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Meningitis is commonly caused by infection with a variety of bacterial or viral pathogens. Acute bacterial meningitis (ABM) can cause severe disease, which can progress rapidly to a critical life-threatening condition. Rapid diagnosis of ABM is critical, as this is most commonly associated with severe sequelae with associated high mortality and morbidity rates compared to viral meningitis, which is less severe and self-limiting. We have designed a microarray for detection and diagnosis of ABM. This has been validated using randomly amplified DNA targets (RADT), comparing buffers with or without formamide, in glass slide format or on the Alere ArrayTubeTM (Alere Technologies GmbH) microarray platform. Pathogen-specific signals were observed using purified bacterial nucleic acids and to a lesser extent using patient cerebral spinal fluid (CSF) samples, with some technical issues observed using RADT and glass slides. Repurposing the array onto the Alere ArrayTubeTM platform and using a targeted amplification system increased specific and reduced nonspecific hybridization signals using both pathogen nucleic and patient CSF DNA targets, better revealing pathogen-specific signals although sensitivity was still reduced in the latter. This diagnostic microarray is useful as a laboratory diagnostic tool for species and strain designation for ABM, rather than for primary diagnosis.

Methicillin-resistant Staphylococcus aureus (MRSA) is defined as S. aureus genetically having the mecA or mecC genes or phenotypically showing minimum inhibitory concentration (MIC) of oxacillin higher than 2 mg/L. However, recently, cefoxitin/oxacillin-susceptible mecA-positive S. aureus (OS-MRSA) has been reported worldwide. Little is known about the prevalence and virulence of these strains among clinically significant isolates in the UK. The aims were to (1) investigate the prevalence of OS-MRSA in seven major hospitals in the Wessex region/UK from a cohort of 500 clinically significant phenotypically identified MSSA isolates, (2) genetically characterise OS-MRSA strains by pulsed-field gel electrophoresis (PFGE) and compare these to common UK epidemic strains; and (3) to determine Panton-Valentine leukocidin (PVL; lukFS) gene carriage rates among these isolates. OS-MRSA was found in six isolates (1.2 %) of phenotypically identified and reported MSSA isolates by conventional methods. PFGE showed OS-MRSA strains to be genetically diverse and distinct from the common UK epidemic strains EMRSA-15 and EMRSA-16. None of these OS-MRSA stains carried the genes encoding PVL; however, overall positivity rate for PVL was 4.4 %, much higher than the nationally reported rates of 2 % in the UK. There are still many unknowns regarding phenotypic and/or genetic characterization of the emerging OS-MRSA isolates in the UK and worldwide. Data regarding their epidemiology and optimal therapy for infection are limited and need further investigation not only in the UK, but also worldwide, as it is likely to have an impact on the empirical treatment of S. aureus infections.

A verification study of wind accuracy is presented for wind nowcasts generated by augmenting Rapid Update Cycle (RUC) wind forecasts with near-real-time aircraft reports using the Integrated Terminal Weather System (ITWS) gridded winds algorithm. Aircraft wind reports collected between the end of the RUC data collection interval and the time each RUC forecasts is valid are available for use in augmenting the RUC wind forecast to form a wind nowcast. The 60-km resolution, hourly RUC-1 wind forecasts are used. ITWS-based nowcast wind errors and RUC forecast wind errors are examined statistically over a 1-yr dataset. The addition of the recent aircraft reports significantly reduces the rms vector error and the 90th percentile vector error. Also reduced is the number of hours of sustained large errors and the correlation among errors. The errors increase with increasing wind speed, in part due to an underestimation of wind speed that increases with increasing wind speed. The errors in the augmented w...

Genetic typing of small round structured viruses (SRSVs) by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing has been confined to analysis of the RNA polymerase because of the considerable genome variability outside of this region. To provide capsid sequence data for epidemiological studies and outbreak investigations, a broadly reactive capsid PCR was developed using two sets of degenerate, inosine-containing primers. Primer pairs Capla/Caplb and Caplla/Capllb specifically amplify a 223-bp region of the SRSV capsid open reading frame from SRSV genetic groups I and II, respectively. The capsid PCR was used to investigate SRSVs from nine UK outbreaks of gastroenteritis occurring between 1992 and 1995. Differential amplification by the primer pairs suggested that three strains belonged to genetic group I and six to genetic group II. The capsid amino acid sequences of the group I strains were 75.9% to 79.3% identical with Sot/91/UK (group I), while those of the group II strains were 75.9% to 98.3% identical with Bri/93/UK (group II). Phylogenetic comparison of the capsid region from the outbreak strains and 13 previously characterised SRSVs revealed clusters of strains closely related to Bri/93/UK and Tor/77/C within genetic group II. With the exception of some Bri/93/UK-like strains, there was no correlation between capsid sequence and the geographical origin of SRSVs. UK strains were found with greater than 90% capsid sequence identity to SRSVs from various locations worldwide including Australia (Cam/94/A), Canada (Tor/77/C), Hawaii (Haw/71/US), and Saudi Arabia (DSV395/90/SA) together with group I (B447/92/UK) and group II (Yat/94/UK) strains that were genetically distinct from known SRSV capsids. Three SRSVs very closely related to Bri/93/UK were from recent UK hospital outbreaks. These Bri/93/UK-like strains appear to be prevalent in the UK.

The clinical significance of the presence of Clostridium difficile in children's faeces remains uncertain using current diagnostic procedures. Clostridium difficile is a relatively common finding in infants with no symptoms of gastrointestinal disease, suggesting it may be an incidental finding and form part of the normal gut micro-flora in this age group. On the other hand, particularly in older children or those with significant co-morbidity, there are examples where C. difficile causes disease and exerts considerable morbidity and even mortality (C. difficile infection, CDI). Between these extremes lie a substantial group of children who have both diarrhoea and C. difficile in their stools but where the nature of the association is not clear: Clostridium difficile associated disease (CDAD). We review the significance of C. difficile in children presenting recently uncovered paediatric data from a large UK epidemiological study that informs some key unanswered questions.

The complete nucleotide sequence of genome segment 4 from the human group C rotavirus (Bristol strain) was determined. Comparison of the nucleotide sequences of the genome termini with the consensus 5′ and 3′ terminal non-coding sequences of the human group C rotavirus genome revealed characteristic 5′ and 3′ sequence motifs. Human group C rotavirus genome segment 4 is 2, 166bp long and encodes a single open reading frame of 2,082 nucleotides (693 amino acids) starting at nucleotide 55 and terminating at nucleotide 2,136 giving a 3′ untranslated region of 30 nucleotides. Alignment with the porcine group C VP3 equivalent gene showed the human gene is one amino acid longer, and that the proteins have 84.1% amino acid sequence identity. A conserved potential nucleotide binding motif shared with the porcine VP3 sequence was identified. Analogy with the group A rotaviruses suggested that the genome segment 4 encodes the group C rotavirus guanylyltransferase.

The clinical significance of the presence of Clostridium difficile in children's faeces remains uncertain using current diagnostic procedures. Clostridium difficile is a relatively common finding in infants with no symptoms of gastrointestinal disease, suggesting it may be an incidental finding and form part of the normal gut micro-flora in this age group. On the other hand, particularly in older children or those with significant co-morbidity, there are examples where C. difficile causes disease and exerts considerable morbidity and even mortality (C. difficile infection, CDI). Between these extremes lie a substantial group of children who have both diarrhoea and C. difficile in their stools but where the nature of the association is not clear: Clostridium difficile associated disease (CDAD). We review the significance of C. difficile in children presenting recently uncovered paediatric data from a large UK epidemiological study that informs some key unanswered questions.

Sequence comparison of the RNA-dependent RNA polymerases of small round-structured viruses (SRSVs) from 10 recent U.K. outbreaks of gastroenteritis revealed significant genetic variation. Computer analyses indicated that these viruses can be divided into two discrete groups. SRSV group I contains the previously characterized antigenic type 1 Norwalk and type 3 Southampton viruses. The amino acid sequences of the RNA polymerase, capsid and ORF3 of these two viruses are relatively similar (about 92%, 69% and 72% amino acid identity, respectively). A representative member of group II SRSVs, Bristol virus, was subjected to a detailed genetic analysis. Bristol virus is a recent antigenic type 2 isolate from a U.K. hospital outbreak of gastroenteritis. Using a single clinical sample the 3'-terminal 3881 nucleotide cDNA sequence [excluding the poly(A) tail] of this virus was determined. Analysis of the sequence revealed significant differences from those of group I viruses with the RNA polymerase region, capsid and ORF3 showing only about 62%, 43% and 30% amino acid identity respectively with the equivalent proteins of the Norwalk and Southampton viruses. These data suggest that the morphologically identical SRSVs belong to at least two genetically distinct groups.

Treatment of Clostridium difficile is a major problem as a hospital-associated infection which can cause severe, recurrent diarrhea. The currently available antibiotics are not effective in all cases and alternative treatments are required. In the present study, an ovine antibody-based platform for passive immunotherapy of C. difficile infection is described. Antibodies with high toxin-neutralizing titers were generated against C. difficile toxins A and B and were shown to neutralize three sequence variants of these toxins (toxinotypes) which are prevalent in human C. difficile infection. Passive immunization of hamsters with a mixture of toxin A and B antibodies protected them from a challenge with C. difficile spores in a dose-dependent manner. Antibodies to both toxins A and B were required for protection. The administration of toxin A and B antibodies up to 24 h postchallenge was found to reduce significantly the onset of C. difficile infection compared to nonimmunized controls. Protection from infection was also demonstrated with key disease isolates (ribotypes 027 and 078), which are members of the hypervirulent C. difficile clade. The ribotype 027 and 078 strains also have the capacity to produce an active binary toxin and these data suggest that neutralization of this toxin is unnecessary for the management of infection induced by these strains. In summary, the data suggest that ovine toxin A and B antibodies may be effective in the treatment of C. difficile infection; their potential use for the management of severe, fulminant cases is discussed.

Staphylococcus aureus infections are a burden to healthcare systems. There remains a lack of understanding on the relative contributions of S. aureus infection in the healthcare and community settings. In this study, 59 S. aureus isolates were selected for molecular analysis. The mobile variant staphylococcal cassette chromosome mec type IV was present in both healthcare-associated meticillin-resistant S. aureus (HA-MRSA) and community-associated MRSA (CA-MRSA), as was the Panton–Valentine leukocidin gene. PFGE identified 24 distinct clonal groups whilst multi-locus sequence typing identified 26 different sequence types, including four with new combinations of alleles. This is the first time, to our knowledge, that a selection of CA and HA MSSA and MRSA strains have been subjected to molecular analysis and comparison in the UK. Definitions for CA-MRSA need further debate as the movement of strains between healthcare and community settings is confounding the use of epidemiological definitions.

To this day the words "we tried that before and it didn't work then either" are too often heard when a procedural problem occurs. This is arguably because work records have historically been stored on a well by well basis in filing cabinets or on field service engineer's hard drives with no centralized, globally accessible capability to allow gathering and comparison of data pertaining to similar operations anywhere in the world. As a result a means of making cross reference to what happened on other wells when a given procedure or product application is being considered can be difficult, if not impossible. There is clearly a need for a different approach to procedural and product performance record keeping that will facilitate comparison with previous applications on a global basis and provide a means of evaluating the risk and the chances of success. The adoption of the Internet by business provided the opportunity to create a single database from a multitude of desktop databases and spreadsheets. With this end in mind the Performance Tracking System (PTS) was conceived to provide a method of storing product utilization and performance data on a global basis that could provide instant access to the results obtained, allowing a means to evaluate and plan any given procedure based on historical performance. This in turn allows an evaluation to be made as to how the product or procedure could be expected to perform when used in a given set of parameters. This system, which has been in use now since 2002 and is now implemented company wide, provides a means of cataloging performance across a full spectrum of product lines and well conditions and as the body of accumulated data has grown so it can provide comparative analysis for any given worldwide operational area, regardless of end user, to the benefit of all operations. It utilizes the features of a simple "asset tracking system" while providing a means of resolving the myths and perceptions associated with the phrase. The PTS provides a means of answering thousands of independent questions but perhaps foremost among them, "has this been done this way before and how successful was the outcome" can be answered with certainty. In this paper the authors will discuss the concept of the PTS, how it was designed and the methods used to implement the gathering and storage of data in an instantly recoverable manner. They will go on to provide examples of how the system has been used to assist in well planning, to resolve disagreements on procedural matters and to set up a position ahead of the curve in knowledge management to the benefit of working relationships between supplier and customer.

Study objective Most injured patients taken by ambulance to hospital emergency departments do not require emergency surgery, yet most US trauma centers require a surgeon to be present on their arrival. If a clinical decision rule could be developed to accurately identify which injured patients require emergency operative intervention, then such "secondary triage" criteria could permit a trauma center to more efficiently use their surgeons' time. Methods We analyzed 7.5 years of data (8,289 consecutive trauma activations) in our prospectively maintained Level I trauma center registry. We used classification and regression tree analyses to generate clinical decision rules using standard out-of-hospital variables to identify emergency operative intervention (within 1 hour) by a general surgeon (for adults) or a pediatric surgeon (if ≤14 years). Results Emergency operative intervention occurred in 3.0% of adults and 0.35% of children. For adults, summoning a surgeon for any one of 3 criteria (penetrating mechanism, systolic blood pressure 104 beats/min) could reduce surgeon calls by 51.2% while failing to identify emergency operative intervention in only 0.08% (rule sensitivity 97.2% and specificity 48.6%). For children, no rule at all (ie, never automatically summoning a surgeon) would fail to identify emergency operative intervention in only 0.35% of patients, and use of a single criterion (penetrating mechanism) would reduce surgeon calls by 96.2% while failing to identify emergency operative intervention in only 0.09% (rule sensitivity 75.0% and specificity 96.5%). Conclusion We have derived simple decision rules for trauma centers that, if validated, could substantially reduce the need for routine surgeon presence on trauma patient arrival. These rules demonstrate low false-negative rates.

Parainfluenza viruses (PIV) are an important cause of respiratory morbidity. Conventional diagnostic methods for detection of PIV are time consuming or lack sensitivity. A multiplex PCR that detects PIV 1-3 was developed using novel primers for PIV viruses 1 and 2 and primers for PIV 3 described previously. Following RNA extraction a single multiplex reverse transcription was undertaken using antisense primers specific for each virus type. This was followed by a 40-cycle multiplex PCR using primers directed towards the haemagglutinin-neuraminidase coding region of each virus type. Products were probed with type-specific fluorescein labelled internal probes and detected by chemiluminescence. Cultured PIV viruses were detectable to a sensitivity of 1 TCID50. The technique was applied to 57 nasal aspirates taken from children presenting with various acute respiratory conditions and analysed previously by culture, immunofluorescence and/or serology. It was possible to detect PIV 1, 2 or 3 in 13/13 samples found previously positive for PIV by tissue culture, 13/15 found previously positive by immunofluorescence and 6/10 that coincided with positive serology. None of the samples found previously positive for other viruses (26) or negative to virus detection (6) were found positive by RT-PCR. It is concluded that this method is as sensitive as combined immunofluorescence and tissue culture for the detection of the PIV viruses 1-3 and should be useful for rapid diagnosis of PIV 1-3 infections.

Studies of antigenic variation between small round-structured viruses (SRSVs) using immune electron microscopy have revealed 3 antigenic types currently circulating in the UK represented by the strains SRSV/Bri/93/UK, SRSV/Sot/91/UK and SRSV/Mel/89/UK. Mel/89/UK RNA was isolated from a 1989 school outbreak of gastroenteritis. The 3'-terminal 3435 nucleotides (excluding the poly(A) tail) were determined by RT-PCR and cDNA sequencing, completing our molecular characterization of antigenically diverse SRSVs. Coding regions for the calicivirus RNA polymerase and capsid protein were found together with a 3' open reading frame of unknown function. The polymerase region was most highly conserved between Mel/89/UK and the other two SRSVs while the 3' open reading frame exhibited extreme variation. Phylogenetic analysis of SRSV capsids showed that Mel/89/UK differed significantly from Bri/93/UK and Sot/91/UK (62 and 39% identity, respectively) and was distinct from 6 other non-UK SRSVs that had been previously characterized. This was consistent with the designation of Mel/89/UK as a novel antigenic variant. Comparison of the capsid amino acid sequences of the 3 UK strains together with the antigenically distinct SRSV/Nor/68/US revealed a hypervariable region that could be surface-exposed and contain the SRSV antigenic determinants.

Two outbreaks of gastroenteritis in the UK which occurred nine days apart at Lymington and Southampton hospitals were investigated. The clinical and epidemiological features of both outbreaks were characteristic of small round-structured virus (SRSV) infection with rapid onset of diarrhoea and/or nausea and vomiting and propagation of the outbreaks by secondary spread. SRSV particles were observed by immune electron microscopy (EM) in 60% of faecal samples from both outbreaks and no other pathogens were detected. The index case for the second outbreak was a patient who was admitted with diarrhoea and vomiting after being discharged from Lymington hospital during the first outbreak. The possibility that the two outbreaks were caused by the same strain of SRSV was investigated by the polymerase chain reaction (PCR). New inosine-containing PCR primers were designed to amplify the RNA polymerase region of SRSV cDNA from genetic groups I and II. The PCR using the group II primers achieved a higher detection rate for SRSVs in faecal samples (68% of samples positive from both outbreaks) than immune EM. SRSVs were not detected using the group I primers or using conventional degenerate PCR primers. The nucleotide sequences of PCR amplicons from both outbreaks were identical providing molecular epidemiological evidence for the involvement of a single SRSV strain. Comparison of the RNA polymerase region of this virus with the equivalent regions of genetic group I (69.4-75.0% amino acid identify) and genetic group II (88.9-100% amino acid and 77.1-88.1% nucleotide identity) SRSVs revealed that the causative SRSV was a distinct member of genetic group II.

Skin biopsies taken from UK cases of erythema migrans rash were cultured for Borrelia burgdorferi sensu lato. Reverse line blotting was used to type the infecting genospecies in PCR-positive cultures and biopsies. B. garinii or B. afzelii was identified in 56% (5/9) of biopsies/cultures tested. All patients were tested by conventional serology. PCR confirmed infection in two patients where serological testing failed to detect antibody.