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International audience; Nosocomial Candida albicans infections are a significant problem in neonatal intensive care units (NICUs). We investigated the clonality of C. albicans isolates recovered over an 8-year period from neonates at a NICU. We also validated multilocus sequence typing (MLST) compared with pulsed-field gel electrophoresis (PFGE) for the genotyping of C. albicans strains from the same NICU. A total of 43 clinical isolates (10 blood, 19 urine, and 14 other) were obtained from 43 neonates between 2005 and 2012. Clonal strains were defined as the isolation of two or more strains with identical or similar genotypes as determined with both MLST and PFGE. Using MLST, the 43 isolates yielded 25 diploid sequence types (DSTs) and 10 DSTs were shared by 28 isolates (65.1%). Among the 28 isolates sharing 10 DSTs, isolates from each of seven DSTs had the same or similar PFGE pattern. In addition, two sets of isolates that differed by MLST at only one locus had the same or similar PFGE pattern. Overall, when the MLST and PFGE results were combined, 22 isolates (51.2%) shared eight genotypes, suggesting clonal strains. Strains from each of seven genotypes (total, 19 isolates) were isolated among the 22 clonal strains within a 6-month period, whereas three strains of one genotype were obtained over a 3-year interval. Our findings suggest that horizontal transmission of C. albicans may occur more frequently than vertical transmission among NICU patients and that MLST appears to be a useful method for genotyping C. albicans strains isolated from NICU patients.

International audience; The black yeast Exophiala dermatitidis is a frequent agent of colonization of the lungs of patients with cystic fibrosis (CF). A total of 71 clinical isolates of Exophiala from 13 patients were identified at the species level by sequencing the internal transcribed spacer (ITS) regions 1 and 2 of the rDNA genes and typed by random amplification of polymorphic DNA (RAPD), using two different primers, BG-2 and ERIC-1. In vitro susceptibility of these isolates to some systemic antifungal drugs was investigated using the CLSI method. Almost all the isolates were identified as E. dermatitidis, but long-term colonization with the closely related species E. phaeomuriformis was observed in one patient. No clustering was found according to the geographical origin of the isolates, the isolation date or the antifungal susceptibility. Variations were seen in the susceptibility of studied isolates to antifungals but most of them exhibited low susceptibility to amphotericin B and although some patients were successively colonized by two distinct genotypes, most of the isolates were distributed in patient-specific clusters. This phenomenon may be due to genomic variations of E. dermatitidis in the lung environment of CF patients. These results are typical of colonization of the airways of patients by a poorly distributed environmental fungus, which occupies particular reservoirs that need to be defined.

International audience; When Candida dubliniensis isolates obtained from seabird excrement and from humans in Ireland were compared by using multilocus sequence typing, 13 of 14 avian isolates were genetically distinct from human isolates. The remaining avian isolate was indistinguishable from a human isolate, suggesting that transmission may occur between humans and birds.

Cryptococcus neoformans var. neoformans (serotype D) represents about 30% of the clinical isolates in Europe and is present less frequently in the other continents. It is the prevalent etiological agent in primary cutaneous cryptococcosis as well as in cryptococcal skin lesions of disseminated cryptococcosis. Very little is known about the genotypic diversity of this Cryptococcus subtype. The aim of this study was to investigate the genotypic diversity among a set of clinical and environmental C. neoformans var. neoformans isolates and to evaluate the relationship between genotypes, geographical origin and clinical manifestations. A total of 83 globally collected C neoformans var. neoformans isolates from Italy, Germany, France, Belgium, Denmark, Greece, Turkey, Thailand, Japan, Colombia, and the USA, recovered from different sources (primary and secondary cutaneous cryptococcosis, disseminated cryptococcosis, the environment, and animals), were included in the study. All isolates were confirmed to belong to genotype VNIV by molecular typing and they were further investigated by MLST analysis. Maximum likelihood phylogenetic as well as network analysis strongly suggested the existence of a recombinant rather than a clonal population structure. Geographical origin and source of isolation were not correlated with a specific MLST genotype. The comparison with a set of outgroup C neoformans var. grubii isolates provided clear evidence that the two varieties have different population structures. United States Department of Health & Human Services - Z99 AI999999 - P41 RR001646 National Institutes of Health (NIH) - USA NIH National Center for Research Resources (NCRR)

International audience; BACKGROUND: The aim of our study was to assess the detection of circulating DNA from the most common species of Mucorales for early diagnosis of mucormycosis in at-risk patients. METHODS: We retrospectively evaluated a combination of 3 quantitative polymerase chain reaction (qPCR) assays using hydrolysis probes targeting Mucor/Rhizopus, Lichtheimia (formerly Absidia), and Rhizomucor for circulating Mucorales detection. Serial serum samples from 10 patients diagnosed with proven mucormycosis (2-9 samples per patient) were analyzed. RESULTS: No cross-reactivity was detected in the 3 qPCR assays using 19 reference strains of opportunistic fungi, and the limit of detection ranged from 3.7 to 15 femtograms/10 µL, depending on the species. DNA from Mucorales was detected in the serum of 9 of 10 patients between 68 and 3 days before mucormycosis diagnosis was confirmed by histopathological examination and/or positive culture. All the qPCR results were concordant with culture and/or PCR-based identification of the causing agents in tissue (Lichtheimia species, Rhizomucor species, and Mucor/Rhizopus species in 4, 3, and 2 patients, respectively). Quantitative PCR was negative in only 1 patient with proven disseminated mucormycosis caused by Lichtheimia species. CONCLUSION: Our study suggests that using specific qPCR targeting several species of Mucorales according to local ecology to screen at-risk patients could be useful in a clinical setting. The cost and efficacy of this strategy should be evaluated. However, given the human and economic cost of mucormycosis and the need for rapid diagnosis to initiate prompt directed antifungal therapy, this strategy could be highly attractive.

EA MERS CT3 Enjeu 3; International audience; The dimorphic yeast Candida albicans is a component of the normal microflora at the mucosal surfaces of healthy individuals. It possesses an array of phenotypic properties considered as virulence traits that contribute to pathogenicity of the yeast in immuno-compromised patients. We addressed the question of the pathogenicity of lineages of C. albicans with regard to their genotype in three series of C. albicans isolates (a series of commensal isolates collected in healthy individuals, a group of bloodstream isolates and a group of non-bloodstream clinical isolates) using a Multi-Locus Microsatellite Typing (MLMT) approach based on the analysis of the polymorphism of 11 microsatellite loci. The MLMT analysis of the three series, corresponding to 174 C. albicans isolates, gave a 100% typability to the method, with a DP index of 0.999. The UPGMA analysis showed that the isolates segregated in eight phylogenetic groups. Interestingly, the clustering was comparable when using NJ and MS-tree algorithms and a good concordance index of the clustering was observed with MLST. All in all our data strongly indicated MLMT as a reliable tool for DNA-typing studies in C. albicans. Isolates from healthy and non-healthy individuals segregated at the same proportions into the eight phylogenetic groups, suggesting that isolates of different origin share the same overall pathogenicity. Surprisingly allelic frequencies at the HIS3 microsatellite differed significantly in commensal isolates (group A) from pooled groups B and C (clinical isolates), raising the possibility that some individual alleles at the HIS3 microsatellite may be associated with distinct pathogenic profiles in C. albicans.

International audience; Black yeasts including Exophiala species are increasingly recognized as agents of human disease. In recent years, progress in molecular phylogeny and taxonomy of the genus Exophiala has led to the description of numerous novel species. The 'classical' but highly variable species Exophiala jeanselmei was split into a number of morphological siblings, which, however, were phylogenetically and clinically remote from each other. E. jeanselmei was restricted to an uncommon species causing subcutaneous infections. Hence only limited information is available on the segregants, among which is E. xenobiotica. We describe a case of an HIV-patient presenting with fungal arthritis and subcutaneous nodules caused by the latter species, which was identified by means of phenotypic and molecular methods.

Pneumocystis carriage was detected in 12.5% of 104 cystic fibrosis (CF) patients during a prospective multicenter French study, with a prevalence of genotype 85C/248C and geographic variations. It was significantly associated with the absence of Pseudomonas aeruginosa colonization and a greater forced expiratory volume in 1 s. Results are discussed considering the natural history of CF.

Multilocus sequence typing (MLST) has been successfully applied to the epidemiology of Candida albicans isolates not only within the hospital setting but also in multiple locations nationwide. We performed MLST to investigate the genetic relatedness among bloodstream infection (BSI) isolates of C. albicans recovered from 10 Korean hospitals over a 12-month period. The 156 isolates yielded 112 unique diploid sequence types (DSTs). While 95 DSTs were each derived from a single isolate, 17 DSTs were shared by 61 isolates (39.1%). Interestingly, 111 (71.1%) isolates clustered within previously known clades, and 29 (18.6%) clustered within a new clade that includes strains of Asian origin previously typed as singletons. This MLST study was complemented by restriction endonuclease analysis of genomic DNA using BssHII (REAG-B) in order to evaluate whether strains with identical DSTs and originating from the same hospital corresponded to nosocomial clusters. Importantly, only those isolates with a strong epidemiological relationship showed ≥95% identical REAG-B types. Our results indicate that REAG-B typing can be complementary to MLST but should be limited to the investigation of isolates of identical DSTs and when interhuman transmission is suspected.

International audience; Recent changes in the epidemiology of candidiasis highlighted an increase in non- Candida albicans species emphasizing the need for reliable identification methods. Molecular diagnostics in fungal infections may improve species characterization, particularly in cases of the closely related species in the Candida complexes. We developed two PCR/restriction fragment length polymorphism assays, targeting either a part of the intergenic spacer 2 or the entire intergenic spacer (IGS) of ribosomal DNA using a panel of 270 isolates. A part of the intergenic spacer was used for discrimination between C. albicans and C. dubliniensis and between species of the C. glabrata complex (C. glabrata/C. bracarensis/C. nivariensis). The whole IGS was applied to C. parapsilosis, C. metapsilosis, and C. orthopsilosis, and to separate C. famata (Debaryomyces hansenii) from C. guilliermondii (Pichia guilliermondii) and from the other species within this complex (ie, C. carpophila, C. fermentati and C. xestobii). Sharing similar biochemical patterns, Pichia norvegensis and C. inconspicua exhibited specific IGS profiles. Our study confirmed that isolates of C. guilliermondii were frequently mis-identified as C. famata. As much as 67% of the clinical isolates phenotypically determined as C. famata were recognized mostly as true P. guilliermondii. Conversely, 44% of the isolates initially identified as C. guilliermondii were corrected by the IGS fingerprints as C. parapsilosis, C. fermentati, or C. zeylanoides. These two PCR/restriction fragment length polymorphism methods may be used as reference tools [either alternatively or adjunctively to the existing ribosomal DNA (26S or ITS) sequence comparisons] for unambiguous determination of the Candida species for which phenotypic characterization remains problematic.

Saksenaea is a monotypic genus belonging to the order Mucorales and capable of producing severe human infections. Through a polyphasic study based on analysis of the sequences of the internal transcribed spacer (ITS) region, domains D1 and D2 of the 28S rRNA gene, and the elongation factor 1α (EF-1α) gene, as well as by evaluation of relevant morphological and physiological characteristics of a set of clinical and environmental strains, we have demonstrated that Saksenaea vasiformis is a complex of species. We propose as new species Saksenaea oblongispora , characterized by oblong sporangiospores and unable to grow at 42°C, and Saksenaea erythrospora , characterized by large sporangiophores and sporangia and by ellipsoid sporangiospores, biconcave in the lateral view. Itraconazole, posaconazole, and terbinafine were active against all isolates included in the study, while amphotericin B, voriconazole, and the echinocandins showed low activity.

International audience; Poorly sporulating Aspergillus isolates from patients with cystic fibrosis (CF) are generally identified in routine procedures as Aspergillus spp. In this study, we identified and characterized 11 isolates belonging to two unusual Aspergillus species of the section Fumigati (A. lentulus and Neosartorya pseudofischeri) recovered from four different patients. Aspergillus lentulus was found occasionally during a 10-year follow-up study of one CF patient colonized by A. fumigatus. Neosartorya pseudofischeri was isolated from three patients followed in different European hospitals. This species was recovered from two sputum samples of one patient, and from four successive samples of the two other patients, suggesting that it may be responsible for chronic colonization. Both species were isolated together with A. fumigatus. Isolates from both species did not grow at 50°C, and DNA sequence analysis, together with further morphological observations permitted identification at the species level. Growth at different temperatures and antifungal susceptibility were also investigated. All the isolates of N. pseudofischeri exhibited a very low susceptibility to voriconazole (VRZ) whereas a very low susceptibility to VRZ and amphotericin B was seen with the A. lentulus isolates.

International audience; Risk factors discovery and prevention is an active research field within the biomedical domain. Despite abundant existing information on risk factors, as found in bibliographical databases or on several websites, accessing this information may be difficult. Methods from Natural Language Processing and Information Extraction can be helpful to access it more easily. Specifically, we show a procedure for analyzing massive amounts of scientific literature and for detecting linguistically marked associations between pathologies and risk factors. This approach allowed us to extract over 22,000 risk factors and associated pathologies. The performed evaluations pointed out that (1) over 88% of risk factors for coronary heart disease are correct, (2) associated pathologies, when they could be compared to MeSH indexing, are correct in about 70%, and (3) in existing terminologies links between risk factors and their pathologies are seldom recorded.

Thirty-eight isolates (including 28 isolates from patients) morphologically identified as Lichtheimia corymbifera (formerly Absidia corymbifera ) were studied by sequence analysis (analysis of the internal transcribed spacer [ITS] region of the ribosomal DNA, the D1-D2 region of 28S, and a portion of the elongation factor 1α [EF-1α] gene). Phenotypic characteristics, including morphology, antifungal susceptibility, and carbohydrate assimilation, were also determined. Analysis of the three loci uncovered two well-delimited clades. The maximum sequence similarity values between isolates from both clades were 66, 95, and 93% for the ITS, 28S, and EF-1α loci, respectively, with differences in the lengths of the ITS sequences being detected (763 to 770 bp for isolates of clade 1 versus 841 to 865 bp for isolates of clade 2). Morphologically, the shapes and the sizes of the sporangiospores were significantly different among the isolates from both clades. On the basis of the molecular and morphological data, we considered isolates of clade 2 to belong to a different species named Lichtheimia ramosa because reference strains CBS 269.65 and CBS 270.65 (which initially belonged to Absidia ramosa ) clustered within this clade. As neotype A. corymbifera strain CBS 429.75 belongs to clade 1, the name L. corymbifera was conserved for clade 1 isolates. Of note, the amphotericin B MICs were significantly lower for L. ramosa than for L. corymbifera ( P < 0.005) but were always ≤0.5 μg/ml for both species. Among the isolates tested, the assimilation of melezitose was positive for 67% of the L. ramosa isolates and negative for all L. corymbifera isolates. In conclusion, this study reveals that two Lichtheimia species are commonly associated with mucormycosis in humans.

International audience; A series of 256 Aspergillus fumigatus isolates, recovered from eight patients with cystic fibrosis (CF), were genotyped using microsatellite-based typing. Only a limited number of genotypes were shared between patients and co-colonisation with multiple strains was indicated for all patients. Additionally, some genotypes were isolated recurrently, indicating that they are capable of prolonged colonisation.

A multiple-locus variable-number tandem-repeat analysis (MLVA) using six microsatellite markers was assessed in 127 Candida glabrata isolates. Thirty-seven different genotypes, stable both in vitro and in vivo, were observed. The highest discriminatory power ( D = 0.902) was reached by using only four markers. MLVA seems to be relevant for C. glabrata typing.

Aims: To assess suitability of Multi Locus Sequence Typing (MLST) for investigating the biodiversity of wine yeast strains. This method was compared with established ones like microsatellite analysis or amplification of genomic regions flanked by repeated (delta) elements. Methods and Results: DNA fragments were amplified and sequenced for 26 loci representing housekeeping genes, open reading frames (ORFs) of unknown functions or intergenic regions. A set of seven loci was tested on 84 Saccharomyces cerevisiae strains, including 65 strains isolated from traditional wineries in Lebanon, commercial wine strains and Asian isolates. An overall sequence diversity of 2·05% was observed, consisting of single nucleotide polymorphisms, 60% of them occurring in a heterozygous state. The number of polymorphic sites per locus varied between 4 and 14. The same set of strains was analysed by microsatellite typing on six polymorphic loci and by interdelta amplification. Conclusions: Clustering of MLST profiles clearly differentiated the Asian group of strains from Lebanese and European commercial strains that appear closely related. The current MLST scheme appears less discriminatory (92·27%) on closely related wine yeasts than microsatellite or interdelta typing (>99%). Significance and Impact of the Study: MLST is a highly reliable method for relatedness inference and promising for wine yeast typing.