Birth mode is associated with earliest strain-conferred gut microbiome functions and immunostimulatory potential.

The rate of caesarean section delivery (CSD) is increasing worldwide. It remains unclear whether disruption of mother-to-neonate transmission of microbiota through CSD occurs and whether it affects human physiology. Here we perform metagenomic analysis of earliest gut microbial community structures and functions. We identify differences in encoded functions between microbiomes of vaginally delivered (VD) and CSD neonates. Several functional pathways are over-represented in VD neonates, including lipopolysaccharide (LPS) biosynthesis. We link these enriched functions to individual-specific strains, which are transmitted from mothers to neonates in case of VD. The stimulation of primary human immune cells with LPS isolated from early stool samples of VD neonates results in higher levels of tumour necrosis factor (TNF-α) and interleukin 18 (IL-18). Accordingly, the observed levels of TNF-α and IL-18 in neonatal blood plasma are higher after VD. Taken together, our results support that CSD disrupts mother-to-neonate transmission of specific microbial strains, linked functional repertoires and immune-stimulatory potential during a critical window for neonatal immune system priming.

Colonization and Succession within the Human Gut Microbiome by Archaea, Bacteria, and Microeukaryotes during the First Year of Life.

Perturbations to the colonization process of the human gastrointestinal tract have been suggested to result in adverse health effects later in life. Although much research has been performed on bacterial colonization and succession, much less is known about the other two domains of life, archaea, and eukaryotes. Here we describe colonization and succession by bacteria, archaea and microeukaryotes during the first year of life (samples collected around days 1, 3, 5, 28, 150, and 365) within the gastrointestinal tract of infants delivered either vaginally or by cesarean section and using a combination of quantitative real-time PCR as well as 16S and 18S rRNA gene amplicon sequencing. Sequences from organisms belonging to all three domains of life were detectable in all of the collected meconium samples. The microeukaryotic community composition fluctuated strongly over time and early diversification was delayed in infants receiving formula milk. Cesarean section-delivered (CSD) infants experienced a delay in colonization and succession, which was observed for all three domains of life. Shifts in prokaryotic succession in CSD infants compared to vaginally delivered (VD) infants were apparent as early as days 3 and 5, which were characterized by increased relative abundances of the genera Streptococcus and Staphylococcus, and a decrease in relative abundance for the genera Bifidobacterium and Bacteroides. Generally, a depletion in Bacteroidetes was detected as early as day 5 postpartum in CSD infants, causing a significantly increased Firmicutes/Bacteroidetes ratio between days 5 and 150 when compared to VD infants. Although the delivery mode appeared to have the strongest influence on differences between the infants, other factors such as a younger gestational age or maternal antibiotics intake likely contributed to the observed patterns as well. Our findings complement previous observations of a delay in colonization and succession of CSD infants, which affects not only bacteria but also archaea and microeukaryotes. This further highlights the need for resolving bacterial, archaeal, and microeukaryotic dynamics in future longitudinal studies of microbial colonization and succession within the neonatal gastrointestinal tract.

Integrated multi-omics of the human gut microbiome in a case study of familial type 1 diabetes.

The gastrointestinal microbiome is a complex ecosystem with functions that shape human health. Studying the relationship between taxonomic alterations and functional repercussions linked to disease remains challenging. Here, we present an integrative approach to resolve the taxonomic and functional attributes of gastrointestinal microbiota at the metagenomic, metatranscriptomic and metaproteomic levels. We apply our methods to samples from four families with multiple cases of type 1 diabetes mellitus (T1DM). Analysis of intra- and inter-individual variation demonstrates that family membership has a pronounced effect on the structural and functional composition of the gastrointestinal microbiome. In the context of T1DM, consistent taxonomic differences were absent across families, but certain human exocrine pancreatic proteins were found at lower levels. The associated microbial functional signatures were linked to metabolic traits in distinct taxa. The methodologies and results provide a foundation for future large-scale integrated multi-omic analyses of the gastrointestinal microbiome in the context of host-microbe interactions in human health and disease.

Identification, Recovery, and Refinement of Hitherto Undescribed Population-Level Genomes from the Human Gastrointestinal Tract.

Linking taxonomic identity and functional potential at the population-level is important for the study of mixed microbial communities and is greatly facilitated by the availability of microbial reference genomes. While the culture-independent recovery of population-level genomes from environmental samples using the binning of metagenomic data has expanded available reference genome catalogs, several microbial lineages remain underrepresented. Here, we present two reference-independent approaches for the identification, recovery, and refinement of hitherto undescribed population-level genomes. The first approach is aimed at genome recovery of varied taxa and involves multi-sample automated binning using CANOPY CLUSTERING complemented by visualization and human-augmented binning using VIZBIN post hoc. The second approach is particularly well-suited for the study of specific taxa and employs VIZBIN de novo. Using these approaches, we reconstructed a total of six population-level genomes of distinct and divergent representatives of the Alphaproteobacteria class, the Mollicutes class, the Clostridiales order, and the Melainabacteria class from human gastrointestinal tract-derived metagenomic data. Our results demonstrate that, while automated binning approaches provide great potential for large-scale studies of mixed microbial communities, these approaches should be complemented with informative visualizations because expert-driven inspection and refinements are critical for the recovery of high-quality population-level genomes.

Tools to overcome potential barriers to chlamydia screening in general practice: Qualitative evaluation of the implementation of a complex intervention.

BACKGROUND: Chlamydia trachomatis remains a significant public health problem. We used a complex intervention, with general practice staff, consisting of practice based workshops, posters, computer prompts and testing feedback and feedback to increase routine chlamydia screening tests in under 25 year olds in South West England. We aimed to evaluate how intervention components were received by staff and to understand what determined their implementation into ongoing practice. METHODS: We used face-to-face and telephone individual interviews with 29 general practice staff analysed thematically within a Normalisation Process Theory Framework which explores: 1. Coherence (if participants understand the purpose of the intervention); 2. Cognitive participation (engagement with and implementation of the intervention); 3. Collective action (work actually undertaken that drives the intervention forwards); 4. Reflexive monitoring (assessment of the impact of the intervention). RESULTS: Our results showed coherence as all staff including receptionists understood the purpose of the training was to make them aware of the value of chlamydia screening tests and how to increase this in their general practice. The training was described by nearly all staff as being of high quality and responsible for creating a shared understanding between staff of how to undertake routine chlamydia screening. Cognitive participation in many general practice staff teams was demonstrated through their engagement by meeting after the training to discuss implementation, which confirmed the role of each staff member and the use of materials. However several participants still felt unable to discuss chlamydia in many consultations or described sexual health as low priority among colleagues. National targets were considered so high for some general practice staff that they didn't engage with the screening intervention. Collective action work undertaken to drive the intervention included use of computer prompts which helped staff remember to make the offer, testing rate feedback and having a designated lead. Ensuring patients collected samples when still in the general practice was not attained in most general practices. Reflexive monitoring showed positive feedback from patients and other staff about the value of screening, and feedback about the general practices testing rates helped sustain activity. CONCLUSIONS: A complex intervention including interactive workshops, materials to help implementation and feedback can help chlamydia screening testing increase in general practices.

A qualitative study exploring the acceptability of the McNulty-Zelen design for randomised controlled trials evaluating educational interventions.

BACKGROUND: Traditional randomised controlled trials evaluating the effect of educational interventions in general practice may produce biased results as participants know they are being evaluated. We aimed to explore the acceptability of a McNulty-Zelen Cluster Randomised Control Trial (CRT) design which conceals from educational participants that they are in a RCT. Consent is obtained from a trusted third party considered appropriate to give consent on participants' behalf, intervention practice staff then choose whether to attend the offered education as would occur with normal continuing professional development. METHODS: We undertook semi structured telephone interviews in England with 16 general practice (GP) staff involved in a RCT evaluating an educational intervention aimed at increasing chlamydia screening tests in general practice using the McNulty-Zelen design, 4 Primary Care (PC) Research Network officers, 5 Primary Care Trust leads in Public or sexual health, and one Research Ethics committee Chair. Interviews were undertaken by members of the original intervention evaluation McNulty-Zelen design RCT study team. These experienced qualitative interviewers used an agreed semi-structured interview schedule and were careful not to lead the participants. To further mitigate against bias, the data analysis was undertaken by a researcher (CR) not involved in the original RCT. RESULTS: We reached data saturation and found five main themes; Support for the design: All found the McNulty-Zelen design acceptable because they considered that it generated more reliable evidence of the value of new educational interventions in real life GP settings. Lack of familiarity with study design: The design was novel to all. GP staff likened the evaluation using the McNulty-Zelen design to audit of their activities with feedback, which were to them a daily experience and therefore acceptable. Ethical considerations: Research stakeholders considered the consent procedure should be very clear and that these trial designs should go through at least a proportionate ethical review. GP staff were happy for the PCT leads to give consent on their behalf. GP research capacity and trial participation: GP staff considered the design increased generalisability, as staff who would not normally volunteer to participate in research due to perceived time constraints and paperwork might do so. Design 'worth it': All interviewees agreed that the advantages of the "more accurate" or "truer" results and information gained about uptake of workshops within Primary Care Trusts (PCTs) outweighed any disadvantages of the consent procedure. DISCUSSION: Our RCT was evaluating the effect of an educational intervention to increase chlamydia screening tests in general practices where there was routine monitoring of testing rates; our participants may have been less enthusiastic about the design if it had been evaluating a more controversial educational area, or if data monitoring was not routine. IMPLICATIONS: The McNulty-Zelen design should be considered for the evaluation of educational interventions, but these designs should have clear consent protocols and proportionate ethical review. TRIAL REGISTRATION: The trial was registered on the UK Clinical Research Network Study Portfolio database. UKCRN9722 .

Contact tracing for influenza A(H1N1)pdm09 virus-infected passenger on international flight.

In April 2009, influenza A(H1N1)pdm09 virus infection was confirmed in a person who had been symptomatic while traveling on a commercial flight from Mexico to the United Kingdom. Retrospective public health investigation and contact tracing led to the identification of 8 additional confirmed cases among passengers and community contacts of passengers.

International flight-related transmission of pandemic influenza A(H1N1)pdm09: an historical cohort study of the first identified cases in the United Kingdom.

BACKGROUND: Transporting over two billion passengers per year, global airline travel has the potential to spread emerging infectious diseases, both via transportation of infectious cases and through in-flight transmission. Current World Health Organization (WHO) guidance recommends contact tracing of passengers seated within two rows of a case of influenza during air travel. OBJECTIVES: The objectives of this study were to describe flight-related transmission of influenza A(H1N1)pdm09 during a commercial flight carrying the first cases reported in the United Kingdom and to test the specific hypothesis that passengers seated within two rows of an infectious case are at greater risk of infection. METHODS: An historical cohort study, supplemented by contact tracing, enhanced surveillance data and laboratory testing, was used to establish a case status for passengers on board the flight. RESULTS: Data were available for 239 of 278 (86·0%) of passengers on the flight, of whom six were considered infectious in-flight and one immune. The attack rate (AR) was 10 of 232 (4·3%; 95% CI 1·7-6·9%). There was no evidence that the AR for those seated within two rows of an infectious case was different from those who were not (relative risk 0·9; 95% CI 0·2-3·1; P = 1·00). Laboratory testing using PCR and/or serology, available for 118 of 239 (49·4%) of the passengers, was largely consistent with clinically defined case status. CONCLUSIONS: This study of A(H1N1)pdm09 does not support current WHO guidance regarding the contact tracing of passengers seated within two rows of an infectious case of influenza during air travel.

Increasing chlamydia screening tests in general practice: a modified Zelen prospective Cluster Randomised Controlled Trial evaluating a complex intervention based on the Theory of Planned Behaviour.

OBJECTIVE: To determine if a structured complex intervention increases opportunistic chlamydia screening testing of patients aged 15-24 years attending English general practitioner (GP) practices. METHODS: A prospective, Cluster Randomised Controlled Trial with a modified Zelen design involving 160 practices in South West England in 2010. The intervention was based on the Theory of Planned Behaviour (TPB). It comprised of practice-based education with up to two additional contacts to increase the importance of screening to GP staff and their confidence to offer tests through skill development (including videos). Practical resources (targets, posters, invitation cards, computer reminders, newsletters including feedback) aimed to actively influence social cognitions of staff, increasing their testing intention. RESULTS: Data from 76 intervention and 81 control practices were analysed. In intervention practices, chlamydia screening test rates were 2.43/100 15-24-year-olds registered preintervention, 4.34 during intervention and 3.46 postintervention; controls testing rates were 2.61/100 registered patients prior intervention, 3.0 during intervention and 2.82 postintervention. During the intervention period, testing in intervention practices was 1.76 times as great (CI 1.24 to 2.48) as controls; this persisted for 9 months postintervention (1.57 times as great, CI 1.27 to 2.30). Chlamydia infections detected increased in intervention practices from 2.1/1000 registered 15-24-year-olds prior intervention to 2.5 during the intervention compared with 2.0 and 2.3/1000 in controls (Estimated Rate Ratio intervention versus controls 1.4 (CI 1.01 to 1.93). CONCLUSIONS: This complex intervention doubled chlamydia screening tests in fully engaged practices. The modified Zelen design gave realistic measures of practice full engagement (63%) and efficacy of this educational intervention in general practice; it should be used more often. TRIAL REGISTRATION: The trial was registered on the UK Clinical Research Network Study Portfolio database. UKCRN number 9722.

Exploring attitudes and practices of General Practice staff towards offers of opportunistic screening for chlamydia: a UK survey.

AIM: This study applied a theory-based questionnaire to examine the behaviours and beliefs of all practice staff who may be involved in offering chlamydia screens to young people aged 15-25 years old. We aimed to identify potential influencing factors and examine the organisational constraints, which may be amenable to change. BACKGROUND: The National Chlamydia Screening Programme offers opportunistic screening to men and women between 15 and 25 years old who have ever had sexual intercourse and primary care is the second largest source of screens. In England 15.9% of the target group were screened against a target of 17% in 2008. Interventions to improve the frequency of offers have shown effects with volunteer practices. METHODS: A survey of 85 General Practices was completed by 55 doctors, nurses and receptionists. Interviews were conducted with 12 staff from three practices. FINDINGS: Respondents were unable to identify the national screening target. Only half record if a patient is sexually active. Half the sample had some recollection of the frequency of offers they made, with a mean of 4 per month. These were predominantly in consultations concerning sexual health. Perceived social norms are favourable to screening and respondents have strong perceived control over offering screens, including to those under 16 who are sexually active. Attitudes towards screening were positive but disadvantages and barriers related to increased pressure on practice resources for longer consultations and contact tracing. There were no differences in beliefs and practice behaviours between medical and nursing staff. CONCLUSIONS: Future interventions should be targeted at increasing the range of consultations in which offers are made, demonstrating how to make offers without increasing consultation time, providing more complete records of sexual activity, screens and results, and encouraging audit and review within the practice to promote practice wide approaches to increasing opportunistic screening.

Have the public's expectations for antibiotics for acute uncomplicated respiratory tract infections changed since the H1N1 influenza pandemic? A qualitative interview and quantitative questionnaire study.

OBJECTIVE: To investigate the effect of the H1N1 influenza pandemic on the public's expectations for a general practice consultation and antibiotic for acute respiratory illness. DESIGN: Mixed methods. PARTICIPANTS: Qualitative interviews: 17 participants with acute respiratory tract infection (RTI) visiting English pharmacies. Face-to-face survey: about 1700 adults aged 15 years and older were recruited from households in England in January 2008, 2009 and 2011. RESULTS: The qualitative data indicated that the general public had either forgotten about the 'swine flu' (H1N1 influenza) pandemic or it did not concern them as it had not affected them directly or affected their management of their current RTI illness. Between 2009 and 2011, we found that there was little or no change in people's expectations for antibiotics for runny nose, colds, sore throat or cough, but people's expectations for antibiotics for flu increased (26%-32%, p=0.004). Of the 1000 respondents in 2011 with an RTI in the previous 6 months, 13% reported that they took care of themselves without contacting their general practitioners and would not have done so before the pandemic, 9% reported that they had contacted their doctor's surgery and would not have done so before the pandemic and 0.6% stated that they had asked for antibiotics and would not have done so before the pandemic. In 2011, of 123 respondents with a young child (0-4 years) having an RTI in the previous 6 months, 7.4% requested antibiotics and would not have done so before the pandemic. Unprompted, 20% of respondents thought Tamiflu© (oseltamivir) was a vaccine. CONCLUSIONS: Expectations of the general public for a consultation or antibiotics with an RTI are similar now to before the H1N1 influenza pandemic; therefore, public antibiotic campaign messages and general practice advice to patients can remain unchanged. Parents with young children and those with personal experience of the H1N1 influenza are more likely to consult and will need more reassurance. The public need more education about Tamiflu©.

"...they should be offering it": a qualitative study to investigate young peoples' attitudes towards chlamydia screening in GP surgeries.

BACKGROUND: Despite the known health and healthcare costs of untreated chlamydia infection and the efforts of the National Chlamydia Screening Programme (NCSP) to control chlamydia through early detection and treatment of asymptomatic infection, the rates of screening are well below the 2010-2011 target rate of 35%. General Practitioner (GP) surgeries are a key venue within the NCSP however; previous studies indicate that GP surgery staff are concerned that they may offend their patients by offering a screen. This study aimed to identify the attitudes to, and preferences for, chlamydia screening in 15-24 year old men and women attending GP surgeries (the target group). METHODS: We undertook 36 interviews in six surgeries of differing screening rates. Our participants were 15-24 year olds attending a consultation with a staff member. Data were analysed thematically. RESULTS: GP surgeries are acceptable to young people as a venue for opportunistic chlamydia screening and furthermore they think it is the duty of GP surgery staff to offer it. They felt strongly that it is important for surgery staff to have a non-judgmental attitude and they did not want to be singled out as 'needing' a chlamydia screen. Furthermore, our sample reported a strong preference for being offered a screen by staff and providing the sample immediately at the surgery rather than taking home a testing kit. The positive attitude and subjective norms demonstrated by interviewees suggest that young peoples' behaviour would be to accept a screen if it was offered to them. CONCLUSION: Young people attending GP surgeries have a positive attitude towards chlamydia screening and given the right environment are likely to take up the offer in this setting. The right environment involves normalising screening by offering a chlamydia screen to all 15-24 year olds at every interaction with staff, offering screening with a non-judgmental attitude and minimising barriers to screening such as embarrassment. The GP surgery is the ideal place to screen young people for chlamydia as it is not a threatening place for them and our study has shown that they think it is the normal place to go to discuss health matters.

Overcoming the barriers to chlamydia screening in general practice--a qualitative study.

BACKGROUND: There is low uptake of chlamydia screening in general practices registered with the English National Chlamydia Screening Programme (NCSP). Aims. To explore staff's attitudes and behaviour around chlamydia screening and how screening could be optimized in general practice. METHODS: A qualitative study with focus groups and interviews, in general practices in seven NCSP areas. Twenty-five focus groups and 12 interviews undertaken with a purposively selected diverse group of high and low chlamydia-screening practices in 2006-08. Data were collected and analysed using a framework analytical approach. RESULTS: Higher screening practices had more staff with greater belief in patient and population benefits of screening and, as screening was a subjective norm, it was part of every day practice. Many staff in the majority of other practices were uncomfortable raising chlamydia opportunistically and time pressures meant that any extra public health issues covered within a consultation were determined by Quality Outcomes Framework (QOF) targets. All practices would value more training and feedback about their screening rates and results. Practices suggested that use of computer prompts, simplified request forms and more accessible kits could increase screening. CONCLUSION: Practice staff need more evidence of the value of opportunistic chlamydia screening in men and women; staff development to reduce the barriers to broaching sexual health; simpler request forms and easily accessible kits to increase their ability to offer it within the time pressures of general practice. Increased awareness of chlamydia could be attained through practice meetings, computer templates and reminders, targets and incentives or QOF points with feedback.

Diagnosis of Streptococcus pneumoniae infections in adults with bacteremia and community-acquired pneumonia: clinical comparison of pneumococcal PCR and urinary antigen detection.

The diagnosis of severe Streptococcus pneumoniae infection relies heavily on insensitive culture techniques. To improve the usefulness of PCR assays, we developed a dual-PCR protocol (targeted at pneumolysin and autolysin) for EDTA blood samples. This was compared to the Binax NOW S. pneumoniae urine antigen test in patients with bacteremic pneumococcal infections. Patients with nonbacteremic community-acquired pneumonia also were tested by these methods to determine what proportion could be confirmed as pneumococcal infections. A direct comparison was made in a group of patients who each had both tests performed. The Binax NOW S. pneumoniae urine antigen test was positive in 51 of 58 bacteremic pneumococcal cases (sensitivity, 88%; 95% confidence interval [CI], 77 to 95%), whereas the dual PCR was positive in 31 cases (sensitivity, 53.5%; 95% CI, 40 to 67%; P < 0.0001), and all of these had detectable urinary antigens. Both tests gave positive results in 2 of 51 control patients (referred to as other-organism septicemia), giving a specificity of 96% (95% CI, 86.5 to 99.5%). In 77 patients with nonbacteremic community-acquired pneumonia, urinary antigen was detected significantly more often (in 21 patients [27%]) than a positive result by the dual-PCR protocol (6 [8%]) (P = 0.002). The development of a dual-PCR protocol enhanced the sensitivity compared to that of the individual assays, but it is still significantly less sensitive than the Binax NOW urine antigen test, as well as being more time-consuming and expensive. Urinary antigen detection is the nonculture diagnostic method of choice for patients with possible severe pneumococcal infection.

The phosphoinositol 3,4-bisphosphate-binding protein TAPP1 interacts with syntrophins and regulates actin cytoskeletal organization.

Syntrophins are scaffold proteins of the dystrophin glycoprotein complex (DGC), which target ion channels, receptors, and signaling proteins to specialized subcellular domains. A yeast two-hybrid screen of a human brain cDNA library with the PSD-95, Discs-large, ZO-1 (PDZ) domain of gamma1-syntrophin yielded overlapping clones encoding the C terminus of TAPP1, a pleckstrin homology (PH) domain-containing adapter protein that interacts specifically with phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)). In biochemical assays, the C terminus of TAPP1 bound specifically to the PDZ domains of gamma1-, alpha1-, and beta2-syntrophin and was required for syntrophin binding and for the correct subcellular localization of TAPP1. TAPP1 is recruited to the plasma membrane of cells stimulated with platelet-derived growth factor (PDGF), a motogen that produces PI(3,4)P(2). Cell migration in response to PDGF stimulation is characterized by a rapid reorganization of the actin cytoskeleton, which gives rise to plasma membrane specializations including peripheral and dorsal circular ruffles. Both TAPP1 and syntrophins were localized to PDGF-induced circular membrane ruffles in NIH-3T3 cells. Ectopic expression of TAPP1 potently blocked PDGF-induced formation of dorsal circular ruffles, but did not affect peripheral ruffling. Interestingly, coexpression of alpha1- or gamma1-syntrophin with TAPP1 prevented the blockade of circular ruffling. In addition to syntrophins, several other proteins of the DGC were enriched in circular ruffles. Collectively, our results suggest syntrophins regulate the localization of TAPP1, which may be important for remodeling the actin cytoskeleton in response to growth factor stimulation.