Evaluation of an in-house Pan-Malassezia quantitative PCR in human clinical samples.

Althought Malassezia spp. have been involved in various pathologies, they are integral part of the cutaneous, gut, oral, ears, nose and throat (ENT) mycobiota. Since Malassezia are difficult to grow in culture, unexhaustive molecular biology methods have been developed to detect them. The aim of the study was to evaluate an in-house pan-Malassezia quantitative PCR (panM-qPCR) on various clinical human samples and determine Malassezia burden in various human mycobiota. The panM-qPCR was designed to target the repeated 28S rDNA gene from all Malassezia species. We used the assay to quantify the Malassezia burden on 361 samples from 161 subjects (80 skin swabs from 10 healthy volunteers (HV), 13 samples from 2 seborrheic dermatitis patients (SD), 90 skin samples from 19 burned patients, 119 stools samples from 89 immunocompromised patients, 59 ENT samples from 41 patients). For HV, the amount of Malassezia was different according to the swabbed areas. Cq in SD are lower than in HV. In burned patients, Cq was significantly lower compared to HV. In stool samples, 6.7% were positive for Malassezia spp. with a high Cq. For the ENT area, a higher proportion of positive specimens were detected in ears samples than in nose samples. Our findings emphasized the importance of qPCR, confirming elevated Malassezia spp. levels on individuals' faces and scapls, increased burden in SD patients and in severely burnt patients than in HV. The pan-MqPCR appears to be a promising tool for studying Malassezia in various human mycobiota.

Malassezia species are ubiquitous members of various human mycobiomes, including cutaneous and mucosal sites. While these fungi have been implicated in several pathologies, their presence as commensals complicates their study, especially due to difficulties in culturing them in vitro. This has necessitated the development of molecular techniques to detect and quantify Malassezia species directly from clinical samples.

Unveiling Trichosporon austroamericanum sp. nov.: A Novel Emerging Opportunistic Basidiomycetous Yeast Species.

During an epidemiological survey, a potential novel species within the basidiomycetous yeast genus Trichosporon was observed. The clinical strain was obtained from a urine sample taken from a Brazilian kidney transplant recipient. The strain was molecularly identified using the intergenic spacer (IGS1) ribosomal DNA locus and a subsequent phylogenetic analysis showed that multiple strains that were previously reported by other studies shared an identical IGS1-genotype most closely related to that of Trichosporon inkin. However, none of these studies provided an in-depth characterization of the involved strains to describe it as a new taxon. Here, we present the novel clinically relevant yeast for which we propose the name Trichosporon austroamericanum sp. nov. (holotype CBS H-24937). T. austroamericanum can be distinguished from other siblings in the genus Trichosporon using morphological, physiological, and phylogenetic characters.

Echinocandins Susceptibility Patterns of 2,787 Yeast Isolates: Importance of the Thresholds for the Detection of FKS Mutations.

Since echinocandins are recommended as first line therapy for invasive candidiasis, detection of resistance, mainly due to alteration in FKS protein, is of main interest. EUCAST AFST recommends testing both MIC of anidulafungin and micafungin, and breakpoints (BPs) have been proposed to detect echinocandin-resistant isolates. We analyzed MIC distribution for all three available echinocandins of 2,787 clinical yeast isolates corresponding to 5 common and 16 rare yeast species, using the standardized EUCAST method for anidulafungin and modified for caspofungin and micafungin (AM3-MIC). In our database, 64 isolates of common pathogenic species were resistant to anidulafungin, according to the EUCAST BP, and/or to caspofungin, using our previously published threshold (AM3-MIC ≥ 0.5 mg/L). Among these 64 isolates, 50 exhibited 21 different FKS mutations. We analyzed the capacity of caspofungin AM3-MIC and anidulafungin MIC determination in detecting isolates with FKS mutation. They were always identified using caspofungin AM3-MIC and the local threshold while some isolates were misclassified using anidulafungin MIC and EUCAST threshold. However, both methods misclassified four wild-type C. glabrata as resistant. Based on a large data set from a single center, the use of AM3-MIC testing for caspofungin looks promising in identifying non-wild-type C. albicans, C. tropicalis and P. kudiravzevii isolates, but additional multicenter comparison is mandatory to conclude on the possible superiority of AM3-MIC testing compared to the EUCAST method.

Azole Susceptibility Profiles of More than 9,000 Clinical Yeast Isolates Belonging to 40 Common and Rare Species.

Invasive yeast infections represent a major global public health issue, and only few antifungal agents are available. Azoles are one of the classes of antifungals used for treatment of invasive candidiasis. The determination of antifungal susceptibility profiles using standardized methods is important to identify resistant isolates and to uncover the potential emergence of intrinsically resistant species. Here, we report data on 9,319 clinical isolates belonging to 40 pathogenic yeast species recovered in France over 17 years. The antifungal susceptibility profiles were all determined at the National Reference Center for Invasive Mycoses and Antifungals based on the EUCAST broth microdilution method. The centralized collection and analysis allowed us to describe the trends of azole susceptibility of isolates belonging to common species, confirming the high susceptibility for Candida albicans (n = 3,295), Candida tropicalis (n = 641), and Candida parapsilosis (n = 820) and decreased susceptibility for Candida glabrata (n = 1,274) and Pichia kudriavzevii (n = 343). These profiles also provide interesting data concerning azole susceptibility of Cryptococcus neoformans species complex, showing comparable MIC distributions for the three species but lower MIC50s and MIC90s for serotype D (n = 208) compared to serotype A (n = 949) and AD hybrids (n = 177). Finally, these data provide useful information for rare and/or emerging species, such as Clavispora lusitaniae (n = 221), Saprochaete clavata (n = 184), Meyerozyma guilliermondii complex (n = 150), Candida haemulonii complex (n = 87), Rhodotorula mucilaginosa (n = 55), and Wickerhamomyces anomalus (n = 36).

A Short-Tandem-Repeat Assay (MmySTR) for Studying Genetic Variation in Madurella mycetomatis.

Madurella mycetomatis is the major causative agent of eumycetoma, a neglected tropical infection characterized by painless subcutaneous lesions, inflammation, and grains draining from multiple sinuses. To study the epidemiology of mycetoma, a robust discriminatory typing technique is needed. We describe the use of a short-tandem-repeat assay (MmySTR) for genotyping of M. mycetomatis isolates predominantly from Sudan. Eleven microsatellite markers (3 dinucleotides, 4 trinucleotide repeats, and 4 tetranucleotide repeats) were selected from the M. mycetomatis MM55 genome using the Tandem Repeats Finder software. PCR amplification primers were designed for each microsatellite marker using primer3 software and amplified in a multicolor multiplex PCR approach. To establish the extent of genetic variation within the population, a collection of 120 clinical isolates from different regions was genotyped with this assay. The 11 selected MmySTR markers showed a large genotypic heterogeneity. From a collection of 120 isolates, 108 different genotypes were obtained. Simpson's diversity index (D) value for individual markers ranged from 0.081 to 0.881, and the combined panel displayed an overall D value of 0.997. The MmySTR assay demonstrated high stability, reproducibility, and specificity. The MmySTR assay is a promising new typing technique that can be used to genotype isolates of M. mycetomatis Apart from the possible contribution of host factors, the genetic diversity observed among this group of isolates might contribute to the different clinical manifestations of mycetoma. We recommend that the MmySTR assay be used to establish a global reference database for future study of M. mycetomatis isolates.

Central nervous system candidiasis beyond neonates: Lessons from a nationwide study.

Though candidiasis is the most frequent invasive fungal infection, Candida spp. central nervous system (CNS) infections are rare but severe. To further describe clinico-patho-radiological presentations of this entity, we report a retrospective study from January 2005 to December 2018 including patients aged ≥ 28 days with proven or probable CNS candidiasis in France. Twenty-four patients were included. Seventeen patients (70%) had CNS localization secondary to disseminated candidiasis (10 with hematologic malignancies [HM]; the seven other patients had infective endocarditis [IE]). Among patients with HM, seven previously had lumbar puncture for intrathecal chemotherapy, the three others had IE. Among patients with disseminated infection, magnetic resonance imaging (MRI) evidenced meningitis (17%), micro-abscesses (58%), or vascular complications (67%). Seven patients (30%) had isolated CNS involvement related to neurosurgery (n = 2), CARD9 deficiency (n = 2), intravenous drug use, diabetes mellitus, or no identified predisposing condition (n = 1 each). All evaluated patients with isolated CNS involvement had meningitis on cerebrospinal fluid (CSF) and intracranial hypertension. For the latter patients, MRI evidenced meningitis (71%) or abscesses (57%). Among all patients, cerebrospinal fluid (CSF) culture grew Candida spp. in 31% of cases. CSF ßDGlucan or mannan Ag were positive in respectively 86% and 80% of cases. Mortality attributed to CNS candidiasis was 42%: 53% in case of disseminated infection (70% for HM) and 14% in case of localized infection. CNS candidiasis are isolated or occur during disseminated infection in patients with HM and lumbar puncture for intrathecal chemotherapy or during IE. Clinical, radiological finding and outcome highly vary according to CNS localized versus disseminated candidiasis. LAY SUMMARY: Candida is a yeast and is the most common cause of fungal infections worldwide. Candida central nervous system (CNS) infections are rare, severe, and poorly described. We report a retrospective study from January 2005 to December 2018 including patients aged ≥ 28 days with proven or probable CNS candidiasis in France. Twenty-four patients were included (14 men, median age 51 years). Seventeen patients had CNS localization secondary to disseminated candidiasis from blood to CNS (10 with hematologic malignancies [HM], the seven other patients had infective endocarditis [IE]). Seven patients had isolated CNS involvement related to neurosurgery (n = 2), CARD9 deficiency (n = 2), intravenous drug use (n = 1), diabetes mellitus (n = 1), or no identified risk factor (n = 1).During Candida CNS infections, brain lesions were meningitis abscesses or vascular complications. Cerebrospinal fluid (CSF) culture grew Candida spp. in 31% of cases. Forty-two percent of patients died from infection: 53% in case of disseminated infection (70% for HM) and 14% in case of localized infection.

Saprochaete clavata Outbreak Infecting Cancer Center through Dishwasher.

Saprochaete clavata is a pathogenic yeast responsible for rare outbreaks involving immunocompromised patients, especially those with hematologic malignancies. During February 2016-December 2017, we diagnosed S. clavata infections in 9 patients (8 with fungemia), including 3 within 1 month, at a cancer center in Marseille, France. The patients (median age 58 years), 4 of 9 of whom had acute myeloid leukemia, were hospitalized in 3 different wards. Ten environmental samples, including from 2 dishwashers and 4 pitchers, grew S. clavata, but no contaminated food was discovered. The outbreak ended after contaminated utensils and appliances were discarded. Whole-genome sequencing analysis demonstrated that all clinical and environmental isolates belonged to the same phylogenetic clade, which was unrelated to clades from previous S. clavata outbreaks in France. We identified a dishwasher with a deficient heating system as the vector of contamination.

Yarrowia lipolytica causes sporadic cases and local outbreaks of infections and colonisation.

BACKGROUND: Yarrowia lipolytica belongs to the normal human microbiota but is also found in substrates with high contents in lipids and used in biotechnological processes. It is sometimes reported as human pathogen and especially in catheter-related candidaemia. OBJECTIVES: Two apparently grouped cases of infections and/or contamination were reported involving 3 and 9 patients, respectively, in two hospitals. The goal of this study was to design a molecular tool to study the genetic diversity of Y lipolytica and confirm or not the common source of contamination during these grouped cases. METHODS: Given that there is no genotyping method, we used genomic markers assessed on environmental isolates to determine intra-species relationship. We selected five highly polymorphic intergenic regions, totalling more than 3200 bp and sequenced them for clinical (n = 20) and environmental (n = 14) isolates. Antifungal susceptibility was determined by EUCAST broth microdilution method. RESULTS: Multiple alignment of the five sequences revealed divergence of 0%-5.8% between isolates as compared to approximately 0.2%-0.25% after alignment of whole genomes, suggesting their potential usefulness to establish genetic relatedness. The analysis showed the multiple origins of the isolates. It uncovered two grouped case of fungaemia involving 3 and 2 patients, respectively. It also revealed several unrelated sporadic cases despite their temporal relationship and one probable laboratory contamination by a common yet uncovered source, explaining several consecutive positive cultures without infection. All isolates had high minimal inhibitory concentration (MIC) for flucytosine, the majority (14/34) was susceptible to fluconazole, and all to the other antifungal agents tested. CONCLUSION: This method could help elucidate cases related to the opportunistic pathogen Y lipolytica.

Epidemiological investigation for grouped cases of Trichosporon asahii using whole genome and IGS1 sequencing.

BACKGROUND: Trichosporonosis is a rare invasive infection in humans mainly due to Trichosporon asahii, and especially recovered from patients having haematological malignancy. Since 2012, IGS1 region sequencing is used as a genotyping method to distinguish isolates, with high frequency of one haplotype worldwide and a geographic specificity for some haplotypes. OBJECTIVES: We compared the IGS1 genotyping method and whole genome sequencing (WGS) to study the relationship between clinical isolates involved in two grouped cases in France. METHODS: IGS1 sequencing and antifungal susceptibility testing were performed for 54 clinical isolates. Clinical data for 28 isolates included in surveillance programs were analysed. Whole genome was sequenced for 32 clinical isolates and the type strain. RESULTS: All isolates were intrinsically resistant to flucytosine, while voriconazole had the most potent in vitro activity. The majority of the isolates was recovered from patients with haematological malignancies (42.86%), with a high proportion of children (<15 yrs-old, 32.14%) and a high mortality rate at three months (46.15%). Based on the WGS analysis, isolates exhibiting IGS1 haplotype 1, 3 and 7 belonged to different clades. Five isolates recovered during the first grouped cases had the same IGS1 haplotype and shared 99% of SNPs similarity. For the second grouped cases, four isolates had 98.7% of SNPs similarity while the isolate recovered 4 years earlier was totally unlinked. CONCLUSIONS: We confirmed the usefulness of IGS1 sequencing for grouped cases infection of T. asahii. We underlined its limitation for the study of population structure and the utility of WGS analysis for the study of epidemiologically unrelated isolates.

Genotypes and population genetics of cryptococcus neoformans and cryptococcus gattii species complexes in Europe and the mediterranean area.

A total of 476 European isolates (310 Cryptococcus neoformans var. grubii, 150 C. neoformans var. neoformans, and 16 C. gattii species complex) from both clinical and environmental sources were analyzed by multi-locus sequence typing. Phylogenetic and population genetic analyses were performed. Sequence analysis identified 74 sequence types among C. neoformans var. neoformans (VNIV), 65 among C. neoformans var. grubii (56 VNI, 8 VNII, 1 VNB), and 5 among the C. gattii species complex (4 VGI and 1 VGIV) isolates. ST23 was the most frequent genotype (22%) among VNI isolates which were mostly grouped in a large clonal cluster including 50% of isolates. Among VNIV isolates, a predominant genotype was not identified. A high percentage of autochthonous STs were identified in both VNI (71%) and VNIV (96%) group of isolates. The 16 European C. gattii species complex isolates analyzed in the present study originated all from the environment and all belonged to a large cluster endemic in the Mediterranean area. Population genetic analysis confirmed that VNI group of isolates were characterized by low variability and clonal expansion while VNIV by a higher variability and a number of recombination events. However, when VNI and VNIV environmental isolates were compared, they showed a similar population structure with a high percentage of shared mutations and the absence of fixed mutations. Also linkage disequilibrium analysis reveals differences between clinical and environmental isolates showing a key role of PLB1 allele combinations in host infection as well as the key role of LAC1 allele combinations for survival of the fungus in the environment. The present study shows that genetic comparison of clinical and environmental isolates represents a first step to understand the genetic characteristics that cause the shift of some genotypes from a saprophytic to a parasitic life style.

Database establishment for the secondary fungal DNA barcode translational elongation factor 1α (TEF1α) 1.

With new or emerging fungal infections, human and animal fungal pathogens are a growing threat worldwide. Current diagnostic tools are slow, non-specific at the species and subspecies levels, and require specific morphological expertise to accurately identify pathogens from pure cultures. DNA barcodes are easily amplified, universal, short species-specific DNA sequences, which enable rapid identification by comparison with a well-curated reference sequence collection. The primary fungal DNA barcode, ITS region, was introduced in 2012 and is now routinely used in diagnostic laboratories. However, the ITS region only accurately identifies around 75% of all medically relevant fungal species, which has prompted the development of a secondary barcode to increase the resolution power and suitability of DNA barcoding for fungal disease diagnostics. The translational elongation factor 1α (TEF1α) was selected in 2015 as a secondary fungal DNA barcode, but it has not been implemented into practice, due to the absence of a reference database. Here, we have established a quality-controlled reference database for the secondary barcode that together with the ISHAM-ITS database, forms the ISHAM barcode database, available online at http://its.mycologylab.org/ . We encourage the mycology community for active contributions.

Investigating Clinical Issues by Genotyping of Medically Important Fungi: Why and How?

Genotyping studies of medically important fungi have addressed elucidation of outbreaks, nosocomial transmissions, infection routes, and genotype-phenotype correlations, of which secondary resistance has been most intensively investigated. Two methods have emerged because of their high discriminatory power and reproducibility: multilocus sequence typing (MLST) and microsatellite length polymorphism (MLP) using short tandem repeat (STR) markers. MLST relies on single-nucleotide polymorphisms within the coding regions of housekeeping genes. STR polymorphisms are based on the number of repeats of short DNA fragments, mostly outside coding regions, and thus are expected to be more polymorphic and more rapidly evolving than MLST markers. There is no consensus on a universal typing system. Either one or both of these approaches are now available for Candida spp., Aspergillus spp., Fusarium spp., Scedosporium spp., Cryptococcus neoformans, Pneumocystis jirovecii, and endemic mycoses. The choice of the method and the number of loci to be tested depend on the clinical question being addressed. Next-generation sequencing is becoming the most appropriate method for fungi with no MLP or MLST typing available. Whatever the molecular tool used, collection of clinical data (e.g., time of hospitalization and sharing of similar rooms) is mandatory for investigating outbreaks and nosocomial transmission.

Population Structure of Candida parapsilosis: No Genetic Difference Between French and Uruguayan Isolates Using Microsatellite Length Polymorphism.

Candida parapsilosis is a human commensal yeast, frequently involved in infection worldwide and especially in neonates. It is the second species responsible for bloodstream infections in Uruguay and the third species in France. We were interested in knowing whether the population structure of isolates responsible for candidemia in France and in Uruguay was different. Genotyping methods based on microsatellite length polymorphism (MLP) have been described and are especially used for investigation of local outbreaks. We therefore determined the genotypes of 159 C. parapsilosis isolates recovered from 122 patients (84 French patients from 43 hospitals and 38 Uruguayan patients from 10 hospitals) using three microsatellites markers previously described. Our results confirmed that C. parapsilosis population has a high genetic diversity, clonal inheritance and that majority of patients were infected by a single isolate. But we described recurrent infections due to related or unrelated genotypes resulting from isolates harboring loss or gain of heterozygosity. We also described three cases of coinfections due to unrelated genotypes. We did not uncover geographic specificity but observed two linked genotypes that seem to be associated with voriconazole resistance. Finally, among eight isolates involved in grouped cases, the genotypes were similar in six cases supporting the hypothesis of inter-patient transmission. These results confirmed the usefulness of performing MLP genotyping analysis for grouped cases of C. parapsilosis isolates in order to reinforce preventive hygiene measures.

Fundamental niche prediction of the pathogenic yeasts Cryptococcus neoformans and Cryptococcus gattii in Europe.

Fundamental niche prediction of Cryptococcus neoformans and Cryptococcus gattii in Europe is an important tool to understand where these pathogenic yeasts have a high probability to survive in the environment and therefore to identify the areas with high risk of infection. In this study, occurrence data for C. neoformans and C. gattii were compared by MaxEnt software with several bioclimatic conditions as well as with soil characteristics and land use. The results showed that C. gattii distribution can be predicted with high probability along the Mediterranean coast. The analysis of variables showed that its distribution is limited by low temperatures during the coldest season, and by heavy precipitations in the driest season. C. neoformans var. grubii is able to colonize the same areas of C. gattii but is more tolerant to cold winter temperatures and summer precipitations. In contrast, the C. neoformans var. neoformans map was completely different. The best conditions for its survival were displayed in sub-continental areas and not along the Mediterranean coasts. In conclusion, we produced for the first time detailed prediction maps of the species and varieties of the C. neoformans and C. gattii species complex in Europe and Mediterranean area.

Predisposing factors and outcome of uncommon yeast species-related fungaemia based on an exhaustive surveillance programme (2002-14).

Objectives: Using registry data to compare fungaemia caused by uncommon yeast species (UYS; i.e. other than Candida albicans , Candida glabrata , Candida parapsilosis , Candida tropicalis and Candida krusei ) and C. albicans -related fungaemia can reveal specific predisposing factors of UYS with potential impact on treatment strategies. Methods: We analysed 338 episodes of UYS fungaemia prospectively collected from 27 hospitals (Paris, France; 1 October 2002-31 December 2014) and compared these with 1998 single episodes of C. albicans fungaemia using univariate and multivariate analyses. Results: The proportion of UYS fungaemia was stable over time. Thirty-five different species were identified (27 ascomycetes, 8 basidiomycetes), 11 had caspofungin MIC 50 >0.25 mg/L and 15 fluconazole MIC 50 >4 mg/L. Haematological malignancies [OR=2.39 (95% CI 1.79-3.18)] and prior exposure to antifungal drugs [OR=1.87 (1.30-2.69)] were independent predisposing factors for UYS infections upon multivariate analysis. However, when considering the genus/species complex level, only infections due to Candida kefyr -related species [OR=4.01 (2.42-6.64)] and to Trichosporon spp. [OR=5.38 (1.72-16.81)] remained associated with haematological malignancies, those due to the GEOTRICHUM group with acute leukaemia [OR=61.29 (19.23-195.36)], and infections with Trichosporon spp. or the GEOTRICHUM group with prior exposure to caspofungin [OR=15.67 (3.62-67.80) and OR=13.17 (3.33-52.03), respectively] but not to fluconazole. The global mortality at day 30 for UYS was similar to that for C. albicans (35.4%, and 39.9%, respectively), but very divergent results were observed according to the specific UYS. Conclusions: UYS encompass a high diversity of species, each with its own behaviour and predisposing factors for human infections. This variety makes it important to rapidly identify an isolate to the species level in order to optimize antifungal treatment.

Novel Taxa Associated with Human Fungal Black-Grain Mycetomas: Emarellia grisea gen. nov., sp. nov., and Emarellia paragrisea sp. nov.

Eumycetoma is a debilitating, chronic, fungal infection that is endemic in India, Indonesia, and parts of Africa and South and Central America. It remains a neglected tropical disease in need of international recognition. Infections follow traumatic implantation of saprophytic fungi and frequently require radical surgery or amputation in the absence of appropriate treatment. Several fungal species can cause black-grain mycetomas, including Madurella spp. (Sordariales), Falciformispora spp., Trematosphaeria grisea, Biatriospora mackinnonii, Pseudochaetosphaeronema larense, and Medicopsis romeroi (all Pleosporales). We performed phylogenetic analyses based on five loci on 31 isolates from two international culture collections to establish the taxonomic affiliations of fungi that had been isolated from cases of black-grain mycetoma and historically classified as Madurella grisea Although most strains were well resolved to species level and corresponded to known agents of eumycetoma, six independent isolates, which failed to produce conidia under any conditions tested, were only distantly related to existing members of the Pleosporales Five of the six isolates shared >99% identity with each other and are described as Emarellia grisea gen. nov. and sp. nov; the sixth isolate represents a sister species in this novel genus and is described as Emarellia paragrisea. Several E. grisea isolates were present in both United Kingdom and French culture collections and had been isolated independently over 6 decades from cases of imported eumycetoma. Four of the six isolates involved patients that had originated on the Indian subcontinent. All isolates were all susceptible in vitro to the azole antifungals, but had elevated MICs with caspofungin.

Multilocus sequence typing analysis reveals that Cryptococcus neoformans var. neoformans is a recombinant population.

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.

Environmental distribution of Cryptococcus neoformans and C. gattii around the Mediterranean basin.

In order to elucidate the distribution of Cryptococcus neoformans and C. gattii in the Mediterranean basin, an extensive environmental survey was carried out during 2012-2015. A total of 302 sites located in 12 countries were sampled, 6436 samples from 3765 trees were collected and 5% of trees were found to be colonized by cryptococcal yeasts. Cryptococcus neoformans was isolated from 177 trees and C. gattii from 13. Cryptococcus neoformans colonized 27% of Ceratonia, 10% of Olea, Platanus and Prunus trees and a lower percentage of other tree genera. The 13 C. gattii isolates were collected from five Eucalyptus, four Ceratonia, two Pinus and two Olea trees. Cryptococcus neoformans was distributed all around the Mediterranean basin, whereas C. gattii was isolated in Greece, Southern Italy and Spain, in agreement with previous findings from both clinical and environmental sources. Among C. neoformans isolates, VNI was the prevalent molecular type but VNII, VNIV and VNIII hybrid strains were also isolated. With the exception of a single VGIV isolate, all C. gattii isolates were VGI. The results confirmed the presence of both Cryptococcus species in the Mediterranean environment, and showed that both carob and olive trees represent an important niche for these yeasts.

Application of Isothermal Amplification Techniques for Identification of Madurella mycetomatis, the Prevalent Agent of Human Mycetoma.

Appropriate diagnosis and treatment of eumycetoma may vary significantly depending on the causative agent. To date, the most common fungus causing mycetoma worldwide is Madurella mycetomatis. This species fails to express any recognizable morphological characteristics, and reliable identification can therefore only be achieved with the application of molecular techniques. Recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) are proposed as alternatives to phenotypic methods. Species-specific primers were developed to target the ribosomal DNA (rDNA) internal transcribed spacer (ITS) region of M. mycetomatis. Both isothermal amplification techniques showed high specificity and sufficient sensitivity to amplify fungal DNA and proved to be appropriate for detection of M. mycetomatis. Diagnostic performance of the techniques was assessed in comparison to conventional PCR using biopsy specimens from eumycetoma patients. RPA is reliable and easy to operate and has the potential to be implemented in areas where mycetoma is endemic. The techniques may be expanded to detect fungal DNA from environmental samples.

International Society of Human and Animal Mycology (ISHAM)-ITS reference DNA barcoding database--the quality controlled standard tool for routine identification of human and animal pathogenic fungi.

Human and animal fungal pathogens are a growing threat worldwide leading to emerging infections and creating new risks for established ones. There is a growing need for a rapid and accurate identification of pathogens to enable early diagnosis and targeted antifungal therapy. Morphological and biochemical identification methods are time-consuming and require trained experts. Alternatively, molecular methods, such as DNA barcoding, a powerful and easy tool for rapid monophasic identification, offer a practical approach for species identification and less demanding in terms of taxonomical expertise. However, its wide-spread use is still limited by a lack of quality-controlled reference databases and the evolving recognition and definition of new fungal species/complexes. An international consortium of medical mycology laboratories was formed aiming to establish a quality controlled ITS database under the umbrella of the ISHAM working group on "DNA barcoding of human and animal pathogenic fungi." A new database, containing 2800 ITS sequences representing 421 fungal species, providing the medical community with a freely accessible tool at http://www.isham.org/ and http://its.mycologylab.org/ to rapidly and reliably identify most agents of mycoses, was established. The generated sequences included in the new database were used to evaluate the variation and overall utility of the ITS region for the identification of pathogenic fungi at intra-and interspecies level. The average intraspecies variation ranged from 0 to 2.25%. This highlighted selected pathogenic fungal species, such as the dermatophytes and emerging yeast, for which additional molecular methods/genetic markers are required for their reliable identification from clinical and veterinary specimens.