Next-Generation Sequencing of Coccidioides immitis Isolated during Cluster Investigation

Next-generation sequencing enables use of whole-genome sequence typing (WGST) as a viable and discriminatory tool for genotyping and molecular epidemiologic analysis. We used WGST to confirm the linkage of a cluster of Coccidioides immitis isolates from 3 patients who received organ transplants from a single donor who later had positive test results for coccidioidomycosis. Isolates from the 3 patients were nearly genetically identical (a total of 3 single-nucleotide polymorphisms identified among them), thereby demonstrating direct descent of the 3 isolates from an original isolate. We used WGST to demonstrate the genotypic relatedness of C. immitis isolates that were also epidemiologically linked. Thus, WGST offers unique benefits to public health for investigation of clusters considered to be linked to a single source

Whole Genome Sequence Analysis of Cryptococcus gattii from the Pacific Northwest Reveals Unexpected Diversity

A recent emergence of Cryptococcus gattii in the Pacific Northwest involves strains that fall into three primarily clonal molecular subtypes: VGIIa, VGIIb and VGIIc. Multilocus sequence typing (MLST) and variable number tandem repeat analysis appear to identify little diversity within these molecular subtypes. Given the apparent expansion of these subtypes into new geographic areas and their ability to cause disease in immunocompetent individuals, differentiation of isolates belonging to these subtypes could be very important from a public health perspective. We used whole genome sequence typing (WGST) to perform fine-scale phylogenetic analysis on 20 C. gattii isolates, 18 of which are from the VGII molecular type largely responsible for the Pacific Northwest emergence. Analysis both including and excluding (289,586 SNPs and 56,845 SNPs, respectively) molecular types VGI and VGIII isolates resulted in phylogenetic reconstructions consistent, for the most part, with MLST analysis but with far greater resolution among isolates. The WGST analysis presented here resulted in identification of over 100 SNPs among eight VGIIc isolates as well as unique genotypes for each of the VGIIa, VGIIb and VGIIc isolates. Similar levels of genetic diversity were found within each of the molecular subtype isolates, despite the fact that the VGIIb clade is thought to have emerged much earlier. The analysis presented here is the first multi-genome WGST study to focus on the C. gattii molecular subtypes involved in the Pacific Northwest emergence and describes the tools that will further our understanding of this emerging pathogen

New Methods To Assess Susceptibilities of Aspergillus Isolates to Caspofungin

Echinocandins are a group of antifungal agents that target 1,3-β-glucan synthase, causing disruption of mold growth at cells compromising tips and branch points. In part because echinocandins do not induce clear growth inhibition end points using broth dilution techniques, methods to test susceptibility have not yet been standardized. We developed a novel susceptibility assay that measures growth of Aspergillus species on solid agar media that contain serial dilutions of caspofungin (agar dilution). Results of agar dilution testing of multiple isolates were compared to results obtained by broth microdilution (MIC), microscopic evaluation (minimal effective concentration [MEC]), and a new method to measure fungal burden, quantification of secreted hyphal antigen. MICs obtained by the agar dilution method were within 1 dilution of MECs for 85% of the Aspergillus isolates; the highest agreement was observed for isolates of Aspergillus niger (95%), which were particularly susceptible to caspofungin. Agar dilution MICs were also consistent with those obtained by quantifying antigen secretion. MICs obtained by broth microdilution were different than MICs by any other method. Several Aspergillus isolates with decreased susceptibility to caspofungin were identified. Agar dilution is simple and reproducible, and results were consistent with the results of more technically demanding techniques. This method may be appropriate for use in the clinical laboratory

Conidial Viability Assay for Rapid Susceptibility Testing of Aspergillus Species

Antifungal susceptibility testing of filamentous fungi has become more important given the recognition of drug-resistant organisms and the availability of therapies other than amphotericin B (AMB). As current microdilution and E-test methods are limited by a 2 to 3 day incubation time required to obtain results, a more rapid method for susceptibility testing of fungi is needed. We report here a flow cytometric assay that relies on conidial metabolism of the viability dye FUN-1. Conidia are incubated in media containing increasing concentrations of AMB for 3 h, exposed to FUN-1, and then analyzed by flow cytometry. Relative susceptibility to AMB can be measured both by forward and side scatter characteristics of the conidial population and by mean fluorescence intensity (MFI) of the dye. MIC, calculated as the concentration of AMB to yield 90% reduction in MFI relative to growth controls, was determined for 27 clinical isolates Aspergillus species and correlated well with the standard (i.e., NCCLS) method. The results of these studies illustrate a method by which AMB susceptibility can be rapidly and reproducibly determined by measuring conidial viability

Aspergillus Section Fumigati Typing by PCR-Restriction Fragment Polymorphism▿

Recent studies have shown that there are multiple clinically important members of the Aspergillus section Fumigati that are difficult to distinguish on the basis of morphological features (e.g., Aspergillus fumigatus, A. lentulus, and Neosartorya udagawae). Identification of these organisms may be clinically important, as some species vary in their susceptibilities to antifungal agents. In a prior study, we utilized multilocus sequence typing to describe A. lentulus as a species distinct from A. fumigatus. The sequence data show that the gene encoding β-tubulin, benA, has high interspecies variability at intronic regions but is conserved among isolates of the same species. These data were used to develop a PCR-restriction fragment length polymorphism (PCR-RFLP) method that rapidly and accurately distinguishes A. fumigatus, A. lentulus, and N. udagawae, three major species within the section Fumigati that have previously been implicated in disease. Digestion of the benA amplicon with BccI generated unique banding patterns; the results were validated by screening a collection of clinical strains and by in silico analysis of the benA sequences of Aspergillus spp. deposited in the GenBank database. PCR-RFLP of benA is a simple method for the identification of clinically important, similar morphotypes of Aspergillus spp. within the section Fumigati

Molecular Studies Reveal Frequent Misidentification of Aspergillus fumigatus by Morphotyping

Aspergillus fumigatus has been understood to be the most common cause of invasive aspergillosis (IA) in all epidemiological surveys. However, recent studies have uncovered a large degree of genetic heterogeneity between isolates morphologically identified as A. fumigatus, leading to the description of a new species, Aspergillus lentulus. Here, we examined the genetic diversity of clinical isolates identified as A. fumigatus using restriction enzyme polymorphism analysis and sequence-based identification. Analysis of 50 clinical isolates from geographically diverse locations recorded the presence of at least three distinct species: A. lentulus, Aspergillus udagawae, and A. fumigatus. In vitro, A. lentulus isolates demonstrated decreased susceptibility to antifungal drugs currently used for IA, including amphotericin B, voriconazole, and caspofungin; A. udagawae isolates demonstrated decreased in vitro susceptibility to amphotericin B. Results of the present study demonstrate that current phenotypic methods to identify fungi do not differentiate between genetically distinct species in the A. fumigatus group. Differential antifungal susceptibilities of these species may account for some of the reported poor outcomes of therapy in clinical studies

Atypical Aspergillus flavus Isolates Associated with Chronic Azole Therapy▿

A case of chronic sinus disease due to morphologically atypical Aspergillus flavus is described. Multiple fungal isolates sporulated poorly or not at all, displaying unusual color and microscopic morphology, including the absence of typical vesicles and phialides, which caused the isolates to resemble several other fungal genera superficially. The patient received multiple antifungal therapies over at least 10 years with various azole drugs, including voriconazole, itraconazole, and posaconazole. We speculate that this lengthy exposure to azole antifungal drugs may have caused or promoted the atypical morphology seen in these isolates

Aspergillus fumigatus Variant with Decreased Susceptibility to Multiple Antifungals

Isolates of Aspergillus fumigatus that demonstrate resistance to itraconazole (ITZ) have been described previously; however, the prevalence and clinical significance of ITZ resistance are not completely understood. In this study we assessed the ITZ susceptibilities of 128 A. fumigatus isolates that caused invasive infection in 82 stem cell transplant patients before and after the use of ITZ in our institution (study period, 1991 to 2000). The MICs for 10 isolates obtained from seven patients were high, ≥1 μg/ml. The average ITZ MIC increased after institutional use of the drug began in 1995. The majority of the isolates for which MICs were high (6 of 10) and one isolate for which the MIC was low (0.06 μg/ml) demonstrated an unusual phenotype, appearing as predominantly white colonies. For all seven atypical isolates, voriconazole MICs were high (≥2 μg/ml), and minimal effective concentrations of caspofungin were high (≥4 μg/ml). For two of the seven atypical isolates, amphotericin B MICs were high (≥2 μg/ml). The isolates appeared white due to slow sporulation; however, after prolonged incubations, the isolates sporulated with no difference in conidial color or conidiophore morphology compared with typical isolates. Randomly amplified polymorphic DNA-PCR patterns of these isolates were distinct compared with those of other A. fumigatus isolates. Sequencing of 18S rRNA genes confirmed that all were A. fumigatus; however, the mitochondrial cytochrome b gene sequences of all the atypical isolates were unique. These data suggest the potential presence of a genetically unique, poorly sporulating variant of A. fumigatus that demonstrates decreased susceptibilities to several antifungals