Dermatology mycology diagnostics in Ireland: National deficits identified in 2022 that are relevant internationally.

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.

Comparative assessment of regulated methods and PCR in the diagnosis of trichophytosis in veterinary mycology.

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.

Cell Cycle Synchrony Methods for Fission Yeast, Schizosaccharomyces pombe.

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.

Environment-specificity and universality of the microbial growth law.

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.

All You Need To Know and More about the Diagnosis and Management of Rare Yeast Infections.

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.

A simple method for the cultivation of the "unculturable" asterinaceous fungi (Asterinales/Dothideomycetes).

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.

Sulfur transfer from the endophytic fungus Serendipita indica improves maize growth and requires the sulfate transporter SiSulT.

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.

Candida auris: A Quick Review on Identification, Current Treatments, and Challenges.

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.

IX Fórum Micológico-Asociación Española de Micología (AEM): Comunicaciones / IX Mycologic Forum- Asociación Española de Micología

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Effect of cultural conditions on the production of radicinin, a specific fungal phytotoxin for buffelgrass (Cenchrus ciliaris) biocontrol, by different Cochlioboulus australiensis strains.

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.

Direct examination, histopathology and fungal culture for the diagnosis of onychomycosis: A retrospective, comparative study on 2245 specimens.

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.

European confederation of medical mycology quality of clinical candidaemia management score: A review of the points based best practice recommendations.

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.

FastFung: A novel medium for the culture and isolation of fastidious fungal species from clinical samples.

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.

Slow Adaptive Response of Budding Yeast Cells to Stable Conditions of Continuous Culture Can Occur without Genome Modifications.

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.

Beauveria bassiana Culturing and Harvesting for Bioassays With House Flies.

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.

Automated quantification of Candida albicans biofilm-related phenotypes reveals additive contributions to biofilm production.

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.

Identification of antibiotics for use in selection of the chytrid fungi Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans.

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.

3-Methylxanthine production through biodegradation of theobromine by Aspergillus sydowii PT-2.

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 Fe2+ 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.

[Update on the diagnosis of parasitic and fungal infections]. / Actualités du diagnostic des infections parasitaires et fongiques.

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.

Primary Cutaneous Coccidioidomycosis: An Update.

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.