The distribution of Mycobacterium bovis infection in naturally infected badgers.

Populations of Eurasian badgers (Meles meles) with tuberculosis (Mycobacterium bovis infection) are a significant reservoir of infection for cattle in Ireland and the United Kingdom. In this study the distribution of infection, histological lesions and gross lesions was determined in a sample of 132 culled badgers from naturally-infected wild populations. Badgers were culled when an epidemiological investigation following a tuberculosis breakdown in a cattle herd implicated badgers as the probable source of infection. The definition of tuberculosis infection was based on the isolation of M. bovis from tissues or clinical samples. An accurate diagnosis of infection was achieved by culturing a wide range of lymph nodes (LN) and organ tissues (mean 32.1) and clinical samples (faeces and urine) from each badger. Infection was detected in 57/132 badgers (43.2%). Histological lesions consistent with tuberculosis were seen in 39/57 (68.4%) culture-positive and 7/75 (9.3%) culture-negative animals. Gross lesions were seen in only 30/57 (52.6%) infected badgers, leaving a high proportion (47.4%) of infected animals with latent infection (no grossly visible lesions). The most frequently infected tissues were the lungs and axillary LN, followed by the deep cervical LN, parotid LN and tracheobronchial LN. The data support the hypotheses that in badgers there are only two significant routes of infection, namely, the lower respiratory tract and bite wounds, and that badgers are very susceptible to infection but resistant to the development and progression of the disease. At all levels of disease severity, infection was found in widely dispersed anatomical locations suggesting that there is early dissemination of infection in the period preceding the development of active immunity.

The prevalence and distribution of Mycobacterium bovis infection in European badgers (Meles meles) as determined by enhanced post mortem examination and bacteriological culture.

The accurate diagnosis of Mycobacterium bovis infection in badgers is key to understanding the epidemiology of tuberculosis in this species and has significant implications for devising strategies to limit spread of the disease. In this study, badgers (n=215) in the Republic of Ireland were examined at post mortem and tissues were collected from a range of anatomical locations and pooled into groups for bacterial culture of M. bovis. By assessing confirmed gross visible lesions (VL) alone, infection was detected in 12.1% of badgers. However, by including the results of all culture positive pooled samples, the overall infection prevalence increased significantly to 36.3%. Two-thirds (66.7%) of infected animals had no visible lesions (NVL). While the thoracic cavity (lungs and pulmonary lymph nodes) was found to be the most common site of infection, in a proportion of animals infection was absent from the lungs and draining lymph nodes and was confined to the lymph nodes of the carcase or the head. This may indicate an early extrapulmonary dissemination of infection or alternatively, in the case of the head lymph nodes, a secondary pathogenic pathway involving the lymphoid tissues of the upper respiratory tract (URT).

Experimental tuberculosis in the European badger (Meles meles) after endobronchial inoculation with Mycobacterium bovis: II. Progression of infection.

The aim of the study was to describe, over a period of 24 weeks, the pathological and bacteriological changes in badgers experimentally infected with Mycobacterium bovis. The badgers were infected by endobronchial instillation of 2.5 x10(4) colony forming units (cfu) M. bovis. After infection, the badgers were examined at 3 weekly intervals when blood and tracheal aspirates were collected. At 6, 12, 18 and 24 weeks post-infection (pi) three animals were euthanized and a detailed pathological and bacteriological examination was performed to assess the nature of the experimental disease. During the course of the study only one badger developed clinical signs of disease: a subcutaneous swelling on its head, first observed at 18 weeks pi. At post-mortem examination gross and histological lesions of tuberculosis were observed and M. bovis was recovered from all, except one badger. In the majority of badgers the endobronchial route of inoculation resulted in the establishment of infection that over 24 weeks was non-progressive with limited dissemination of infection from the thoracic cavity, mainly to the hepatic and mesenteric lymph nodes. However, in one of the badgers examined at 18 weeks pi and one at 24 weeks pi, infection was widely disseminated. The disease induced by the endobronchial inoculation displayed the characteristics of disease observed in naturally infected badgers.

Experimental tuberculosis in the European badger (Meles meles) after endobronchial inoculation of Mycobacterium bovis: I. Pathology and bacteriology.

The aim was to develop an endobronchial infection procedure for the study of Mycobacterium bovis infection in badgers. The badgers were anaesthetised and a cannula was passed per os to the tracheal bifurcation. When in place 1 ml of M. bovis suspension was inoculated. Three concentrations of M. bovis suspension were used; <10 colony forming units (cfu), approximately 10(2) cfu and approximately 3 x 10(3) cfu. The badgers were examined at three weekly intervals for clinical signs of disease and a tracheal aspirate was collected at each examination. The badgers were euthanased 17 weeks post infection (pi) and at the post mortem examination a wide range of tissues were examined for gross and histopathological lesions of tuberculosis and cultured for M. bovis. A sample of bronchial alveolar lavage (BAL) fluid was collected at post mortem for culture. At post mortem examination 17 weeks after infection, gross and histopathological lesions of tuberculosis were observed in all badgers inoculated with the high and medium dose and 1/3 inoculated with the low dose. M. bovis was recovered from all inoculated badgers. Infection in the high dose group was more widely disseminated than in the other groups. The number of sites with gross and histopathological lesions increased with increasing dose of M. bovis. All tracheal aspirates were negative on culture and only one BAL, collected from a badger of the high dose group, was positive on culture. No clinical signs due to the experimental infection were observed. The endobronchial route of inoculation is an effective route for establishing experimental infection, and could be used for studies of tuberculosis pathogenesis, immunology of M. bovis infection in badgers and for challenging badgers in vaccine protection studies. Badgers appeared to be very susceptible to infection by this procedure even with a dose of < 10 cfu but appear to control and limit the resulting infection.

Functional handgrip test to determine the coefficient of static friction at the hand/handle interface.

The aim was to devise a method of measuring friction at the hand/handle interface during a functional handgrip task. No descriptions of methods of this kind was found in the literature. An indirect technique of measuring normal grip force was employed to determine friction at the hand/handle interface while performing a functional handgrip action with a grabrail. The coefficient of static friction was calculated between palmar skin (dry, wet, and soapy hands) and five grabrail materials (stainless steel, powder-coated steel, chrome, textured aluminium and knurled steel). Thirty subjects participated (15 female, 15 male), who were aged from 17 to 45 years with a mean age of 30 years. Knurled steel produced a significantly larger mean coefficient of static friction than chrome, powder-coated steel and stainless steel, and textured aluminium had a significantly larger coefficient of static friction than stainless steel. Soapy hands produced the lowest mean coefficients (0.46+/-0.04), significantly less then dry (1.72+/-0.16, p <0.001) and wet hands (1.42+/-0.16, p <0.001). This study has demonstrated the influence of grabrail material and palmar skin treatments on static friction at the hand/handle interface. The use of a functional test that incorporates an indirect determination of normal handgrip force has provided a quantitative method of observing stability at the hand/handle interface.

Static friction properties between human palmar skin and five grabrail materials.

The purpose of this study was to investigate the static friction properties between human palmar skin and five grabrail materials (chrome, stainless steel, power-coated steel, textured aluminium and knurled steel) for dry, wet and soapy hands. Thirty subjects (15 female, 15 male) participated in this study, their ages ranging from 19 to 45 years with a mean age of 28 years. The normal force, friction force, and coefficient of static friction were determined by measuring three-dimensional forces while slipping the palm of the hand over the surface of a grabrail. A repeated measures ANOVA indicated that gender, age, hand size and trial effect had no significant influence (p>0.05) on these results. The coefficient of friction (p<0.001) and friction force (p<0.001) were significantly lower when the hand was soapy than when it was dry or wet. The normal force applied when the hand was soapy was significantly greater (p<0.001) than when it was dry or wet. No significant difference was found between dry and wet hands. The two textured materials displayed superior friction properties when the hand was soapy, while the smooth materials performed best when the hand was dry.

Monitoring resistance to human immunodeficiency virus type 1 protease inhibitors by pyrosequencing.

The emergence of drug-resistant viral variants is the inevitable consequence of incomplete suppression of human immunodeficiency virus type 1 (HIV-1) replication during treatment with antiretroviral drugs. Sequencing to determine the resistance profiles of these variants has become increasingly important in the clinical management of HIV-1 patients, both in the initial design of a therapeutic plan and in selecting a salvage regimen. Here we have developed a pyrosequencing assay for the rapid characterization of resistance to HIV-1 protease inhibitors (PIs). Twelve pyrosequencing primers were designed and were evaluated on the MN strain and on viral DNA from peripheral blood mononuclear cells from eight untreated HIV-1-infected individuals. The method had a limit of detection of 20 to 25% for minor sequence variants. Pattern recognition (i.e., comparing actual sequence data with expected wild-type and mutant sequence patterns) simplified the identification of minor sequence variants. This real-time pyrosequencing method was applied in a longitudinal study monitoring the development of PI resistance in plasma samples obtained from four patients over a 2 1/2-year period. Pyrosequencing identified eight primary PI resistance mutations as well as several secondary mutations. This sequencing approach allows parallel analysis of 96 reactions in 1 h, facilitating the monitoring of drug resistance in eight patients simultaneously and, in combination with viral load analysis, should be a useful tool in the future to monitor HIV-1 during therapy.

Genotyping by apyrase-mediated allele-specific extension.

This report describes a single-step extension approach suitable for high-throughput single-nucleotide polymorphism typing applications. The method relies on extension of paired allele-specific primers and we demonstrate that the reaction kinetics were slower for mismatched configurations compared with matched configurations. In our approach we employ apyrase, a nucleotide degrading enzyme, to allow accurate discrimination between matched and mismatched primer-template configurations. This apyrase-mediated allele-specific extension (AMASE) protocol allows incorporation of nucleotides when the reaction kinetics are fast (matched 3'-end primer) but degrades the nucleotides before extension when the reaction kinetics are slow (mismatched 3'-end primer). Thus, AMASE circumvents the major limitation of previous allele-specific extension assays in which slow reaction kinetics will still give rise to extension products from mismatched 3'-end primers, hindering proper discrimination. It thus represents a significant improvement of the allele-extension method. AMASE was evaluated by a bioluminometric assay in which successful incorporation of unmodified nucleotides is monitored in real-time using an enzymatic cascade.

Cooperative oligonucleotides in purification of cycle sequencing products.

Nucleic acid hybridization is an essential component in many of today's standard molecular biology techniques. In a recent study, we investigated whether nucleic acid capture could be improved by taking advantage of stacking hybridization, which refers to the stabilizing effect that exists between oligonucleotides when they hybridize in a contiguous tandem fashion. Here, we describe a specific approach for purification of sequencing products using cooperative probes that hybridize to single-strand targets where one of the probes has been coupled to a magnetic bead. This approach has been developed for standard sequencing primers and has been applied to shotgun plasmid libraries. The cooperative probes have been designed to anneal within the common vector sequence and to avoid co-purification of nonextended sequencing primers and misprimed sequencing products. The reuse of magnetic beads, together with salt independent elution, makes the approach suitable for high-capacity capillary electrophoresis instruments.

Quantitative investigation of the modular primer effect for DNA and peptide nucleic acid hexamers.

The effect on oligonucleotide-template duplex stability upon cohybridization of adjacently annealing oligonucleotides, the modular primer effect, was studied with biosensor technology. DNA and peptide nucleic acid (PNA) hexamer modules and sensor chip-immobilized template DNA strands were designed for analysis of nick, overlap, and gap modular hybridization situations. The fast hybridization kinetics for such hexamer modules allowed for the determination of apparent duplex affinities from equilibrium responses. The results showed that the hybridizational stability of modular hexamer pairs is strongly dependent on the positioning, concentration, and inherent affinity of the adjacently annealing hexamer module. Up to 80-fold increases in apparent affinities could be observed for adjacent modular oligonucleotide pairs compared to affinities determined for single hexamer oligonucleotide hybridizations. Interestingly, also for coinjections of different module combinations where DNA hexamer modules were replaced by their PNA counterparts, a modular primer effect was observed. The introduction of a single base gap between two hexamer modules significantly reduced the stabilization effect, whereas a gap of two bases resulted in a complete loss of the effect. The results suggest that the described biosensor-based methodology should be useful for the selection of appropriate modules and working concentrations for use in different modular hybridization applications.

[Modelling of intraperitoneal chemohyperthermia: experimental study and identification of certain thermal aspects]. / Modélisation de la chimiohyperthermie intrapéritonéale: étude expérimentale et identification de certains aspects thermiques.

Intraperitoneal chemohyperthermia is more and more considered as an interesting therapeutic option in cases of some abdominal cancers, particularly of digestive origin. However, many technical aspects of this treatment remain far from being mastered, particularly the homogeneous dispatch of temperatures within the abdomen cavity. This work consists, first of all, in an experimental study, which is being carried out on a physical "prototype" of the abdomen, on which different hot fluid flows and injection conditions (configurations) are investigated. The results of this experimental study are prospected in two ways. First, an a priori thermal model is proposed, based on physical equations (heat transfer, etc.). Then a "black box" model is identified from the measured temperatures evolutions, so as to obtain a model of the "system" behaviour. Finally, the two modelling approaches are being compared, and the results converge to a simple expression of a few parameters, either physical or identified. These modelling results have helped to optimize the injection circuit and its running parameters while applied to the human treatment.

Cooperative oligonucleotides mediating direct capture of hepatitis C virus RNA from serum.

A novel method for direct capture of hepatitis C virus (HCV) RNA from clinical samples has been developed. This approach takes advantage of the cooperative interactions between adjacently hybridized oligonucleotides. Here, this cooperative effect was combined with solid-phase technology, whereby a capture probe was covalently coupled to magnetic beads and a second probe, which anneals adjacent to the capture probe site, was prehybridized in solution to the target. When these contiguously hybridized probes were used for the extraction of HCV RNA from clinical samples, the capture efficiency was increased up to 25-fold in comparison to capture with a single probe. The applicability of this sample preparation assay was further investigated by performing a comparative study with both a conventional guanidinium extraction method and a commercial quantitative assay.

Capture of single-stranded DNA assisted by oligonucleotide modules.

Real-time biospecific interaction analysis was employed to monitor direct capture of a hepatitis C virus (HCV) derived polymerase chain reaction (PCR) product by nucleic acid hybridization. Different formats for hybridization were used to study the interaction between a single-stranded HCV PCR product and capture oligonucleotides immobilized on a sensor chip via streptavidin-biotin chemistry. By employing a prehybridization step in solution with nonbiotin oligonucleotides complementary to the single-stranded target and adjacent to the immobilized probe, a significant capture was achieved in comparison to the low capture efficiency obtained using single immobilized probes (9-36 mer). High capture efficiencies were also observed when shorter immobilized probes were used in combination with strings of adjacently positioned prehybridized probes (i.e., modules). Interestingly, the introduction of single nucleotide gaps between prehybridized and/or immobilized probes dramatically reduced the capture efficiency. These results suggest that flexible systems for capture could be designed from libraries of short oligonucleotides (9 mers) used in module fashion, taking advantage of stacking interactions between the oligonucleotides. The potential applications of such oligonucleotide-assisted capture systems are discussed.

NLRB to issue landmark decision on nurse practitioners.

The National Labor Relations Board is poised to issue a decision in Saints Memorial Medical Center. The narrow legal issue to be decided is with which of the eight acute care bargaining units nurse practitioners should be placed. The practical consequences of the decision, however, will be profound. The NLRB will determine, among other matters, the ease or difficulty of organizing nurse practitioners into labor organizations. Numerous friend-of-the-court briefs have been filed by various health care labor organizations, employer groups, and professional groups, taking contrary positions. The authors favor placement of the nurse practitioners and other advanced practice nurses within the physicians bargaining unit, because as a practical matter, this will lead to the creation of more nurse practitioner positions. Such a ruling, however, will make it more difficult to organize nurse practitioners.

Detection of the heat-stable toxin coding gene (ST-gene) in enterotoxigenic Escherichia coli: development of a colour amplified PCR detection system.

Screening biological samples using the polymerase chain reaction (PCR) has obvious advantages compared with current molecular analytical methods based on gel electrophoresis and/or hybridisation, both of which are expensive and time-consuming, therefore the development of a PCR assay format that is applicable to large sample numbers and that can readily use equipment commonly found in diagnostic laboratories would be advantageous. This report describes the development of a colour amplified PCR detection system which is simple in design and could be universally applied to the detection of any DNA template. As an example, the system has been applied in the detection of the heat-stable toxin coding gene (ST-gene) from enterotoxigenic Escherichia coli (ETEC). The assay is sensitive, detecting 10 fg of a purified DNA template and 270 cfu of an ST-gene-positive ETEC strain.

Colorimetric detection of heat-labile toxin-encoding gene of enterotoxigenic Escherichia coli by PCR.

In the developing world, enterotoxigenic Escherichia coli (ETEC) strains which produce enterotoxins are a significant cause of morbidity and mortality. Heat-labile (LT) toxin PCR detection methods have been described, but they have limited applications in a routine laboratory setting. A colorimetric DNA method for the rapid amplification and detection of the LT toxin gene in ETEC strains is described. Target amplification together with colorimetric detection would overcome many of the limitations of conventional PCR. This paper describes a colorimetric PCR detection method specific for LT-gene-encoding ETEC strains. DNA was extracted from two representative colonies from each bacterial isolate and amplified by PCR. Digoxigenin was incorporated into the amplification product, permitting a one-step direct detection using anti-digoxigenin alkaline phosphatase-conjugated antibody. This technique was applied to the investigation of 70 E. coli isolates derived from clinical fecal samples obtained from an Irish population. Eleven percent of the samples were LT positive, confirming the applicability of this method. All LT-positive ETEC strains (controls and clinical isolates) were detected, and no false-positive results occurred.