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Biomedical subjects

A J Fox

Publications and source records attributed to A J Fox.

At least 55 records · Page 3Linked to original sources
PubMed2000

Improved methods of detection of meningococcal DNA from oropharyngeal swabs from cases and contacts of meningococcal disease.

In the UK the increasing use of pre-admission parenteral antibiotic therapy in meningococcal disease has lessened the value of routine cultures as a tool to confirm diagnosis, and laboratory confirmation of invasive meningococcal infection is achieved increasingly by non-culture, nucleic acid amplification methods. The purpose of this study was to evaluate a DNA extraction and meningococcal-specific DNA amplification methodology for detection of meningococci from oropharyngeal swabs. One hundred and six swabs from suspected or confirmed cases of meningococcal disease, and 94 swabs from contacts of meningococcal disease cases were examined. Of laboratory-confirmed cases, 38/65 (58.5%) yielded a positive oropharyngeal swab PCR result and 5/24 (20.8%) swabs from suspected but laboratory-unconfirmed cases were PCR positive. No significant differences in PCR positivity rates were found between the types of swab transport systems utilized, but transport time to the testing laboratory was found to affect PCR positivity (P < 0.05). Application of meningococcus-specific PCR to oropharyngeal swabs, in addition to routine culture of swabs, can provide valuable epidemiological information as well as case confirmation for contact management. PCR amplification of meningococcal PCR from oropharyngeal swabs will also increase the ascertainment in swabbing surveys carried out as part of meningococcal disease outbreak investigation and management.

DNA, Bacterial↗
PubMed2000

Comparison of different doses of remifentanil on the cardiovascular response to laryngoscopy and tracheal intubation.

We have compared three bolus and infusion regimens of remifentanil on the cardiovascular response to laryngoscopy and orotracheal intubation in three groups of 20 ASA I-II female patients, in a randomized, double-blind study. Patients in group 1 received glycopyrolate 200 micrograms i.v. followed by a bolus dose of remifentanil 1 microgram kg-1 over 30 s and an infusion of remifentanil at a rate of 0.5 microgram kg-1 min-1. The other patients received remifentanil 0.5 microgram kg-1 over 30 s and an infusion of 0.25 microgram kg-1 min-1 with (group 2) or without (group 3) pretreatment with glycopyrrolate 200 micrograms. All patients then received a sleep dose of propofol, rocuronium 0.6 mg kg-1 and 1% isoflurane with 67% nitrous oxide in oxygen. Laryngoscopy and tracheal intubation were performed 3 min later. Heart rate and arterial pressure were recorded at 1-min intervals from before induction of anaesthesia until 5 min after intubation. Baseline heart rate was similar in all groups, but decreased in group 3 (no glycopyrrolate) after induction and remained significantly lower after intubation compared with the other groups (P < 0.05). Heart rate and arterial pressure increased slightly after intubation in each group but there were no significant differences in mean arterial pressure between groups at any time. The incidence of bradycardia (one patient in group 2) and hypotension (two patients in groups 1 and 2 and three patients in group 3) was low.

Adult↗
PubMed2000

Rapid discrimination between methicillin-sensitive and methicillin-resistant Staphylococcus aureus by intact cell mass spectrometry.

Rapid, accurate discrimination between methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) strains is essential for appropriate therapeutic management and timely intervention for infection control. A rapid method involving intact cell mass spectrometry (ICMS) is presented that shows promise for identification, discrimination of MSSA from MRSA and typing. In ICMS, cells from a bacterial colony are emulsified in a chemical matrix, added to a sample slide, dried and analysed by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). This technique examines the chemistry of the intact bacterial cell surface, yielding spectra consisting of a series of peaks from 500 to 10000, which represent the mass:charge (m:z) ratios. Each peak corresponds to a molecular fragment released from the cell surface during laser desorption. Specimens can be prepared in a few seconds from plate cultures and a spectrum can be obtained within 2 min. ICMS spectra for 20 staphylococcal isolates showed characteristic peaks, some of which were conserved at species level, some at strain level and some were characteristic of the methicillin susceptibility status of the strain. ICMS may have potential for MRSA identification and typing, and may improve infection control measures.

Cluster Analysis↗
PubMed2000

Evaluation of the Applied Biosystems automated Taqman polymerase chain reaction system for the detection of meningococcal DNA.

In a period where the proportion of culture confirmed cases in the UK has been steadily declining, diagnosis by PCR has been used to increase the number of confirmed cases and provide additional epidemiological data. This report presents a comparative evaluation of the fluorogenic probe-based 5' exonuclease assay (Taqman) using the Perkin-Elmer Applied Biosystems automated sequence detection system 7700 with previously reported polymerase chain reaction enzyme-linked immunosorbent (PCR ELISA) assays for the detection of meningococcal DNA in CSF, plasma and serum samples. Taqman assays developed were based on the detection of a meningococcal capsular transfer gene (ctrA), the insertion sequence IS1106 and the sialytransferase gene (siaD) for serogroup B and C determination and compared with similar assays in a PCR ELISA format. The Taqman ctrA assay was specific for Neisseria meningitidis, however the IS1106 assay gave false positive reactions with a number of non-meningococcal isolates. Sensitivity of the Taqman ctrA, IS1106 and siaD assays testing samples from culture-confirmed cases were 64, 69 and 50%, respectively, compared with 26, 67 and 43% for the corresponding PCR ELISA assays. Improvements to the DNA extraction procedure has increased the sensitivity to 93 and 91% for the TaqMan ctrA and siaD assays, respectively, compared to culture confirmed cases. Since the introduction of Taqman PCR a 56% increase in laboratory confirmed cases of meningococcal disease has been observed compared to culture only confirmed cases. The developed Taqman assays for the diagnosis of meningococcal disease enables a high throughput, rapid turnaround of samples with considerable reduced risk of contamination.

Bacterial Proteins↗
PubMed2000

Contamination and sensitivity issues with a real-time universal 16S rRNA PCR.

A set of universal oligonucleotide primers specific for the conserved regions of the eubacterial 16S rRNA gene was designed for use with the real-time PCR Applied Biosystems 7700 (TaqMan) system. During the development of this PCR, problems were noted with the use of this gene as an amplification target. Contamination of reagents with bacterial DNA was a major problem exacerbated by the highly sensitive nature of the real-time PCR chemistry. This was compounded by the use of a small amplicon of approximately 100 bases, as is necessary with TaqMan chemistry. In an attempt to overcome this problem, several methodologies were applied. Certain treatments were more effective than others in eliminating the contaminating DNA; however, to achieve this there was a decrease in sensitivity. With UV irradiation there was a 4-log reduction in PCR sensitivity, with 8-methoxypsoralen activity facilitated by UV there was between a 5- and a 7-log reduction, and with DNase alone and in combination with restriction digestion there was a 1.66-log reduction. Restriction endonuclease treatment singly and together did not reduce the level of contaminating DNA. Without the development of ultrapure Taq DNA polymerase, ultrapure reagents, and plasticware guaranteed to be free of DNA, the implementation of a PCR for detection of eubacterial 16S rRNA with the TaqMan system will continue to be problematical.

Base Sequence↗
PubMed2000

Angiographically defined collateral circulation and risk of stroke in patients with severe carotid artery stenosis. North American Symptomatic Carotid Endarterectomy Trial (NASCET) Group.

BACKGROUND AND PURPOSE: Blood supply through collateral pathways improves regional cerebral blood flow and may protect against ischemic events. The effect of collaterals on the risk of stroke and transient ischemic attack (TIA), in the presence of angiographic severe internal carotid artery (ICA) stenosis, was assessed. METHODS: Angiographic collateral filling through anterior communicating and posterior communicating arteries and retrograde filling through ophthalmic arteries were determined in all patients at entry into the North American Symptomatic Carotid Endarterectomy Trial. Kaplan-Meier event-free survival analyses were performed on 339 medically treated and 342 surgically treated patients. RESULTS: The presence of collaterals supplying the symptomatic ICA increased with severity of stenosis. Two-year risk of hemispheric stroke in medically treated patients with severe ICA stenosis was reduced in the presence of collaterals: 27.8% to 11.3% (P=0.005). Similar reductions were observed for hemispheric TIA (36.1% versus 19.1%; P=0.008) and disabling or fatal strokes (13.3% versus 6.3%; P=0.11). For surgically treated patients, the perioperative risk of hemispheric stroke was 1.1% in the presence of collaterals versus 4. 9% when absent. The 2-year stroke risks for surgical patients with and without collaterals were 5.9% versus 8.4%, respectively. Neither comparison in the surgical group was statistically significant. The observed reductions were independent of the degree of ICA stenosis and other vascular risk factors. CONCLUSIONS: Collaterals are associated with a lower risk of hemispheric stroke and TIA, both long term and perioperatively. Angiographic identification of collaterals assists in identifying patients with severe ICA stenosis at lower risk of stroke and TIA.

Aged↗
PubMed2000

Internal borderzone infarction: a marker for severe stenosis in patients with symptomatic internal carotid artery disease. For the North American Symptomatic Carotid Endarterectomy (NASCET) Group.

BACKGROUND AND PURPOSE: Among subcortical infarctions, internal borderzone infarcts (IBI) are considered to be separate entities from perforating artery infarcts (PAI). The purpose of the present study is to examine the relationship between the presence of IBI and the degree of angiographically defined internal carotid artery (ICA) stenosis in symptomatic patients. METHODS: A review of 1253 brain CTs from patients recruited by the North American Symptomatic Carotid Endarterectomy Trial was performed, using templates for the identification of subcortical and cortical vascular territories. RESULTS: A total of 413 patients had visible ischemic lesions on the side ipsilateral to their symptomatic ICA. Of these, 138 had PAI, 108 had IBI, 122 had cortical infarcts, and 45 had a combination of different lesions. Mean (+/-SD) lesion diameter was larger for IBI (11.0+/-5.9 mm) than for PAI (7.1+/-4.7 mm) (P<0.001 for comparing 2 means). IBI was associated with higher degrees of ICA stenosis (P<0. 001). Sixty-three percent of the patients with IBI had severe (70% to 99%) ICA stenosis compared with 42% of patients with PAI; 18% of the IBI patients had stenosis of 90% or more compared with 8% of the patients with PAI. Multiple logistic regression did not identify any patient characteristics as confounders. CONCLUSIONS: Among subcortical infarctions, IBI are associated with higher degrees of ICA stenosis in symptomatic patients. Differentiating between internal borderzone and perforating artery infarcts is important, because each may arise from different mechanisms, namely, carotid disease and small-vessel disease, respectively.

Adult↗
PubMed2000

Post-transcriptional regulation of bradykinin B1 and B2 receptor gene expression in human lung fibroblasts by tumor necrosis factor-alpha: modulation by dexamethasone.

The cellular and molecular mechanisms governing bradykinin B1 and B2 receptor expression and function are poorly understood. We investigated the regulation of both B1 and B2 receptors in human embryonic lung fibroblasts (HEL 299) by the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin 1beta (IL-1beta). TNF-alpha and IL-1beta both induced a rapid and transient increase in B1 and B2 receptor mRNA expression that was maximal by 2 h, accompanied by an increase in B1 and B2 receptor protein, as measured by radioligand binding assay with [(3)H]des-Arg(10)-kallidin, and [(3)H]bradykinin, respectively. The induced B1 receptors were functionally coupled, because the B1 agonist, des-Arg(10)-kallidin, induced an increase in arachidonic acid release in TNF-alpha-stimulated cells but not in control cells. The induction of B1 and the up-regulation of B2 receptors by TNF-alpha was partly mediated through activation of p38 mitogen-activated protein kinase and that of B2 receptor by protein kinase A. TNF-alpha and IL-1beta regulation of both B1 and B2 receptors was inhibited by dexamethasone. When compared with vehicle-treated cells, dexamethasone increased the rate of decline of both B1 and B2 receptor mRNAs. Nuclear run-on experiments demonstrate that the induction of B1 and the up-regulation of B2 receptors as well as the inhibitory effect of dexamethasone are entirely mediated through post-transcriptional mechanisms.

Anti-Inflammatory Agents↗
PubMed2000

Endovascular therapy of a large vertebral artery aneurysm using stent and coils.

BACKGROUND: Endovascular therapy is becoming an increasingly popular treatment for cerebral aneurysms. Total angiographic occlusion of small-necked aneurysms (<4 mm) can be obtained in a high percentage of cases. The endovascular treatment of wide-necked or fusiform aneurysms remains a challenge with complete angiographic occlusion reported in <15% of cases. CASE REPORT: We describe the combined use of a flexible coronary stent and platinum coils to treat a wide-necked aneurysm of the distal left vertebral artery, in a patient with Grade IV subarachnoid hemorrhage. RESULTS: The procedure was technically successful as the parent artery was protected by the stent while coils were deposited in the aneurysm lumen. Although angiographic aneurysm occlusion was incomplete, the dome was packed with coils. No further hemorrhage has occurred. CONCLUSION: Combined endovascular stent and coil therapy is a promising technique for the treatment of wide-necked cerebral aneurysms.

Cerebral Angiography↗
PubMed2000

Deoxyribonucleic acid restriction digest patterns in Campylobacter species: a comparison with Penner serotype.

Diversity, based on restriction fragment length polymorphism (RFLP) analysis, was studied in 48 strains of Campylobacter, comprising 27 chicken and 21 human strains of C. jejuni and C. coli, using genomic Southern hybridisation. Restriction digests of chromosomal DNA were prepared by treating with HaeIII and probed using a C. jejuni DNA probe. Nineteen distinct hybridisation patterns were identified, and differences in hybridisation pattern between members of the two species, and in individual strains of the same species, were seen. The method described proved more discriminatory than the Penner serotype, as strains from the same serotype were distinguished. The relative simplicity of the patterns obtained, together with the apparent diversity identified among individual strains and species, suggests that DNA fingerprinting using the C. jejuni DNA probe could be a useful identification method in epidemiological studies of Campylobacter infection in Nigeria.

Animals↗
PubMed2000

Genotyping of clinical and chicken isolates of Campylobacter jejuni and Campylobacter coli.

Genomic DNA from 58 strains of Campylobacter made up of 48 Campylobacter jejuni and ten Campylobacter coli were digested with Sma I and analysed by pulsed-field gel electrophoresis (PFGE). The cleavage of DNA by Sma I gave 22 distinct hybridization patterns. The two Campylobacter species were subtyped by PFGE. The average genomic size for C. jejuni by Sma I digestion was 1.73 Mb, while that of C. coli gave 1.7 Mb. Results from this study indicate that PFGE analysis by Sma I digested genomic DNA provides a reliable means of differentiating between and within species of Campylobacter and provides a practical approach to epidemiological studies of Campylobacter.

Animals↗
PubMed1999

Anaesthesia.

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Anesthesia↗
PubMed1999

The development of a quantitative PCR ELISA to determine HCMV DNAaemia levels in heart, heart/lung and lung transplant recipients.

The adaptation of the polymerase chain reaction (PCR) enzyme-linked immunosorbent assay (ELISA) using a co-amplified DNA standard to quantitate the human cytomegalovirus (HCMV) glycoprotein B (gB) gene in clinical samples is described. The PCR ELISA is a solution hybridisation system with colorimetric end stage detection of amplicons. A DNA internal standard (IS) was designed by replacing a probe sequence used currently within the gB region with a heterogeneous sequence, allowing co-amplification with the same oligonucleotide primer sets and differentiation by probe hybridisation. Two DNA fragments homologous to the gB and IS sequences were generated and used for co-amplification studies to construct a standard curve. From this the copy number of the gB gene present in clinical samples could be interpolated. Co-amplification with 1000 IS copies allowed quantitation of 10-1000000 gB DNA copies in a single PCR. This assay was validated subsequently using blood samples tested by the HCMV antigenaemia assay and showed a trend of increasing HCMV DNAaemia with increasing antigenaemia levels. This rapid assay avoids using gel electrophoresis and cumbersome quantitative systems. It has the potential for early identification of patients at high risk of developing HCMV disease, and for therapeutic monitoring.

Base Sequence↗
PubMed1999

Improved recognition of MRSA case clusters by the application of molecular subtyping using pulsed-field gel electrophoresis.

Methicillin-resistant Staphylococcus aureus (MRSA) is increasingly common in hospital and community populations, making the recognition of true nosocomial outbreaks more difficult. We have used pulsed-field gel electrophoresis (PFGE) with Sma I digestion to analyse retrospectively two perceived outbreaks of epidemic methicillin-resistant Staphylococcus aureus 15 (EMRSA 15) colonization. The first cluster of cases in patients and staff on a general ward (ward D) revealed three different antibiograms based on differences in ciprofloxacin and rifampicin sensitivities. All isolates typed using PFGE, which was more discriminatory than phage-typing. One PFGE banding profile labelled type 5 was predominant, but 12 isolates proved to be subtypes of type 5 and two were PFGE type 11. Four staff members carried a strain not found in patients, three carried strains found in patients and transient carriage was highlighted as a problem when screening staff. PFGE enhanced the epidemiological data and proved that the cases on this ward did not comprise one large outbreak but numerous sporadic cases and smaller clusters. In contrast, isolates from a second cluster of cases which occurred on ward F were indistinguishable using antibiograms, phage-typing and PFGE, confirming this was more likely to be a true outbreak of colonization. We conclude that PFGE usefully augments epidemiological information and allows more logical infection control decisions to be made, with better utilization of scarce resources.

Bacterial Typing Techniques↗
PubMed1999

Salivary antibodies following parenteral immunization of infants with a meningococcal serogroup A and C conjugated vaccine.

Bacterial and viral salivary antibody testing is proving sensitive and specific, useful for epidemiological studies, and is simple and non-invasive. Salivary serogroup C polysaccharide-specific (SC PS-S) IgA and IgG were determined as a proportion of total salivary IgA and IgG in a group of UK infants who were recipients of a conjugated A/C meningococcal PS vaccine. Geometric mean concentrations (GMCs) of salivary SC PS-S IgG per mg of total IgG (microg/mg) were 0.1 pre-vaccination, rising to 8.2 post first, 16.1 post second and 29.3 post third dose of vaccine. For IgA, the corresponding GMCs in ng/mg were 0.1, 82.8, 69.6 and 91.2. Significant correlations (P < 0.0001) were found between serum Ig and salivary IgG SC PS-S antibody for pre-vaccine and 1 month post each dose of vaccine suggesting that SC PS-S IgG in saliva was largely derived from serum. Of the five infants whose sera were analysed for isotype-specific responses, only traces of IgM and IgA were measurable suggesting that the SC PS-S IgA was locally produced. These findings suggests that the widespread use of meningococcal conjugate vaccines is likely to reduce nasopharyngeal carriage and may thereby induce herd immunity in the vaccinated population.

Antibodies, Bacterial↗