Gene expression profiling in the human pathogenic dermatophyte Trichophyton rubrum during growth on proteins.

Dermatophytes are highly specialized filamentous fungi which cause the majority of superficial mycoses in humans and animals. The high secreted proteolytic activity of these microorganisms during growth on proteins is assumed to be linked to their particular ability to exclusively infect keratinized host structures such as the skin stratum corneum, hair, and nails. Individual secreted dermatophyte proteases were recently described and linked with the in vitro digestion of keratin. However, the overall adaptation and transcriptional response of dermatophytes during protein degradation are largely unknown. To address this question, we constructed a cDNA microarray for the human pathogenic dermatophyte Trichophyton rubrum that was based on transcripts of the fungus grown on proteins. Profiles of gene expression during the growth of T. rubrum on soy and keratin protein displayed the activation of a large set of genes that encode secreted endo- and exoproteases. In addition, other specifically induced factors potentially implicated in protein utilization were identified, including heat shock proteins, transporters, metabolic enzymes, transcription factors, and hypothetical proteins with unknown functions. Of particular interest is the strong upregulation of key enzymes of the glyoxylate cycle in T. rubrum during growth on soy and keratin, namely, isocitrate lyase and malate synthase. This broad-scale transcriptional analysis of dermatophytes during growth on proteins reveals new putative pathogenicity-related host adaptation mechanisms of these human pathogenic fungi.

The genome of the leaf-cutting ant Acromyrmex echinatior suggests key adaptations to advanced social life and fungus farming.

We present a high-quality (>100× depth) Illumina genome sequence of the leaf-cutting ant Acromyrmex echinatior, a model species for symbiosis and reproductive conflict studies. We compare this genome with three previously sequenced genomes of ants from different subfamilies and focus our analyses on aspects of the genome likely to be associated with known evolutionary changes. The first is the specialized fungal diet of A. echinatior, where we find gene loss in the ant's arginine synthesis pathway, loss of detoxification genes, and expansion of a group of peptidase proteins. One of these is a unique ant-derived contribution to the fecal fluid, which otherwise consists of "garden manuring" fungal enzymes that are unaffected by ant digestion. The second is multiple mating of queens and ejaculate competition, which may be associated with a greatly expanded nardilysin-like peptidase gene family. The third is sex determination, where we could identify only a single homolog of the feminizer gene. As other ants and the honeybee have duplications of this gene, we hypothesize that this may partly explain the frequent production of diploid male larvae in A. echinatior. The fourth is the evolution of eusociality, where we find a highly conserved ant-specific profile of neuropeptide genes that may be related to caste determination. These first analyses of the A. echinatior genome indicate that considerable genetic changes are likely to have accompanied the transition from hunter-gathering to agricultural food production 50 million years ago, and the transition from single to multiple queen mating 10 million years ago.

Mechanisms of antifungal resistance in the opportunistic fungus Aspergillus fumigatus

ABSTRACT Aspergillus fumigatus is one of the most prevalent airbone fungal pathogen and can cause severe fatal invasive aspergillosis in immunocompromised patients. Several antifungal agents are available to treat these infections but with limited success. These agents include polyenes (amphotericin B), echinocandins (caspofungin) and azoles, which constitute the most important class with itraconazole (ITC) and voriconazole as major active compounds. Azole-derived antifungal agents target the ergosterol biosynthesis pathway via the inhibition of the lanosterol 14α-demethylase (cyp51/ERG1 1), a cytochrome P450 responsible for the conversion of lanosterol to ergosterol, which is the main component of cell membrane in fungi. A. fumigatus is also found in the environment as a contaminant of rotting plant or present in composting of organic waste. Among antifungal agents used in the environment for crop protection, the class of azoles is also widely used with propiconazole or prochloraz as examples. However, other agents such as dicarboximide (iprodione), phenylamide (benalaxyl) or strobilurin (azoxystrobin) are also used. Emergence of clinical azole-resistant isolates has been described in several European countries. However the incidence of antifungal resistance has not been yet reported in details in Switzerland. In this study, the status of antifungal resistance was investigated on A. fumigatus isolates collected from Swiss hospitals and from different environmental sites and. tested for their susceptibility to several currently used antifungal agents. The data showed a low incidence of resistance for all tested agents among clinical and environmental isolates. Only two azole-resistant environmental isolates were detected and none among the clinical tested isolates. In general, A. fumigatus was susceptible to all antifungals tested in our study, except to azoxystrobin which was the less active agent against all isolates. Since mechanisms of antifungal resistance have been poorly investigated until now in A. fumigatus, this work was aimed 1) to identify A. fumigatus genes involved in antifungal resistance and 2) to test their involvement in the development of resistance in sampled isolates. Therefore, this work proposed to isolate A. fumigatus genes conferring resistance to a drug-hypersusceptible Saccharomyces cerevisiae strain due to a lack of multidrug transporter genes. Several genes were recovered including three distinct efflux transporters (atrF, atrH and mdrA) and a bZip transcription factor, yapA. The inactivation of each transporter in A. fumigatus indicated that the transporters were involved in the basal level of azole susceptibility. The inactivation of YapA led to a hypersusceptibility to H2O2, thus confirming the involvement of this gene in the oxidative stress response of A. fumigatus. The involvement of the abovementioned transporters genes and of other transporters genes identified by genome analysis in azole resistance was tested by probing their expression in some ITC-resistant isolates. Even if upregulation of some transporters genes was observed in some investigated isolates, the correlation between azole resistance and expression levels of all these transporters genes could not be clearly established for all tested isolates. Given these results, the present work addressed 1) alteration in the expression of cyp51A encoding for the azole target enzyme, and 2) mutation(s) in the cyp51A sequence as potential mechanisms of azote resistance in A. . However, overexpression of cyp51A in the investigated isolates was not linked with azote resistance. Since it was reported that mutation(s) in cyp51A were participating in azote resistance in A. fumigatus, a functional complementation of cyp51A cDNAs from ITC-resistant A. fumigatus strains in S. cerevisiae ergl 1 Δ mutant strain was attempted. Expression in S. cerevisiae allowed the testing of these cDNAs with regards to their functionality and involvement in resistance to specific azote compounds. We could demonstrate that Cyp51A protein with a G54E or M220K mutations conferred resistance to specific azoles in S. cerevisiae, therefore suggesting that these mutations were important for the development of azote resistance in A. fumigatus. In conclusion, this work showed a correlation between ITC resistance and mechanisms involving overexpression of transporters and cyp51A mutations in A. fumigatus isolates. However, azole resistance of some isolates has not been solved and thus it will be necessary to approach the study of resistance mechanisms in this fungal species using alternative methodologies. RESUME Aspergillus fumigatus est un champignon opportuniste répandu et est la cause d'aspergilloses invasives le plus souvent fatales chez des patients immunodéprimés. Plusieurs antifongiques sont disponibles afin de traiter ces infections, cependant avec un succès limité. Ces agents incluent les polyènes (amphotericin B), les échinocandines (caspofungin) et les azoles, qui représentent la plus importante classe d'antifongiques avec l'itraconazole (ITC) et le voriconazole comme principaux agents actifs. Les dérivés azolés ciblent la voie de biosynthèse de l'ergostérol via l'inhibition de la lanostérol 14α-demethylase (cyp51/ERG11), un cytochrome P450 impliqué dans la conversion du lanostérol en ergostérol, qui est un composant important de la membrane chez les champignons. A. fumigatus est également répandu dans l'environnement. Parmi les antifongiques employés en agriculture afin de protéger les cultures, les azoles sont aussi largement utilisés. Cependant, d'autres agents tels que les dicarboximides (iprodione), les phenylamides (benalaxyl) et les strobilurines (azoxystrobin) peuvent être également utilisés. L'émergence de souches cliniques résistantes aux azoles a été décrite dans différents pays européens. Cependant, l'incidence d'une telle résistance aux azoles n'a pas encore été reportée en détails en Suisse. Dans ce travail, l'émergence de la résistance aux antifongiques a été étudiée par analyse de souches d'A. fumigatus provenant de milieux hospitaliers en Suisse et de différents sites et leur susceptibilité testée envers plusieurs antifongiques couramment utilisés. Les données obtenues ont montré une faible incidence de la résistance parmi les souches cliniques et environnementales pour les agents testés. Seulement deux souches environnementales résistantes aux azoles ont été détectées et aucune parmi les souches cliniques. Les mécanismes de résistance aux antifongiques ayant été très peu étudiés jusqu'à présent chez A. fumigatus , ce travail a eu aussi pour but 1) d'identifier les gènes d' A. fumigatus impliqués dans la résistance aux antifongiques et 2) de tester leur implication dans la résistance de certaines souches. Ainsi, il a été proposé d'isoler les gènes d' A. fumigatus pouvant conférer une résistance aux antifongiques à une souche de Saccharomyces cerevisiae hypersensible aux antifongiques. Trois transporteurs à efflux (atrF, atrH et mdrA) et un facteur de transcription appartenant à la famille des bZip (YapA) ont ainsi été isolés. L'inactivation, dans une souche d'A. fumigatus, de chacun des ces transporteurs a permis de mettre en évidence leur implication dans la susceptibilité d'A. fumigatus aux antifongiques. L'inactivation de YapA a engendré une hypersusceptibilité à l' H2O2, confirmant ainsi le rôle de ce gène dans la réponse au stress oxydatif chez A . fumigatus. La participation dans la résistance aux antifongiques des gènes codant pour des transporteurs ainsi que d'autres gènes identifiés par analyse du génome a été déterminée en testant leur niveau d'expression dans des souches résistantes à l'ITC. Bien qu'une surexpression de transporteurs ait été observée dans certaines souches, une corrélation entre la résistance à l'ITC et les niveaux d'expression de ces transporteurs n'a pu être clairement établie. Ce présent travail s'est donc porté sur l'étude de 2 autres mécanismes potentiellement impliqués dans la résistance aux azoles : 1) la surexpression de cyp51A codant pour l'enzyme cible et 2) des mutations dans cyp51A. Cependant, la surexpression de cyp51A dans les souches étudiées n'a pas été constatée. L'effet des mutations de cyp51A dans la résistance aux azoles a été testée par complémentation fonctionnelle d'une souche S. cerevisiae déletée dans son gène ERG11. L'expression de ces gènes chez S. cerevisiae a permis de démontrer que les protéines Cyp51Ap contenant une mutation G54E ou M220K pouvaient conférer une résistance spécifique à certains azoles, ainsi suggérant que ces mutations pourraient être importantes dans le développement d'une résistance aux azoles chez A. fumigatus. En conclusion, ce travail a permis de mettre en évidence, dans des souches d'A. fumigatus , une corrélation entre leur résistance à l' ITC et les mécanismes impliquant une surexpression de transporteurs et des mutations dans cyp51A. Cependant, ces mécanismes n'ont pu expliquer la résistance aux azoles de certaines souches et c'est pourquoi de nouvelles approches doivent être envisagées afin d'étudier ces mécanismes.

Genetic exchange in an arbuscular mycorrhizal fungus results in increased rice growth and altered mycorrhiza-specific gene transcription.

Arbuscular mycorrhizal fungi (AMF) are obligate symbionts with most terrestrial plants. They improve plant nutrition, particularly phosphate acquisition, and thus are able to improve plant growth. In exchange, the fungi obtain photosynthetically fixed carbon. AMF are coenocytic, meaning that many nuclei coexist in a common cytoplasm. Genetic exchange recently has been demonstrated in the AMF Glomus intraradices, allowing nuclei of different Glomus intraradices strains to mix. Such genetic exchange was shown previously to have negative effects on plant growth and to alter fungal colonization. However, no attempt was made to detect whether genetic exchange in AMF can alter plant gene expression and if this effect was time dependent. Here, we show that genetic exchange in AMF also can be beneficial for rice growth, and that symbiosis-specific gene transcription is altered by genetic exchange. Moreover, our results show that genetic exchange can change the dynamics of the colonization of the fungus in the plant. Our results demonstrate that the simple manipulation of the genetics of AMF can have important consequences for their symbiotic effects on plants such as rice, which is considered the most important crop in the world. Exploiting natural AMF genetic variation by generating novel AMF genotypes through genetic exchange is a potentially useful tool in the development of AMF inocula that are more beneficial for crop growth.

Tissue-adapted invasion strategies of the rice blast fungus Magnaporthe oryzae.

Magnaporthe oryzae causes rice blast, the most serious foliar fungal disease of cultivated rice (Oryza sativa). During hemibiotrophic leaf infection, the pathogen simultaneously combines biotrophic and necrotrophic growth. Here, we provide cytological and molecular evidence that, in contrast to leaf tissue infection, the fungus adopts a uniquely biotrophic infection strategy in roots for a prolonged period and spreads without causing a loss of host cell viability. Consistent with a biotrophic lifestyle, intracellularly growing hyphae of M. oryzae are surrounded by a plant-derived membrane. Global, temporal gene expression analysis used to monitor rice responses to progressive root infection revealed a rapid but transient induction of basal defense-related gene transcripts, indicating perception of the pathogen by the rice root. Early defense gene induction was followed by suppression at the onset of intracellular fungal growth, consistent with the biotrophic nature of root invasion. By contrast, during foliar infection, the vast majority of these transcripts continued to accumulate or increased in abundance. Furthermore, induction of necrotrophy-associated genes during early tissue penetration, previously observed in infected leaves, was not seen in roots. Collectively, our results not only report a global characterization of transcriptional root responses to a biotrophic fungal pathogen but also provide initial evidence for tissue-adapted fungal infection strategies.

Comparative genomics suggests that the human pathogenic fungus Pneumocystis jirovecii acquired obligate biotrophy through gene loss.

Pneumocystis jirovecii is a fungal parasite that colonizes specifically humans and turns into an opportunistic pathogen in immunodeficient individuals. The fungus is able to reproduce extracellularly in host lungs without eliciting massive cellular death. The molecular mechanisms that govern this process are poorly understood, in part because of the lack of an in vitro culture system for Pneumocystis spp. In this study, we explored the origin and evolution of the putative biotrophy of P. jirovecii through comparative genomics and reconstruction of ancestral gene repertoires. We used the maximum parsimony method and genomes of related fungi of the Taphrinomycotina subphylum. Our results suggest that the last common ancestor of Pneumocystis spp. lost 2,324 genes in relation to the acquisition of obligate biotrophy. These losses may result from neutral drift and affect the biosyntheses of amino acids and thiamine, the assimilation of inorganic nitrogen and sulfur, and the catabolism of purines. In addition, P. jirovecii shows a reduced panel of lytic proteases and has lost the RNA interference machinery, which might contribute to its genome plasticity. Together with other characteristics, that is, a sex life cycle within the host, the absence of massive destruction of host cells, difficult culturing, and the lack of virulence factors, these gene losses constitute a unique combination of characteristics which are hallmarks of both obligate biotrophs and animal parasites. These findings suggest that Pneumocystis spp. should be considered as the first described obligate biotrophs of animals, whose evolution has been marked by gene losses.

Recombination in Glomus intraradices, a supposed ancient asexual arbuscular mycorrhizal fungus

Background: Arbuscular mycorrhizal fungi (AMF) are important symbionts of most plant species, promoting plant diversity and productivity. This symbiosis is thought to have contributed to the early colonisation of land by plants. Morphological stasis over 400 million years and the lack of an observed sexual stage in any member of the phylum Glomeromycota led to the controversial suggestion of AMF being ancients asexuals. Evidence for recombination in AMF is contradictory. Results: We addressed the question of recombination in the AMF Glomus intraradices by sequencing 11 polymorphic nuclear loci in 40 morphologically identical isolates from one field. Phylogenetic relationships among genotypes showed a reticulate network pattern providing a rationale to test for recombination. Five statistical tests predicted multiple recombinant regions in the genome of a core set of isolates. In contrast, five clonal lineages had fixed a large number of differences. Conclusion: Our data show that AMF from one field have undergone recombination but that clonal lineages coexist. This finding has important consequences for understanding AMF evolution, co-evolution of AMF and plants and highlights the potential for commercially introduced AMF inoculum recombining with existing local populations. Finally, our results reconcile seemingly contradictory studies on whether AMF are clonal or form recombining populations.

Remorins form a novel family of coiled coil-forming oligomeric and filamentous proteins associated with apical, vascular and embryonic tissues in plants.

Remorins form a superfamily of plant-specific plasma membrane/lipid-raft-associated proteins of unknown structure and function. Using specific antibodies, we localized tomato remorin 1 to apical tissues, leaf primordia and vascular traces. The deduced remorin protein sequence contains a predicted coiled coil-domain, suggesting its participation in protein-protein interactions. Circular dichroism revealed that recombinant potato remorin contains an alpha-helical region that forms a functional coiled-coil domain. Electron microscopy of purified preparations of four different recombinant remorins, one from potato, two divergent isologs from tomato, and one from Arabidopsis thaliana , demonstrated that the proteins form highly similar filamentous structures. The diameters of the negatively-stained filaments ranged from 4.6-7.4 nm for potato remorin 1, 4.3-6.2 nm for tomato remorin 1, 5.7-7.5 nm for tomato remorin 2, and 5.7-8.0 nm for Arabidopsis Dbp. Highly polymerized remorin 1 was detected in glutaraldehyde-crosslinked tomato plasma membrane preparations and a population of the protein was immunolocalized in tomato root tips to structures associated with discrete regions of the plasma membrane.

Diagnostic and identification in situ of fungal pathogens in superficial mycosis

Fungi are divided in 3 groups in the field of medical mycology. The dermatophytes are filamentous fungi able to grow on keratinized tissues from human or animals. They are the main cause of superficial and cutaneous mycoses of the skin and its appendix (hair and nail). The yeasts, or dimorphic fungi, can be responsible of diverse types of infections (superficial to deep mycoses). The moulds include all Non-dermatophyte Filamentous Fungi (NDF). In medical mycology, the most representative moulds are Aspergillus spp., Fusarium spp. and Mucor spp. Diagnosis of mycosis is currently based on direct mycological examination of biological samples, as well as macroscopic and microscopic identification of the infectious fungus in culture assay. However, culture assays were found to remain sterile in roughly 40% of cases otherwise positive by direct mycological examinations. Additionally, results from culture assays are often difficult to interpret as various NDF are sometimes isolated. This thesis work is composed of three projects focusing on the development of new assays for direct in situ identification of fungi from dermatological samples. Part 1. A Polymerase Chain Reaction - Terminal Restriction Fragment Length Polymorphism assay (PCR-TRFLP) targeting the 28S rDNA was developed to identify dermatophytes and NDF in nails with suspected onychomycosis. This method is faster and more efficient than culture. It further enables the distinction of more than one agent in case of mixed infection. A fast and reliable assay for the identification of dermatophytes and NDF in onychomycosis was found to be highly relevant since onychomycosis with Fusarium spp. or other NDF are weakly responsive or unresponsive to standard onychomycosis treatments with oral terbinafine and itraconazole. Part 2. A nested PCR-sequencing assay targeting the 28S rDNA was developed to identify dermatophyte species in skin and hair samples. This method is especially suitable for tinea capitis where dermatophytes identification is critical for subsequently prescribing the adequate treatment. The challenge presented when performing direct PCR fungi identification in skin and hair differs from that seen in onychomycosis as small amount of material is generally collected, few fungal elements are present in the clinical sample and one dermatophyte among a dozen species must be identified. Part 3. Fusarium spp. is currently isolated from nails with a frequency of 15% of that of dermatophytes in the laboratory of Mycology of the CHUV (2005-2012). The aim of this work was to examine if the intensive use of terbinafine and itraconazole could be a cause of the high incidence of Fusarium nail infections. For that purpose, two different methods, specific PCR and TRFLP, were used to detect both Fusarium spp. and Trichophyton spp. in nails of previously treated or untreated patients. TRFLP assay was found to be less sensitive than classical PCR assays specifically detecting Fusarium spp. or Trichophyton spp. Independently of the detection method used, the prevalence of Fusarium spp. appears not to be higher in patients previously treated by oral standard treatment with terbinafine and azoles which are highly effective to fight Trichophyton spp. in nails. In many cases Fusarium sp. was detected in samples of patients not previously subjected to antifungal therapy. Therefore, these treatments do not appear to favor the establishment of Fusarium spp. after elimination of a dermatophyte in nail infection. - En mycologie médicale, les champignons sont classés en 3 groupes. Les dermatophytes sont des champignons filamenteux capables de se développer dans les tissus kératinisés des hommes et des animaux, ils représentent la principale cause des mycoses superficielles et cutanées de la peau et de ses appendices (ongles et cheveux). Les levures, ou champignons dimorphiques, peuvent être responsables de divers types d'infections (superficielles à profondes). Les moisissures incluent tous les champignons filamenteux non-dermatophytes (NDF), les Aspergillus spp., les Fusarium spp. et les Mucor spp. sont les principales espèces rencontrées. Le diagnostic d'une mycose est basé sur un examen mycologique direct des prélèvements biologiques ainsi que sur l'identification macroscopique et microscopique du champignon infectieux isolé en culture. Cependant, dans environ 40% des cas, l'identification de l'agent pathogène est impossible par cette méthode car la culture reste stérile, bien que l'examen direct soit positif. De plus, la croissance de moisissures et/ou autres contaminants peut rendre l'interprétation de l'examen difficile. Ce travail de thèse est composé de trois projets focalisés sur le développement de nouvelles méthodes d'identification des champignons directement à partir d'échantillons dermatologiques. Projet 1. Une méthode de Réaction en chaîne de polymérase couplée à du polymorphisme de longueur des fragments de restriction terminaux (PCR-TRFLP), en ciblant l'ADN ribosomal 28S, a été développée pour l'identification des dermatophytes et moisissures dans les ongles avec suspicion d'onychomycoses. Cette technique s'est avérée plus rapide et plus efficace que la culture, permettant l'identification de plusieurs champignons en même temps. Posséder une méthode d'identification rapide et fiable des dermatophytes et des NDF dans les onychomycoses a été jugée nécessaire du fait que les Fusarium et d'autres NDF sont peu ou pas sensibles aux traitements oraux standards à la terbinafine et à Γ itraconazole. Projet 2. Une PCR nichée couplée au séquençage d'un fragment de l'ADN ribosomal 28S a été développée afin de différencier les dermatophytes dans la peau et les cheveux. Cette méthode est particulièrement adaptée au cas de tinea capitis, où l'identification du dermatophyte est essentielle afin de prescrire le traitement adéquat. Le problème de l'identification du pathogène fongique dans les cheveux et la peau diffère des onychomycoses car de petites quantités sont prélevées chez les patients, peu d'éléments fongiques sont présents et il faut discriminer un dermatophyte parmi une douzaine d'espèces potentielles. Projet 3. Au laboratoire de Mycologie du CHUV, les Fusarium ont été isolé dans les ongles à une fréquence de 15% pour la période 2005-2012. Le but de ce travail était d'examiner si l'utilisation intensive de terbinafine et d'itraconazole pouvait être une des causes de la forte incidence des infections des ongles par Fusarium. A cet effet, deux méthodes ont été utilisées pour détecter à la fois Fusarium spp. et Trichophyton spp., la PCR spécifique et le TRFLP. Indépendamment de la méthode choisie, il en résulte que la prévalence des Fusarium η'apparaît pas liée à un traitement au préalable des patients avec de la terbinafine ou des azoles, thérapies très efficaces contre les Trichophyton spp. dans les ongles. De plus, il existe de nombreux cas où Fusarium était détecté chez des patients non traités.

Characterization of the surface hydrophobicity of filamentous fungi.

A method for the quantitative analysis of the hydrophobicity of the mycelial mat of filamentous fungi based on contact angle measurements is presented. It was tested for a range of fungi belonging to the classes of basidiomycetes, ascomycetes and deuteromycetes. The measured contact angles of the mycelial mats ranged between hydrophilic (<30 degrees) for the deuteromycetes Fusarium oxysporum Fo47 GUS1 and Trichoderma harzianum P1[pZEGA1] and hydrophobic (>60 degrees) for the ascomycete Cladosporium sp. DSE48.1b and the basidiomycetes Paxillus involutus WSL 37.7, Hebeloma crustiliniforme WSL 6.2, Suillus bovinus WSL 48.1 and Laccaria bicolor WSL 73.1. For some fungi, variations in the hydrophobicity of the mycelium depending on the growth medium, the physiological state and the exposure to water were distinguished.

Evaluation of a polymerase chain reaction-restriction fragment length polymorphism assay for dermatophyte and nondermatophyte identification in onychomycosis.

BACKGROUND: Dermatophytes are the main cause of onychomycoses, but various nondermatophyte filamentous fungi are often isolated from abnormal nails. The correct identification of the aetiological agent of nail infections is necessary in order to recommend appropriate treatment. OBJECTIVE: To evaluate a rapid polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay based on 28S rDNA for fungal identification in nails on a large number of samples in comparison with cultures. METHODS: Infectious fungi were analysed using PCR-RFLP in 410 nail samples in which fungal elements were observed in situ by direct mycological examination (positive samples). The results were compared with those previously obtained by culture of fungi on Sabouraud agar from the same nail samples. RESULTS: PCR-RFLP identification of fungi in nails allowed validation of the results obtained in culture when Trichophyton spp. grew from infected samples. In addition, nondermatophyte filamentous fungi could be identified with certainty as the infectious agents in onychomycosis, and discriminated from dermatophytes as well as from transient contaminants. The specificity of the culture results relative to PCR-RFLP appeared to be 81%, 71%, 52% and 63% when Fusarium spp., Scopulariopsis brevicaulis, Aspergillus spp. and Candida spp., respectively, grew on Sabouraud agar. It was also possible to identify the infectious agent when direct nail mycological examination showed fungal elements, but negative results were obtained from fungal culture. CONCLUSIONS: Improved sensitivity for the detection of fungi in nails was obtained using the PCR-RFLP assay. Rapid and reliable molecular identification of the infectious fungus can be used routinely and presents several important advantages compared with culture in expediting the choice of appropriate antifungal therapy.

Fungus ball in the urinary tract: A rare entity.

A fungal mass in the urinary tract (fungus ball), mainly occurring in compromised patients, is a rare and dangerous complication of candiduria. We report 2 cases of fungus ball associated with hydronephrosis and sepsis. As reported in the literature, we treated the first patient by prompt relief of obstruction by nephrostomy and local and systemic antifungal agent. The second patient failed to respond to this treatment due to a distal ureteral stenosis and required open surgery with fungus ball removal and ureteral reimplantation. Despite a large success in urinary tract drainage with antifungal treatments, some cases need a modified approach due to anatomical modification.

Which Fungus Originally was Trichophyton mentagrophytes? Historical Review and Illustration by a Clinical Case.

Several dermatophytes producing numerous pyriform or round microconidia were called Trichophyton mentagrophytes. Among these dermatophytes are the teleomorph species Arthroderma benhamiae, Arthroderma vanbreuseghemii and Arthroderma simii, and other species such as Trichophyton interdigitale, Trichophyton erinacei and Trichophyton quinckeanum for which only the anamorph is known. Confusion exists about which fungus should be really called T. mentagrophytes and about the rational use of this name in practice. We report a case of beard ringworm (tinea barbae) with A. vanbreuseghemii. According to both clinical signs and the type of hair parasitism, this case was exactly compatible to the first description of a non-favic dermatophytosis by Gruby under the name of "mentagrophyte" from which was derived the dermatophyte epithet mentagrophytes. In addition, the phenotypic characters of the isolated fungus in cultures perfectly matched with those of the first description of a dermatophyte under T. mentagrophytes by Blanchard (Parasites animaux et parasites végétaux à l'exclusion des Bactéries, Masson, Paris, 1896). In conclusion, T. mentagrophytes corresponds to the fungus later named A. vanbreuseghemii. However, because the neotype of T. mentagrophytes was not adequately designated in regard to the ancient literature, we would privilege the use of A. vanbreuseghemii and abandon the name of T. mentagrophytes.

RNA enrichment method for quantitative transcriptional analysis of pathogens in vivo applied to the fungus Candida albicans.

UNLABELLED: In vivo transcriptional analyses of microbial pathogens are often hampered by low proportions of pathogen biomass in host organs, hindering the coverage of full pathogen transcriptome. We aimed to address the transcriptome profiles of Candida albicans, the most prevalent fungal pathogen in systemically infected immunocompromised patients, during systemic infection in different hosts. We developed a strategy for high-resolution quantitative analysis of the C. albicans transcriptome directly from early and late stages of systemic infection in two different host models, mouse and the insect Galleria mellonella. Our results show that transcriptome sequencing (RNA-seq) libraries were enriched for fungal transcripts up to 1,600-fold using biotinylated bait probes to capture C. albicans sequences. This enrichment biased the read counts of only ~3% of the genes, which can be identified and removed based on a priori criteria. This allowed an unprecedented resolution of C. albicans transcriptome in vivo, with detection of over 86% of its genes. The transcriptional response of the fungus was surprisingly similar during infection of the two hosts and at the two time points, although some host- and time point-specific genes could be identified. Genes that were highly induced during infection were involved, for instance, in stress response, adhesion, iron acquisition, and biofilm formation. Of the in vivo-regulated genes, 10% are still of unknown function, and their future study will be of great interest. The fungal RNA enrichment procedure used here will help a better characterization of the C. albicans response in infected hosts and may be applied to other microbial pathogens. IMPORTANCE: Understanding the mechanisms utilized by pathogens to infect and cause disease in their hosts is crucial for rational drug development. Transcriptomic studies may help investigations of these mechanisms by determining which genes are expressed specifically during infection. This task has been difficult so far, since the proportion of microbial biomass in infected tissues is often extremely low, thus limiting the depth of sequencing and comprehensive transcriptome analysis. Here, we adapted a technology to capture and enrich C. albicans RNA, which was next used for deep RNA sequencing directly from infected tissues from two different host organisms. The high-resolution transcriptome revealed a large number of genes that were so far unknown to participate in infection, which will likely constitute a focus of study in the future. More importantly, this method may be adapted to perform transcript profiling of any other microbes during host infection or colonization.