2020 computing: science in an exponential world.
Explore the source record for details and available documents.
Computers↗
Biomedical subjects
Jim Gray
Publications and source records attributed to Jim Gray.
Molecular detection and characterization of human enteroviruses directly from clinical samples using RT-PCR and DNA sequencing.
Enteroviruses are common human pathogens associated with a wide spectrum of symptoms ranging from asymptomatic infection to acute flaccid paralysis and neonatal multi-organ failure. Molecular methods that provide rapid diagnosis and increased sensitivity have been developed for the diagnosis of enterovirus infection using oligonucleotide primers complementary to conserved sequences located in the 5' untranslated region (UTR), but data generated from these regions are not sufficiently discriminatory for typing due to the lack of correlation between their nucleic acid sequence and serotype specificity. Sequences derived from the gene encoding the capsid VP1 correlate with serotype, and therefore provide the opportunity for the development of molecular typing methods consistent with present serogical methods. In this study, oligonucleotide primers that amplify a region of the 5'UTR to detect enterovirus RNA, and the region encoding the enterovirus VP1 N-terminus to characterize virus strains were used in nested and semi-nested RT-PCRs, respectively. The ability of the VP1 RT-PCR to amplify diverse viruses within genotypes and genogroups was confirmed by the correct identification of both prototype strains, and strains circulating currently of the same genotypes. The molecular methods proved their utility through the detection of enteroviruses that failed to grow in cell culture, their subsequent characterization and the characterization of strains that failed to serotype in neutralization assays. Molecular methods increased significantly the sensitivity of detection (P < 0.001) and of characterization (P < 0.01) of enteroviruses when compared to classical methods.
Amino Acid Sequence↗
Rotavirus vaccines: considerations for successful implementation in Europe.
A group of European experts in infectious diseases and vaccinology has met on several occasions to assess the rationale for universal vaccination against rotavirus infection of infants in Europe. On the basis of the available data, we concluded that vaccination was the best approach to prevent severe rotavirus gastroenteritis, and that European countries should consider implementing rotavirus vaccination in their routine immunisation programmes. The main barrier to the implementation of rotavirus vaccination in Europe is a general lack of awareness of stakeholders, policymakers, health-care professionals, and parents about rotavirus disease and the advantages of vaccination. Further studies on the cost of the disease and the benefit of vaccination, together with raising awareness are necessary steps to ensure successful implementation of rotavirus vaccination in Europe.
Europe↗
Human rotavirus G9 and G3 as major cause of diarrhea in hospitalized children, Spain.
In Spain, diarrhea remains a major cause of illness among infants and young children. To determine the prevalence of rotavirus genotypes and temporal and geographic differences in strain distribution, a structured surveillance study of hospitalized children <5 years of age with diarrhea was initiated in different regions of Spain during 2005. Rotavirus was detected alone in samples from 362 (55.2%) samples and as a coinfection with other viruses in 41 samples (6.3%). Enteropathogenic bacterial agents were detected in 4.9% of samples; astrovirus and norovirus RNA was detected in 3.2% and 12.0% samples, respectively; and adenovirus antigen was detected in 1.8% samples. Including mixed infections, the most predominant G type was G9 (50.6%), followed by G3 (33.0%) and G1 (20.2%). Infection with multiple rotavirus strains was detected in >11.4% of the samples studied during 2005.
Base Sequence↗
Detection of viral, bacterial, and parasitological RNA or DNA of nine intestinal pathogens in fecal samples archived as part of the english infectious intestinal disease study: assessment of the stability of target nucleic acid.
Fecal samples were collected from cases and controls as part of the Infectious Intestinal Disease (IID) study in England and were stored as frozen suspensions for 8 to 12 years. The purpose of this study was to apply PCR-based procedures to assess the stability of pathogen-specific nucleic acid sequences present in this archive. Samples from which Cryptosporidium, Giardia, Salmonella, Campylobacter, enteroaggregative Escherichia coli (EAggEC), enterotoxigenic Clostridium perfringens, rotaviruses, noroviruses, or sapoviruses had been previously detected during the IID study using conventional methods were selected from the archive. A generic nucleic acid extraction method to recover RNA or DNA was used. Complementary DNA was generated from RNA by reverse transcription with random priming. Block-based and real-time PCR assays were used to amplify and detect gene fragments from each of these pathogens. The percentage reconfirmation of target was as follows: Giardia duodenalis 68%, Cryptosporidium 96%, Campylobacter 98%, Salmonella 98%, enterotoxigenic C perfringens 34%, EAggEC 93.3%, rotavirus 95%, norovirus 73%, and sapovirus 85%. This study has shown that nucleic acid can be extracted and specific sequences amplified and detected from archived fecal samples. The IID archive therefore represents a valuable resource for further studies, especially the investigation of the samples from which no pathogens had previously been detected.
Cryopreservation↗
An epidemic of enterovirus 71 infection among HIV-1-infected orphans in Nairobi.
Explore the source record for details and available documents.
Child↗
Increase in viral gastroenteritis outbreaks in Europe and epidemic spread of new norovirus variant.
BACKGROUND: Highly publicised outbreaks of norovirus gastroenteritis in hospitals in the UK and Ireland and cruise ships in the USA sparked speculation about whether this reported activity was unusual. METHODS: We analysed data collected through a collaborative research and surveillance network of viral gastroenteritis in ten European countries (England and Wales were analysed as one region). We compiled data on total number of outbreaks by month, and compared genetic sequences from the isolated viruses. Data were compared with historic data from a systematic retrospective review of surveillance systems and with a central database of viral sequences. FINDINGS: Three regions (England and Wales, Germany, and the Netherlands) had sustained epidemiological and viral characterisation data from 1995 to 2002. In all three, we noted a striking increase in norovirus outbreaks in 2002 that coincided with the detection and emergence of a new predominant norovirus variant of genogroup II4, which had a consistent mutation in the polymerase gene. Eight of nine regions had an annual peak in 2002 and the new genogroup II4 variant was detected in nine countries. Also, the detection of the new variant preceded an atypical spring and summer peak of outbreaks in three countries. INTERPRETATION: Our data from ten European countries show a striking increase and unusual seasonal pattern of norovirus gastroenteritis in 2002 that occurred concurrently with the emergence of a novel genetic variant. In addition to showing the added value of an international network for viral gastroenteritis outbreaks, these observations raise questions about the biological properties of the variant and the mechanisms for its rapid dissemination.
Caliciviridae Infections↗
Quantitation of group A rotavirus by real-time reverse-transcription-polymerase chain reaction: correlation with clinical severity in children in South India.
The epidemiology and pathogenesis of rotaviruses are not completely understood, although recent developments in polymerase chain reaction (PCR) techniques now make it possible to quantify the viral load during an infective episode and investigate its relevance to clinical features of the disease. We studied rotavirus-positive stool samples collected from 10 children without symptoms of gastroenteritis and from 81 children with acute gastroenteritis and in whom the clinical severity of disease was recorded. A semi-quantitative real-time reverse-transcription (RT)-PCR was used to estimate the rotavirus load and to assess its correlation with the Vesikari score for severity of diarrhoea. There was a significant negative correlation (r = -0.80, P < 0.001) between severity and the PCR cycle at which the PCR amplicons were detectable (crossing point) on the assay, indicating that children with more severe diarrhoea excrete more virus than children with less severe disease.
Base Sequence↗
Pharmacokinetics of intravenous flucloxacillin and amoxicillin in neonatal and infant cardiopulmonary bypass surgery.
OBJECTIVES: To determine the blood and tissue concentrations of flucloxacillin and amoxicillin during cardiopulmonary bypass (CPB) in infants weighing less than 5 kg. METHODS: Eleven patients aged between 3 and 60 days and weighing <5 kg. Intravenous flucloxacillin 30 mg kg(-1) and amoxicillin 30 mg kg(-1) were administered at time of anaesthesia. Blood and muscle samples were collected at four stages of the operation: immediately before commencement of CPB; before cross-clamping; after weaning of CPB; and at the time of skin closure. Concentrations, half-lives (t(1/2)), clearance and volume of distribution were calculated for both antibiotics in serum and tissue. RESULTS: After connection to CPB the mean serum concentration of flucloxacillin decreased by 42.5% from 75.5 to 43.4 mg l(-1) (P=0.003) and that of amoxicillin decreased by 36.2% from 73.3 to 46.7 mg l(-1) (P=0.021). Serum concentrations of the two antibiotics decreased by a further 16.5 and 14.5% during the remainder of the surgery, but remained >15-fold above the expected minimum inhibitory concentration (MIC) for target pathogens. Muscle concentrations of both antibiotics reached MIC values by the time of the first sample and there was no decrease associated with connection to CPB. Levels remained >8-fold above MIC for target pathogens throughout the procedure. The t(1/2) for flucloxacillin was 2.64(+/-0.23)h and for amoxicillin was 3.16(+/-0.29)h, both of which are more than double the values in normal adults. There was an equivalent reduction in clearance for both antibiotics. CONCLUSIONS: Single doses of flucloxacillin and amoxicillin at 30 mg kg(-1) maintain serum and muscle concentrations well above the MIC throughout cardiac surgery. This is partly due to a prolonged t(1/2) and reduced clearance of both antibiotics in infants.
Amoxicillin↗
Rotavirus genotyping: keeping up with an evolving population of human rotaviruses.
The use of molecular methods for rotavirus characterisation provides not only increased sensitivity for typing, but also allows accurate and more complete characterisation of strains, and the identification of putative reassortant strains. However, due to the constant accumulation of point mutations through genetic drift, and to the emergence of novel genotypes, possibly zoonotic transmission and subsequent reassortment, the reagents and methods used require close monitoring and updating. Methods and oligonucleotide primers are described to overcome failures to type G9, G10 and P[11] rotavirus strains, and cross-reactivity identified between G10 and G3 rotaviruses.
Capsid Proteins↗
The zoonotic potential of rotavirus.
Rotaviruses are generally species-specific, but cross-species transmission is possible, as has been demonstrated experimentally. Several case studies have indicated infection of humans by animal rotaviruses. Comparison of genetic sequences of human and animal rotaviruses often reveals close identity. Surveillance of circulating rotaviruses in the human population has revealed the presence of several uncommon genotypes. Many of these have been found in domestic animals, and it is possible that they arose in the human population through zoonotic transmission. The low incidence of uncommon strains would suggest that such transmission, or at least the establishment of an animal rotavirus or a human/animal reassortant virus in the human population, does not happen with any great frequency. However, many millions of people will be exposed year on year to animal rotaviruses. This happens within farming communities, and potentially to visitors to the countryside. There may be some measure of environmental contamination through livestock excrement. This exposure may not result in high levels of infection, but some infection could occur. There may be a continual input of rotavirus strains or sequences into the human population from the animal population albeit at a very low level.
Animals↗
Seroepidemiology of group C rotavirus infection in England and Wales.
A total of 3199 serum samples collected in 1993--1994 from two population cohorts from England and Wales were tested for the prevalence of IgG antibodies specifically directed against group C rotavirus VP6. Seroprevalence was 39% (95% confidence intervals: 37.0-40.4%). Seroprevalence was highest (46.0%) in the oldest age group (61-70 years of age). Infection with group C rotaviruses occurred at an earlier age and with higher incidence in rural than in urban populations. These results may suggest transmission from animals to humans, however further work is required to identify the reservoir of group C rotavirus for human infection.
Adolescent↗
Contamination of primary total hip replacements in standard and ultra-clean operating theaters detected by the polymerase chain reaction.
BACKGROUND: Many organisms that are responsible for low-grade infection after total hip replacement (THR) are not recognized by routine culture. PATIENTS AND METHODS: We examined wound contamination during primary total hip replacement performed in standard and ultra-clean operating theaters. 20 THRs were performed in each type of theater. Paired tissue specimens taken at the beginning and end of surgery were analyzed by bacterial culture and for the presence of bacterial DNA by the polymerase chain reaction (PCR). In total, 160 specimens (80 for culture, 80 for PCR) from 40 THRs were tested. RESULTS: In standard theaters, none of the 20 specimens taken at the start of surgery were positive by culture, but 3 were positive by PCR (15%). Of the 20 specimens taken at the end of surgery, 2 were positive by enriched culture and 9 were positive by PCR. All specimens positive by culture were also positive by PCR. In ultra-clean theaters, none of the 20 specimens taken at the start of surgery were positive by culture, but 2 were positive by PCR. Of the 20 specimens taken at the end of surgery, none were positive by culture, but 6 were positive by PCR. All specimens that were positive by culture were positive by PCR. INTERPRETATION: Wound contamination of primary THR occurs frequently in both standard and ultra-clean operating theaters and contamination is greater at the end of surgery than at the beginning (p=0.04). In this small series, we found no differences in wound contamination between standard and ultra-clean theaters (p=0.1).
Arthroplasty, Replacement, Hip↗
Polymerase chain reaction can detect bacterial DNA in aseptically loose total hip arthroplasties.
Identifying low-grade infection in failed total hip arthroplasties is an important but difficult task. This study investigated the ability of the polymerase chain reaction to identify low-grade infection during revision of total hip arthroplasties that failed from aseptic causes. One hundred thirteen specimens from 31 total hip arthroplasties revised for aseptic loosening were compared with 105 control specimens from 28 primary total hip arthroplasties. All surgeries were done in laminar flow operating rooms. No primary or revision specimen had positive microbiologic cultures. No revision specimen had histologic evidence suggestive of infection. Using the polymerase chain reaction with a detection threshold of 10 organisms per cubic centimeter of specimen, bacterial DNA was identified in 39 of 85 revision tissue specimens (46%) compared with 18 of 84 primary tissue specimens (21.4%). Bacterial DNA was identified in the synovial fluid of three specimens taken from 28 revision total hip arthroplasties (10.7%) and in two specimens taken from 21 primary total hip arthroplasties (9.5%). As multiple specimens were sent for each hip, a maximum of 16 of 31 revision total hip arthroplasties (52%) and eight of 28 primary total hip arthroplasties (29%) were considered to be infected. Bacterial DNA can be found in many specimens obtained from revised total hip arthroplasties considered to be aseptically loose. Because bacterial DNA identified at primary total hip arthroplasty was assumed to be attributable to contamination rather than present in healthy tissues, the overall specimen contamination rate of 19% and case contamination rate of 29% indicate that the polymerase chain reaction has poor specificity at this sensitivity level for diagnosing infection in revision total hip arthroplasty.
DNA, Bacterial↗
Characterization of G10P[11] rotaviruses causing acute gastroenteritis in neonates and infants in Vellore, India.
Rotavirus G10P[11] strains, which are commonly found in cattle, have frequently been associated with asymptomatic neonatal infections in India. We report the finding of G10P[11] strains associated with severe disease in neonates in Vellore, southern India. Rotavirus strains from 43 fecal samples collected from neonates with or without gastrointestinal symptoms between 1999 and 2000 were genotyped by reverse transcription-PCR. Forty-one neonates (95%) were infected with G10P[11] rotavirus strains, and 63% of the infections were in children who had gastrointestinal symptoms, including acute watery diarrhea. G10P[11] strains were also seen infecting older children with dehydrating gastroenteritis in Vellore. Characterization of the genes encoding VP7, VP4, VP6, and NSP4 of these strains revealed high sequence homology with the corresponding genes of the asymptomatic neonatal strain I321, which in turn is very closely related to bovine G10P[11] strains circulating in India. No significant differences were seen in the sequences obtained from strains infecting symptomatic neonates or children and asymptomatic neonates.
Acute Disease↗
Incidence and risk factors of paediatric rotavirus diarrhoea in northern Ghana.
We measured the type-specific incidence of paediatric rotavirus diarrhoea in an area of northern Ghana. Over 1 year, diarrhoea 1717 episodes were identified, of which 677 (39%) were positive for rotavirus. Risk factors for rotavirus infection included old age, wasting, high Vesikari score and the episode occurring in the dry season. Rotavirus-positive episodes tended to be more acute, causing vomiting and greater dehydration, and were more likely to require hospitalization. The incidence was 0.089 episodes per person-year for all diarrhoea, and 0.035 for rotavirus diarrhoea. The observed incidence decreased markedly with distance from the nearest health centre, suggesting a large unobserved burden. G2P[6], G3P[4] and G9P[8] made up more than half the genotypes detected, but the remainder were diverse. There is a large burden of rotavirus diarrhoea, but the effectiveness of future vaccines could be diluted by the high polymorphism of the virus, and the difficulty of reaching remote populations.
Child, Preschool↗
Evidence for genetic linkage between the gene segments encoding NSP4 and VP6 proteins in common and reassortant human rotavirus strains.
NSP4-encoding genes of 78 human rotavirus strains of common or reassortant genotypes were characterized by reverse transcription-PCR followed by sequencing and phylogenetic analysis. It was found that all the human strains characterized clustered into only two of the five known NSP4 genotypes. Linkage between NSP4 genotypes and VP6 subgroups was 100%, NSP4 genotype A being linked to VP6 of subgroup I (SGI) and NSP4 of genotype B being linked to VP6 of SGII. The diversity among the NSP4- and VP6-encoding genes was significantly less than that among the VP7 and VP4 genes in cocirculating human rotavirus strains. Whereas G and P types appear to be shared among different animal species and humans, the NSP4- and VP6-encoding genes appear to segregate according to their host of origin, suggesting that these two proteins may be host restriction determinants. The NSP4-VP6 association may be structurally determined during rotavirus replication (morphogenesis).
Animals↗
Rotavirus subgroup characterisation by restriction endonuclease digestion of a cDNA fragment of the VP6 gene.
Human rotavirus strains characterised with subgroup-specific monoclonal antibodies were amplified using a VP6-specific RT-PCR and amplicons of 379 bp size (nt 747-1126) were analysed by restriction fragment length polymorphism (RFLP) using the restriction endonuclease AciI. The restriction patterns were compared to the subgroups determined serologically and to VP6 genogroups determined through sequence analysis. RFLP discriminated successfully between VP6 genogroups I and II. None of the strains characterised serologically as either subgroup nonI,nonII or subgroup I+II were distinguishable from subgroup II on the basis of their restriction pattern or sequence analysis. The results obtained by RFLP correlated well with the VP6 genogrouping results, providing a more reliable method for subgrouping of rotaviruses than ELISAs using subgroup-specific antibodies.
Antigens, Viral↗