversities. Three patients had a within-host diversity that ranged from 4 to 10 SNPs and two patients ranged from 2 to 16 977 SNPs. Findings revealed highly diverse A. fumigatus strains among patients and two patterns of diversity for isolates that come from the same patient, low and extremely high diversity.

BACKGROUND Candida auris, a multidrug-resistant yeast, can spread rapidly in ventilator-capable skilled-nursing facilities (vSNFs) and long-term acute care hospitals (LTACHs). In 2018, a laboratory serving LTACHs in southern California began identifying species of Candida that were detected in urine specimens to enhance surveillance of C auris, and C auris was identified in February 2019 in a patient in an Orange County (OC), California, LTACH. Further investigation identified C auris at 3 associated facilities. OBJECTIVE To assess the prevalence of C auris and infection prevention and control (IPC) practices in LTACHs and vSNFs in OC. DESIGN Point prevalence surveys (PPSs), postdischarge testing for C auris detection, and assessments of IPC were done from March to October 2019. SETTING All LTACHs (n = 3) and vSNFs (n = 14) serving adult patients in OC. PARTICIPANTS Current or recent patients in LTACHs and vSNFs in OC. INTERVENTION In facilities where C auris was detected, PPSs were repeated every 2 weeks. Ongoing IPC support was provided. MEASUREMENTS Antifungal susceptibility testing and whole-genome sequencing to assess isolate relatedness. RESULTS Initial PPSs at 17 facilities identified 44 additional patients with C auris in 3 (100%) LTACHs and 6 (43%) vSNFs, with the first bloodstream infection reported in May 2019. By October 2019, a total of 182 patients with C auris were identified by serial PPSs and discharge testing. Of 81 isolates that were sequenced, all were clade III and highly related. Assessments of IPC identified gaps in hand hygiene, transmission-based precautions, and environmental cleaning. The outbreak was contained to 2 facilities by October 2019. LIMITATION Acute care hospitals were not assessed, and IPC improvements over time could not be rigorously evaluated. CONCLUSION Enhanced laboratory surveillance and prompt investigation with IPC support enabled swift identification and containment of C auris. PRIMARY FUNDING SOURCE Centers for Disease Control and Prevention.

Coccidioidomycosis is a fungal infection endemic to hot, arid regions of the western United States, northern Mexico, and parts of Central and South America. Sporadic cases outside these regions are likely travel-associated; alternatively, an infection could be acquired in as-yet unidentified newly endemic locales. A previous study of cases in nonendemic regions with patient self-reported travel history suggested that infections were acquired during travel to endemic regions. We sequenced 19 Coccidioides isolates from patients with known travel histories from that earlier investigation and performed phylogenetic analysis to identify the locations of potential source populations. Our results show that those isolates were phylogenetically linked to Coccidioides subpopulations naturally occurring in 1 of the reported travel locales, confirming that these cases were likely acquired during travel to endemic regions. Our findings demonstrate that genomic analysis is a useful tool for investigating travel-related coccidioidomycosis.

Background Outbreaks of healthcare-associated mucormycosis (HCM), a life-threatening fungal infection, have been attributed to multiple sources, including contaminated healthcare linens. In 2020, staff at Hospital A in Arkansas alerted public health officials of a potential HCM outbreak. Methods We collected data on patients at Hospital A who had invasive mucormycosis during January 2017–June 2021 and calculated annual incidence of HCM (defined as mucormycosis diagnosed within ≥7 days after hospital admission). We performed targeted environmental assessments, including linen sampling at the hospital, to identify potential sources of infection. Results During the outbreak period (June 2019–June 2021), 16 patients had HCM; clinical features were similar between HCM patients and non-HCM patients. Hospital-wide HCM incidence (per 100 000 patient-days) increased from 0 in 2018 to 3 in 2019 and 6 in 2020. For the 16 HCM patients, the most common underlying medical conditions were hematologic malignancy (56%) and recent traumatic injury (38%); 38% of HCM patients died in-hospital. Healthcare-associated mucormycosis cases were not epidemiologically linked by common procedures, products, units, or rooms. At Hospital A and its contracted offsite laundry provider, suboptimal handling of laundered linens and inadequate environmental controls to prevent mucormycete contamination were observed. We detected Rhizopus on 9 (9%) of 98 linens sampled at the hospital, including on linens that had just arrived from the laundry facility. Conclusions We describe the largest, single-center, HCM outbreak reported to date. Our findings underscore the importance of hospital-based monitoring for HCM and increased attention to the safe handling of laundered linens.

S6.5 Efforts of improving the management of mycetoma: working towards the 2030 goals, September 22, 2022, 4:45 PM - 6:15 PM Mycetoma is a debilitating disease recognized as a neglected tropical disease by the World Health Organization. The etiology of mycetoma is poorly understood; ∼ 60% of cases are caused by fungi and the rest are bacterial, although this varies by region. The pathogenic fungus, Madurella mycetomatis, is most frequently identified in mycetoma cases. Here, we present a high-quality genome assembly of M. mycetomatis and the results of the whole genome sequence analysis of 25 isolates from Sudan. We demonstrate evidence of at least seven genetically diverse lineages and extreme clonality among isolates within these lineages. Shotgun metagenomic analysis of DNA from mycetoma grains confirmed that M. mycetomatis was the predominant causative agent of eumycetoma Sudan; however, 10% of grains also contained bacterial reads suggestive of secondary infections. A thorough understanding of the genetic structure and diversity of fungi causing mycetoma is essential for the development of new diagnostic methods and for identifying potential drug targets.

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM Objective The epidemic of antifungal-resistant dermatophytosis in India has been reported. These infections are associated with severe morbidity, resistance to oral itraconazole and terbinafine, and the widespread misuse of topical steroids. Trichophyton indotineae has emerged as the predominant causative agent. In this study, we investigated 162 dermatophytosis infections from eight Indian states using genomic sequencing. The primary objective was to determine whether a clonal outbreak strain is responsible for the current epidemic. Methods A total of 161 T. indotineae and one T. rubrum isolates from skin scrapings collected from India in 2017-2019 and previously reported were sent to the U.S. Centers for Disease Control and Prevention (CDC) for genomic analysis. After species identification, genomic DNA was extracted and sequenced using Illumina NovaSeq. Single-nucleotide (SNP) analysis was performed using the portable workflow MycoSNP (v0.21). Briefly, MycoSNP prepared the reference genome, performed pre-processing, aligned sample reads to the reference using the BWA (v 0.7.17) alignment algorithm, and called variants using GATK (v4.1.4.1). High-quality SNPs were used for constructing phylogenetic trees using neighbor-joining (NJ) and maximum likelihood (ML) methods. Further, to understand if infections are genetically clustered by state or region, multi-dimensional scaling (MDS) was applied using the ML tree in R. Result SNP analysis identified 1259 450 variant sites which were used to construct an NJ and ML tree. The tree topology from both NJ and ML methods showed consensus. All 161 T. indotineae isolates from India clustered together forming a large, well-supported clade. SNP differences between the samples varied from 0-160 SNPs. Historical isolates available at CDC were included as controls and clustered over 40 000 SNPs from the clade comprising isolates from India. The MDS plot revealed that isolates did not cluster by state or region. Conclusion Antifungal-resistant dermatophytosis is an emerging threat with cases of chronic, recurrent infection reported from several countries including India. Additionally, the rapid spread of infections involves person-to-person spread. Our results suggest that a clonal outbreak of a T. indotineae strain is circulating in multiple states in India. Current plans are to expand the geographic scope of the study by including over 10 countries from Europe, Middle East, and the Americas. This work will allow the public health community to better understand the emergence and transmission of antifungal-resistant dermatophytosis worldwide.

Candida auris is an urgent public health threat characterized by high drug-resistant rates and rapid spread in healthcare settings worldwide. As part of the C. auris response, molecular surveillance has helped public health officials track the global spread and investigate local outbreaks. Here, we describe whole-genome sequencing analysis methods used for routine C. auris molecular surveillance in the United States; methods include reference selection, reference preparation, quality assessment and control of sequencing reads, read alignment, and single-nucleotide polymorphism calling and filtration. We also describe the newly developed pipeline MycoSNP, a portable workflow for performing whole-genome sequencing analysis of fungal organisms including C. auris.

Candida auris is a yeast that is difficult to eradicate and has caused outbreaks in health care facilities. We report a cluster of 5 patients in 1 intensive care unit who were colonized or infected in 2017. The initial 2 patients were recipients of liver transplants who had cultures that grew C auris within 3 days of each other in June 2017 (days 43 and 30 posttransplant). Subsequent screening cultures identified 2 additional patients with C auris colonization. Respiratory and urine cultures from a fifth patient yielded C auris. All isolates were fluconazole resistant but susceptible to echinocandins. Whole genome sequencing showed the strains were clonal, suggesting in-hospital transmission, and related but distinct from New York/New Jersey strains, consistent with a separate introduction. However, no source or contact was found. Two of the 5 patients died. C auris infection likely contributed to 1 patient death by infecting a vascular aneurysm at the graft anastomosis. Strict infection control precautions were initiated to control the outbreak. Our experience reveals that although severe disease from C auris can occur in transplant recipients, outbreaks can be controlled using recommended infection control practices. We have had no further patients infected with C auris to date.

BACKGROUND Candida auris is a difficult-to-diagnose multidrug-resistant yeast that can cause invasive infections with high mortality. Since emerging in 2009, this pathogen has been associated with numerous outbreaks around the world. Whole genome sequencing (WGS) is instrumental for understanding the emergence and local transmission of this pathogen. OBJECTIVES To describe the clinical, molecular characteristics of Candida auris infection and clinical outcome in our centre. PATIENTS AND METHODS Patients with positive cultures for Candida auris were identified in a microbiology database. Clinical characteristics and antifungal susceptibility were obtained. Isolates were sent to the US CDC for whole genome sequencing. RESULTS Seven unique patients with eight different isolates were identified. Seven isolates were sent to the US CDC for whole genome sequencing. None of the patients had bloodstream infection. Thirty-day mortality was higher in infected patients compared with those who were colonised. Seven of the eight isolates were resistant to both fluconazole, and five were resistant to amphotericin B. WGS analysis demonstrated that the seven isolates belonged to the South Asian clade but formed two distinct subclades suggesting two independent introductions and ongoing transmission within the facility. CONCLUSIONS Candida auris is associated with a high mortality rate in infected patients. Strict infection control measures and surveillance for asymptomatic cases are warranted to halt ongoing transmission.

Coccidioides immitis and Coccidioides posadasii are causative agents of Valley fever, a serious fungal disease endemic to regions with hot, arid climate in the United States, Mexico, and Central and South America. The environmental niche of Coccidioides spp. is not well defined, and it remains unknown whether these fungi are primarily associated with rodents or grow as saprotrophs in soil. To better understand the environmental reservoir of these pathogens, we used a systematic soil sampling approach, quantitative PCR (qPCR), culture, whole-genome sequencing, and soil chemical analysis to identify factors associated with the presence of C. immitis at a known colonization site in Washington State linked to a human case in 2010. We found that the same strain colonized an area of over 46,000 m2 and persisted in soil for over 6 years. No association with rodent burrows was observed, as C. immitis DNA was as likely to be detected inside rodent holes as it was in the surrounding soil. In addition, the presence of C. immitis DNA in soil was correlated with elevated levels of boron, calcium, magnesium, sodium, and silicon in soil leachates. We also observed differences in the microbial communities between C. immitis-positive and -negative soils. Our artificial soil inoculation experiments demonstrated that C. immitis can use soil as a sole source of nutrients. Taken together, these results suggest that soil parameters need to be considered when modeling the distribution of this fungus in the environment. IMPORTANCE Coccidioidomycosis is considered a highly endemic disease for which geographic range is likely to expand from climate change. A better understanding of the ecological niche of Coccidioides spp. is essential for generating accurate distribution maps and predicting future changes in response to the changing environment. Our study used a systematic sampling strategy, advanced molecular detection methods, and soil chemical analysis to identify environmental factors associated with the presence of C. immitis in soil. Our results demonstrate the fungus can colonize the same areas for years and is associated with chemical and microbiological soil characteristics. Our results suggest that in addition to climate parameters, soil characteristics need to be considered when building habitat distribution models for this pathogen.

To assess the prevalence ofPoint prevalence surveys (PPSs), postdischarge testing forAll LTACHs (Current or recent patients in LTACHs and vSNFs in OC.In facilities whereAntifungal susceptibility testing and whole-genome sequencing to assess isolate relatedness.Initial PPSs at 17 facilities identified 44 additional patients withAcute care hospitals were not assessed, and IPC improvements over time could not be rigorously evaluated.Enhanced laboratory surveillance and prompt investigation with IPC support enabled swift identification and containment ofCenters for Disease Control and Prevention.

Azole resistance in pathogenic Aspergillus fumigatus has become a global public health issue threatening the use of medical azoles. The environmentally occurring resistance mutations, TR34/L98H (TR34) and TR46/Y121F/T289A (TR46), are widespread across multiple continents and emerging in the United States. We used whole-genome single nucleotide polymorphism (SNP) analysis on 179 nationally represented clinical and environmental A. fumigatus genomes from the United States along with 18 non-U.S. genomes to evaluate the genetic diversity and foundation of the emergence of azole resistance in the United States. We demonstrated the presence of clades of A. fumigatus isolates: clade A (17%) comprised a global collection of clinical and environmental azole-resistant strains, including all strains with the TR34/L98H allele from India, The Netherlands, the United Kingdom, and the United States, and clade B (83%) consisted of isolates without this marker mainly from the United States. The TR34/L98H polymorphism was shared among azole-resistant A. fumigatus strains from India, The Netherlands, the United Kingdom, and the United States, suggesting the common origin of this resistance mechanism. Six percent of azole-resistant A. fumigatus isolates from the United States with the TR34 resistance marker had a mixture of clade A and clade B alleles, suggestive of recombination. Additionally, the presence of equal proportions of both mating types further suggests the ongoing presence of recombination. This study demonstrates the genetic background for the emergence of azole resistance in the United States, supporting a single introduction and subsequent propagation, possibly through recombination of environmentally driven resistance mutations. IMPORTANCE Aspergillus fumigatus is one of the most common causes of invasive mold infections in patients with immune deficiencies and has also been reported in patients with severe influenza and severe acute respiratory syndrome coronavirus 2 (SARs-CoV-2). Triazole drugs are the first line of therapy for this infection; however, their efficacy has been compromised by the emergence of azole resistance in A. fumigatus, which was proposed to be selected for by exposure to azole fungicides in the environment [P. E. Verweij, E. Snelders, G. H. J. Kema, E. Mellado, et al., Lancet Infect Dis 9:789–795, 2009, https://doi.org/10.1016/S1473-3099(09)70265-8]. Isolates with environmentally driven resistance mutations, TR34/L98H (TR34) and TR46/Y121F/T289A (TR46), have been reported worldwide. Here, we used genomic analysis of a large sample of resistant and susceptible A. fumigatus isolates to demonstrate a single introduction of TR34 in the United States and suggest its ability to spread into the susceptible population is through recombination between resistant and susceptible isolates.

Whole-genome sequencing confirmed the presence of a Malassezia pachydermatis outbreak among neonates in a neonatal intensive care unit. This technology supports the importance of adhering to infection prevention measures.

Coccidioidomycosis is an emerging fungal infection in Washington, USA, and the epidemiology of the disease in this state is poorly understood. We used whole-genome sequencing to differentiate locally acquired cases in Washington on the basis of the previously identified phylogeographic population structure of Coccidioides spp. Clinical isolates from coccidioidomycosis cases involving possible Washington soil exposure were included. Of 17 human infections with epidemiologic evidence of possible local acquisition, 4 were likely locally acquired infections and 13 were likely acquired outside Washington. Isolates from locally acquired cases clustered within the previously established Washington clade of C. immitis. Genetic differences among these strains suggest multiple environmental reservoirs of C. immitis in the state.

Summary Background Transmission of multidrug-resistant Candida auris infection has been reported in the USA. To better understand its emergence and transmission dynamics and to guide clinical and public health responses, we did a molecular epidemiological investigation of C auris cases in the USA. Methods In this molecular epidemiological survey, we used whole-genome sequencing to assess the genetic similarity between isolates collected from patients in ten US states (California, Connecticut, Florida, Illinois, Indiana, Maryland, Massachusetts, New Jersey, New York, and Oklahoma) and those identified in several other countries (Colombia, India, Japan, Pakistan, South Africa, South Korea, and Venezuela). We worked with state health departments, who provided us with isolates for sequencing. These isolates of C auris were collected during the normal course of clinical care (clinical cases) or as part of contact investigations or point prevalence surveys (screening cases). We integrated data from standardised case report forms and contact investigations, including travel history and epidemiological links (ie, patients that had shared a room or ward with a patient with C auris ). Genetic diversity of C auris within a patient, a facility, and a state were evaluated by pairwise differences in single-nucleotide polymorphisms (SNPs). Findings From May 11, 2013, to Aug 31, 2017, isolates that corresponded to 133 cases (73 clinical cases and 60 screening cases) were collected. Of 73 clinical cases, 66 (90%) cases involved isolates related to south Asian isolates, five (7%) cases were related to South American isolates, one (1%) case to African isolates, and one (1%) case to east Asian isolates. Most (60 [82%]) clinical cases were identified in New York and New Jersey; these isolates, although related to south Asian isolates, were genetically distinct. Genomic data corroborated five (7%) clinical cases in which patients probably acquired C auris through health-care exposures abroad. Among clinical and screening cases, the genetic diversity of C auris isolates within a person was similar to that within a facility during an outbreak (median SNP difference three SNPs, range 0–12). Interpretation Isolates of C auris in the USA were genetically related to those from four global regions, suggesting that C auris was introduced into the USA several times. The five travel-related cases are examples of how introductions can occur. Genetic diversity among isolates from the same patients, health-care facilities, and states indicates that there is local and ongoing transmission. Funding US Centers for Disease Control and Prevention

Candida auris is an emerging fungal pathogen of rising concern due to global spread, the ability to cause healthcare-associated outbreaks, and antifungal resistance. Genomic analyses revealed that early contemporaneously detected cases of C. auris were geographically stratified into four major clades. While Clades I, III, and IV are responsible for ongoing outbreaks of invasive and multidrug-resistant infections, Clade II, also termed the East Asian clade, consists primarily of cases of ear infection, is often susceptible to all antifungal drugs, and has not been associated with outbreaks. Here, we generate chromosome-level assemblies of twelve isolates representing the phylogenetic breadth of these four clades and the only isolate described to date from Clade V. This Clade V genome is highly syntenic with those of Clades I, III, and IV, although the sequence is highly divergent from the other clades. Clade II genomes appear highly rearranged, with translocations occurring near GC-poor regions, and large subtelomeric deletions in most chromosomes, resulting in a substantially different karyotype. Rearrangements and deletion lengths vary across Clade II isolates, including two from a single patient, supporting ongoing genome instability. Deleted subtelomeric regions are enriched in Hyr/Iff-like cell-surface proteins, novel candidate cell wall proteins, and an ALS-like adhesin. Cell wall proteins from these families and other drug-related genes show clade-specific signatures of selection in Clades I, III, and IV. Subtelomeric dynamics and the conservation of cell surface proteins in the clades responsible for global outbreaks causing invasive infections suggest an explanation for the different phenotypes observed between clades.

Histoplasmosis, one of the most frequent endemic mycoses in the Americas, is caused by the inhalation of airborne conidia of Histoplasma capsulatum. Better understanding of the distribution of this fungus in the environment is important for the development of appropriate public health measures to prevent human infections. Previously, we used Hc100 nested polymerase chain reaction (PCR) to identify H. capsulatum DNA in 10% of environmental samples in Colombia. Here, we validate a 100-kDa real-time PCR assay for the detection of this fungus in the environment. Using this method, we identified H. capsulatum DNA in 80% of samples of raw organic materials, such as chicken manure, soil from caves, and bird and bat guano, as well as in 62% of samples of organic fertilizer that underwent the composting process. We demonstrated that 100-KDa real-time PCR is a useful tool for environmental surveillance that can be used to identify the potential reservoirs of H. capsulatum and to prevent outbreaks, especially in people with the higher risk of exposure, such as spelunkers, farmers, poultry manure collectors, and anyone who handle organic fertilizers or bat and bird excreta.

Whole-genome sequencing confirmed the presence of a Malassezia pachydermatis outbreak among neonates in a neonatal intensive care unit. This technology supports the importance of adhering to infection prevention measures.

In less than a decade, C. auris has emerged in health care settings worldwide; this species is capable of colonizing skin and causing outbreaks of invasive candidiasis. In contrast to other Candida species, C. auris is unique in its ability to spread via nosocomial transmission and its high rates of drug resistance. As part of the public health response, whole-genome sequencing has played a major role in characterizing transmission dynamics and detecting new C. auris introductions. Through a global collaboration, we assessed genome evolution of isolates of C. auris from 19 countries. Here, we described estimated timing of the expansion of each C. auris clade and of fluconazole resistance, characterized discrete phylogeographic population structure of each clade, and compared genome data to sensitivity measurements to describe how antifungal resistance mechanisms vary across the population. These efforts are critical for a sustained, robust public health response that effectively utilizes molecular epidemiology., Candida auris has emerged globally as a multidrug-resistant yeast that can spread via nosocomial transmission. An initial phylogenetic study of isolates from Japan, India, Pakistan, South Africa, and Venezuela revealed four populations (clades I, II, III, and IV) corresponding to these geographic regions. Since this description, C. auris has been reported in more than 30 additional countries. To trace this global emergence, we compared the genomes of 304 C. auris isolates from 19 countries on six continents. We found that four predominant clades persist across wide geographic locations. We observed phylogeographic mixing in most clades; clade IV, with isolates mainly from South America, demonstrated the strongest phylogeographic substructure. C. auris isolates from two clades with opposite mating types were detected contemporaneously in a single health care facility in Kenya. We estimated a Bayesian molecular clock phylogeny and dated the origin of each clade within the last 360 years; outbreak-causing clusters from clades I, III, and IV originated 36 to 38 years ago. We observed high rates of antifungal resistance in clade I, including four isolates resistant to all three major classes of antifungals. Mutations that contribute to resistance varied between the clades, with Y132F in ERG11 as the most widespread mutation associated with azole resistance and S639P in FKS1 for echinocandin resistance. Copy number variants in ERG11 predominantly appeared in clade III and were associated with fluconazole resistance. These results provide a global context for the phylogeography, population structure, and mechanisms associated with antifungal resistance in C. auris.

Background:Candida auris is an emerging pathogen that has recently disseminated globally and caused challenging outbreaks in healthcare facilities (HCFs), in part because it is commonly multidrug-resistant. Candida auris remains rare in Canada, with ~20 known cases to date. We describe the emergence of a novel subclade of C. auris in Ontario, Canada, using whole-genome sequencing (WGS). Methods: In Ontario, many HCFs submit yeast isolates from sterile sites requiring species-level characterization and antifungal susceptibility testing (AFST) to the provincial reference laboratory. Yeasts were identified using a combination of standard methods (morphology, API 20C, MALDI-ToF MS) including ITS2 sequencing. Sensititre YO9 panels were used for AFST. Genomic analysis of C. auris was performed using an Illumina HiSeq platform with at least 50× coverage; variants were called against the reference genome by using the previously published North Arizona SNP pipeline (NASP). Phylogenetic trees were produced by maximum parsimony method (MEGA7.0). Results: Between 2014 and 2018, yeast isolates from 5 different patients from 4 HCFs in the same region of Ontario were confirmed to be C. auris by ITS2 PCR and sequence analysis (Table 1). Based on interim CDC criteria for antifungal drug break points, all isolates were pansusceptible to common antifungals. WGS analysis demonstrated that the C. auris isolates were part of the South American clade (IV) and formed an isolated subclade that is well supported by bootstrap analysis, indicating clonal relationships among these isolates (Fig. 1). Conclusions: Although C. auris isolates are usually drug resistant, all 5 initial Ontario isolates were pansusceptible. WGS determined that these isolates clustered within clade IV and were clonal. This cluster of C. auris appears to represent a new subclade of the South American clade that has been transmitted among patients within a region of Ontario. C. auris may have been present in Ontario for some time, escaping earlier detection due to lack of screening programs in HCFs, historical challenges with microbiologic detection of C. auris, and the antifungal susceptibility of the circulating isolates. Investigations are underway to determine clinical features and epidemiologic relatedness among patients in this cluster.Funding: NoneDisclosures: Susy Hota, Contracted Research - Finch Therapeutics

Candida auris is an emerging fungal pathogen of rising concern due to its increasing incidence, its ability to cause healthcare-associated outbreaks and antifungal resistance. Genomic analysis revealed that early cases of C. auris that were detected contemporaneously were geographically stratified into four major clades. Clade II, also termed East Asian clade, consists of the initial isolates described from cases of ear infection, is less frequently resistant to antifungal drugs and to date, the isolates from this group have not been associated with outbreaks. Here, we generate nearly complete genomes (“telomere-to-telomere”) of an isolate of this clade and of the more widespread Clade IV. By comparing these to genome assemblies of the other two clades, we find that the Clade II genome appears highly rearranged, with 2 inversions and 9 translocations resulting in a substantially different karyotype. In addition, large subtelomeric regions have been lost from 10 of 14 chromosome ends in the Clade II genomes. We find that shorter telomeres and genome instability might be a consequence of a naturally occurring loss-of-function mutation in DCC1 exclusively found in Clade II isolates, resulting in a hypermutator phenotype. We also determine that deleted subtelomeric regions might be linked to clade-specific adaptation as these regions are enriched in Hyr/Iff-like cell surface proteins, novel candidate cell surface proteins, and an ALS-like adhesin. The presence of these cell surface proteins in the clades responsible for global outbreaks causing invasive infections suggests an explanation for the different phenotypes observed between clades.IMPORTANCECandida auris was unknown prior to 2009 and since then it has quickly spread around the world, causing outbreaks in healthcare facilities and representing a high fraction of candidemia cases in some regions. The emergence of C. auris is a major concern, since it is often multidrug-resistant, easily spread between patients, and causes invasive infections. While isolates from three global clades cause invasive infections, isolates from Clade II primarily cause ear infections and have not been implicated in outbreaks, though cases of Clade II infections have been reported on different continents. Here, we describe genetic differences between Clade II and Clades I, III and IV, including a loss-of-function mutation in a gene associated with telomere length maintenance and genome stability, and the loss of cell wall proteins involved in adhesion and biofilm formation, that may suggest an explanation for the lower virulence and potential for transmission of Clade II isolates.

Coccidioidomycosis is a debilitating fungal disease caused by inhalation of arthroconidia. We developed a novel approach for detection of airborne Coccidioides and used it to investigate the distribution of arthroconidia across the Phoenix, Arizona, metropolitan area. Air filters were collected daily from 21 stationary air-sampling units across the area: the first set collected before, during and after a large dust storm on August 25, 2015, and the second over the 45-day period September 25–November 8, 2016. Analysis of DNA extracted from the filters demonstrated that the day of the dust storm was not associated with increase of Coccidioides in air samples, although evidence of the low-level polymerase chain reaction (PCR) inhibition was observed in DNA extracted from samples collected on the day of the dust storm. Testing over 45 days identified uneven geographic distribution suggesting Coccidioides hot spots. In 2016, highest daily concentration of arthroconidia was observed between September 25–October 20, and only sporadic low levels were detected after that. These results provide evidence of seasonality and uneven spatial distribution of Coccidioides in the air. Our results demonstrate that routine air monitoring for arthroconidia is possible and provides an important tool for Coccidioides surveillance, which can address important questions about environmental exposure and human infection.

Background Candida auris is a multidrug-resistant yeast associated with hospital outbreaks worldwide. During 2015-2016, multiple outbreaks were reported in Colombia. We aimed to understand the extent of contamination in healthcare settings and to characterize the molecular epidemiology of C. auris in Colombia. Methods We sampled patients, patient contacts, healthcare workers, and the environment in 4 hospitals with recent C. auris outbreaks. Using standardized protocols, people were swabbed at different body sites. Patient and procedure rooms were sectioned into 4 zones and surfaces were swabbed. We performed whole-genome sequencing (WGS) and antifungal susceptibility testing (AFST) on all isolates. Results Seven of the 17 (41%) people swabbed were found to be colonized. Candida auris was isolated from 37 of 322 (11%) environmental samples. These were collected from a variety of items in all 4 zones. WGS and AFST revealed that although isolates were similar throughout the country, isolates from the northern region were genetically distinct and more resistant to amphotericin B (AmB) than the isolates from central Colombia. Four novel nonsynonymous mutations were found to be significantly associated with AmB resistance. Conclusions Our results show that extensive C. auris contamination can occur and highlight the importance of adherence to appropriate infection control practices and disinfection strategies. Observed genetic diversity supports healthcare transmission and a recent expansion of C. auris within Colombia with divergent AmB susceptibility.

Candida haemulonii is an emerging multidrug-resistant yeast that can cause invasive candidiasis. Here, we report the first genome sequence of C. haemulonii (isolate B11899) generated using PacBio sequencing technology. The estimated genome size was 13.3 Mb, with a GC content of 45.19%.

Candida aurisis an emergent fungal pathogen of rising public health concern due to increasing reports of outbreaks in healthcare settings and resistance to multiple classes of antifungal drugs. While distantly related to the more common pathogensC. albicansandC. glabrata,C. aurisis closely related to three rarely observed and often multidrug-resistant species,C. haemulonii, C. duobushaemulonii and C. pseudohaemulonii. Here, we generated and analyzed near complete genome assemblies and RNA-Seq-guided gene predictions for isolates from each of the four majorC. aurisclades and forC. haemulonii, C. duobushaemulonii and C. pseudohaemulonii. Our analyses mapped seven chromosomes and revealed chromosomal rearrangements betweenC. aurisclades and related species. We found conservation of genes involved in mating and meiosis and identified bothMTLaandMTLαC. aurisisolates, suggesting the potential for mating between clades. Gene conservation analysis highlighted that many genes linked to drug resistance and virulence in other pathogenicCandidaspecies are conserved inC. aurisand related species including expanded families of transporters and lipases, as well as mutations and copy number variants inERG11that confer drug resistance. In addition, we found genetic features of the emerging species that likely underlie differences in virulence and drug response between these and otherCandidaspecies, including genes involved in cell wall structure. To begin to characterize the species-specific genes important for antifungal response, we profiled the gene expression ofC. aurisin response to voriconazole and amphotericin B and found induction of several transporters and metabolic regulators that may play a role in drug resistance. This study provides a comprehensive view of the genomic basis of drug resistance, potential for mating, and virulence in this emerging fungal clade.

Candida duobushaemulonii is a drug-resistant yeast that can cause invasive candidiasis. Here, we report the first genome sequence of C. duobushaemulonii , isolate B09383, generated using PacBio sequencing technology. The estimated genome size was 12.5 Mb with a GC content of 46.84%.

Coccidioidomycosis (CM), a serious life-threatening fungal infection endemic to arid regions of the western United States and Mexico, can be challenging to diagnose in a timely manner. Commercially developed enzyme immunoassays (EIAs) (from Meridian Biosciences and Immuno-Mycologics [IMMY]) have provided faster, simpler means for serodiagnosis; however, independent evaluations have questioned EIA specificity, particularly IgM-positive/IgG-negative results. This study was conducted to evaluate EIA specificity among persons residing in Puerto Rico ( n = 534), where CM is not endemic (who were not likely to have been exposed to Coccidioides spp.), compared to blood bank donors residing in Arizona ( n = 1,218), where CM is endemic. Upon comparing serum reactivity between Puerto Rico and Arizona, the Meridian EIA showed a significant difference in IgG reactivity (0.37% versus 3.6%; P < 0.001) but not IgM reactivity (3.4% versus 2.4%; P = 0.31). No IgM-/IgG-reactive sera were detected among sera from Puerto Rico, compared to 7 (0.57%) sera from Arizona. Similar results were observed using the IMMY EIA, although significantly ( P = 0.03) fewer IgM-reactive sera from Arizona were observed, compared to the Meridian EIA. EIA-reactive sera were also evaluated by immunodiffusion before and after 3- to 4-fold concentration of the sera. These results demonstrate that elevated IgG EIA reactivity is present in sera from healthy individuals in regions of endemicity and that IgM EIA reactivity observed in sera from individuals residing outside regions of endemicity is most likely nonspecific. Other criteria, including clinical and microbiological evaluations, should be taken into account when interpreting results from surveillance studies and other reporting measures.