Aflatoxin profiles of Aspergillus flavus isolates in Sudanese fungal rhinosinusitis.

Aspergillus flavus is a commonly encountered pathogen responsible for fungal rhinosinusitis (FRS) in arid regions. The species is known to produce aflatoxins, posing a significant risk to human health. This study aimed to investigate the aflatoxin profiles of A. flavus isolates causing FRS in Sudan. A total of 93 clinical and 34 environmental A. flavus isolates were studied. Aflatoxin profiles were evaluated by phenotypic (thin-layer and high-performance chromatography) and genotypic methods at various temperatures and substrates. Gene expression of aflD and aflR was also analyzed. A total of 42/93 (45%) isolates were positive for aflatoxin B1 and AFB2 by HPLC. When the incubation temperature changed from 28°C to 36°C, the number of positive isolates decreased to 41% (38/93). Genetic analysis revealed that 85% (79/93) of clinical isolates possessed all seven aflatoxin biosynthesis-associated genes, while 27% (14/51) of non-producing isolates lacked specific genes (aflD/aflR/aflS). Mutations were observed in aflS and aflR genes across both aflatoxin-producers and non-producers. Gene expression of aflD and aflR showed the highest expression between the 4th and 6th days of incubation on the Sabouraud medium and on the 9th day of incubation on the RPMI (Roswell Park Memorial Institute) medium. Aspergillus flavus clinical isolates demonstrated aflatoxigenic capabilities, influenced by incubation temperature and substrate. Dynamic aflD and aflR gene expression patterns over time enriched our understanding of aflatoxin production regulation. The overall findings underscored the health risks of Sudanese patients infected by this species, emphasizing the importance of monitoring aflatoxin exposure.

Aspergillus flavus, mainly causing fungal rhinosinusitis in Sudan, poses health risks due to aflatoxin production. This study revealed diverse levels of aflatoxin and gene expression of clinical isolates by pheno- and genotypic methods, emphasizing the need for vigilant monitoring in the region.

A conceptual framework for nomenclatural stability and validity of medically important fungi: a proposed global consensus guideline for fungal name changes supported by ABP, ASM, CLSI, ECMM, ESCMID-EFISG, EUCAST-AFST, FDLC, IDSA, ISHAM, MMSA, and MSGERC.

The rapid pace of name changes of medically important fungi is creating challenges for clinical laboratories and clinicians involved in patient care. We describe two sources of name change which have different drivers, at the species versus the genus level. Some suggestions are made here to reduce the number of name changes. We urge taxonomists to provide diagnostic markers of taxonomic novelties. Given the instability of phylogenetic trees due to variable taxon sampling, we advocate to maintain genera at the largest possible size. Reporting of identified species in complexes or series should where possible comprise both the name of the overarching species and that of the molecular sibling, often cryptic species. Because the use of different names for the same species will be unavoidable for many years to come, an open access online database of the names of all medically important fungi, with proper nomenclatural designation and synonymy, is essential. We further recommend that while taxonomic discovery continues, the adaptation of new name changes by clinical laboratories and clinicians be reviewed routinely by a standing committee for validation and stability over time, with reference to an open access database, wherein reasons for changes are listed in a transparent way.

Antifungal Susceptibility and Mutations in the Squalene Epoxidase Gene in Dermatophytes of the Trichophyton mentagrophytes Species Complex.

During the past decade, a prolonged and serious outbreak of dermatophytosis due to a terbinafine-resistant novel species in the Trichophyton mentagrophytes-T. interdigitale complex has been ongoing in India, and it has spread to several European countries. The objective of this study was to investigate the molecular background of the squalene epoxidase (SQLE) gene in order to understand the risk of emergence and spread of multiresistance in dermatophytes. Antifungal susceptibility to fluconazole, griseofulvin, itraconazole, ketoconazole, miconazole, naftifine, sertaconazole, and terbinafine was tested in 135 isolates from India, China, Australia, Germany, and The Netherlands. Based on the latest taxonomic insights, strains were identified as three species: T. mentagrophytes sensu stricto (n = 35), T. indotineae (n = 64, representing the Indian clone), and T. interdigitale sensu stricto (n = 36). High MICs of terbinafine (>16 mg/liter) were found in 34 (53%) T. indotineae isolates. These isolates showed an amino acid substitution in the 397th position of the SQLE gene. Elevated MICs of terbinafine (0.5 mg/liter) were noted in 2 (3%) T. indotineae isolates; these isolates lead to Phe415Val and Leu393Ser of the SQLE gene. The stability of the effect of the mutations was proven by serial transfer on drug-free medium. Lys276Asn and Leu419Phe substitutions were found in susceptible T. mentagrophytes strains. The Phe377Leu/Ala448Thr double mutant showed higher MIC values for triazoles. High MICs of terbinafine are as yet limited to T. indotineae and are unlikely to be distributed throughout the T. mentagrophytes species complex by genetic exchange.

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

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

Taxonomy of the Trichophyton mentagrophytes/T. interdigitale Species Complex Harboring the Highly Virulent, Multiresistant Genotype T. indotineae.

A severe outbreak of highly virulent and multi-resistant dermatophytosis by species in the Trichophyton mentagrophytes/T. interdigitale complex is ongoing in India. The correct identity of the etiologic agent is a much-debated issue. In order to define species limits, a taxonomic study was undertaken combining molecular, morphological, and physiological characteristics as evidence of classification. Molecular characteristics show that T. mentagrophytes s. str. and T. interdigitale s. str. can be distinguished with difficulty from each other, but are unambiguously different from the Indian genotype, T. indotineae by sequences of the HMG gene. The entities were confirmed by multilocus analysis using tanglegrams. Phenotypic characters of morphology and physiology are not diagnostic, but statistically significant differences are observed between the molecular siblings. These properties may be drivers of separate evolutionary trends. Trichophyton mentagrophytes represents the ancestral, homothallic cloud of genotypes with a probable geophilic lifestyle, while T. indotineae and T. interdigitale behave as anthropophilic, clonal offshoots. The origin of T. indotineae, which currently causes a significant public health problem, is zoonotic, and its emergence is likely due to widespread misuse of antifungals.

Molecular and Phenotypic Characterization of Nannizzia (Arthrodermataceae).

Phylogenetic studies of the family Arthrodermataceae have revealed seven monophyletic dermatophyte clades representing the genera Trichophyton, Epidermophyton, Nannizzia, Lophophyton, Paraphyton, Microsporum, and Arthroderma. Members of the genus Nannizzia are geo- or zoophiles that occasionally infect humans. With the newly proposed taxonomy, the genus Nannizzia comprises thirteen species, i.e., Nannizzia aenigmatica, N. corniculata, N. duboisii, N. fulva, N. graeserae, N. gypsea, N. nana, N. incurvata, N. perplicata, N. persicolor, N. praecox, and two novel species. Nannizzia polymorpha sp. nov. was isolated from a skin lesion of a patient from French Guiana. For the strain originally described as Microsporum racemosum by Borelli in 1965, we proposed Nannizzia lorica nom. nov. The species are fully characterized with five sequenced loci (ITS, LSU, TUB2, RP 60S L1 and TEF3), combined with morphology of the asexual form and physiological features. A key to the species based on phenotypic and physiological characters is provided.

Species Distinction in the Trichophyton rubrum Complex.

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

Revision of the medically relevant species of the yeast genus Diutina.

Diutina (Candida) rugosa is emerging as a causative agent of human infections. Recently some close relatives have been described, that is, D. mesorugosa, D. pseudorugosa, and D. neorugosa, some of which have also been implicated in human infection. Phylogenetic relationships of 24 clinical isolates of the D. rugosa complex are reconstructed using multilocus sequence analysis of five housekeeping genes, supplemented with phenotypic studies of CandiSelect™ 4 Agar and nutritional physiology. Diutina mesorugosa could not meaningfully be distinguished from D. rugosa and is regarded as a synonym. Diutina neorugosa and D. pseudorugosa represent separate, distantly related species within the genus Diutina, but have as yet not been encountered in clinical settings.

A Comparison of Isolation Methods for Black Fungi Degrading Aromatic Toxins.

The prevalence of black fungi in the order Chaetothyriales has often been underestimated due to the difficulty of their isolation. In this study, three methods which are often used to isolate black fungi are compared. Enrichment on aromatic hydrocarbon appears effective in inhibiting growth of cosmopolitan microbial species and allows appearance of black fungi. We miniaturized the method for high-throughput purposes. The new procedure saves time, consumes less space and can process multiple samples simultaneously.

Nigrograna mackinnonii, Not Trematosphaeria grisea (syn., Madurella grisea), Is the Main Agent of Black Grain Eumycetoma in Latin America.

Mycetoma, a chronic and mutilating subcutaneous infection recognized by the WHO as a neglected tropical disease, has been reported in >25 countries in Africa, Asia, and South America. In Latin America, Trematosphaeria grisea is assumed to be the prevalent fungal agent. Recent molecular studies have shown that this is an environmental saprobe in Europe, where it is rarely implicated in human diseases. The aim of the present paper is to establish the identity of Latin American cases ascribed to Trematosphaeria grisea Three cases analyzed were caused by Nigrograna mackinnonii Data on an additional 21 strains in the literature revealed that N. mackinnonii rather than T. grisea is responsible for most cases of black grain eumycetoma in Latin America.

Fusarium metavorans sp. nov.: The frequent opportunist 'FSSC6'.

The Fusarium solani species complex (FSSC) is the most common group of fusaria associated with superficial and life-threatening infections in humans. Here we formally introduce Fusarium metavorans sp. nov., widely known as FSSC6 (Fusarium solani species complex lineage 6), one of the most frequent agents of human opportunistic infections. The species is described with multilocus molecular data including sequences of internal transcribed spacer region (ITS), portions of the translation elongation factor 1-a gene (TEF1), and the partial RNA polymerase II gene (rPB2). A phylogenetic approach was used to evaluate species delimitation. Topologies of the trees were concordant. Phylogenetic analyses suggest that the FSSC consists of three major clades encompassing a large number of phylogenetic species; Fusarium metavorans corresponds to phylogenetic species 6 within FSSC clade 3. The species has a global distribution and a wide ecological amplitude, also including strains from soil and agents of opportunistic plant disease; it was also isolated from the gut of the wood-boring cerambycid beetle Anoplophora glabripennis.

Two new species of the Fusarium solani species complex isolated from compost and hibiscus (Hibiscus sp.).

Two new species in the Fusarium solani species complex (FSSC) are described and introduced. The new taxa are represented by German isolates CBS 142481 and CBS 142480 collected from commercial yard waste compost and vascular tissue of a wilting branch of hibiscus, respectively. The phylogenetic relationships of the collected strains to one another and within the FSSC were evaluated based on DNA sequences of 6 gene loci. Due to the limited sequence data available for reference strains in GenBank, however, a multi-gene phylogenetic analysis included partial sequences for the internal transcribed spacer region and intervening 5.8S nrRNA gene (ITS), translation elongation factor 1-alpha (tef1) and the RNA polymerase II second largest subunit (rpb2). Morphological and molecular phylogenetic data independently showed that these strains are distinct populations of the FSSC, nested within Clade 3. Thus, we introduce Fusarium stercicola and Fusarium witzenhausenense as novel species in the complex. In addition, 19 plant species of 7 legume genera were evaluated for their potential to host the newly described taxa. Eighteen plant species were successfully colonized, with 6 and 9 of these being symptomatic hosts for F. stercicola and F. witzenhausenense, respectively. As plants of the family Fabaceae are very distant to the originally sourced material from which the new taxa were recovered, our results suggest that F. stercicola and F. witzenhausenense are not host-specific and are ecologically fit to sustain stable populations in variety of habitats.

Two new species of the Fusarium fujikuroi species complex isolated from the natural environment.

Two new species in the Fusarium fujikuroi species complex (FFSC) are introduced. One of these, represented by strain CBS 454.97 was isolated from plant debris (Striga hermonthica) in the Sudan, while the second, represented by strains CBS 119850 and CBS 483.94, which originated from soil in Australia. Molecular analyses were performed including TEF1 spanning 576 bp region, 860 bp region of rPB2, and 500 bp BT2 region. Phylogenetic trees based on these regions showed that the two species are clearly distinct from all known taxa in the F. fujikuroi species complex. Based on phenotypic, physiological characters and molecular data, we introduce Fusarium sudanense and Fusarium terricola as novel species in the complex.

Fusarium species causing eumycetoma: Report of two cases and comprehensive review of the literature.

Recently, mycetoma was added to the World Health Organization's list of neglected tropical disease priorities. Fusarium as a genus has been reported to cause eumycetoma, but little is known about the species involved in this infection and their identification. In this study, molecular tools were applied to identify Fusarium agents from human eumycetoma cases. The partial translation elongation factor 1-alpha (TEF-1α) gene was used as diagnostic parameter. Two additional cases of eumycetoma, due to F. keratoplasticum and F. pseudensiforme, respectively, are presented. A systematic literature review was performed to assess general features, identification, treatment and outcome of eumycetoma infections due to Fusarium species. Of the 20 reviewed patients, the majority (75%) were male. Most agents belonged to the F. solani species complex, ie F. keratoplasticum, F. pseudensiforme, and an undescribed lineage of F. solani. In addition, F. thapsinum, a member of Fusarium fujikuroi species complex was encountered. The main antifungal drugs used were itraconazole, ketoconazole and amphotericin B, but cure rates were low (15%). Partial response or relapse was observed in some cases, and a case ended in amputation. Clinical management of eumycetoma due to Fusarium is complex and combination therapy might be required to increase cure rates.

Phaeohyphomycosis Caused by a Novel Species, Pseudochaetosphaeronema martinelli.

Among the opportunistic mycoses that are emerging in patients with immunosuppression or severe underlying illness, many isolates lack of characteristic sporulation and until recently could not be identified. Clinical signs are mostly nonspecific and therefore such infections have often been disregarded. In the present paper we describe a novel, nonsporulating fungal species causing subcutaneous phaeohyphomycosis in two patients of different origin. One is a 73-year-old female from Martinique who suffered from rheumatoid arthritis, while the other case concerns a 72-year-old male from Mexico who had a history of type 2 diabetes mellitus. Sequencing of the partial ribosomal operon revealed that in both cases a member of the order Pleosporales was concerned which could not be affiliated to any family within this order. Multilocus analysis revealed that the fungus was related to another, unaffiliated agent of human mycetoma, Pseudochaetosphaeronema larense, and therefore the name Pseudochaetosphaeronema martinelli was introduced.

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

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

Seventeen years of subcutaneous infection by Aspergillus flavus; eumycetoma confirmed by immunohistochemistry.

Chronic subcutaneous infections caused by Aspergillus species are considered to be extremely rare. Because these fungi are among the most common laboratory contaminants, their role as eumycetoma causative agents is difficult to ascertain. Here, we report the first case of A. flavus eumycetoma confirmed by isolation, molecular identification and immunohistochemical analysis. Patient was a 55-year-old male from Sudan suffering from eumycetoma on his left foot for a period of 17 years. He developed swelling, sinuses and white grain discharge was observed. He has been operated nine times and was treated with several regimens of ketoconazole and itraconazole without improvement. Initial diagnosis based on histology and radiology was Scedosporium eumycetoma. However, examination of the biopsy revealed A. flavus, which was identified by molecular analysis and MALDI-TOF MS. Immunohistochemistry using antibody directed against Aspergillus species was positive. Because of the earlier treatment failures with ketoconazole and itraconazole, therapy with voriconazole was initiated. However, in vitro susceptibility testing yielded a lower Minimum Inhibitory Concentration (MIC) value for itraconazole (0.25 µg ml(-1) ) than for voriconazole (1 µg ml(-1) ). Based on the presented results, A. flavus can be considered as one of the agents of white-grain eumycetoma.

Roussoella percutanea, a novel opportunistic pathogen causing subcutaneous mycoses.

We report the isolation of a novel agent of subcutaneous mycosis from a 45-year-old Indian male immigrant in the United States. Phylogenetic analysis of partial small ribosomal subunit and large ribosomal subunit, internal transcribed spacer, partial translation elongation factor (TEF1), and RNA polymerase second largest subunit (rPB2) loci revealed that the strain was identical to another isolate previously reported as "Madurella mycetomatis." Both strains clustered in the order Pleosporales, nested in the family Arthopyreniaceae/Roussoellaceae. The fungus differed from known species and hence a new taxon, Roussoella percutanea, is introduced, typified by a strain that showed delayed production of pycnidial conidiomata. Antifungal susceptibility testing suggested that the new species is resistant to echinocandins and flucytosine, with variable results with azoles and amphotericin B.

Description of new micro-colonial fungi species Neophaeococcomyces mojavensis, Coniosporium tulheliwenetii, and Taxawa tesnikishii cultured from biological soil crusts.

Black yeasts and relatives comprise Micro-Colonial Fungi (MCFs) which are slow-growing stress-tolerant micro-eukaryotes that specialize in extreme environments. MCFs are paraphyletic and found in the Orders Chaetothyriales (Eurotiomycetes) and Dothideales (Dothidiomycetes). We have isolated and described three new MCFs species from desert biological soil crusts (BSCs) collected from two arid land regions: Joshua Tree National Park (Mojave Desert) and UC Natural Reserve at Boyd Deep Canyon (confluence of Mojave and Sonoran Deserts). BSCs are composite assemblages of cyanobacteria, eukaryotic algae, fungi, lichens, and bryophytes embedded into the surface of desert soils, providing a protective buffer against the harsh desert environment. Our work focused on one type of desert BSC, the cyanolichen crust dominated by Collema sp. Using culture-dependent protocols, three MCFs were axenically isolated from their respective samples along with the extracted DNA. Their genomes were sequenced using Illumina and Nanopore, and finally assembled and annotated using hybrid assembly approaches and established bioinformatics pipelines to conduct final taxonomic phylogenetic analysis and placement. The three species described here are unique specimen from desert BSCs, here we introduce, Neophaeococcomyces mojavensis (Chaetothyriales), Cladosporium tulheliwenetii (Dothideales), and Taxawa tesnikishii (Dothideales).