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Introduction: Advances in diagnostic technologies, particularly Point-of-Care Diagnostics (POCDs), have revolutionized clinical practice by providing rapid, user-friendly, and affordable testing at or near the patient's location. POCDs have been increasingly introduced in medical mycology and hold promise to improve patient outcomes in a variety of important human fungal diseases., Areas Covered: This review focuses on validated POCDs, particularly lateral flow assays (LFAs), for various fungal diseases. Additionally, we discuss emerging innovative techniques such as body fluid analysis, imaging methods, loop-mediated isothermal amplification (LAMP), microfluidic systems, clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics, and the emerging role of artificial intelligence., Expert Opinion: Compact and user-friendly POCDs have been increasingly introduced in medical mycology, and some of these tests (e.g. Cryptococcus and Histoplasma antigen LFAs) have become mainstream diagnostics, while others, such as LFA in invasive aspergillosis show promise to become part of our routine diagnostic armamentarium. POCDs offer immense benefits such as timely and accurate diagnostic results, reduced patient discomfort, and lower healthcare costs and might contribute to antifungal stewardship. Integrated fluidics combined with microtechnology having multiplex capabilities will be pivotal in medical mycology.

Due to large outbreaks observed worldwide, Candida auris has emerged as a major threat to healthcare facilities. To prevent these phenomena, a systematic screening should be performed in patients transferred from regions where the pathogen is highly endemic. In this study, we recorded and analyzed French mycologists' current knowledge and practice regarding C. auris screening and diagnosis. Thirty-six centers answered an online questionnaire. Only 11 (30.6 %) participants were aware of any systematic screening for C. auris for patients admitted to their hospital. In the case of post-admission screening, axillae/groins (n = 21), nares (n = 7), rectum (n = 9), and mouth (n = 6) alone or various combinations were the body sites the most frequently sampled. Only six centers (8.3 %) reported using a commercially available plate allowing the differentiation of C. auris colonies from that of other Candida species, while five laboratories (13.8 %) had implemented a C. auris-specific qPCR. Considering the potential impact on infected patients and the risk of disorganization in the care of patients, it is crucial to remember to biologists and clinicians the utmost importance of systematic screening on admission., Competing Interests: Declaration of competing interest The authors declared no conflict of interest, (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: dermatophytoses are a current fungal infection, caused by keratinophilic fungi (dermatophytes) able to invade the nails, hair and skin of humans and animals., Aim: the aim of this study was to establish the epidemiological and mycological profile of dermatophytes isolated in the parasitology-mycology laboratory of the Mohammed VI University Hospital in Oujda., Methods: this is a 48-month retrospective study from January 2019 to December 2022.The study includes samples taken or sent to our parasitology-mycology laboratory for mycological study. A direct examination and culture were performed on each biological specimen. Species identification was based on macroscopic and microscopic colony criteria., Results: in the present report we reviewed 950 mycological samples. Dermatophytes were isolated in 505 (53.15%) cases. The most common infections were tinea unguium (n=353; 69.90%), followed by tinea corporis (n=123; 5.74%) and tinea capitis (n=29; 5.98%). Trichophyton rubrum was the most frequently incriminated species., Conclusion: dermatophytes are the most frequent mycoses in humans. They are generally benign and often develop in a chronic and frequently recurrent pattern. Mycological examination is essential. It confirms the fungal origin and isolates the species responsible, in order to identify the source of contamination and implement an appropriate treatment.

This was a cross-sectional study on the availability of laboratory infrastructure and capacity for the diagnosis of invasive fungal diseases in 24 public hospitals in Vietnam in 2023. Among the hospitals surveyed, 66.7% (14/21) had specialized personnel assigned for mycology testing, and 95.8% (23/24) had a separate microbiology laboratory space. Microscopy and culture methods are available in nearly all laboratories for isolate identification. Antifungal susceptibility testing is only performed for yeasts in 16/24 (66.7%) laboratories. Non-culture methods are hardly used in laboratories. Strengthening local laboratory capacities is essential to meeting health needs in these endemic regions., (© The Author(s) 2024. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Forensic microbiology is a relatively new area of forensic sciences. It considers the potential of microorganisms to be used in criminal investigations. As most studies involve the role of bacteria in fields like post-mortem interval estimation, personal identification or geolocation, the data on the role of fungi is comparatively scarce. Forensic mycology involves the application of fungi and their structures in forensic cases. The aim of this review is the evaluation of the current state of knowledge on fungi associated with human cadavers and their possible role in estimating the time since death. In accordance with the available reports, we focused on the relation between microscopic fungi isolated from human corpses and the cadaver condition e.g., the stage of decomposition. We also emphasised the contrast between the reported methodologies and attempted to standardise research methods in forensic mycology from sample collection to its storage, mycological analysis and identification of the obtained fungal cultures. Moreover, the potential usage of microscopic fungi in criminal cases was discussed based on various case reports., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest. Moreover, they declare that no competing interests exist., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Clinicians may not request fungal culture when it is indicated. For sterile specimens without a specific request for fungal culture, in addition to bacteriology media, we routinely incubated a Sabouraud dextrose agar (SAB) plate for 4 weeks. From 44635 sterile specimens from years 2011 to 2016, 2722 (6.1%) had fungal request. Fungi were identified from 1037 (2.3%) specimens, 292 (0.6%) from specimens with specific fungal request, 574 (1.3%) from bacteriology media, and 171 (0.4%) solely from SAB plate (of 171, 77 were deemed clinically significant and 55 were treated). Relying on request for fungal studies from sterile specimens underdiagnosed fungal infection. Routine fungal culture had a modest incremental yield at a moderately high cost. Lay Summary Routine fungal culture of sterile specimens had a modest incremental yield to routine bacteriology culture and specific fungal request at a moderately high financial cost. [ABSTRACT FROM AUTHOR]

Background: There is an increase in the incidence of human and animal infectious skin diseases of fungal etiology in the world. The main source of infecting the population has become agricultural and stray animals., Aim: The objective of this study was to examine the morphophysiological and microbiological characteristics of pathogenic fungi belonging to the species Trichophyton verrucosum . This species is known to cause diseases in both humans and livestock in Kazakhstan. In addition, the study aimed to assess the feasibility of using the polymerase chain reaction (PCR) method for detecting T. verrucosum . This assessment was conducted in comparison to the outcomes of conventional laboratory diagnostic tests commonly employed for trichophytosis., Methods: The research focused on analyzing 141 samples of pathological material obtained from calves in Almaty, Turkestan, and Kyzylorda regions. These calves exhibited clinical symptoms of skin disease. The study aimed to identify the causative agent using various techniques, including microscopic examination, microbiological methods involving the isolation of pure cultures, and PCR., Results: The detection of the causative agent of dermatophytosis using conventional methods was relatively low, 86% for the microscopic method, and 79% for the microbiological method with the isolation of the culture of the pathogen. Extraction and detection of the genetic material of the causative agent of the disease for PCR was carried out according to the method developed by the authors. The effectiveness of the PCR method was 97.9%, which is significantly higher ( p < 0.05) compared with the diagnostic effectiveness of conventional methods. The PCR method using specific primers identified the causative agent in 98% of cases, which significantly ( p < 0.05) exceeded the results obtained using conventional diagnostic methods. Accordingly, the PCR method had better sensitivity and specificity indicators., Conclusion: The conducted study recommends the method of PCR diagnosis of dermatophytosis for fast and reliable confirmation of the diagnosis of dermatophytosis in humans and animals in Kazakhstan., Competing Interests: The authors declare that there is no conflict of interest.

Background: Conventional testing methods for dermatophytes are time-consuming, and resource limitations in our institution have prompted curtailed access to these diagnostics., Objectives: Evaluation of our hospital's dermatological mycology diagnostic services and similar services nationally., Methods: This was a retrospective observational study on skin, hair and nail mycology samples in our institution comparing twenty five-year periods (2011-2015 and 2016-2021), including analysis of dermatology clinic data and correspondence related to fungal infection. A survey of national public hospitals' laboratories was conducted to evaluate their mycology testing capabilities., Results: The total 5 year test count prior to curtailment was 4851 specimens comprising 90% (n = 4344) from general practice and 6% (n = 290) from dermatology clinics. For the 5 years post curtailment, 64.5% (582/903) of specimens were from dermatology clinics. Dermatology clinic data demonstrated doubling of attendances (for all conditions) and of correspondence related to fungal infection. During this time also, national dermatological antifungal purchasing increased 11%. Ten of 28 Irish public hospital laboratories reported the provision of in-house dermatological mycology testing, and none had routine availability of susceptibility or molecular testing of dermatophytes., Conclusion: This study is the first to report an appraisal of dermatological fungal diagnostic services in Ireland. Insufficient testing capacity implies that patients are either being treated for fungal infection without appropriate diagnostic confirmation, or being left untreated because of the lack of access to diagnostics. The introduction of molecular detection methods and susceptibility systems would enhance testing capabilities and reduce the requirement for the external referral., (© 2022 The Authors. Mycoses published by Wiley-VCH GmbH.)

As the nutrient quality changes, the fractions of ribosomal proteins in the proteome are usually positively correlated with the growth rates due to the auto-catalytic nature of ribosomes. While this growth law is observed across multiple organisms, the relation between the ribosome fraction and growth rate is often more complex than linear, beyond models assuming a constant translation speed. Here, we propose a general framework of protein synthesis considering heterogeneous translation speeds and protein degradations. We demonstrate that the growth law curves are generally environment-specific, e.g., depending on the correlation between the translation speeds and ribosome allocations among proteins. Our predictions of ribosome fractions agree quantitatively with data of Saccharomyces cerevisiae. Interestingly, we find that the growth law curve of Escherichia coli nevertheless appears universal, which we prove must exhibit an upward bending in slow-growth conditions, in agreement with experiments. Our work provides insights on the connection between the heterogeneity among genes and the environment-specificity of cell behaviors., (© 2022. The Author(s).)

The fission yeast, Schizosaccharomyces pombe, is a genetically tractable model organism for cell cycle and molecular genetics research. We describe methods to synchronize S. pombe cultures, and the benefits and limitations of each. Drug-induced synchrony is a convenient method to arrest the cell cycle. An example of the drug hydroxyurea is shown, which arrests cells in S-phase. Environmental modulation through media composition or growth conditions may also be used to synchronize cultures, most commonly with nitrogen depletion to arrest in G1-phase. Finally, examples of temperature-sensitive conditional alleles are shown which arrest the cell cycle at key transition points. Each of these methods must be assessed relative to the desired effect and the process being studied, providing the best synchrony with the fewest off-target effects., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Clinicians may not request fungal culture when it is indicated. For sterile specimens without a specific request for fungal culture, in addition to bacteriology media, we routinely incubated a Sabouraud dextrose agar (SAB) plate for 4 weeks. From 44635 sterile specimens from years 2011 to 2016, 2722 (6.1%) had fungal request. Fungi were identified from 1037 (2.3%) specimens, 292 (0.6%) from specimens with specific fungal request, 574 (1.3%) from bacteriology media, and 171 (0.4%) solely from SAB plate (of 171, 77 were deemed clinically significant and 55 were treated). Relying on request for fungal studies from sterile specimens underdiagnosed fungal infection. Routine fungal culture had a modest incremental yield at a moderately high cost., Lay Summary: Routine fungal culture of sterile specimens had a modest incremental yield to routine bacteriology culture and specific fungal request at a moderately high financial cost., (© The Author(s) 2021. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Invasive infections with emerging yeasts such as Geotrichum , Saprochaete/Magnusiomyces, Trichosporon , and other species are associated with high morbidity and mortality rates. Due to the rarity and heterogeneity of these yeasts, medical mycology has lacked guidance in critical areas affecting patient management. Now, physicians and life scientists from multiple disciplines and all world regions have united their expertise to create the "Global guideline for the diagnosis and management of rare yeast infections: an initiative of the European Confederation of Medical Mycology in cooperation with the International Society for Human and Animal Mycology and the American Society for Microbiology." Recommendations are stratified for high- and low-resource settings and are therefore applicable worldwide. The advantages and disadvantages of various diagnostic methods and treatment options are outlined. This guideline reflects the current best-practice management for invasive rare yeast infections in a range of settings, with the intent of establishing a global standard of care for laboratorians and clinicians alike.

Although asterinaceous fungi have been studied for many years, all previous attempts to isolate, cultivate, and propagate these fungi in vitro have failed. This paper provides the first reports of in vitro isolation of representative strains of species belonging to five fungi from different genera belonging to Asterinales. To confirm if the sequences of DNA obtained from the mycelia are the same obtained in the direct extraction, a phylogenetic analysis of nuc LSU rDNA was performed. This paper reports for the first time the success of in vitro culturing of asterinaceous fungi using the ascospores ejection technique, opening perspectives of studies of genetics, physiology, among other aspects of the biology for this very understudied group of fungi., (Copyright © 2021 Elsevier B.V. All rights reserved.)

A deficiency of the essential macronutrient sulfur leads to stunted plant growth and yield loss; however, an association with a symbiotic fungus can greatly improve nutrient uptake by the host plant. Here, we identified and functionally characterized a high-affinity sulfate transporter from the endophytic fungus Serendipita indica. SiSulT fulfills all the criteria expected of a functional sulfate transporter responding to sulfur limitation: SiSulT expression was induced when S. indica was grown under low-sulfate conditions, and heterologous expression of SiSulT complemented a yeast mutant lacking sulfate transport. We generated a knockdown strain of SiSulT by RNA interference to investigate the consequences of the partial loss of this transporter for the fungus and the host plant (maize, Zea mays) during colonization. Wild-type (WT) S. indica, but not the knockdown strain (kd-SiSulT), largely compensated for low-sulfate availability and supported plant growth. Colonization by WT S. indica also allowed maize roots to allocate precious resources away from sulfate assimilation under low-sulfur conditions, as evidenced by the reduction in expression of most sulfate assimilation genes. Our study illustrates the utility of the endophyte S. indica in sulfur nutrition research and offers potential avenues for agronomically sound amelioration of plant growth in low-sulfate environments., (� American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Candida auris is a novel and major fungal pathogen that has triggered several outbreaks in the last decade. The few drugs available to treat fungal diseases, the fact that this yeast has a high rate of multidrug resistance and the occurrence of misleading identifications, and the ability of forming biofilms (naturally more resistant to drugs) has made treatments of C. auris infections highly difficult. This review intends to quickly illustrate the main issues in C. auris identification, available treatments and the associated mechanisms of resistance, and the novel and alternative treatment and drugs (natural and synthetic) that have been recently reported.

Background: Onychomycosis affects 5.5% of the general population and represents up to 50% of all nail diseases. Diagnosis and pathogen identification are essential in order to plan an adequate treatment. Many diagnostic techniques are available, and however, no solid data regarding comparison between different techniques over a large number of specimens are available to date., Objectives: To compare sensitivity and specificity of direct examination, histopathology and fungal culture in our referral mycology laboratory., Methods: Nail specimens received at the cutaneous pathology and mycology laboratory of the University Hospital Saint-Pierre (Brussels, Belgium) between 1 January and 15 May 2018 were retrospectively analysed. All specimens were submitted to direct examination and culture. In cases of adequate specimen size, histopathology was performed. Fungal culture was considered the gold standard for diagnosis., Results: A total of 2245 nail samples were included in the study. Onychomycosis was diagnosed in 1266 specimens. Sensitivity and positive predictive value were found to be higher for direct examination compared to histopathology, while sensitivity of direct examination was found to be lower. Combined approach with all the three techniques showed the highest rate of positivity, followed by the association of direct examination and histopathology., Conclusions: To our knowledge, this study included the largest number of nail specimens to date, allowing a comparison between direct examination, culture and histopathology. Direct examination showed to be the most performing technique in routine practice. Histopathology represents the most effective option in cases where both specimen size and laboratory resources are adequate. Our paper adds to the literature the 'real-life' experience of the mycology laboratory of a referral centre for nail diseases., (© 2020 Wiley-VCH GmbH.)

Candidaemia is associated with high mortality. In the last few years, several guidelines have been published on the management of Candida bloodstream infection. However, adherence to the practice guidelines has been suboptimal. In order to facilitate and objectively measure the adherence to good practice recommendations, a scoring criterion was published by the European Confederation of Medical Mycology (ECMM). The ECMM Quality (EQUAL) of Clinical Candidaemia Management is an audit tool that comprises of 10 quality indicators. Each quality indicator is allotted between 1 and 3 points. The maximum achievable score is 22 or 19 in patients with or without a central venous catheter, respectively. This paper reviews each of the 10 quality indicators and provides the context for improving quality within the individual domains. The review also suggests areas that are in need of further clarity or areas which merit attention in the future updates of the EQUAL scoring system so that clinicians are able to derive maximum benefit from the audit tool. The EQUAL scoring tool is an important milestone in the quality improvement aspect of the management of candidaemia and contributes to the various components of clinical governance in the management of Candida infection of the bloodstream., (© 2020 Wiley-VCH GmbH.)

We developed a novel culture medium, referred to FastFung medium as suitable for the culture of clinical fungi, including fastidious ones, for both research and diagnostic studies. It is based on Schædler agar supplemented with many essential components for the growth of fastidious fungi. It also contains selective antibacterial agents for the inhibition of contaminant bacteria growth. In this preliminary study, the FastFung medium was compared to the gold standard Sabouraud medium for 98 fungal and 20 bacterial strains. The fungal strain positive culture rate was 100% vs. 95% and the bacterial strain inhibition was 100% vs. 20%, for the FastFung and Sabouraud media, respectively. When compared to the Sabouraud medium on 120 clinical samples, the FastFung medium displayed both a higher fungal colonies count, and a lower culture contamination rate. Storage at 4 °C for 4 weeks did not alter the FastFung culture medium performances for the six isolates of Candida, Cryptococcus, and Penicillium tested. These encouraging results suggest future development of using the FastFung medium in clinical mycology and in mycobiome characterization. Further prospective evaluation aiming at assessing whether implementing the FastFung medium in the routine workflow simplifies and strengthen fungal isolation capacities in the clinical laboratory is warranted., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Radicinin is a phytotoxic fungal dihydropyranopyran-4,5-dione under evaluation for the development of a target-specific bioherbicide for invasive buffelgrass ( Cenchrus ciliaris ) control. It has already demonstrated high toxicity on host plants, low toxicity to native plants and no negative effects on zebrafish embryos. To continue these studies at the whole-plant level there is a need to obtain much larger quantities of radicinin, either by optimizing its large-scale production by fungal fermentation or through its total stereoselective synthesis. A rapid and sensitive HPLC method for quantification of radicinin in complex mixtures has been developed in order to evaluate its production by different Cochliobolus australiensis strains and in different cultural conditions. The analysis proved that radicinin is not produced by all the strains tested and its synthesis is strongly affected by cultural conditions. The HPLC method could be useful in selecting the best fungal source for the production of this promising potential bioherbicide.

Continuous cultures assure the invariability of environmental conditions and the metabolic state of cultured microorganisms, whereas batch-cultured cells undergo constant changes in nutrients availability. For that reason, continuous culture is sometimes employed in the whole transcriptome, whole proteome, or whole metabolome studies. However, the typical method for establishing uniform growth of a cell population, i.e., by limited chemostat, results in the enrichment of the cell population gene pool with mutations adaptive for starvation conditions. These adaptive changes can skew the results of large-scale studies. It is commonly assumed that these adaptations reflect changes in the genome, and this assumption has been confirmed experimentally in rare cases. Here we show that in a population of budding yeast cells grown for over 200 generations in continuous culture in non-limiting minimal medium and therefore not subject to selection pressure, remodeling of transcriptome occurs, but not as a result of the accumulation of adaptive mutations. The observed changes indicate a shift in the metabolic balance towards catabolism, a decrease in ribosome biogenesis, a decrease in general stress alertness, reorganization of the cell wall, and transactions occurring at the cell periphery. These adaptive changes signify the acquisition of a new lifestyle in a stable nonstressful environment. The absence of underlying adaptive mutations suggests these changes may be regulated by another mechanism.

The entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin (Hypocreales: Cordycipitaceae) has been widely studied against a wide range of arthropod pests, including many of medical and veterinary importance. New investigators must sort through a wide array of published methods for the production, harvest, storage, and bioassay methods for this pathogen. Simplified methods for production of conidia using Sabouraud dextrose agar with yeast (SDYA) plates and two conidial harvesting methods are described. Dry harvesting yields conidia that are ready to incorporate into dusts and food baits, but the fungal product includes mycelial debris that can hamper quantification and introduces variable amounts of unwanted bulk. Wet harvesting with filtration produces a cleaner product that is immediately ready for testing in liquid formulations. Examples of bioassays with house flies are presented that include conidia applied topically to the dorsal thorax for dose-mortality assays and conidial suspensions applied to filter paper disks for concentration mortality assays., (Published by Oxford University Press on behalf of Entomological Society of America 2020.)

Global amphibian populations are being decimated by chytridiomycosis, a deadly skin infection caused by the fungal pathogens Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal). Although ongoing efforts are attempting to limit the spread of these infections, targeted treatments are necessary to manage the disease. Currently, no tools for genetic manipulation are available to identify and test specific drug targets in these fungi. To facilitate the development of genetic tools in Bd and Bsal, we have tested five commonly used antibiotics with available resistance genes: Hygromycin, Blasticidin, Puromycin, Zeocin, and Neomycin. We have identified effective concentrations of each for selection in both liquid culture and on solid media. These concentrations are within the range of concentrations used for selecting genetically modified cells from a variety of other eukaryotic species., Competing Interests: The authors have declared that no competing interests exist.

Biofilms are organized communities of microbial cells that promote persistence among bacterial and fungal species. Biofilm formation by host-associated Candida species of fungi occurs on both tissue surfaces and implanted devices, contributing to host colonization and disease. In C. albicans, biofilms are built sequentially by adherence of yeast to a surface, invasion into the substrate, the formation of aerial hyphal projections, and the secretion of extracellular matrix. Measurement of these biofilm-related phenotypes remains highly qualitative and often subjective. Here, we designed an informatics pipeline for quantifying filamentation, adhesion, and invasion of Candida species on solid agar media and utilized this approach to determine the importance of these component phenotypes to C. albicans biofilm production. Characterization of 23 C. albicans clinical isolates across three media and two temperatures revealed a wide range of phenotypic responses among isolates in any single condition. Media profoundly altered all biofilm-related phenotypes among these isolates, whereas temperature minimally impacted these traits. Importantly, the extent of biofilm formation correlated significantly with the additive score for its component phenotypes under some conditions, experimentally linking the strength of each component to biofilm mass. In addition, the response of the genome reference strain, SC5314, across these conditions was an extreme outlier compared to all other strains, suggesting it may not be representative of the species. Taken together, development of a high-throughput, unbiased approach to quantifying Candida biofilm-related phenotypes linked variability in these phenotypes to biofilm production and can facilitate genetic dissection of these critical processes to pathogenesis in the host.

Coccidioidomycosis is an endemic mycosis of the southern United States, Northern Mexico, and South America. Primary cutaneous coccidioidomycosis, despite being a very rare clinical presentation, has shown an increasing incidence. An extensive literature search for cutaneous coccidioidomycosis cases was performed using the OLDMEDLINE, PubMed, Cochrane, LILACS and Google Scholar databases for studies published from January 1927 through December 21, 2019. Forty-two observational studies were included totaling 82 cases of primary cutaneous coccidioidomycosis. Narrative reviews, systematic reviews, and meta-analyses were also included. Additionally, an original case was included. Patients with primary cutaneous coccidioidomycosis share the same geographical and epidemiological characteristics as those with pulmonary or disseminated coccidioidomycosis. Most of the imported cases came from endemic areas. A large portion of cases had prior local skin trauma. Tissue culture is still the leading diagnostic method; nevertheless, molecular techniques such as polymerase chain reaction (PCR) are currently relevant to differentiate between species. First-line treatment consists of azoles; however, it has an excellent prognosis even without treatment. Primary cutaneous coccidioidomycosis should be considered a differential diagnosis of unusual infections or neoformations in any part of the body in resident populations of endemic areas or in patients with a previous history of travel to these areas.

Background: Methylxanthines, including caffeine, theobromine and theophylline, are natural and synthetic compounds in tea, which could be metabolized by certain kinds of bacteria and fungi. Previous studies confirmed that several microbial isolates from Pu-erh tea could degrade and convert caffeine and theophylline. We speculated that these candidate isolates also could degrade and convert theobromine through N-demethylation and oxidation. In this study, seven tea-derived fungal strains were inoculated into various theobromine agar medias and theobromine liquid mediums to assess their capacity in theobromine utilization. Related metabolites with theobromine degradation were detected by using HPLC in the liquid culture to investigate their potential application in the production of 3-methylxanthine., Results: Based on theobromine utilization capacity, Aspergillus niger PT-1, Aspergillus sydowii PT-2, Aspergillus ustus PT-6 and Aspergillus tamarii PT-7 have demonstrated the potential for theobromine biodegradation. Particularly, A. sydowii PT-2 and A. tamarii PT-7 could degrade theobromine significantly (p < 0.05) in all given liquid mediums. 3,7-Dimethyluric acid, 3-methylxanthine, 7-methylxanthine, 3-methyluric acid, xanthine, and uric acid were detected in A. sydowii PT-2 and A. tamarii PT-7 culture, respectively, which confirmed the existence of N-demethylation and oxidation in theobromine catabolism. 3-Methylxanthine was common and main demethylated metabolite of theobromine in the liquid culture. 3-Methylxanthine in A. sydowii PT-2 culture showed a linear relation with initial theobromine concentrations that 177.12 ± 14.06 mg/L 3-methylxanthine was accumulated in TLM-S with 300 mg/L theobromine. Additionally, pH at 5 and metal ion of Fe 2+ promoted 3-methylxanthine production significantly (p < 0.05)., Conclusions: This study is the first to confirm that A. sydowii PT-2 and A. tamarii PT-7 degrade theobromine through N-demethylation and oxidation, respectively. A. sydowii PT-2 showed the potential application in 3-methylxanthine production with theobromine as feedstock through the N-demethylation at N-7 position.

T2Candida enables detection of five Candida species in whole blood within approximately 5 hours. Routinely drawn EDTA blood samples were prospectively stored and tested with T2Candida in patients with invasive candidiasis identified by routine index blood or sterile site cultures. T2Candida was compared to diagnostic blood and sterile site cultures and also performed with samples obtained prior and after collection of index cultures. T2Candida was evaluated with 133 samples of 32 patients with candidemia and 22 patients with deep-seated invasive candidiasis. In the candidemic group 28/32 (87.5%) patients had at least one positive T2Candida result at any time point. A total of 17/25 (68%) candidemic patients had a positive T2Candida sample that was drawn concurrently to the index blood culture. In the per patient analysis 17/18 (94.4%) candidemic patients with matched T2Candida samples and peripheral blood cultures at any timepoint had a positive T2Candida test. T2Candida revealed discordant Candida species identification in two candidemic patients. Six of 22 (27.3%) deep-seated IC patients had a positive T2Candida result. Despite advanced time-to-results the clinical value of T2Candida in diagnosing candidemia seems to be limited by missing blood culture positive cases. Positivity rates of T2Candida increased when serial T2Candida samples were tested. In patients with suspected deep-seated invasive candidiasis T2Candida might act as a blood based adjunct to sterile site cultures., (© The Author(s) 2019. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) is routinely used in mycology laboratories to rapidly identify pathogenic yeasts. Various methods have been proposed to perform routine MS-based identification of clinically relevant species. In this study, we focused on Bruker technology and assessed the identification performance of three protocols: two pretreatment methods (rapid formic acid extraction directly performed on targets and full extraction using formic acid/acetonitrile in tubes) and a direct deposit protocol that omits the extraction step. We also examined identification performance using three target types (ground-steel, polished-steel, and biotargets) and two databases (Bruker and online MSI [biological-mass-spectrometry-identification application]) in a multicenter manner. Ten European centers participated in the study, in which a total of 1511 yeast isolates were analyzed. The 10 centers prospectively performed the three protocols on approximately 150 yeast isolates each, and the corresponding spectra were then assessed against two reference spectra databases (MSI and Bruker), with appropriate thresholds. Three centers evaluated the impact of the targets. Scores were compared between the various combinations, and identification accuracy was assessed. The protocol omitting the extraction step was inappropriate for yeast identification, while the full extraction method yielded far better results. Rapid formic acid extraction yielded variable results depending on the target, database and threshold. Selecting the optimal extraction method in combination with the appropriate target, database and threshold may enable simple and accurate identification of clinically relevant yeast samples. Concerning the widely used polished-steel targets, the full extraction method still ensured better scores and better identification rates., (© The Author(s) 2019. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Background: Candida glabrata is a common pathogen that causes invasive candidiasis. Among non-albicans Candida infections, C glabrata infections are associated with the highest fatality rates. Candida glabrata sensu stricto, Candida nivariensis, and Candida bracarensis have been identified and together form the C glabrata species complex. It is difficult to detect the two rare species by traditional laboratory methods. This study established a method for the rapid identification of members of the C glabrata species complex based on high-resolution melting curve (HRM) analysis and evaluated its practical application., Methods: The internal transcribed spacer (ITS) region was used as target gene region to design specific primers. HRM analysis was performed with three subspecies of the C glabrata species complex and negative controls to test its specificity and sensitivity. To evaluate its practical application, the HRM technique was tested with clinical isolates, and the results were compared with the DNA sequencing results., Results: Differences were detected among the melting profiles of the members of the C glabrata species complex. The negative controls were not amplified, indicating the high specificity of the method. The minimum detection limits of C glabrata sensu stricto, C nivariensis, and C bracarensis were approximately 1 × 10 1  copies/µL or less. The results of the HRM analysis of the clinical isolates were consistent with the DNA sequencing results., Conclusions: The HRM method is sensitive and can be used to rapidly identify the members of the C glabrata species complex. The method can allow early and targeted treatment of patients with invasive candidiasis., (© 2020 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals, Inc.)

A survey of mycology laboratories for antifungal susceptibility testing (AFST) was undertaken in France in 2018, to better understand the difference in practices between the participating centers and to identify the difficulties they may encounter as well as eventual gaps with published standards and guidelines. The survey captured information from 45 mycology laboratories in France on how they perform AFST (number of strains tested, preferred method, technical and quality aspects, interpretation of the MIC values, reading and interpretation difficulties). Results indicated that 86% of respondents used Etest as AFST method, with a combination of one to seven antifungal agents tested. Most of the participating laboratories used similar technical parameters to perform their AFST method and a large majority used, as recommended, internal and external quality assessments. Almost all the participating mycology laboratories (98%) reported difficulties to interpret the MIC values, especially when no clinical breakpoints are available. The survey highlighted that the current AFST practices in France need homogenization, particularly for MIC reading and interpretation., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

The diagnosis of parasitic and fungal infections, historically based on the detection of these pathogens using direct diagnosis (macro/microscopic examination, culture) or serological methods, has considerably evolved in the last decades, especially with the development of molecular approaches and mass spectrometry. These techniques, as well as most analyses of parasitic and fungal serology, are mostly the preserve of Hospital University Centers Parasitology-Mycology laboratories. In 2016, the French association of medical parasitology and mycology teachers and hospital practitioners (Anofel) has provided a Catalogue of rare analyses, regularly updated and freely accessible on the Anofel website (https://anofel.net/). This tool, which hinges on 4 parts (parasitology, parasitic serology, mycology, and fungal serology), aims to provide information on all available analyses, and a list of hospital laboratories able to undertake them. It is complementary to the other reference works that were developed by our association, including the Guide of analyses and methods in parasitology and mycology, published in 2018, and the eANOFEL pictures and videos database, freely accessible online (http://www.eanofel.fr). In this article, we draw-up a state-of-the-art of the most specialized techniques available in the parasitology-mycology laboratories and presented in the Catalogue of rare analyses of the Anofel collegium, and their interest for the diagnosis of these infections.

Objectives: Difficult-to-treat invasive fungal infections require infectious diseases expert consultation to improve treatment outcome and increase survival rates., Methods: The European Confederation of Medical Mycology (ECMM) intends to provide expert help free of charge by a newly founded ECMM Expert Consultation Service for medical centres around the globe seeking advice when there is no fungal infection consultant available. The expert consult will provide recommendations and broad expertise on difficult-to-treat invasive fungal infections (eg azole-resistant Aspergillus species, Candida auris, mucormycosis) to improve diagnostic and therapeutic management and outcome., Results: The initiative plans global outreach through video conferencing between ECMM Excellence Centers and treating physicians. FungiScope ® registries will be used to structure case information and to evaluate the impact of the collegial advice system at regular intervals. Advice will follow recent guidelines, and EQUAL Scores will be used to measure guideline adherence., Conclusions: Infectious diseases expert consultation should be an integral component of care for patients with difficult-to-treat invasive fungal infections. The ECMM Expert Consult will attend to this matter on a global scale., (© 2020 The Authors. Mycoses published by Blackwell Verlag GmbH.)

Background: Diversified etiology of lower respiratory tract infection renders diagnosis challenging. The mainstay microbial culture is time-consuming and constrained by variable growth requirements. In this study, we explored the use of Nanopore sequencing as a supplementary tool to alleviate this diagnostic bottleneck., Methods: We developed a targeted Nanopore method based on amplification of bacterial 16S rRNA gene and fungal internal transcribed spacer region. The performance was compared with routine infectious disease workups on 43 respiratory specimens., Results: Nanopore successfully identified majority of microbes (47/54, 87.04%) and 7 possible pathogens not detected by routine workups, which were attributable to the content of microbiological investigations (n = 5) and negative culture (n = 2). The average sequencing time for first target reads was 7 min (1-43 min) plus 5 h of pre-sequencing preparation., Conclusions: The Nanopore method described here was rapid, economical and hypothesis-free, which might provide valuable hints to further microbiological follow-up for opportunistic pathogens missed or not detectable by conventional tests.

Trichophyton mentagrophytes complex is the most frequent agent found in dermatophyte isolates after Trichophyton rubrum. It is divided into 3main groups: (1) Trichophyton benhamiae and related species; (2) Trichophyton simii and 2related species, Trichophyton quinckeanum and Trichophyton schoenleinii; and (3) T. mentagrophytes, T. interdigitale, and related species. They are all difficult to identify by morphophysiology. With the aim of classifying them at the species level, a descriptive study was performed on 17 isolated clinical strains identified in the Mycology Laboratory of the Universidad de Valparaíso as belonging to the T. mentagrophytes complex. They were compared with 3 reference strains (T. mentagrophytes CBS 318.56, T. interdigitale CBS 428.63, Trichophyton erinacei CBS 511.73). Morphophysiological and molecular tests were performed by high resolution melting analysis curves and ITS regions sequencing. Phenotypically, 3 species of the complex were identified. Molecular analyses reclassified all the species as belonging to T. interdigitale. In conclusion, no reliable morphophysiological patterns were established to differentiate between the species of the complex. Molecularly, all the strains studied were classified as T. interdigitale., (Copyright © 2019 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.)

Background: Trichophyton rubrum is an obligate human parasitic fungus and responsible for approximately 80-90% of dermatomycosis in human. Molecular genetic manipulations of this pathogen are challenging and available tools and protocols are only rudimentary. We adapt molecular genetics methods of well established fungal model organism, to knock out genes in T. rubrum. For the adaptation, crucial modifications are necessary. With the implementation of in vitro synthesized Cas9-sgRNA ribonucleoprotein complex, it is possible to adapt molecular genetic methods, to knock out genes in T. rubrum., Results: The gene knock-out method is based on integration of a selection marker into the target site, to interrupt the gene translation. The target gene gets preassigned by the homologous sequence of the in vitro synthesized Cas9-sgRNA ribonucleoprotein complex. To develop the method, we first isolated and characterized a T. rubrum strain with a high amount of microconidia. Next, we developed a transformation protocol, whereby the Cas9-sgRNA ribonucleoprotein gets delivered into the fungal protoplast by the PEG method. We knocked out the URA3 gene and resulted, as predicted, uracil auxotrophic strains. These strains can be used for specific gene knock-outs by reintegrating the URA3 fragment and selection on uracil lacking cultivation media. Exemplary, we knocked out the TRP3 gene and got the predicted phenotype, tryptophan auxotrophic strains. The mutation had been verified by sequencing., Conclusions: We developed a method, based on in vitro synthesized Cas9-sgRNA ribonucleoprotein complex, for target specific gene knock-outs in T. rubrum. We knocked out the Ura3 gene and resulted uracil auxotrophic strains. These strains were used for target specific gene knock-outs by reintegrating the Ura3 fragment into the target gene site to interrupt the gene transcription. The developed method allows to adapt sophisticate gene manipulation methods of model fungal species to non-model species.

Fungal pathogens represent a major human threat affecting more than a billion people worldwide. Invasive infections are on the rise, which is of considerable concern because they are accompanied by an escalation of antifungal resistance. Deciphering the mechanisms underlying virulence traits and drug resistance strongly relies on genetic manipulation techniques such as generating mutant strains carrying specific mutations, or gene deletions. However, these processes have often been time-consuming and cumbersome in fungi due to a number of complications, depending on the species (e.g., diploid genomes, lack of a sexual cycle, low efficiency of transformation and/or homologous recombination, lack of cloning vectors, nonconventional codon usage, and paucity of dominant selectable markers). These issues are increasingly being addressed by applying clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 mediated genetic manipulation to medically relevant fungi. Here, we summarize the state of the art of CRISPR-Cas9 applications in four major human fungal pathogen lineages: Candida spp., Cryptococcus neoformans, Aspergillus fumigatus, and Mucorales. We highlight the different ways in which CRISPR has been customized to address the critical issues in different species, including different strategies to deliver the CRISPR-Cas9 elements, their transient or permanent expression, use of codon-optimized CAS9, and methods of marker recycling and scarless editing. Some approaches facilitate a more efficient use of homology-directed repair in fungi in which nonhomologous end joining is more commonly used to repair double-strand breaks (DSBs). Moreover, we highlight the most promising future perspectives, including gene drives, programmable base editors, and nonediting applications, some of which are currently available only in model fungi but may be adapted for future applications in pathogenic species. Finally, this review discusses how the further evolution of CRISPR technology will allow mycologists to tackle the multifaceted issue of fungal pathogenesis., Competing Interests: The authors have declared that no competing interests exist.

Background: Despite availability of rapid fungal potassium hydroxide (KOH) tests, many care providers rely on visual assessment to determine the diagnosis of monilial diaper dermatitis (MDD)., Purpose: To determine whether a KOH test, when MDD is suspected, would result in more accurate diagnoses, with decreased antifungal medication prescription and exposure., Methods: Quality improvement project from 2016 through 2017 with protocol implemented in 2017 for treatment of MDD after positive KOH testing. If monilial rash suspected, after 2 negative KOH tests, then antifungal ordered (considered false negative). χ testing and cost determination were performed., Sample: Neonates in 2 level III neonatal intensive care units., Outcome Variables: KOH test results, use of antifungal medication, and cost., Results: The patient census included 1051 and 1015 patients in the year before and after the protocol initiation. The medical orders for antifungal medication decreased from 143 to 36 (P < .001; 95% odds ratio confidence interval, 2.24-4.38). There was a 75% reduction in both use and cost, as charged, of antifungal agents. Overall charges, including KOH test costs, decreased by 12%. Three infants received multiple negative KOH tests, then a positive one. These met the definition of false-negative tests, per protocol. There were no cases of fungal sepsis., Implications for Practice: Use of a quality improvement protocol, in which the use of KOH testing is required, before antifungal agents are prescribed, results in decreased exposure and costs., Implications for Research: To test the feasibility of bedside "point-of-care" KOH testing, and whether KOH testing and reduced antifungal medication use affects antimicrobial resistance or invasive fungal sepsis.

Rice false smut, caused by the ascomycete Ustilaginoidea virens, is a serious disease of rice worldwide. Conidia are very important infectious propagules of U. virens, but the ability of pathogenic isolates to produce conidia frequently decreases in culture, which influences pathogenicity testing. Here, we developed tissue media with rice leaves or panicles that stimulate conidiation of U. virens. Among the tested media, 0.10 g/ml panicle medium was most efficient for conidiation. Whereas, some rice leaf media more effectively increased conidiation than panicle media except 0.10 g/ml panicle medium, and certain non-filtered tissue media were better than their filtered counterparts. Although the conidia induced in rice tissue media were smaller, they were able to germinate on potato sucrose agar medium and infect rice normally. The rice tissue medium is also workable in inducing conidia for conidiation-defective isolates. This method provides a foundation for the production of conidia by U. virens that will be widely applicable in pathogenicity testing as well as in genetic analyses for false smut resistance in rice cultivars., Competing Interests: The authors have declared that no competing interests exist.

Subculturing is frequently used for the preservation of basidiomycetes. In this study, to assess and verify the risks of repeated subculturing on the long-term preservation of strains of culture collections, we performed single nucleotide polymorphism (SNP) analysis in genes encoding enzymes of the mevalonate pathway, 1,3-β-glucan synthesis, lignin degradation, and the tricarboxylic acid (TCA) cycle of mycelia before and after preserving for a 4-year period by the subculturing 30 times every 45 days of Ganoderma lucidum NBRC 8346. As a result of analyzing 60 genes of the strain, SNPs were found in 18 genes, and 14 of them were found in the exon region. Nonsynonymous coding SNPs were found in two genes ( atoB&#95;2 , hmgr ) encoding enzymes of mevalonate pathway and five genes ( lcc1&#95;9 , lcc1&#95;11 , lcc1&#95;13 , dslcc6 , aa5&#95;1&#95;9 ) encoding enzymes of lignin degradation after subculturing of G. lucidum NBRC 8346.

Podospora anserina is a model ascomycetous fungus which shows pronounced phenotypic senescence when grown on solid medium but possesses unlimited lifespan under submerged cultivation. In order to study the genetic aspects of adaptation of P. anserina to submerged cultivation, we initiated a long-term evolution experiment. In the course of the first 4 years of the experiment, 125 single-nucleotide substitutions and 23 short indels were fixed in eight independently evolving populations. Six proteins that affect fungal growth and development evolved in more than one population; in particular, in the G-protein alpha subunit FadA, new alleles fixed in seven out of eight experimental populations, and these fixations affected just four amino acid sites, which is an unprecedented level of parallelism in experimental evolution. Parallel evolution at the level of genes and pathways, an excess of nonsense and missense substitutions, and an elevated conservation of proteins and their sites where the changes occurred suggest that many of the observed fixations were adaptive and driven by positive selection., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Aspergillus fumigatus is an opportunistic human fungal pathogen, capable of causing invasive aspergillosis in patients with compromised immune systems. The fungus was long considered a purely asexual organism. However, a sexual cycle was reported in 2009, with methods described to induce mating under laboratory conditions. The presence of a sexual cycle now offers a valuable tool for classical genetic analysis of the fungus, such as allowing determination of whether traits of interest are mono- or poly-genic in nature. For example, the sexual cycle is currently being exploited to determine the genetic basis of traits of medical importance such as resistance to azole antifungals and virulence, and to characterize the genes involved. The sexual cycle can also be used to assess the possibility of gene flow between isolates. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. This unit describes protocols for culturing of A. fumigatus and for inducing sexual reproduction between compatible MAT1-1 and MAT1-2 isolates of the species. The unit also provides working methods for harvesting sexual structures, isolating single-spore progeny and confirming whether sexual recombination has occurred. © The Authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited., (© 2019 The Authors.)

Background: Talaromyces marneffei is a thermally dimorphic fungus endemic in south-east Asia. It predominantly occurs in both immunocompromised and immunosuppressed patients and can be fatal if diagnosis and treatment are delayed. The clinical manifestations of T. marneffei infection are nonspecific and rapid diagnosis of T. marneffei infection remains challenging., Case Presentation: A 24-year-old man came to our outpatient department with the sign of common skin lesions. The lesions were cuticolor follicular papules with or without central umbilication, nodules and acne-like lesions, which are common in syringoma, steatocystoma multiplex and trichoepithelioma. A dermatoscopy examination was performed to differentiate these skin lesions. The dermatoscopic images revealed circular or quasi-circular whitish amorphous structure with a central brownish keratin plug, providing the diagnostic clues of T. marneffei infection. Therefore, a skin scrapings culture, skin biopsy and serological detection for human immunodeficiency virus (HIV) were performed. The final diagnosis of this patient was T. marneffei and HIV co-infection., Conclusion: Rapid diagnosis of T. marneffei infection is clinically challenging since presenting clinical manifestations are nonspecific with significant overlap with other common conditions. This case highlights that dermatoscopy is a promising tool for the rapid diagnosis of T. marneffei infection in patients with nonspecific skin lesions, assisting clinicians to avoid delayed diagnosis or misdiagnosis.

Background: Many dermatologists do not understand the perpetual adjustments in the dermatophyte nomenclature., Objectives: The aim is to explain the background and the development of methods that have led to previous and current changes of dermatophyte taxonomy and to the detection of new dermatophytes., Methods: In this article we evaluate the recent literature on this topic and our own results in the fields of dermatophyte identification, their detection, and of the associated taxonomic developments., Results: Today, the phylogenetic species concept is the basis of taxonomic classification, including that of dermatophytes. Genetic techniques have decisively advanced this and are state of the art nowadays. The detection of new dermatophyte species was often triggered by clinical observations and by morphologically conspicuous cultures that prompted their subsequent exact mycological characterization. Even today not all species of dermatophytes are unequivocally defined., Conclusions: By exclusively using selected genetic characteristics for the construction of phylogenetic trees additional taxonomically relevant features are neglected. Therefore it is necessary to better integrate data derived from morphologic, physiologic, ecologic and pathophysiologic observations into phylogenetic analyses. Dermatologists are still asked to contribute such information.

Background: The Guatemalan Highlands is a region of great but so far poorly known mycological diversity. People living in this area have long used wild fungi as a source of food and income. However, our knowledge of the ethnomycological practices of the Mayan peoples of Guatemala is still rudimental, especially if compared with information reported for the neighboring region of Mexico. Among the main indigenous groups of the Maya people inhabiting the highlands of Central Guatemala, stand the Kaqchikel, accounting for nearly 8% of the entire Guatemalan population. The main aim of this study was to record the traditional knowledge and use of edible wild mushrooms by inhabitants of the municipality of San Juan Sacatepéquez that lies at the heart of the Kaqchikel area in the central highlands of Guatemala, also describing the relevant selling practices and dynamics. A secondary aim was to compare the diversity and composition of the mushroom assemblage offered at the market with the macrofungal diversity of woods in the area., Methodology: This study is the result of 4 years of ethnomycological research, conducted through continuous visits to the municipal market and focused interviews with collectors and vendors. Field sampling in pine-oak forested areas surrounding San Juan Sacatepéquez, from where the mushrooms sold at the market are foraged, were also conducted, in the presence of local collectors., Results: The results show a significant richness of species sold in the market, a network of commerce of purchase, sale, and resale of several species, with relatively stable prices, and knowledge about edible and inedible species that is transmitted mainly within the family nucleus. The business of selling mushrooms in the market is an exclusive activity of women, who are supplied by collectors or by other vendors. Fungi are sold and bought only as food, while no consumption of hallucinogenic mushrooms or medicinal mushrooms was recorded. Several species of Amanita, Cantharellus, Boletus, Lactarius, and Russula were those most commercialized in the 4 years of the study, but we also spotted fungi never reported before as consumed in the country, including Gastropila aff. fumosa (= Calvatia fumosa) and several species of Cortinarius. Field sampling in nearby pine-oak forests confirmed an elevated local macrofungal diversity., Conclusion: Our study unveiled the contemporary wealth of Kaqchikel culture for what concerns mushrooms, demonstrating that mushrooms continue to be culturally and economically important for these communities despite the erosion of traditional knowledge. Our results also confirmed the need to investigate in greater detail the Guatemalan mycodiversity that is vast and poorly known.

Reference fungal cultures (RFCs) are essential for the internal quality control of laboratories. The production of these cultures requires standardized procedures (IRAM 14950:2016 and ISO 17034:2016 standards) carried out by a recognized and accredited laboratory. The aim of this work was to produce RFC in paper disks of autochthonous strains, characterized by two, homogeneous and stable reference methods traceable at species level. RFC were produced using 14 regional species (7 yeasts and 7 filamentous fungi) from the fungal culture collection (DMic). Paper disks were impregnated with a culture suspension, dried and packed. Homogeneity, viability, identity and purity were verified. Short- and long-term stability at different temperatures and storage times were studied. Characterization of each strain allowed to confirm its identity and to ensure its traceability at international level. Produced batches were homogeneous and stable at -20±5°C for 30 months. This method of production was adequate to produce homogeneous and stable RFC with phenotypic and genotypic characteristics correctly defined and internationally traceable. Standardized procedures were developed for the production of certified RFC that could be transferred to other microorganisms. Providing RFC that represent regional strains allows laboratories to produce more reliable results with a favorable impact on medical diagnosis, the environment or the food industry., (Copyright © 2018 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.)

The Cryptococcus pathogenic species complex is a group of opportunistic human fungal pathogens that cause cryptococcal meningoencephalitis, an infection associated with unacceptably high mortality rates. The public health relevance of these pathogens has galvanized extensive research over the past several decades and led to characterization of their sexual cycles. This research has allowed several Cryptococcus species to develop into model fungal organisms for both pathogenesis and basic science studies. Many of these studies require observation of the meiotic process and its associated mating structures as well as generation of meiotic progeny with novel phenotypes and genotypes. Herein, we describe how to set up genetic crosses between Cryptococcus strains and observe their mating phenotypes as well as how to recover progeny from these crosses for further analysis. © 2019 by John Wiley & Sons, Inc., (© 2019 John Wiley & Sons, Inc.)

Cordyceps, a type of Chinese herbal medicine that exhibits anti-angiogenesis and tumor growth suppression effects, has recently gained increasing popularity. However, high-quality, natural Cordyceps, such as Ophiocordyceps sinensis, is very rare and difficult to obtain in large amounts. Cordyceps is cultured instead of harvested from natural sources, but the quality with respect to the ingredients has not been fully studied. In this study, we performed an NMR metabolic profiling of aqueous extracts of Cordyceps without any sample treatment to evaluate the proper species and medium and influence of two different disinfection methods. It was discovered that Cordyceps militaris fungus and silkworm chrysalis medium were suitable for cultivation of Cordyceps. Furthermore, cordycepin, a Cordyceps-specific functional compound, was produced at different growth stages during different cultivation processes, even at the mycelial stage, and was found at three times higher concentrations in cultured C. militaris compared to that in naturally occurring C. militaris.

Background: The gold standard for the sporotrichosis diagnosis is culture; however, serologic approaches have been recently implemented to aid in the sporotrichosis diagnosis. Nevertheless, the clinical consequences of the introduction of serologic tests are poorly addressed., Aims: To correlate the results of culture and serology of patients with suspected sporotrichosis., Methods: A retrospective study of 198 patients with suspected sporotrichosis was conducted. Information about culture isolation of Sporothrix from clinical samples and antibody detection by an enzyme-linked immunosorbent assay (ELISA) were obtained from the medical records of the patients., Results: Positive culture and antibody detection was observed in the samples of 84 patients (42.4%). Forty-one samples (20.7%) showed negative results with both techniques and divergent results were obtained in the samples of 73 patients (36.9%). False negative results in the ELISA were observed with 23 patients (31.5%), 78.3% of them with less than 30 days of infection (p=0.0045). Among the initial false positive ELISA in the sera of 50 patients, four samples in culture yielded the growth of Sporothrix, and 27 improved with itraconazole. At the end of follow-up, a diagnosis of proven or probable sporotrichosis was established in 139 patients, and possible sporotrichosis in 11 patients. The treatment of the patients with probable sporotrichosis with antifungal drugs resulted in clinical cure for these individuals., Conclusions: These two techniques are complementary in the diagnosis of sporotrichosis, making diagnosis and clinical decision more precise., (Copyright © 2019 Asociación Española de Micología. Publicado por Elsevier España, S.L.U. All rights reserved.)

Regular protocols for the isolation of fungal extracellular vesicles (EVs) are time-consuming, hard to reproduce, and produce low yields. In an attempt to improve the protocols used for EV isolation, we explored a model of vesicle production after growth of Cryptococcus gattii and Cryptococcus neoformans on solid media. Nanoparticle tracking analysis in combination with transmission electron microscopy revealed that C. gattii and C. neoformans produced EVs in solid media. The properties of cryptococcal vesicles varied according to the culture medium used and the EV-producing species. EV detection was reproduced with an acapsular mutant of C. neoformans , as well as with isolates of Candida albicans , Histoplasma capsulatum , and Saccharomyces cerevisiae Cryptococcal EVs produced in solid media were biologically active and contained regular vesicular components, including the major polysaccharide glucuronoxylomannan (GXM) and RNA. Since the protocol had higher yields and was much faster than the regular methods used for the isolation of fungal EVs, we asked if it would be applicable to address fundamental questions related to cryptococcal secretion. On the basis that polysaccharide export in Cryptococcus requires highly organized membrane traffic culminating with EV release, we analyzed the participation of a putative scramblase (Aim25; CNBG&#95;3981) in EV-mediated GXM export and capsule formation in C. gattii EVs from a C. gattii aim25 Δ strain differed from those obtained from wild-type (WT) cells in physical-chemical properties and cargo. In a model of surface coating of an acapsular cryptococcal strain with vesicular GXM, EVs obtained from the aim25 Δ mutant were more efficiently used as a source of capsular polysaccharides. Lack of the Aim25 scramblase resulted in disorganized membranes and increased capsular dimensions. These results associate the description of a novel protocol for the isolation of fungal EVs with the identification of a previously unknown regulator of polysaccharide release. IMPORTANCE Extracellular vesicles (EVs) are fundamental components of the physiology of cells from all kingdoms. In pathogenic fungi, they participate in important mechanisms of transfer of antifungal resistance and virulence, as well as in immune stimulation and prion transmission. However, studies on the functions of fungal EVs are still limited by the lack of efficient methods for isolation of these compartments. In this study, we developed an alternative protocol for isolation of fungal EVs and demonstrated an application of this new methodology in the study of the physiology of the fungal pathogen Cryptococcus gattii Our results describe a fast and reliable method for the study of fungal EVs and reveal the participation of scramblase, a phospholipid-translocating enzyme, in secretory processes of C. gattii ., (Copyright © 2019 Reis et al.)