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ABSTRACT(1-3)-ß-d-Glucan (BDG) from cerebrospinal fluid (CSF) is a promising marker for diagnostic and prognostic aid of central nervous system (CNS) fungal infection, but its relationship to serum values has not been studied. Herein, we detected BDG from CSF at levels 2-fold lower than those in serum in patients without evidence of fungal disease but 25-fold higher than those in in serum in noncryptococcal CNS fungal infections. CSF BDG may be a useful biomarker in the evaluation of fungal CNS disease.

ABSTRACTCandida kefyris an emerging pathogen among patients with hematologic malignancies (HM). We performed a retrospective study at Johns Hopkins Hospital to evaluate the epidemiology of C. kefyrcolonization and infection in HM patients between 2004 and 2010. Eighty-three patients were colonized and/or infected with C. kefyr, with 8 (9.6%) having invasive candidiasis (IC). The yearly incidence of C. kefyrcolonization and candidemia increased over the study period (P< 0.01), particularly after 2009. In 2010, C. kefyrcaused 16.7% of candidemia episodes. The monthly incidence of C. kefyrwas higher during the summer throughout the study. In a cohort of patients with acute myelogenic leukemia receiving induction chemotherapy, risks for C. kefyrcolonization included the summer season (odds ratio [OR], 3.1; P= 0.03); administration of an azole (OR, 0.06; P< 0.001) or amphotericin B (OR, 0.35; P= 0.05) was protective. Fingerprinting of 16 isolates by repetitive sequence-based PCR showed that all were different genotypes. The epidemiology of C. kefyrcandidemia was evaluated in another hospital in Montreal, Canada; data confirmed higher rates of C. kefyrinfection in the summer. C. kefyrappears to be increasing in HM patients, with prominent summer seasonality. These findings raise questions about the effect of antifungal agents and health care exposures (e.g., yogurt) on the epidemiology of this yeast.

ABSTRACTA large aggregate collection of clinical isolates of aspergilli (n= 218) from transplant patients with proven or probable invasive aspergillosis was available from the Transplant-Associated Infection Surveillance Network, a 6-year prospective surveillance study. To determine the Aspergillusspecies distribution in this collection, isolates were subjected to comparative sequence analyses by use of the internal transcribed spacer and ß-tubulin regions. Aspergillus fumigatuswas the predominant species recovered, followed by A. flavusand A. niger. Several newly described species were identified, including A. lentulusand A. calidoustus; both species had high in vitro MICs to multiple antifungal drugs. Aspergillus tubingensis, a member of the A. nigerspecies complex, is described from clinical specimens; all A. tubingensisisolates had low in vitro MICs to antifungal drugs.

ABSTRACTWe analyzed antifungal susceptibilities of 274 clinical Aspergillusisolates from transplant recipients with proven or probable invasive aspergillosis collected as part of the Transplant-Associated Infection Surveillance Network (TRANSNET) and examined the relationship between MIC and mortality at 6 or 12 weeks. Antifungal susceptibility testing was performed by the Clinical and Laboratory Standards Institute (CLSI) M38-A2 broth dilution method for amphotericin B (AMB), itraconazole (ITR), voriconazole (VOR), posaconazole (POS), and ravuconazole (RAV). The isolate collection included 181 Aspergillus fumigatus, 28 Aspergillus niger, 27 Aspergillus flavus, 22 Aspergillus terreus, seven Aspergillus versicolor, five Aspergillus calidoustus, and two Aspergillus nidulansisolates and two isolates identified as Aspergillusspp. Triazole susceptibilities were =4 µg/ml for most isolates (POS, 97.6%; ITR, 96.3%; VOR, 95.9%; RAV, 93.5%). The triazoles were not active against the five A. calidoustusisolates, for which MICs were =4 µg/ml. AMB inhibited 93.3% of isolates at an MIC of =1 µg/ml. The exception was A. terreus, for which 15 (68%) of 22 isolates had MICs of >1 µg/ml. One of 181 isolates of A. fumigatusshowed resistance (MIC = 4 µg/ml) to two of three azoles tested. Although there appeared to be a correlation of higher VOR MICs with increased mortality at 6 weeks, the relationship was not statistically significant (R2= 0.61; P= 0.065). Significant relationships of in vitro MIC to all-cause mortality at 6 and 12 weeks for VOR or AMB were not found.

ABSTRACTRecent studies have shown that there are multiple clinically important members of the Aspergillussection Fumigatithat 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. lentulusas a species distinct from A. fumigatus. The sequence data show that the gene encoding ß-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 Fumigatithat have previously been implicated in disease. Digestion of the benAamplicon with BccI generated unique banding patterns; the results were validated by screening a collection of clinical strains and by in silico analysis of the benAsequences of Aspergillusspp. deposited in the GenBank database. PCR-RFLP of benAis a simple method for the identification of clinically important, similar morphotypes of Aspergillusspp. within the section Fumigati.

ABSTRACTInvasive fungal infections caused by Neosartorya pseudofischeriS. W. Peterson [anamorph Aspergillus thermomutatus(Paden) S. W. Peterson] are extremely rare. Phenotypically, the anamorphic state of N. pseudofischeriresembles Aspergillus fumigatus, the predominant agent of invasive aspergillosis in immunocompromised hosts. We report the recovery of three clinical isolates of N. pseudofischeri, all initially misidentified by morphological characteristics as A. fumigatus. All three isolates were correctly identified by sequencing portions of the ß-tubulin and the rodlet A genes. Only one of the three isolates produced the confirmatory fruiting bodies and was thus classified as N. pseudofischeri; the other isolates did not produce asci and were therefore identified as A. thermomutatus. All three isolates had higher MICs to voriconazole in vitro compared to A. fumigatusAf293. This report emphasizes that phenotypic identification of filamentous fungi may not identify morphologically similar, but genetically distinct, members of the genus Aspergillussection Fumigati. Accurate identification of these organisms may be clinically meaningful, given their potential differences in antifungal susceptibilities.

ABSTRACTInvasive pulmonary aspergillosis (IPA) is frequent and often fatal in hematopoietic stem cell transplant patients. Diagnosis requires microbiological or histopathologic demonstration of the organism in tissues; however, cultivation of Aspergillusspecies from respiratory secretions has low diagnostic sensitivity. Assays to detect Aspergillusantigen or DNA in bronchoalveolar lavage (BAL) fluid could facilitate earlier diagnosis, thereby guiding optimal therapy and obviating the need for additional costly and potentially morbid diagnostic evaluation. We evaluated the performance of a galactomannan enzyme immunoassay (GM EIA; Bio-Rad) by using a range of index cutoffs to define positivity and a quantitative PCR (qPCR) assay for the detection of Aspergillusspecies from BAL samples of patients with proven and probable IPA (case patients; n= 49) and without IPA (control patients; n= 50). The sensitivity of the GM EIA was 61% with an index cutoff of 1.0 and 76% with an index cutoff of 0.5; the corresponding specificities were 98 and 94%, respectively. The sensitivity and specificity of qPCR assay were 67 and 100%, respectively. The sensitivity with 22 culture-negative BAL specimens from patients with IPA was 41% for GM EIA with an index cutoff of 1.0, 59% for GM EIA with an index cutoff of 0.5, and 36% for qPCR assay. GM EIA indices and DNA quantities corresponded to BAL fungal burdens, with culture-positive samples having larger amounts of antigen and DNA compared to culture-negative samples. GM EIA and qPCR assay add to the sensitivity of BAL for diagnosing IPA in high-risk patients, with excellent specificity. Adjunctive use of these tests may reduce dependence on invasive diagnostic procedures.

ABSTRACTEchinocandins are a group of antifungal agents that target 1,3-ß-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 Aspergillusspecies 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 Aspergillusisolates; 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 Aspergillusisolates 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.

ABSTRACTPrimarily saprophytic in nature, fungi of the genus Acremoniumare a well-documented cause of mycetoma and other focal diseases. More recently, a number of Acremoniumspp. have been implicated in invasive infections in the setting of severe immunosuppression. During the course of routine microbiological studies involving a case of fatal mycosis in a nonmyeloablative hematopoietic stem cell transplant patient, we identified a greater-than-expected variation among strains previously identified as Acremonium strictumby clinical microbiologists. Using DNA sequence analysis of the ribosomal DNA intergenic transcribed spacer (ITS) regions and the D1-D2 variable domain of the 28S ribosomal DNA gene (28S), the case isolate and four other clinical isolates phenotypically identified as A. strictumwere found to have <99% homology to the A. strictumtype strain, CBS 346.70, at the ITS and 28S loci, while a sixth isolate phenotypically identified only as Acremoniumsp.had >99% homology to the type strain at both loci. These results suggest that five out of the six clinical isolates belong to species other than A. strictumor that the A. strictumtaxon is genetically diverse. Based upon these sequence data, the clinical isolates were placed into three genogroups.

ABSTRACTThis case series highlights our experience with use of the Fungitell assay for quantifying (1,3)-ß-d-glucan in cerebrospinal fluid during the current U.S. outbreak of fungal meningitis related to contaminated methylprednisolone acetate. This test may prove a useful adjunct in diagnosis and management of exposed patients.

ABSTRACTWe report a case of cryptococcosis due to C. gattiiwhich appears to have been acquired in the Puget Sound region, Washington State. Genotyping confirmed identity to the predominant Vancouver Island genotype. This is the first documented case of human disease by the major Vancouver Island emergence strain acquired within the United States.