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Background Cryptococcal meningitis is most commonly found in HIV-infected patients. In HIV-negative patients, its low incidence can lead to prolonged time to diagnosis. Detailed case reports of chronic cryptococcal meningitis are scarce, but could provide clues for earlier diagnosis in this patient category. Case presentation A 60-year old man presented June 2015 with intermittent headaches for several months without any fever. Initial work-up showed a leukocytosis, raised CSF opening pressure and raised leukocytes and protein in the CSF. An MRI revealed leptomeningeal contrast enhancement and cerebellar oedema. While malignancy and various infectious causes were excluded, the patient had a spontaneous clinical and radiological recovery. One year later, the patient returned with complaints of headaches. Also, cerebellar oedema and leptomeningeal contrast enhancement had recurred. Eventually in March 2017, the novel cryptococcal antigen lateral flow assay (CrAg LFA) was positive on CSF, and one colony of Cryptococcus neoformans was cultured from CSF. The patient was treated with the standard antifungal regimen which resulted in resolution of his headaches. In retrospect, the cryptococcal antigen test was already positive on a serum sample from June 2015. Interestingly, post-treatment immunological analysis revealed both a low mannose-binding lectin (MBL) concentration and low naïve CD4 counts. Conclusions We present a patient with cryptococcal meningitis in an HIV-negative patient with low MBL and low naïve CD4 count suffering a chronic relapsing meningo-encephalitis with relatively mild symptoms for around 2 years. In patients with an unexplained meningo-encephalitis such as this case, early performance of CrAg LFA on serum and/or CSF is an inexpensive and rapid method to reduce time-to diagnosis.

Autosomal recessive (AR) CARD9 (caspase recruitment domain-containing protein 9) deficiency underlies invasive infections by fungi of the ascomycete phylum in previously healthy individuals at almost any age. Although CARD9 is expressed mostly by myeloid cells, the cellular basis of fungal infections in patients with inherited CARD9 deficiency is unclear. Therapy for fungal infections is challenging, with at least 20% premature mortality. We report two unrelated patients from Brazil and Morocco with AR CARD9 deficiency, both successfully treated with hematopoietic stem cell transplantation (HSCT). From childhood onward, the patients had invasive dermatophytic disease, which persisted or recurred despite multiple courses of antifungal treatment. Sanger sequencing identified homozygous missense CARD9 variants at the same residue, c.302G>T (p.R101L) in the Brazilian patient and c.301C>T (p.R101C) in the Moroccan patient. At the ages of 25 and 44 years, respectively, they received a HSCT. The first patient received a HLA-matched HSCT from his CARD9-mutated heterozygous sister. There was 100% donor chimerism at D + 100. The other patient received a T cell-depleted haploidentical HSCT from his CARD9-mutated heterozygous brother. A second HSCT from the same donor was performed due to severe amegakaryocytic thrombocytopenia despite achieving full donor chimerism (100%). At last follow-up, more than 3 years after HSCT, both patients have achieved complete clinical remission and stopped antifungal therapy. HSCT might be a life-saving therapeutic option in patients with AR CARD9 deficiency. This observation strongly suggests that the pathogenesis of fungal infections in these patients is largely due to the disruption of leukocyte-mediated CARD9 immunity.

Background Progressive lung injury in Cystic Fibrosis (CF) patients can lead to chronic colonization with bacteria and fungi. Fungal colonization is obtained from the environment which necessitates locally performed epidemiology studies. We prospectively analyzed respiratory samples of CF patients during a 3-year period, using a uniform fungal culture protocol, focusing on filamentous fungi and azole resistance in Aspergillus fumigatus. Methods Over a 3-year period, all respiratory specimens collected from CF patients in 5 Dutch CF centers, were analyzed. Samples were inoculated onto the fungal culture media Sabouraud dextrose agar (SDA) and Medium B+. All fungal isolates were collected and identified in one centre, using Amplified Fragment Length Polymorphism (AFLP) fingerprinting, rDNA PCR and ITS, calmodulin and β-tubulin sequencing. Azole resistance was assessed for all A. fumigatus using a qPCR assay followed by phenotypic confirmation. Results Filamentous fungi were recovered from 699 patients from at least one respiratory sample, corresponding with 3787 cultured fungal species. A. fumigatus was cultured most often with a mean prevalence of 31.7%, followed by Penicillium species (12.6%), non-fumigatus Aspergillus species (5.6%), Scedosporium species (4.5%) and Exophiala dermatitidis and Cladosporium species (1.1% each). In total 107 different fungal species were identified, with 39 Penicillium species and 15 Aspergillus species. Azole resistance frequency in A. fumigatus was 7.1%, with TR34/L98H being the dominant resistance mechanism. Conclusion A vast diversity of filamentous fungi was demonstrated, dominated by Aspergillus and Penicillium species. We observed a mean azole resistance prevalence of 7.1% of A. fumigatus culture positive patients.

Objectives: Historically, the Brazilian Central-West region has had high numbers of paracoccidioidomycosis (PCM) cases caused by the dimorphic fungus Paracoccidioides lutzii. Methods: This epidemiological, observational, analytical, cross-sectional study was performed to investigate the clinical and laboratory data of 44 PCM patients with a culture-proven P. lutzii infection. All patients were referred to the Systemic Mycosis Center, Júlio Muller University Hospital, Cuiabá, Brazil, during January 2017 to March 2020. The neutrophil to lymphocyte ratio (NLR) was calculated and dichotomized by its median value to include in the identification of factors associated with severity. Results: At admission, 13 (31.7%) patients showed the disseminated multifocal chronic form of PCM and 16 (36.4%) patients met the clinical severity criteria. Treatment prescribed on admission did not follow the recommendations of the Brazilian Guideline for the Clinical Management of Paracoccidioidomycosis in 26% of the severe PCM cases (prevalence ratio 0.26, 95% confidence interval 0.14–0.49; P < 0.0001). Patients with severe PCM had a higher NLR that was greater than the median (≥4.11). Conclusions: The NLR biomarker complements the criteria for PCM severity. Applying the low-cost NLR test can greatly increase the diagnostic sensitivity when screening patients for PCM and contribute to better control of the disease, management of complications, and therapeutic strategies.

BACKGROUND PCM is a neglected systemic mycosis endemic in Brazil. The middle-west region of Brazil has shown the highest number of PCM by Paracoccidioides lutzii (P lutzii) cases. Differentiating cases of severe PCM from non-severe ones should be a concern at the bedside. Diagnosis of severe PCM by P lutzii is based on the subjectivity of clinical manifestations, which can result in a delay in starting its treatment and, consequently evolution to severe sequelae. There is not laboratory biomarker available to support the early diagnosis of severe PCM that is feasible for all the realities that coexist in Brazil. OBJECTIVES The aim of this study was to investigate the usefulness of laboratory biomarkers as erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and neutrophil/lymphocyte ratio (NLR) in the diagnosis of severe PCM. PATIENTS/METHODS ESR, CRP and NLR were analysed for 44 patients with PCM by P lutzii and a Receiver Operation Characteristic (ROC) curve were generated to identify the NLR cut-off point and point out the presence of severe PCM. RESULTS Sixteen (36.4%) had severe PCM and 28 (63.6%) had non-severe PCM. The mean NLR was higher and statistically significant among patients with severe PCM than among those with non-severe PCM. The area under the ROC curve was 0.859 for the diagnosis of severe PCM. The cut-off point for NLR for the diagnosis of severe PCM was 3.318 (sensitivity of 100%, specificity of 77%). CONCLUSIONS According to results, it is plausible to conclude that NLR represents a potential biomarker for the diagnosis of severe PCM.

More than a decade ago, the first cryptococcal lateral flow assay (LFA) became available. This affordable point-of-care test has undeniably led to tremendous improvements in early detection of cryptococcosis ([1][1], [2][2]). However, from several studies, it must be concluded that the reliability

International audience; The 2020 COVID-19 pandemic had a profound impact on the publishing landscape. The 'pre-peer-review' publication model is likely to become common as a lag in publishing is not acceptable in a pandemic or other time! Mycopathologia is well placed to adopt such changes with its improved editorial processes, article formats, author engagements, and published articles' access and citation. Mycopathologia had an improved journal impact factor and article downloads in 2018-2019. A limited sampling suggested a slight decrease in the total submissions in 2019 (352 articles) compared to 2018 (371 articles). However, the acceptance rate improved to 30% in 2019 from 19% in 2018. Nearly half of all submissions in 2019 were rejected before peer-review or transferred to other Springer Nature journals. The published articles were contributed from 34 different countries, with authors from China, the USA, and Brazil among the top three contributors. An enhanced editorial oversight allowed peer-reviewers to focus on fewer articles that were well-matched to their expertise, which led to lower rejection rates post-peer-review. The introduction of MycopathologiaGENOME and MycopathologiaIMAGE article types received a good reception with notable downloads and citations.

Interlaboratory evaluations of Mucorales qPCR assays were developed to assess the reproducibility and performance of methods currently used. The participants comprised 12 laboratories from French university hospitals (nine of them participating in the Modimucor study) and 11 laboratories participating in the Fungal PCR Initiative. For panel 1, three sera were each spiked with DNA from three different species (Rhizomucor pusillus, Lichtheimia corymbifera, Rhizopus oryzae). For panel 2, six sera with three concentrations of R. pusillus and L. corymbifera (1, 10, and 100 genomes/ml) were prepared. Each panel included a blind negative-control serum. A form was distributed with each panel to collect results and required technical information, including DNA extraction method, sample volume used, DNA elution volume, qPCR method, qPCR template input volume, qPCR total reaction volume, qPCR platform, and qPCR reagents used. For panel 1, assessing 18 different protocols, qualitative results (positive or negative) were correct in 97% of cases (70/72). A very low interlaboratory variability in Cq values (SD = 1.89 cycles) were observed. For panel 2 assessing 26 different protocols, the detection rates were high (77–100%) for 5/6 of spiked serum. There was a significant association between the qPCR platform and performance. However, certain technical steps and optimal combinations of factors may also impact performance. The good reproducibility and performance demonstrated in this study support the use of Mucorales qPCR as part of the diagnostic strategy for mucormycosis.

The arthroconidial yeasts Magnusiomyces capitatus and M. clavatus are emerging opportunistic pulmonary pathogens. They are closely related and difficult to distinguish based on morphological and physiological traits. We applied an SYBR® green-based quantitative PCR (qPCR) assay to identify the species. We analyzed 30 reference strains originating from clinical and environmental sources by targeting the Rpb2 gene encoding the second largest subunit of RNA polymerase II. The qPCR assays were tested by direct identification of M. capitatus and M. clavatus in spiked sputum and household dishwasher swabs, respectively, as models for clinical and environmental samples. The assays were proved to be reliable for species-level identification of both species, with 100% sensitivity and 100% specificity, lowest inter-assay deviations (RSDr ? 1.65%, R2 values >0.99), detection limit of 10 theoretical copy number of target DNA, and detection cell limit of ?5000 yeast cells from spiked sputum samples. The developed qPCR assay is a practical molecular approach for the detection of M. capitatus and M. clavatus that can be used as a stand-alone assay or in conjunction with culture-dependent approaches. © 2021, The Author(s), under exclusive licence to Springer Nature B.V. part of Springer Nature.

Mucormycosis is a life-threatening invasive fungal infection, most commonly described in severely immunocompromised patients. It is characterized by rapid invasive growth of the fungus and often with fatal outcome. We report a case of a renal transplant recipient diagnosed with a donor-derived invasive mucormycosis. In this patient, we used a step-wise approach of withdrawal of immunosuppressants, antifungal induction therapy, extensive surgical debridement of all (potentially) infected tissue, abdominal irrigation of liposomal amphotericin B and interferon gamma. Due to rapid diagnosis and intensive therapy the patient survived.

Contains fulltext : 229620.pdf (Publisher’s version ) (Closed access) Emmonsia crescens is known as an environmental pathogen causing adiaspiromycosis in small rodents. As the generic name Emmonsia is no longer available for this species, its taxonomic position is re-evaluated. The intraspecific variation of Emmonsia crescens was analyzed using molecular, morphological, and physiological data, and the relationship between frequency of adiaspiromycosis and body temperature of host animals was explored. A North American and a pan-global lineage could be discerned, each with subclusters at low genetic distance. European strains produced the classical type of very large adiaspores, while in the North American lineage adiaspores relatively small, resembling the broad-based budding cells of Blastomyces. Members of the closely related genus Emergomyces may exhibit large, broad-based in addition to small, narrow-based budding cells. We conclude that the morphology of the pathogenic phase in these fungi differs gradationally between species and even populations, and is therefore less suitable as a diagnostic criterion for generic delimitation. Two Emmonsia species are reclassified in Emergomyces.

Clonal expansion of fluconazole resistant (FLZ-R) Candida parapsilosis isolates is increasingly being identified in many countries, while there is no study exploring the antifungal susceptibility pattern, genetic diversity, and clinical information for Iranian C. parapsilosis blood isolates. Candida parapsilosis species complex blood isolates (n = 98) were recovered from nine hospitals located in three major cities, identified by MALDI-TOF MS, and their genetic relatedness was examined by AFLP fingerprinting. Antifungal susceptibility testing followed CLSI-M27-A3 and ERG11, MRR1 and hotspots 1/2 (HS1/2) of FKS1 were sequenced to assess the azole and echinocandin resistance mechanisms, respectively. Ninety-four C. parapsilosis and four Candida orthopsilosis isolates were identified from 90 patients. Only 43 patients received systemic antifungal drugs with fluconazole as the main antifungal used. The overall mortality rate was 46.6% (42/90) and death mostly occurred for those receiving systemic antifungals (25/43) relative to those not treated (17/47). Although, antifungal-resistance was rare, one isolate was multidrug-resistant (FLZ = 16 μg/ml and micafungin = 8 μg/ml) and the infected patient showed therapeutic failure to FLZ prophylaxis. Mutations causing azole and echinocandin resistance were not found in the genes studied. AFLP revealed five genotypes (G) and G1 was the main one (59/94; 62.7%). Clinical outcome was significantly associated with city (P = 0.02, α < 0.05) and Mashhad was significantly associated with mortality (P = 0.03, α < 0.05). Overall, we found a low level of antifungal resistance for Iranian C. parapsilosis blood isolates, but the noted MDR strain can potentially become the source of future infections and challenge the antifungal therapy in antifungal-naïve patients. AFLP typing results warrants confirmation using other resolutive typing methods.

Background Candida glabratais the third leading cause of candidaemia in Turkey; however, the data regarding antifungal resistance mechanisms and genotypic diversity in association with their clinical implication are limited. Objectives To assess genotypic diversity, antifungal susceptibility and mechanisms of drug resistance ofCglabratablood isolates and their association with patients' outcome in a retrospective multicentre study. Patients/Methods Isolates from 107 patients were identified by ITS sequencing and analysed by multilocus microsatellite typing, antifungal susceptibility testing, and sequencing ofPDR1andFKS1/2hotspots (HSs). Results Candida glabrataprevalence in Ege University Hospital was twofold higher in 2014-2019 than in 2005-2014. Six of the analysed isolates had fluconazole MICs >= 32 mu g/mL; of them, five harboured uniquePDR1mutations. Although echinocandin resistance was not detected, three isolates had mutations in HS1-Fks1 (S629T, n = 1) and HS1-Fks2 (S663P, n = 2); one of the latter was also fluconazole-resistant. All patients infected with isolates carrying HS-FKSmutations and/or demonstrating fluconazole MIC >= 32 mu g/mL (except one without clinical data) showed therapeutic failure (TF) with echinocandin and fluconazole; seven such isolates were collected in Ege (n = 4) and Gulhane (n = 3) hospitals and six detected recently. Among 34 identified genotypes, none were associated with mortality or enriched for fluconazole-resistant isolates. Conclusion Antifungal susceptibility testing should be supplemented with HS-FKSsequencing to predict TF for echinocandins, whereas fluconazole MIC >= 32 mu g/mL may predict TF. Recent emergence ofC glabrataisolates associated with antifungal TF warrants future comprehensive prospective studies in Turkey., Major National R&D Projects of the National Health Department [2018ZX10101003]; National Natural Science Foundation of ChinaNational Natural Science Foundation of China (NSFC) [31770161]; Shanghai Science and Technology CommitteeShanghai Science & Technology Committee [17DZ2272900, 14495800500]; Shanghai Municipal Commission of Health and Family Planning [2017ZZ01024-001]; Shanghai Sailing Program [19YF1448000]; Chinese Academy of Engineering [2019-XY-33]; NIHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [AI10902], This work was supported by the Major National R&D Projects of the National Health Department [2018ZX10101003], National Natural Science Foundation of China [31770161], Shanghai Science and Technology Committee [17DZ2272900 and 14495800500], Shanghai Municipal Commission of Health and Family Planning [2017ZZ01024-001], Shanghai Sailing Program [19YF1448000], and the Chinese Academy of Engineering [2019-XY-33]. DSP was supported by NIH grant #AI10902.

The anthropophilic dermatophyte Trichophyton tonsurans and its zoophilic counterpart T. equinum are phylogenetically closely related. The barcoding marker rDNA internal transcribed spacer (ITS) shows limited variation between these two species. In the current study, we combined molecular approaches with phenotypic data to determine the species boundaries between T. tonsurans (n = 52) and T. equinum (n = 15) strains originating from humans (n = 40), horses (n = 26), and a mouse (n = 1). Culture characteristics and physiology on Trichophyton agar media 1 and 5 were evaluated. Multi-locus sequencing involving ITS, partial large rDNA subunit (LSU), ß-tubulin (TUB), 60S ribosomal protein (RPB), and translation elongation factor-3 (TEF3) genes, and the mating-type (MAT) locus was performed. Amplified fragment length polymorphism data were added. None of the test results showed complete mutual correspondence. With the exception of strains from New Zealand, strains of equine origin required niacin for growth, whereas most strains from human origin did not show this dependence. It is concluded that T. tonsurans and T. equinum incompletely diverged from a common lineage relatively recently. MAT1-1 and MAT1-2 are the main distinguishing genes between the two species. © 2019, Springer Nature B.V. Federation of European Microbiological Societies: FEMS-RG-2016-0067 HK was supported by a Grant from the Federation of European Microbiological Societies (FEMS-RG-2016-0067). The funders had no influence on the study design; on the collection, analysis, and interpretation of data; on the preparation of the manuscript; or the decision to publish.

Contains fulltext : 226024.pdf (Publisher’s version ) (Closed access) Quantitative real-time PCR (qPCR) is increasingly used to detect Pneumocystis jirovecii for the diagnosis of Pneumocystis pneumonia (PCP), but there are differences in the nucleic acids targeted, DNA only versus whole nucleic acid (WNA), and also the target genes for amplification. Through the Fungal PCR Initiative, a working group of the International Society for Human and Animal Mycology, a multicenter and monocenter evaluation of PCP qPCR assays was performed. For the multicenter study, 16 reference laboratories from eight different countries, performing 20 assays analyzed a panel consisting of two negative and three PCP positive samples. Aliquots were prepared by pooling residual material from 20 negative or positive- P. jirovecii bronchoalveolar lavage fluids (BALFs). The positive pool was diluted to obtain three concentrations (pure 1:1; 1:100; and 1:1000 to mimic high, medium, and low fungal loads, respectively). The monocenter study compared five in-house and five commercial qPCR assays testing 19 individual BALFs on the same amplification platform. Across both evaluations and for all fungal loads, targeting WNA and the mitochondrial small sub-unit (mtSSU) provided the earliest Cq values, compared to only targeting DNA and the mitochondrial large subunit, the major surface glycoprotein or the beta-tubulin genes. Thus, reverse transcriptase-qPCR targeting the mtSSU gene could serve as a basis for standardizing the P. jirovecii load, which is essential if qPCR is to be incorporated into clinical care pathways as the reference method, accepting that additional parameters such as amplification platforms still need evaluation.

Item does not contain fulltext Candida auris is an emerging multidrug resistant pathogenic fungus that causes candidaemia with high mortality rates and exhibits the ability to persist within the hospital environment. Candida auris is phylogenetically closely related to Candida haemulonii, C. lusitaniae, C. pseudohaemulonii and C. duobushaemulonii and is frequently misidentified by commercial identification methods. In the present study, the GPS() MONODOSE dtec-qPCR kit (dried single-dose PCR tubes) for the detection of C. auris was validated following the guidelines of the UNE/EN ISO/IEC 17025:2005, the French Standard NF T90-471:2010 and using fast-cycling protocols. Validation terms included in vitro specificity (inclusivity/exclusivity), quantitative phase analysis (10-10(6) standard DNA copies), reliability (repeatability/reproducibility) and sensitivity (detection/quantification limits). GPS() dtec-qPCR kits passed validation with strict acceptance criteria (n >/= 10 repetitions). In silico specificity was proven by the designer (GPS() ). Experimental inclusiveness was achieved in two independent laboratories by testing strain JCM15448(T) and 117 clinical C. auris isolates from Asia, the Middle-East Africa, Latin America and Europe. Exclusiveness was evaluated with 25 strains of closely related Candida spp. Use of the MONODOSE format provided considerable advantages allowing the detection of C. auris to be accurately achieved in less than an hour. The GPS() MONODOSE dtec-qPCR kit is ready to undergo clinical evaluation.

Objective Cryptococcosis is a major opportunistic fungal infection caused by members of the genus Cryptococcus, mainly those belonging to the Cryptococcus neoformans/Cryptococcus gattii species complexes. Here, we report a comprehensive molecular epidemiological study of the environmental distribution of Cryptococcus isolates in Shiraz, Iran with review of litreature. Method A total of 406 samples were obtained from Eucalyptus trees and 139 samples from pigeon droppings. Cryptococcus species identification and genotyping were performed by amplified fragment length polymorphism (AFLP) fingerprinting sequencing and sequencing of the ITS rDNA region. Results Majority of the isolates belonged to the Naganishia taxon (n = 69) including N. albida (formerly C. albidus, n = 62), N. globosa (formerly C. saitoi, n = 4), N. adeliensis (formerly C. adeliensis, n = 2), N. diffluens (formerly C. diffluens, n = 1), and the identified C. neoformans isolates (n = 25) belonged to genotype AFLP1/VNI (n = 22) and AFLP1B/VNII (n = 3). Conclusion More research efforts should be employed to isolate C. gattii species complex from environmental niches in Iran and provide additional evidence related to novel molecular types.

Item does not contain fulltext Influenza virus infection is now recognised as a risk factor for invasive pulmonary aspergillosis (IPA). Delays in diagnosis contribute to delayed commencement of antifungal therapy. In addition, the emergence of resistance to first-line triazole antifungal agents puts emphasis on early detection to prevent adverse outcomes. We present 2 allogeneic stem cell transplant patients who developed IPA due to triazole-resistant Aspergillus fumigatus following influenza infection. We underline the challenges faced in the management of these cases, the importance of early diagnosis and need for surveillance given the emergence of triazole resistance.

Knowledge of the environmental prevalence of members of the Cryptococcus neoformans/Cryptococcus gattii species complex is important, since cryptococcal infection is acquired from the environment. We determined whether trees located in two South African recreational areas harboured pathogenic cryptococci and compared the isolates to clinical isolates obtained from Western Cape hospitals with molecular typing techniques. The majority of isolates originating from trees in a public park in Cape Town (PPCT) were C. gattii sensu stricto, followed by C. neoformans sensu stricto genotype AFLP1/VNI. The PPCT trees might be a source of infection, since all genotype AFLP1/VNI isolates from these trees and one clinical isolate belonged to the same sequence type (ST), i.e. ST23. Recombination and basidiospore production might be occurring in PPCT trees that contained C. gattii s.s. isolates belonging to both mating types. The presence of C. gattii s.s. in PPCT trees might therefore pose a risk to human health.

Colletotrichum gloeosporioides is an economically important fungal pathogen causing substantial yield losses indifferent host plants. To understand the genetic diversity and molecular epidemiology of this fungus, we have developed a novel, high-resolution multi-locus microsatellite typing (MLMT) method. Bioinformatic analysis of C. gloeosporioides unannotated genome sequence yielded eight potential microsatellite loci, of which five, CG1 (GT)n, CG2 (GT1)n, CG3 (TC)n, CG4 (CT)n, and CG5 (CT1)n were selected for further study based on their universal amplification potential, reproducibility, and repeat number polymorphism. The selected microsatellites were used to analyze 31 strains of C. gloeosporioides isolated from 20 different host plants from India. All microsatellite loci were found to be polymorphic, and the approximate fragment sizes of microsatellite loci CG1, CG2, CG3, CG4, and CG5 were in ranges of 213-241, 197-227, 231-265, 209-275, and 132-188, respectively. Among the 31 isolates, 55 different genotypes were identified. The Simpson's index of diversity (D) values for the individual locus ranged from 0.79 to 0.92, with the D value of all combined five microsatellite loci being 0.99. Microsatellite data analysis revealed that isolates from Ocimum sanctum, Capsicum annuum (chili pepper), and Mangifera indica (mango) formed distinct clusters, therefore exhibited some level of correlation between certain genotypes and host. The developed MLMT method would be a powerful tool for studying the genetic diversity and any possible genotype-host correlation in C. gloeosporioides.

Background and Purpose: Candida parapsilosis is a predominant species found in nosocomial infection, particularly in hospitalized patients. The molecular epidemiology of the clinical strains of this species has not been well studied. The present study was performed with the aim of investigating the microsatellite genotyping of Candida parapsilosis among the Iranian clinical isolates. Materials and Methods: This study was conducted on 81 independent clinical C. parapsilosis isolates that were genotyped by using a panel of six microsatellite markers. Results: The short tandem repeat (STR) typing of clinical C. parapsilosis isolates demonstrated 68 separate genotypes, among which 57 genotypes were observed once and the remaining 11 cases were identified for multiple times. The Simpson’s diversity index for the panel of combined six markers yielded a diversity index of 0.9951. The heterogeneity was observed among the Iranian and the Netherlands clinical C. parapsilosis isolates. Conclusion: As the findings indicated, the clinical C. parapsilosis isolates from Iran showed a high genetic diversity. It can be concluded that molecular epidemiology could be useful for screening during outbreak investigation where C. parapsilosis is involved.&nbsp

Background During the past decades there has been an increase in cryptococcal infections caused by the basidiomycetous yeast species Cryptococcus gattii sensu lato, among humans and animals that live in endemic regions in Australia, Europe and the Americas. Unlike human cryptococcosis, little epidemiological data are available about C. gattii sensu lato infections in horses. Case report A fatal case of a disseminated C. gattii sensu lato infection in an 11-year-old Arabian gelding imported from South Africa into the United Arab Emitares is reported. Tissue samples were studied by conventional mycology procedures and the obtained cryptococcal isolate was molecularly characterized by mating-type determination, amplified fragment length polymorphism (AFLP) fingerprinting, and multi-locus sequence typing (MLST). Phylogenetic analysis was performed to investigate the geographic origin of the cryptococcal isolate. The isolate was identified as Cryptococcus deuterogattii (AFLP6/VGII), mating-type α. Phylogenetic analysis showed that it was closely related to another C. deuterogattii isolate from the Middle East. Conclusions A second case of a C. deuterogattii infection in the Middle East is described. It is likely that the horse acquired the infection in the Middle East, as the isolate is closely related to that of a recent human case from that region.

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.

The pathogenic yeast Cryptococcus gattii was isolated from a tree hollow of a Castanopsis argyrophylla King ex Hook.f. (Fagaceae) in Chiang Mai, Thailand. Molecular characterization with amplified fragment length polymorphism analysis and multi-locus sequence typing showed that this isolate belonged to genotype AFLP4/VGI representing C. gattii sensu stricto. Subsequent comparison of the environmental isolate with those from clinical samples from Thailand showed that they grouped closely together in a single cluster.

Item does not contain fulltext OBJECTIVE: A multiplex extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E) quantitative polymerase chain reaction (qPCR), performed directly on rectal swabs, was compared with a culture-based protocol to study the discrepancies between the two methods, and identify existing challenges to apply this assay in routine clinical practice. The secondary objective was to assess the performance of the qPCR. MATERIALS AND METHODS: In two Dutch teaching hospitals, 573 rectal swabs were collected prospectively. Culture with additional testing with the Check-MDR CT103XL (Check-Points) was compared with the Check-Direct ESBL Screen for BD MAX (Check-Points), which detects the presence of the ESBL gene families CTX-M1, CTX-M2, CTX-M9 and SHV2/5-ESBL. The culture-based protocol (with Brilliance agar) was considered as the gold standard to assess the performance of the qPCR. RESULTS: Of the 573 rectal swabs, 74 (12.9%) were culture-positive. Eighty-four (14.7%) were qPCR-positive. There were eight culture-positive/qPCR-negative discrepancies and 18 culture-negative/qPCR-positive discrepancies. Sensitivity and specificity of qPCR vs culture were 87.7% [95% confidence interval (CI) 79.7-95.7] and 96.3% (95% CI 94.6-98.0), respectively. CONCLUSION: The Check-Direct ESBL Screen for the BD MAX is an easy-to-perform, quick molecular diagnostic test with the potential to significantly speed up screening for rectal ESBL-E carriage. Discrepancies were observed between the culture-based protocol and the qPCR in 4.5% of tested samples. Existing challenges for implementing qPCR are its limited sensitivity, the need for thorough knowledge of the local ESBL-E genes, and interpretation of culture-negative but qPCR-positive samples. It is believed that the limited sensitivity of qPCR could be optimized by including blaTEM as a molecular target, and improving the limit of detection.

Item does not contain fulltext We here show adequate species identification for bacterial isolates of the genus Nocardia spp. through VITEK mass spectrometry. Application of a specific sample preparation method in combination with a robust matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) database leads to 94% accurate identification to the species level on a set of 164 isolates. The possibility to identify Nocardia spp. using MALDI-TOF MS will be available in the next release of VITEK MS update (IVD Version 3.0).

BACKGROUND: Cryptococcus neoformans is the leading cause of cryptococcal meningitis in HIV/AIDS patients. As infections in humans are predominantly caused by the inhalation of basidiospores from environmental sources, therefore, analysing the population structure of both clinical and environmental populations of C neoformans can increase our understanding of the molecular epidemiology of cryptococcosis. OBJECTIVE: To investigate the genotypic diversity and antifungal susceptibility profile of a large collection of C neoformans isolates (n = 523) from clinical and environmental sources in India between 2001 and 2014. MATERIALS AND METHODS: Cryptococcus neoformans isolates were genotyped by AFLP, microsatellite typing (MLMT) and MLST. In vitro antifungal susceptibility for standard antifungals was undertaken using CLSI M27-A3. RESULTS: All isolates were C neoformans, AFLP1/VNI and exhibited mating-type MATα. MLMT revealed that the majority of isolates belonged to microsatellite cluster (MC) MC3 (49%), followed by MC1 (35%), and the remaining isolates fell in 11 other MC types. Interestingly, two-thirds of clinical isolates were genotype MC3 and only 17% of them were MC1, whereas majority of environmental strains were MC1 (54%) followed by MC3 (16%). Overall, MLST assigned 5 sequence types (STs) among all isolates and ST93 was the most common (n = 76.7%), which was equally distributed in both HIV-positive and HIV-negative patients. Geometric mean MICs revealed that isolates in MC1 were significantly less (P < .05) susceptible to amphotericin B, 5-flucytosine, itraconazole, posaconazole and isavuconazole than isolates in MC3. CONCLUSIONS: The study shows a good correlation between MLMT and MLST genotyping methods. Further, environmental isolates were genetically more diverse than clinical isolates.

Candida vulturna is a new member of the Candida haemulonii species complex that recently received much attention as it includes the emerging multidrug-resistant pathogen Candida auris. Here, we describe the high-quality genome sequence of C. vulturna type strain CBS 14366T to cover all genomes of pathogenic C. haemulonii species complex members.

Although Cyberlindnera fabinaii is a rare opportunist yeast species, its ability to cause septicemia, produce biofilm, and rapid acquisition of resistance to fluconazole and voriconazole, reinforced the urge for its identification from its closely related species. Widely used biochemical assays mainly identify Cyberlindnera fabinaii as Cyberlindnera jadinii and Wickerhamomyces anomalus, resulting in underestimation of this yeast in clinical settings. Moreover, the urge for a reliable molecular means of identification remains unsolved for 28 years. In order to unequivocally differentiate Cy. fabianii, Cy. mississipiensis, Cy. jadinii, and W. anomalus, we designed a dual-function multiplex polymerase chain reaction (PCR) assay. Challenging our dual-function multiplex PCR assay with 30 most clinically important yeast species, proved its specificity. Although conventional PCR could differentiate four target species, the real-time PCR counterpart due to Tm overlap misidentified Cy. mississipiensis as Cy. jadinii. Alongside of presenting a comprehensive literature review of published cases of Cy. fabianii from 1990 to 2018, we collected various clinical isolates from Tehran, Shiraz, and Fasa (July 1, 2017, to December 31, 2017) to find a passive relative distribution of these closely-related species in Iran. Subjecting our Iranian collection of yeast isolates to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS and LSU and ITS rDNA sequencng revealed six isolates of Cy. fabianii (central venous catheter n = 2 and vaginal swabs n = 4) and one isolate of Cy. jadinii (vaginal swabs). Due to the use of biochemical assays in global ARTEMIS study, we encourage reidentification of clinical isolates of Cy. jadinii and Cy. jadinii using MALDI-TOF or Sanger sequencing that might lead to correcting the distribution of this fungus.

The Trichophyton rubrum species complex comprises commonly encountered dermatophytic fungi with a worldwide distribution. The members of the complex usually have distinct phenotypes in culture and cause different clinical symptoms, despite high genome similarity. In order to better delimit the species within the complex, molecular, phenotypic, and physiological characteristics were combined to reestablish a natural species concept., The Trichophyton rubrum species complex comprises commonly encountered dermatophytic fungi with a worldwide distribution. The members of the complex usually have distinct phenotypes in culture and cause different clinical symptoms, despite high genome similarity. In order to better delimit the species within the complex, molecular, phenotypic, and physiological characteristics were combined to reestablish a natural species concept. Three groups, T. rubrum, T. soudanense, and T. violaceum, could be distinguished based on the sequence of the internal transcribed spacer (ITS) ribosomal DNA barcode gene. On average, strains within each group were similar by colony appearance, microscopy, and physiology, but strains between groups showed significant differences. Trichophyton rubrum strains had higher keratinase activity, whereas T. violaceum strains tended to be more lipophilic; however, none of the phenotypic features were diagnostic. The results of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and amplified fragment length polymorphism (AFLP) were partially consistent with the ITS data but failed to distinguish the species unambiguously. Despite their close similarity, T. violaceum, T. soudanense, and T. rubrum can be regarded as independent species with distinct geographical distributions and clinical predilections. Trichophyton soudanense is pheno- and genotypically intermediate between T. rubrum and T. violaceum. For routine diagnostics, ITS sequencing is recommended.

Despite the increasing occurrence of Candida orthopsilosis and Candida metapsilosis in clinical settings, little is known about their microbiological and clinical properties. Herein, we conducted a national retrospective study (2014–2019) from multiple centers in Iran. Among the 1,770 Candida isolates collected, we identified 600 Candida parapsilosis species complex isolates. Isolate identification was performed by 9-plex PCR, matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS), and rDNA sequencing, and antifungal susceptibility testing (AFST) followed CLSI M27-A3/S4; genotyping was performed by amplified fragment length polymorphism (AFLP) analysis; and clinical information was mined. Thirty-one isolates of C. orthopsilosis from various clinical sources, one mixed sample (blood) concurrently containing C. orthopsilosis and C. parapsilosis and one isolate of C. metapsilosis from a nail sample were identified. Although both 9-plex PCR and MALDI-TOF successfully identified all isolates, only 9-plex PCR could identify the agents in a mixed sample. For the C. orthopsilosis isolates, resistance (non-wild type) was noted only for itraconazole (n = 4; 12.5%). Anidulafungin and fluconazole showed the highest and voriconazole had the lowest geometric mean values. AFLP analysis showed three main and four minor genotypes. Interestingly, 90% of nail isolates clustered with 80% of the blood isolates within two clusters, and four blood isolates recovered from four patients admitted to a hospital clustered into two genotypes and showed a high degree of similarity (>99.2%), which suggests that C. orthopsilosis disseminates horizontally. Supported by our data and published case studies, C. orthopsilosis and C. metapsilosis can be linked to challenging clinical failures, and successful outcomes are not always mirrored by in vitro susceptibility. Accordingly, conducting nationwide studies may provide more comprehensive data, which is required for a better prognosis and clinical management of patients.

Within a decade after its first description, the multidrug-resistant yeast Candida auris has emerged globally as a nosocomial pathogen causing difficult to control outbreaks. This, together with the alarmingly high mortality rate of up to 66% associated with C. auris candidemia, calls for a better understanding of its virulence traits and routes of transmission. Unlike other clinically relevant Candida species, C. auris seems to have the unique ability to be easily transmitted between patients. Although initially thought to express fewer virulence traits than Candida albicans, recent genomic insights suggest C. auris to possess these traits to a much more similar extent. This review highlights the virulence traits C. auris expresses to attack the host, defend itself against antimicrobial agents and to persist within the healthcare environment.

Purpose. Identification of the emerging yeast species Candida nivariensis among presumptively identified Iranian Candida glabrata isolates. Methodology. Clinical C. glabrata species complex isolates from blood (n=100; 46.9%), vaginal swabs (n=20; 9.4%), bronchoalveolar lavage (n=10; 4.7%) and sputum (n=12; 5.6%) from 68 patients from Iran were investigated. Isolates were characterized by CHROMagar, multiplex PCRs, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), amplified fragment length polymorphism (AFLP) fingerprinting, internal transcribed spacer (ITS)/large subunit (LSU) rDNA and FKS1/FKS2 sequencing, and the European Committee on Antimicrobial Susceptibility Testing broth microdilution method. A comprehensive literature review was conducted and all the relevant clinical and microbiological data were collected.Results. Four C. nivariensis isolates were recovered from blood samples of three subjects and were all consistently identified by nine-plex PCR, Bruker MALDI-TOF MS, and LSU and ITS rDNA sequencing. AFLP genotyping clustered the isolates into two groups. Sequencing of the FKS1 and FKS2 hotspots showed no accountable amino acid substitutions. All isolates were susceptible to amphotericin B, fluconazole, itraconazole, posaconazole, voriconazole, anidulafungin and micafungin.Conclusion. In total, 4 out of 213 clinical C. glabrata species complex candidemia isolates were C. nivariensis. Improvement of the BioMerieux Vitek MS database is required to accurately identify C. nivariensis and it is advised to alternatively use CHROMagar and/or PCR-based techniques. As other species within the Nakaseomyces clade may cause infection and showed high MIC values for antifungals, inclusion of their spectra into the MALDI-TOF MS database seems relevant. Due to developing resistance to fluconazole and insufficient efficacy of caspofungin, the combination of catheter removal plus treatment with caspofungin, or voriconazole, or micafungin might be effective for patients.

BACKGROUND: Studies on Aspergillus fumigatus azole resistance in cystic fibrosis patients are scarce despite the fact that it is the most frequently isolated fungus from respiratory samples from these individuals. OBJECTIVES: To evaluate resistance prevalence, investigate mechanisms of resistance and explore the relationship between resistant isolates by genotyping. METHODS: We conducted a prospective 1 year study (from 1 January to 31 December 2015), based on the investigation of up to five colonies per sample from cystic fibrosis patients. RESULTS: Twenty-three (6.5%) isolates among the 355 tested were resistant to at least one triazole drug, using the EUCAST reference method, leading to a prevalence of 6.8% (6/88 patients). Analysis of resistance mechanisms highlighted TR34/L98H (n = 10), TR46/Y121F/T289A (n = 1), WT cyp51A (n = 11) and F46Y/M172V/N248T/D255E/E427K (n = 1). No genotype was shared between patients. CONCLUSIONS: This study showed a relatively stable resistance prevalence in comparison with the previous study conducted in 2010-11 (8%), although resistance mechanisms varied between the two studies.

Aim: Presenting the first clinical case of Wickerhamomyces myanmarensis. Patients & methods: Yeast cells were isolated from blood and central venous catheter of a 5.5-year-old male subject. API 20C AUX, MALDI-TOF MS, ITS and LSU rDNA sequencing, and our qPCR assay were used for identification and the MIC values were determined by CLSI M27-A3. Results: ITS and LSU rDNA sequencing identified both isolates as W. myanmarensis, while API 20C AUX and MALDI-TOF MS did not identify them correctly. Our qPCR specifically distinguished W. myanmarensis from W. anomalus. Isolate obtained from blood showed a higher MIC value for fluconazole, voriconazole and posaconazole. Conclusion: Utilization of reliable identification tools might reveal the genuine spectrum of opportunistic yeast species.

Candida parapsilosis causes ~35% of all candidemia cases in neonates. High-resolution fingerprinting of C. parapsilosis isolates from neonatal intensive care unit (NICU) patients in Maternity Hospital (MH) was performed to identify epidemiologically related strains. Sixty-eight bloodstream/colonizing strains isolated from 59 NICU patients, two isolates from health care workers (HCWs) from MH and 18 bloodstream isolates from two other hospitals were used. Six microsatellite markers were employed, isolates were assigned a numerical microsatellite genotype (MSG), dendrogram was constructed and similarities between genotypes were visualized by minimum spanning tree. Fifty bloodstream isolates from MH yielded 37 MSGs with 20 isolates clustering in 7 MSGs. Duplicate isolates and colonizing strains yielded same/highly similar MSG as bloodstream isolates. Colonizing strains from two non-candidemia patients yielded unique MSGs while others belonged to a cluster. All isolates from HCWs and from two other hospitals belonged to unique MSGs. Cluster isolates came from patients in NICU-1 or from neonates in NICU-1 and other NICUs. Clonal complexes comprising closely related genotypes indicative of microevolution were also detected. Our data show that some C. parapsilosis strains have persisted in MH environment over several years and these endemic genotypes were transmitted to other patients in NICU-1 and/or other nearby NICUs.

Background: Oral candidiasis (OC) has a profound effect on the life quality of immunocompromised patients, such as those undergoing chemotherapy. Objective: Systematic investigation of clinical outcome and microbiological features of yeast isolates recovered from the oral cavity of 150 Iranian patients with hematological malignancies. Design: MALDI-TOF MS, 21-plex PCR, and rDNA sequencing were used for identification. Antifungal susceptibility testing (broth microdilution, CLSI M27-A3/S4) and genotypic diversity of yeast isolates (amplified fragment length polymorphism) were assessed. Results: Nystatin treatment resulted in 70% therapeutic failure and administration of 150 mg fluconazole (FLZ) + nystatin for patients with OC relapse showed 70% clinical failure. Previous history of OC was significantly correlated with FLZ treatment requirement and nystatin failure (P = 0.005, α Candida albicans (80.3%) and Kluyveromyces marxianus (C. kefyr) (12.7%) were the two most prevalent yeast species isolated. FLZ and AMB exhibited the highest geometric mean values. 21-PCR showed 98.9% agreement with MALDI-TOF MS. K. marxianus isolates had the same genotype, while C. albicans isolates grouped in 15 genotypes. Conclusions: Marked rate of therapeutic failure of nystatin necessitated OC treatment with systemic antifungals. K. marxianus was the second most prevalent yeast and 21-plex PCR could be considered as an inexpensive identification tool.

Identification of opportunistic yeasts in developing countries is mainly performed by phenotypic assays, which are time-consuming and prone to errors. Wrong species identification may result in suboptimal treatment and inaccurate epidemiological data. To improve rapidity and accuracy of species identification, a diagnostic strategy using a stepwise "YEAST PANEL multiplex PCR assays" targeting 21 clinically important yeast species of Candida, Trichosporon, Rhodotorula, Cryptococcus, and Geotrichum was designed. Four hundred CBS reference strains were used for optimization and specificity testing. Eight hundred clinical species were prepared in blinded sets for multiplex polymerase chain reaction (PCR) and matrix-assisted laser desorption time of flight mass spectrophotometry (MALDI-TOF MS) investigation. Results obtained from YEAST PANEL multiplex PCR assay were 100% consistent with those of MALDI-TOF MS. Utilization of pure colony testing showed distinct amplicons for each species, thus eliminating the need for DNA extraction. The targeted yeast species of this assay are responsible for 95% of the yeast infections. In conclusion, due to the high accuracy and coverage of a broad range of yeasts, this assay could be useful for identification in routine laboratories and epidemiological studies.

Candida auris is an emerging antifungal resistant yeast species causing nosocomial and invasive infections, emphasising the need of improved diagnostics and epidemiological typing methods. We show that MALDI-TOF VITEK-MS followed by amplified length polymorphisms allows for accurate species identification and subsequent epidemiological characterisation of strains encountered during potential outbreaks.

Triazole antifungal agents are the mainstay of aspergillosis treatment. As highlighted in numerous studies, the global increase in the prevalence of triazole resistance could hamper the management of aspergillosis. In the present three-year study, 513 samples (213 clinical and 300 environmental samples) from 10 provinces of Iran were processed and screened in terms of azole resistance (4 and 1 mg l-1 of itraconazole and voriconazole, respectively), using selective plates. Overall, 150 A. fumigatus isolates (71 clinical and 79 environmental isolates) were detected. The isolates were confirmed by partial sequencing of the β-tubulin gene. Afterwards, in vitro antifungal susceptibility tests against triazole agents were performed, based on the Clinical and Laboratory Standards Institute (CLSI) M38-A2 document. The CYP51A gene was sequenced in order to detect mutations. The MIC of itraconazole against 10 (6.6 %) strains, including clinical (n=3, 4.2 %) and environmental (n=7, 8.8 %) strains, was higher than the breakpoint and epidemiological cut-off value. Based on the findings, the prevalence of azole-resistant A. fumigatus in Iran has increased remarkablyfrom 3.3 % to 6.6 % in comparison with earlier epidemiological research. Among resistant isolates, TR34/L98H mutations in the CYP51A gene were the most prevalent (n=8, 80 %), whereas other point mutations (F46Y, G54W, Y121F, G138C, M172V, F219C, M220I, D255E, T289F, G432C and G448S mutations) were not detected. Although the number of patients affected by azole-resistant A. fumigatus isolates was limited, strict supervision of clinical azole-resistant A. fumigatus isolates and persistent environmental screening of azole resistance are vital to the development of approaches for the management of azole resistance in human pathogenic fungi.

Item does not contain fulltext Talaromyces amestolkiae is a common cosmopolitan species that has been cultured from indoor house dust, sputum and lungs from cystic fibrosis patients, indoor air, wheat, soil, pineapple, sculptures and manure. It was described as an asexual Talaromyces species and was reported to produce black sclerotia. In this study we report on the induction of sexual reproductive structures in T. amestolkiae. The mating type of 18 T. amestolkiae strains was determined with MAT-specific primers. Subsequently opposite mating types were inoculated on oatmeal agar and malt-extract agar and incubated 6-20 wk at 25 and 30 C in darkness. After incubation single ascospore isolations were made and evidence of recombination in the offspring was examined by amplified fragment length polymorphism and pairwise homoplasy index test, which is implemented in Splitstree4. The offspring displayed clear evidence of recombination on a genetic level as shown in the variations observed between banding patterns in the amplified fragment length polymorphism. Also a net-like and reticulated NeighborNet was observed and the pairwise homoplasy index test for recombination supported the presence of recombination (P = 0.003372). The distribution of MAT1-1 and MAT1-2 genes in the progeny showed a close to 1:1 ratio. Talaromyces amestolkiae is only the second heterothallic Talaromyces species to produce ascomata and ascospores under laboratory conditions.

Item does not contain fulltext Aspergillus terreus species complex is recognized as a frequent agent of invasive aspergillosis in Tyrol. The reason for this specific epidemiological situation is unclear. Aspergillus terreus strains isolated from environmental and clinical sources were genotyped using a novel panel of short tandem repeats and were evaluated for virulence. Three major endemic genotypes collected from the Inn region and its side valleys were found to cause the majority of invasive A. terreus infections. All of these genotypes were of the same mating type, which suggests that a mating barrier is present between these geographically well-adapted strains which is found to persist for at least 11 years. The three major genotypes were prevalent in both human infections and the environment. No major differences in virulence were observed using Galleria mellonella as model. Our data suggest a specific environmental exposure being responsible for the high incidence of A. terreus infections in Innsbruck, the Inn valley and side valleys (Tyrol, Austria).

The Fusarium fujikuroi species complex (FFSC) is one of the most common group of fusaria associated with plant diseases, mycotoxins production and traumatic and disseminated human infections. Here we present the description and taxonomy of a new taxon, Fusarium ficicrescens sp. nov., collected from contaminated fig fruits in Iran. Initially this species identified as F. andiyazi by morphology. In the present study the species was studied by multilocus sequence analysis, amplified fragment length polymorphism (AFLP), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and phenotypic characters. Multilocus analyses were based on translation elongation factor 1α (TEF1), RNA polymerase subunit (RPB2) and beta-tubulin (BT2) and proved F. ficicrescens as a member of the FFSC. Phylogenetic analysis showed that the fungus is closely related to F. lactis, F. ramigenum, and F. napiforme; known plant pathogens, mycotoxin producers, and occasionally occurring multidrug resistant opportunists. The new species differed by being able to grow at 37ºC and by the absence of mycotoxin production. TEF1 was confirmed as an essential barcode for identifying Fusarium species. In addition to TEF1, we evaluated BT2 and RPB2 in order to provide sufficient genetic and species boundaries information for recognition of the novel species. DNA barcoding, MALDI-TOF and AFLP data support Fusarium ficicrescens as a distinct species within the F. fujikuroi species complex (PDF Download Available). Available from: https://www.researchgate.net/publication/280444385_DNA_barcoding_MALDI-TOF_and_AFLP_data_support_Fusarium_ficicrescens_as_a_distinct_species_within_the_F_fujikuroi_species_complex [accessed Jan 11, 2016].

Sporotrichosis is a chronic subcutaneous mycosis caused by Sporothrix species, of which the main aetiological agents are S. brasiliensis, S. schenckii, and S. globosa. Infection occurs after a traumatic inoculation of Sporothrix propagules in mammals’ skin and can follow either a classic route through traumatic inoculation by plant debris (e.g., S. schenckii and S. globosa) or an alternative route through zoonotic transmission from animals (e.g., S. brasiliensis). Epizootics followed by a zoonotic route occur in Brazil, with Rio de Janeiro as the epicenter of a recent cat-transmitted epidemic. DNA-based markers are needed to explore the epidemiology of these Sporothrix expansions using molecular methods. This paper reports the use of amplified-fragment-length polymorphisms (AFLP) to assess the degree of intraspecific variability among Sporothrix species. We used whole-genome sequences from Sporothrix species to generate 2,304 virtual AFLP fingerprints. In silico screening highlighted 6 primer pair combinations to be tested in vitro. The protocol was used to genotype 27 medically relevant Sporothrix. Based on the overall scored AFLP markers (97–137 fragments), the values of polymorphism information content (PIC = 0.2552–0.3113), marker index (MI = 0.002–0.0039), effective multiplex ratio (E = 17.8519–35.2222), resolving power (Rp = 33.6296–63.1852), discriminating power (D = 0.9291–0.9662), expected heterozygosity (H = 0.3003–0.3857), and mean heterozygosity (Havp = 0.0001) demonstrated the utility of these primer combinations for discriminating Sporothrix. AFLP markers revealed cryptic diversity in species previously thought to be the most prevalent clonal type, such as S. brasiliensis, responsible for cat-transmitted sporotrichosis, and S. globosa responsible for large sapronosis outbreaks in Asia. Three combinations (#3 EcoRI-FAM-GA/MseI-TT, #5 EcoRI-FAM-GA/MseI-AG, and #6 EcoRI-FAM-TA/MseI-AA) provide the best diversity indices and lowest error rates. These methods make it easier to track routes of disease transmission during epizooties and zoonosis, and our DNA fingerprint assay can be further transferred between laboratories to give insights into the ecology and evolution of pathogenic Sporothrix species and to inform management and mitigation strategies to tackle the advance of sporotrichosis., Author summary Sporotrichosis is a subacute or chronic infection characterized by nodular lesions of the (sub)cutaneous tissues and adjacent lymphatics. Sporothrix brasiliensis, S. schenckii, and S. globosa are the main agents of sporotrichosis in humans and other mammals. Sporothrix propagules gain entrance by traumatic implantation in the skin following two main routes of infection, which include animal transmission (e.g., cat-cat and cat-human) and plant origin. In recent years there has been a significant increase in the number of atypical and more severe cases of sporotrichosis, along with the expansion of the area of occurrence of Sporothrix species, such as the highly virulent S. brasiliensis. We investigated the usefulness of the AFLP technology, a DNA fingerprinting technique, which is based on the selective amplification of genomic restriction fragments by PCR to explore genetic diversity and population structure in Sporothrix during ongoing outbreaks. We report six highly effective sets of AFLP markers to discriminate Sporothrix at species and strain level, thus allowing tracking the spread of sporotrichosis. Adding molecular data in an outbreak response context can reveal better ways to improve public policies to contain the advance of sporotrichosis, by early detection, response, intervention, and follow-up.

Invasive mycoses present a global challenge with expansion into new hosts, emergence of new pathogens, and development of multidrug resistance. In parallel, new antifungal agents and advanced laboratory diagnostic systems are being developed. In response to these evolving challenges, the European Confederation of Medical Mycology (ECMM) is committed to providing international expertise, guidance, and leadership with the key objectives of improving diagnosis, treatment, outcome, and survival of persons with invasive fungal diseases. Representing 25 affiliated National Medical Mycology Societies, the ECMM has developed several major ways to achieving these critical objectives: (a) tasking specific medical mycology working groups; (b) founding the ECMM Academy and Fellow program (FECMM); (c) expanding the goals of ECMM beyond the European region; (d) implementing the ECMM Excellence Centre Initiative in Europe; and (e) the ECMM Global Guidelines and Neglected Orphan Disease Guidance Initiatives focusing on mucormycosis, rare mould diseases, rare yeast diseases, and endemic mycoses. We believe that these important initiatives and other strategies of the ECMM will advance the field of medical mycology and improve the outcome of patients with invasive mycoses worldwide. ispartof: MYCOSES vol:61 issue:11 pages:885-894 ispartof: location:Germany status: published

Trichophyton schoenleinii is an anthropophilic dermatophyte usually causing tinea favosa. Only few studies have provided data on molecular epidemiology and antifungal profiles of this fungus due to its limited prevalence after 1950s. Forty-nine strains from Asia (n = 27), Africa (n = 10), Europe (n = 10) and from unknown regions (n = 2) were analysed with amplified fragment length polymorphism fingerprinting (AFLP) to reveal intraspecific genetic diversity in this dataset. Amplified fragment length polymorphism fingerprinting genotyping revealed five clusters which did not correspond to geographic origins or clinical characteristics. Additionally, in vitro antifungal susceptibility to seven antifungals was provided for all strains. Terbinafine, ketoconazole, miconazole and itraconazole proved to be the most effective drugs, followed by griseofulvin. No correlation between genotypes and differences in antifungal susceptibility was observed. It is concluded that the AFLP groups are lineages within a single species.

Cryptococcosis is a life‐threatening mycosis primarily occurring in adult patients particularly those with immunosuppression such as HIV infection/AIDS. The number of reported cases of paediatric cryptococcosis has increased in the last decade around the world, including China. However, current information on the characteristics of cryptococcosis in children, particularly the genotypic diversity and antifungal susceptibility of the isolates, is limited. In the present study, a total of 25 paediatric isolates of Cryptococcus neoformans were genotyped using the ISHAM‐MLST scheme. In vitro susceptibility to antifungal agents of the 22 isolates was tested using the CLSI M27‐A3 method. Our analyses revealed that the genotypic diversity of C. neoformans isolates from Chinese paediatric patients was low, with ST 5 (80%) and ST 31 (12%) being the two major sequence types. Reduced susceptibility to fluconazole (FLU), 5‐flucytosine (5‐FC) and itraconazole (ITR) was observed among C. neoformans isolates from Chinese paediatric patients, particularly among the ST5 isolates, which was similar to observations made on C. neoformans isolates from Chinese adult patients. In addition, the majority of isolates (3/4, 75%) obtained from deceased patients showed decreased antifungal susceptibility, which indicates that further monitoring of antifungal susceptibility of Cryptococcus isolates is warranted in management of paediatric cryptococcosis.

IntroductionCryptococcal meningitis is a neglected disease and an AIDS-defining illness, responsible for 15% of all AIDS-related deaths globally. In 2014, the estimated number of incident cryptococcal meningitis cases was 223 100, with 73% of them occurring in Africa. Currently available data on the prevalence, incidence, aetiologies and mortality of cryptococcal meningitis across Africa are sparse and of limited quality. We propose to conduct the first systematic review to summarise the epidemiological data available on cryptococcal meningitis and its aetiological causes in Africa.Methods and analysisWe will search PubMed, MEDLINE, Excerpta Medica Database, ISI Web of Science, Africa Index Medicus, Cumulative Index to Nursing and Allied Health for studies on cryptococcal meningitis published between 1st January 1950 and 31st December 2017, involving adults and/or children residing in Africa. After study selection, full text paper acquisition and data extraction, we will use validated tools and checklists to assess the quality of reporting and risk of bias for each study. Heterogeneity across studies will be assessed using the χ2test on Cochrane’s Q statistic and a random effect meta-analysis will be used to estimate the overall prevalence, incidence density and mortality of cryptococcal meningitis across studies with similar characteristics. This protocol is prepared and presented in accordance with the 2015 Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols guidelines. Reporting of the results will be compliant with the Meta-Analysis Of Observational Studies in Epidemiology (MOOSE) guidelines.Ethics and disseminationThere is no requirement for ethical approval since we will be using data from published studies. The final report will be published in a peer-reviewed journal and further presented at conferences. This study is expected to provide useful contextual estimates needed to inform treatment policies on the African continent and assess the impact of diagnostic and prevention strategies on the burden of cryptococcal meningitis in the post antiretroviral therapy era.PROSPERO registration numberCRD42017081312.

Candida auris, C. haemulonii, C. duobushaemulonii, and C. pseudohaemulonii are closely related and highly multidrug resistant yeast pathogens. The high cost and low accuracy of current diagnostics may underestimate their prevalence, especially in medical resource-limited regions. In this study, we used 172 C. auris stains and its relatives and 192 other fungal strains to establish and validate a novel multiplex end-point PCR. A prospective and a retrospective clinical screenings using this assay were further performed in China and Iran respectively. We identified the first isolate of C. pseudohaemulonii in China and the first isolate of C. haemulonii in Iran from 821 clinical isolates in total, without any false positive. Animal models of C. auris and C. haemulonii were established for validation. The overall positive rates of the assay for mice blood and tissue were 28.6 and 92.9%, respectively. Compared with previously developed assays, our assay is more available and affordable to the developing countries, and may contribute to a better understanding of the epidemiology of C. auris and its relatives in these regions.