Search

Timely and accurate identification of yeasts is essential for adequate treatment, considering the increase in antifungal resistance of some species, particularly for C. auris . Current matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) manufacturer's protocol for identification of yeasts requires 24- to 72-h cultivation on Sabouraud dextrose media (SAB), but not some of the mainstay primary culture media used in mycology such as inhibitory mold agar (IMA), Mycosel, CHROMagar Candida Plus, and CHROMagar Candida. As culture media can influence MALDI-TOF MS identification results, this study evaluated the accuracy and performance of identification of clinically relevant yeasts on these first-line media using the VITEK-MS MALDI-TOF MS system.IMPORTANCEIn this study, a panel of 140 strains (21 species) was used to assess the performance of the selected media. Although not in the manufacturer's list of accepted media, IMA and chromogenic media are suitable for the identification of yeasts on the VITEK-MS systems. CHROMagar Candida Plus allowed the identification of 135/140 isolates tested after 24-h incubation similar to SAB reference media (137/140). Yeast isolates that grew on Mycosel selective media were also reliably identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. VITEK-MS system with IVD database V3.2 correctly identified C. auris strains to the species level on CHROMagar Candida Plus alleviating the need for subcultivation and reduced turnaround time (24-72 h) to identification for patient screening., Competing Interests: The authors declare no conflict of interest.

Alternaria alternata is part of a genus comprised of over 600 different species that occur all over the world and cause damage to humans, plants and thereby to the economy. Yet, even though some species are causing tremendous issues, the past years have shown that assigning newly found isolates to known species was rather inconsistent. Most identifications are usually done on the basis of spore morphology, chemotype and molecular markers. In this work we used strains isolated from the wild as well as commercial strains of the DSMZ (German collection of microorganisms and cell cultures) as a reference, to show, that the variation within the Alternaria alternata species is comparable to the variation between different species of the genus Alternaria in regards to spore morphology and chemotype. We compared the different methods of identification and discerned the concatenation of multiple molecular markers as the deciding factor for better identification. Up until this point, usually a concatenation of two or three traditional molecular markers was used. Some of those markers being stronger some weaker. We show that the concatenation of five molecular markers improves the likeliness of a correct assignment, thus a better distinction between the different Alternaria species., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

During construction work (2017-2019), an increase in Aspergillus flavus infections was noted among pediatric patients, the majority of whom were receiving amphotericin B prophylaxis. Microsatellite genotyping was used to characterize the outbreak. A total of 153 A . flavus isolates of clinical and environmental origin were included. Clinical isolates included 140 from 119 patients. Eight patients were outbreak-related patients, whereas 111 were outbreak-unrelated patients from Danish hospitals (1994-2023). We further included four control strains. Nine A. flavus isolates were from subsequent air sampling in the outbreak ward (2022-2023). Typing followed Rudramurthy et al.(S. M. Rudramurthy, H. A. de Valk, A. Chakrabarti, J. Meis, and C. H. W. Klaassen, PLoS One 6:e16086, 2011, https://doi.org/10.1371/journal.pone.0016086). Minimum spanning tree (MST) and discriminant analysis of principal components (DAPC) were used for cluster analysis. DAPC analysis placed all 153 isolates in five clusters. Microsatellite marker pattern was clearly distinct for one cluster compared to the others. The same cluster was observed in an MST. This cluster included all outbreak isolates, air-sample isolates, and additional patient isolates from the outbreak hospital, previously undisclosed as outbreak related. The highest air prevalence of A. flavus was found in two technical risers of the outbreak ward, which were then sealed. Follow-up air samples were negative for A. flavus . Microsatellite typing defined the outbreak as nosocomial and facilitated the identification of an in-hospital source. Six months of follow-up air sampling was without A. flavus . Outbreak-related/non-related isolates were easily distinguished with DAPC and MST, as the outbreak clone's distinct marker pattern was delineated in both statistical analyses. Thus, it could be a variant of A. flavus , with a niche ability to thrive in the outbreak-hospital environment., Importance: Aspergillus flavus can cause severe infections and hospital outbreaks in immunocompromised individuals. Although lack of isogeneity does not preclude an outbreak, our study underlines the value of microsatellite genotyping in the setting of potential A. flavus outbreaks. Microsatellite genotyping documented an isogenic hospital outbreak with an internal source. This provided the "smoking gun" that prompted the rapid allocation of resources for thorough environmental sampling, the results of which guided immediate and relevant cleaning and source control measures. Consequently, we advise that vulnerable patients should be protected from exposure and that genotyping be included early in potential A. flavus outbreak investigations. Inspection and sampling are recommended at any site where airborne spores might disperse from. This includes rarely accessed areas where air communication to the hospital ward cannot be disregarded., Competing Interests: The authors declare no conflict of interest.

Fungal infections pose an increasing threat to public health. New pathogens and changing epidemiology are a pronounced risk for nosocomial outbreaks. To investigate clonal transmission between patients and trace the source, genotyping is required. In the last decades, various typing assays have been developed and applied to different medically important fungal species. While these different typing methods will be briefly discussed, this review will focus on the development and application of short tandem repeat (STR) genotyping. This method relies on the amplification and comparison of highly variable STR markers between isolates. For most common fungal pathogens, STR schemes were developed and compared to other methods, like multilocus sequence typing (MLST), amplified fragment length polymorphism (AFLP) and whole genome sequencing (WGS) single nucleotide polymorphism (SNP) analysis. The pros and cons of STR typing as compared to the other methods are discussed, as well as the requirements for the development of a solid STR typing assay. The resolution of STR typing, in general, is higher than MLST and AFLP, with WGS SNP analysis being the gold standard when it comes to resolution. Although most modern laboratories are capable to perform STR typing, little progress has been made to standardize typing schemes. Allelic ladders, as developed for Aspergillus fumigatus, facilitate the comparison of STR results between laboratories and develop global typing databases. Overall, STR genotyping is an extremely powerful tool, often complimentary to whole genome sequencing. Crucial details for STR assay development, its applications and merit are discussed in this review., (© 2024. The Author(s).)

Objective: This study investigated C. albicans strain diversity and maintenance in the oral cavity of HIV positive women over a 6 month period., Study Design: C. albicans strains were isolated from 17 HIV positive women at Charlotte Maxeke Academic Hospital, Johannesburg at 3 intervals over a 6 month period. Strains were genotyped using ABC and Multilocus Sequence Typing (MLST) techniques. In the MLST technique, for each strain, a Diploid Sequence Type (DST) number was obtained. Using cluster analysis, an Unweighted Pair Group Method with Arithmetic Mean (UPGMA) dendrogram and a matrix of strain similarities were generated. Strains were also compared to the previous South African isolates documented in the MLST database., Results: Ninety four percent of women carried the same ABC genotype for 6 months. MLST technique, showed that ten women (58.8%) carried the same DST at 2 visits, while seven (41.2%) carried different DST at all visits. Further analysis showed that 64.7% of women were recolonised with different strains and 35.3% carried the same strains of C. albicans with heterozygosity. A total of 40 diploid sequence types were identified of which 27 DSTs were unique to this study group that were added to the MLST database. Most of the strains were closely related to previously isolated strains from South Africa., Conclusion: Recolonization of the oral cavity with different strains and microevolution of the original strains of C. albicans can occur, which can be a potential problem for HIV patients, in whom highly virulent and drug resistant strains can emerge., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: Candida auris is increasingly being isolated from patients all over the world. It has five clades. In this study, it was aimed to compare the results of biochemical tests obtained using different methods and the antifungal susceptibility profiles of C. auris strains isolated from the first seven cases reported in Türkiye, and evaluate whether this information could be useful as preliminary data in determining the clade of strains in centers that lack the opportunity to apply molecular methods., Methods: Identification test results obtained using API ID 32 C, API 20 C AUX, VITEK-2 YST, and MALDI-TOF MS; colony color and morphology on Chromagar Candida, CHROMagar Candida Plus media, and cornmeal-Tween 80 agar; susceptibility to antifungals were tested and compared. Antifungal susceptibility test was studied using microdilution method according to the recommendations of EUCAST. Additionally, a pilot study was conducted to investigate the value of CHROMagar Candida Plus., Results: All seven strains were identified as Lachancea kluyveri with API ID 32 C, Rhodotorula glutinis; Cryptococcus neoformans with API 20 C AUX, and C. auris with both VITEK-2 YST and MALDI-TOF MS. MIC values for fluconazole were very high (≥64 mg/L) for all seven strains. It was observed that 11 (37.9%) of 29 Candida parapsilosis strains formed colonies with morphology similar to C. auris on CHROMagar Candida Plus medium, leading to false positivity., Conclusions: Although there have been many isolations of C. auris in our country in recent years, clade distribution of only a small number of strains is known yet. In this study, when the biochemical properties and antifungal susceptibility profiles of the seven strains were evaluated, it was concluded that they exhibited some characteristics compatible with clade I. It was also observed that strains 1 and 2 may belong to a different clade., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Indian Association of Medical Microbiologists. Published by Elsevier B.V. All rights reserved.)

The infection proportion of Candida orthopsilosis , a member of the C. parapsilosis complex, has increased globally in recent years, and nosocomial outbreaks have been reported in several countries. This study aimed to establish microsatellite loci-based typing method that was able to effectively distinguish among C. orthopsilosis isolates. Three reference C. orthopsilosis genome sequences were analyzed to identify repeat loci. DNA sequences containing over eight bi- or more nucleotide repeats were selected. A total of 51 loci were initially identified, and locus-specific primers were designed and tested with 20 epidemiologically unrelated isolates. Four loci with excellent reproducibility, specificity, and resolution for molecular typing purposes were identified, and the combined discriminatory power (DP, based on 20 epidemiologically unrelated isolates) of these four loci was 1.0. Reproducibility was demonstrated by consistently testing three strains each in triplicate, and stability, demonstrated by testing 10 successive passages. Then, we collected 48 C . orthopsilosis non-duplicate clinical isolates from the China Hospital Invasive Fungal Surveillance Net study to compare the DP of the microsatellite-based typing with internal transcribed spacer (ITS) and amplified fragment length polymorphism (AFLP) typing analyses, using ATCC 96139 as a reference strain. These 49 isolates were subdivided into 12 microsatellite types (COMT1-12), six AFLP types, and three ITS types, while all the isolates with the same COMT belonged to consistent AFLP and ITS type, demonstrating the high DP of our microsatellite-type method. According to our results, COMT12 was found to be the predominant type in China, and COMT5 was the second largest and responsible for causing a nosocomial outbreak. This microsatellite-type method is a valuable tool for the differentiation of C. orthopsilosis and could be vital for epidemiological studies to determine strain relatedness and monitor transmission., Competing Interests: The authors declare no conflict of interest.

Trichosporon asteroides is an emerging yeast-like pathogen commonly misidentified by commercial biochemical identification systems. We evaluated the performance of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the identification of 21 clinical T. asteroides strains using the Bruker Daltonics database (BDAL) and an in-house developed library. Mass spectra were obtained by the FlexControl system v.3.4, and characterizations were performed in the Biotyper BDAL database v.4.1 and the developed in-house library. Species identification for T. asteroides failed as all 21 strains were misidentified as T. japonicum (log-scores 1.89-2.19). Extending the existing database was crucial to achieving 100% correct species-level identification and accurate distinction between species. Our results indicate that the commercial BDAL database has no discriminatory power to distinguish between T. japonicum and T. asteroides. Whereas improvement of the current BDAL database is pending, we strongly advise system users not to exclude the possibility of the failure to report T. asteroides., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Cryptococcus neoformans species complex (CNSC) is a globally distributed human opportunistic yeast pathogen consisting of five major molecular types (VNI, VNII, VNB, VNIII and VNIV) belonging to two species, C. neoformans (VNI, VNII and VNB, collectively called serotype A) and C. deneoformans (VNIV, commonly called serotype D), and their hybrids (VNIII, serotype AD). Over the years, many studies have analyzed the geographical distribution and genetic diversity of CNSC. However, the global population structure and mode of reproduction remain incompletely described. In this study, we analyze the published multilocus sequence data at seven loci for CNSC. The combined sequences at the seven loci identified a total of 657 multilocus sequence types (STs), including 296 STs with known geographic information, representing 4200 non-redundant isolates from 31 countries and four continents. Among the 296 STs, 78 and 52 were shared among countries and continents, respectively, representing 3643 of the 4200 isolates. Except for the clone-corrected serotype D sample among countries, our analysis of the molecular variance of the 4200 isolates revealed significant genetic differentiations among countries and continents in populations of CNSC, serotype A, and serotype D. Phylogenetic analyses of the concatenated sequences of all 657 STs revealed several large clusters corresponding to the major molecular types. However, several rare but distinct STs were also found, representing potentially novel molecular types and/or hybrids of existing molecular types. Phylogenetic incompatibility analyses revealed evidence for recombination within all four major molecular types-VNI, VNII, VNIV and VNB-as well as within two VNB subclades, VNBI and VNBII, and two ST clusters around the most common STs, ST5 and ST93. However, linkage disequilibrium analyses rejected the hypothesis of random recombination across most samples. Together, our results suggest evidence for historical differentiation, frequent recent gene flow, clonal expansion and recombination within and between lineages of the global CNSC population.

Introduction: Candida dubliniensis was first identified by Sullivan et al. (1995) in Dublin, Ireland. Its clinical significance is associated with development of fluconazole-resistance and invasive diseases in immunocompromised hosts. C. dubliniensis share many features with C. albicans so has been overlooked and misidentified for a long time., Aims: Evaluation of various phenotypic tests with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) as a gold standard to find out the best method/methods for identifying C. dubliniensis., Materials and Methods: First PCR-RFLP was performed on 186C. albicans and 14C. dubliniensis strains and then five phenotypic tests were performed simultaneously on all the strains., Results: The results of salt tolerance test at 48 h, colony color on HiCrome candida differential agar (HCDA) at 72 h, heat tolerance test at 48 h, xylose assimilation using discs at 72 h and growth on xylose based agar medium (XAM) at 48 h are completely concordant with PCR-RFLP. Colony color on Tobacco agar could differentiate accurately 100% test strains while peripheral hyphal fringes and chlamydosporulation on this agar was seen in only 86% and 87% respectively. Our routine methods proved to be cost effective than PCR-RFLP but the turnaround time was same or more than PCR-RFLP., Conclusion: For routine identification of C. dubliniensis we recommend use of colony color on HCDA and growth on XAM as simple, reliable and inexpensive method., (Copyright © 2021. Published by Elsevier B.V.)

In Aspergillus fumigatus, the repetitive region of the csp1 gene is one of the most frequently used loci for intraspecies typing of this human pathogenic mold. Using PCR amplification and Sanger sequencing of only a single marker, csp1 typing is readily available to most laboratories and highly reproducible. Here, I evaluate the usefulness of the csp1 marker for resistance detection and epidemiologic stratification among A. fumigatus isolates. After resolving nomenclature conflicts from published studies and adding novel csp1 types, the number of known types now adds up to 38. Their distribution mostly correlates with A. fumigatus population structure, and they are also meaningful for narrowly defined cases of azole resistance phenotypes. Isolates carrying the pandemic resistance allele TR 34 /L98H show signs of interclade crossing of strains with t02 or t04A, into the t11 clade. Furthermore, absolute differences in voriconazole MIC values between t02/t04B versus t11 TR 34 /L98H isolates indicate that the genetic background of resistance mutations may have a pivotal role in cross-resistance phenotypes and, thus, clinical outcome and environmental selection. Despite the general genetic similarity of isolates with identical csp1 types, outcrossing into other clades is also observed. The csp1 type alone, therefore, does not sufficiently discriminate genetic clades to be used as the sole marker in epidemiologic studies. IMPORTANCE Aspergillus fumigatus is a ubiquitously distributed saprophytic mold and a leading cause of invasive aspergillosis in human hosts. Pandemic azole-resistant strains have emerged on a global scale, which are thought to be propagated through use of azole-based fungicides in agriculture. To perform epidemiologic studies, genetic typing of large cohorts is key. Here, I evaluate the usefulness of the frequently used csp1 marker for resistance detection and epidemiologic stratification among A. fumigatus isolates. The phylogenetic distribution of csp1 types mostly correlates with A. fumigatus population structure and is also meaningful for narrowly defined cases of azole resistance phenotypes. Nevertheless, outcrossing of csp1 into other clades is also observed. The csp1 type alone, therefore, does not sufficiently discriminate genetic clades and should not be used as the sole marker in epidemiologic studies.

Neonothopanus gardneri, also known as coconut flower mushroom (flor-de-coco), is a Brazilian bioluminescent basidiomycete found in Palm Forest, a transitional biome between the Amazonian Forest and Caatinga (Savanna-like vegetation) in Northeast Brazil, especially in Piauí State. Recent advances toward the elucidation of fungal bioluminescence have contributed to the discovery of four genes (hisps, h3h, luz and cph) involved with the bioluminescence process, the so-called Caffeic Acid Cycle (CAC) and to develop biotechnological applications such autoluminescent tobacco plants and luciferase-based reporter genes. High-yield and -quality RNA-extraction methods are required for most of these purposes. Herein, four methods for RNA isolation from the mycelium of N. gardneri were evaluated: RNeasy® kit (QIAGEN), TRI+, TRI18G+, and TRI26G+. Highest RNA yield was observed for TRI18G+ and TRI26G+ methods, an increase of ~130% in comparison to the RNeasy® method and of ~40% to the TRI+ protocol. All the RNA samples showed good purity and integrity, except by gDNA contamination in RNA samples produced with the RNeasy® method. High quality of RNA samples was confirmed by successful cDNA synthesis and PCR amplification of the coding sequence of h3h gene, responsible for the hydroxylation of the precursor of fungal luciferin (3-hydroxyhispidin). Similarly, RT-qPCR amplification of ef-tu gene, related to the protein biosynthesis in the cell, was demonstrated from RNA samples. This is the first report of a reproducible, time-saving and low-cost optimized method for isolation of high-quality and -yield, DNA-free RNA from a bioluminescent fungus, but that can also be useful for other basidiomycetes., (Copyright © 2021 Elsevier B.V. All rights reserved.)

An investigation of members of the soil keratinophilic fungi community in China resulted in the identification of one new monotypic genus, Zongqia , and 10 new species, 2 of which are affiliated with Solomyces , 1 with the new genus Zongqia , 4 with Pseudogymnoascus , and 3 with Scedosporium . These novel taxa form an independent lineage distinct from other species, based on morphological and multilocus phylogenetic analyses. Descriptions, illustrations, and notes are provided for each taxon. These new taxa of the soil keratinophilic fungi add to the increasing number of fungi known from China, and it is now evident that numerous novel taxa are waiting to be described. IMPORTANCE Keratinophilic fungi are a group that can degrade and utilize keratin-rich material. It is also because of this ability that many taxa can cause infections in animals or humans but remain poorly studied. In this study, we reported a novel genus and 10 novel species, 7 novel species belonging to the order Thelebolales and 3 to the genus Scedosporium , based on multilocus phylogenetic analyses combined with morphological characteristics. Our study significantly updates the taxonomy of Thelebolales and Scedosporium and enhances our understanding of this group of the keratin-degrading fungal community. The findings also encourage future studies on the artificially constructed keratin-degrading microbial consortia.

Rapid yeast identification is of particular importance in monitoring wine fermentation and assessing strain application in winemaking. We used MALDI-TOF MS analysis supported by 26 S rRNA gene sequence analysis and Saccharomyces-specific PCR testing to differentiate reference and field strains recovered from organic wine production facilities in Waipara, New Zealand, in which Pinot Noir wine was produced by spontaneous fermentations in the vineyard and in the winery. Strains were isolated from each of four key stages of each ferment to evaluate changes in taxonomic diversity. MALDI-TOF MS analysis was confirmed as an excellent yeast identification method, with even closely related Saccharomyces species readily distinguished. A total of 13 indigenous species belonging to eight genera were identified from Pinot Noir ferments, with taxonomic diversity generally reducing as fermentation progressed. However, differences between the taxa recovered were observed between the vineyard and winery ferments, despite the grapes used being from the same batch. Furthermore, some consistent proteomic differences between strains of S. cerevisiae, Hanseniasporum uvarum, Candida californica, Pichia membranifaciens and Starmerella bacillaris correlated with the different fermentation systems used. The high speed, low cost, taxonomic resolution and ability to characterise subtle changes in phenotype that may result from variations in environmental conditions makes MALDI-TOF analysis an attractive tool for further and wider applications in the wine industry. Such applications may include monitoring wine fermentation to actively support the consistency of high-quality wine products, and potentially for the development of such products too., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Fungi of the genus Aspergillus are ubiquitously distributed in nature, and some cause invasive aspergillosis (IA) infections in immunosuppressed individuals and contamination in agricultural products. Because microscopic observation and molecular detection of Aspergillus species represent the most operator-dependent and time-intensive activities, automated and cost-effective approaches are needed. To address this challenge, a deep convolutional neural network (CNN) was used to investigate the ability to classify various Aspergillus species. Using a dissecting microscopy (DM)/stereomicroscopy platform, colonies on plates were scanned with a 35× objective, generating images of sufficient resolution for classification. A total of 8,995 original colony images from seven Aspergillus species cultured in enrichment medium were gathered and autocut to generate 17,142 image crops as training and test datasets containing the typical representative morphology of conidiophores or colonies of each strain. Encouragingly, the Xception model exhibited a classification accuracy of 99.8% on the training image set. After training, our CNN model achieved a classification accuracy of 99.7% on the test image set. Based on the Xception performance during training and testing, this classification algorithm was further applied to recognize and validate a new set of raw images of these strains, showing a detection accuracy of 98.2%. Thus, our study demonstrated a novel concept for an artificial-intelligence-based and cost-effective detection methodology for Aspergillus organisms, which also has the potential to improve the public's understanding of the fungal kingdom.

Cryptococcus species is an opportunistic yeast pathogen and classified into different molecular types according to typing techniques including multilocus sequence typing (MLST). The study aimed to investigate the genotypes of environmental Cryptococcus isolates using MLST and the relationship between the in vitro antifungal susceptibility and sequence types of isolates. Genotyping Cryptococcus isolates was performed by the MLST method at seven nuclear loci. Antifungal susceptibility was determined by using CLSI broth micro-dilution method for amphotericin B, fluconazole, itraconazole, voriconazole, flucytosine, and luliconazole. Seven sequence types (ST) were detected using MLST analysis, with the most frequent (50%) ST77, followed by ST4 (16.7%) among 30 C. neoformans isolates. All antifungals demonstrated excellent activity against isolates, except for itraconazole and amphotericin B that were non-wild type against 53.3% and 10% of isolates, respectively. Although seven sequence types belonging to C. neoformans isolates were detected, ST77 was the main sequence type in Ahvaz. Also, non-wild type isolates were only found against itraconazole and amphotericin B.

Snow and ice present challenging substrates for cellular growth, yet microbial snow communities not only exist, but are diverse and ecologically impactful. These communities are dominated by green algae, but additional organisms, such as fungi, are also abundant and may be important for nutrient cycling, syntrophic interactions, and community structure in general. However, little is known about these non-algal community members, including their taxonomic affiliations. An example of this is Chionaster nivalis, a unicellular fungus that is morphologically enigmatic and frequently observed in snow communities globally. Despite being described over one hundred years ago, the phylogeny and higher-level taxonomic classifications of C. nivalis remain unknown. Here, we isolated and sequenced the internal transcribed spacer (ITS) and the D1-D2 region of the large subunit ribosomal RNA gene of C. nivalis, providing a molecular barcode for future studies. Phylogenetic analyses using the ITS and D1-D2 region revealed that C. nivalis is part of a novel lineage in the class Tremellomycetes (Basidiomycota, Agaricomycotina) for which a new order Chionasterales ord. nov. (MB838717) and family Chionasteraceae fam. nov. (MB838718) are proposed. Comparisons between C. nivalis and sequences generated from environmental surveys revealed that the Chionasterales are globally distributed and probably psychrophilic, as they appear to be limited to the high alpine and arctic regions. These results highlight the unexplored diversity that exists within these extreme habitats and emphasize the utility of single-cell approaches in characterizing these complex algal-dominated communities., Competing Interests: The authors have declared that no competing interests exist.

Rationale: The microsporidia are obligate intracellular pathogenic fungi that parasitize a wide range of invertebrate and vertebrate hosts and have important impacts on health, food security and the economy. In this paper, we focus on Nosema ceranae and N. apis, which chronically infect the digestive tract of honeybees, altering their physiology and lifespan., Methods: We applied matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for rapid molecular profiling of extracts of Nosema spores in order to identify the species and the geographical origin, and assess the viability status of Nosema microsporidia in conjunction with a flow cytometric approach. Pure solutions of spores were prepared for flow cytometric analysis and MALDI-MS profiling. A mechanical extraction of viable or heat-killed Nosema spores was conducted to obtain mass fingerprints of peptides/proteins for samples of microsporidia from different geographical origins (MBO.NC01, MBO.NC02 and MBO.NA01)., Results: A distinction in the peptide/protein profiles between two isolates with different geographical origins was observed. Mass fingerprints of viable and experimentally killed spores were also clearly distinguishable, regardless of Nosema species. Finally, using our computational models on the different Nosema species, we were able to classify five independent isolates of Nosema microsporidia., Conclusions: We have shown that MALDI-MS is a rapid, cost-effective and simple method for identifying Nosema species. We demonstrated that MALDI Biotyping could represent a valuable surveillance tool of nosemosis in apiaries for sanitary services and beekeepers., (© 2020 John Wiley & Sons, Ltd.)

Identification of moulds is crucial for the clinical management of patients. The goal of this study was to evaluate the new ID-FUNGI plate (IDFP) for the identification of moulds by MALDI Biotyper. IDFP was compared with Sabouraud with gentamicin and chloramphenicol plate (SAB) for the identification of 80 moulds from respiratory samples and eight reference strains. With the direct transfer method, species identification rose from 6% with SAB to 68% with IDFP using score cut-off 2 and from 20 to 75% using cut-off 1.7 (p < 0.001). Our study highlights that the new IDFP improves mycological diagnostic and workflow in laboratories.

Introduction. Histopathological examination (HPE) of tissue helps in the diagnosis of invasive fungal infections (IFIs) but cannot identify the fungus to the genus/species level Gap Statement Available protocols for the molecular identification of fungi from formalin-fixed and paraffin-embedded (FFPE) tissues have limitations in terms of extraction and target selection, and standardisation. Aim. Development of sequence-based fungal identification protocol after extraction of DNA from formalin-fixed and paraffin-embedded (FFPE) tissues. Methodology. A total of 63 FFPE tissues from histopathology proven IFI cases were used to standardize the DNA extraction (commercial QIAamp kit-based extraction and conventional phenol-chloroform-isoamyl alcohol [PCI] method) and sequence-based fungal identification protocols. The PCR targeted different ribosomal DNA (rDNA) regions including complete internal transcribed spacer (ITS1-5.8S-ITS2), separate ITS1 and ITS2, 18S and D1/D2 of 28S regions. Semi-nested PCR targeting Mucorales-specific 18S rDNA region was performed in tissues having aseptate hyphae. The optimized ITS1-PCR protocol was evaluated in 119 FFPE tissues containing septate hyphae or yeast, and Mucorales-specific semi-nested PCR in 126 FFPE tissues containing aseptate hyphae. Results. The DNA yield by conventional PCI method was significantly higher ( P <0.0001) than commercial kit, though the quality of DNA was similar by both protocols. The test accuracy was best while using ITS1 (61.9 %) as the target compared to 7.9, 29.9 and 22.2 % on targeting ITS1-5.8S-ITS2, ITS2, the D1/D2 region of 28S, respectively. The test accuracies of ITS1-PCR in tissues containing septate hyphae, aseptate hyphae and yeasts were 75.5, 18.7 and 100 %, respectively. The amplification (targeting ITS1 region) improved by increasing the thickness of tissue section (up to 50 µm) used for DNA extraction. ITS1-PCR protocol could amplify fungal DNA in 76 (63.8 %) tissues and Mucorales-specific semi-nested PCR in 86 (68.3 %) tissues. Conclusion. Conventional PCI-based DNA extraction from thick tissue (50 µm) may be used until optimal commercial fungal DNA extraction kit is developed. Subsequent ITS1-PCR for septate fungi and yeast, and semi-nested PCR targeting 18S rDNA for Mucorales are recommended to identify the fungus in FFPE tissues.

Background: The pathogen Candida auris is rapidly gaining clinical importance because of its resistance to antifungal treatments and its persistence in hospital environments. Early and accurate diagnosis of C. auris infections is crucial, and however, the fungus has often been misidentified by commercial systems., Objectives: To develop conventional and real-time PCR methods for accurate and rapid identification of C. auris and its discrimination from closely related species by exploiting the uniqueness of certain glycosylphosphatidylinositol (GPI)-modified protein-encoding genes., Methods: Species-specific primers for two unique putative GPI protein-encoding genes per species were designed for C. auris, C. haemulonii, C. pseudohaemulonii, C. duobushaemulonii, C. lusitaniae and C. albicans. Primers were blind tested for their specificity and efficiency in conventional and real-time multiplex PCR set-up., Results: All primers combinations showed excellent species specificity. In multiplex mode, correct identification was aided by different-sized amplicons for each species. Efficiency of the C. auris primers was validated using a panel of 155 C. auris isolates, including all known genetically diverse clades. In real-time multiplex PCR, different melting points of the amplicons allowed the distinction of C. auris from four related species. C. auris limit of detection was 5 CFU/reaction with a threshold value of 32. The method was also able to detect C. auris in spiked blood and serum., Conclusions: PCR identification based on unique GPI protein-encoding genes allows for accurate and rapid species identification of C. auris and related species without need for expensive equipment when applied in conventional PCR set-up., (© 2020 Wiley-VCH GmbH.)

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) holds promise as a potential tool for clinical identification of filamentous fungi. However, due to the lack of an appropriate extraction protocol and the difficulty of database building, the identification power of each system differs. In this study, we selected 126 clinical mould isolates comprising 28 species identified using internal transcribed spacer (ITS) sequencing as the reference method to evaluate three MALDI-TOF MS systems. When using cultures and sample preparation as recommended by the respective vendors, of the 126 strains tested, VITEK MS identified 121 (96.0%) to species-level and 124 (98.4%) to genus-level; Biotyper identified 53 (42.1%) to species-level and 54 (42.9%) to genus-level; Autof identified 74 (58.7%) to species-level and 76 (60.3%) to genus-level. For the Autof system, the tube extraction method recommended by the vendor performed better (59%) than the on-plate lysis (51%). Our study demonstrates that MALDI-TOF MS systems can successfully identify most clinically relevant fungi, while performance is still highly dependent on the database and sample preparation protocol., (© The Author(s) 2020. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Aims: Accurate identification of dermatophytes is essential for implementing appropriate antifungal treatment and epidemiological analysis. However, the limitations of conventional diagnostics are a frequently discussed topic, and new diagnostic techniques are constantly expanding. In this study, we assess the suitability of conventional diagnostic techniques in comparison to the real-time PCR assay and MALDI-TOF MS in detection and identification of dermatophytes., Methods and Results: Strains included in this study were obtained from human and animals with symptomatic, and asymptomatic infection. A direct examination revealed that 31·7 and 60·9% of samples from symptomatic patients, and 25·7 and 60% from asymptomatic animals were positive, as shown by light and fluorescence microscopy respectively. In turn, dermatophytes were isolated from 90·2 and 71·4% of these samples. The pan-dermatophyte primers in real-time PCR assay facilitated detection in 85·3 and 82·9% of the symptomatic and asymptomatic dermatophytoses respectively. Additionally, species-specific PCR assays were positive in 70·7 and 37·1% of these samples. The MALDI-TOF MS analysis yielded positive results consistent with conventional techniques in 97·2 and 72% of symptomatic and asymptomatic infections respectively., Conclusions: Our study revealed that there is no universal diagnostic method that would be ideal in each of the cases considered. Nonetheless, conventional techniques are still the most effective and reliable tools for mycological diagnostics., Significance and Impact of the Study: Dermatologists and veterinarians have difficulties in making a diagnosis of dermatophytoses based only on observed symptoms of fungal infections, as they mimic symptoms of other dermatoses. In this context, a comparative analysis of the results of diagnostics performed using conventional methods and new technologies are crucial for implementing these pioneer methods into routine laboratory practice., (© 2020 The Society for Applied Microbiology.)

Invasive fungal sinusitis is a morbid pathology that typically affects immunocompromised patients and may quickly progress to fulminant disease. The purpose of this study was to measure the sensitivity and specificity of touch preparation of nasal debridement specimens as a rapid diagnostic tool for invasive fungal sinusitis. A retrospective chart review was performed of 22 patients undergoing nasal debridement due to suspicion for invasive fungal sinusitis over a 10-year period. Thirteen patients had touch preparation of nasal specimens followed by routine histologic processing; two of these patients underwent 2, and 1 patient had 3 separate debridements, for a total of 17 touch preparations performed. The sensitivity and specificity of touch preparation were calculated by correlating the initial results with the presence of fungal invasion on final pathologic analysis. The sensitivity of touch preparation was 56% (95% confidence interval [CI]: 0.23-0.85), specificity was 100% (95% CI: 0.60-1.00), positive predictive value was 100% (95% CI: 0.46-1.00), and negative predictive value was 67% (95% CI: 0.35-0.89). This procedure may be a useful adjunct in situations requiring rapid diagnosis of invasive fungal sinusitis but should not be used as the sole criteria for determining the need for surgical intervention.

During bloodstream infections, rapid adaptation of empirical treatment according to the microorganism identified is essential to decrease mortality. The aim of the present study was to assess the microbiological performances of a new rapid version of the Sepsityper® kit (Bruker Daltonics) allowing identification of bacteria and yeast by MALDI-TOF mass spectrometry directly from positive blood cultures in 10 min and of the specific MBT-Sepsityper module for spectra analysis, designed to increase identification performance. Identification rates were determined prospectively on 350 bacterial and 29 fungal positive blood cultures, and compared to conventional diagnostic method. Our rapid diagnosis strategy (Rapid Sepsityper® protocol: one spot with and one without formic acid extraction step) combined to MBT-Sepsityper module provided 65.4%, 78.9% and 62% reliable identification to the species level of monomicrobial positive blood cultures growing respectively Gram-positive, Gram-negative bacteria or yeast. Importantly, identification rates of Gram-positive bacteria were higher in anaerobic than in aerobic bottles (77.8% vs 22.2%; p = 0.004), if formic acid extraction step was performed (60.8% vs 39.2%; p = 1.8e -6 ) and if specific MBT-Sepsityper module was used (76.2% vs 61.9%, p = 0.041) while no significant differences were observed for Gram-negative bacteria. For yeasts identification, formic acid extraction step improved rapid identification rate by 37.9% while the specific MBT-Sepsityper module increased overall performances by 38%, providing up to 89.7% reliable identification if associated with the standard Sepsityper® protocol. These performances, associated with a reduce turnaround time, may help to implement a rapid identification strategy of bloodstream infections in the routine workflow of microbiology laboratories.

Candida nivariensis is a cryptic fungal species classified within the Candida glabrata complex. It was described for the first time in 2005 by the means of DNA sequencing. We report a rare case of C. nivariensis deep-seated infection occurring in a 77-year-old man hospitalized for cysto-prostatectomy. Phenotypic testing based on the direct examination and the macroscopic features of the in vitro culture initially suggested C. glabrata species, while MALDI-TOF mass spectrometry enables correct identification. The isolate was found resistant to fluconazole, like in almost 20% of the reported cases. Herein, we present our practical strategy to reliably characterize this rare cryptic species. To date, MALDI-TOF mass spectrometry-based analysis showed very good results for such a purpose., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Background and Objectives: Matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry (MALDI-TOF MS) is a novel technique for identifying dermatophytes. This study aimed to detect the limitation of MALDI-TOF MS applied to dermatophytes., Methods: A total of 113 DNA-sequenced dermatophyte isolates preserved at the Research Center for Medical Mycology of Peking University were selected for this study. Forty-two isolates were selected as reference strains used to create a supplementary database. Seventy-one isolates (Trichophyton rubrum series, T benhamiae series, T mentagrophytes series species and T schoenleinii) were used to evaluate the suitability of the MALDI-TOF MS Biotyper system. MALDI Biotyper 4.0 software was employed to construct the main spectrum profile (MSP) dendrograms., Results: Correct identification rates at the species and genus levels were 90.1% and 91.5%, respectively, using Bruker Filamentous Fungi Library 1.0 combined with the novel database. The MSP dendrogram of the T rubrum series showed unambiguous separation of T rubrum and T violaceum and that of the T benhamiae series distinguished T verrucosum, T benhamiae and T erinacei. Conversely, the MSP dendrogram of the T mentagrophytes series did not successfully distinguish T mentagrophytes, T interdigitale and T tonsurans., Conclusion: MALDI-TOF MS showed good performance in the identification and delineation of the T rubrum series and T benhamiae series, but showed poor performance in T mentagrophytes series., (© 2020 Wiley-VCH GmbH.)

Candida albicans is a common mucosal colonizer, as well as a cause of lethal invasive fungal infections. The major predisposing factor for invasive fungal disease is a compromised immune system. One component of the host immune response to fungal infection is the activation of the inflammasome, a multimeric protein complex that is critical for regulating host pro-inflammatory responses. Here, we describe methods for investigating the interactions between C. albicans and host macrophages, with a focus on the inflammasome. C. albicans isolates differ in the degree to which they activate the inflammasome due to differences in internalization, morphogenic switching, and inflammasome priming. Therefore, we include protocols for identifying these factors. This simple in vitro model can be used to elucidate the contributions of specific C. albicans strains or mutants to different aspects of interactions with macrophages. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Measuring inflammasome priming in response to Candida albicans Basic Protocol 2: Measuring inflammasome activation in response to Candida albicans Support Protocol: Controlling for phagocytosis., (© 2020 Wiley Periodicals LLC.)

A shift to Candida non-albicans infections has been noted during the last years, and the emergence of multi-resistant Candida auris has complicated their management. The aim of this study was first to compare the performance of the novel chromogenic medium CHROMagar™ Candida Plus (CHROMagar, France) with CHROMagar™ Candida (Becton Dickinson, Germany) for the presumptive identification of Candida species; and then, to evaluate its utility in the detection of C. auris in surveillance samples. CHROMagar™ Candida Plus showed a good performance compared with the reference medium CHROMagar™ Candida. Sensitivity and specificity were 100% in both media for tested species at 48 h of incubation, except for Candida glabrata and Candida lusitaniae. Furthermore, the new medium allows a reliable presumptive identification of C. auris, as a new specific color for this species is assigned (light blue with a blue halo), obtaining a sensitivity and specificity of 100% at 36 h of incubation., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The order Mucorales is an ancient group of fungi classified in the subphylum Mucoromycotina. Mucorales are mainly fast-growing saprotrophs that belong to the first colonizers of diverse organic materials and represent a permanent part of the human environment. Several species are able to cause human infections (mucormycoses) predominantly in patients with impaired immune system, diabetes, or deep trauma. In this review, we compiled 32 reports on community- and hospital-acquired outbreaks caused by Mucorales. The most common source of mucoralean outbreaks was contaminated medical devices that are responsible for 40.7% of the outbreaks followed by contaminated air (31.3%), traumatic inoculation of soil or foreign bodies (9.4%), and the contact (6.2%) or the ingestion (6.2%) of contaminated plant material. The most prevalent species were Rhizopus arrhizus and R. microsporus causing 57% of the outbreaks. The genus Rhizomucor was dominating in outbreaks related to contaminated air while outbreaks of Lichtheimia species and Mucor circinelloides were transmitted by direct contact. Outbreaks with the involvement of several species are reported. Subtyping of strains revealed clonality in two outbreaks and no close relation in two other outbreaks. Based on the existing data, outbreaks of Mucorales can be caused by heterogeneous sources consisting of different strains or different species. Person-to-person transmission cannot be excluded because Mucorales can sporulate on wounds. For a better understanding and prevention of outbreaks, we need to increase our knowledge on the physiology, ecology, and population structure of outbreak causing species and more subtyping data.

The yeast Candida glabrata has emerged, second only to Candida albicans, to be one of the most frequently isolated fungi in clinical specimen from human. Its frequent resistance towards azole antifungal drugs and the high capacity to form biofilms on indwelling catheters of individual isolates render it an often difficult to treat pathogen. Hence, there is a notably increasing scientific and clinical interest in this species. This has led to the development of a variety of molecular tools for genetic modification, strain collections, and last but not least different approaches to analyse the population structure among isolates of different geographical and clinical contexts. Often, these are used to study correlations (or the absence thereof) with different pathogenicity, virulence, or drug resistance traits. Three molecular methods have been used to type within the C. glabrata population on a genetic level by multiple studies: multi-locus sequence typing, microsatellite length polymorphisms, and clustering of whole-genome sequencing data, and these are subject of this review.

To determine fungal species distribution of interdigital intertrigo among seafarers in Dakar, Senegal, a cross-sectional study was carried out from May to August 2017 among seafarers clinically diagnosed with interdigital intertrigo. A questionnaire was filled to each patient before sampling the affected folds and transporting to Aristide Le Dantec University Hospital where mycological analyses were realized. Species identification by MALDI-TOF MS was performed in Marseille, France. In total, 169 men (21-66 years) were included. Few of them (3%) had a high level of education and the duration of the mycosis exceed 10 years for 88% of patients. Direct microscopic examination (ME) was positive in 34.3%. Among samples with positive ME, 58.6% had positive culture. An overall incidence of 30.2% was found. Patients with confirmed cases aged between 28 and 66 years. Among them, those between 36-50 years were predominant (52.9%). Those with a high level of education were less representative (2%). For 52.1% of patients, the duration of the mycosis was superior to 10 years. Furthermore, 57% of cases were significantly associated with other types of tinea pedis and/or onychomycosis (P=0.03). Culture was positive in 23.7% isolating 43 strains successfully identified at the species level by MALDI-TOF MS for 31 isolates: 20 Candida and 11 dermatophytes. The rest was identified only at the genus level belonged to Fusarium. In definitive, MALDI-TOF MS could be a useful tool for routine and fast identification of dermatophytes, yeasts and NDFF in clinical mycology laboratories., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Background: The number of invasive Candida infections has significantly increased in recent decades. For the successful treatment of fungal infections, rapid identification at the species level, particularly in polyfungal infections, is a key factor. In this study, four commercially available chromogenic media, CandiSelect™ 4 (CS4), chromID™ Candida Agar (CCA), BBL™ CHROMagar™ Candida Medium (BBL) and Brilliance™ Candida Agar (BCA) were evaluated for Candida identification., Material/methods: Overall, 181 bronchial secretion samples from intensive care patients were analysed prospectively. In addition, 18 primarily sterile materials, previously tested positive for Candida, were investigated retrospectively. All samples were cultured as recommended by the manufacturer and visually inspected after 24 and 48 hours by three independent investigators. As a control, colonies were identified by MALDI-TOF MS. Specificity and sensitivity were determined for C albicans identification prospectively., Results: CS4 and BCA showed the best overall consensus with the identification results reached by MALDI-TOF MS for Candida albicans and species. A clear differentiation between the species could be ascertained via easily identifiable, species-specific coloration in contrast to BBL and CCA. Sensitivity for C albicans (n = 73) identification varied between 32% (BCA) and 69% (CS4 and CCA) after 24 hours and 68% (BBL) and 82% (BCA) after 48 hours incubation, while specificity ranged between 62% (BBL) and 81% (CCA) after 24 hours and 82% (BBL) and 85% (CS4) after 48 hours., Conclusion: CS4 and BCA are recommended for routine identification of Candida species in human samples., (© 2020 Blackwell Verlag GmbH.)

There are approximately 1 million cryptococcal infections per year among HIV+ individuals, resulting in nearly 625,000 deaths. Cryptococcus neoformans and Cryptococcus gattii are the two most common species that cause human cryptococcosis. These two species of Cryptococcus have differences in pathogenicity, diagnosis, and treatment. Cryptococcal infections are usually difficult to identify because of their slow growth in vitro. In addition, the long detection cycle of Cryptococcus in clinical specimens makes the diagnosis of Cryptococcal infections difficult. Here, we used positively charged silver nanoparticles (AgNPs + ) as a substrate to distinguish between C. neoformans and C. gattii in clinical specimens directly via surface-enhanced Raman scattering (SERS) and spectral analysis. The AgNPs + self-assembled on the surface of the fungal cell wall via electrostatic aggregation, leading to enhanced SERS signals that were better than the standard substrate negatively charged silver nanoparticles (AgNPs). The SERS spectra could also be used as a sample database in the multivariate analysis via orthogonal partial least-squares discriminant analysis. This novel SERS detection method can clearly distinguish between the two Cryptococcus species using principal component analysis. The accuracy of the training data and test data was 100% after a tenfold crossover validation.

The aim of this study was to evaluate the performance of the DiaSorin Molecular PJ-CMV multiplex real-time PCR (PJ-CMV PCR) assay (DiaSorin Molecular LLC, USA) in bronchoalveolar lavage (BAL) samples compared to direct immunofluorescence assay (IFA) for the detection of Pneumocystis jirovecii and assess CMV and P. jirovecii co-infection rate in immunosuppressed patients with suspected pneumonia. A total of 125 BAL samples from immunosuppressed patients submitted for PJP-IFA were tested. Surplus samples were saved and further tested by using the PJ-CMV PCR assay. Among the 125 samples, P. jirovecii was detected in 31.2% (39/125) and in 40% (50/125) of the specimens using IFA and PJ-CMV PCR respectively. Eleven of the PJ-CMV PCR positive samples were negative by direct IFA for P. jirovecii. All samples positive by direct IFA were also positive by PJ-CMV PCR. Using the direct IFA as a gold standard, the PJ-CMV PCR sensitivity, specificity, positive predictive value and negative predictive value for detection of P. jirovecii were 100%, 87.2%, 78% and 100%, respectively. However, after reviewing the clinical diagnosis, the specificity and PPV increased to 100%. Of the 50 P. jirovecii samples positive by PJ-CMV PCR, 18 (36%) were also positive for CMV by the PJ-CMV PCR. The co-infection rate was found to be 37.5% (6/16) and 35.2% (12/34) in HIV infected and non-HIV infected patients. This study indicated that the DiaSorin Molecular PJ-CMV multiplex real-time PCR assay has higher sensitivity than direct IFA for detection of P. jirovecii and provides rapid detection of PJ and CMV infection in BAL samples., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Background: Candida parapsilosis is the second or third most frequently isolated Candida species related to nosocomial infections, even overtaking Candida albicans in some hospitals. C. parapsilosis constitutes a complex of closely related species: Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis. Accurate detection of these species is of importance, as the incidence of C. orthopsilosis has been reported to surpass that of Candida krusei., Objective: To evaluate the diagnostic utility of two PCR-RFLP methods targeting the SADH and FKS1 genes and to determine the prevalence of cryptic species in 96 bloodstream isolates of C. parapsilosis from 93 patients., Methods: Restriction patterns of the SADH and FKS1 genes were analysed, and sequencing of the D1/D2 regions of the ribosomal RNA was used to evaluate the reliability of both PCR-RFLP methods., Results: In our study, 77 C. parapsilosis sensu stricto, 13 C. orthopsilosis and five C. metapsilosis were identified by sequencing. Both PCR-RFLP methods demonstrated strong agreement with D1/D2 sequencing in the identification of C. parapsilosis and C. orthopsilosis, while both methods were unable to identify the C. metapsilosis isolates. Moreover, unexpected restriction patterns were observed for two isolates on SADH PCR-RFLP and for four isolates on FKS1 PCR-RFLP. Mixed bloodstream infections of C. parapsilosis sensu stricto and C. orthopsilosis were detected for three patients, for which differential growth characteristics were observed., Conclusion: The molecular method chosen for identification could have an impact on determination of the real prevalence of C. metapsilosis in candidaemia, and mixed fungaemias can remain undetected., (© 2020 Blackwell Verlag GmbH.)

Background: To report a case of fungal keratitis caused by Colletotrichum gloeosporioides in an east coast city of China, which are rare pathogens that cause fungal keratitis in humans., Methods: A 52-year-old man whose right eye was injured by a branch of an apple tree during farm work was referred to our Hospital. He was examined by Slit-lamp and the HRT II-RCM confocal scanning microscope, thus suggesting filamentous. Orneal scrapings were acquired and then inoculated into Sabouraud medium incubated at 28°C and 37°C. In vitro antifungal susceptibility tests were performed following the CLSI M38-A2 for Filamentous Fungi. Surgical intervention was advised because the abscess in the anterior chamber of the right eye was not completely absorbed., Results: The Colletotrichum gloeosporioides isolate was identified by MALDI-TOF mass spectrometry and the BLAST after DNA sequencing of the amplified internal transcribed spacer (ITS) in rRNA. The patient's eye condition is under control and the patient's vision remains at the level of light perception (LP)., Conclusions: We report the rare keratitis caused by C. gloeosporioides in eastern China, which has not been published. Suddenly ocular trauma and old surgical intervention may be the risk factors associated with Colletotrichum keratitis., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Onychomycosis is considered a fungal nail infection caused mainly by dermatophytes, yeasts and non-dermatophyte molds including dematiaceous fungi. Onychomycosis caused by non-dermatophyte molds is a health problem in the medical environment as the patients frequently return to outpatient clinics seeking new therapeutic modalities. Here, we report the first case of onychomycosis caused by a black fungus, Neoscytalidium novaehollandiae, in the right hand finger nail of a 52-year-old Iranian female with no history of immunodeficiency and underlying disease. The pattern of nail involvement was recognized as total dystrophic onychomycosis. Examination of nail scrapings with potassium hydroxide revealed brown, septate and branching sub-hyaline to dark-colored hyphae. The black fungus isolated in culture was identified as Neoscytalidium novaehollandiae by molecular analysis. The patient received oral terbinafine plus ciclopirox nail lacquer twice a week and began responding to the treatment three months after initial antifungal therapy. Additional four weeks' use of terbinafine plus ciclopirox nail lacquer completely resolved the clinical manifestations of onychomycosis. After four months, both microscopy and culture were negative., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Sporotrichosis is considered a neglected disease of humans and animals in many regions of the world and is the most frequent implantation mycosis in Latin America., Objectives: To illustrate the zoonotic importance of the disease, describing a case involving a veterinarian and an infant that acquired the disease from a domestic cat and to describe, genotype and characterize these new isolates., Methods: Direct examination of tissue samples from the two patients and feline lesions revealed the presence of Sporothrix yeast-like organisms. Fungal cultures and molecular identification of the strains were performed. Since antifungal susceptibility data of animal-borne isolates are scarce, the in vitro susceptibility testing by a microdilution reference method was determined against azoles, amphotericin B and terbinafine., Results: Fungal culture and sequence analysis of the ITS region of rDNA and calmodulin and β-tubulin genes confirmed the diagnosis and the causative agent as Sporothrix brasiliensis. In all cases, terbinafine was the most active drug, followed by posaconazole, itraconazole and voriconazole; the least active drugs were amphotericine B and fluconazole. Lack of clinical response in the veterinarian and in the infant to itraconazole and potassium iodide, respectively was observed., Conclusions: This study contributed to the molecular epidemiology of Sporothrix species in Argentina and the characterization of the in vitro susceptibility pattern of S. brasiliensis isolates recovered from a cat and two humans involved in this case of zoonotic sporotrichosis. Bearing in mind the "One Health" concept, the experience described in the present study highlights the need for future strategies for sporotrichosis treatment, control and prevention., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Filamentous fungi are one of the main causes of food losses worldwide and their ability to produce mycotoxins represents a hazard for human health. Their correct and rapid identification is thus crucial to manage food safety. In recent years, MALDI-TOF emerged as a rapid and reliable tool for fungi identification and was applied to typing of bacteria and yeasts, but few studies focused on filamentous fungal species complex differentiation and typing. Therefore, the aim of this study was to evaluate the use of MALDI-TOF to identify species of the Aspergillus section Flavi, and to differentiate Penicillium roqueforti isolates from three distinct genetic populations. Spectra were acquired from 23 Aspergillus species and integrated into a database for which cross-validation led to more than 99% of correctly attributed spectra. For P. roqueforti, spectra were acquired from 63 strains and a two-step calibration procedure was applied before database construction. Cross-validation and external validation respectively led to 94% and 95% of spectra attributed to the right population. Results obtained here suggested very good agreement between spectral and genetic data analysis for both Aspergillus species and P. roqueforti, demonstrating MALDI-TOF applicability as a fast and easy alternative to molecular techniques for species complex differentiation and strain typing of filamentous fungi., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Fifty four Trichoderma strains were isolated from soil samples collected from garlic and onion crops in eight different sites in Brazil and were identified using phylogenetic analysis based on combined ITS region, tef1-α, cal, act and rpb2 sequences. The genetic variability of the recovered Trichoderma species was analysed by AFLP and their phenotypic variability determined using MALDI-TOF. The strain clusters from both typing techniques coincided with the taxonomic determinations made from phylogenetic analysis. The phylogenetic analysis showed the occurrence of Trichoderma asperellum, Trichoderma asperelloides, Trichoderma afroharzianum, Trichoderma hamatum, Trichoderma lentiforme, Trichoderma koningiopsis, Trichoderma longibrachiatum and Trichoderma erinaceum, in the soil samples. We also identified and describe two new Trichoderma species, both in the harzianum clade of section Pachybasium, which we have named Trichoderma azevedoi sp. nov. and Trichoderma peberdyi sp. nov. The examined strains of both T. azevedoi (three strains) and T. peberdyi (12 strains) display significant genotypic and phenotypic variability, but form monophyletic clades with strong bootstrap and posterior probability support and are morphologically distinct from their respective most closely related species., Competing Interests: The authors have declared that no competing interests exist.

Background: The identification of foodborne pathogenic bacteria types plays a crucial role in food safety and public health. In consideration of long culturing times, tedious operations and the desired specific recognition elements in conventional methods, the alternative fluorescent sensor arrays can offer a high-effective approach in bacterial identification by using multiple cross-reactive receptors. Herein, we achieve this goal by constructing an upconversion fluorescent sensor array based on anti-stokes luminogens featuring a series of functional lanthanide-doped upconversion nanoparticles (UCNPs) with phenylboronic acid, phosphate groups, or imidazole ionic liquid. The prevalent spotlight effect of microorganism and the electrostatic interaction between UCNPs and bacteria endow such sensor array an excellent discrimination property., Results: Seven common foodborne pathogenic bacteria including two Gram-positive bacteria (Staphylococcus aureus and Listeria monocytogenes) and five Gram-negative bacteria (Escherichia coli, Salmonella, Cronobacter sakazakii, Shigella flexneri and Vibrio parahaemolyticus) are precisely identified with 100% accuracy via linear discriminant analysis (LDA). Furthermore, blends of bacteria have been identified accurately. Bacteria in real samples (tap water, milk and beef) have been effectively discriminated with 92.1% accuracy., Conclusions: Current fluorescence sensor array is a powerful tool for high-throughput bacteria identification, which overcomes the time-consuming bacteria culture and heavy dependence of specific recognition elements. The high efficiency of whole bacterial cell detection and the discrimination capability of life and death bacteria can brighten the application of fluorescence sensor array.

Background: Invasive candidiasis is the most important health-care-associated fungal infection worldwide. In the last two decades, the cause of the increase of fungal infections is immunosuppression or serious underlying diseases. Additionally, Rhodotorula species, Blastoschizomyces capitatus, and Trichosporon species are emerging as important human pathogens in immunocompromised patients with hematological malignancy., Methods: Between January 2012 and January 2018, a total of 603 fungal organisms were isolated from blood culture samples and included in the study. All of the isolates were identified by using standard mycological methods, MALDI TOF MS system, and the Phoenix system. Sequence analysis was performed on yeasts that could not be definitively identified by using SMM and incompatible according to the results with Phoenix and MALDI-TOF MS analysis., Results: 603 fungal isolates including 594 Candida spp. and 9 other yeasts like species were analyzed. C. albicans was the most frequently isolated species. The results of identification by conventional methods and MALDI TOF MS were compared to the results of the Phoenix system. The observed concordance was 99.2%. The compatibility with other systems of the Phoenix system was 100%, 100%, 97.3%, 100%, and 96.9% for C. albicans, C. parapsilosis, C. tropicalis, C. glabrata, and C. krusei, respectively., Conclusions: The BD Phoenix system was found to be a simple, reliable, and effective method to identify the main species of the genus Candida in our study.

With the continual improvement in high-throughput sequencing technology and constant updates to fungal reference databases, the use of amplicon-based DNA markers as a tool to reveal fungal diversity and composition in various ecosystems has become feasible. However, both primer selection and the experimental procedure require meticulous verification. Here, we computationally and experimentally evaluated the accuracy and specificity of three widely used or newly designed internal transcribed spacer (ITS) primer sets (ITS1F/ITS2, gITS7/ITS4 and 5.8S-Fun/ITS4-Fun). In silico evaluation revealed that primer coverage varied at different taxonomic levels due to differences in degeneracy and the location of primer sets. Using even and staggered mock community standards, we identified different proportions of chimeric and mismatch reads generated by different primer sets, as well as great variation in species abundances, suggesting that primer selection would affect the results of amplicon-based metabarcoding studies. Choosing proofreading and high-fidelity polymerase (KAPA HiFi) could significantly reduce the percentage of chimeric and mismatch sequences, further reducing inflation of operational taxonomic units. Moreover, for two types of environmental fungal communities, plant endophytic and soil fungi, it was demonstrated that the three primer sets could not reach a consensus on fungal community composition or diversity, and that primer selection, not experimental treatment, determines observed soil fungal community diversity and composition. Future DNA marker surveys should pay greater attention to potential primer effects and improve the experimental scheme to increase credibility and accuracy., (© 2019 John Wiley & Sons Ltd.)

Objective: Patients with primary sclerosing cholangitis (PSC) were previously shown to display a bacterial gut dysbiosis but fungal microbiota has never been examined in these patients. The aim of this study was to assess the fungal gut microbiota in patients with PSC., Design: We analysed the faecal microbiota of patients with PSC and concomitant IBD (n=27), patients with PSC and no IBD (n=22), patients with IBD and no PSC (n=33) and healthy subjects (n=30). Bacterial and fungal composition of the faecal microbiota was determined using 16S and ITS2 sequencing, respectively., Results: We found that patients with PSC harboured bacterial dysbiosis characterised by a decreased biodiversity, an altered composition and a decreased correlation network density. These alterations of the microbiota were associated with PSC, independently of IBD status. For the first time, we showed that patients with PSC displayed a fungal gut dysbiosis, characterised by a relative increase in biodiversity and an altered composition. Notably, we observed an increased proportion of Exophiala and a decreased proportion of Saccharomyces cerevisiae . Compared with patients with IBD and healthy subjects, the gut microbiota of patients with PSC exhibited a strong disruption in bacteria-fungi correlation network, suggesting an alteration in the interkingdom crosstalk., Conclusion: This study demonstrates that bacteria and fungi contribute to gut dysbiosis in PSC., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

The contamination of foods and beverages by fungi is a severe health hazard. The rapid identification of fungi species in contaminated goods is important to avoid further contamination. To this end, we developed a fungal discrimination method based on the bioimage informatics approach of colony fingerprinting. This method involves imaging and visualizing microbial colonies (referred to as colony fingerprints) using a lens-less imaging system. Subsequently, the quantitative image features were extracted as discriminative parameters and subjected to analysis using machine learning approaches. Colony fingerprinting has been previously found to be a promising approach to discriminate bacteria. In the present proof-of-concept study, we tested whether this method is also useful for fungal discrimination. As a result, 5 fungi belonging to the Aspergillus, Penicilium, Eurotium, Alternaria, and Fusarium genera were successfully discriminated based on the extracted parameters, including the number of hyphae and their branches, and their intensity distributions on the images. The discrimination of 6 closely-related Aspergillus spp. was also demonstrated using additional parameters. The cultivation time required to generate the fungal colonies with a sufficient size for colony fingerprinting was less than 48 h, shorter than those for other discrimination methods, including MALDI-TOF-MS. In addition, colony fingerprinting did not require any cumbersome pre-treatment steps prior to discrimination. Colony fingerprinting is promising for the rapid and easy discrimination of fungi for use in the ensuring the safety of food manufacturing., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Cranberry fruit rot (CFR) is arguably one of the most limiting factors of cranberry ( Vaccinium macrocarpon ) production throughout its growing areas. The disease is caused by a group of closely related fungi that require identification using long and cumbersome steps of isolation and microscopic observations of structural features. The objective of this study was to develop a molecular assay to simultaneously detect and discriminate 12 of the most important fungal species reported to be pathogenic on cranberry fruit to facilitate the diagnosis of CFR. As the first approach, internal transcribed spacers and large subunit regions of all fungi were sequenced and confirmed with sequences available in the NCBI database. These data were used to develop primers able to differentiate seven of the 12 species. The five remaining species, including three in the Phacidiaceae family and two in the Glomerellaceae family, were differentiated on the basis of a more discriminant marker, the translation elongation factor 1-α. Two PCR reactions were optimized to clearly delineate the 12 species. The multiplex test was first validated using pure fungal cultures; it was subsequently validated using fruit collected in cranberry beds in eastern Canada. In the latter case, the test was rigorous enough to clearly discriminate the fungal pathogens from contaminants. Within the tested samples, Physalospora vaccinii and Coleophoma empetri were most commonly found. This molecular test offers scientists, diagnosticians, and growers a powerful tool that can rapidly and precisely identify fungi causing CFR so they can implement appropriate control methods.

Backgound: Conventional diagnosis of invasive fungal disease from blood cultures is often notoriously delayed and inadequately sensitive. We aimed to develop a universal primers-based polymerase chain reaction (PCR) assay and restriction fragment length polymorphisms (RFLP) for rapid identification of invasive fungal disease (IFD)., Methods: We evaluated 16 clinical fungal species using a combination of PCR assays with 3 different restriction endonucleases targeting various internal transcribed spacer (ITS) regions and high resolution melting analysis (HRMA). Serial samples from 75 patients suspected to have IFD were analyzed for clinical verification., Results: We have designed a universal PCR capable of amplifying a portion of the 18S rRNA gene of 16 clinically important fungal species. The restriction patterns of most PCR products generated by EcoRI or double digested by ClaI and AvaI were different, except Aspergillus niger and Aspergillus flavus had a similar pattern, and Aspergillus fumigatus and Aspergillus terreus had a similar pattern. All these species had a unique melting curve shape using the HRMA. Both HRMA and universal PCR had adequate sensitivity, and all sixteen reference fungal species can be clearly distinguished by the universal PCR-RFLP-HRMA assay. With a reference library of 176 clinically relevant fungal strains, and 75 clinical samples from patients with suspicious IFD were tested, our assay identified 100% and 61.1% of isolates from the reference library and clinical samples, respectively., Conclusions: Universal PCR and RFLP coupled with HRMA could be a highly discriminative and useful molecular diagnostic that could enhance the current diagnostic, treatment, and surveillance methods of invasive fungal disease., (Copyright © 2019. Published by Elsevier B.V.)

An increasing number of infections originating from probiotic use are reported worldwide, with the majority of such cases caused by the yeast Saccharomyces 'boulardii', a subtype of S. cerevisiae. Reliably linking infectious cases to probiotic products requires unequivocal genotyping data, however, these techniques are often time-consuming and difficult to implement in routine diagnostics. This leads to a widespread lack of genetic data regarding the origin of Saccharomyces infections. We propose a quick and reliable PCR-based protocol for the identification of S. 'boulardii' based on a combined analysis of interdelta fingerprinting and microsatellite typing. By applying various typing methods and our proposed method to the clinical yeast collection of a Hungarian hospital we show that probiotic origin is common among clinical Saccharomyces, and that the new multiplex method enables rapid and unequivocal identification of probiotic yeast infections. This method can be applied for the identification of yeast infection sources, helping decisions on probiotic use., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Enterocytozoon bieneusi is one of the most frequently diagnosed Microsporidia of humans and most animals. However, there is no information on E. bieneusi infection of pigs in Tibet and Henan, China. In this study, 1,190 fecal samples were collected from pigs in Tibet and Henan and screened for the presence of E. bieneusi. The overall prevalence of E. bieneusi infection was 54.2% (645/1,190), with differences in prevalence observed among geographical areas, ages, and pig breeds. Moreover, 10 E. bieneusi genotypes were identified based on internal transcribed spacer region genotyping, including eight known genotypes (EbpC, EbpA, CHG19, CHC5, Henan-III, I, D, and H) and two novel genotypes (XZP-I and XZP-II). Multilocus sequence typing revealed 18, 7, 17, and 13 genotypes at minisatellite/microsatellite loci MS1, MS3, MS4, and MS7, respectively. Strong linkage disequilibrium (LD) and few numbers of recombination events, suggest a clonal structure of the E. bieneusi population examined in this study. The low pairwise genetic distance (F ST ) and gene flow (Nm) values indicated limited gene flow in the E. bieneusi population from different hosts, with phylogenetic, structure, and median-joining network analyses all indicating the existence of host and geographical isolation. The identification of isolates belonging to nine human-pathogenic genotypes indicates that pigs play an important role in the dissemination of E. bieneusi, improving our present understanding of E. bieneusi epidemiology in the studied region., (© 2019 International Society of Protistologists.)