Effects of antibacterial agents and drugs monitored by atomic force microscopy.

Originally invented for topographic imaging, atomic force microscopy (AFM) has evolved into a multifunctional biological toolkit, enabling to measure structural and functional details of cells and molecules. Its versatility and the large scope of information it can yield make it an invaluable tool in any biologically oriented laboratory, where researchers need to perform characterizations of living samples as well as single molecules in quasi-physiological conditions and with nanoscale resolution. In the last 20 years, AFM has revolutionized the characterization of microbial cells by allowing a better understanding of their cell wall and of the mechanism of action of drugs and by becoming itself a powerful diagnostic tool to study bacteria. Indeed, AFM is much more than a high-resolution microscopy technique. It can reconstruct force maps that can be used to explore the nanomechanical properties of microorganisms and probe at the same time the morphological and mechanical modifications induced by external stimuli. Furthermore it can be used to map chemical species or specific receptors with nanometric resolution directly on the membranes of living organisms. In summary, AFM offers new capabilities and a more in-depth insight in the structure and mechanics of biological specimens with an unrivaled spatial and force resolution. Its application to the study of bacteria is extremely significant since it has already delivered important information on the metabolism of these small microorganisms and, through new and exciting technical developments, will shed more light on the real-time interaction of antimicrobial agents and bacteria.

Dermatophyte identification in skin and hair samples using a simple and reliable nested polymerase chain reaction assay.

BACKGROUND: Dermatophyte identification in tinea capitis is essential for choosing the appropriate treatment and in tinea infections to identify the possible source. The failure of fungi to grow in cultures frequently occurs, especially in cases of previous antifungal therapy. OBJECTIVES: To develop a rapid polymerase chain reaction (PCR) sequencing assay for dermatophyte identification in tinea capitis and tinea corporis. MATERIAL AND METHODS: Fungal DNA was extracted from hair and skin samples that were confirmed to be positive by direct mycological examination. Dermatophytes were identified by the sequence of a 28S ribosomal DNA subunit amplicon generated by nested PCR. RESULTS: Nested PCR was found to be necessary to obtain amplicons in substantial amounts for dermatophyte identification by sequencing. The results agreed with those of classical mycological identification in 14 of 23, 6 of 10, and 20 of 23 cases of tinea capitis, tinea corporis and tinea pedis, respectively, from which a dermatophyte was obtained in culture. In seven of the 56 cases, another dermatophyte was identified, revealing previous misidentification. A dermatophyte was identified in 12 of 18, three of five, and four of nine cases of tinea capitis, tinea corporis and tinea pedis, respectively, in cases in which no dermatophyte grew in culture. CONCLUSIONS: Although the gold standard dermatophyte identification from clinical samples remains fungal cultures, the assay developed in the present study is especially suitable for tinea capitis. Improved sensitivity for the identification of dermatophyte species was obtained as it is possible to identify the dermatophyte when the fungus fails to grow in cultures.

Devolatilization-induced pressure build-up: Implications for reaction front movement and breccia pipe formation

Generation of fluids during metamorphism can significantly influence the fluid overpressure, and thus the fluid flow in metamorphic terrains. There is currently a large focus on developing numerical reactive transport models, and with it follows the need for analytical solutions to ensure correct numerical implementation. In this study, we derive both analytical and numerical solutions to reaction-induced fluid overpressure, coupled to temperature and fluid flow out of the reacting front. All equations are derived from basic principles of conservation of mass, energy and momentum. We focus on contact metamorphism, where devolatilization reactions are particularly important owing to high thermal fluxes allowing large volumes of fluids to be rapidly generated. The analytical solutions reveal three key factors involved in the pressure build-up: (i) The efficiency of the devolatilizing reaction front (pressure build-up) relative to fluid flow (pressure relaxation), (ii) the reaction temperature relative to the available heat in the system and (iii) the feedback of overpressure on the reaction temperature as a function of the Clapeyron slope. Finally, we apply the model to two geological case scenarios. In the first case, we investigate the influence of fluid overpressure on the movement of the reaction front and show that it can slow down significantly and may even be terminated owing to increased effective reaction temperature. In the second case, the model is applied to constrain the conditions for fracturing and inferred breccia pipe formation in organic-rich shales owing to methane generation in the contact aureole.

Detection and differentiation of Cryptosporidium by real-time polymerase chain reaction in stool samples from patients in Rio de Janeiro, Brazil

This study reports the first genetic characterisation of Cryptosporidium isolates in Brazil using real-time polymerase chain reaction (RT-PCR). A total of 1,197 faecal specimens from children and 10 specimens from human immunodeficiency virus-infected patients were collected between 1999-2010 and screened using microscopy. Forty-eight Cryptosporidium oocyst-positive isolates were identified and analysed using a generic TaqMan assay targeting the 18S rRNA to detect Cryptosporidium species and two other TaqMan assays to identify Cryptosporidium hominis and Cryptosporidium parvum. The 18S rRNA assay detected Cryptosporidium species in all 48 of the stool specimens. The C. parvum TaqMan assay correctly identified five/48 stool samples, while 37/48 stool specimens were correctly amplified in the C. hominis TaqMan assay. The results obtained in this study support previous findings showing that C. hominis infections are more prevalent than C. parvum infections in Brazil and they demonstrate that the TaqMan RT-PCR procedure is a simple, fast and valuable tool for the detection and differentiation of Cryptosporidium species.

Vectorial capacity, basic reproduction number, force of infection and all that: formal notation to complete and adjust their classical concepts and equations

A dimensional analysis of the classical equations related to the dynamics of vector-borne infections is presented. It is provided a formal notation to complete the expressions for the Ross' Threshold Theorem, the Macdonald's basic reproduction "rate" and sporozoite "rate", Garret-Jones' vectorial capacity and Dietz-Molineaux-Thomas' force of infection. The analysis was intended to provide a formal notation that complete the classical equations proposed by these authors.

La force motrice hydraulique au service du développement économique helvétique : L'exemple du réseau d'eau sous pression à Lausanne 1868-1914

The success story of hydroelectricity long influenced and dominated Swiss scholarly literature devoted to the history of technology. This means of conducting power, which emerged at the end of the 19th century and is still dominating today, has attracted much more attention than technologies that have been shadowed by its success. In spite of their important contribution to Swiss economic development, the distribution networks of pressurized water have been neglected by scholars. This article contributes to close this historiographic gap by analyzing the introduction of pressurized water distribution in 1876 in Lausanne, in the context of the building of the first Swiss cable funicular between Lausanne and Ouchy. This article shows how pressurized water distribution transformed socio-economic practices in the urban areas in which it was adopted. Indeed, this innovation, which allowed the use of distant hydraulic resources, enabled the rationalization of industrial and artisanal production as well as improved the density of the urban industrial base. By facilitating the introduction of electric lighting, pressurized water networks played a key role in the early development, and further successes, of the Swiss hydroelectric industry.

Polymerase chain reaction for the evaluation of Schistosoma mansoni infection in two low endemicity areas of Minas Gerais, Brazil

This study aimed to evaluate the occurrence of schistosomiasis in areas with low endemicity using polymerase chain reaction (PCR) as a diagnostic method. We analysed faecal samples from 219 individuals residing in Piau and Coronel Pacheco, state of Minas Gerais, Brazil, using a single faecal sample from each individual and two slides of the Kato-Katz technique as a gold standard. Fifteen out of the 219 samples were positive with both methods of diagnosis. One sample was diagnosed as positive by the Kato-Katz technique only and 61 were diagnosed only by PCR. The positivity rates were 7.3% with the Kato-Katz method and 34.7% with PCR. When both techniques were assumed to have 100% specificity and positive individuals were identified by both methods, the sensitivity of the Kato-Katz method was 20.8% and the PCR sensitivity was 98.7%. The Kappa index between the two techniques was 0.234, suggesting weak agreement. The assessment of a single faecal sample by PCR detected more cases of infection than the analysis of one sample with two slides using the Kato-Katz technique, suggesting that PCR can be a useful diagnostic tool, particularly in areas with low endemicity.

Melt topology and chemistry during partial melting in contact aureoles : an experimental study and field example

This study was initiated to investigate partial melting within the high-grade metamorphic rocks beneath the Little Cottonwood contact aureole (Utah, USA), in order to understand the melt generation, melt migration, and geometry of initial melt distribution on grain scale during crustal anatexis. The emplacement of the Little Cottonwood stock produced a contact aureole in the pelitic host rocks of the Big Cottonwood formation (BC). Metamorphic isogrades in pelitic rocks range form biotite to 2nd sillimanite grade as a function of distance from the contact. Migmatites are restricted to the highest grade and resulted form partial melting of the BC formation rocks. First melt was produced by a combined muscovite/biotite dehydration reaction in the sillimanite + k-feldspar stability field. Melt extraction from the pelites resulted in restites (magnetite + cordierite + alumosilicate ± biotite) surrounded by feldspar enriched quartzite zones. This texture is the result of gradual infiltration of partial melts into the quartzite. Larger, discrete melt accumulation occurred in extensional or transpressional domains such as boudin necks, veins, and ductile shear zones. Melt composition are Si02- rich, crystallized as pegmatites, and apparently were very mobile. They were able to infiltrate the quartzite pervaisivly. These melts are similar in composition to first melts produced in the hydrothermal partial melt experiments at 2kbar between 700 - 800°C on fine grained high metamorphic rocks (andalusite-cordierited-biotite-zone) of the BC formation. The experimental melts are water rich and in disequilibrium with the melting rock. Initial melt composition is heterogeneous for short run duration, reflective a lack of chemical equilibrium between individual melt pools. Rock core scale heterogeneity decreased with time indicating partial homogenization of melt compositions. A simultaneous shift of melt composition to higher silica content with time was observed. The silica content of the melt increased due to local melt/mineral reactions. Melt textures indicate that reactive melt transport is most efficient along grain boundaries rimmed by dissimilar grains. Melt heterogeneity resulted in chemical potential gradients which are major driving forces for initial melt migration and govern melt distribution during initial melting. An additional subject of the thesis is the crystal size distributions of opaque minerals in a fine-grained, high-grade meta-pelite of the Big Cottonwood which were obtained from 3D X-ray tomography (uCT) and 2D thin section analysis. µCT delivers accurate size distributions within a restricted range (~ a factor of 20 in size in a single 3D image), while the absolute number of crystals is difficult to obtain from these sparsely distributed, small crystals on the basis of 2D images. Crystal size distributions obtained from both methods are otherwise similar. - Ce travail de recherche a été entrepris dans le but d'étudier les processus de fusion partielle dans les roches fortement métamorphiques de l'auréole de contact de Little Cottonwood (Utah, USA) et ceci afin de comprendre la génération de liquide de fusion, la migration de ces liquides et la géométrie de la distribution initiale des liquides de fusion à l'échelle du grain durant l'anatexie de la croûte. L'emplacement du petit massif intrusif de Little Cottonwood a produit une auréole de contact dans les roches pélitiques encaissantes appartenant à la Foimation du Big Cottonwood (BC). Les isogrades métamorphiques dans les roches pélitiques varient de l'isograde de la biotite à la deuxième isograde de la sillimanite en fonction de la distance par rapport au massif intrusif. Les migmatites sont restreintes aux zones montrant le plus haut degré métamorphique et résultent de la fusion partielle des roches de la Formation de BC. Le premier liquide de fusion a été produit par la réaction de déshydratation combinée de la muscovite et de la biotite dans le champ de stabilité du feldspath potassique Pt de la sillimanite. L'extraction du liquide de fusion des pélites forme des restites (magnétites + cordiérite + aluminosilicate ± biotite) entourées par des zones de quartzites enrichies en feldspath. Cette texture est le résultat de l'infiltration graduelle du liquide de fusion partielle dans les quartzites. Des accumulations distinctes et plus larges de liquide de fusion ont lieu dans des domaines d'extension ou de transpression tels que les boudins, les veines, et les zones de cisaillement ductile. La composition des liquides de fusion est similaire à celle des liquides pegmatoïdes, et ces liquides sont apparemment très mobiles et capables d'infiltrer les quartzites. Ces liquides de fusion ont la même composition que les premiers liquides produits dans les expériences hydrotheunales de fusion partielle à 2kbar et entre 700-800° C sur les roches finement grenues et hautement métamorphiques (andalousite-cordiérite-biotite zone) de la Formation de BC. Les liquides de fusion obtenus expérimentalement sont riches en eau et sont en déséquilibre avec la roche en fusion. La composition initiale des liquides de fusion est hétérogène pour les expériences de courte durée et reflète l'absence d'équilibre chimique entre les différentes zones d'accumulation des liquides de fusion. L'hétérogénéité à l'échelle du noyau s'estompe avec le temps et témoigne de l'homogénéisation de la composition des liquides de fusion. Par ailleurs, on observe parallèlement un décalage de la composition des liquides vers des compositions plus riches en silice au cours du temps. Le contenu en silice des liquides de fusion évolue vers un liquide pegmatitique en raison des réactions liquides/minéraux. Les textures des liquides de fusion indiquent que le transport des liquides est plus efficace le long des bordures de grains bordés par des grains différents. Aucun changement apparent du volume total n'est visible. L'hétérogénéité des liquides s'accompagne d'un gradient de potentiel chimique qui sert de moteur principal à la migration des liquides et à la distribution des liquides durant la fusion. Un sujet complémentaire de ce travail de thèse réside dans l'étude de la distribution de la taille des cristaux opaques dans les pélites finement grenues et fortement métamorphiques de la Formation de Big Cottonwood. Les distributions de taille ont été obtenues suite à l'analyse d'images 3D acquise par tomographie ainsi que par analyse de lames minces. La microtomographie par rayon X fournit une distribution de taille précise sur une marge restreinte (- un facteur de taille 20 dans une seule image 3D), alors que le nombre absolu de cristaux est difficile à obtenir sur la base d'image 2D en raison de la petite taille et de la faible abondance de ces cristaux. Les distributions de taille obtenues par les deux méthodes sont sinon similaire. Abstact: Chemical differentiation of the primitive Earth was due to melting and separation of melts. Today, melt generation and emplacement is still the dominant process for the growth of the crust. Most granite formation is due to partial melting of the lower crust, followed by transport of magma through the crust to the shallow crust where it is emplaced. Partial melting and melt segregation are essential steps before such a granitic magma can ascent through the crust. The chemistry and physics of partial melting and segregation is complex. Hence detailed studies, in which field observations yield critical information that can be compared to experimental observations, are crucial to the understanding of these fundamental processes that lead and are leading to the chemical stratification of the Earth. The research presented in this thesis is a combined field and experimental study of partial melting of high-grade meta-pelitic rocks of the Little Cottonwood contact aureole (Utah, USA). Contact metamorphic rocks are ideal for textural studies of melt generation, since the relatively short times of the metamorphic event prevents much of the recrystallization which plagues textural studies of lower crustal rocks. The purpose of the study is to characterize melt generation, identify melting reactions, and to constrain melt formation, segregation and migration mechanisms. In parallel an experimental study was undertaken to investigate melt in the high grade meta pelitic rocks, to confirm melt composition, and to compare textures of the partial molten rock cores in the absence of deformation. Results show that a pegmatoidal melt is produced by partial melting of the pelitic rocks. This melt is highly mobile. It is capable of pervasive infiltration of the adjacent quartzite. Infiltration results in rounded quartz grains bordered by a thin feldspar rim. Using computed micro X-ray tomography these melt networks can be imaged. The infiltrated melt leads to rheological weakening and to a decompaction of the solid quartzite. Such decompaction can explain the recent discovery of abundant xenocrysts in many magmas, since it favors the isolation of mineral grains. Pervasive infiltration is apparently strongly influenced by melt viscosity and melt-crystal wetting behavior, both of which depend on the water content of melt and the temperature. In all experiments the first melt is produced on grain boundaries, dominantly by the local minerals. Grain scale heterogeneity of a melting rock leads thus to chemical concentration gradients in the melt, which are the driving force for initial melt migration. Pervasive melt films along grain boundaries leading to an interconnected network are immediately established. The initial chemical heterogeneities in the melt diminish with time. Résumé large public: La différenciation chimique de la Terre primitive est la conséquence de la fusion des roches et de la séparation des liquides qui en résultent. Aujourd'hui, la production de liquide magmatique est toujours le mécanisme dominant pour la croissance de la croûte terrestre. Ainsi la formation de la plupart des granites est un processus qui implique la production de magma par fusion partielle de la croûte inférieure, la migration de ces magmas à travers la croûte et finalement son emplacement dans les niveaux superficielle de la croûte terrestre. Au cours de cette évolution, les processus de fusion partielle et de ségrégation sont des étapes indispensables à l'ascension des granites à travers la croûte. Les conditions physico-chimiques nécessaires à la fusion partielle et à l'extraction de ces liquides sont complexes. C'est pourquoi des études détaillées des processus de fusion partielle sont cruciales pour la compréhension de ces mécanismes fondamentaux responsables de la stratification chimique de la Terre. Parmi ces études, les observations de terrain apportent notamment des informations déterminantes qui peuvent être comparées aux données expérimentales. Le travail de recherche présenté dans ce mémoire de thèse associe études de terrain et données expérimentales sur la fusion partielle des roches pélitiques de haut degré métamorphiques provenant de l'auréole de contact de Little Cottonwood (Utah, USA). Les roches du métamorphisme de contact sont idéales pour l'étude de la folination de liquide de fusion. En effet, la durée relativement courte de ce type d'événement métamorphique prévient en grande partie la recristallisation qui perturbe les études de texture des roches dans la croûte inférieure. Le but de cette étude est de caractériser la génération des liquides de fusion, d'identifier les réactions responsables de la fusion de ces roches et de contraindre la formation de ces liquides et leur mécanisme de ségrégation et de migration. Parallèlement, des travaux expérimentaux ont été entrepris pour reproduire la fusion partielle de ces roches en laboratoire. Cette étude a été effectuée dans le but de confirmer la composition chimique des liquides, et de comparer les textures obtenues en l'absence de déformation. Les résultats montrent qu'un liquide de fusion pegmatoïde est produit par fusion partielle des roches pélitiques. La grande mobilité de ce liquide permet une infiltration pénétrative dans les quarzites. Ces infiltrations se manifestent par des grains de quartz arrondis entourés par une fine bordure de feldspath. L'utilisation de la tomography à rayons X a permis d'obtenir des images de ce réseau de liquide de fusion. L'infiltration de liquide de fusion entraîne un affaiblissement de la rhéologie de la roche ainsi qu'une décompaction des quartzites massifs. Une telle décompaction peut expliquer la découverte récente d'abondants xénocristaux dans beaucoup de magmas, puisque elle favorise l'isolation des minéraux. L'infiltration pénétrative est apparemment fortement influencée par la viscosité du fluide de fusion et le comportement de la tension superficielle entre les cristaux et le liquide, les deux étant dépendant du contenu en eau dans le liquide de fusion et de la température. Dans toutes les expériences, le premier liquide est produit sur les bordures de grains, principalement par les minéraux locaux. L'hétérogénéité à l'échelle des grains d'une roche en fusion conduit donc à un gradient de concentration chimique dans le liquide, qui sert de moteur à l'initiation de la migration du liquide. Des fines couches de liquide de fusion le long de bordures de grains formant un réseau enchevêtré s'établit immédiatement. Les hétérogénéités chimiques initiales dans le liquide s'estompent avec le temps.

Detection of replication-defective hepatitis A virus based on the correlation between real-time polymerase chain reaction and ELISA in situ results

ELISA in situ can be used to titrate hepatitis A virus (HAV) particles and real-time polymerase chain reaction (RT-PCR) has been shown to be a fast method to quantify the HAV genome. Precise quantification of viral concentration is necessary to distinguish between infectious and non-infectious particles. The purpose of this study was to compare cell culture and RT-PCR quantification results and determine whether HAV genome quantification can be correlated with infectivity. For this purpose, three stocks of undiluted, five-fold diluted and 10-fold diluted HAV were prepared to inoculate cells in a 96-well plate. Monolayers were then incubated for seven, 10 and 14 days and the correlation between the ELISA in situ and RT-PCR results was evaluated. At 10 days post-incubation, the highest viral load was observed in all stocks of HAV via RT-PCR (10(5) copies/mL) (p = 0.0002), while ELISA revealed the highest quantity of particles after 14 days (optical density = 0.24, p < 0.001). At seven days post-infection, there was a significant statistical correlation between the results of the two methods, indicating equivalents titres of particles and HAV genome during this period of infection. The results reported here indicate that the duration of growth of HAV in cell culture must be taken into account to correlate genome quantification with infectivity.

Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting

Quantitative polymerase chain reaction-high-resolution melting (qPCR-HRM) analysis was used to screen for mutations related to drug resistance in Mycobacterium tuberculosis. We detected the C526T and C531T mutations in the rifampicin resistance-determining region (RRDR) of the rpoB gene with qPCR-HRM using plasmid-based controls. A segment of the RRDR region from M. tuberculosis H37Rv and from strains carrying C531T or C526T mutations in the rpoB were cloned into pGEM-T vector and these vectors were used as controls in the qPCR-HRM analysis of 54 M. tuberculosis strains. The results were confirmed by DNA sequencing and showed that recombinant plasmids can replace genomic DNA as controls in the qPCR-HRM assay. Plasmids can be handled outside of biosafety level 3 facilities, reducing the risk of contamination and the cost of the assay. Plasmids have a high stability, are normally maintained in Escherichia coli and can be extracted in large amounts.

Molecular diagnosis of eosinophilic meningitis due to Angiostrongylus cantonensis (Nematoda: Metastrongyloidea) by polymerase chain reaction-DNA sequencing of cerebrospinal fluids of patients

Cerebrospinal fluid (CSF) samples from clinically diagnosed patients with detectable Angiostrongylus canto-nensis-specific antibodies (n = 10), patients with clinically suspected cases that tested negative for A. cantonensis-an-tibodies (n = 5) and patients with cerebral gnathostomiasis (n = 2) and neurocysticercosis (n = 2) were examined by a single-step polymerase chain reaction (PCR) method using the AC primers for the 66-kDa native protein gene. The PCR method detected A. cantonensis DNA in CSF samples from four of 10 serologically confirmed angiostrongyliasis cases. The PCR results were negative for the remaining CSF samples. The nucleotide sequences of three positive CSF-PCR samples shared 98.8-99.2% similarity with the reference sequence of A. cantonensis. These results indicate the potential application of this PCR assay with clinical CSF samples for additional support in the confirmation of eosinophilic meningitis due to A. cantonensis.

Influence of Bacille Calmette-Guérin vaccination on size of tuberculin skin test reaction : to what size ?

RESUME Le diagnostic d'infection tuberculeuse repose essentiellement sur le test tuberculinique (test de Mantoux). Cependant, le résultat de ce dernier est également influencé par d'autres facteurs, le plus important étant la vaccination par le Bacille Calmette-Guérin (BCG), interaction connue depuis de nombreuses années. Il est généralement admis que l'effet de la vaccination peut entraîner des réactions positives jusqu'à un diamètre d'induration de 15 mm. Au-delà, la positivité du test est en général attribuée à une primo-infection tuberculeuse. Peu d'études se sont réellement penchées sur le sujet. Chez le personnel de soins soumis à des Mantoux répétés, cette notion revêt une importance particulière pour interpréter correctement une réaction fortement positive en l'absence de facteurs de risque tuberculeux, dans un pays à faible endémie tuberculeuse. Notre étude a cherché à déterminer si le diamètre transversal de l'induration du Mantoux était un critère fiable pour distinguer une positivité associée à une infection tuberculeuse de celle associée à une ancienne vaccination. Elle s'est attachée à rechercher un seuil au-delà duquel l'infection tuberculeuse pourrait être considérée comme probable. Entre janvier 1991 et mars 1998, tous les nouveaux employés du CHUV ont été invités à recevoir un test tuberculinique à l'occasion de leur visite d'entrée à la Médecine du personnel. En cas de réponse négative, un deuxième test a été pratiqué une semaine plus tard, pour détecter un éventuel effet booster. Lors de la première visite, l'infirmière a rempli un questionnaire comprenant les données démographiques usuelles, des informations concernant les facteurs pouvant influencer la positivité du test, notamment les antécédents de vaccination par le BCG, les expositions à la tuberculose et l'existence d'antécédents d'infection tuberculeuse. Parmi les 5117 sujets inclus dans l'étude, nous avons trouvé que l'influence de la vaccination variait en fonction de l'âge. Chez les sujets de moins de 40 ans, la vaccination par le BCG était le prédicteur le plus important d'un Mantoux positif inférieur à 18 mm, de loin supérieur aux facteurs de risque habituels pour une infection tuberculeuse, eux aussi significatifs. L'effet du BCG était présent pour des réactions allant jusqu'à 20 mm. Pour les Mantoux supérieurs à 20 mm, l'odds ratio (OR) relatif au BCG demeure clairement élevé (supérieur à 3,4) bien que non significatif. Par contre, pour les employés âgés de plus de 40 ans, le BCG est un facteur prédictif pour les tests supérieurs à 10 mm (OR 2.4) mais n'est plus un facteur significatif pour une taille supérieure à 15 mm. Ces résultats montrent que l'interprétation d'un test tuberculinique même fortement positif, doit être faite avec prudence et discernement. En effet, notre étude démontre que chez les sujets vaccinés de moins de 40 ans, dans les zones de faible endémie tuberculeuse particulièrement en l'absence de facteurs de risque pour une infection tuberculeuse, un Mantoux positif jusqu'à 18 mm est dû, le plus probablement, à une ancienne vaccination par le BCG, plutôt qu'à une infection par M tuberczilosis. L'interprétation des Mantoux de taille inférieure à 18 mm et les Mantoux effectués chez des sujets de moins de 40 ans, doit prendre en compte l'existence d'un BCG antérieur. En conséquence, la mise en évidence d'une réaction de Mantoux fortement positive ne devrait pas conduire systématiquement à un traitement préventif. L'absence de spécificité du test Mantoux, utilisé pour le dépistage de la tuberculose depuis bientôt une centaine d'année, est un problème connu. Nous démontrons que la taille de l'induration ne peut pas être utilisée de façon fiable comme critère pour identifier une infection tuberculeuse chez une personne vaccinée avec le BCG, avec le risque de sui-traiter un nombre important de sujets. Dans notre étude, 21% des sujets avaient un Mantoux supérieur ou égal à 15 mm et auraient dû être traités selon les recommandations en vigueur en Suisse si l'on ne tenait pas compte du BCG antérieur. Des tests plus spécifiques sont actuellement à l'étude et permettront vraisemblablement, à l'avenir, de palier au problème de l'absence de spécificité du test de Mantoux. Abstract : Background. Previous bacillus Calmette-Guerin (BCG) vaccination can confound the results of a tuberculin skin test (TST). We sought to determine a cutoff diameter of TST induration beyond which the influence of BCG vaccination was negligible in evaluating potential Mycobacterium tuberculosis infection in a population of health care workers with a high vaccination rate and low incidence of tuberculosis. Methods. From 1991 through 1998, all new employees at the University Hospital of Lausanne, Switzerland, underwent a 2-step TST at entry visit. We also gathered information on demographic characteristics, along with factors commonly associated with tuberculin positivity, including previous BCG vaccination, history of latent M. tuberculosis infection, and predictors for M. tuberculosis infection. Results. Among the 5117 investigated subjects, we found that influence of BCG vaccination on TST results varied across categories of age (likelihood ratio test, 0.0001). Prior BCG vaccination had a strong influence on skin test results of mm in diameter among persons <40 years old, compared with the influence of factors predictive of M. tuberculosis infection. Prior latent M. tuberculosis infection and travel or employment in a country in which tuberculosis is endemic also had significant influences. Conclusions. Interpretation of TST reactions of mm among BCG-vaccinated persons <40 years of age must be done with caution in areas with a low incidence of tuberculosis. In such a population, except for persons who have never been vaccinated, TST reactions of ---518 mm are more likely to be the result of prior vaccination than infection and should not systematically lead to preventive treatment.

Use of sodC versus ctrA for real-time polymerase chain reaction-based detection of Neisseria meningitidis in sterile body fluids

We evaluated the use of a newly described sodC-based real-time-polymerase chain reaction (RT-PCR) assay for detecting Neisseria meningitidis in normally sterile sites, such as cerebrospinal fluid and serum. The sodC-based RT-PCR assay has an advantage over ctrA for detecting nongroupable N. meningitidis isolates, which are commonly present in asymptomatic pharyngeal carriage. However, in our study, sodC-based RT-PCR was 7.5% less sensitive than ctrA. Given the public health impact of possible false-negative results due to the use of the sodC target gene alone, sodC-based RT-PCR for the diagnosis of meningococcal meningitis should be used with caution.

Multiplex polymerase chain reaction to identify and determine the toxigenicity of Corynebacterium spp with zoonotic potential and an overview of human and animal infections

Corynebacterium diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis constitute a group of potentially toxigenic microorganisms that are related to different infectious processes in animal and human hosts. Currently, there is a lack of information on the prevalence of disease caused by these pathogens, which is partially due to a reduction in the frequency of routine laboratory testing. In this study, a multiplex polymerase chain reaction (mPCR) assay that can simultaneously identify and determine the toxigenicity of these corynebacterial species with zoonotic potential was developed. This assay uses five primer pairs targeting the following genes: rpoB (Corynebacterium spp), 16S rRNA (C. ulcerans and C. pseudotuberculosis), pld (C. pseudotuberculosis), dtxR (C. diphtheriae) and tox [diphtheria toxin (DT) ]. In addition to describing this assay, we review the literature regarding the diseases caused by these pathogens. Of the 213 coryneform strains tested, the mPCR results for all toxigenic and non-toxigenic strains of C . diphtheriae, C. ulcerans and C. pseudotuberculosis were in 100% agreement with the results of standard biochemical tests and PCR-DT. As an alternative to conventional methods, due to its advantages of specificity and speed, the mPCR assay used in this study may successfully be applied for the diagnosis of human and/or animal diseases caused by potentially toxigenic corynebacterial species.