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.

Combined effect of natural antimicrobials and high pressure processing to prevent Listeria monocytogenes growth after a cold chain break during storage of cooked ham

The effect of high pressure processing (400 MPa for 10 min) and natural antimicrobials 2 (enterocins and lactate-diacetate) on the behaviour of L. monocytogenes in sliced cooked ham 3 during refrigerated storage (1ºC and 6ºC) was assessed. The efficiency of the treatments after a 4 cold chain break was evaluated. Lactate-diacetate exerted a bacteriostatic effect against L. 5 monocytogenes during the whole storage period (3 months) at 1ºC and 6ºC, even after 6 temperature abuse. The combination of low storage temperature (1ºC), high pressure 7 processing (HPP) and addition of lactate-diacetate reduced the levels of L. monocytogenes 8 during storage by 2.7 log CFU/g. The most effective treatment was the combination of HPP, 9 enterocins and refrigeration at 1ºC, which reduced the population of the pathogen to final counts 10 of 4 MPN/g after 3 months of storage, even after the cold chain break.

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.

Estudi de la capacitat del suro com a material adsorbent de pesticides

La presència de pesticides en el medi ambient pot comportar efectes nocius pel propi medi i la salut humana, fet que, en alguns casos, converteix en necessària la seva eliminació. Un dels mètodes utilitzats per dur a terme aquesta eliminació és la sorció de contaminats sobre materials adsorbents. Per tal de fer d’aquest mètode un procés sostenible cal investigar nous materials capaços de retenir els contaminants. El suro és la part més externa de l’escorça de l’alzina surera: Quercus Suber L. S’extreu cada 5- 10 anys depenent de la regió i es caracteritza per ser una font natural, renovable i biodegradable amb una heterogènia composició química que el converteix en un material potencialment apte com a adsorbent d’un ampli rang de contaminants. En aquest context, l’objectiu principal d’aquest treball és investigar l’afinitat d’adsorció del suro amb quatre pesticides de diferent hidrofobicitat i estructura química i estudiar el paper que hi juguen els seus compostos químics (extractius, suberina, lignina i polisacàrids) en aquest procés de sorció. Els pesticides investigats han estat: Metamitron: poc hidrofòbic (logKow = 0.83) i de caràcter molecular, Alaclor: moderadament hidrofòbic (logKow = 2.80) i de caràcter iònic (pKa = 0.62), 2,4-D: moderadament hidrofòbic (logKow = 2.81) i de caràcter iònic (pKa = 2.64) i Clorpirifos: molt hidrofòbic (logKow = 4.92) i de caràcter molecular

La cultura material de Tarraco - Tarracona (Hispania Tarraconensis - Regnum Visigothorum): cerámica común y ánforas

Síntesis actualizada de los estudios monográficos realizados sobre las cerámicas comunes y ánforas de la ciudad de Tarraco desde finales del s. IV hasta inicios del s. VIII. Se hace especial hincapié en los últimos hallazgos del siglo VII que reflejan una intensificación de la presencia de materiales orientales en la ciudad.

Wound-healing gene family expression differences between fetal and foreskin cells used for bioengineered skin substitutes.

For tissue engineering, several cell types and tissues have been proposed as starting material. Allogenic skin products available for therapeutic usage are mostly developed with cell culture and with foreskin tissue of young individuals. Fetal skin cells offer a valuable solution for effective and safe tissue engineering for wounds due to their rapid growth and simple cell culture. By selecting families of genes that have been reported to be implicated in wound repair and particularly for scarless fetal wound healing including transforming growth factor-beta (TGF-beta) superfamily, extracellular matrix, and nerve/angiogenesis growth factors, we have analyzed differences in their expression between fetal skin and foreskin cells, and the same passages. Of the five TGF-beta superfamily genes analyzed by real-time reverse transcription-polymerase chain reaction, three were found to be significantly different with sixfold up-regulated for TGF-beta2, and 3.8-fold for BMP-6 in fetal cells, whereas GDF-10 was 11.8-fold down-regulated. For nerve growth factors, midkine was 36-fold down-regulated in fetal cells, and pleiotrophin was 4.76-fold up-regulated. We propose that fetal cells present technical and therapeutic advantages compared to foreskin cells for effective cell-based therapy for wound management, and overall differences in gene expression could contribute to the degree of efficiency seen in clinical use with these cells.

Systemic exposure to tobramycin after local antibiotic treatment with calcium sulphate as carrier material.

INTRODUCTION: Osteoset(®) T is a calcium sulphate void filler containing 4% tobramycin sulphate, used to treat bone and soft tissue infections. Despite systemic exposure to the antibiotic, there are no pharmacokinetic studies in humans published so far. Based on the observations made in our patients, a model predicting tobramycin serum levels and evaluating their toxicity potential is presented. METHODS: Following implantation of Osteoset(®) T, tobramycin serum concentrations were monitored systematically. A pharmacokinetic analysis was performed using a non-linear mixed effects model based on a one compartment model with first-degree absorption. RESULTS: Data from 12 patients treated between October 2006 and March 2008 were analysed. Concentration profiles were consistent with the first-order slow release and single-compartment kinetics, whilst showing important variability. Predicted tobramycin serum concentrations depended clearly on both implanted drug amount and renal function. DISCUSSION AND CONCLUSION: Despite the popularity of aminoglycosides for local antibiotic therapy, pharmacokinetic data for this indication are scarce, and not available for calcium sulphate as carrier material. Systemic exposure to tobramycin after implantation of Osteoset(®) T appears reassuring regarding toxicity potential, except in case of markedly impaired renal function. We recommend in adapting the dosage to the estimated creatinine clearance rather than solely to the patient's weight.

Quantification of thermotolerant campylobacter in Swiss water treatment plants, by real-time quantitative polymerase chain reaction

A total of 49 wastewater samples from 23 different wastewater treatment plants (WWTPs) were analyzed using real-time quantitative polymerase chain reaction for the presence and quantity of thermotolerant campylobacters. Thermotolerant campylobacters were detected in 87.5% (21/24) and 64% (16/25) of untreated and treated wastewater samples, respectively. Their concentration was sufficiently high to be quantified in 20.4% (10/49) of the samples. In these samples, the concentration ranged from 68 000 to 2292 000 cells/L in untreated wastewater and from 10 800 to 28 000 cells/L in treated water. We conclude that thermotolerant campylobacters present a health hazard for workers at WWTPs in Switzerland. [Authors]

Epoxy Thermoplastic Traffic Marking Material, HR-1033, 1982

This Implementation Package summarizes the result of an effort to develop a more durable traffic marking material-Epoxy Thermoplastic (ETP). The report includes background information on the development of ETP, a discussion of the field tests and evaluations, the material composition and equipment modifications for applying ETP. The package also includes material specifications for purchasing ETP and specifications for the application of ETP by contract.

Laboratory Study of the Leachate from Crushed Portland Cement Concrete Base Material, MLR-96-04, 1999

Since the 1980s, the Iowa Department of Transportation has increased its use of recycled Portland Cement Concrete (PCC) as drainable base material below some new pavements. Water flowing out of the longitudinal drains on projects having recycled PCC drainable bases was found to have a high pH value. The high pH water impedes vegetation growth and becomes a contributing factor to soil erosion at the drain outlet. In addition, the high pH water contributes to the growth of crystalline deposits on the drain outlet wire mesh rodent guard and in some cases caused it to become completely blocked. This research determined which of three choices of recycled PCC drainable base material, gradation, and design would give the lowest pH value in the drain discharge water. The drainable base material having its fines separated out and placed as a 2-in. (5.1-mm) bottom layer, below the remaining coarse material, generally gave pH values around 11.2 while other designs tested gave pH values around 11.5.

Diseño e implementación de un serious game para el aprendizaje del material quirúrgico y desarrollo de una operación

En este trabajo se recoge información útil que se debe tener en cuenta para desarrollar un serious game pertinente para el entrenamiento de profesionales de la salud, que aporte destrezas y habilidades necesarias para afrontar las cirugías reales. Se propone un serious game a modo de simulador de diferentes operaciones quirúrgicas traumatológicas, que permite al usuario las etapas preoperatorias, intraoperatorias y postoperatorias de una cirugía, en las que se debe describir qué acciones se llevan a cabo, quiénes la realizan y si es necesario algún material específico.

Peptide-antibody conjugates for tumour therapy: a MHC-class-II-restricted tetanus toxin peptide coupled to an anti-Ig light chain antibody can induce cytotoxic lysis of a human B-cell lymphoma by specific CD4 T cells.

Anti-idiotype antibody therapy of B-cell lymphomas, despite numerous promising experimental and clinical studies, has so far met with limited success. Tailor-made monoclonal anti-idiotype antibodies have been injected into a large series of lymphoma patients, with a few impressive complete tumour remissions but a large majority of negative responses. The results presented here suggest that, by coupling to antilymphoma idiotype antibodies a few molecules of the tetanus toxin universal epitope peptide P2 (830-843), one could markedly increase the efficiency of this therapy. We show that after 2-hr incubation with conjugates consisting of the tetanus toxin peptide P2 coupled by an S-S bridge to monoclonal antibodies directed to the lambda light chain of human immunoglobulin, human B-lymphoma cells can be specifically lysed by a CD4 T-lymphocyte clone specific for the P2 peptide. Antibody without peptide did not induce B-cell killing by the CD4 T-lymphocyte clone. The free cysteine-peptide was also able to induce lysis of the B-lymphoma target by the T-lymphocyte clone, but at a molar concentration 500 to 1000 times higher than that of the coupled peptide. Proliferation assays confirmed that the antibody-peptide conjugate was antigenically active at a much lower concentration than the free peptide. They also showed that antibody-peptide conjugates required an intact processing function of the B cell for peptide presentation, which could be selectively inhibited by leupeptin and chloroquine.(ABSTRACT TRUNCATED AT 250 WORDS)