Abstract 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.

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 m(2) 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.

SummaryBackgroundTwo species complexes (SC) cause the majority of human Cryptococcus infections: Cryptococcus neoformans SC and Cryptococcus gattii SC. Infection is typically thought to be acquired following environmental exposure. In an urban setting, parks and other public spaces are a likely source of contact with C. gattii SC.ObjectivesThe goals of this study were to describe the genetic diversity of C. gattii SC in the California environment, to determine the extent of environmental exposure in publicly accessed areas and to correlate the genotypes of environmental C. gattii SC isolates with those from patients in southern California.MethodsSpecimens from trees and soil from 13 parks and public areas of seven California counties were examined for C. gattii SC isolates. Isolates were sequence typed and compared to sequence types from human clinical isolates from the same area.ResultsMultilocus sequence typing identified C. gattii sensu stricto (VGI molecular type) as well as Cryptococcus bacillisporus (VGIII molecular type). Several C. bacillisporus but none of the C. gattii sensu stricto isolates shared sequence types with human clinical isolates from southern California.ConclusionsC. gattii SC colonies exist in some California public parks. The presence of identical STs in environmental and human isolates of C. bacillisporus is suggestive of an arboreal origin of human infections. Two new tree species were documented as hosts for C. gattii SC in California, adding to the four species previously identified.

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.

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.

ABSTRACTCoccidioidomycosis (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 toCoccidioidesspp.), 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.

ABSTRACT Exserohilum rostratum was the cause of most cases of fungal meningitis and other infections associated with the injection of contaminated methylprednisolone acetate produced by the New England Compounding Center (NECC). Until this outbreak, very few human cases of Exserohilum infection had been reported, and very little was known about this dematiaceous fungus, which usually infects plants. Here, we report using whole-genome sequencing (WGS) for the detection of single nucleotide polymorphisms (SNPs) and phylogenetic analysis to investigate the molecular origin of the outbreak using 22 isolates of E. rostratum retrieved from 19 case patients with meningitis or epidural/spinal abscesses, 6 isolates from contaminated NECC vials, and 7 isolates unrelated to the outbreak. Our analysis indicates that all 28 isolates associated with the outbreak had nearly identical genomes of 33.8 Mb. A total of 8 SNPs were detected among the outbreak genomes, with no more than 2 SNPs separating any 2 of the 28 genomes. The outbreak genomes were separated from the next most closely related control strain by ∼136,000 SNPs. We also observed significant genomic variability among strains unrelated to the outbreak, which may suggest the possibility of cryptic speciation in E. rostratum .

We used real-time polymerase chain reaction and culture to demonstrate persistent colonization of soils by Coccidioides immitis, an agent of valley fever, in Washington State linked to recent human infections and located outside the endemic range. Whole-genome sequencing confirmed genetic identity between isolates from soil and one of the case-patients.

Coccidioidomycosis ("valley fever") is caused by inhaling spores of the soil-dwelling fungi Coccidioides immitis or Coccidioides posadasii. Most infections are subclinical. When clinical manifestations do occur (typically 1-4 weeks after exposure), they are similar to those associated with influenza or community-acquired pneumonia. Disseminated disease is rare. Residual pulmonary nodules can lead to chronic lung disease. Fluconazole or other triazoles often are used for treatment, but mild cases often resolve without specific therapy. A total of 17,802 cases were reported in the United States in 2012.

Infections caused by Histoplasma capsulatum are found most often in endemic regions of North, Central, and South America. H. capsulatum has been divided into eight geographic clades by multi-locus sequence typing (MLST). Recently, one isolate and five formalin-fixed paraffin-embedded (FFPE) tissue samples were received from six of 15 suspected cases of histoplasmosis in cats residing in areas not known to be endemic for H. capsulatum. Polymerase chain reaction (PCR) amplification and sequence analysis of the rDNA ITS-2 region confirmed the diagnosis of H. capsulatum. Since these cases were not, as noted, from the accepted endemic areas, it was of interest to understand the molecular epidemiology of these isolates. Results of molecular analysis indicated that the H. capsulatum recovered from the cats were most closely related to the North American-1 clade, but clustered separately outside this clade, suggesting that the H. capsulatum infecting the animals may represent a separate clade or phylogenetic species. This study also demonstrated the utility of obtaining valuable molecular subtype data directly from archived FFPE tissue blocks, particularly when a fungus culture was not performed or is otherwise unavailable.

BACKGROUND:Invasive fusariosis (IF) is a rare but often fatal fungal infection in immunosuppressed patients. In 2007, cases of IF above the expected epidemiologic baseline were detected in the hematology ward of a hospital in Rio de Janeiro, Brazil. Possible sources of infection were investigated by performing environmental sampling and patient isolate collection, followed by molecular typing. Isolates from dermatology patients with superficial fusariosis were included in the study for comparison to molecular types found in the community.METHODS:Environmental sampling focused on water-related sources in and around the hematology ward. Initially, we characterized 166 clinical and environmental isolates using the Fusarium translation elongation factor 1α (EF-1α) genetic locus. Isolates included 68 collected from water-related sources in the hospital environment, 55 from 18 hematology patients, and 43 from the skin/nails of 40 outpatients seen at the hospital dermatology clinic. Multi-locus sequence typing was performed on Fusarium solani species complex (FSSC) species 1 and 2 isolates to investigate their relatedness further.RESULTS:Most of the hematology samples were FSSC species 2, with species type FSSC 2-d the most commonly isolated from these patients. Most of the outpatient dermatology samples were also FSSC 2, with type 2-d again predominating. In contrast, environmental isolates from water sources were mostly Fusarium oxysporum species complex (FOSC) and those from air samples mostly Fusarium incarnatum-equiseti species complex (FIESC). A third of the environmental samples were FSSC, with species types FSSC 1-a and FSSC 1-b predominating.CONCLUSIONS:Fusarium isolate species types from hematology patient infections were highly similar to those recovered from dermatology patients in the community. Four species types (FSSC 1-a, 1-b, 2-d and 2-f) were shared between hematology patients and the environment. Limitations in environmental sampling do not allow for nosocomial sources of infection to be ruled out. Future studies will focus on environmental factors that may have influenced the prevalence of FSSC fusariosis in this hematology ward.

A 2.5 yr old sexually intact male vizsla was admitted to the Iowa State University Veterinary Teaching Hospital for persistent diarrhea, weight loss, and panhypoproteinemia. Examination revealed an emaciated condition and melena. Two masses were palpated in the cranial abdomen. Hematology and serum biochemistry exhibited a regenerative anemia and confirmed the presence of panhypoproteinemia, suggestive of a protein-losing eneteropathy. Distinct areas of thickened intestinal wall and enlarged mesenteric lymph nodes were found on abdominal ultrasound. Cytology from those nodes showed the presence of suspected Cryptococcus spp., and infection was confirmed utilizing a cryptococcal antigen titer. Medical therapy with lipid-complexed amphotericin B and fluconazole was unsuccessful. Two surgical procedures were performed to remove the affected areas of intestine and lymph nodes, but the disease persisted as evidenced by a persistently elevated cryptococcal antigen titer. Terbinafine was prescribed, which resulted in complete resolution of clinical signs and a steadily decreasing cryptococcal antigen titer. Very few cases of intestinal cryptococcosis have been reported. In this case, infection resulted in a protein-losing enteropathy. In addition, this article describes the use of terbinafine in the treatment of intestinal cryptococcal infection in the dog, which has not been previously reported.

A 67 year-old Caucasian male from Arizona presented with indolent symptoms of intestinal obstruction and hydronephrosis, found at surgery to be caused by a mass involving the terminal ileum and cecum, extending into the posterior abdominal wall and obstructing the right ureter. Histopathology was diagnostic of basidiobolomycosis. PCR of tissue and sequencing identified the fungus as, Basidiobolus ranarum. During one year of posaconazole treatment, the residual mass shrank, hydronephrosis was relieved and peripheral eosinophilia resolved.

Mycotic aortic aneurysm is a local, irreversible dilatation of the aorta associated with destruction of the vessel wall by infection and is a grave clinical condition associated with high morbidity and mortality in humans. Rupture of aortic aneurysms can be spontaneous, idiopathic, or due to severe trauma, and the condition has been associated with bacterial and, rarely, fungal infections in humans and animals. Here, we describe a case of ruptured spontaneous aortic aneurysm associated with zygomycetic infection in a 21-y-old female sooty mangabey. The animal did not present with any significant clinical signs before being found dead. At necropsy, she was in good body condition, and the thoracic cavity had a large amount of clotted blood filling the left pleural space and surrounding the lung lobes. Near the aortic arch, the descending thoracic aorta was focally perforated (diameter, approximately 0.15 cm), and clotted blood adhered to the tunica adventitia. The aortic intima had multiple, firm, pale-yellow nodules (diameter, 0.25 to 0.5 cm). Histopathologically, these nodules consisted of severe multifocal pyogranulomatous inflammation intermixed with necrosis, fibrin, and broad, infrequently septate, thin-walled fungal hyphae. Immunohistochemistry revealed fungal hyphae characteristic of Mucormycetes (formerly Zygomycetes), and PCR analysis identified the organism as Basidiobolus spp. Dissemination of the fungus beyond the aorta was not noted. Spontaneous aortic aneurysms have been described in nonhuman primates, but this is the first reported case of a ruptured spontaneous aortic aneurysm associated with entomophthoromycetic infection in a sooty mangabey.

Background: Aspergillus terreus causes invasive aspergillosis (IA) in immunocompromised individuals and can be the leading cause of IA in certain medical centers. We examined a large isolate collection (n = 117) for the presence of cryptic A. terreus species and employed a genome scanning method, Inter-Simple Sequence Repeat (ISSR) PCR to determine A. terreus population structure.Results: Comparative sequence analyses of the calmodulin locus revealed the presence of the recently recognized species A. alabamensis (n = 4) in this collection. Maximum parsimony, Neighbor joining, and Bayesian clustering of the ISSR data from the 113 sequence-confirmed A. terreus isolates demonstrated that one clade was composed exclusively of isolates from Europe and another clade was enriched for isolates from the US.Conclusions: This study provides evidence of a population structure linked to geographical origin in A. terreus.

Seven international laboratories tested the recently proposed single-locus typing strategy for Aspergillus fumigatus subtyping for interlaboratory reproducibility. Comparative sequence analyses of portions of the locus AFUA_3G08990, encoding a putative cell surface protein (denoted CSP), was performed with a panel of Aspergillus isolates. Each laboratory followed very different protocols for extraction of DNA, PCR, and sequencing. Results revealed that the CSP typing method was a reproducible and portable strain typing method.

ABSTRACT The detection of urinary Histoplasma capsulatum polysaccharide antigen (HPA) by enzyme immunoassay (EIA) has proven useful for the presumptive diagnosis of histoplasmosis in AIDS patients. Assay limitations include (i) detection of a largely uncharacterized antigen and (ii) difficulty in reproducibly generating antibodies for use in the EIA. To improve antibody production for use in this test and to better understand the antigen being detected, we compared rabbit antibodies elicited using various immunization schedules, routes, and H. capsulatum -derived antigens. Antibodies were evaluated by EIA for their ability to detect purified H. capsulatum C antigen (C-Ag) and antigenuria. Reported as enzyme immunoassay (EI) units (the A 450 with antigen divided by the A 450 without antigen), results demonstrated that intravenous immunization of rabbits with whole, killed yeast-phase cells (yeast-i.v. regimen) produced antibodies giving the highest EI values in the C-Ag EIA (mean EI units ± standard deviation, 14.9 ± 0.6 versus 6.4 ± 0.4 for rabbits immunized with C-Ag versus 2.4 ± 0.3 for all other regimens combined). Yeast-i.v. antibodies were highly sensitive for the detection of antigenuria in patients with histoplasmosis, as shown by the following results: 12/12 patients compared to 10/12, 6/12, 3/12, and 3/12, respectively, for antibodies from rabbits immunized with (i) C-Ag; (ii) whole, killed yeast-phase cells administered subcutaneously and intramuscularly; (iii) yeast-phase culture filtrates; and (iv) HPA-positive urine. Rabbits immunized using the yeast-i.v. regimen also gave higher peak antibody titers than rabbits immunized by any other regimen ( P < 0.03), and their antibodies were most comparable in reactivity to antibodies produced for use in the standard HPA-EIA test ( P < 0.001). Therefore, rabbits immunized using the yeast-i.v. regimen produced the most sensitive antibodies with the highest titers for detection of C-Ag and antigenuria in histoplasmosis patients.

Lindsley and colleagues ([5][1]) reported an antigen assay for diagnosis of histoplasmosis. Although they do not claim to have compared their test to that at MiraVista Diagnostics ([9][2]), the reader may draw that conclusion since the authors note that I provided antibodies, vaccine strain, and