Antifungal Susceptibility of 182 Fusarium Species Isolates from 20 European Centers: Comparison between EUCAST and Gradient Concentration Strip Methods.

We determined the susceptibility of 182 Fusarium species isolates to five antifungal drugs (amphotericin B, voriconazole, posaconazole, isavuconazole, and terbinafine) by the EUCAST method. Based on the latest taxonomic insights, isolates collected from 20 European centers were distributed into seven complexes and 27 species. The susceptibility was variable, depending on the species. Comparison with the gradient concentration strip method, which was used for 77 isolates, showed essential agreement values for voriconazole, posaconazole, isavuconazole, and amphotericin B of 17%, 91%, 83%, and 70%, respectively.

Identification of Molds with Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry: Performance of the Newly Developed MSI-2 Application in Comparison with the Bruker Filamentous Fungi Database and MSI-1.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) represents a promising tool for the rapid and efficient identification of molds, but improvements are still necessary to achieve satisfactory results when identifying cryptic species. Here, we aimed to validate a new web application, MSI-2, which replaces MSI-1, an application that was built and deployed online in 2017. For the evaluation, we gathered 633 challenging isolates obtained from daily hospital practice that were first identified with DNA-based methods, and we submitted their corresponding mass spectra to three identification programs (Bruker, MSI-1, and MSI-2). The MSI-2 application had a better identification performance at the species level than MSI-1 and Bruker, reaching 83.25% correct identifications, compared with 63.19% (MSI-1), 38.07% (Bruker with a 1.7 threshold), and 21.8% (Bruker with a 2.0 threshold). The MSI-2 application performed especially well for Aspergillus and Fusarium species, including for many cryptic species, reaching 90% correct identifications for Aspergillus species and 78% for Fusarium species compared to 69% and 43% with MSI-1. Such an improvement may have a positive impact on patient management by facilitating the identification of cryptic species potentially associated with a specific antifungal resistance profile.

Lineages Within the Trichophyton rubrum Complex.

The most important species of the Trichophyton rubrum group are T. rubrum, causing mainly skin and nail infections, and T. violaceum which is mostly scalp-associated. The status of a third species, T. soudanense, has been under debate. With a polyphasic approach, using molecular phylogenetic techniques, MALDI-TOF mass spectrometry and physiological and morphological analysis, we re-evaluated the T. rubrum complex. Our results support four genetic lineages within the complex each with a distinct morphology and identifiable via MALDI-TOF MS: T. rubrum, T. violaceum, T. soudanense and the T. yaoundei clade. However, ITS and Bt2 sequencing data could not confirm these taxa as four monophyletic species. Our results also suggest that strains formerly identified as T. kuryangei and T. megninii should be considered in future taxonomic studies.

Black aspergilli: A remaining challenge in fungal taxonomy?

Aspergillus section Nigri is a taxonomically difficult but medically and economically important group. In this study, an update of the taxonomy of A. section Nigri strains within the BCCM/IHEM collection has been conducted. The identification accuracy of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was tested and the antifungal susceptibilities of clinical isolates were evaluated. A total of 175 strains were molecularly analyzed. Three regions were amplified (ITS, benA, and caM) and a multi-locus phylogeny of the combined loci was created by using maximum likelihood analysis. The in-house MALDI-TOF MS reference database was extended and an identification data set of 135 strains was run against a reference data set. Antifungal susceptibility was tested for voriconazole, itraconazole, and amphotericin B, using the EUCAST method. Phylogenetic analysis revealed 18 species in our data set. MALDI-TOF MS was able to distinguish between A. brasiliensis, A. brunneoviolaceus, A. neoniger, A. niger, A. tubingensis, and A. welwitschiae of A. sect. Nigri. In the routine clinical lab, isolates of A. sect. Nigri are often identified as A. niger. However, in the clinical isolates of our data set, A. tubingensis (n = 35) and A. welwitschiae (n = 34) are more common than A. niger (n = 9). Decreased antifungal susceptibility to azoles was observed in clinical isolates of the /tubingensis clade. This emphasizes the importance of identification up to species level or at least up to clade level in the clinical lab. Our results indicate that MALDI-TOF MS can be a powerful tool to replace classical morphology.

Multi-centric evaluation of the online MSI platform for the identification of cryptic and rare species of Aspergillus by MALDI-TOF.

The taxonomy of Aspergillus species has recently been revolutionized with the introduction of cryptic species and section concepts. However, their species-level identification in routine laboratories remains a challenge. The aim of this study was to prospectively assess the identification accuracy of cryptic species of Aspergillus in various laboratories using the mass spectrometry identification (MSI) platform, an independent and freely accessible online mass spectrometry database. Over a 12-month period, when a select set of MSI users identified cryptic species, they were contacted and requested to send the isolates to our laboratory for sequence-based identification. Sequence and MSI identification results were then compared. During the study period, 5108 Aspergillus isolates were identified using MSI including 1477 (28.9%) cryptic species. A total of 245 isolates that corresponded to 56 cryptic species and 13 sections were randomly selected for DNA sequencing confirmation. Agreement between the two methods was 99.6% at the section level and 66.1% at the species level. However, almost all discrepancies (72/83, 86.7%) were misidentifications between closely related cryptic species belonging to the same section. Fifty-one isolates from noncryptic species were also identified, thus yielding 100% and 92.2% agreement at the section and species level, respectively. Although the MSI fungus database is a reliable tool to identify Aspergillus at the section level, the database still requires adjustment to correctly identify rare or cryptic species at the species level. Nevertheless, the application properly differentiated between cryptic and sensu stricto species in the same section, thus alerting on possible specific isolate characteristics.

Toward a Novel Multilocus Phylogenetic Taxonomy for the Dermatophytes.

Type and reference strains of members of the onygenalean family Arthrodermataceae have been sequenced for rDNA ITS and partial LSU, the ribosomal 60S protein, and fragments of ß-tubulin and translation elongation factor 3. The resulting phylogenetic trees showed a large degree of correspondence, and topologies matched those of earlier published phylogenies demonstrating that the phylogenetic representation of dermatophytes and dermatophyte-like fungi has reached an acceptable level of stability. All trees showed Trichophyton to be polyphyletic. In the present paper, Trichophyton is restricted to mainly the derived clade, resulting in classification of nearly all anthropophilic dermatophytes in Trichophyton and Epidermophyton, along with some zoophilic species that regularly infect humans. Microsporum is restricted to some species around M. canis, while the geophilic species and zoophilic species that are more remote from the human sphere are divided over Arthroderma, Lophophyton and Nannizzia. A new genus Guarromyces is proposed for Keratinomyces ceretanicus. Thirteen new combinations are proposed; in an overview of all described species it is noted that the largest number of novelties was introduced during the decades 1920-1940, when morphological characters were used in addition to clinical features. Species are neo- or epi-typified where necessary, which was the case in Arthroderma curreyi, Epidermophyton floccosum, Lophophyton gallinae, Trichophyton equinum, T. mentagrophytes, T. quinckeanum, T. schoenleinii, T. soudanense, and T. verrucosum. In the newly proposed taxonomy, Trichophyton contains 16 species, Epidermophyton one species, Nannizzia 9 species, Microsporum 3 species, Lophophyton 1 species, Arthroderma 21 species and Ctenomyces 1 species, but more detailed studies remain needed to establish species borderlines. Each species now has a single valid name. Two new genera are introduced: Guarromyces and Paraphyton. The number of genera has increased, but species that are relevant to routine diagnostics now belong to smaller groups, which enhances their identification.

Use of matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of molds of the Fusarium genus.

The rates of infection with Fusarium molds are increasing, and a diverse number of Fusarium spp. belonging to different species complexes can cause infection. Conventional species identification in the clinical laboratory is time-consuming and prone to errors. We therefore evaluated whether matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a useful alternative. The 289 Fusarium strains from the Belgian Coordinated Collections of Microorganisms (BCCM)/Institute of Hygiene and Epidemiology Mycology (IHEM) culture collection with validated sequence-based identities and comprising 40 species were used in this study. An identification strategy was developed, applying a standardized MALDI-TOF MS assay and an in-house reference spectrum database. In vitro antifungal testing was performed to assess important differences in susceptibility between clinically relevant species/species complexes. We observed that no incorrect species complex identifications were made by MALDI-TOF MS, and 82.8% of the identifications were correct to the species level. This success rate was increased to 91% by lowering the cutoff for identification. Although the identification of the correct species complex member was not always guaranteed, antifungal susceptibility testing showed that discriminating between Fusarium species complexes can be important for treatment but is not necessarily required between members of a species complex. With this perspective, some Fusarium species complexes with closely related members can be considered as a whole, increasing the success rate of correct identifications to 97%. The application of our user-friendly MALDI-TOF MS identification approach resulted in a dramatic improvement in both time and accuracy compared to identification with the conventional method. A proof of principle of our MALDI-TOF MS approach in the clinical setting using recently isolated Fusarium strains demonstrated its validity.

International Society of Human and Animal Mycology (ISHAM)-ITS reference DNA barcoding database--the quality controlled standard tool for routine identification of human and animal pathogenic fungi.

Human and animal fungal pathogens are a growing threat worldwide leading to emerging infections and creating new risks for established ones. There is a growing need for a rapid and accurate identification of pathogens to enable early diagnosis and targeted antifungal therapy. Morphological and biochemical identification methods are time-consuming and require trained experts. Alternatively, molecular methods, such as DNA barcoding, a powerful and easy tool for rapid monophasic identification, offer a practical approach for species identification and less demanding in terms of taxonomical expertise. However, its wide-spread use is still limited by a lack of quality-controlled reference databases and the evolving recognition and definition of new fungal species/complexes. An international consortium of medical mycology laboratories was formed aiming to establish a quality controlled ITS database under the umbrella of the ISHAM working group on "DNA barcoding of human and animal pathogenic fungi." A new database, containing 2800 ITS sequences representing 421 fungal species, providing the medical community with a freely accessible tool at http://www.isham.org/ and http://its.mycologylab.org/ to rapidly and reliably identify most agents of mycoses, was established. The generated sequences included in the new database were used to evaluate the variation and overall utility of the ITS region for the identification of pathogenic fungi at intra-and interspecies level. The average intraspecies variation ranged from 0 to 2.25%. This highlighted selected pathogenic fungal species, such as the dermatophytes and emerging yeast, for which additional molecular methods/genetic markers are required for their reliable identification from clinical and veterinary specimens.

Banana infecting fungus, Fusarium musae, is also an opportunistic human pathogen: are bananas potential carriers and source of fusariosis?

During re-identification of Fusarium strains in the BCCM™/IHEM fungal collection by multilocus sequence-analysis we observed that five strains, previously identified as Fusarium verticillioides, were Fusarium musae, a species described in 2011 from banana fruits. Four strains were isolated from blood samples or biopsies of immune-suppressed patients and one was isolated from the clinical environment, all originating from different hospitals in Belgium or France, 2001-2008. The F. musae identity of our isolates was confirmed by phylogenetic analysis using reference sequences of type material. Absence of the gene cluster necessary for fumonisin biosynthesis, characteristic to F. musae, was also the case for our isolates. In vitro antifungal susceptibility testing revealed no important differences in their susceptibility compared to clinical F. verticillioides strains and terbinafine was the most effective drug. Additional clinical F. musae strains were searched by performing BLAST queries in GenBank. Eight strains were found, of which six were keratitis cases from the U.S. multistate contact lens-associated outbreak in 2005 and 2006. The two other strains were also from the U.S., causing either a skin infection or sinusitis. This report is the first to describe F. musae as causative agent of superficial and opportunistic, disseminated infections in humans. Imported bananas might act as carriers of F. musae spores and be a potential source of infection with F. musae in humans. An alternative hypothesis is that the natural distribution of F. musae is geographically a lot broader than originally suspected and F. musae is present on different plant hosts.

Is Trichophyton simii endemic to the Indian subcontinent?

Trichophyton simii is considered to be prevalent only in the Indian subcontinent where it was isolated from soil, as well as from infections of humans and animals. We have investigated a case of onychomycosis caused by this exotic dermatophyte, not traceable to endemic areas. This case, as in others due to this fungus in man or animals, that have been previously and sporadically reported worldwide, suggests infections caused by T. simii might be underestimated, especially outside its primary geographic areas. Indeed, there are isolates that do not show species-specific morphology, as in our case isolate, and as a result may be misidentified by classical methods. By checking the identity of some strains preserved in the collection BCCM/IHEM, we found several that proved to be T. simii, originating from non-endemic areas (Belgium, France and Ivory Coast). Therefore, the natural distribution of T. simii is probably not as restricted as has previously been proposed.

An atypical, pigment-producing Metschnikowia strain from a leukaemia patient.

A yeast strain was isolated from the sputum sample of a leukaemia patient in the Spirito Santo Hospital of Pescara, Italy. The fungus produced a pigment that formed a reddish halo around colonies, and was identified and deposited as a Metschnikowia spp. (accession number IHEM 25107-GenBank accession number JQ921016) in the BCCM/IHEM collection of biomedical fungi and yeasts (Bruxelles, Belgium). Although the physiology of the strain was close to that of Metschnikowia sinensis, the D1/D2 sequence did not correspond to any previously described Metschnikowia species. Phylogeny of the genus Metschnikowia is complex and requires far more analysis. We present the first non-M. pulcherrima Metschnikowia spp. isolate recovered from a human, and emphasize the role of man as a transient carrier of environmental yeasts, the pathogenicity of which still needs to be defined.

Remarkable fungal biodiversity on northern Belgium bats and hibernacula.

Bats can be affected by fungal pathogens such as Pseudogymnoascus destructans, the causative agent of the white-nose syndrome. Their body surface can also be colonized by fungal commensals or carry transient fungal species and participate in their dispersal. In this study, 114 bat specimens belonging to seven species were sampled from various locations in northern Belgium. Culture-based methods revealed an important mycological diversity, with a total of 209 different taxa out of the 418 isolates. Overall, a mean of 3.7 taxa per bat was recorded, but significant differences were observed between sampling sites and seasons. The mycobiomes were dominated by cosmopolitan and plant-associated species, in particular from the genera Cladosporium, Penicillium, and Aspergillus. Other species known to be related to bats or their environment, such as Apiotrichum otae, were also retrieved. Sampling of hibernacula indicated that diverse fungal species can inhabit these sites, including a yet undescribed Pseudogymnoascus species, distinct from Ps. destructans, namely, Ps. cavicola.

The taxonomic status of Trichophyton quinckeanum and T. interdigitale revisited: a multigene phylogenetic approach.

Trichophyton quinckeanum, known as the causative agent of mouse favus, has been a subject of controversy since its discovery, 125 years ago. The purpose of this study was to examine the phylogenetic relationships between this fungus and related taxa. To achieve this objective, the ITS rDNA region, as well as actin and ß-tubulin gene regions of various isolates were sequenced. Bayesian inference and maximum likelihood analyses were conducted with T. rubrum as outgroup. Our study showed that strains identified as T. quinckeanum and others identified as T. schoenleinii are part of the complex T. mentagrophytes, and that their genotype cannot be confused with any other dermatophytes. Furthermore, this study demonstrates that the choice of the neotype of T. mentagrophytes was inappropriate. The beta-tubulin topology also revealed that isolates of T. interdigitale form a genetically distinct population from the type strains of Arthroderma vanbreuseghemii. Therefore, contrary to what is generally accepted, the anthropophilic species T. interdigitale cannot be considered as the anamorph associated with the latter.

Lactarius subg. Plinthogalus: the European taxa and American varieties of L. lignyotus re-evaluated.

The European species Lactarius subg. Plinthogalus were subjected to a molecular phylogenetic analysis based on ITS, LSU and rpb2 sequences. Morphological characters of the species are discussed in the light of the phylogenetic results. In addition to a broad sampling within Europe, some Asian and North American taxa also were included in the analysis. Eight European species are confirmed molecularly: L. lignyotus, L. acris, L. azonites, L. pterosporus, L. ruginosus, L. romagnesii, L. fuliginosus and L. picinus. Except the sibling species L. fuliginosus and L. picinus, all are morphologically distinct. Our results suggest that L. fuliginosus is associated exclusively with broadleaf trees and L. picinus with conifers, but this putative difference in host specificity needs to be investigated further. Lactarius subruginosus turns out to be a synonym of either L. pterosporus or L. ruginosus. The position of Lactarius terenopus remains to be clarified. The North American taxa that are closely related to the European L. lignyotus (L. fallax, L. lignyotus var. canadensis, var. nigroviolascens, var. marginatus) are not resolved. Intercontinental conspecificity was demonstrated between Europe and northern Asia but was not found between Europe and southern Asia or between Europe and North America. A taxonomic subdivision of L. subg. Plinthogalus based on the height of the spore ornamentation should be rejected.

A new species of Lactarius (subgenus Gerardii) from two relict Fagus grandifolia var. mexicana populations in Mexican montane cloud forests.

A new milkcap species, Lactarius fuscomarginatus, was found in the subtropical region of central Veracruz (eastern Mexico) associated with two relict populations of Fagus grandifolia var. mexicana. The species is characterized macroscopically by its dark pileus and stipe and by its distant and whitish lamellae with blackish to blackish brown edges. A molecular phylogenetic analyses based on ITS and LSU nucDNA sequences confirms the delimitation of this new taxon and places L. fuscomarginatus in subgenus Gerardii. A detailed morphological comparison is given with similar species.

A Polyphasic Approach to Classification and Identification of Species within the Trichophyton benhamiae Complex.

In recent years, considerable advances have been made in clearing up the phylogenetic relationships within the Arthrodermataceae family. However, certain closely related taxa still contain poorly resolved species boundaries. Here, we tried to elucidate the species composition of the Trichophyton benhamiae species complex using a combined approach consisting of multi-gene phylogenetic analysis based on internal transcribed spacer (ITS) and beta-tubulin (BT) gene regions, morphological analysis, and spectral comparison using MALDI-ToF. We confirmed the existence of 11 different monophyletic clades within the complex representing either species or genetically distinct groups within species. MALDI-ToF spectrometry analysis revealed that most of these clades were readily distinguishable from one another; however, some closely related sister clades, such as T. europaeum and T. japonicum, were often misidentified as their counterpart. The distinct "yellow" and "white" phenotypes of T. benhamiae do not have a clear genetic basis and should thus be considered as different morphotypes of the same species. Strains traditionally considered T. benhamiae can be divided into three main clades: (i) T. benhamiae, (ii) T. europaeum/T. japonicum, and (iii) the phylogenetically distant T. africanum. While T. europaeum and T. japonicum are distinguishable based on their genotype, spectral and morphological analysis did not provide clear delimiting characteristics.

The taxonomy of the Trichophyton rubrum complex: a phylogenomic approach.

The medically relevant Trichophyton rubrum species complex has a variety of phenotypic presentations but shows relatively little genetic differences. Conventional barcodes, such as the internal transcribed spacer (ITS) region or the beta-tubulin gene, are not able to completely resolve the relationships between these closely related taxa. T. rubrum, T. soudanense and T. violaceum are currently accepted as separate species. However, the status of certain variants, including the T. rubrum morphotypes megninii and kuryangei and the T. violaceum morphotype yaoundei, remains to be deciphered. We conducted the first phylogenomic analysis of the T. rubrum species complex by studying 3105 core genes of 18 new strains from the BCCM/IHEM culture collection and nine publicly available genomes. Our analyses revealed a highly resolved phylogenomic tree with six separate clades. Trichophyton rubrum, T. violaceum and T. soudanense were confirmed in their status of species. The morphotypes T. megninii, T. kuryangei and T. yaoundei all grouped in their own respective clade with high support, suggesting that these morphotypes should be reinstituted to the species-level. Robinson-Foulds distance analyses showed that a combination of two markers (a ubiquitin-protein transferase and a MYB DNA-binding domain-containing protein) can mirror the phylogeny obtained using genomic data, and thus represent potential new markers to accurately distinguish the species belonging to the T. rubrum complex.

Identification of fungal isolates by MALDI-TOF mass spectrometry in veterinary practice: validation of a web application.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a reliable method to identify fungal isolates. The success of this approach relies on the availability of exhaustive databases, but the latter were built with a focus on human pathogens. We assessed a large in-house database of reference spectra and a dedicated web application for their suitability for use in veterinary laboratories. A panel of 290 mold and yeast isolates representing 69 different fungal species was isolated from various animals (including pets, cattle, and zoo animals) and identified using both MALDI-TOF MS and conventional techniques. The performance of the 2 methods was compared, and identifications were confirmed by DNA sequencing. MALDI-TOF MS allowed distinction between some closely related species and achieved 89% correct identification at the species level. In comparison, only 60% of the isolates were correctly identified with conventional approaches. Using this online application, MALDI-TOF MS thus appears to be a relevant alternative for the identification of fungal isolates encountered by animal health professionals.

Novel diversity in Lactifluus section Gerardii from Asia: five new species with pleurotoid or small agaricoid basidiocarps.

The ectomycorrhizal milkcap genus Lactifluus (Russulaceae) is commonly found in tropical and subtropical forests of Southeast Asia. During several sampling expeditions in Thailand, multiple collections of Lactifluus (Lf.) species with pleurotoid or small agaricoid basidiocarps were found. A molecular study was combined with a morphological study, in which the collections were compared with herbarium material. The molecular study indicated that four Thai collections belonged to undescribed species within Lactifluus section Gerardii, as was also the case for herbarium collections of a pleurotoid species from Nepal. One other collection from Thailand appeared closely related to Lf. uyedae, known only from Japan. Five species are described as new in the genus: Lf. auriculiformis, Lf. gerardiellus, Lf. bhandaryi, Lf. pulchrellus, and Lf. raspei.