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Wastewater surveillance holds great promise as a sensitive method to detect spillover of zoonotic infections and early pandemic emergence, thereby informing risk mitigation and public health response. Known viruses with pandemic potential are shed in human stool or urine, or both, and the experiences with SARS-CoV-2, monkeypox virus, and Zika virus highlight the feasibility of community-based wastewater surveillance for pandemic viruses that have different transmission routes. We reviewed human shedding and wastewater surveillance data for prototype viruses representing viral families of concern to estimate the likely sensitivity of wastewater surveillance compared with that of clinical surveillance. We examined how data on wastewater surveillance detection, together with viral genetic sequences and animal faecal biomarkers, could be used to identify spillover infections or early human transmission and adaptation. The opportunities and challenges associated with global wastewater surveillance for the prevention of pandemics are described in this Personal View, focusing on low-income and middle-income countries, where the risk of pandemic emergence is the highest. We propose a research and public health agenda to ensure an equitable and sustainable solution to these challenges., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Widespread surveillance, rapid detection, and appropriate intervention will be critical for successful eradication of poliovirus. Using deployable next-generation sequencing (NGS) approaches, such as Oxford Nanopore Technologies' MinION, the time from sample to result can be significantly reduced compared to cell culture and Sanger sequencing. We developed piranha (poliovirus investigation resource automating nanopore haplotype analysis), a 'sequencing reads-to-report' solution to aid routine poliovirus testing of both stool and environmental samples and alleviate the bioinformatic bottleneck that often exists for laboratories adopting novel NGS approaches. Piranha can be used for efficient intratypic differentiation of poliovirus serotypes, for classification of Sabin-like polioviruses, and for detection of wild-type and vaccine-derived polioviruses. It produces interactive, distributable reports, as well as summary comma-separated values files and consensus poliovirus FASTA sequences. Piranha optionally provides phylogenetic analysis, with the ability to incorporate a local database, processing from raw sequencing reads to an interactive, annotated phylogeny in a single step. The reports describe each nanopore sequencing run with interpretable plots, enabling researchers to easily detect the presence of poliovirus in samples and quickly disseminate their results. Poliovirus eradication efforts are hindered by the lack of real-time detection and reporting, and piranha can be used to complement direct detection sequencing approaches., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press.)

Background: In 2015, the UK government established the Fleming Fund with the aim to address critical gaps in surveillance of antimicrobial resistance (AMR) in low- and middle-income countries in Asia and Africa. Among a large portfolio of grants, the Capturing Data on Antimicrobial Resistance Patterns and Trends in Use in Regions of Asia (CAPTURA) project was awarded with the specific objective of expanding the volume of historical data on AMR, consumption (AMC), and use (AMU) in the human healthcare sector across 12 countries in South and Southeast Asia., Methods: Starting in early 2019, the CAPTURA consortium began working with local governments and >100 relevant data-holding facilities across the region to identify, assess for quality, prioritize, and subsequently retrieve data on AMR, AMC, and AMU. Relevant and shared data were collated and analyzed to provide local overviews for national stakeholders as well as regional context, wherever possible., Results: From the vast information resource generated on current surveillance capacity and data availability, the project has highlighted gaps and areas for quality improvement and supported comprehensive capacity-building activities to optimize local data-collection and -management practices., Conclusions: The project has paved the way for expansion of surveillance networks to include both the academic and private sector in several countries and has actively engaged in discussions to promote data sharing at the local, national, and regional levels. This paper describes the overarching approach to, and emerging lessons from, the CAPTURA project, and how it contributes to other ongoing efforts to strengthen national AMR surveillance in the region and globally., Competing Interests: Potential conflicts of interest. A. G. S., P. F. G., A. T. S., and D. B. report grant funding from the Public Health Surveillance Group. H. S. J. reports being an employee with the International Vaccine Institute since 2021; they also received consultancy fees from the CAPTURA project between 2019 and 2021. J. S. reports salary support through a subcontract from the International Vaccine Institute. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Wastewater surveillance (WWS) was developed in the early 1960s for the detection of poliovirus (PV) circulation in the population. It has been used to monitor several pathogens, including non-polio enteroviruses (NPEVs), which are increasingly recognised as causes of morbidity in children. However, when applying WWS to a new pathogen, it is important to consider the purpose of such a study as well as the suitability of the chosen methodology. With this purpose, the European Non-Polio Enterovirus Network (ENPEN) organised an expert webinar to discuss its history, methods, and applications; its evolution from a culture-based method to molecular detection; and future implementation of next generation sequencing (NGS). The first simulation experiments with PV calculated that a 400 mL sewage sample is sufficient for the detection of viral particles if 1:10,000 people excrete poliovirus in a population of 700,000 people. If the method is applied correctly, several NPEV types are detected. Despite culture-based methods remaining the gold standard for WWS, direct methods followed by molecular-based and sequence-based assays have been developed, not only for enterovirus but for several pathogens. Along with case-based sentinel and/or syndromic surveillance, WWS for NPEV and other pathogens represents an inexpensive, flexible, anonymised, reliable, population-based tool for monitoring outbreaks and the (re)emergence of these virus types/strains within the general population.

Timely detection of outbreaks is needed for poliovirus eradication, but gold standard detection in the Democratic Republic of the Congo takes 30 days (median). Direct molecular detection and nanopore sequencing (DDNS) of poliovirus in stool samples is a promising fast method. Here we report prospective testing of stool samples from suspected polio cases, and their contacts, in the Democratic Republic of the Congo between 10 August 2021 and 4 February 2022. DDNS detected polioviruses in 62/2,339 (2.7%) of samples, while gold standard combination of cell culture, quantitative PCR and Sanger sequencing detected polioviruses in 51/2,339 (2.2%) of the same samples. DDNS provided case confirmation in 7 days (median) in routine surveillance conditions. DDNS enabled confirmation of three serotype 2 circulating vaccine-derived poliovirus outbreaks 23 days (mean) earlier (range 6-30 days) than the gold standard method. The mean sequence similarity between sequences obtained by the two methods was 99.98%. Our data confirm the feasibility of implementing DDNS in a national poliovirus laboratory., (© 2023. The Author(s).)

Clostridium perfringens is an anaerobic toxin-producing bacterium associated with intestinal diseases, particularly in neonatal humans and animals. Infant gut microbiome studies have recently indicated a link between C. perfringens and the preterm infant disease necrotizing enterocolitis (NEC), with specific NEC cases associated with overabundant C. perfringens termed C. perfringens-associated NEC (CPA-NEC). In the present study, we carried out whole-genome sequencing of 272 C. perfringens isolates from 70 infants across 5 hospitals in the United Kingdom. In this retrospective analysis, we performed in-depth genomic analyses (virulence profiling, strain tracking and plasmid analysis) and experimentally characterized pathogenic traits of 31 strains, including 4 from CPA-NEC patients. We found that the gene encoding toxin perfringolysin O, pfoA, was largely deficient in a human-derived hypovirulent lineage, as well as certain colonization factors, in contrast to typical pfoA-encoding virulent lineages. We determined that infant-associated pfoA + strains caused significantly more cellular damage than pfoA - strains in vitro, and further confirmed this virulence trait in vivo using an oral-challenge C57BL/6 murine model. These findings suggest both the importance of pfoA + C. perfringens as a gut pathogen in preterm infants and areas for further investigation, including potential intervention and therapeutic strategies., (© 2023. The Author(s).)

Direct detection by PCR of poliovirus RNA in stool samples provides a rapid diagnostic and surveillance tool that can replace virus isolation by cell culture in global polio surveillance. The sensitivity of direct detection methods is likely to depend on the choice of RNA extraction method and sample volume. We report a comparative analysis of 11 nucleic acid extraction methods (7 manual and 4 semiautomated) for poliovirus molecular detection using stool samples ( n  = 59) that had been previously identified as poliovirus positive by cell culture. To assess the effect of RNA recovery methods, extracted RNA using each of the 11 methods was tested with a poliovirus-specific reverse transcription-quantitative PCR (RT-qPCR), a pan-poliovirus RT-PCR (near-whole-genome amplification), a pan-enterovirus RT-PCR (entire capsid region), and a nested VP1 PCR that is the basis of a direct detection method based on nanopore sequencing. We also assessed extracted RNA integrity and quantity. The overall effect of extraction method on poliovirus PCR amplification assays tested in this study was found to be statistically significant ( P < 0.001), thus indicating that the choice of RNA extraction method is an important component that needs to be carefully considered for any diagnostic based on nucleic acid amplification. Performance of the methods was generally consistent across the different assays used. Of the 11 extraction methods tested, the MagMAX viral RNA isolation kit used manually or automatically was found to be the preferable method for poliovirus molecular direct detection considering performance, cost, and processing time. IMPORTANCE Poliovirus, the causative agent of poliomyelitis, is a target of global eradication led by the World Health Organization since 1988. Direct molecular detection and genomic sequencing without virus propagation in cell culture is arguably a critical tool in the final stages of polio eradication. Efficient recovery of good-quality viral RNA from stool samples is a prerequisite for direct detection by nucleic acid amplification. We tested 11 nucleic acid extraction methods to identify those facilitating sensitive, fast, simple, and cost-effective extraction, with flexibility for manual and automated protocols considered. Several different PCR assays were used to compare the recovered viral RNA to test suitability for poliovirus direct molecular detection. Our findings highlight the importance of choosing a suitable RNA extraction protocol and provide useful information to diagnostic laboratories and researchers facing the choice of RNA extraction method for direct molecular virus detection from stool.

Background: The gastrointestinal (GI) microbiota has been linked to health consequences throughout life, from early life illnesses (e.g. sepsis and necrotising enterocolitis) to lifelong chronic conditions such as obesity and inflammatory bowel disease. It has also been observed that events in early life can lead to shifts in the microbiota, with some of these changes having been documented to persist into adulthood. A particularly extreme example of a divergent early GI microbiota occurs in premature neonates, who display a very different GI community to term infants. Certain characteristic patterns have been associated with negative health outcomes during the neonatal period, and these patterns may prove to have continual damaging effects if not resolved., Results: In this study we compared a set of premature infants with a paired set of term infants (n = 37 pairs) at 6 weeks of age and at 2 years of age. In the samples taken at 6 weeks of age we found microbial communities differing in both diversity and specific bacterial groups between the two infant cohorts. We identified clinical factors associated with over-abundance of potentially pathogenic organisms (e.g. Enterobacteriaceae) and reduced abundances of some beneficial organisms (e.g. Bifidobacterium). We contrasted these findings with samples taken at 2 years of age, which indicated that despite a very different initial gut microbiota, the two infant groups converged to a similar, more adult-like state. We identified clinical factors, including both prematurity and delivery method, which remain associated with components of the gut microbiota. Both clinical factors and microbial characteristics are compared to the occurrence of childhood wheeze and eczema, revealing associations between components of the GI microbiota and the development of these allergic conditions., Conclusions: The faecal microbiota differs greatly between infants born at term and those born prematurely during early life, yet it converges over time. Despite this, early clinical factors remain significantly associated with the abundance of some bacterial groups at 2 years of age. Given the associations made between health conditions and the microbiota, factors that alter the makeup of the gut microbiota, and potentially its trajectory through life, could have important lifelong consequences., (© 2023. The Author(s).)

Background and Aims: Necrotizing enterocolitis (NEC) is a life-threatening disease and the most common gastrointestinal emergency in premature infants. Accurate early diagnosis is challenging. Modified Bell's staging is routinely used to guide diagnosis, but early diagnostic signs are nonspecific, potentially leading to unobserved disease progression, which is problematic given the often rapid deterioration observed. We investigated fecal cytokine levels, coupled with gut microbiota profiles, as a noninvasive method to discover specific NEC-associated signatures that can be applied as potential diagnostic markers., Methods: Premature babies born below 32 weeks of gestation were admitted to the 2-site neonatal intensive care unit (NICU) of Imperial College hospitals (St. Mary's or Queen Charlotte's & Chelsea) between January 2011 and December 2012. During the NICU stay, expert neonatologists grouped individuals by modified Bell's staging (healthy, NEC1, NEC2/3) and fecal samples from diapers were collected consecutively. Microbiota profiles were assessed by 16S rRNA gene amplicon sequencing and cytokine concentrations were measured by V-Plex multiplex assays., Results: Early evaluation of microbiota profiles revealed only minor differences. However, at later time points, significant changes in microbiota composition were observed for Bacillota (adj. P  = .0396), with Enterococcus being the least abundant in Bell stage 2/3 NEC. Evaluation of fecal cytokine levels revealed significantly higher concentrations of IL-1α ( P  = .045), IL-5 ( P  = .0074), and IL-10 ( P  = .032) in Bell stage 1 NEC compared to healthy individuals., Conclusion: Differences in certain fecal cytokine profiles in patients with NEC indicate their potential use as diagnostic biomarkers to facilitate earlier diagnosis. Additionally, associations between microbial and cytokine profiles contribute to improving knowledge about NEC pathogenesis., (© 2023 The Authors.)

Background: The international spread of poliovirus exposes all countries to the risk of outbreaks and is designated a Public Health Emergency of International Concern by WHO. This risk can be exacerbated in countries using inactivated polio vaccine, which offers excellent protection against paralysis but is less effective than oral vaccine against poliovirus shedding, potentially allowing circulation without detection of paralytic cases for long periods of time. Our study investigated the molecular properties of type 2 poliovirus isolates found in sewage with an aim to detect virus transmission in the community., Methods: We performed environmental surveillance in London, UK, testing sewage samples using WHO recommended methods that include concentration, virus isolation in cell culture, and molecular characterisation. We additionally implemented direct molecular detection and determined whole-genome sequences of every isolate using novel nanopore protocols., Findings: 118 genetically linked poliovirus isolates related to the serotype 2 Sabin vaccine strain were detected in 21 of 52 sequential sewage samples collected in London between Feb 8 and July 4, 2022. Expansion of environmental surveillance sites in London helped localise transmission to several boroughs in north and east London. All isolates have lost two key attenuating mutations, are recombinants with a species C enterovirus, and an increasing proportion (20 of 118) meet the criterion for a vaccine-derived poliovirus, having six to ten nucleotide changes in the gene coding for VP1 capsid protein., Interpretation: Environmental surveillance allowed early detection of poliovirus importation and circulation in London, permitting a rapid public health response, including enhanced surveillance and an inactivated polio vaccine campaign among children aged 1-9 years. Whole-genome sequences generated through nanopore sequencing established linkage of isolates and confirmed transmission of a unique recombinant poliovirus lineage that has now been detected in Israel and the USA., Funding: Medicines and Healthcare products Regulatory Agency, UK Health Security Agency, Bill & Melinda Gates Foundation, and National Institute for Health Research Medical Research Council., Competing Interests: Declaration of interests We declare no competing interests., (© 2022 Crown Copyright claimed by UK, Canadian or Australian Government employee. This is an Open Access article under the OGL 3.0 license.)

Background: Detection of poliovirus outbreaks relies on a complex laboratory algorithm of cell-culture, polymerase chain reaction (PCR), and sequencing to distinguish wild-type and vaccine-derived polioviruses (VDPV) from Sabin-like strains. We investigated the potential for direct molecular detection and nanopore sequencing (DDNS) to accelerate poliovirus detection., Methods: We analyzed laboratory data for time required to analyze and sequence serotype-2 VDPV (VDPV2) in stool collected from children with acute flaccid paralysis in Africa (May 2016-February 2020). Impact of delayed detection on VDPV2 outbreak size was assessed through negative binomial regression., Results: VDPV2 confirmation in 525 stools required a median of 49 days from paralysis onset (10th-90th percentile, 29-74), comprising collection and transport (median, 16 days), cell-culture (7 days), intratypic differentiation quantitative reverse transcription PCR (3 days), and sequencing, including shipping if required (15 days). New VDPV2 outbreaks were confirmed a median of 35 days (27-60) after paralysis onset, which we estimate could be reduced to 16 days by DDNS (9-37). Because longer delays in confirmation and response were positively associated with more cases (P < .001), we estimate that DDNS could reduce the number of VDPV2 cases before a response by 28% (95% credible interval, 12%-42%)., Conclusions: DDNS could accelerate poliovirus outbreak response, reducing their size and the cost of eradication., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America.)

Background: Respiratory virus infection is common in early childhood, and children may be symptomatic or symptom-free. Little is known regarding the association between symptomatic/asymptomatic infection and particular clinical factors such as breastfeeding as well as the consequences of such infection., Method: We followed an unselected cohort of term neonates to two years of age (220 infants at recruitment, 159 who remained in the study to 24 months), taking oral swabs at birth and oropharyngeal swabs at intervals subsequently (at 1.5, 6, 9, 12, 18 and 24 months and in a subset at 3 and 4.5 months) while recording extensive metadata including the presence of respiratory symptoms and breastfeeding status. After 2 years medical notes from the general practitioner were inspected to ascertain whether doctor-diagnosed wheeze had occurred by this timepoint. Multiplex PCR was used to detect a range of respiratory viruses: influenza (A&B), parainfluenza (1-4), bocavirus, human metapneumovirus, rhinovirus, coronavirus (OC43, 229E, NL63, HKU1), adenovirus, respiratory syncytial virus (RSV), and polyomavirus (KI, WU). Logistic regression and generalised estimating equations were used to identify associations between clinical factors and virus detection., Results: Overall respiratory viral incidence increased with age. Rhinovirus was the virus most frequently detected. The detection of a respiratory virus was positively associated with respiratory symptoms, male sex, season, childcare and living with another child. We did not observe breastfeeding (whether assessed as the number of completed months of breastfeeding or current feed status) to be associated with the detection of a respiratory virus. There was no association between early viral infection and doctor-diagnosed wheeze by age 2 years., Conclusion: Asymptomatic and symptomatic viral infection is common in the first 2 years of life with rhinovirus infection being the most common. Whilst there was no association between early respiratory viral infection and doctor-diagnosed wheeze, we have not ruled out an association of early viral infections with later asthma, and long-term follow-up of the cohort continues., (© 2022. The Author(s).)

Purpose: Many athletes report consuming alcohol the day before their event, which might negatively affect their performance. However, the effects of previous-day alcohol ingestion on performance are equivocal, in part, due to no standardization of alcohol dose in previous studies. The purpose of this study was to examine the impact of a standardized previous-day alcohol dose and its corresponding impact on morning-after muscular strength, muscular power, and muscular fatigue in a short-duration test and on performance of severe-intensity exercise., Methods: On 2 occasions, 12 recreationally active individuals reported to the Applied Physiology Laboratory in the evening and ingested a beverage containing either 1.09 g ethanol·kg-1 fat-free body mass (ALC condition) or water (PLA condition). The following morning, they completed a hangover symptom questionnaire, vertical jumps, isometric midthigh pulls, biceps curls, and a constant-power cycle ergometer test to exhaustion. The responses from ALC and PLA were compared using paired-means t tests., Results: Time to exhaustion in the cycle ergometer tests was less (P = .03) in the ALC condition (181 [39] s vs 203 [34] s; -11%, Cohen d = 0.61). There was no difference in performance in vertical jump test, isometric midthigh pulls, and biceps curls tests between the ALC and PLA conditions., Conclusions: Previous-day alcohol consumption significantly reduces morning-after performance of severe-intensity exercise. Practitioners should educate their athletes, especially those whose events rely on anaerobic capacity and/or a rapid response of the aerobic pathways, of the adverse effect of previous-day alcohol consumption on performance.

Background: Necrotising enterocolitis (NEC) is a devastating bowel disease, primarily affecting premature infants, with a poorly understood aetiology. Prior studies have found associations in different cases with an overabundance of particular elements of the faecal microbiota (in particular Enterobacteriaceae or Clostridium perfringens), but there has been no explanation for the different results found in different cohorts. Immunological studies have indicated that stimulation of the TLR4 receptor is involved in development of NEC, with TLR4 signalling being antagonised by the activated TLR9 receptor. We speculated that differential stimulation of these two components of the signalling pathway by different microbiota might explain the dichotomous findings of microbiota-centered NEC studies. Here we used shotgun metagenomic sequencing and qPCR to characterise the faecal microbiota community of infants prior to NEC onset and in a set of matched controls. Bayesian regression was used to segregate cases from control samples using both microbial and clinical data., Results: We found that the infants suffering from NEC fell into two groups based on their microbiota; one with low levels of CpG DNA in bacterial genomes and the other with high abundances of organisms expressing LPS. The identification of these characteristic communities was reproduced using an external metagenomic validation dataset. We propose that these two patterns represent the stimulation of a common pathway at extremes; the LPS-enriched microbiome suggesting overstimulation of TLR4, whilst a microbial community with low levels of CpG DNA suggests reduction of the counterbalance to TLR4 overstimulation., Conclusions: The identified microbial community patterns support the concept of NEC resulting from TLR-mediated pathways. Identification of these signals suggests characteristics of the gastrointestinal microbial community to be avoided to prevent NEC. Potential pre- or pro-biotic treatments may be designed to optimise TLR signalling., (© 2021. The Author(s).)

Global poliovirus surveillance involves virus isolation from stool and environmental samples, intratypic differential (ITD) by PCR, and sequencing of the VP1 region to distinguish vaccine (Sabin), vaccine-derived, and wild-type polioviruses and to ensure an appropriate response. This cell culture algorithm takes 2 to 3 weeks on average between sample receipt and sequencing. Direct detection of viral RNA using PCR allows faster detection but has traditionally faced challenges related to poor sensitivity and difficulties in sequencing common samples containing poliovirus and enterovirus mixtures. We present a nested PCR and nanopore sequencing protocol that allows rapid (<3 days) and sensitive direct detection and sequencing of polioviruses in stool and environmental samples. We developed barcoded primers and a real-time analysis platform that generate accurate VP1 consensus sequences from multiplexed samples. The sensitivity and specificity of our protocol compared with those of cell culture were 90.9% (95% confidence interval, 75.7% to 98.1%) and 99.2% (95.5% to 100.0%) for wild-type 1 poliovirus, 92.5% (79.6% to 98.4%) and 98.7% (95.4% to 99.8%) for vaccine and vaccine-derived serotype 2 poliovirus, and 88.3% (81.2% to 93.5%) and 93.2% (88.6% to 96.3%) for Sabin 1 and 3 poliovirus alone or in mixtures when tested on 155 stool samples in Pakistan. Variant analysis of sequencing reads also allowed the identification of polioviruses and enteroviruses in artificial mixtures and was able to distinguish complex mixtures of polioviruses in environmental samples. The median identity of consensus nanopore sequences with Sanger or Illumina sequences from the same samples was >99.9%. This novel method shows promise as a faster and safer alternative to cell culture for the detection and real-time sequencing of polioviruses in stool and environmental samples., (Copyright © 2020 Shaw et al.)

Genetic sequencing of polioviruses detected through clinical and environmental surveillance is used to confirm detection, identify their likely origin, track geographic patterns of spread, and determine the appropriate vaccination response. The critical importance of genetic sequencing and analysis to the Global Polio Eradication Initiative has grown with the increasing incidence of vaccine-derived poliovirus (VDPV) infections in Africa specifically (470 reported cases in 2019), and globally, alongside persistent transmission of serotype 1 wild-type poliovirus in Pakistan and Afghanistan (197 reported cases in 2019). Adapting what has been learned about the virus genetics and evolution to address these threats has been a major focus of recent work. Here, we review how phylogenetic and phylogeographic methods have been used to trace the spread of wild-type polioviruses and identify the likely origins of VDPVs. We highlight the analysis methods and sequencing technology currently used and the potential for new technologies to speed up poliovirus detection and the interpretation of genetic data. At a pivotal point in the eradication campaign with the threat of anti-vaccine sentiment and donor and public fatigue, innovation is critical to maintain drive and overcome the last remaining circulating virus., (© The Author(s) 2020. Published by Oxford University Press.)

Background: Clostridium perfringens forms part of the human gut microbiota and has been associated with life-threatening necrotising enterocolitis (NEC) in premature infants. Whether specific toxigenic strains are responsible is unknown, as is the extent of diversity of strains in healthy premature babies. We investigated the C. perfringens carrier status of premature infants in the neonatal intensive care unit, factors influence this status, and the toxic potential of the strains., Methods: C. perfringens was isolated by culture from faecal samples from 333 infants and their toxin gene profiles analysed by PCR. A survival analysis was used to identify factors affecting probability of carriage. Competitive growth experiments were used to explore the results of the survival analysis., Results: 29.4% of infants were colonized with C. perfringens before they left hospital. Three factors were inversely associated with probability of carriage: increased duration of maternal milk feeds, CPAP oxygen treatment and antibiotic treatment. C. perfringens grew poorly in breast milk and was significantly outperformed by Bifidobacterium infantis, whether grown together or separately. Toxin gene screening revealed that infants carried isolates positive for collagenase, perfringolysin O, beta 2, beta, becA/B, netB and enterotoxin toxin genes, yet none were observed to be associated with the development of NEC., Conclusions: Approximately a third of preterm infants are colonised 3 weeks after birth with toxin gene-carrying C. perfringens. We speculate that increased maternal breast milk, oxygen and antibiotic treatment creates an environment in the gut hostile to growth of C. perfringens. Whilst potentially toxigenic C. perfringens isolates were frequent, no toxin type was associated with NEC., Trial Registration: clinicaltrials.gov NCT01102738, registered 13th April 2010.

Background: A critical window in infancy has been proposed, during which the microbiota may affect subsequent health. The longitudinal development of the oropharyngeal microbiota is under-studied and may be associated with early-life wheeze. We aimed to investigate the temporal association of the development of the oropharyngeal microbiota with early-life wheeze., Methods: A population-based birth cohort based in London, UK was followed for 24 months. We collected oropharyngeal swabs at six time-points. Microbiota was determined using sequencing of the V3-V5 region of the 16S rRNA-encoding gene. Medical records were reviewed for the outcome of doctor diagnosed wheeze. We used a time-varying model to investigate the temporal association between the development of microbiota and doctor-diagnosed wheeze., Findings: 159 participants completed the study to 24 months and for 98 there was complete sequencing data at all timepoints and outcome data. Of these, 26 had doctor-diagnosed wheeze. We observed significant increase in the abundance of Neisseria between 9 and 24 months in children who developed wheeze (p = 0∙003), while in those without wheezing there was a significant increment in the abundance of Granulicatella (p = 0∙012) between 9 and 12 months, and of Prevotella (p = 0∙018) after 18 months., Interpretation: A temporal association between the respiratory commensal Granulicatella and also Prevotella with wheeze (negative), and between Neisseria and wheeze (positive) was identified in infants prior to one year of age. This adds to evidence for the proposed role of the microbiota in the development of wheeze. FUND: Research funding from the Winnicott Foundation, Meningitis Now and Micropathology Ltd., (Copyright © 2019. Published by Elsevier B.V.)

Objectives: Inflammatory bowel disease states are associated with gastrointestinal dysbiosis. Mucosal biopsy sampling, retrieving the bacterial community that most directly interacts with the host, is an invasive procedure that, we hypothesis, may be sufficiently approximated by other sampling methods. We investigate the relatedness of samples obtained by different methods and the effects of bowel preparation on the gastrointestinal community in a paediatric population., Methods: We recruited a cohort of patients undergoing colonoscopy, collecting serial samples via differing methods (rectal swabs, biopsies, and faecal matter/luminal contents) prebowel preparation, during colonoscopy and after colonoscopy. Next-generation sequencing was used to determine the structure of the microbial community., Results: The microbial community in luminal contents collected during colonoscopy was found to be more similar to that of mucosal biopsies than rectal swabs. Community traits of the mucosal biopsies could be used to segregate patients with inflammatory bowel disease from other patients, and the similarity of the communities in the luminal contents was sufficient for the segregation to be reproduced. Microbial communities sampled by rectal swabs and prebowel preparation faeces were less similar to mucosal biopsies. Bowel preparation was found to have no significant long-term effects on the microbial community, despite the transient effects evident during colonoscopy., Conclusions: A clinically relevant description of the mucosal microbial community can be obtained via the noninvasive collection of luminal contents after bowel cleansing. Bowel preparation in a paediatric population results in no consistent sustained alterations to the gastrointestinal microbiota.

Background: Few studies have investigated the gut microbiome of infants, fewer still preterm infants. In this study we sought to quantify and interrogate the resistome within a cohort of premature infants using shotgun metagenomic sequencing. We describe the gut microbiomes from preterm but healthy infants, characterising the taxonomic diversity identified and frequency of antibiotic resistance genes detected., Results: Dominant clinically important species identified within the microbiomes included C. perfringens , K. pneumoniae and members of the Staphylococci and Enterobacter genera. Screening at the gene level we identified an average of 13 antimicrobial resistance genes per preterm infant, ranging across eight different antibiotic classes, including aminoglycosides and fluoroquinolones. Some antibiotic resistance genes were associated with clinically relevant bacteria, including the identification of mecA and high levels of Staphylococci within some infants. We were able to demonstrate that in a third of the infants the S. aureus identified was unrelated using MLST or metagenome assembly, but low abundance prevented such analysis within the remaining samples., Conclusions: We found that the healthy preterm infant gut microbiomes in this study harboured a significant diversity of antibiotic resistance genes. This broad picture of resistances and the wider taxonomic diversity identified raises further caution to the use of antibiotics without consideration of the resident microbial communities., Competing Interests: The authors declare there are no competing interests.

Background: In this manuscript, we investigate the "stones best left unturned" of sample storage and preparation and their implications for the next-generation sequencing of infant faecal microbial communities by the 16S ribosomal ribonucleic acid (rRNA) gene. We present a number of experiments that investigate the potential effects of often overlooked methodology factors, establishing a "normal" degree of variation expected between replica sequenced samples. Sources of excess variation are then identified, as measured by observation of alpha diversity, taxonomic group counts and beta diversity magnitudes between microbial communities., Results: Extraction of DNA from samples on different dates, by different people and even using varied sample weights results in little significant difference in downstream sequencing data. A key assumption in many studies is the stability of samples stored long term at -80 °C prior to extraction. After 2 years, we see relatively few changes: increased abundances of lactobacilli and bacilli and a reduction in the overall OTU count. Where samples cannot be frozen, we find that storing samples at room temperature does lead to significant changes in the microbial community after 2 days. Mailing of samples during this time period (a common form of sample collection from outpatients for example) does not lead to any additional variation., Conclusions: Important methodological standards can be drawn from these results; painstakingly created archives of infant faecal samples stored at -80 °C are still largely representative of the original community and varying factors in DNA extraction methodology have comparatively little effect on overall results. Samples taken should ideally be either frozen at -80 °C or extracted within 2 days if stored at room temperature, with mail samples being mailed on the day of collection.

Background: Late-onset bloodstream infection (LO-BSI) is a common complication of prematurity, and lack of timely diagnosis and treatment can have life-threatening consequences. We sought to identify clinical characteristics and microbial signatures in the gastrointestinal microbiota preceding diagnosis of LO-BSI in premature infants., Method: Daily faecal samples and clinical data were collected over two years from 369 premature neonates (<32 weeks gestation). We analysed samples from 22 neonates who developed LO-BSI and 44 matched control infants. Next-generation sequencing of 16S rRNA gene regions amplified by PCR from total faecal DNA was used to characterise the microbiota of faecal samples preceding diagnosis from infants with LO-BSI and controls. Culture of selected samples was undertaken, and bacterial isolates identified using MALDI-TOF. Antibiograms from bloodstream and faecal isolates were compared to explore strain similarity., Results: From the week prior to diagnosis, infants with LO-BSI had higher proportions of faecal aerobes/facultative anaerobes compared to controls. Risk factors for LO-BSI were identified by multivariate analysis. Enterobacteriaceal sepsis was associated with antecedent multiple lines, low birth weight and a faecal microbiota with prominent Enterobacteriaceae. Staphylococcal sepsis was associated with Staphylococcus OTU faecal over-abundance, and the number of days prior to diagnosis of mechanical ventilation and of the presence of centrally-placed lines. In 12 cases, the antibiogram of the bloodstream isolate matched that of a component of the faecal microbiota in the sample collected closest to diagnosis., Conclusions: The gastrointestinal tract is an important reservoir for LO-BSI organisms, pathogens translocating across the epithelial barrier. LO-BSI is associated with an aberrant microbiota, with abundant staphylococci and Enterobacteriaceae and a failure to mature towards predominance of obligate anaerobes.

Background: Necrotizing enterocolitis (NEC) is a devastating inflammatory bowel disease of premature infants speculatively associated with infection. Suspected NEC can be indistinguishable from sepsis, and in established cases an infant may die within hours of diagnosis. Present treatment is supportive. A means of presymptomatic diagnosis is urgently needed. We aimed to identify microbial signatures in the gastrointestinal microbiota preceding NEC diagnosis in premature infants., Methods: Fecal samples and clinical data were collected from a 2-year cohort of 369 premature neonates. Next-generation sequencing of 16S ribosomal RNA gene regions was used to characterize the microbiota of prediagnosis fecal samples from 12 neonates with NEC, 8 with suspected NEC, and 44 controls. Logistic regression was used to determine clinical characteristics and operational taxonomic units (OTUs) discriminating cases from controls. Samples were cultured and isolates identified using matrix-assisted laser desorption/ionization-time of flight. Clostridial isolates were typed and toxin genes detected., Results: A clostridial OTU was overabundant in prediagnosis samples from infants with established NEC (P = .006). Culture confirmed the presence of Clostridium perfringens type A. Fluorescent amplified fragment-length polymorphism typing established that no isolates were identical. Prediagnosis samples from NEC infants not carrying profuse C. perfringens revealed an overabundance of a Klebsiella OTU (P = .049). Prolonged continuous positive airway pressure (CPAP) therapy with supplemental oxygen was also associated with increased NEC risk., Conclusions: Two fecal microbiota signatures (Clostridium and Klebsiella OTUs) and need for prolonged CPAP oxygen signal increased risk of NEC in presymptomatic infants. These biomarkers will assist development of a screening tool to allow very early diagnosis of NEC. Clinical Trials Registration. NCT01102738., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) Staphylococcus aureus colonization is associated with increased rates of infection. Rapid and reliable detection methods are needed to identify colonization of nares and extra-nare sites, particularly given recent reports of oropharynx-only colonization. Detection methods for MRSA/MSSA colonization include culture, PCR, and novel methods such as PCR/electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS)., Methods: We evaluated 101 healthy military members for S. aureus colonization in the nares, oropharynx, axilla, and groin, using CHROMagar S. aureus medium and Xpert SA Nasal Complete PCR for MRSA/MSSA detection. The same subjects were screened in the nares, oropharynx, and groin using PCR/ESI-TOF-MS., Results: By culture, 3 subjects were MRSA-colonized (all oropharynx) and 34 subjects were MSSA-colonized (all 4 sites). PCR detected oropharyngeal MRSA in 2 subjects, which correlated with culture findings. By PCR, 47 subjects were MSSA-colonized (all 4 sites); however, 43 axillary samples were invalid, 39 of which were associated with deodorant/anti-perspirant use (93%, p < 0.01). By PCR/ESI-TOF-MS, 4 subjects were MRSA-colonized, 2 in the nares and 2 in the oropharynx; however, neither of these correlated with positive MRSA cultures. Twenty-eight subjects had MSSA by PCR/ESI-TOF-MS, and 41 were found to have possible MRSA (S. aureus with mecA and coagulase-negative Staphylococcus (CoNS))., Conclusion: The overall 3% MRSA colonization rate is consistent with historical reports, but the oropharynx-only colonization supports more recent findings. In addition, the use of deodorant/anti-perspirant invalidated axillary PCR samples, limiting its utility. Defining MRSA positivity by PCR/ESI-TOF-MS is complicated by co-colonization of S. aureus with CoNS, which can also carry mecA.

The Raf/ERK (Extracellular Signal Regulated Kinase) signal transduction pathway controls numerous cellular processes, including growth, differentiation, cellular transformation and senescence. ERK activation is thought to involve complex spatial and temporal regulation, to achieve a high degree of specificity, though precisely how this is achieved remains to be confirmed. We report here that prolonged activation of a conditional form of c-Raf-1 (BXB-ER) leads to profound changes in the level and distribution of a heterochromatic histone mark. In mouse fibroblasts, the heterochromatic trimethylation of lysine 9 in histone H3 (H3K9Me3) is normally confined to pericentromeric regions. However, following ERK activation a genome-wide redistribution of H3K9Me3 correlates with loss of the histone modification from chromocentres and the appearance of numerous punctuate sites throughout the interphase nucleus. These epigenetic changes during interphase correlate with altered chromosome structure during mitosis, where robust H3K9Me3 signals appear within telomeric heterochromatin. This pattern of heterochromatinization is distinct from previously described oncogene induced senescence associated heterochromatin foci (SAHF), which are excluded from telomeres. The H3K9Me3 histone mark is known to bind the major heterochromatin protein HP1 and we show that the alterations in the distribution of this histone epistate correlate with redistribution of HP1β throughout the nucleus. Interestingly while ERK activation is fully reversible, the observed chromatin changes induced by epigenetic modifications are not reversible once established. We describe for the first time a link from prolonged ERK activation to stable changes in genome organization through redistribution of heterochromatic domains involving the telomeres. These epigenetic changes provide a possible mechanism through which prolonged activation of Raf/ERK can lead to growth arrest or the induction of differentiation, senescence and cancer.

Objective: It has recently been reported that up to one-third of patients with nonmetastatic distal rectal cancer managed with neoadjuvant chemoradiation therapy (CRT) had a complete clinical response (cCR) to treatment. In the selected cases, this has been used as the sole treatment. The aim of this study was to determine the frequency of complete pathological response for patients receiving CRT in one centre in the UK., Method: Patients receiving 6 weeks of neoadjuvant CRT were identified using the two cancer audit databases in two different tertiary hospitals from January 2002 to November 2007. Pathology was reviewed and the histopathological response of the resected specimen to CRT was evaluated using the Mandard classification (1 = complete response, 5 = no response), Results: One hundred and thirty-two consecutive patients [median age 61 (range 44-86) years, 90 men] with nonmetastatic locally advanced rectal cancer received neoadjuvant chemo radiotherapy between 2002 and 2007 followed by resection of the tumour. Data were available from 129 patients., Conclusion: Only 13 out of 132 (10%) of patients had a complete pathological response. This is one-third of the cCR previously reported. Nonsurgical therapy for rectal cancer using the Habr-Gama treatment algorithm may only be effective in a very small proportion of patients with rectal cancer in the UK and nonoperative treatment would not be recommended.

Background: High circulating platelet counts have been associated with poor prognosis in a variety of solid tumours such as breast, renal and lung cancer. We investigated the significance of a high pre-operative platelet count on overall survival in patients with stages I-IV colorectal cancer., Patients and Methods: 630 Consecutive patients who colorectal cancer resection between 2004 and 2007 with a full blood count taken 14 days prior to the surgery were assessed. Male:female 7:5, median (range) age 73 (40-99 years). Thrombocytosis was defined as platelet count of ≥450 × 10(9)/L. The relationship between platelet count, pathological features and overall survival was assessed., Results: : Mantel-Cox regression showed that platelet count does not predict survival on multivariate analysis (p = 0.067). Thrombocytosis was present in 51/627 (8.1%) of cases. There was no statistically significant difference in mean survival (p = 0.067) observed in patients with platelet count <450 × 10(9)/L (n = 576; 95%CI: 1550.5-1405.4 SE 37.0) versus ≥450 × 10(9)/L (n = 51, CI: 1261.6-955.0, SE 78.2). There was also no correlation between Dukes stage and thrombocytosis., Conclusion(s): In our study, pre-operative thrombocytosis is not a prognostic indicator of survival in colorectal cancer patients regardless of pathological stage., (Copyright © 2010 Surgical Associates Ltd. Published by Elsevier Ltd. All rights reserved.)

Introduction: A rectus sheath haematoma is a rare condition that arises from the accumulation of blood within the rectus sheath from either muscular tear or rupture of the epigastric vessels. It is a known complication of either blunt abdominal trauma, anticoagulation therapy or the repetitive contraction of the rectus muscle such as paroxysms of coughing. It remains an uncommon and elusive entity and is often clinically misdiagnosed., Case Presentation: An 80-year-old British man with a known aortic aneurysm was admitted with sudden onset of right iliac fossa pain. The patient was hemodynamically stable and underwent a computed tomography scan which revealed an intact aorta and an acute rectus sheath hematoma., Conclusion: To the best of our knowledge, no case has previously been reported involving the diagnostic challenge of a rectus sheath hematoma in a patient with a known aortic aneurysm. Here we discuss the symptoms and signs of a rectus sheath hematoma, as well as the radiological modalities that could be utilized to reach the diagnosis.

Aim: To find the proportion of patients with a faecal occult blood (FOB) performed as part of the referral for the lower gastrointestinal two week wait (2WW) referral system, and whether this correlates with the cancer status., Method: All patients referred to the colorectal cancer service using the 2WW referral criteria, between August 2005 and August 2007, were identified using the hospital's cancer audit database. Faecal occult bloods and cancer status were recorded for each patient., Results: Two thousand one hundred and fifty-nine patients (1177 female: 903 male; median age 58; age range 18-98) were referred by general practitioners. The FOBT was only performed on three samples in all cases. In total, 172 of 2159 patients (7.9%) had an FOB performed prior to their referral, with 55 of 172 patients (31.9%) as part of the referral for 2WW. Sixteen of 172 patients (9.3%) had an FOB performed in the presence of overt rectal bleeding. In only 2 of 172 patients (1.1%) the FOB correlated with a colorectal cancer. Unnecessary testing for FOB costs has cost pound4072.96 in total., Discussion: A significant number of faecal occult bloods are being performed and the detection rate, even in this symptomatic group of patients, is very low. It is evident that the test is being performed in the community on three samples and not six. This, combined with the high false positive rate, leads to patients not only undergoing unnecessary psycho-social consequences but could potentially lead to significant risks from unnecessary invasive investigation as well as the added financial burden of test itself.

Introduction: Heterotopic bone is rarely found in the gastrointestinal tract. Here we report a rare case of metaplastic ossification within a benign ulcerating adenoma and review the literature concerning the aetiology., Case Presentation: A 63-year-old woman, who presented with a history of melaena, was found at colonoscopy to have a pedunculated ulcerating polyp. Histological examination demonstrated multiple areas of osseous metaplasia within the polyp stroma., Conclusion: Heterotopic ossification in colonic adenomas is a particularly rare phenomenon, with the majority of cases occurring within malignant lesions. The suggested mechanisms for its aetiology still remain unclear.

Objective: One of the 2-week wait (2WW) criteria for suspected lower gastrointestinal cancer states that patients should be referred who have iron deficiency anaemia (IDA) without obvious cause [Haemoglobin (Hb) <11 g/dl men, <10 g/dl postmenopausal women]., Aim: Our aim was to find the proportion of patients referred as a 2WW not meeting the criteria, and the cost accrued by unnecessary referral., Method: Patients referred over 1 year were identified using the hospitals cancer database. Haematology, haematinics, coeliac serology and cancer status were recorded for each patient., Results: A total of 204 patients were referred. In total, only 22/204 patients (10.8%) met all the necessary criteria for diagnosis and investigation of IDA prior to referral. As only 43/204 (21.1%) had been assessed for coeliac serology, this accounted for the majority of incomplete referrals. Excluding coeliac serology, only 127 (62.3%) met 2WW criteria for IDA. Of the remaining 77 patients, 57 (74%) patients did not meet the 2WW criteria on Hb alone and 35/77 were referred with no evidence of IDA. 12/127 (9.4%) patients were diagnosed with colorectal cancer. No cancers were detected in patients without BSG evidence of IDA, although one patient did not meet the criteria on Hb level alone., Conclusion: Although iron deficiency is a good marker for gastrointestinal cancer, it is evident that 2WW referral guidelines are not being followed. 89.2% of referrals are inappropriate according to guidelines. This not only has considerable workload and financial implications but could be potentially detrimental to patient health.

We report a case of a 79 year old man with a known benign anastomotic stricture presenting with large bowel obstruction. At laparotomy the obstruction was found to be caused by a large sesame seed bezoar. Seed bezoars are well known to cause impaction in the rectum but have never been previously reported to cause large bowel obstruction. We recommend that patients with known large bowel strictures should be advised not to eat seeds as this could ultimately lead to obstruction, ischaemia or perforation.

The thermosteric contribution of the North Atlantic Oscillation (NAO) to the North Sea sea-level for the winter period is investigated. Satellite sea surface temperature as well as in situ measurements are used to define the sensitivity of winter water temperature to the NAO as well as to determine the trends in temperature. The sea surface temperature sensitivity to the NAO is about 0.85 degrees C per unit NAO, which results in thermosteric sea-level changes of about 1-2 cm per unit NAO. The sensitivity of sea surface temperatures to the NAO is strongly time-dependent. Model data from a two-dimensional hydrodynamic tide+surge model are used in combination with the estimated thermosteric anomalies to explain the observed sea-level changes and, in particular, the sensitivity of the datasets to the NAO variability. The agreement between the model and the observed data is improved by the inclusion of the thermosteric effect.

Within the framework of a Tyndall Centre research project, sea level and wave changes around the UK and in the North Sea have been analysed. This paper integrates the results of this project. Many aspects of the contribution of the North Atlantic Oscillation (NAO) to sea level and wave height have been resolved. The NAO is a major forcing parameter for sea-level variability. Strong positive response to increasing NAO was observed in the shallow parts of the North Sea, while slightly negative response was found in the southwest part of the UK. The cause of the strong positive response is mainly the increased westerly winds. The NAO increase during the last decades has affected both the mean sea level and the extreme sea levels in the North Sea. The derived spatial distribution of the NAO-related variability of sea level allows the development of scenarios for future sea level and wave height in the region. Because the response of sea level to the NAO is found to be variable in time across all frequency bands, there is some inherent uncertainty in the use of the empirical relationships to develop scenarios of future sea level. Nevertheless, as it remains uncertain whether the multi-decadal NAO variability is related to climate change, the use of the empirical relationships in developing scenarios is justified. The resulting scenarios demonstrate: (i) that the use of regional estimates of sea level increase the projected range of sea-level change by 50% and (ii) that the contribution of the NAO to winter sea-level variability increases the range of uncertainty by a further 10-20cm. On the assumption that the general circulation models have some skill in simulating the future NAO change, then the NAO contribution to sea-level change around the UK is expected to be very small (<4cm) by 2080. Wave heights are also sensitive to the NAO changes, especially in the western coasts of the UK. Under the same scenarios for future NAO changes, the projected significant wave-height changes in the northeast Atlantic will exceed 0.4m. In addition, wave-direction changes of around 20 degrees per unit NAO index have been documented for one location. Such changes raise the possibility of consequential alteration of coastal erosion.

Background: Indigenous Australians are at high risk for cardiovascular disease and type 2 diabetes. Carotid artery intimal medial thickness (CIMT) and brachial artery flow-mediated vasodilation (FMD) are ultrasound imaging based surrogate markers of cardiovascular risk. This study examines the relative contributions of traditional cardiovascular risk factors on CIMT and FMD in adult Indigenous Australians with and without type 2 diabetes mellitus., Method: One hundred and nineteen Indigenous Australians were recruited. Physical and biochemical markers of cardiovascular risk, together with CIMT and FMD were measured for all subjects., Results: Fifty-three Indigenous Australians subjects (45%) had type 2 diabetes mellitus. There was a significantly greater mean CIMT in diabetic versus non-diabetic subjects (p=0.049). In the non-diabetic group with non-parametric analyses, there were significant correlations between CIMT and: age (r=0.64, p<0.001), systolic blood pressure (r=0.47, p<0.001) and non-smokers (r=-0.30, p=0.018). In the diabetic group, non-parametric analysis showed correlations between CIMT, age (r=0.36, p=0.009) and duration of diabetes (r=0.30, p=0.035) only. Adjusting for age, sex, smoking and history of cardiovascular disease, Hb(A1c) became the sole significant correlate of CIMT (r=0.35, p=0.01) in the diabetic group. In non-parametric analysis, age was the sole significant correlate of FMD (r=-0.31, p=0.013), and only in non-diabetic subjects. Linear regression analysis showed significant associations between CIMT and age (t=4.6, p<0.001), systolic blood pressure (t=2.6, p=0.010) and Hb(A1c) (t=2.6, p=0.012), smoking (t=2.1, p=0.04) and fasting LDL-cholesterol (t=2.1, p=0.04). There were no significant associations between FMD and examined cardiovascular risk factors with linear regression analysis, Conclusions: CIMT appears to be a useful surrogate marker of cardiovascular risk in this sample of Indigenous Australian subjects, correlating better than FMD with established cardiovascular risk factors. A lifestyle intervention programme may alleviate the burden of cardiovascular disease in Indigenous Australians by reducing central obesity, lowering blood pressure, correcting dyslipidaemia and improving glycaemic control. CIMT may prove to be a useful tool to assess efficacy of such an intervention programme.

Type II diabetes is a familial disorder, as evidenced by the increased prevalence in monozygotic cotwins and first-degree relatives of affected subjects; however, its genetic etiology is largely unknown. Well-characterized pedigrees are an essential resource for the study of susceptibility genes for type II diabetes. This study describes a 5-yr search for type II diabetic families in Oxfordshire, U.K. We interviewed 950 type II diabetic subjects concerning the availability of first-degree relatives; 127 Caucasian families ascertained through a proband with type II diabetes were studied, and 589 first-degree relatives were characterized. Three large pedigrees with maturity-onset diabetes of the young, and 8 multiplex multigenerational type II diabetic pedigrees were identified. We identified 12 sib-pairs in which both siblings had type II diabetes; however, only 7 sib-pairs had both parents alive, and 2 of these had both parents affected. If one also considers one sib having diabetes and one sib having glucose intolerance as being an affected sib-pair, we identified 30 sib-pairs of which 7 had both parents affected and probably had bilineal inheritance. We identified 76 complete nuclear families with both parents and offspring available for study, but only 6 were of optimal structure for linkage analysis. In conclusion, multiplex pedigrees and type II diabetic sib-pairs with living parents are uncommon, and their ascertainment requires a substantial investment of resources. Large-scale collaborative multicenter initiatives would be needed to collect a large resource of family material for the study of susceptibility genes for type II diabetes.