PCR-terminal restriction fragment length polymorphism for direct detection and identification of dermatophytes in veterinary mycology.

The biological diagnosis of dermatophytosis in veterinary medicine usually relies on direct microscopic examination and inoculation of the samples on appropriate culture media. However, identification of dermatophytes needs expertise, and cultures which require from days to weeks to be conclusive, may lack of sensitivity because of the quite common overgrowth of contaminants. Here we developed a polymerase chain reaction (PCR) assay based on terminal restriction fragment length polymorphism (TRFLP), which may improve sensitivity of the biological diagnosis and reduce the delay for initiation of treatment. This study was first conducted on pure cultures of various dermatophytes (27 species), yeasts (14 species) and moulds (45 species). After DNA extraction, the internal transcribed spacer (ITS)-28S region of ribosomal DNA was amplified with primers targeting specifically pathogenic dermatophytes, and species of interest were identified by TRFLP with appropriate restriction enzymes. After validation, this assay was applied to veterinary samples and results were compared to those obtained by direct microscopic examination and cultures. All target species were correctly identified, and none of the yeast or mould species was amplified, demonstrating specificity of the assay. Regarding clinical samples, the causative agent was detected by PCR-TRFLP from 97.1% of the samples with both positive direct microscopic examination and cultures. No dermatophytes were detected when both conventional tests were negative. PCR-TRFLP developed here demonstrated to be highly sensitive and specific, allowing rapid detection and direct identification of dermatophytes in veterinary practice. Therefore, this assay is especially suitable for the biological diagnosis of dermatophytosis in different animal species.

How to introduce a program of Enhanced Recovery after Surgery? The experience of the CAPIO group.

The traditional model of hospital care has been challenged by the development of a care-management process that allows early patient autonomy (outpatient surgery, Enhanced Recovery after Surgery). Hospitalization has been transformed in response to this development, based on innovative medical and organizational strategies. Within a surgical service, the deployment of these processes requires the creation of a support structure, with re-organization of existing structures, analysis of potential obstacles, implementation of management tools, and ongoing follow-up of organizational function, clinical results, organizational and patient satisfaction. These will ultimately assess adaptation of structures within these new organizations. In this article, we share our insights based on experience gained over the past six years by surgical teams of the CAPIO group.

Evolutionary Pathways to Break Down the Resistance of Allelic Versions of the PVY Resistance Gene va.

Emergence of viral genotypes can make control strategies based on resistance genes ineffective. A few years after the deployment of tobacco genotypes carrying alleles of the Potato virus Y (PVY) recessive resistance gene va, virulent PVY isolates have been reported, suggesting the low durability of va. To have a broader view of the evolutionary processes involved in PVY adaptation to va, we studied mutational pathways leading to the emergence of PVY resistance-breaking populations. The viral genome-linked protein (VPg) has been described to be potentially involved in va adaptation. Analyses of the VPg sequence of PVY isolates sampled from susceptible and resistant tobacco allowed us to identify mutations in the central part of the VPg. Analysis of the virulence of wild-type isolates with known VPg sequences and of mutated versions of PVY infectious clones allowed us to (i) validate VPg as the PVY virulence factor corresponding to va, (ii) highlight the fact that virulence gain in PVY occurs rapidly and preferentially by substitution at position AA105 in the VPg, and (iii) show that the 101G substitution in the VPg of a PVYC isolate is responsible for cross-virulence toward two resistance sources. Moreover, it appears that the evolutionary pathway of PVY adaptation to va depends on both virus and host genetic backgrounds.

Anthraquinone-2-sulfonic acid (AQ2S) is a novel neurotherapeutic agent.

Anthraquinone derivatives such as emodin have recently been shown to protect in models of beta amyloid ß (Aß) and tau aggregation-induced cell death. The mechanisms of action possibly involve preconditioning effects, anti-aggregation properties, and/or enhancing the phosphatidylinositol-3-kinase (PI3K)/AKT survival mechanism. We studied several natural (emodin, rhein, and aloin) and synthetic (AQ2S) anthraquinones, to screen for post-treatment therapeutic benefit in two models of neuronal death, namely hydrogen peroxide (H(2)O(2)) and staurosporine (STS)-induced injury. Treatment with emodin, rhein, or aloin failed to reduce H(2)O(2) injury. Moreover, consistent with emodin behaving like a mild toxin, it exacerbated oxidative injury at the highest concentration used (50 µM) in our post-treatment paradigm, and potently inhibited AKT. In contrast, AQ2S was neuroprotective. It reduced H(2)O(2) injury at 50 and 75 µM. In addition, AQ2S potently inhibited staurosporine (STS)-induced injury. The mechanisms of action involve caspase inhibition and AKT activation. However, blockade of AKT signaling with LY294002 failed to abolish AQ2S-mediated protection on the STS assay. This is the first study to report that AQ2S is a new neuroprotective compound and a novel caspase inhibitor.

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.

Development of SCAR markers linked to three disease resistances based on AFLP within Nicotiana tabacum L.

Amplified fragment length polymorphism (AFLP) was conducted on a set of 92 Nicotiana tabacum L. accessions from diverse types (flue-cured, dark air-cured, burley, oriental, and cigar wrapper) and breeding origins to identify markers associated with disease resistances. Eleven primer combinations were required to identify 33 polymorphic fragments. This allowed the identification of 92% of these accessions, and yielded sufficient information for building a neighbor joining tree. Clusters of accessions with common traits or breeding origins were observed. An important part of this polymorphism could be related to interspecific introgressions from other Nicotiana species, performed during the breeding history of N. tabacum to confer resistance to pathogens. Seven fragments were associated with three different resistances: two for the blue-mold (Peronospora tabacina Adam) resistance derived from Nicotiana debneyi Domin, two for the Va gene (Potato Virus Y susceptibility), and three for the black root rot (Chalara elegans) resistance of N. debneyi origin. Some of these markers were converted into sequence characterized amplified region markers, and validated on recombinant inbred lines or doubled-haploid lines.

Pathogen-induced elicitin production in transgenic tobacco generates a hypersensitive response and nonspecific disease resistance.

The rapid and effective activation of disease resistance responses is essential for plant defense against pathogen attack. These responses are initiated when pathogen-derived molecules (elicitors) are recognized by the host. We have developed a strategy for creating novel disease resistance traits whereby transgenic plants respond to infection by a virulent pathogen with the production of an elicitor. To this end, we generated transgenic tobacco plants harboring a fusion between the pathogen-inducible tobacco hsr 203J gene promoter and a Phytophthora cryptogea gene encoding the highly active elicitor cryptogein. Under noninduced conditions, the transgene was silent, and no cryptogein could be detected in the transgenic plants. In contrast, infection by the virulent fungus P. parasitica var nicotianae stimulated cryptogein production that coincided with the fast induction of several defense genes at and around the infection sites. Induced elicitor production resulted in a localized necrosis that resembled a P. cryptogea-induced hypersensitive response and that restricted further growth of the pathogen. The transgenic plants displayed enhanced resistance to fungal pathogens that were unrelated to Phytophthora species, such as Thielaviopsis basicola, Erysiphe cichoracearum, and Botrytis cinerea. Thus, broad-spectrum disease resistance of a plant can be generated without the constitutive synthesis of a transgene product.

Bidirectional inferior vena cava-pulmonary artery shunt.

BACKGROUND: Bidirectional superior vena cava-pulmonary shunt is widely used as an interim palliation for patients with univentricular hearts. Bidirectional inferior vena cava-pulmonary artery shunt, as an alternative approach of partial Fontan circulation, may offer the advantage of performing the complete Fontan circulation more easily due to the already constructed inferior vena cava lateral tunnel. METHODS: We used bidirectional inferior vena cava-pulmonary artery shunt in 2 patients. Contraindications to a complete Fontan circulation were due to, respectively, a volume-overloaded systemic ventricle and an irregular pulmonary arterial tree. RESULTS: Postoperative courses were uneventful. There were no significant pleural effusions. Transcutaneous oxygen saturations were 77% and 78%. Pulmonary-to-systemic blood flow ratios were 0.57 and 0.63. A complete Fontan circulation was safely performed 8 and 12 months later, without any "Fontan-related" complications. CONCLUSIONS: Bidirectional inferior vena cava-pulmonary artery shunt can be useful in selected patients with univentricular hearts, although its place in the field of "partial Fontan operations" cannot be determined as yet.

[Pulmonary arborization abnormalities in complex forms of pulmonary atresia with ventricular septal defect: unification, unifocalization and complete repair]. / Défauts d'arborisation pulmonaire des formes complexes d'atrésie pulmonaire à septum ouvert:unification, unifocalisation et réparation complète.

The presence of intrapulmonary arborization abnormalities in patients with pulmonary atresia and ventricular septal defect remains a therapeutic challenge. The aim of this study was to assess the value of procedures of pulmonary unifocalization, i.e. pulmonary unification, remodelling of the central pulmonary arteries and creation of an unifocal pulmonary blood supply, thereby resulting in complete repair. From october 1989 to october 1995, 27 unifocalization procedures were performed in 19 patients. The number of pulmonary segments dependant on non-communicating systemico-pulmonary collaterals was 14.7 +/- 5.4 per patient. The number of non-communicating systemico-pulmonary collaterals was 3.4 +/- 1.2 per patient. The Nakata index was 71 +/- 83 mm2/mm2. There were 3 deaths after an unifocalization procedure (mortality rate 15.8%). In 12 patients (63.2% of cases) a pulmonary arterial tree compatible with a complete repair was obtained. Eight complete repairs, with no mortality, following one or several pulmonary unifocalization procedures with a right to left ventricular systolic pressure ratio of 0.61 +/- 0.12 (range 0.4 to 0.75). Pulmonary unifocalization increases the recruitment of pulmonary segments and thereby the possibilities of complete correction in forms of pulmonary atresia with ventricular septal defect and arborization abnormalities of the pulmonary arterial tree.

Biatrial inferior transseptal approach to the mitral valve.

The standard surgical approach to the mitral valve is via a longitudinal incision in the left atrium. This is applicable in the vast majority of patients. In cases of small left atrium with poor exposure the standard incision may be modified. We report a biatrial inferior transseptal approach that we have employed in 25 patients over a 2-year period. The technique is simple to execute and is without risk to surrounding structures.

Computerized densitometric analysis of interproximal bone levels in a controlled clinical study into the treatment of periodontal bone defects with ceramic hydroxyapatite implant material.

The aim of this controlled clinical study was to utilize computer-assisted densitometric analysis of radiographs to assess the effectiveness of treating periodontal osseous defects with a sintered hydroxyapatite implant material. It was found that over the 2-year period of the study for the osseous defects treated by the implant material, there was a gain in the height of the hard tissue relative to the cemento-enamel junction; this gain was statistically significant compared with the results for the control sites.

[Accessory mitral tissue responsible for left ventricular outflow obstruction. Reports of 7 cases]. / Tissu mitral accessoire responsable d'une gêne à l'éjection du ventricule gauche. A propos de 7 cas.

The authors report the medico-surgical experience of Marie Lannelongue hospital of a rare condition: accessory mitral valve tissue. Seven patients aged 2 to 28 years (average: 8.7 years) had left ventricular outflow obstruction due to accessory mitral valve tissue. The diagnostic was not obvious clinically and was based on the association of echocardiographic and angiographic data. This condition was associated with another intra-cardiac malformation in 6 of the 7 patients. Surgical treatment included resection of the accessory mitral valve tissue by an aortic or combined aorto-left atrial approach, together with correction of the associated intracardiac abnormality. The postoperative results were excellent with the regression of the ventriculo-aortic pressure gradient and the physiological integrity of the mitral valve.

A microcomputer system for the analysis of dental radiographs.

A system for the digital storage and computer analysis of dental radiographs is described. The system is based on popular microcomputer, of a type commonly used for other purposes in general dental practice. The analysis system includes methods to compensate for variations due to exposure and development in serial radiographs. Interactive software allows a detailed analysis of the radiograph, producing qualitative and quantitative data for diagnosis and monitoring. An example of its application is given.

[Analysis of factors related to early mortality in digestive hemorrhage caused by portal hypertension]. / Analyse des facteurs liés à la mortalité précoce au cours des hémorragies digestives dues à l'hypertension portale.

In order to determine immediate criteria of prognosis for patients with portal hypertension hospitalized for digestive hemorrhage, in an intensive care unit, 18 variables were recorded during the 24 hours following admission in 65 patients. Data related to death were age, ascites, hepatic encephalopathy, shock, active hemorrhage, acute pneumonia, decrease in prothrombin time, use of esophageal balloon tamponade, use of mechanical ventilation, number of red blood cell units transfused. Discriminant analysis yielded a linear combination of 4 variables which best separated survivors from non survivors with the following equation: F = 0.330 X hepatic encephalopathy + 0.433 X shock + 0.226 X active hemorrhage + 0.0097 X age - 0.396. The threshold decision of the hemorrhage prognosis index (HPI) was F = 0.57; 80 p 100 of all patients were correctly classified. In order to be validated, HPI was compared with a general (SAPS) and specific (Pugh's classification) scoring system, in a prospective study of 57 episodes of digestive hemorrhage. In this study, sensitivity was better with HPI than with SAPS (0.70 versus 0.45), specificity was higher with HPI than with Pugh's classification (0.86 versus 0.70). Percentage of correctly classified patients was higher using HPI (81 p. 100) than SAPS (77 p. 100) and Pugh's classification (68 p. 100). We suggest that the HPI, determined with 4 easily defined and recorded variables should be used prospectively to compare efficacy of different treatments.

Technical and clinical data on high-performance hemofiltration: twelve patients during one year.

In 12 chronic hemodialysis patients, postdilutional hemofiltration (HF) was substituted for conventional acetate hemodialysis (HD) (4-5 h/session with high-area capillary dialyzers). In HF, the purposes were to obtain (a) no increase in pre-HF uremia compared with pre-HD uremia (high ultrafiltrate volume), (b) an HF duration shorter than that of HD (mean ultrafiltrate rate greater than 120 ml/min), (c) a disposable cost of an HF session identical to that of an HD session (reuse of hemofilters and extemporaneous preparation of substitution fluid). One-year results were (a) an ultrafiltrate volume of 26.8 L/session and a pre-HF uremia of 35.4 mmol/L (pre-HD uremia 34.0 mmol/L), (b) a mean ultrafiltrate rate of 143 ml/min and a mean HF duration of 190 min (mean HD session duration 250 min), and (c) better clinical tolerance and vascular stability in HF than in HD (weight loss 3.5 kg in HF and 3.0 kg in HD). Reuse of filters and extemporaneous preparation of substitution fluid were not responsible for any pyrogen reaction or bacterial contamination. In conclusion, (a) compared with conventional HD, high-flux HF results included identical removal of small molecules, improvement in vascular stability, decrease in session duration, and identical disposable cost; (b) routine high-flux HF is workable in a dialysis unit; (c) vascular access is the most important limiting factor to high-flux HF. Today 30-40% of patients can be treated with this method.

Stereospecific hydrolysis by soluble and immobilized lipases.

Stereospecific hydrolysis of insoluble monoesters by lipases are reported. Among the lipases tested, porcine pancreatic lipase was the most stereospecific when acting on 3-chloro-2-methyl propanol propionate. When the chain length of the acid was enhanced, the stereospecificity decreased. Initial rate measurements analysis concluded that the observed stereospecificity was the result of different catalytic constants rather than different Michaelis constants. From these results, methods were derived for the preparation of l- or d-3-chloro-2-methylpropanol (an intermediary in the synthesis of levomepromasine) based on the hydrolysis of esters by soluble or immobilized lipases.

[Non-ketotic hyperglycaemic coma with diabetic lipaemia and hyponatraemia (author's transl)]. / Coma hyperglycémique non cetosique avec lipémie diabétique et hyponatrémie.

A 68-year old male patients without personal or family history of diabetes mellitus developed diabetic lipaemia with severe hyponatraemia and became deeply comatose. The low blood sodium level (110 mmol/l) was consecutive to hyperglycaemia (45 mmol/l), hyperlipaemia (65 g/l) and true sodium depletion. This rare form of type V hyperlipidaemia is attributed to deficiency of heparin-activated lipoprotein lipase; it rapidly responds to insulin therapy. Hyponatraemia is a prominent feature of the syndrome, but it is partly due to blood dilution resulting from hyperglycaemia and therefore fictitious. For accurate measurement of sodium depletion blood sodium levels must be adjusted taking into account blood lipoprotein and glucose levels.