Identification of infectious agents in onychomycoses by PCR-terminal restriction fragment length polymorphism.

A fast and reliable assay for the identification of dermatophyte fungi and nondermatophyte fungi (NDF) in onychomycosis is essential, since NDF are especially difficult to cure using standard treatment. Diagnosis is usually based on both direct microscopic examination of nail scrapings and macroscopic and microscopic identification of the infectious fungus in culture assays. In the last decade, PCR assays have been developed for the direct detection of fungi in nail samples. In this study, we describe a PCR-terminal restriction fragment length polymorphism (TRFLP) assay to directly and routinely identify the infecting fungi in nails. Fungal DNA was easily extracted using a commercial kit after dissolving nail fragments in an Na(2)S solution. Trichophyton spp., as well as 12 NDF, could be unambiguously identified by the specific restriction fragment size of 5'-end-labeled amplified 28S DNA. This assay enables the distinction of different fungal infectious agents and their identification in mixed infections. Infectious agents could be identified in 74% (162/219) of cases in which the culture results were negative. The PCR-TRFLP assay described here is simple and reliable. Furthermore, it has the possibility to be automated and thus routinely applied to the rapid diagnosis of a large number of clinical specimens in dermatology laboratories.

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.

Targeting Cytokines: Production and Characterization of Anti-TNF-α scFvs by Phage Display Technology.

The antibody display technology (ADT) such as phage display (PD) has substantially improved the production of monoclonal antibodies (mAbs) and Ab fragments through bypassing several limitations associated with the traditional approach of hybridoma technology. In the current study, we capitalized on the PD technology to produce high affinity single chain variable fragment (scFv) against tumor necrosis factor-alpha (TNF- α), which is a potent pro-inflammatory cytokine and plays important role in various inflammatory diseases and malignancies. To pursue production of scFv antibody fragments against human TNF- α, we performed five rounds of biopanning using stepwise decreased amount of TNF-α (1 to 0.1 μ g), a semi-synthetic phage antibody library (Tomlinson I + J) and TG1 cells. Antibody clones were isolated and selected through enzyme-linked immunosorbent assay (ELISA) screening. The selected scFv antibody fragments were further characterized by means of ELISA, PCR, restriction fragment length polymorphism (RFLP) and Western blot analyses as well as fluorescence microscopy and flow cytometry. Based upon binding affinity to TNF-α , 15 clones were selected out of 50 positive clones enriched from PD in vitro selection. The selected scFvs displayed high specificity and binding affinity with Kd values at nm range to human TNF-α . The immunofluorescence analysis revealed significant binding of the selected scFv antibody fragments to the Raji B lymphoblasts. The effectiveness of the selected scFv fragments was further validated by flow cytometry analysis in the lipopolysaccharide (LPS) treated mouse fibroblast L929 cells. Based upon these findings, we propose the selected fully human anti-TNF-α scFv antibody fragments as potential immunotherapy agents that may be translated into preclinical/clinical applications.

Molecular identification of an endemic Alpine mammal, Apodemus alpicola, using a PCR-based RFLP method

The ability of a PCR-based restriction fragment length polymorphism (RFLP) analysis of the cytochrome b (mtDNA) to distinguish Apodemus alpicola from two other Apodemus species was investigated. The partial sequencing of the cytochrome b allowed the identification of one enzyme as being potentially diagnostic. This was supported by an analysis of 131 specimens previously identified using morphometric and/or allozymic data, indicating that the PCR-based RFLP method provides a rapid and reliable tool for distinguishing A. alpicola from its two co-occurring congenerics. The method is applicable to samples taken in the field for ecological studies, and could easily be adapted to the identification of museum samples.

Onychomycosis insensitive to systemic terbinafine and azole treatments reveals non-dermatophyte moulds as infectious agents.

BACKGROUND: Dermatophytes are the main cause of onychomycoses, but various non-dermatophyte filamentous fungi are often isolated from abnormal nails. OBJECTIVE: Our aim was the in situ identification of the fungal infectious agent in 8 cases of onychomycoses which could not be cured after systemic terbinafine and itraconazole treatment. METHODS: Fungal DNA was extracted from nail samples, and infectious fungi were identified by restriction fragment length polymorphism (RFLP) of amplified fungal ribosomal DNA using a previously described PCR/RFLP assay. RESULTS: PCR/RFLP identification of fungi in nails allows the identification of the infectious agent: Fusarium sp., Acremonium sp. and Aspergillus sp. were found as a sole infectious agent in 5, 2 and 1 cases, respectively. CONCLUSIONS: Fusarium spp. and other non-dermatophyte filamentous fungi are especially difficult to cure in onychomycoses utilising standard treatment with terbinafine and itraconazole. PCR fungal identification helps demonstrate the presence of moulds in order to prescribe alternative antifungal treatments.

Linkage arrangement in the vitellogenin gene family of Xenopus laevis as revealed by gene segregation analysis.

Using restriction fragment length polymorphism (RFLP) we have analyzed the segregation of alleles of the different vitellogenin genes of Xenopus laevis. The results demonstrate that the four genes whose expression is controlled by oestrogen, form two linkage groups. The genes A1, A2 and B1 are linked genetically whereas the fourth gene, the gene B2, segregates independently. The possible origin of this unexpected arrangement is discussed.

Association between bipolar disorder and monoamine oxidase A gene polymorphisms: results of a multicenter study

OBJECTIVE: Although genetic factors have been implicated in the etiology of bipolar disorder, no specific gene has been conclusively identified. Given the link between abnormalities in serotonergic neurotransmission and bipolar disorder, a candidate gene association approach was applied to study the involvement of the monoamine oxidase A (MAOA) gene, which codes for a catabolic enzyme of serotonin, in the susceptibility to bipolar disorder. METHOD: In France and Switzerland, 272 patients with bipolar disorder and 122 healthy subjects were typed for three polymorphic markers of the MAOA gene: the MAOA-CA repeat, the MAOA restriction fragment length polymorphism (RFLP), and a repeat directly adjacent to the variable number of tandem repeats (VNTR) locus. RESULTS: A significant difference in the distribution of the alleles for the MAOA-CA repeat was observed between the female bipolar patients and comparison group. CONCLUSIONS: The results obtained in the French and Swiss population confirm findings from two studies conducted in the United Kingdom.

Signature polymorphisms in the internal transcribed spacer region relevant for the differentiation of zoophilic and anthropophilic strains of Trichophyton interdigitale and other species of T. mentagrophytes sensu lato.

Summary Background Dermatophytes are the main cause of superficial mycoses in humans and animals. Molecular research has given useful insights into the phylogeny and taxonomy of the dermatophytes to overcome the difficulties with conventional diagnostics. Objectives The Trichophyton mentagrophytes complex consists of anthropophilic as well as zoophilic species. Although several molecular markers have been developed for the differentiation of strains belonging to T. mentagrophytes sensu lato, correct identification still remains problematic, especially concerning the delineation of anthropophilic and zoophilic strains of T. interdigitale. This differentiation is not academic but is essential for selection of the correct antimycotic therapy to treat infected patients. Methods One hundred and thirty isolates identified by morphological characteristics as T. mentagrophytes sensu lato were investigated using restriction fragment length polymorphism (RFLP) and sequence analysis of the polymerase chain reaction-amplified internal transcribed spacer (ITS) region of the rDNA. Results Species of this complex produced individual RFLP patterns obtained by the restriction enzyme MvaI. Subsequent sequence analysis of the ITS1, 5.8S and ITS2 region of all strains, but of T. interdigitale in particular, revealed single unique polymorphisms in anthropophilic and zoophilic strains. Conclusions Signature polymorphisms were observed to be useful for the differentiation of these strains and epidemiological data showed a host specificity among zoophilic strains of T. interdigitale/Arthroderma vanbreuseghemii compared with A. benhamiae as well as characteristic clinical pictures in humans when caused by zoophilic or anthropophilic strains. The delineation is relevant because it helps in determining the correct treatment and provides clues regarding the source of the infection.

Evaluation of a PCR-RFLP assay for the identification of dermatophytes in situ

Dermatophytes are the main cause of superficial mycoses. These fungi have the capacity to invade keratinized tissue of humans or animals to produce infections that are generally restricted to the corneocytes of the skin, hair, and nails. Nevertheless, it is common to obtain negative results from fungal cultures of dermatological specimens where direct mycological examination showed fungal elements (30-40%). However, correct identification of the isolated dermatophytes from Tinea is important to choose the appropriate treatment. Therefore, we aim to develop a rapid polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay based on 28S rDNA that is able to identify dermatophytes species in positive dermatological samples. PCR-RFLP identification of dermatophytes in skin or hair allowed validation of the results obtained in culture. It was also possible to identify the infectious dermatophytes when direct hair/ skin mycological examination showed fungal elements, but negative results were obtained from fungal culture. As a conclusion, PCR methods may provide significant benefits in the rapid diagnosis of Tinea. First, there is an increase in sensitivity of dermatophytes identification when enough material is available. Secondly, identification of the infecting agent can be obtained in 24 hours with PCR-RFLP or sequencing, whereas results from fungal cultures can take 2-3 weeks.

A genotypic mutation system measuring mutations in restriction recognition sequences.

The RFLP/PCR approach (restriction fragment length polymorphism/polymerase chain reaction) to genotypic mutation analysis described here measures mutations in restriction recognition sequences. Wild-type DNA is restricted before the resistant, mutated sequences are amplified by PCR and cloned. We tested the capacity of this experimental design to isolate a few copies of a mutated sequence of the human c-Ha-ras1 gene from a large excess of wild-type DNA. For this purpose we constructed a 272 bp fragment with 2 mutations in the PvuII recognition sequence 1727-1732 and studied the rescue by RFLP/PCR of a few copies of this 'PvuII mutant standard'. Following amplification with Taq-polymerase and cloning into lambda gt10, plaques containing wild-type sequence, PvuII mutant standard or Taq-polymerase induced bp changes were quantitated by hybridization with specific oligonucleotide probes. Our results indicate that 10 PvuII mutant standard copies can be rescued from 10(8) to 10(9) wild-type sequences. Taq polymerase errors originating from unrestricted, residual wild-type DNA were sequence dependent and consisted mostly of transversions originating at G.C bp. In contrast to a doubly mutated 'standard' the capacity to rescue single bp mutations by RFLP/PCR is limited by Taq-polymerase errors. Therefore, we assessed the capacity of our protocol to isolate a G to T transversion mutation at base pair 1698 of the MspI-site 1695-1698 of the c-Ha-ras1 gene from excess wild-type ras1 DNA. We found that 100 copies of the mutated ras1 fragment could be readily rescued from 10(8) copies of wild-type DNA.

Transition mutation in codon 248 of the p53 tumor suppressor gene induced by reactive oxygen species and a nitric oxide-releasing compound.

Exposing the human bronchial epithelial cell line BEAS-2B to the nitric oxide (NO) donor sodium 1-(N,N-diethylamino)diazen-1-ium-1, 2-diolate (DEA/NO) at an initial concentration of 0.6 mM while generating superoxide ion at the rate of 1 microM/min with the hypoxanthine/xanthine oxidase (HX/XO) system induced C:G-->T:A transition mutations in codon 248 of the p53 gene. This pattern of mutagenicity was not seen by 'fish-restriction fragment length polymorphism/polymerase chain reaction' (fish-RFLP/PCR) on exposure to DEA/NO alone, however, exposure to HX/XO led to various mutations, suggesting that co-generation of NO and superoxide was responsible for inducing the observed point mutation. DEA/NO potentiated the ability of HX/XO to induce lipid peroxidation as well as DNA single- and double-strand breaks under these conditions, while 0.6 mM DEA/NO in the absence of HX/XO had no significant effect on these parameters. The results show that a point mutation seen at high frequency in certain common human tumors can be induced by simultaneous exposure to reactive oxygen species and a NO source.

Characterisation of microbial communities colonising the hyphal surfaces of arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal fungi (AMF) are symbiotic soil fungi that are intimately associated with the roots of the majority of land plants. They colonise the interior of the roots and the hyphae extend into the soil. It is well known that bacterial colonisation of the rhizosphere can be crucial for many pathogenic as well as symbiotic plant-microbe interactions. However, although bacteria colonising the extraradical AMF hyphae (the hyphosphere) might be equally important for AMF symbiosis, little is known regarding which bacterial species would colonise AMF hyphae. In this study, we investigated which bacterial communities might be associated with AMF hyphae. As bacterial-hyphal attachment is extremely difficult to study in situ, we designed a system to grow AMF hyphae of Glomus intraradices and Glomus proliferum and studied which bacteria separated from an agricultural soil specifically attach to the hyphae. Characterisation of attached and non-attached bacterial communities was performed using terminal restriction fragment length polymorphism and clone library sequencing of 16S ribosomal RNA (rRNA) gene fragments. For all experiments, the composition of hyphal attached bacterial communities was different from the non-attached communities, and was also different from bacterial communities that had attached to glass wool (a non-living substratum). Analysis of amplified 16S rRNA genes indicated that in particular bacteria from the family of Oxalobacteraceae were highly abundant on AMF hyphae, suggesting that they may have developed specific interactions with the fungi.

Seasonal fluctuations of bacterial community diversity in agricultural soil and experimental validation by laboratory disturbance experiments.

Natural fluctuations in soil microbial communities are poorly documented because of the inherent difficulty to perform a simultaneous analysis of the relative abundances of multiple populations over a long time period. Yet, it is important to understand the magnitudes of community composition variability as a function of natural influences (e.g., temperature, plant growth, or rainfall) because this forms the reference or baseline against which external disturbances (e.g., anthropogenic emissions) can be judged. Second, definition of baseline fluctuations in complex microbial communities may help to understand at which point the systems become unbalanced and cannot return to their original composition. In this paper, we examined the seasonal fluctuations in the bacterial community of an agricultural soil used for regular plant crop production by using terminal restriction fragment length polymorphism profiling (T-RFLP) of the amplified 16S ribosomal ribonucleic acid (rRNA) gene diversity. Cluster and statistical analysis of T-RFLP data showed that soil bacterial communities fluctuated very little during the seasons (similarity indices between 0.835 and 0.997) with insignificant variations in 16S rRNA gene richness and diversity indices. Despite overall insignificant fluctuations, between 8 and 30% of all terminal restriction fragments changed their relative intensity in a significant manner among consecutive time samples. To determine the magnitude of community variations induced by external factors, soil samples were subjected to either inoculation with a pure bacterial culture, addition of the herbicide mecoprop, or addition of nutrients. All treatments resulted in statistically measurable changes of T-RFLP profiles of the communities. Addition of nutrients or bacteria plus mecoprop resulted in bacteria composition, which did not return to the original profile within 14 days. We propose that at less than 70% similarity in T-RFLP, the bacterial communities risk to drift apart to inherently different states.

Biogeochemical evolution of a marine shore tailings deposit during bioremediation

Summary : Mining activities produce enormous amounts of waste material known as tailings which are composed of fine to medium size particles. These tailings often contain sulfides, which oxidation can lead to acid and metal contamination of water; therefore they need to be remediated. In this work a tailings bioremediation approach was investigated by an interdisciplinary study including geochemistry, mineralogy and microbiology. The aim of the work was to study the effect of the implementation of wetland above oxidizing tailings on the hydrogeology and the biogeochemical element cycles, and to assess the system evolution over time. To reach these goals, biogeochemical processes occurring in a marine shore tailings deposit were investigated. The studied tailings deposit is located at the Bahìa de Ite, Pacific Ocean, southern Peru, where between 1940 and 1996 the tailings were discharged from the two porphyry copper mines Cuajone and Toquepala. After the end of deposition, a remediation approach was initiated in 1997 with a wetland implementation above the oxidizing tailings. Around 90% of the tailings deposits (total 16 km2) were thus remediated, except the central delta area and some areas close to the shoreline. The multi-stable isotope study showed that the tailings were saturated with fresh water in spite of the marine setting, due to the high hydraulic gradient resulting from the wetland implementation. Submarine groundwater discharge (SGD) was the major source of SO4 2-, C1-, Na+, Fe2+, and Mn2+ input into the tailings at the original shelf-seawater interface. The geochemical study (aquatic geochemistry and X-Ray diffraction (XRD) and sequential extractions from the solid fraction) showed that iron and sulfur oxidation were the main processes in the non-remediated tailings, which showed a top a low-pH oxidation zone with strong accumulation of efflorescent salts at the surface due to capillary upward transport of heavy metals (Fe, Cu, Zn, Mn, Cd, Co, and Ni) in the arid climate. The study showed also that the implementation of the wetland resulted in very low concentrations of heavy metals in solution (mainly under the detection limit) due to the near neutral pH and more reducing conditions (100-150 mV). The heavy metals, which were taken from solution, precipitated as hydroxides and sulfides or were bound to organic matter. The bacterial community composition analysis by Terminal Restriction Fragment Length Polymorphism (T-RFLP) and cloning and sequencing of 16S rRNA genes combined with a detailed statistical analysis revealed a high correlation between the bacterial distribution and the geochemical variables. Acidophilic autotrophic oxidizing bacteria were dominating the oxidizing tailings, whereas neutrophilic and heterotrophic reducing bacteria were driving the biogeochemical processes in the remediated tailings below the wetland. At the subsurface of the remediated tailings, an iron cycling was highlighted with oxidation and reduction processes due to micro-aerophilic niches provided by the plant rhizosphere in this overall reducing environment. The in situ bioremediation experiment showed that the main parameter to take into account for the effectiveness was the water table and chemistry which controls the system. The constructed remediation cells were more efficient and rapid in metal removal when saturation conditions were available. This study showed that the bioremediation by wetland implementation could be an effective and rapid treatment for some sulfidic mine tailings deposits. However, the water saturation of the tailings has to be managed on a long-term basis in order to guarantee stability. Résumé : L'activité minière produit d'énormes quantités de déchets géologiques connus sous le nom de « tailings » composées de particules de taille fine à moyenne. Ces déchets contiennent souvent des sulfures dont l'oxydation conduit à la formation d'effluents acides contaminés en métaux, d'où la nécessité d'effectuer une remédiation des sites de stockage concernés. Le but de ce travail est dans un premier temps d'étudier l'effet de la bio-remédiation d'un dépôt de tailings oxydés sur l'hydrogéologie du système et les cycles biogéochimiques des éléments et en second lieu, d'évaluer l'évolution du processus de remédiation dans le temps. Le site étudié dans ce travail est situé dans la Bahía de Ite, au sud du Pérou, au bord de l'Océan Pacifique. Les déchets miniers en question sont déposés dans un environnement marin. De 1940 à 1996, les déchets de deux mines de porphyre cuprifère - Cuajone et Toquepala - ont été acheminés sur le site via la rivière Locumba. En 1997, une première remédiation a été initiée avec la construction d'une zone humide sur les tailings. Depuis, environ 90% de la surface du dépôt (16 km2) a été traité, les parties restantes étant la zone centrale du delta du Locumba et certaines zones proches de la plage. Malgré la proximité de l'océan, les études isotopiques menées dans le cadre de ce travail ont montré que les tailings étaient saturés en eau douce. Cette saturation est due à la pression hydraulique résultant de la mise en place des zones humides. Un écoulement d'eau souterrain sous-marin a été à détecté à l'interface entre les résidus et l'ancien fond marin. En raison de la géologie locale, il constitue une source d'entrée de SO4 2-, Cl-, Na+, FeZ+, et Mn2+ dans le système. L'analyse de la géochimie aquatique, la Diffraction aux Rayons X (XRD) et l'extraction séquentielle ont montré que l'oxydation du fer et .des sulfures est le principal processus se produisant dans les déchets non remédiés. Ceci a entraîné le développement d'une zone d'oxydation à pH bas induisant une forte accumulation des sels efflorescents, conséquence de la migration capillaire des métaux lourds (Fe, Cu, Zn, Mn, Cd, Co et Ni) de la solution vers la surface dans ce climat aride. Cette étude a montré également que la construction de la zone humide a eu comme résultats une précipitation des métaux dans des phases minérales en raison du pH neutre et des conditions réductrices (100-150mV). Les métaux lourds ont précipité sous la forme d'hydroxydes et de sulfures ou sont adsorbés à la matière organique. L'analyse de la composition de la communauté bactérienne à l'aide la technique T-RFLP (Terminal Restriction Fragment Length Polymorphism) et par le clonage/séquençage des gènes de l'ARNr 16S a été combinée à une statistique détaillée. Cette dernière a révélé une forte corrélation entre la distribution de bactéries spécifiques et la géochimie : Les bactéries autotrophes acidophiles dominent dans les déchets oxydés non remédiés, tandis que des bactéries hétérotrophes neutrophiles ont mené les processus microbiens dans les déchets remédiés sous la zone humide. Sous la surface de la zone humide, nos analyses ont également mis en évidence un cycle du fer par des processus d'oxydoréduction rendus possibles par la présence de niches micro-aérées par la rhizosphère dans cet environnement réducteur. L'expérience de bio-remédiation in situ a montré que les paramètres clés qui contrôlent l'efficacité du traitement sont le niveau de la nappe aquifère et la chimie de l'eau. Les cellules de remédiation se sont montrées plus efficaces et plus rapides lorsque le système a pu être saturé en eau. Finalement, cette étude a montré que la bio-remédiation de déchets miniers par la construction de zones humides est un moyen de traitement efficace, rapide et peu coûteux. Cependant, la saturation en eau du système doit être gérée sur le long terme afin de garantir la stabilité de l'ensemble du système.

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.