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While firefighters currently have low smoking rates, rates of smokeless tobacco (SLT) use among this population are remarkably high and substantially greater than similar occupational groups, and the general population. This study explored determinants associated with SLT use, barriers to cessation, and motivators for SLT cessation in the fire service. Key informant interviews were conducted in 23 career firefighters who were current (n = 14) and former (n = 9) SLT users from across the U.S. Discussions were recorded and independently coded according to questions and themes. Major themes that developed among firefighters regarding SLT use determinants included positive perceptions of SLT products, social influences from their peers and family members, acceptability of SLT use in the fire service, and a coping resource for job stress. Firefighters discussed several barriers to SLT cessation, including intrapersonal barriers such as SLT use habits and its dependency, concerns about withdrawal symptoms; and social-environmental barriers including lack of support from health and other services providers, and lack of enforcement of existing tobacco policies regarding SLT use. Firefighters also mentioned both internal and external motivators for cessation. Internal motivators included self-motivation and their health concerns while external motivators included friends and family support, incentives or rewards, and price of SLT products. Findings provide unique perspectives from firefighters on factors that influence SLT use and barriers and motivators to SLT cessation. These are insufficiently assessed and considered by the fire service organizations and their health care providers. Thus, the organizations must understand these issues in order to mitigate barriers and motivate the personnel to quit using SLT. Information gained from firefighters who were current and former SLT users can be used to develop an effective, culturally-tailored intervention that is acceptable to fire service personnel.

The CRISPR/Cas9 system is an RNA guided nuclease system that evolved as a mechanism of adaptive immunity in bacteria. This system has been adopted for numerous genome engineering applications in research and recently, therapeutics. The CRISPR/Cas9 system has been largely implemented by delivery of Cas9 as protein, RNA, or plasmid along with a chimeric crRNA-tracrRNA guide RNA (gRNA) under the expression of a pol III promoter, such as U6. Using this approach, multiplex genome engineering has been achieved by delivering several U6-gRNA plasmids targeting multiple loci. However, this approach is limited due to the efficiently of delivering multiple plasmids to a single cell at one time. To augment the capability and accessibility of multiplexed genome engineering, we developed an efficient golden gate based method to assemble gRNAs linked by optimal Csy4 ribonuclease sequences to deliver up to 10 gRNAs as a single gRNA array transcript. Here we report the optimal expression of our guide RNA array under a strong pol II promoter. This system can be implemented alongside the myriad of CRISPR applications, allowing users to model complex biological processes requiring numerous gRNAs.

The mammalian prion protein (PrP, encoded by Prnp) is most infamous for its central role in prion diseases, invariably fatal neurodegenerative diseases affecting humans, food animals, and animals in the wild. However, PrP is also hypothesized to be an important receptor for toxic protein conformers in Alzheimer's disease, and is associated with other clinically relevant processes such as cancer and stroke. Thus, key insights into important clinical areas, as well as into understanding PrP functions in normal physiology, can be obtained from studying transgenic mouse models and cell culture systems. However, the Prnp locus is difficult to manipulate by homologous recombination, making modifications of the endogenous locus rarely attempted. Fortunately in recent years genome engineering technologies, like TALENs or CRISPR/Cas9 (CC9), have brought exceptional new possibilities for manipulating Prnp. Herein, we present our observations made during systematic experiments with the CC9 system targeting the endogenous mouse Prnp locus, to either modify sequences or to boost PrP expression using CC9-based synergistic activation mediators (SAMs). It is our hope that this information will aid and encourage researchers to implement gene-targeting techniques into their research program.

Dispositional anxiety is a stable personality trait that is a key risk factor for internalizing disorders, and understanding the neural correlates of trait anxiety may help us better understand the development of these disorders. Abnormal cortical folding is thought to reflect differences in cortical connectivity occurring during brain development. Therefore, assessing gyrification may advance understanding of cortical development and organization associated with trait anxiety. Previous literature has revealed structural abnormalities in trait anxiety and related disorders, but no study to our knowledge has examined gyrification in trait anxiety. We utilized a relatively novel measure, the local gyrification index (LGI), to explore differences in gyrification as a function of trait anxiety. We obtained structural MRI scans using a 3T magnetic resonance scanner on 113 young adults. Results indicated a negative correlation between trait anxiety and LGI in the left superior parietal cortex, specifically the precuneus, reflecting less cortical complexity among those high on trait anxiety. Our findings suggest that aberrations in cortical gyrification in a key region of the default mode network is a correlate of trait anxiety and may reflect disrupted local parietal connectivity.

Prion diseases induce neurodegeneration in specific brain areas for undetermined reasons. A thorough understanding of the localization of the disease-causing molecule, the prion protein (PrP), could inform on this issue but previous studies have generated conflicting conclusions. One of the more intriguing disagreements is whether PrP is synthesized by astrocytes. We developed a knock-in reporter mouse line in which the coding sequence of the PrP expressing gene (Prnp), was replaced with that for green fluorescent protein (GFP). Native GFP fluorescence intensity varied between and within brain regions. GFP was present in astrocytes but did not increase during reactive gliosis induced by scrapie prion infection. Therefore, reactive gliosis associated with prion diseases does not cause an acceleration of local PrP production. In addition to aiding in Prnp gene activity studies, this reporter mouse line will likely prove useful for analysis of chimeric animals produced by stem cell and tissue transplantation experiments.

Background: Indigenous youth in settler nations are susceptible to poor mental health due to complex intergenerational systemic inequities. Research has shown benefits of cultural connectedness for improving mental health; however, there are few studies which have evaluated the impact of culturally relevant mental health interventions, particularly among Indigenous youth. The purpose of this study is to assess the impact of a culturally-responsive, land-based, active living initiative on the mental health of Indigenous youth., Methods: This quasi-experimental qualitative study is part of Smart Indigenous Youth (SIY), a mixed-methods 5-year longitudinal digital citizen science initiative. SIY embeds culturally responsive, land-based active living programs into the curricula of high schools in rural Indigenous communities in the western Canadian province of Saskatchewan. In year-1 (Winter 2019), 76 Indigenous youth citizen scientists (13-18 years) from 2 schools participated in the study. At the beginning of the term, each school initiated separate 4-month land-based active living programs specific to their culture, community, geography, and language (Cree and Saulteaux). Before and after the term, focus groups were conducted with the 2 Youth Citizen Scientist Councils, which included students from both participating schools. This study includes data from focus groups of one participating school, with 11 youth citizen scientists (5 boys, 6 girls). Focus group data were transcribed and analyzed by two independent reviewers using Nvivo to identify themes and subthemes. Both reviewers discussed their thematic analysis to reach consensus about final findings., Results: Baseline focus group analyses (before land-based programming) revealed themes demonstrating the importance of Indigenous culture, identity, history, and language. Youth emphasized the impact of loss of language and culture, the importance of being a helper, and the necessity of intergenerational knowledge transfer. Follow-up focus group analyses (post land-based programming) indicated that cultural school programming led to students expressing positive mental health benefits, increased interest in ceremonies, increased participation in physical activity, and greater knowledge of culture, identity, and ceremonial protocol., Conclusions: This novel qualitative quasi-experimental study offers a window into the future of upstream interventions in partnership with Indigenous communities, where Indigenous youth can be engaged in real-time via their digital devices, while participating in culturally-sensitive, land-based school programming that promotes culture, identity, and mental health., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Walker et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

OptiBreech collaborative care is a multi-disciplinary care pathway for breech presentation at term, with continuity from a breech specialist midwife, including where chosen, for vaginal breech birth (VBB). Pilot randomised trial using unblinded 1:1 parallel group allocation to OptiBreech versus standard care, within a cohort. Participants were women with a breech-presenting fetus > 33 weeks, at four sites in England, January-June 2022. A two-stage consent process was used. Participants consented to undergo random selection to be offered a 'new care process', with a choice to accept it, or not. Primary objectives were to identify recruitment, acceptance, and attrition rates. Randomisation procedures and potential primary outcomes for a substantive study were also feasibility-tested. 68 women were randomised between January-June 2022. The consent process was acceptable to participants, but randomisation was unacceptable to women who specifically sought OptiBreech care. Two women withdrew due to concerns about sharing personal information. More women planned a VBB when randomised to OptiBreech Care (23.5% vs 0, p = .002, 95% CI = 9.3%,37.8%). Women randomised to OptiBreech care had: lower rates of cephalic presentation at birth (38.2% vs 54.5%), higher rates of vaginal birth (32.4% vs 24.2%), lower rates of in-labour caesarean birth (20.6% vs 36.4%), lower rates of neonatal intensive care (5.9% vs 9.1%), and lower rates of severe neonatal morbidity (2.9% vs 9.1%). Randomisation was stopped on the advice of the steering committee before the planned sample of 104, as lack of access to VBB within standard care prohibited comparison of outcomes. Demand for VBB is sufficient for a cohort study, but comparison of outcomes by 1:1 randomisation is not feasible. OptiBreech care would be best evaluated using stepped wedge cluster randomisation. Funded by the United Kingdom National Institute for Health and Care Research (NIHR300582). Clinical trial registration: ISRCTN 14521381., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: SW and ES are co-Directors of Breech Birth Network, Community Interest Company, a not-for-profit social enterprise that delivers VBB training and supports research. They and other members of the OptiBreech Collaborative have received teaching fees and expenses for providing breech training. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2023 Walker et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Current agricultural production depends on very limited species grown as monocultures that are highly vulnerable to climate change, presenting a threat to the sustainability of agri-food systems. However, many hundreds of neglected crop species have the potential to cater to the challenges of climate change by means of resilience to adverse climate conditions. Proso millet (Panicum miliaceum L.), one of the underutilised minor millets grown as a rainfed subsistence crop, was selected in this study as an exemplary climate-resilient crop. Using a previously calibrated version of the Agricultural Production Systems Simulator (APSIM), the sensitivity of the crop to changes in temperature and precipitation was studied using the protocol of the Coordinated Climate Crop Modelling Project (C3MP). The future (2040-2069) production was simulated using bias-corrected climate data from 20 general circulation models of the Coupled Model Intercomparison Project (CMIP5) under RCP4.5 and 8.5 scenarios. According to the C3MP analysis, we found a 1°C increment of temperature decreased the yield by 5-10% at zero rainfall change. However, Proso millet yields increased by 5% within a restricted climate change space of up to 2°C of warming with increased rainfall. Simulated future climate yields were lower than the simulated yields under the baseline climate of the 1980-2009 period (mean 1707 kg ha-1) under both RCP4.5 (-7.3%) and RCP8.5 (-16.6%) though these changes were not significantly (p > 0.05) different from the baseline yields. Proso millet is currently cultivated in limited areas of Sri Lanka, but our yield mapping shows the potential for expansion of the crop to new areas under both current and future climates. The results of the study, indicating minor impacts from projected climate change, reveal that Proso millet is an excellent candidate for low-input farming systems under changing climate. More generally, through this study, a framework that can be used to assess the climate sensitivity of underutilized crops was also developed., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Wimalasiri et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Diffuse malignant peritoneal mesothelioma (DMPM) is a rare variant of malignant mesothelioma, representing 10-15% of malignant mesothelioma cases. The preferred therapeutic approach is cytoreductive surgery (CRS) accompanied by hyperthermic intraperitoneal chemotherapy (HIPEC); the role of systemic chemotherapy is not well established. While some limited retrospective studies report worse outcomes with neoadjuvant chemotherapy, our institution has favored the use of neoadjuvant chemotherapy for symptom relief and surgical optimization. The aim of our study was to assess the outcomes of patients receiving neoadjuvant chemotherapy, compared to those receiving adjuvant or no perioperative chemotherapy., Patients and Methods: We conducted a single-center retrospective cohort study of treatment-naïve, non-papillary DMPM patients seen at our institution between 1/1/2009 and 9/1/2019. We explored the effect of type of systemic therapy on clinical outcomes and estimated median overall survival (mOS) using Kaplan-Meier curves. Hazard ratios (HR) calculated by Cox proportional hazard model were used to estimate effect of the exposures on overall survival., Results: 47 patients were identified with DMPM (median age at diagnosis 61.2 years, 76.6% epithelioid histology, 74.5% white race, 55.3% known asbestos exposure). CRS was performed in 53.2% of patients (25/47); 76.0% of surgical patients received HIPEC (19/25). The majority received systemic chemotherapy (37/47, 78.7%); among patients receiving both CRS and chemotherapy, neoadjuvant chemotherapy was more common than adjuvant chemotherapy (12 neoadjuvant, 8 adjuvant). Overall mOS was 84.1 months. Among neoadjuvant patients, 10/12 underwent surgery, and 2 were lost to follow-up; the majority (9/10) had clinically stable or improved disease during the pre-operative period. There were numerical more issues with chemotherapy with the adjuvant patients (4/8: 2 switches in platinum agent, 2 patients stopped therapy) than with the neoadjuvant patients (2/10: 1 switch in platinum agent, 1 delay due to peri-procedural symptoms). Neoadjuvant chemotherapy was not associated with worse mOS compared to adjuvant chemotherapy (mOS NR vs 95.1 mo, HR 0.89, 95% CI 0.18-4.5, p = 0.89)., Conclusions: When used preferentially, the use of neoadjuvant chemotherapy in DMPM patients was not associated with worse outcomes compared to adjuvant chemotherapy. It was well-tolerated and did not prevent surgical intervention., Competing Interests: Melina E. Marmarelis reports researching funding from Eli Lilly (Inst), Trizell (Inst), AstraZeneca (Inst); consulting role with Novocure, Boehringer Ingelheim; stock in Gilead Sciences, Portola Pharmaceuticals, Merck, Bluebird Bio, Johnson & Johnson, Pfizer; and previous medical writing support from Novartis. Christine A. Ciunci reports research funding from Celgene (Inst), Merck (Inst), Bristol-Myers Squibb (Inst), MacroGenics (Inst); and honoraria from Imedex. Corey J. Langer reports research funding from Merck (Inst), Advantagene (Inst), Clovis Oncology (Inst), Celgene (Inst), Inovio Pharmaceuticals (Inst), ARIAD (Inst), GlaxoSmithKline (Inst), Genentech/Roche (Inst), Stem CentRx (Inst), Lilly, Trizell (Inst); honoraria from Bristol-Myers Squibb, Genentech/Roche, Lilly/ImClone, AstraZeneca, Takeda Science Foundation; consulting roles with Genentech/Roche, Lilly/ImClone, Merck, Abbott Biotherapeutics, Bayer/Onyx, Clarient, Clovis Oncology, Celgene, Cancer Support Community, Bristol-Myers Squibb, ARIAD, Takeda, AstraZeneca, Pfizer, Novocure, Gilead Sciences; and other relationships with Lilly, Amgen, Peregrine Pharmaceuticals, Synta. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Evidence for parenting programs to improve wellbeing in children under three is inconclusive. We investigated the fidelity, impact, and cost-effectiveness of two parenting programs delivered within a longitudinal proportionate delivery model ('E-SEE Steps')., Methods: Eligible parents with a child ≤ 8 weeks were recruited into a parallel two-arm, assessor blinded, randomized controlled, community-based, trial with embedded economic and process evaluations. Post-baseline randomization applied a 5:1 (intervention-to-control) ratio, stratified by primary (child social-emotional wellbeing (ASQ:SE-2)) and key secondary (maternal depression (PHQ-9)) outcome scores, sex, and site. All intervention parents received the Incredible Years® Baby Book (IY-B), and were offered the targeted Infant (IY-I)/Toddler (IY-T) program if eligible, based on ASQ:SE-2/PHQ-9 scores. Control families received usual services. Fidelity data were analysed descriptively. Primary analysis applied intention to treat. Effectiveness analysis fitted a marginal model to outcome scores. Cost-effectiveness analysis involved Incremental Cost-Effectiveness Ratios (ICERs)., Results: The target sample (N = 606) was not achieved; 341 mothers were randomized (285:56), 322 (94%) were retained to study end. Of those eligible for the IY-I (n = 101), and IY-T (n = 101) programs, 51 and 21 respectively, attended. Eight (of 14) groups met the 80% self-reported fidelity criteria. No significant differences between arms were found for adjusted mean difference scores; ASQ:SE-2 (3.02, 95% CI: -0.03, 6.08, p = 0.052), PHQ-9 (-0.61; 95% CI: -1.34, 0.12, p = 0.1). E-SEE Steps had higher costs, but improved mothers' Health-related Quality of Life (0.031 Quality Adjusted Life Year (QALY) gain), ICER of £20,062 per QALY compared to control. Serious adverse events (n = 86) were unrelated to the intervention., Conclusions: E-SEE Steps was not effective, but was borderline cost-effective. The model was delivered with varying fidelity, with lower-than-expected IY-T uptake. Changes to delivery systems and the individual programs may be needed prior to future evaluation., Trial Registration: International Standard Randomized Controlled Trial Number: ISRCTN11079129., Competing Interests: All authors were supported by the grant, National Institute for Health Research (NIHR) Public Health Research (PHR) (ref 13/93/10). TB, SB, KP and GR were also supported by the NIHR Yorkshire and Humber Applied Research Collaboration (ARC-YH; Ref: NIHR200166, see https://www.arc-yh.nihr.ac.uk,); VB by the South West Peninsula ARC (PenARC, Ref: NIHR200167, see https://arc-swp.nihr.ac.uk). Public Health England supported research sites’ excess treatment costs (no award number). This above funding does not alter our adherence to PLOS ONE policies on sharing data and materials. In addition, TB is a voluntary Trustee for Children’s Early Intervention Trust (CEIT). Early Intervention Wales Training (EIWT) is owned by CEIT and offers training courses, including Incredible Years® (IY). Trustees do not benefit financially from trainings or CEIT/EIWT activities. ESEE study trainings were arranged via the IY developer in the U.S., not via EIWT.

Objective: To assess the feasibility of a problem-solving skills training intervention in improving psychological outcomes in mothers of infants with sickle cell disease (SCD)., Design and Methods: This parallel randomized controlled trial recruited 64 babies with SCD, 6 to 12 months of age, and their mothers. Baseline measurements assessed mothers' coping and problem-solving skills, depression, and parental stress before random assignment to intervention or control groups (n = 32 each). Problem-solving skills intervention was delivered through 6 monthly sessions, when babies attended for routine penicillin prophylaxis. All measurements were repeated for both groups at the end of the intervention period. Intention to treat analysis used repeated measures mixed models with the restricted estimation maximum likelihood approach., Results: The problem-solving intervention had no significant effect on mothers' problem-solving skills (adjusted treatment effect: -1.69 points (95% CI:-5.62 to 2.25)), coping behaviours (adjusted treatment effect: 0.65 points (95% CI:- -7.13 to 8.41)) or depressive symptoms (adjusted treatment effect: -0.41 (95% CI: -6.00 to 5.19)). It reduced mothers' level of difficulty in managing stressful events by 9.5 points (95% CI (-16.86 to -2.16); effect size: 0.21 SD). In the subgroup of mothers at risk of depression (n = 31 at baseline), the intervention reduced depression scores with treatment effect of 10.4 points (95%CI: -18.83 to -1.88; effect size: 0.67 SD)., Conclusion: This problem-solving skills intervention study suggests feasibility and possible efficacy in improving some maternal outcomes. Further refinement and culturally appropriate adaptations of the intervention could lead to stronger effects., Competing Interests: The authors have declared that no competing interests exist.

Power declines at a greater rate during ageing and is more relevant for functional deterioration than either loss of maximum strength or muscle mass. Human movement typically consists of stretch-shortening cycle action. Therefore, plyometric exercises, using an eccentric phase quickly followed by a concentric phase to optimize power production, should resemble daily function more than traditional resistance training, which primarily builds force production capacity in general. However, it is unclear whether older adults can sustain such high-impact training. This study compared the effects of plyometric exercise (PLYO) on power, force production, jump and functional performance to traditional resistance training (RT) and walking (WALK) in older men. Importantly, feasibility was investigated. Forty men (69.5 ± 3.9 years) were randomized to 12-weeks of PLYO (N = 14), RT (N = 12) or WALK (N = 14). Leg press one-repetition maximum (1-RM), leg-extensor isometric maximum voluntary contraction (MVC) and rate of force development (RFD), jump and functional performance were evaluated pre- and post-intervention. One subject in RT (low back pain) and three in PLYO (2 muscle strains, 1 knee pain) dropped out. Adherence to (91.2 ± 4.4%) and acceptability of (≥ 7/10) PLYO was high. 1-RM improved more in RT (25.0 ± 10.0%) and PLYO (23.0 ± 13.6%) than in WALK (2.9 ± 13.7%) (p < 0.001). PLYO improved more on jump height, jump power, contraction time of jumps and stair climbing performance compared to WALK and/or RT (p < 0.05). MVC improved in RT only (p = 0.028) and RFD did not improve (p > 0.05). To conclude, PLYO is beneficial over RT for improving power, jump and stair climbing performance without compromising gains in strength. This form of training seems feasible, but contains an inherent higher risk for injuries, which should be taken into account when designing programs for older adults., Competing Interests: The authors have declared that no competing interests exist.

Objectives: To estimate the adult health burden and costs in California during 2013 associated with adults' prior Adverse Childhood Experiences (ACEs)., Methods: We analyzed five ACEs-linked conditions (asthma, arthritis, COPD, depression, and cardiovascular disease) and three health risk factors (lifetime smoking, heavy drinking, and obesity). We estimated ACEs-associated fractions of disease risk for people aged 18+ for these conditions by ACEs exposure using inputs from a companion study of California Behavioral Risk Factor Surveillance System data for 2008-2009, 2011, and 2013. We combined these estimates with published estimates of personal healthcare spending and Disability-Adjusted-Life-Years (DALYs) in the United States by condition during 2013. DALYs captured both the years of healthy life lost to disability and the years of life lost to deaths during 2013. We applied a published estimate of cost per DALY., Results: Among adults in California, 61% reported ACEs. Those ACEs were associated with $10.5 billion in excess personal healthcare spending during 2013, and 434,000 DALYs valued at approximately $102 billion dollars. During 2013, the estimated health burden per exposed adult included $589 in personal healthcare expenses and 0.0224 DALYs valued at $5,769., Conclusions: Estimates of the costs of childhood adversity are far greater than previously understood and provide a fiscal rationale for prevention efforts., Competing Interests: The authors have declared that no competing interests exist.

Liver cancer is a poor prognosis cancer with limited treatment options. To develop a new therapeutic approach, we derived HCC cells from a known model of murine hepatocellular carcinoma (HCC). We treated adiponectin (APN) knock-out mice with the carcinogen diethylnitrosamine, and the resulting tumors were 7-fold larger than wild-type controls. Tumors were disassociated from both genotypes and their growth characteristics evaluated. A52 cells from APN KO mice had the most robust growth in vitro and in vivo, and presented with pathology similar to the parental tumor. All primary tumors and cell lines exhibited activity of the mammalian target of Rapamycin (mTOR) and Src pathways. Subsequent combinatorial treatment, with the mTOR inhibitor Rapamycin and the Src inhibitor Dasatinib reduced A52 HCC growth 29-fold in vivo. Through protein and histological analyzes we observed activation of these pathways in human HCC, suggesting that targeting both mTOR and Src may be a novel approach for the treatment of HCC., Competing Interests: The authors have declared that no competing interests exist.

Background: The WHO HIV Treatment Guidelines suggest routine viral-load monitoring can be used to differentiate antiretroviral therapy (ART) delivery and reduce the frequency of clinic visits for patients stable on ART. This recommendation was informed by economic analysis that showed the approach is very likely to be cost-effective, even in the most resource constrained of settings. The health benefits were shown to be modest but the costs of introducing and scaling up viral load monitoring can be offset by anticipated reductions in the costs of clinic visits, due to these being less frequent for many patients., Key Issues for Economic Evaluation: The cost-effectiveness of introducing viral-load informed differentiated care depends upon whether cost reductions are possible if the number of clinic visits is reduced and/or how freed clinic capacity is used for alternative priorities. Where freed resources, either physical or financial, generate large health gains (e.g. if committed to patients failing ART or to other high value health care interventions), the benefits of differentiated care are expected to be high; if however these freed physical resources are already under-utilized or financial resources are used less efficiently and would not be put to as beneficial an alternative use, the policy may not be cost-effective. The implication is that the use of conventional unit costs to value resources may not well reflect the latter's value in contributing to health improvement. Analyses intended to inform resource allocated decisions in a number of settings may therefore have to be interpreted with due consideration to local context. In this paper we present methods of how economic analyses can reflect the real value of health care resources rather than simply applying their unit costs. The analyses informing the WHO Guidelines are re-estimated by implementing scenarios using this framework, informing how differentiated care can be prioritized to generate greatest gains in population health., Implications: The findings have important implications for how economic analyses should be undertaken and reported in HIV and other disease areas. Results provide guidance on conditions under which viral load informed differentiated care will more likely prove to be cost effective when implemented.

Background: The pharynx is a common site of gonorrhoea among men who have sex with men (MSM) and may serve as a reservoir for infection, with saliva implicated in transmission possibly through oral sex, kissing, and rimming. Reducing sexual activities involving saliva may reduce pharyngeal gonorrhoea. This study aimed to explore MSM's views and knowledge of pharyngeal gonorrhoea and their willingness to change saliva transmitting sexual practices. MSM were also asked their views on using alcohol-containing mouthwash to potentially reduce transmission., Methods: Using a qualitative descriptive approach, 30 MSM who were part of a larger study (GONE) conducted at the Melbourne Sexual Health Centre agreed to take part in semi-structured interviews between 14th May and 8th September 2015. The 10 interviews conducted face to face and 20 by telephone, lasted between 20-45 minutes. Data were analysed using qualitative content analysis., Results: Most men considered pharyngeal gonorrhoea to be a non-serious sexually transmitted infection and attributed transmission primarily to oral sex. Almost all men reported they would not stop kissing, oral sex, or consider using condoms for oral sex to reduce their risk of pharyngeal gonorrhoea. Kissing and oral sex were commonly practised and considered enjoyable low risk sexual activities. Men were more likely to consider stopping sexual activities they did not enjoy or practice often, in particular insertive rimming. If proven effective, the majority of men reported they would use alcohol-containing mouthwash to reduce or prevent their risk of pharyngeal gonorrhoea., Conclusion: Findings from this study suggest MSM are unlikely to stop saliva transmitting sexual practices they enjoy and consider low risk. Men would, however, consider using alcohol-containing mouthwash if found to be effective, highlighting the importance of exploring innovative strategies to reduce pharyngeal gonorrhoea., Competing Interests: The authors have declared that no competing interests exist.

Internalin A is an essential virulence gene involved in the uptake of the foodborne pathogen Listeria monocytogenes into host cells. It is intact in clinical strains and often truncated due to Premature Stop Codons (PMSCs) in isolates from processed foods and processing facilities. Less information is known about environmental isolates. We sequenced the inlA alleles and did Multi Locus Variable Number Tandem Repeat Analysis (MLVA) on 112 L. monocytogenes isolates from a 3-year period from naturally contaminated watersheds near a leafy green growing area in Central California. The collection contained 14 serotype 1/2a, 12 serotype 1/2b, and 86 serotype 4b strains. Twenty-seven different inlA alleles were found. Twenty-three of the alleles are predicted to encode intact copies of InlA, while three contain PMSCs. Another allele has a 9-nucleotide deletion, previously described for a clinical strain, indicating that it is still functional. Intact inlA genes were found in 101 isolates, and 8 isolates contained the allele predicted to contain the 3-amino acid deletion. Both allele types were found throughout the 3-year sampling period. Three strains contained inlA alleles with PMSCs, and these were found only during the first 3 months of the study. SNP analysis of the intact alleles indicated clustering of alleles based on serotype and lineage with serotypes 1/2b and 4b (lineage I strains) clustering together, and serotype 1/2a (lineage II strains) clustering separately. The combination of serotype, MLVA types, and inlA allele types indicate that the 112 isolates reflect at least 49 different strains of L. monocytogenes. The finding that 90% of environmental L. monocytogenes isolates contain intact inlA alleles varies significantly from isolates found in processing plants. This information is important to public health labs and growers as to the varieties of L. monocytogenes that could potentially contaminate fresh produce in the field by various means., Competing Interests: The authors have declared that no competing interests exist.

Avian influenza viruses of H5 subtype can cause highly pathogenic disease in poultry. In March 2014, a new reassortant H5N6 subtype highly pathogenic avian influenza virus emerged in Lao People's Democratic Republic. We have assessed the pathogenicity, pathobiology and immunological responses associated with this virus in chickens. Infection caused moderate to advanced disease in 6 of 6 chickens within 48 h of mucosal inoculation. High virus titers were observed in blood and tissues (kidney, spleen, liver, duodenum, heart, brain and lung) taken at euthanasia. Viral antigen was detected in endothelium, neurons, myocardium, lymphoid tissues and other cell types. Pro-inflammatory cytokines were elevated compared to non-infected birds. Our study confirmed that this new H5N6 reassortant is highly pathogenic, causing disease in chickens similar to that of Asian H5N1 viruses, and demonstrated the ability of such clade 2.3.4-origin H5 viruses to reassort with non-N1 subtype viruses while maintaining a fit and infectious phenotype. Recent detection of influenza H5N6 poultry infections in Lao PDR, China and Viet Nam, as well as six fatal human infections in China, demonstrate that these emergent highly pathogenic H5N6 viruses may be widely established in several countries and represent an emerging threat to poultry and human populations., Competing Interests: The authors have declared that no competing interests exist.

Mycoplasma genitalium is a cause of non-gonoccocal urethritis (NGU) in men and cervicitis and pelvic inflammatory disease in women. Recent international data also indicated that the first line treatment, 1 gram stat azithromycin therapy, for M. genitalium is becoming less effective, with the corresponding emergence of macrolide resistant strains. Increasing failure rates of azithromycin for M. genitalium has significant implications for the presumptive treatment of NGU and international clinical treatment guidelines. Assays able to predict macrolide resistance along with detection of M. genitalium will be useful to enable appropriate selection of antimicrobials to which the organism is susceptible and facilitate high levels of rapid cure. One such assay recently developed is the MG 23S assay, which employs novel PlexZyme™ and PlexPrime™ technology. It is a multiplex assay for detection of M. genitalium and 5 mutations associated with macrolide resistance. The assay was evaluated in 400 samples from 254 (186 males and 68 females) consecutively infected participants, undergoing tests of cure. Using the MG 23S assay, 83% (331/440) of samples were positive, with 56% of positives carrying a macrolide resistance mutation. Comparison of the MG 23S assay to a reference qPCR method for M. genitalium detection and high resolution melt analysis (HRMA) and sequencing for detection of macrolide resistance mutations, resulted in a sensitivity and specificity for M. genitalium detection and for macrolide resistance of 99.1/98.5% and 97.4/100%, respectively. The MG 23S assay provides a considerable advantage in clinical settings through combined diagnosis and detection of macrolide resistance.

Background: Few data are available on how women manage recurring bacterial vaginosis (BV) and their experiences of the clinical care of this condition. This study aimed to explore women's recurrent BV management approaches and clinical care experiences, with a view to informing and improving the clinical management of BV., Methods: A descriptive, social constructionist approach was chosen as the framework for the study. Thirty-five women of varying sexual orientation who had experienced recurrent BV in the past 5 years took part in semi-structured interviews., Results: The majority of women reported frustration and dissatisfaction with current treatment regimens and low levels of satisfaction with the clinical management of BV. Overall, women disliked taking antibiotics regularly, commonly experienced adverse side effects from treatment and felt frustrated at having symptoms recur quite quickly after treatment. Issues in clinical care included inconsistency in advice, misdiagnosis and inappropriate diagnostic approaches and insensitive or dismissive attitudes. Women were more inclined to report positive clinical experiences with sexual health physicians than primary care providers. Women's frustrations led most to try their own self-help remedies and lifestyle modifications in an attempt to treat symptoms and prevent recurrences, including well-known risk practices such as douching., Conclusion: In the face of considerable uncertainty about the cause of BV, high rates of recurrence, unacceptable treatment options and often insensitive and inconsistent clinical management, women are trying their own self-help remedies and lifestyle modifications to prevent recurrences, often with little effect. Clinical management of BV could be improved through the use of standardised diagnostic approaches, increased sensitivity and understanding of the impact of BV, and the provision of evidence based advice about known BV related risk factors.

Background: Bacterial vaginosis (BV) is the most common vaginal infection affecting women of childbearing age. While the aetiology and transmissibility of BV remain unclear, there is strong evidence to suggest an association between BV and sexual activity. This study explored women's views and experiences of the triggers for BV onset and factors associated with recurrence., Methods: A descriptive, social constructionist approach was chosen as the framework for the study. Thirty five women of varying sexual orientation who had experienced recurrent BV in the past five years took part in semi-structured interviews., Results: The majority of women predominantly reported sexual contact triggered the onset of BV and sexual and non-sexual factors precipitated recurrence. Recurrence was most commonly referred to in terms of a 'flare-up' of symptoms. The majority of women did not think BV was a sexually transmitted infection however many reported being informed this by their clinician. Single women who attributed BV onset to sex with casual partners were most likely to display self-blame tendencies and to consider changing their future sexual behaviour. Women who have sex with women (WSW) were more inclined to believe their partner was responsible for the transmission of or reinfection with BV and seek partner treatment or change their sexual practices., Conclusion: Findings from this study strongly suggest women believe that BV onset is associated with sexual activity, concurring with epidemiological data which increasingly suggest BV may be sexually transmitted. Exacerbating factors associated with recurrence were largely heterogeneous and may reflect the fact it is difficult to determine whether recurrence is due to persistent BV or a new infection in women. There was however evidence to suggest possible transmission and reinfection among WSW, reinforcing the need for new approaches to treatment and management strategies including male and female partner treatment trials.

Background and Aims: In human adults, and animals, the Apelin-APJ ligand-receptor system is emerging as having a role in the pathogenesis of cardiovascular function and heart failure. The aim was to investigate expression, and regulation by oxygen, of the Apelin APJ receptor (APJ) in myocardium obtained from children undergoing corrective surgery with cardiopulmonary bypass for repair of congenital heart defects., Methods: Western blotting and Real-time PCR were used to determine if APJ was expressed in the infant myocardium, if expression was influenced by the duration of myocardial ischemia and if any relationship existed between APJ expression and early post-operative outcome. The next aim was to determine if there was a difference in mRNA expression of APJ in myocardium from cyanotic patients compared with acyanotic patients and if re-perfusing myocardium in vitro with either hypoxic, normoxic or hyperoxic oxygen affected APJ mRNA expression., Results: APJ was expressed in all myocardial samples and myocardium exposed to longer durations of ischemia and cardioplegia expressed higher levels of APJ (p<0.05). There was a significant correlation between APJ expression in myocardium resected after 10 min with both oxygen extraction ratio (p=0.021, rho= -0.523) and mixed venous oxygen saturation (p=0.028, rho 0.52). This association did not exist for myocardium collected before 10 min. There was no difference in APJ expression between cyanotic and acyanotic patients. No difference was found in APJ expression whether re-perfused with low, normal or high oxygen., Conclusions: Changes in APJ expression were observed during cardiopulmonary bypass in children and the reasons for this require further investigation.

Background: Human papillomavirus (HPV) is a causative agent in oropharyngeal squamous cell carcinoma. The natural history of oral HPV in HIV-positive men who have sex with men (MSM) is unclear., Methods: Detection of oral human papillomavirus in 173 HIV-positive MSM using oral rinse samples 3 years apart was investigated. HPV DNA was detected by polymerase chain reaction, and genotyped by Roche Linear Array., Results: Of 173 men tested in 2010, 30 had at least one HPV genotype (17%, 95% CI: 12-23), 15 at least one hr-HPV (9%, 95% CI: 5-14) and 8 had HPV 16 (5%, 95% CI: 2-9) detected. In 2013, 33 had at least one HPV genotype (19%, 95% CI: 14-26), 20 had at least one hr-HPV (12%, 95% CI: 7-17) and 7 had HPV 16 (4%, 95% CI: 2-8) detected. Of 30 men at baseline (2010) with any HPV detected, 14 (47%, 95% CI: 28-66) had at least one persistent genotype. Of the 15 men in 2010 with high risk (hr-) HPV, 6 men (40%, 95% CI: 16-68) had at least one persistent hr-HPV genotype. The incidence rate of detection of at least one new HPV genotype was 4.8 per 100 person years (95% CI: 3.1-7.0), of at least one hr-HPV genotype was 3.2 per 100 person years (95% CI: 1.8-5.1) and of HPV 16 was 0.8 per 100 person years (95% CI: 0.2-2.0). The clearance rate was 14.9 per 100 person years (95% CI: 8.2-24.2) for any HPV, 18.2 per 100 person years (95% CI: 8.2-32.7) for hr-HPV and 17.4 per 100 person years (95% CI: 5.0-38.8) for HPV-16. Persistent HPV detection was associated with duration of HIV (OR 1.13 (per additional year), 95% CI: 1.00-1.26) and tonsillectomy (OR 8.17, 95% CI: 1.30-51.40)., Conclusion: The same oral HPV genotype was detected again after 3 years in nearly half of HIV-positive men who have sex with men.

Two enrichment methods for Listeria monocytogenes using Immuno Magnetic Separation (IMS) were tested to determine if they selected the same subtypes of isolates. Both methods used a non-selective primary enrichment and one included subculture in Fraser Broth, while the other involved direct plating of IMS beads. Sixty-two naturally contaminated watershed samples from the Central California Coast were used as a source of L. monocytogenes, and subtype diversity was measured by serotype and Multiple Number Variable Tandem Repeat Analysis (MLVA). Three different serotypes were detected from both methods with serotype 4b strains making up 87% of the isolates, serotype 1/2a making up 8%, and serotype 1/2b making up 5%. The data suggest that serotype 1/2a strains were more likely to be isolated from the Fraser Broth culture method. Sixty-two different MLVA types were detected and the more common MLVA types were detected by both culture methods. Forty-three MLVA types were detected only from one culture method or the other, while 19 types were detected from both culture methods. The most common MLVA type-12 was detected in 33 of the 62 water samples, and represented 31% of the isolates from both culture methods. This limited study provides evidence that using both enrichment culture methods allowed for detection of a greater diversity of isolates among the samples than the use of one method alone, and that a wide diversity of L. monocytogenes strains exist in this watershed.

During B cell activation, the DNA lesions that initiate somatic hypermutation and class switch recombination are introduced by activation-induced cytidine deaminase (AID). AID is a highly mutagenic protein that is maintained in the cytoplasm at steady state, however AID is shuttled across the nuclear membrane and the protein transiently present in the nucleus appears sufficient for targeted alteration of immunoglobulin loci. AID has been implicated in epigenetic reprogramming in primordial germ cells and cell fusions and in induced pluripotent stem cells (iPS cells), however AID expression in non-B cells is very low. We hypothesised that epigenetic reprogramming would require a pathway that instigates prolonged nuclear residence of AID. Here we show that AID is completely re-localised to the nucleus during drug withdrawal following etoposide treatment, in the period in which double strand breaks (DSBs) are repaired. Re-localisation occurs 2-6 hours after etoposide treatment, and AID remains in the nucleus for 10 or more hours, during which time cells remain live and motile. Re-localisation is cell-cycle dependent and is only observed in G2. Analysis of DSB dynamics shows that AID is re-localised in response to etoposide treatment, however re-localisation occurs substantially after DSB formation and the levels of re-localisation do not correlate with γH2AX levels. We conclude that DSB formation initiates a slow-acting pathway which allows stable long-term nuclear localisation of AID, and that such a pathway may enable AID-induced DNA demethylation during epigenetic reprogramming.

The reconstitution of anti-viral cellular immunity following hematopoietic stem cell transplantation (HSCT) is crucial in preventing cytomegalovirus (CMV)-associated complications. Thus immunological monitoring has emerged as an important tool to better target pre-emptive anti-viral therapies. However, traditional laboratory-based assays are too cumbersome and complicated to implement in a clinical setting. Here we conducted a prospective study of a new whole blood assay (referred to as QuantiFERON-CMV®) to determine the clinical utility of measuring CMV-specific CD8+ T-cell responses as a prognostic tool. Forty-one evaluable allogeneic HSCT recipients underwent weekly immunological monitoring from day 21 post-transplant and of these 21 (51.2%) showed CMV reactivation and 29 (70.7%) developed acute graft-versus-host disease (GvHD). Patients with acute GvHD (grade ≥ 2) within 6 weeks of transplant showed delayed reconstitution of CMV-specific T-cell immunity (p = 0.013) and a higher risk of CMV viremia (p = 0.026). The median time to stable CMV-specific immune reconstitution was 59 days and the incidence of CMV reactivation was lower in patients who developed this than those who did not (27% versus 65%; p = 0.031). Furthermore, a failure to reconstitute CMV-specific immunity soon after the onset of CMV viraemia was associated with higher peak viral loads (5685 copies/ml versus 875 copies/ml; p = 0.002). Hence, QuantiFERON-CMV® testing in the week following CMV viremia can be useful in identifying HSCT recipients at risk of complicated reactivation.

Background: Bacterial vaginosis is a common vaginal infection, causing an abnormal vaginal discharge and/or odour in up to 50% of sufferers. Recurrence is common following recommended treatment. There are limited data on women's experience of bacterial vaginosis, and the impact on their self-esteem, sexual relationships and quality of life. The aim of this study was to explore the experiences and impact of recurrent bacterial vaginosis on women., Methods: A social constructionist approach was chosen as the framework for the study. Thirty five women with male and/or female partners participated in semi-structured interviews face-to-face or by telephone about their experience of recurrent bacterial vaginosis., Results: Recurrent bacterial vaginosis impacted on women to varying degrees, with some women reporting it had little impact on their lives but most reporting it had a moderate to severe impact. The degree to which it impacted on women physically, emotionally, sexually and socially often depended on the frequency of episodes and severity of symptoms. Women commonly reported that symptoms of bacterial vaginosis made them feel embarrassed, ashamed, 'dirty' and very concerned others may detect their malodour and abnormal discharge. The biggest impact of recurrent bacterial vaginosis was on women's self-esteem and sex lives, with women regularly avoiding sexual activity, in particular oral sex, as they were too embarrassed and self-conscious of their symptoms to engage in these activities. Women often felt confused about why they were experiencing recurrent bacterial vaginosis and frustrated at their lack of control over recurrence., Conclusion: Women's experience of recurrent bacterial vaginosis varied broadly and significantly in this study. Some women reported little impact on their lives but most reported a moderate to severe impact, mainly on their self-esteem and sex life. Further support and acknowledgement of these impacts are required when managing women with recurrent bacterial vaginosis.

Objective: To examine the association between hormonal contraception (HC) and bacterial vaginosis (BV) by systematic review and meta-analysis., Methods: Medline, Web of Science and Embase databases were searched to 24/1/13 and duplicate references removed. Inclusion criteria 1) >20 BV cases; 2) accepted BV diagnostic method; 3) measure of HC-use either as combined oestrogen-progesterone HC (combined), progesterone-only contraception (POC) or unspecified HC (u-HC); 4) ≥ 10% of women using HC; 5) analysis of the association between BV and HC-use presented; 6) appropriate control group. Data extracted included: type of HC, BV diagnostic method and outcome (prevalent, incident, recurrent), and geographical and clinic-setting. Meta-analyses were conducted to calculate pooled effect sizes (ES), stratified by HC-type and BV outcome. This systematic review is registered with PROSPERO (CRD42013003699)., Results: Of 1713 unique references identified, 502 full-text articles were assessed for eligibility and 55 studies met inclusion criteria. Hormonal contraceptive use was associated with a significant reduction in the odds of prevalent BV (pooled effect size by random-effects [reES] = 0.68, 95%CI0.63-0.73), and in the relative risk (RR) of incident (reES = 0.82, 95%CI:0.72-0.92), and recurrent (reES = 0.69, 95%CI:0.59-0.91) BV. When stratified by HC-type, combined-HC and POC were both associated with decreased prevalence of BV and risk of incident BV. In the pooled analysis of the effect of HC-use on the composite outcome of prevalent/incident/recurrent BV, HC-use was associated with a reduced risk of any BV (reES = 0.78, 95%CI:0.74-0.82)., Conclusion: HC-use was associated with a significantly reduced risk of BV. This negative association was robust and present regardless of HC-type and evident across all three BV outcome measures. When stratified by HC-type, combined-HC and POC were both individually associated with a reduction in the prevalence and incidence of BV. This meta-analysis provides compelling evidence that HC-use influences a woman's risk of BV, with important implications for clinicians and researchers in the field.

During a 2.5-year survey of 33 farms and ranches in a major leafy greens production region in California, 13,650 produce, soil, livestock, wildlife, and water samples were tested for Shiga toxin (stx)-producing Escherichia coli (STEC). Overall, 357 and 1,912 samples were positive for E. coli O157:H7 (2.6%) or non-O157 STEC (14.0%), respectively. Isolates differentiated by O-typing ELISA and multilocus variable number tandem repeat analysis (MLVA) resulted in 697 O157:H7 and 3,256 non-O157 STEC isolates saved for further analysis. Cattle (7.1%), feral swine (4.7%), sediment (4.4%), and water (3.3%) samples were positive for E. coli O157:H7; 7/32 birds, 2/145 coyotes, 3/88 samples from elk also were positive. Non-O157 STEC were at approximately 5-fold higher incidence compared to O157 STEC: cattle (37.9%), feral swine (21.4%), birds (2.4%), small mammals (3.5%), deer or elk (8.3%), water (14.0%), sediment (12.3%), produce (0.3%) and soil adjacent to produce (0.6%). stx1, stx2 and stx1/stx2 genes were detected in 63%, 74% and 35% of STEC isolates, respectively. Subtilase, intimin and hemolysin genes were present in 28%, 25% and 79% of non-O157 STEC, respectively; 23% were of the "Top 6″ O-types. The initial method was modified twice during the study revealing evidence of culture bias based on differences in virulence and O-antigen profiles. MLVA typing revealed a diverse collection of O157 and non-O157 STEC strains isolated from multiple locations and sources and O157 STEC strains matching outbreak strains. These results emphasize the importance of multiple approaches for isolation of non-O157 STEC, that livestock and wildlife are common sources of potentially virulent STEC, and evidence of STEC persistence and movement in a leafy greens production environment.

Background: To determine prevalence and incidence of bacterial vaginosis (BV) and risk factors in young sexually-active Australian women., Methods: 1093 women aged 16-25 years were recruited from primary-care clinics. Participants completed 3-monthly questionnaires and self-collected vaginal smears 6-monthly for 12-months. The primary endpoint was a Nugent Score = 7-10 (BV) and the secondary endpoint was a NS = 4-10 (abnormal flora [AF]). BV and AF prevalence estimates and 95% confidence intervals (95%CI) were derived, and adjusted odds ratios (AOR) calculated to explore epidemiological associations with prevalent BV and AF. Proportional-hazards regression models were used to examine factors associated with incident BV and AF., Results: At baseline 129 women had BV [11.8% (95%CI: 9.4-14.2)] and 188 AF (17.2%; 15.1-19.5). Prevalent BV was associated with having a recent female partner [AOR = 2.1; 1.0-4.4] and lack of tertiary-education [AOR = 1.9; 1.2-3.0]; use of an oestrogen-containing contraceptive (OCC) was associated with reduced risk [AOR = 0.6; 0.4-0.9]. Prevalent AF was associated with the same factors, and additionally with >5 male partners (MSP) in 12-months [AOR = 1.8; 1.2-2.5)], and detection of C.trachomatis or M.genitalium [AOR = 2.1; 1.0-4.5]. There were 82 cases of incident BV (9.4%;7.7-11.7/100 person-years) and 129 with incident AF (14.8%; 12.5-17.6/100 person-years). Incident BV and AF were associated with a new MSP [adjusted rate ratio (ARR) = 1.5; 1.1-2.2 and ARR = 1.5; 1.1-2.0], respectively. OCC-use was associated with reduced risk of incident AF [ARR = 0.7; 0.5-1.0]., Conclusion: This paper presents BV and AF prevalence and incidence estimates from a large prospective cohort of young Australian women predominantly recruited from primary-care clinics. These data support the concept that sexual activity is strongly associated with the development of BV and AF and that use of an OCC is associated with reduced risk.

Objective: Differences between women and men in political and economic empowerment, education, and health risks are well-documented. Similar gender inequities in access to care and medicines have been hypothesized but evidence is lacking., Methods: We analyzed 2002 World Health Survey data for 257,922 adult respondents and 80,932 children less than 5 years old from 53 mostly low and middle-income countries. We constructed indicators of need for, access to, and perceptions of care, and we described the number of countries with equal and statistically different proportions of women and men for each indicator. Using multivariate logistic regression models, we estimated effects of gender on our study outcomes, overall and by household poverty., Findings: Women reported significantly more need for care for three of six chronic conditions surveyed, and they were more likely to have at least one of the conditions (OR 1.41 [95% CI 1.38, 1.44]). Among those with reported need for care, there were no consistent differences in access to care between women and men overall (e.g., treatment for all reported chronic conditions, OR 1.00 [0.96, 1.04]) or by household poverty. Of concern, access to care for chronic conditions was distressingly low among both men and women in many countries, as was access to preventive services among boys and girls less than 5 years old., Conclusions: These cross-country results do not suggest a systematic disadvantage of women in access to curative care and medicines for treating selected chronic conditions or acute symptoms, or to preventive services among boys and girls.

Functional magnetic resonance imaging (fMRI) is a powerful method for exploring emotional and cognitive brain responses in humans. However rodent fMRI has not previously been applied to the analysis of learned behaviour in awake animals, limiting its use as a translational tool. Here we have developed a novel paradigm for studying brain activation in awake rats responding to conditioned stimuli using fMRI. Using this method we show activation of the amygdala and related fear circuitry in response to a fear-conditioned stimulus and demonstrate that the magnitude of fear circuitry activation is increased following early life stress, a rodent model of affective disorders. This technique provides a new translatable method for testing environmental, genetic and pharmacological manipulations on emotional and cognitive processes in awake rodent models.

Background: To determine if oral metronidazole (MTZ-400 mg bid) with 2% vaginal clindamycin-cream (Clind) or a Lactobacillus acidophilus vaginal-probiotic containing oestriol (Prob) reduces 6-month bacterial vaginosis (BV) recurrence., Methods: Double-blind placebo-controlled parallel-group single-site study with balanced randomization (1:1:1) conducted at Melbourne Sexual Health Centre, Australia. Participants with symptomatic BV [Nugent Score (NS) = 7-10 or ≥3 Amsel's criteria and NS = 4-10], were randomly allocated to MTZ-Clind, MTZ-Prob or MTZ-Placebo and assessed at 1,2,3 and 6 months. MTZ and Clind were administered for 7 days and Prob and Placebo for 12 days. Primary outcome was BV recurrence (NS of 7-10) on self-collected vaginal-swabs over 6-months. Cumulative BV recurrence rates were compared between groups by Chi-squared statistics. Kaplan-Meier, log rank and Cox regression analyses were used to compare time until and risk of BV recurrence between groups., Results: 450 18-50 year old females were randomized and 408 (91%), equally distributed between groups, provided ≥1 NS post-randomization and were included in analyses; 42 (9%) participants with no post-randomization data were excluded. Six-month retention rates were 78% (n = 351). One-month BV recurrence (NS 7-10) rates were 3.6% (5/140), 6.8% (9/133) and 9.6% (13/135) in the MTZ-Clind, MTZ-Prob and MTZ-Placebo groups respectively, p = 0.13. Hazard ratios (HR) for BV recurrence at one-month, adjusted for adherence to vaginal therapy, were 0.43 (95%CI 0.15-1.22) and 0.75 (95% CI 0.32-1.76) in the MTZ-Clind and MTZ-Prob groups compared to MTZ-Plac respectively. Cumulative 6-month BV recurrence was 28.2%; (95%CI 24.0-32.7%) with no difference between groups, p = 0.82; HRs for 6-month BV recurrence for MTZ-Clind and MTZ-Prob compared to MTZ-Plac, adjusted for adherence to vaginal therapy were 1.09(95% CI = 0.70-1.70) and 1.03(95% CI = 0.65-1.63), respectively. No serious adverse events occurred., Conclusion: Combining the recommended first line therapies of oral metronidazole and vaginal clindamycin, or oral metronidazole with an extended-course of a commercially available vaginal-L.acidophilus probiotic, does not reduce BV recurrence., Trial Registration: ANZCTR.org.au ACTRN12607000350426.

Background: This study aimed to estimate rates of chlamydia incidence and re-infection and to investigate the dynamics of chlamydia organism load in prevalent, incident and re-infections among young Australian women., Methods: 1,116 women aged 16 to 25 years were recruited from primary care clinics in Australia. Vaginal swabs were collected at 3 to 6 month intervals for chlamydia testing. Chlamydia organism load was measured by quantitative PCR., Results: There were 47 incident cases of chlamydia diagnosed and 1,056.34 person years of follow up with a rate of 4.4 per 100 person years (95% CI: 3.3, 5.9). Incident infection was associated with being aged 16 to 20 years [RR = 3.7 (95%CI: 1.9, 7.1)], being employed [RR = 2.4 (95%CI: 1.1, 4.9)] and having two or more new sex partners [RR = 5.5 (95%CI: 2.6, 11.7)]. Recent antibiotic use was associated with a reduced incidence [RR:0.1 (95%CI: 0.0, 0.5)]. There were 14 re-infections with a rate of 22.3 per 100 person years (95%CI: 13.2, 37.6). The median time to re-infection was 4.6 months. Organism load was higher for prevalent than incident infections (p<0.01) and for prevalent than re-infections (p<0.01)., Conclusions: Chlamydia is common among young women and a high proportion of women are re-infected within a short period of time, highlighting the need for effective partner treatment and repeat testing. The difference in organism load between prevalent and incident infections suggests prevalent infection may be more important for ongoing transmission of chlamydia.

HIV rapid diagnostic tests (RDTs) are now used widely in non-laboratory settings by non-laboratory-trained operators. Quality assurance programmes are essential in ensuring the quality of HIV RDT outcomes. However, there is no cost-effective means of supplying the many operators of RDTs with suitable quality assurance schemes. Therefore, it was examined whether photograph-based RDT results could be used and correctly interpreted in the non-laboratory setting. Further it was investigated if a single training session improved the interpretation skills of RDT operators. The photographs were interpreted, a 10-minute tutorial given and then a second interpretation session was held. It was established that the results could be read with accuracy. The participants (n=75) with a range of skills interpreted results (>80% concordance with reference results) from a panel of 10 samples (three negative and seven positive) using four RDTs. Differences in accuracy of interpretation before and after the tutorial were marked in some cases. Training was more effective for improving the accurate interpretation of more complex results, e.g. results with faint test lines or for multiple test lines, and especially for improving interpretation skills of inexperienced participants. It was demonstrated that interpretation of RDTs was improved using photographed results allied to a 10-minute training session. It is anticipated that this method could be used for training but also for quality assessment of RDT operators without access to conventional quality assurance or training schemes requiring wet samples.

Mutations in the LGI1 gene predispose to a hereditary epilepsy syndrome and is the first gene associated with this disease which does not encode an ion channel protein. In zebrafish, there are two paralogs of the LGI1 gene, lgi1a and lgi1b. Knockdown of lgi1a results in a seizure-like hyperactivity phenotype with associated developmental abnormalities characterized by cellular loss in the eyes and brain. We have now generated knockdown morphants for the lgi1b gene which also show developmental abnormalities but do not show a seizure-like behavior. Instead, the most striking phenotype involves significant enlargement of the ventricles (hydrocephalus). As shown for the lgi1a morphants, however, lgi1b morphants are also sensitized to PTZ-induced hyperactivity. The different phenotypes between the two lgi1 morphants support a subfunctionalization model for the two paralogs.

Astrocytes express a wide range of receptors for neurotransmitters and hormones that are coupled to increases in intracellular Ca(2+) concentration, enabling them to detect activity in both neuronal and vascular networks. There is increasing evidence that astrocytes are able to discriminate between different Ca(2+)-linked stimuli, as the efficiency of some Ca(2+) dependent processes--notably release of gliotransmitters--depends on the stimulus that initiates the Ca(2+) signal. The spatiotemporal complexity of Ca(2+) signals is substantial, and we here tested the hypothesis that variation in the kinetics of Ca(2+) responses could offer a means of selectively engaging downstream targets, if agonists exhibited a "signature shape" in evoked Ca(2+) response. To test this, astrocytes were exposed to three different receptor agonists (ATP, glutamate and histamine) and the resultant Ca(2+) signals were analysed for systematic differences in kinetics that depended on the initiating stimulus. We found substantial heterogeneity between cells in the time course of Ca(2+) responses, but the variation did not correlate with the type or concentration of the stimulus. Using a simple metric to quantify the extent of difference between populations, it was found that the variation between agonists was insufficient to allow signal discrimination. We conclude that the time course of global intracellular Ca(2+) signals does not offer the cells a means for distinguishing between different neurotransmitters.

Amniotic fluid stem (AFS) cells are broadly multipotent, can be expanded extensively in culture, are not tumorigenic and can be readily cryopreserved for cell banking. Mesenchymal stem cells (MSC) show immunomodulatory activity and secrete a wide spectrum of cytokines and chemokines that suppress inflammatory responses, block mixed lymphocyte reactions (MLR) and other immune reactions, and have proven therapeutic against conditions such as graft-versus-host disease. AFS cells resemble MSCs in many respects including surface marker expression and differentiation potential. We therefore hypothesized that AFS cells may exhibit similar immunomodulatory capabilities. We present data to demonstrate that direct contact with AFS cells inhibits lymphocyte activation. In addition, we show that cell-free supernatants derived from AFS cells primed with total blood monocytes or IL-1β, a cytokine released by monocytes and essential in mediation of the inflammatory response, also inhibited lymphocyte activation. Further investigation of AFS cell-free supernatants by protein array revealed secretion of multiple factors in common with MSCs that are known to be involved in immune regulation including growth related oncogene (GRO) and monocyte chemotactic protein (MCP) family members as well as interleukin-6 (IL-6). AFS cells activated by PBMCs released several additional cytokines as compared to BM-MSCs, including macrophage inflammatory protein-3α (MIP-3α), MIP-1α and Activin. AFS cells also released higher levels of MCP-1 and lower levels of MCP-2 compared to BM-MSCs in response to IL-1β activation. This suggests that there may be some AFS-specific mechanisms of inhibition of lymphocyte activation. Our results indicate that AFS cells are able to suppress inflammatory responses in vitro and that soluble factors are an essential component in the communication between lymphocytes and AFS cells. Their extensive self-renewal capacity, possibility for banking and absence of tumorigenicity may make AFS cells a superior source of stable, well characterized "off the shelf" immunomodulatory cells for a variety of immunotherapies.

Background: Despite a substantial burden of non-bacteraemic methicillin resistant Staphylococcus aureus (MRSA) disease, most MRSA surveillance schemes are based on bacteraemias. Using bacteraemia as an outcome, trends at hospital level are difficult to discern, due to random variation. We investigated rates of nosocomial bacteraemic and non-bacteraemic MRSA infection as surveillance outcomes., Methods and Findings: We used microbiology and patient administration system data from an Oxford hospital to estimate monthly rates of first nosocomial MRSA bacteraemia, and nosocomial MRSA isolation from blood/respiratory/sterile site specimens ("sterile sites") or all clinical samples (screens excluded) in all patients admitted from the community for at least 2 days between April 1998 and June 2006. During this period there were 441 nosocomial MRSA bacteraemias, 1464 MRSA isolations from sterile sites, and 3450 isolations from clinical specimens (8% blood, 15% sterile site, 10% respiratory, 59% surface swabs, 8% urine) in over 2.6 million patient-days. The ratio of bacteraemias to sterile site and all clinical isolations was similar over this period (around 3 and 8-fold lower respectively), during which rates of nosocomial MRSA bacteraemia increased by 27% per year to July 2003 before decreasing by 18% per year thereafter (heterogeneity p<0.001). Trends in sterile site and all clinical isolations were similar. Notably, a change in rate of all clinical MRSA isolations in December 2002 could first be detected with conventional statistical significance by August 2003 (p = 0.03). In contrast, when monitoring MRSA bacteraemia, identification of probable changes in trend took longer, first achieving p<0.05 in July 2004., Conclusions: MRSA isolation from all sites of suspected infection, including bacteraemic and non-bacteraemic isolation, is a potential new surveillance method for MRSA control. It occurs about 8 times more frequently than bacteraemia, allowing robust statistical determination of changing rates over substantially shorter times or smaller areas than using bacteraemia as an outcome.