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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.

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.

Background Azole resistance in isolates of the fungus Aspergillus fumigatus has been associated with agricultural use of azole fungicides. Environmental isolation of resistant isolates has been reported in Asia, Africa, Europe and South America. Objectives To determine whether A. fumigatus isolates containing TR34/L98H or TR46/Y121F/T289A can be found in fields in the USA treated with agricultural azoles. Methods Crop debris was collected and screened for A. fumigatus. All A. fumigatus isolates were screened for azole resistance. The CYP51A gene of azole-resistant isolates was sequenced. The population structure of a subset of isolates was determined using microsatellite typing. Results This article identifies azole-resistant A. fumigatus isolates containing the TR34/L98H mutation in an experimental peanut field that had been treated with azole fungicides. Conclusions These findings suggest the development of resistance to azole antifungals in A. fumigatus may be present where agricultural azoles are used in the USA.

Candida auris is an emerging, multidrug-resistant yeast that can spread rapidly in healthcare settings. Phenotypic switching has been observed in other Candida species and can potentially interfere with correct identification. The aim of this study is to address misidentification of C. auris by describing alternate phenotypes after broth enrichment and subculturing on CHROMagar Candida. Each isolate displayed different frequencies of phenotypic switching, suggesting a strain to strain variability. Increased knowledge of the multiple phenotypes of C. auris increases the chance of isolating and identifying C. auris by reducing the risk of discarding false negative alternate colony morphologies.

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.

Since September 2012, the Centers for Disease Control and Prevention (CDC), together with state and local health departments, has been investigating an outbreak of fungal meningitis and other infections associated with injection of contaminated methylprednisolone acetate (MPA) produced by the New England Compounding Center (NECC). By October 2013, 751 cases were identified with 64 deaths, making this one of the largest recorded healthcare-associated outbreaks in US history [1, 2]. Although several molds were isolated from cerebrospinal fluid (CSF) and tissues of case patients, Exserohilum rostratum has been the predominant pathogen [3, 4]. Invasive infection due to E. rostratum, a fungus found in soil associated with plants [5], is rare and has previously been described only in persons with impaired immune systems [6, 7]. Furthermore, until this outbreak no cases of meningitis or encephalitis caused by this fungus had been reported. As part of the response to this public health disaster, the CDC developed a rapid polymerase chain reaction (PCR) test for detection of fungal DNA in human fluids and tissues [8]. This assay is estimated to have 29% diagnostic sensitivity and 100% specificity. (1,3)-β-d-glucan (BDG) is a glucose polymer that is part of the fungal cell wall [9]. The Fungitell assay (Associates of Cape Cod, Inc, Falmouth, Massachusetts) has been approved by the US Food and Drug Administration (FDA) for detection of BDG in human serum and used in diagnosis of fungal infections [10–12]. The potential drawbacks of this assay include (1) inability to differentiate among fungal species causing infections, (2) cross-reactivity with certain bacteria and drugs, and (3) false positivity due to specimen contamination [10]. Therefore, results of this assay must be interpreted in combination with clinical and epidemiological findings. There are few data on the Fungitell assay using CSF in patients with fungal meningitis [13, 14]; one recent report using this assay in 5 patients from this outbreak demonstrated that 3 had elevated CSF levels of BDG [15]. Here, we explore BDG as a supplemental diagnostic marker for fungal meningitis in this outbreak, and assess its utility for evaluating patients' response to treatment.

Background. Candida auris is a multidrug-resistant yeast that can cause invasive fungal infections. It was first described in Japan in 2009, and has since been reported in other parts of the world. We established an international collaboration to understand the epidemiology of this emerging pathogen. Methods. We evaluated a convenience sample of C. auris infections from India, Pakistan, South Africa, and Venezuela during 2012–2015. Antifungal susceptibility testing was conducted according to CLSI standards; breakpoints were defined using those from other Candida spp. Whole genome sequencing typing (WGST) was performed. Patient information was obtained using a standardized case report form. Results. Isolates from 54 patients were evaluated (Pakistan 19, India 19, South Africa 10, Venezuela 5, and 1 reference isolate from Japan). All were resistant to fluconazole, 54% to voriconazole, and 7% to echinocandins; resistance to amphotericin B (MIC ≥2) was found in 29% of 21 isolates tested. At least 24% of isolates were resistant to ≥2 antifungal classes. Isolates grouped into distinct South Asian, East Asian, African, and South American clades by WGST. Clades were separated by several thousand single nucleotide polymorphisms (SNPs), but within each clade, isolates were clonal (0–70 SNP differences). Among 38 patients with clinical information, median age was 54 years and 62% were male. Possible risk factors included central venous catheter (CVC) use (78%), surgery in the previous 90 days (50%), recent antifungal therapy (47%), and diabetes (42%). Median time from admission to C. auris culture was 17 days; 63% of patients had bloodstream infections, 16% had urinary tract infections, and 61% died. Conclusion. C. auris is an emerging healthcare-associated pathogen most commonly identified in patients with CVCs and recent surgery and antifungal therapy. It is associated with higher mortality than other Candida spp., and treatment options are limited because of the high prevalence of antifungal resistance. Puzzlingly, WGST analysis suggests emergence at least 4 distinct clades with high genomic similarity within geographical regions. Further exploration of risk factors for infection and transmission mechanisms is needed to inform control measures.

PCR fingerprinting and multilocus sequence typing were applied to determine the major molecular types of the Cryptococcus neoformans/Cryptococcus gattii species complex in the Republic of Korea. Of the 78 strains isolated from patients diagnosed with cryptococcosis between 1990 and 2008, 96% were C. neoformans serotype A, mating type MATα and molecular type VNI. The remaining 4% were C. gattii, serotype B, mating type MATα and either molecular type VGIIb or VGIII. Of the 62 strains with known HIV status, only 14 (22.6%) were isolated from HIV-positive patients and belonged to molecular type VNI. Remarkably, 93% of the C. neoformans isolates had identical PCR fingerprint profiles with the VNIc genotype that has been identified recently as the major genotype among C. neoformans strains in China. Most strains (81.8%) of the VNIc genotype were associated with non-HIV patients compared with strains of the non-VNIc genotype (20%) (P=0.009). Unlike the Chinese strains, a majority (60%) of the non-HIV patients infected with strains of the VNIc genotype in the Republic of Korea had serious underlying conditions, with cancer and liver disease being the most common. This study affirms VNIc to be the most prevalent genotype of C. neoformans isolated from non-HIV patients with cryptococcosis.

We applied multilocus sequence typing (MLST) to investigate the population structure and mode of reproduction of Cryptococcus neoformans var. grubii (serotype A). This MLST system utilizes 12 unlinked polymorphic loci, which are dispersed on nine different chromosomes, and allows the unambiguous identification of closely related strains of serotype A. We compared MLST analyses with the conventional genotyping method of detecting amplified fragment length polymorphisms (AFLPs), and there was excellent correlation between the MLST and AFLP results. However, MLST differentiated a larger number of strains. We analyzed a global collection of isolates of serotype A using both methods, and the results identified at least three genetically distinct subpopulations, designated groups VNI, VNII, and VNB. Groups VNI and VNII are widespread, dominated by isolates with the MATα mating type, and predominantly clonal. Conversely, isolates of group VNB are unique to Botswana, include a significant proportion of fertile strains with the MATa mating type, and manifest compelling evidence of recombination. We have AFLP genotyped >1000 strains of serotype A from different parts of the world, including isolates from several African countries, and, to date, haploid serotype A isolates of group VNB have been found only in Botswana.

Molecular methods of detection based on DNA-sequencing of the internal transcribed spacer 1 and 2 (ITS1 and ITS2) or 5΄ end region of 28S (D1-D2 region) of ribosomal RNA gene (rDNA) have been used extensively for molecular identification and detection of fungal infections. However, these regions are not always informative for identification of mucormycetes and other rare fungal pathogens as they often contain large introns, heterogenic regions, and/or cannot be PCR-amplified using broad range fungal PCR primers. In addition, because of the difficulties of recovering intact fungal DNA from human specimens, smaller regions of DNA are more useful for the direct detection of fungal DNA in tissues and fluids. In this study, we investigated the utility of 12F/13R PCR primers targeting a 200-230 bp region of the extended 28S region of rDNA for molecular identification of fungal DNA in formalin fixed paraffin embedded tissues and other clinical specimens. We demonstrated that this region can be successfully used for identification of all genera and some species of clinically relevant mucormycetes, as well as other medically important fungi, such as Aspergillus, Fusarium, Coccidioides, and Cryptococcus. We also demonstrated that PCR amplification and direct sequencing of the extended 28S region of rDNA was more sensitive compared to targeting the ITS2 region, as we were able to detect and identify mucormycetes and other fungal pathogens in tissues from patients with histopathological and/or culture evidence of fungal infections that were negative with PCR using ITS-specific primers.