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Biomedical subjects

I B Vipond

Publications and source records attributed to I B Vipond.

At least 19 recordsLinked to original sources
PubMed2005

Institutional risk factors for outbreaks of nosocomial gastroenteritis: survival analysis of a cohort of hospital units in South-west England, 2002-2003.

Nosocomial outbreaks of gastroenteritis are a major burden on hospital inpatient services, costing an estimated pound115 million annually to the English National Health Service. We actively followed-up 171 inpatient units from four major acute hospitals and 11 community hospitals in South-west England for one year. Outbreaks of gastroenteritis were ascertained through an active surveillance network using standard clinical definitions. Survival analysis Cox regression models using an outbreak of gastroenteritis as the endpoint were fitted to identify institutional and operational attributes related to increased outbreak rates at the level of the care unit. Greater number of beds in unit [hazard ratio (HR) 1.22 (per 10 additional beds), 95% confidence intervals (CI) 0.96-1.55] was associated with increased hazard, as were geriatric (HR 2.6, 95%CI 1.6-4.3) and general medical (HR 1.7, 95%CI 1.1-2.6) care units. The average length of stay on a unit was inversely associated with outbreak incidence [HR=0.89 (per additional week of stay), 95%CI 0.80-0.99]. Larger care units and those with higher throughput have increased rates of gastroenteritis outbreaks. These results should guide infection control policy and support the design of hospitals with smaller care units.

Caliciviridae Infections↗
PubMed2004

National epidemic of Lordsdale Norovirus in the UK.

BACKGROUND: In early 2002 reports of outbreaks of gastroenteritis reached unprecedented levels in the UK. Forty five Norovirus outbreaks were reported in January 2002. OBJECTIVES: The objective of the study was to determine whether the outbreaks were Noroviral in origin and if so whether they represented a homogeneous or heterogeneous collection of Noroviruses by applying EIA and sequence analysis to representative faecal samples. STUDY DESIGN: Faecal specimens were collected during the week of highest incidence from 21 outbreaks in a variety of health care settings including hospitals and nursing homes. The outbreaks occurred in geographically distinct regions of the UK and samples were collected by reference laboratories in Glasgow, Manchester, Bristol and Southampton. RESULTS: The samples were all positive for Noroviruses by negative stain electron microscopy (EM) and Lordsdale virus (LV) EIA, therefore reverse transcriptase polymerase chain reaction (RT-PCR) amplification and nucleotide sequencing of the Norovirus RNA polymerase gene was performed on amplicons from samples of each of the 21 outbreaks to investigate the nature and extent of diversity. All samples were very closely related to the reference Lordsdale virus genome sequence. LV was first discovered during an hospital outbreak of gastroenteritis in Southampton General Hospital in March 1993. CONCLUSIONS: Noroviruses are a major cause of outbreaks of gastroenteritis in health care settings. LV is the predominant Norovirus in the UK and was detected in outbreaks that occurred during the national peak of gastroenteritis reports in January 2002.

Amino Acid Sequence↗
PubMed2004

Effects of cleaning and disinfection in reducing the spread of Norovirus contamination via environmental surfaces.

A reverse transcriptase polymerase chain reaction assay was used to study the transfer of Norovirus (NV) from contaminated faecal material via fingers and cloths to other hand-contact surfaces. The results showed that, where fingers come into contact with virus-contaminated material, NV is consistently transferred via the fingers to melamine surfaces and from there to other typical hand-contact surfaces, such as taps, door handles and telephone receivers. It was found that contaminated fingers could sequentially transfer virus to up to seven clean surfaces. The effectiveness of detergent- and disinfectant-based cleaning regimes typical of those that might be used to decontaminate faecally contaminated surfaces and reduce spread of NV was also compared. It was found that detergent-based cleaning with a cloth to produce a visibly clean surface consistently failed to eliminate NV contamination. Where there was faecal soiling, although a combined hypochlorite/detergent formulation at 5000 ppm of available chlorine produced a significant risk reduction, NV contamination could still be detected on up to 28% of surfaces. In order consistently to achieve good hygiene, it was necessary to wipe the surface clean using a cloth soaked in detergent before applying the combined hypochlorite/detergent. When detergent cleaning alone or combined hypochlorite/detergent treatment failed to eliminate NV contamination from the surface and the cleaning cloth was then used to wipe another surface, the virus was transferred to that surface and to the hands of the person handling the cloth. In contrast, were surfaces where contaminated with NV-infected faecal suspension diluted to 1 in 10 and 1 in 80, intended to simulate surfaces that have become contaminated after secondary transfer, treatment with a combined bleach/detergent formulation, without prior cleaning, was sufficient to decontaminate surfaces and prevent transfer.

Caliciviridae Infections↗
PubMed2004

Influence of cytomegalovirus (CMV) sero-positivity on CMV infection, lymphocyte recovery and non-CMV infections following T-cell-depleted allogeneic stem cell transplantation: a comparison between two T-cell depletion regimens.

We compared the incidence and outcome of preemptively treated cytomegalovirus (CMV) infection, lymphocyte recovery and non-CMV infections between two different TCD modalities, one employing CD34+ selection and T-cell add-back (TCAB), preceded by Campath-1H in vivo (CD34+/TCAB group, n=29), and the other using grafts incubated with Campath-1H in vitro (Campath-1H in vitro group, n=32). The probabilities of CMV reactivation and recurrence were 67 and 83.6% in the CD34+/TCAB group and 42.9 and 20% in the Campath-1H group (P=0.07 and 0.02). Donor sero-positivity reduced CMV reactivation in the Campath-1H group, but not in the CD34+/TCAB group. The durations of positive PCR/antigenemia positivity and antiviral therapy were also significantly longer in the CD34+/TCAB group. However, only two patients developed CMV disease in each group. The mean absolute lymphocyte counts (x 10(9)/l) at 30 days (0.27 vs 0.4, P=0.03) and 100 days (0.77 vs 1.4, P=0.01) were significantly lower in the CD34+/TCAB group along with a higher incidence of non-CMV infections in CMV at-risk patients, but not in the CMV low-risk group. These findings suggest that the modality of TCD should be tailored according to the CMV risk status, and CMV sero-positive patients should receive a less extensively T-cell-depleted graft and a CMV sero-positive graft if possible.

Adoptive Transfer↗
PubMed2003

A school outbreak of Norwalk-like virus: evidence for airborne transmission.

An outbreak of gastroenteritis affected a school attended by children aged 4-11 years. Epidemiological features suggested this was due to Norwalk-like virus (NLV) and this was confirmed by polymerase chain reaction (PCR). Nucleotide sequence analysis of the PCR amplicons revealed identical strains in all five positive stool samples. Pupils were significantly more likely to become ill following an episode of vomiting within their classroom (adjusted odds ratio 4.1, 95% CI 1.8-9.3). The times from exposure to illness were consistent with direct infection from aerosolized viral particles where exposure to vomiting was high. Cleaning with quaternary ammonium preparations made no impact on the course of the outbreak. However, the outbreak stopped after the school closed for 4 days and was cleaned using chlorine-based agents. This study confirms the importance of vomiting in the transmission of NLV and provides evidence that direct infection with aerosolized viral particles occurs.

Aerosols↗
PubMed2002

Early detection of cytomegalovirus (CMV) infection in bone marrow transplant patients by reverse transcription-PCR for CMV spliced late gene UL21.5: a two site evaluation.

BACKGROUND: Bone marrow transplant (BMT) patients at risk of developing cytomegalovirus (CMV) pneumonitis are identified routinely by the early detection of virus in blood. For early diagnosis of CMV infection, the RNA-based approach demonstrates advantages when compared with the current CMV antigen and DNA detection methods. OBJECTIVES: We have evaluated our previously developed reverse transcription-polymerase chain reaction (RT-PCR) to a spliced late CMV gene (SLG; J. Virol. Methods 56 (1996), 139) to monitor CMV infection in BMT patients at two clinical sites. The diagnostic value of the SLG RT-PCR was compared with the routine CMV antigen and DNA detection methods. STUDY DESIGN: Weekly blood samples from BMT patients were tested for CMV during the first 3 months post-transplant. The qualitative SLG RT-PCR, semiquantitative DNA PCR, and viral antigen tests were compared. The RNA and DNA PCR results were analysed in terms of their temporal relationship and consistency of CMV detection and compared with CMV infection diagnosed by viral antigen tests. RESULTS: Of the 101 BMT recipients studied, 25 developed CMV antigenemia and/or DNAemia resulting in symptomatic infection in two patients. All CMV PCR-positive patients were either CMV seropositive pretransplant or received marrow from seropositive donor. The highest incidence of CMV infection was seen in seropositive recipients (R+) irrespective of the donor's status. Detection of CMV infection by SLG RNA preceded CMV DNA detection by 0-2 weeks (median 1 week) and CMV antigen detection by 0-8 weeks (median 3 weeks). Once detected, the SLG RNA remained consistently positive before antiviral treatment was commenced. Both the SLG RNA and CMV DNA detection methods had the same clinical sensitivity, specificity, positive and negative predictive values of 100, 94, 80 and 100%, respectively. CONCLUSIONS: The RT-PCR for SLG RNA proved to be the earliest indicator of CMV infection in BMT patients demonstrating a sustained pattern of CMV detection during the 3 months post-transplant period. Although very similar in its diagnostic performance to CMV DNA PCR the SLG RNA RT-PCR does not require quantitation and provides an efficient and ongoing indication of active CMV infection.

Adolescent↗
PubMed2001

Molecular epidemiology of outbreak of respiratory syncytial virus within bone marrow transplantation unit.

During the winter of 1995-1996, eight of nine bone marrow transplantation (BMT) unit patients were infected with the same strain of respiratory syncytial virus (RSV). This RSV strain was not detected in 20 hospitalized patients from the community, suggesting that the BMT unit infections did not occur by independent incidents of transmission from the community.

Bone Marrow Transplantation↗
PubMed2000

A diagnostic EIA for detection of the prevalent SRSV strain in United Kingdom outbreaks of gastroenteritis.

Small round structured viruses (SRSVs) are the major cause of outbreaks of gastroenteritis in the UK. Diagnosis is problematic due to insensitive electron microscopy (EM) or technically demanding reverse transcription polymerase chain reaction (RT-PCR) techniques. We have studied outbreaks of non-bacterial gastroenteritis using an EIA based upon recombinant capsid protein from the currently prevalent circulating strain of SRSV (Lordsdale Genotype II) and compared its performance against EM and RT-PCR assays. Faecal specimens sent to the Bristol Public Health Laboratory for outbreak investigation from December 1996 to December 1997 were applied retrospectively to the SRSV EIA and results compared with the routine EM and RT-PCR that had been carried out prospectively. Overall, the three tests identified SRSVs in specimens from 70% of the outbreaks (213/305) investigated. Of the 213 total positive outbreaks, the EIA identified 71%, that compared favourably with EM (63%) and RT-PCR (84%). The Lordsdale Genotype II SRSV EIA provides a simple cost-effective assay that will for the first time make detection of currently circulating SRSV strains associated with UK outbreaks available to all routine laboratories. The EIA format makes the assay widely applicable to non-specialist laboratories, unlike the RT-PCR assay, and the improved sensitivity over EM will allow successful screening of UK outbreaks alongside commercial EIAs currently available for adenovirus, astrovirus and rotavirus. Furthermore, the assay will allow rapid identification of emerging SRSV strains.

Capsid↗
PubMed2000

Evidence for airborne transmission of Norwalk-like virus (NLV) in a hotel restaurant.

An outbreak of gastroenteritis followed a meal in a large hotel during which one of the diners vomited. The clinical features of the illness suggested Norwalk-like virus (NLV, small round structured virus) infection, and this was confirmed by electron microscopy and reverse transcriptase polymerase chain reaction (RT-PCR) of stool samples. Further characterization of the virus by nucleotide sequence analysis of the PCR amplicons revealed identical strains in all the affected individuals. The foods served at the meal could not be demonstrated to be the cause of the outbreak. Analysis of attack rates by dining table showed an inverse relationship with the distance from the person who vomited. No one eating in a separate restaurant reported illness. Transmission from person-to-person or direct contamination of food seems unlikely in this outbreak. However, the findings are consistent with airborne spread of NLV with infection by inhalation with subsequent ingestion of virus particles.

Adult↗
PubMed1999

A community outbreak of food-borne small round-structured virus gastroenteritis caused by a contaminated water supply.

In August 1994, 30 of 135 (23%) bakery plant employees and over 100 people from South Wales and Bristol in the United Kingdom, were affected by an outbreak of gastroenteritis. Epidemiological studies of employees and three community clusters found illness in employees to be associated with drinking cold water at the bakery (relative risk 3.3, 95%, CI 1.6-7.0), and in community cases with eating custard slices (relative risk 19.8, 95%, CI 2.9-135.1) from a variety of stores supplied by one particular bakery. Small round-structured viruses (SRSV) were identified in stool specimens from 4 employees and 7 community cases. Analysis of the polymerase and capsid regions of the SRSV genome by reverse transcription-polymerase chain reaction (RT-PCR) demonstrated viruses of both genogroups (1 and 2) each with several different nucleotide sequences. The heterogeneity of the viruses identified in the outbreak suggests that dried custard mix may have been inadvertently reconstituted with contaminated water. The incident shows how secondary food contamination can cause wide-scale community gastroenteritis outbreaks, and demonstrates the ability of molecular techniques to support classical epidemiological methods in outbreak investigations.

Adolescent↗
PubMed1999

Absence of human astrovirus RNA in sewage and environmental samples.

Over a period of several weeks during the summer of 1996, samples of sewage, sea water, river water, sand and silt were collected from a sewage works at Weston-super-Mare, England and from coastal areas nearby. A sensitive reverse-transcriptase polymerase chain reaction (RT-PCR) was used to search for human astrovirus (HAstV) RNA in concentrates of the samples. No evidence of astrovirus was found in any sample, which suggests that contamination with these viruses is not a problem in this area during the summer holiday season. Furthermore, the single case of astrovirus diarrhoea diagnosed in this laboratory in the summer occurred at the end of the sampling period, and not in the survey area. The primers used sometimes yielded a product two-thirds the expected size but bearing no sequence homology with HAstV. The confirmation that poliovirus adsorbs to sand and silt shows that these materials might be able to concentrate other enteric viruses in water to a level which could be a threat to the health of people coming into contact with it.

Astroviridae Infections↗
PubMed1996

Random mutagenesis targeted to the active site of the EcoRV restriction endonuclease.

Two segments of the gene for the EcoRV restriction endonuclease, each encoding 10 amino acids at the active site, were subjected to random mutagenesis with degenerate oligonucleotides. Mutations that abolished the activity of the EcoRV endonuclease were selected by viability in a strain of Escherichia coli that lacks the EcoRV methyltransferase, under conditions where the gene for the wild-type endonuclease is lethal to the cell. Sixty-five mutants were isolated and analyzed by DNA sequencing to identify the mutations. The collection of null mutants contained 49 with single amino acid substitutions, 15 with double substitutions, and one with a triple substitution. The single substitutions were located at many different positions within the two 10-amino acid segments, though several hot-spots gave rise to null mutants at high frequencies. Some hot-spots were readily explained by reference to the crystal structure of EcoRV since they were at the amino acids immediately adjacent to the scissile phosphodiester bond: for example, Asp90 and Lys92. These residues may be directly involved in the catalytic mechanism. Other hot-spots, such as Gln69, Tyr72, and Ala88, were at unexpected positions that appear to have no direct role in DNA binding or catalysis. At some of the unexpected hot-spots, the side chain of the amino acid lies distant from the DNA, yet the enzyme was still inactivated by conservative substitutions at these positions. The sensitivity of the EcoRV endonuclease to conservative substitutions may be due to its requirement to take up one particular conformation at the DNA-protein interface out of a large number of alternative conformations.

Amino Acid Sequence↗
PubMed1996

An isoleucine to leucine mutation that switches the cofactor requirement of the EcoRV restriction endonuclease from magnesium to manganese.

The EcoRV restriction endonuclease cleaves DNA at its recognition sequence more readily with Mg2+ as the cofactor than with Mn2+ but, at noncognate sequences that differ from the EcoRV site by one base pair, Mn2+ gives higher rates than Mg2+. A mutant of EcoRV, in which an isoleucine near the active site was replaced by leucine, showed the opposite behavior. It had low activity with Mg2+, but, in the presence of Mn2+ ions, it cleaved the recognition site faster than wild-type EcoRV with either Mn2+ or Mg2+. The mutant was also more specific for the recognition sequence than the native enzyme: the noncognate DNA cleavages by wild-type EcoRV and Mn2+ were not detected with the mutant. Further mutagenesis showed that the protein required the same acidic residues at its active site as wild-type EcoRV. The Ile-->Leu mutation seems to perturb the configuration of the metal-binding ligands at the active site so that the protein has virtually no affinity for Mg2+ yet it can still bind Mn2+ ions, though the latter only occurs when the protein is at the recognition site. This contrasts to wild-type EcoRV, where Mn2+ ions bind readily to complexes with either cognate and noncognate DNA and only Mg2+ shows the discrimination between the complexes. The structural perturbation is a specific consequence of leucine in place of isoleucine, since mutants with valine or alanine were similar to wild-type EcoRV.

Base Sequence↗
PubMed1995

Specific DNA recognition by EcoRV restriction endonuclease induced by calcium ions.

In the presence of Mg2+, the EcoRV restriction endonuclease cleaves DNA specifically at its recognition sequence, but in the absence of divalent metal ions, it binds DNA without any specificity: gel-shift experiments had revealed multiple EcoRV-DNA complexes, due to the binding of one, two, three, or more molecules of protein per molecule of DNA, with the same equilibrium constant for each association. In this study, the binding of EcoRV to DNA was measured by gel shift in the presence of Ca2+, an ion that perturbs the Mg(2+)-dependent activity of EcoRV but that fails to support DNA cleavage. With Ca2+, and at a lower concentration of EcoRV protein than that required for binding in the absence of divalent metal ions, a single complex was observed with DNA containing the EcoRV recognition site. This complex was not formed with DNA that had been methylated at the EcoRV site nor with an isogenic DNA lacking the EcoRV recognition site. The single complex thus is due to the specific binding of EcoRV to its recognition site on the DNA. From gel shifts with a permuted set of DNA fragments, the degree of DNA bending by EcoRV at its recognition site was estimated to be 53 degrees +/- 4 degrees. This angle is similar to that seen in the crystal structure of the cognate DNA-protein complex. Calcium ions thus appear to mimic the role of Mg2+ in generating a specific protein-metal-DNA complex, but in contrast to Mg2+, Ca2+ gives a stable ternary complex in which the DNA-bound nuclease cannot cleave the DNA.

Calcium↗
PubMed1995

Divalent metal ions at the active sites of the EcoRV and EcoRI restriction endonucleases.

Restriction enzymes cannot cleave DNA without a metal ion cofactor. The specificities of the EcoRV and EcoRI endonucleases for metals were studied by measuring DNA cleavage rates with several metal ions and with combinations of metal ions. Both EcoRV and EcoRI had optimal activities with Mg2+, were less active with several other ions including Mn2+, and had virtually no activity with Ca2+. But the activities of EcoRV and EcoRI with either Mg2+ or Mn2+ were perturbed by Ca2+. For EcoRI, both Mg2+- and Mn(2+)-dependent activities, at both cognate and noncognate sites, were all inhibited by Ca2+. The activity of EcoRV at its recognition site with Mg2+ was also inhibited by Ca2+. But the Mn(2+)-dependent reaction at the EcoRV recognition site was stimulated by Ca2+. EcoRV activities at noncognate sites with either Mg2+ or Mn2+ displayed a biphasic response to Ca2+: stimulation at low concentrations of Ca2+ and inhibition at high concentrations. These observations, together with the known structures of the proteins, indicate that EcoRI needs only one metal ion per active site and is inactive when Mg2+ is displaced by Ca2+, while EcoRV needs two and that the displacement of one by Ca2+ can enhance activity. We propose a mechanism for phosphodiester hydrolysis by EcoRV that involves two metal ions.

Binding Sites↗