Development and application of MLVA methods as a tool for inter-laboratory surveillance.

Multiple-locus variable-number of tandem-repeats analysis (MLVA) has emerged as a valuable method for subtyping bacterial pathogens and has been adopted in many countries as a critical component of their laboratory-based surveillance. Lack of harmonisation and standardisation of the method, however, has made comparison of results generated in different laboratories difficult, if not impossible, and has therefore hampered its use in international surveillance. This paper proposes an international consensus on the development, validation, nomenclature and quality control for MLVA used for molecular surveillance and outbreak detection based on a review of the current state of knowledge.

Toll-like receptor 2 is present in the microenvironment of oral squamous cell carcinoma.

BACKGROUND: The aim of this study was to investigate the expression of toll-like receptor 2 (TLR2) on cells associated with oral squamous cell carcinoma, epithelial dysplasia and irritative hyperplasia, using immunohistochemistry. RESULTS: More immune cells expressed TLR2 in carcinoma and dysplasia than in hyperplasia (P<0.001). No hyperplastic samples showed positive TLR2 staining on keratinocytes, whereas keratinocytes in 64% of cases of carcinoma and 74% of cases of dysplasia were TLR2 positive. CONCLUSION: Positive TLR2 expression in the microenvironment suggests activation of immune surveillance against the altered epithelium, whereas TLR2 expression by malignant keratinocytes may be indicative of resistance to apoptosis as a pro-survival mechanism.

Surveillance for Listeria monocytogenes and listeriosis, 1995-2004.

Canadian cases and outbreaks of illness caused by Listeria monocytogenes between 1995 and 2004 were assessed. Isolates (722 total) were characterized by serotyping, and pulsed-field gel electrophoresis (PFGE) was performed to provide a means of detecting case clusters. Rates of listeriosis remained fairly consistent during the period of study, and patient characteristics were similar to those seen in studies of other populations. Most isolates were obtained from blood and cerebrospinal fluid, although during some outbreak investigations isolates were also obtained from stools. Serotype 1/2a predominated in isolates from patients in Canada, followed by serotypes 4b and 1/2b. Outbreaks caused by L. monocytogenes that occurred during the period of study were caused by isolates with serotypes 1/2a and 4b. A retrospective analysis of PFGE data uncovered several clusters that might have represented undetected outbreaks, suggesting that comprehensive prospective PFGE analysis coupled with prompt epidemiological investigations might lead to improved outbreak detection and control.

Characterization of antimicrobial resistance, molecular and phage types of Salmonella enterica serovar Typhi isolations.

Isolation rates in Canada of Salmonella enterica serovar Typhi increased from 0.29 to 0.55 isolations/100,000 population during 2000-2006. Although no ciprofloxacin resistance was detected, nalidixic acid resistance increased from 41% to 80%. Multidrug-resistant S. Typhi represented 18% of the strains tested. Pulsed-field gel electrophoresis (PFGE) analysis of 222 isolates resulted in 91 distinct patterns clustering into four major genetic similarity groups. The five most frequently occurring PFGE patterns accounted for 46% of the isolates. Drug-resistant isolates predominantly occurred in one PFGE similarity group. There were 39 phage types identified in 826 isolates analysed with 60% described by five phage types; 134 were untypable. The phage types associated with multidrug resistance were phage types 53, B1, D1, E1, E9, G3 and M1. Improved integration of epidemiological and laboratory case data will facilitate the protection of public health in Canada during an era of increasing travel and globalization.

Characterization of Canadian cefoxitin-resistant non-typhoidal Salmonella isolates, 2005-06.

OBJECTIVES: Resistance to extended-spectrum cephalosporins has increased in Salmonella worldwide, and is a concern in both hospital and community settings. The aim of this report was to investigate cefoxitin-resistant Salmonella isolates identified from human clinical cases across Canada. METHODS: Cefoxitin-resistant isolates, defined as having an MIC > or = 32 mg/L, were screened for the ampC classes DHA, FOX, ENT and CIT in a multiplex PCR followed by sequence analysis. Plasmid analysis by restriction fragment length polymorphism (RFLP) and replicon typing was performed on a convenience sample of cefoxitin-resistant Salmonella. RESULTS: In 2005, 5.3% (181/3442) and in 2006, 3.1% (102/3250) of Salmonella isolates collected from all provinces across Canada displayed cefoxitin resistance. Seventy-one out of 283 (25.1%) were multidrug resistant (MDR), as defined by resistance to at least three different antibiotic classes. The bla(CMY-2) gene was harboured by 96.8% (274/283) of the cefoxitin-resistant isolates. Analysis of CMY-2 plasmids revealed that 19.7% contained genes conferring resistance to multiple antimicrobials. Replicon typing of transformant CMY-2 plasmid DNA revealed the predominance of I1-Igamma and A/C. Of the MDR CMY-2 plasmids, 75% contained replicon type A/C. RFLP patterns of CMY-2 plasmids revealed clusters corresponding to the I1-Igamma and A/C replicon types. CONCLUSIONS: Incompatibility group I1-Igamma is the most prevalent of the Salmonella CMY-2 plasmids, while A/C is associated with MDR CMY-2 plasmids.

Delta 9 -tetrahydrocannabinol and ethanol: differential effects on sympathetic activity in differing environmental setting.

Serum dopamine beta-hydroxylase activity, a useful biochemical index of peripheral sympathetic nervous activity, was measured in rats treated with Delta(9)-tetrahydrocannabinol or ethanol or both substances. After 7 days of treatment with either substance, serum dopamine beta-hydroxylase activity decreased significantly. Combined treatment with both agents enhanced the effects of each given alone. In rats subjected to immobilization stress, treatment with Delta(9)- tetrahydrocannabinol appeared to potentiate the stress-induced increase in serum enzyme activity. Treatment with ethanol, with or without Delta(9)-tetrahydrocannabinol, effectively blocked this increase in enzyme activity. These results show that both substances have significant effects on the sympathetic nervous system which are critically influenced by environmental setting.

Monozygotic dichorionic twins heterokaryotypic for duplication chromosome 2q13-q23.3.

We present an evaluation of the diagnosis, management and outcome of a pair of heterokaryotypic monozygotic dichorionic twins. The heterokaryotype was an incidental finding from an amniocentesis performed for prenatal diagnosis of beta-thalassaemia major in a pair of dichorionic twins. Monozygocity was revealed by QF-PCR showing identical short tandem repeat markers on chromosomes 21, 18, 13, X and Y. The twins were heterokaryotypic for duplication chromosome 2q13-q23.3, as shown by array comparative genomic hybridization. Selective foeticide was performed. This case demonstrates that heterokaryotypic monozygotic dichorionic twins are a genetic possibility that does occur.

Occurrence and characterization of Salmonella from chicken nuggets, strips, and pelleted broiler feed.

Raw, frozen chicken nuggets and strips have been identified as a significant risk factor in contracting foodborne salmonellosis. Cases of salmonellosis as a result of consuming partly cooked chicken nuggets may be due in part to Salmonella strains originating in broiler feed. This study was undertaken to determine the occurrence and characterize the strains of Salmonella contaminating chicken nuggets, strips, and pelleted feeds, in an attempt to demonstrate whether the same Salmonella strains present in broiler feed could be isolated from raw, frozen chicken nuggets and strips available for human consumption. Salmonellae were recovered using the Health Canada MFHPB-20 method for the isolation and identification of Salmonella from foods. Strains were characterized by serotyping, phage typing, antimicrobial resistance typing (R-typing), and by pulsed-field gel electrophoresis (PFGE). Salmonellae were isolated from 25-g samples in 27% (n=92) of nugget and strip samples, 95% (n=20) of chicken nugget meat samples, and from 9% (n=111) of pelleted feed samples. Salmonella Heidelberg, Salmonella Enteritidis, and Salmonella Orion were the most commonly isolated serovars from chicken nuggets and strips, nugget and strip meat, and pelleted broiler feeds, respectively. Salmonella Enteritidis phage type (PT) 13a with PFGE pattern SENXAI.0006 and R-type sensitive as well as Salmonella Enteritidis PT13a with PFGE pattern SENXAI.0068 and R-type sensitive were isolated from pelleted feed, and chicken nugget and strip meat in two separate instances. Data showed that Salmonella strains isolated from broiler feed were indistinguishable from strains isolated from packaged raw, frozen chicken nuggets and strips. However, results did not rule out the possibility that breeding stock or contamination during processing may have contributed to chicken meat contamination by Salmonella.

Modification of morphine-withdrawal syndrome in rats following transauricular electrostimulation: an experimental paradigm for auricular electroacupuncture.

The experimental paradigm reported here provides a useful animal model with which to further study the effects of regional electrostimulation and its possible relationship with electrical acupuncture. What similarities there may be between electrical acupuncture and the technique of regional electrocerebral stimulation used for production of electrosleep and electronarcosis in animals and man (Wageneder et al., 1966) remain to be elucidated. Further research studies, both clinical and basic, are clearly needed to answer some of the questions relating to the safety, efficacy, and possible mechanisms by which a technique such as electrical acupuncture may exert its effects.

Isolation and characterization of Campylobacter pyloridis from gastric biopsies.

Gastric biopsy specimens were examined microbiologically and histologically for the presence of Campylobacter pyloridis. Of 51 randomly selected patients, 22 (43%) were found to harbor C. pyloridis in the gastric mucosa. The histologic demonstration of spiral organisms observed by staining with hematoxylin and eosin correlated well with microbiologic isolation of the organisms. There was a strong association (95.5%) between C. pyloridis in the gastric mucosa and histologically defined gastritis. However, there was no obvious association between C. pyloridis and ulcers. All C. pyloridis strains isolated exhibited uniform biochemical characteristics and had almost identical protein profiles, which indicated that they belong to a relatively homogeneous group distinct from other Campylobacter species. All C. pyloridis isolates were uniformly susceptible to ampicillin, amoxicillin, cephalothin, gentamicin, kanamycin, tetracycline, coumermycin, ciprofloxacin, novobiocin, clorobiocin, and nitrofurantoin. They were moderately resistant to nalidixic acid.

Mankind, its numbers, health, and family arrangements: group report 3.

PIP: The population problem is an imbalance between numbers of people and limited resources. The resources of food, energy, and living space have been well documented as finite in a world with population expanding geometrically. Terrorism, breakdown of social order, and other social symptoms may also stem from this overcrowding. The economically more affluent nations are attempting to deal with unregulated fertility by developing and spreading information on means of conception control and granting equality of status to women to compensate for the reduction in the childbearing role. Social systems are very complex and changing the desire for children is quite difficult. In addition, people have a great capacity for ignoring potential disaster when surrounded by it. Liberty of the individual and control must be kept in balance. It is recommended that more effort be made to understand the motivations behind human reproduction and what social incentives exist to reduce family size. There is also need for contraceptive technology and for more effective dissemination thereof. Values and attitudes need to be changed and the mechanisms of communication, the institutional and organizational structures operating in the field of reproductive behavior, and the role of policy determination by the nation need to be studied. There is need for fertility control in conjunction with mortality control. The committee believes that basic minimum components of the quality of life may be imposed upon a people if necessary in order to preserve the diversity of cultural and individual differences.^ieng

Plasmid analysis and antimicrobial susceptibilities of Peptostreptococcus species.

There are no published methods on plasmid isolation from Peptostreptococcus spp., therefore two methods of plasmid isolation from this genera were analysed: the boiling and alkaline-SDS methods. Plasmid DNA was not recovered by the boiling method, however, with the alkaline-SDS method, cryptic plasmid DNA was detected in two P. asaccharolyticus and one P. magnus strains. To achieve optimum lysis, Peptostreptococcus cells were treated with lysozyme (2 mg/ml) for 15 min. at 37 degrees C followed by proteinase K (0.2 mg/ml) for 1 h at 37 degrees C. In addition we report, the occurrence of clindamycin or metronidazole-resistant peptostreptococci, but these phenotypes were not correlated with plasmid carriage.

Strategies for molecular characterisation of methicillin- and gentamicin-resistant Staphylococcus aureus in a Canadian nosocomial outbreak.

Sixteen methicillin-resistant Staphylococcus aureus (MRSA) isolates, from a single nosocomial outbreak, were tested for molecular and phenotypic relationships. Two of the 16 outbreak strains were gentamicin resistant (Gmr) and the plasmids that they carried were characterised by reverse field electrophoresis, restriction endonuclease analysis and gene hybridisation. The gentamicin-resistant (Gmr) strains harboured two plasmids, a Gmr plasmid of 36.5 kb and a cryptic plasmid of 25.4 kb, whereas the other 14 isolates contained only the cryptic plasmid. Gentamicin resistance was encoded by a 2.5-kb HindIII fragment of the 32.8-kb plasmid and is similar to the 2.5-kb HindIII fragment also described for S. aureus Gmr plasmids from Australia and the USA. The Gmr plasmid was non-conjugal and was cured by ethidium bromide at a frequency of 4%. Two MRSA strains isolated subsequently from the same hospital were also Gmr and had identical plasmid and restriction endonuclease profiles to the two Gmr strains studied initially. Two other S. aureus isolates from the original carrier detected in this study and from his son were methicillin and gentamicin susceptible and had novel profiles. Since large plasmids show anomalous migration in agarose gels, more definitive analyses than simple plasmid identification should be considered when studying nosocomial outbreaks.

Hematoma from arteriovenous malformation producing hydrocephalus and simulating a thalamic tumor. Report of two cases.

Two patients with a history of progressive unilateral neurological symptoms and signs, and evidence of obstructive hydrocephalus from a mass lesion adjacent to the third ventricle as demonstrated by pneumography, were each found to have an intracerebral hematoma secondary to remote hemorrhage from a small vascular malformation. One patient died shortly after surgical exploration and the other after ventriculography. The pathophysiology of hydrocephalus associated with a vascular malformation is discussed and the need for considering a benign cause for obstructive hydrocephalus from a mass deep in the brain substance is emphasized.

Sine-wave auricular TENS produces frequency-dependent hypesthesia in the trigeminal nerve.

OBJECTIVE: To evaluate the effects of different frequencies of auricular (ear-to-ear) sine-wave transcutaneous electrical nerve stimulation (TENS), administered at subliminal intensity, on trigeminal nerve sensitivity. DESIGN: In a double-blind protocol, healthy volunteer subjects were administered one of three different frequencies of active TENS (5, 100, or 2,000 Hz) or placebo TENS (no current was passed) for 30 min. SETTING: Department of Psychology, City University of New York. SUBJECTS: 72 healthy undergraduate volunteers with no preexisting pain problems (16 men and 56 women), from the Department of Psychology, City University of New York. OUTCOME MEASURE: Pretreatment to posttreatment changes were measured in sensation threshold for a 250-Hz electrical stimulus applied transcutaneously to an area 1 cm anterior to the tragus of the ear (mandibular division of the trigeminal nerve). RESULTS: Analysis of variance indicated no group differences in baseline trigeminal sensation threshold, but there were significant group differences in pretreatment to posttreatment changes in sensation threshold (p < 0.001). A postiori analysis showed significant increases in trigeminal sensation threshold after active TENS as compared to placebo TENS (p < 0.05), with 5- and 100-Hz TENS producing significantly greater hypesthesia than 2,000-Hz TENS (p < 0.05). CONCLUSIONS: Sine-wave auricular TENS produces frequency-dependent trigeminal hypesthesia that is not a placebo effect.

Gas chromatographic-mass spectrometric analysis of acids and phenols in distilled alcohol beverages. Application of anion-exchange disk extraction combined with in-vial elution and silylation.

A GC-MS protocol for profiling spirits, based on 19 acids and phenolic compounds, has been proposed and evaluated. The method combined a simple preconcentration procedure based on solid-phase (anion-exchange) disk extraction, and in-vial elution and silylation of the analytes. The derivatized extract was directly injected into the GC-MS system. These analytes were: C6, C8, C10, C12 acids, pyruvic acid, 2-furoic acid, succinic acid, fumaric acid, glutaric acid, lactic acid, glycolic acid, malic acid, tartaric acid, citric acid, vanillin, syringaldehyde, coniferaldehyde, vanillic acid and gallic acid. The profiles of six different spirits were found reproducible from day-to-day with <20% RSD for measurements of most of the analytes at different concentrations. Recoveries of individual analytes appear to be affected by the level of tannins in the spirits, and they varied from sample to sample. The method of standard addition was used to quantify age-related analytes. Good linearity of response with correlation coefficients in the range of 0.992-0.999 was obtained. The results of the study indicate that for spirits of the same brand but of different ages, the amounts of these analytes appear to increase with the ageing period.

Characterization of cigar tobaccos by gas chromatographic/mass spectrometric analysis of nonvolatile organic acids: application to the authentication of Cuban cigars.

A reliable method based on gas chromatographic/mass spectrometric (GC/MS) profiling of nonvolatile organic acids is described for the characterization of cigars. The method involves an aqueous extraction of ground tobacco and selective isolation of the acids by simply stirring strong anion exchange (SAX) disks in the aqueous tobacco extract. The acids are then directly silylated on the disk with N-methyl-N-trimethylsilyl-trifluroacetamide (MSTFA) in acetonitrile in an autosampler vial. Elution of the derivatized acids in situ allows the sample to be directly analyzed by GC/MS without further sample handling. Compared to the conventional disk-extraction technique using a vacuum manifold, this method is much less labor intensive, and is desirable for multiple sample analysis. Nicotinic acid, succinic acid, glyceric acid, malic acid, pyroglutamic acid, threonic acid, citric acid, uracil, and an unidentified acid were reproducibly quantified in tobacco samples. Principal component analysis (PCA) of the acid profiles of the filler tobaccos of 18 Cuban cigars and 31 non-Cuban cigars shows separation of the two groups, indicating that the acid profiles are potentially useful in the authentication of Cuban cigars.

The effects of cranial TENS on measures of autonomic somatic and cognitive activity.

Peripheral and central nervous system effects of the cranial application of sub-threshold transcutaneous electrical nerve stimulation (TENS), of sinusoid waveform passed between earlobe electrodes at an AC frequency of 100 Hertz, were investigated. In a single-blind study, each of thirty healthy volunteer subjects was administered one 30 minute treatment of either active TENS, placebo TENS or no treatment. Pretreatment to post-treatment changes in measures of autonomic activity (blood pressure, pulse rate, peripheral vasomotor activity), somatic activity (skeletal muscle tension), and anxiety were evaluated. Significant reductions in systolic blood pressure (p less than .05), diastolic blood pressure (p less than .01), pulse rate (p less than .05), peripheral vasomotor activity (marginally significant: p less than .07) and anxiety (p less than .05) were observed subsequent to active. TENS as compared to both placebo TENS and no TENS. No significant placebo TENS effect was observed. Possible mechanisms of action of this form of cranial TENS on the peripheral and central nervous system are discussed.

Structural elucidation of the O-antigen of the Shigella flexneri provisional serotype 88-893: structural and serological similarities with S. flexneri provisional serotype Y394 (1c).

The structure of the repeating unit of the O-antigen polysaccharide from Shigella flexneri provisional serotype 88-893 has been determined. (1)H and (13)C NMR spectroscopy as well as 2D NMR experiments were employed to elucidate the structure. The carbohydrate part of the hexasaccharide repeating unit is identical to the previously elucidated structure of the O-polysaccharide from S. flexneri prov. serotype Y394. The O-antigen of S. flexneri prov. serotype 88-893 carries 0.7 mol O-acetyl group per repeating unit located at O-2 of the 3-substituted rhamnosyl residue, as identified by H2BC and BS-CT-HMBC NMR experiments. The O-antigen polysaccharide is composed of hexasaccharide repeating units with the following structure: →2)-α-L-Rhap-(1→2)-α-L-Rhap-(1→3)-α-L-Rhap2Ac-(1→3)[α-D-Glcp-(1→2)-α-D-Glcp-(1→4)]-ß-D-GlcpNAc-(1→. Serological studies showed that type antigens for the two provisional serotypes are identical; in addition 88-893 expresses S. flexneri group factor 6 antigen. We propose that provisional serotypes Y394 and 88-893 be designated as two new serotypes 7a and 7b, respectively, in the S. flexneri typing scheme.