Prevalence of non-communicable diseases are a challenging problems among menopausal women specially in a least developed country like Bangladesh, where majority of women suffering from at least one chronic diseases after menopausal age. So, the main objective of this study was to determine the prevalence of metabolic syndrome and related risk factors in Bangladeshi pre- and post-menopausal women living in the rural setting.This study is based on a community based cross-sectional survey among 1802 rural women aged ≥15 years. Metabolic syndrome was defined according to the criteria of NCEP-ATP III. Logistic regression was used to estimate the association between menopausal status and metabolic syndrome and its components.P = 0.001). Prevalence of high blood pressure, elevated fasting blood glucose, and high triglyceride were significantly higher in post-menopausal women than pre-menopausal women (P < 0.05). However, prevalence of low high-density lipoprotein cholesterol was significantly lower in post-menopausal women than pre-menopausal women (P < 0.001).Metabolic syndrome was presented in 25.6% respondents and it was more prevalent among post-menopausal (39.3%) as compared to pre-menopausal (16.8%) women. Logistic regression analysis reveals that prevalence of metabolic syndrome was 1.78 times higher in post-menopausal women than pre-menopausal women (Metabolic syndrome seems to be a major health problem among post-menopausal women in many developing countries like Bangladesh and proper policy emphasis should be given on its prevention and control. The prevalence of chronic non-communicable diseases such as cardiovascular diseases and type 2 diabetes is increasing significantly in Bangladesh. This inPrevious studies have showed that MS and cardiovascular diseases are more common in women above 55 years of age with significant increase in individual risk factors in the postmenopausal phase,7. ChangPrevalence of MS among pre- and post-menopausal women has varied according to different population. To date, several previous studies found a significance difference in prevalence of MS among pre- and post-menopausal women-17. HoweIn a community based cross-sectional study of rural women, a total of 1802 participants aged ≥15 years were selected using stratified multistage random sampling. This sample size was sufficient to test all the research hypotheses at the 5% level of significance with a power of 80% (β = 0.20). Subjects were excluded having chronic illness such as hypothyroidism, pregnant women, those on hormone replacement therapy (HRT) as well as women with known illness such as ischaemic heart disease (IHD), diabetes and hypertension. The study uses the World Health Organization’s STEPS approach (modified) which entails stepwise collection of the risk factor data based on standardized questionnaires covering demographic characteristics, somatic illnesses, somatic and mental symptoms, medications, life style, and health-related behavior (step 1), basic physical measures (step 2) and basic biochemical investigations such as blood glucose and cholesterol (step 3). Women were defined as post-menopausal if they had reported their last menses to be at least 12 months previously; pre-menopausal if they had an unchanged and regular menstrual pattern during the last five years, without typical climacteric complaints; and peri-menopausal if last menses to be within or before 12 months and were not regular. In our study we excluded peri-menopausal women. A standard questionnaire on health and menopausal status written in the local language was used to identify the pre- and post-menopausal subjects.Upazilla (subdistrict) of Gaibandha district, a poor district of the northern division of Rajshahi, the poorest region of the country. The respondents were selected randomly after selecting division, district, Upazila and villages. Women were recruited through local announcement (loudspeaker) at community level and surveyors visits. The details of the study procedure have been described before in our previous study were ascertained. Fasting plasma glucose [glucose with the Hexokinase G-6-PDH kit  were also measured. After labeling blood sample vials, a part of blood were transported to National Centre for Global Health and Medicine (NCGM), Japan for advanced biochemical assessment processing.We defined the metabolic syndrome (MS) using the National Cholesterol Education Program Adult Treatment Panel III (NCEP-ATP III). As deta2) test for categorical variables. Logistic regression analyses were used to assess the association between menopausal status and MS and each of its components. Two types of models were used. The first model was crude model without any adjustment and the second model was adjusted for age. Statistical significance was set at the conventional P < 0.05. All analysis were performed using statistical software STATA version 12.0 .The clinical and laboratory characteristic of study population are presented according to pre- and post-menopausal women. The significant differences between pre- and post-menopausal women were assessed by using the Student’s “t” test for continuous variables and chi-square (χP < 0.001).The clinical and laboratory characteristics of the study population stratified by menopausal status and MS are presented in Table P < 0.001). Prevalence of metabolic risk factors including elevated fasting blood glucose, high blood pressures, and high triglycerides were significantly higher in post-menopausal women than pre-menopausal women (P < 0.001). However, prevalence of low HDL cholesterol was significantly lower in post-menopausal women than pre-menopausal women (P = 0.001).The prevalence of MS and metabolic risk factors are presented in Table P < 0.001). After adjustment for age the association was slightly attenuated but remains statistically significant . Prevalence of elevated fasting blood glucose, high blood pressures, and high triglycerides were also significantly higher but prevalence of low HDL cholesterol were significantly lower in post-menopausal women than pre-menopausal women even after adjustment for age .Table In this cross-sectional study of Bangladesh we compare prevalence of MS and risk variables of MS in pre- and post-menopausal women and also identified how these variables relate to one another. This is the first report in Bangladesh investigating the prevalence of MS and each component of MS risk factors among rural women according to their menopausal status.The prevalence of MS was higher in post-menopausal (39.3%) women than pre-menopausal 16.8%) women in our study and this prevalence was more likely in post-menopausal women even after adjustment for age. Our findings are consistent with many of previous studies,13-16,24.8% womenThe transition from pre- to post-menopause may be associated with each feature of the MS. In our study metabolic risk factors including high blood pressures, high triglyceride, and elevated fasting blood glucose are significantly higher among post-menopausal women than pre-menopausal women. However, prevalence of low HDL cholesterol is significantly lower in post-menopausal women than pre-menopausal women. In agreement with the results of our study, many previous studies have reported higher concentrations of hypertension,15,26, hThe emergence of metabolic risk factors in post-menopausal phase may be a direct result of ovarian failure with estrogen deficiency. Decrease in estrogen production is thought to be responsible for substantial proportion of increased cardio-metabolic risk factors in post-menopausal women. Changes in metabolic risk factors during pre- and post-menopause may be due to a combination of factors of the aging process. Although previous studies showed age as the predominant variable associated with increased prevalence of MS, but higher prevalence of MS among women >50 years compared with men of the same age has been reported. In our In our study we found no significant difference in obesity component including BMI and waist circumference. Our findings are consistent with two previous studies of India and IranTo the best of our knowledge, our study is the first to demonstrate the prevalence of MS and its components among pre- and post-menopausal women in a low-income country like Bangladesh. One of the major strengths of the present study is that it is based on the largest community-based and comprehensive MS survey. However, our study has several limitations that need to be mentioned. First, an association derived from a cross-sectional study does not necessarily indicate causality. Second, menopausal status was self-reported and could have recall bias. Third, our study may have selection bias during cases recruitment due to selection of rural women from lower socioeconomic class and may not be generalized to the whole Bangladeshi women.In conclusion, the results of our study suggest that the prevalence of MS in post-menopausal women was significantly higher than pre-menopausal women. Low HDL cholesterol, elevated fasting blood glucose, and high blood pressures were the most frequent features in comparison to the others. Prevention through changes in lifestyle, or early detection and treatment of elevated fasting blood glucose, hypertension, and hyperlipidemia are necessary for prevention of many chronic diseases in Bangladeshi women reaching after menopausal status. Health professionals should consider the post-menopausal women as a major target group for prevention of MS, which is an underlying condition of many non-communicable diseases.MS: Metabolic syndrome; BMI: Body mass index; HDL cholesterol: High-density lipoprotein cholesterolThe authors declare that they have no competing interests.SJ developed the idea for this paper, wrote the first draft of the manuscript and extensively edited the manuscript; AMSI and SA contributed to statistical analysis and writing results; MMI, and SNS performed all searches and compiled the text; NY, MH, MM and TW provided conceptual and editorial input. All authors equally contributed to discussion and approved the final version of manuscript.


Purpose. We examine factors associated with self-care, use of practitioner-based complementary and alternative medicine (CAM), and their timing in a cohort of women with breast cancer. Methods. Study participants were women with breast cancer who participated in the Long Island Breast Cancer Study Project. Self-care is defined as the use of multivitamins, single vitamins, botanicals, other dietary supplements, mind-body practices, special diets, support groups, and prayer. Within each modality, study participants were categorized as continuous users (before and after diagnosis), starters (only after diagnosis), quitters (only before diagnosis), or never users. Multivariable logistic regression was used for the main analyses. Results. Of 764 women who provided complete data, 513 (67.2%) initiated a new form of self-care following breast cancer diagnosis. The most popular modalities were those that are ingestible, and they were commonly used in combination. The strongest predictor of continuous use of one type of self-care was continuous use of other types of self-care. Healthy behaviors, including high fruit/vegetable intake and exercise, were more strongly associated with continuously using self-care than starting self-care after diagnosis. Conclusions. Breast cancer diagnosis was associated with subsequent behavioral changes, and the majority of women undertook new forms of self-care after diagnosis. Few women discontinued use of modalities they used prior to diagnosis. Over the past two decades, the use of complementary and alternative medicine (CAM) practices—modalities used for health and wellbeing that are considered outside the realm of conventional medicine—has been steadily rising in the United States –3. PatieThe frequency of CAM use among breast cancer patients is not surprising in the light of the physical and emotional burden that breast cancer entails. Breast cancer patients report using CAM for recovery, healing, improving health, strengthening the immune system, reducing side effects of cancer treatments, reducing physical and psychological distress , and incDue to the fact that large numbers of cancer patients use CAM, it is increasingly important to develop epidemiological methods to both describe and analyze CAM use patterns. Historically, most analyses of CAM use among cancer populations have focused on use after diagnosis. However, it is important to consider postdiagnosis CAM use in context of the patient's behavior prior to a cancer diagnosis. In this paper, we present a novel approach to describe changes in CAM use before and after breast cancer diagnosis. Furthermore, since CAM encompasses a wide variety of individual modalities that have different implications on pathophysiology and treatment course, we propose that it is important to consider changes in CAM use across specific categories of CAM. Using follow-up data from the Long Island Breast Cancer Study Project, we analyzed predictors of self-care practices and practitioner-based modalities of CAM before and after a first breast cancer diagnosis. Most studies of CAM use do not separate self-care  from practitioner-based modalities. Our primary aim was to analyze the determinants of self-care versus practitioner-based modalities; other aims were to determine how use of these modalities changed after the breast cancer diagnosis and how use of self-care and practitioner-based modalities might be associated with health-related behaviors such as exercise, smoking, and alcohol intake. The Long Island Breast Cancer Study Project (LIBCSP) originated as a population-based, case-control study of women residing in Nassau or Suffolk county, New York, USA . The LIBBetween 2002 and 2004, a follow-up study was conducted among the LIBCSP cases . Of the Institutional review boards at all participating institutions approved the study protocol, and all participants provided written informed consent prior to the in-person baseline interview and verbal consent prior to the telephone follow-up interview. The baseline questionnaire was administered to cases by a trained interviewer in the respondent's home soon after a first primary breast cancer diagnosis (mean = 89 days among the subsample with a full follow-up telephone interview). Women responded to a detailed questionnaire that took on average 100 minutes to complete and covered known and suspected risk factors for breast cancer, including environmental exposures, reproductive and menstrual histories, medical history, and demographic characteristics. Women were also asked about lifestyle and health behaviors across the life course, including cigarette and alcohol use  and recrThe full-length follow-up questionnaire, which was administered by telephone by a trained interviewer and lasted about 45 minutes, assessed treatment details for the first primary breast cancer diagnosis, factors associated with breast cancer prognosis, postdiagnosis medical history, and health behaviors. The follow-up interview included detailed questions about CAM used, specifically, before breast cancer diagnosis, after diagnosis, and during breast cancer treatment. The CAM questionnaire covered use of multivitamins, single vitamins, minerals, herbs and botanicals, other dietary supplements, mind-body practices, support groups, prayer and special diets, and visits to CAM practitioners .  Table n = 598), information abstracted from the records was in excellent agreement (κ = 0.92–0.96) with the women's responses to questions about treatment in the follow-up questionnaire; we therefore relied on the follow-up questionnaire data for information about cancer treatment [Medical records obtained at the time of baseline and follow-up were used to confirm disease characteristics and treatment course. For case women for whom complete medical records were available  were included in the full models so that the odds ratios would be comparable across all models. The independent variables tested in univariate regression models that were not included in the final multivariable models (P > 0.05) were oral contraceptive use, hormone replacement therapy, stage at diagnosis, and hormone receptor status. The final models were adjusted for age at diagnosis, race, income, education, body mass index , health behaviors  as reported at baseline, continuous use of other CAM categories, and first course of breast cancer treatment type, as reported at the follow-up interview. First, univariate regression analyses were performed to identify the variables that were independently associated with use of self-care/practitioner-based CAM before and after diagnosis. Then, 2 sets of multivariable logistic regression models were developed to identify factors independently associated with (1) continuously using and (2) starting to use a category of modality after diagnosis . Age, inn = 764), mean age at diagnosis was 56.3 years; 94% were non-Hispanic white; 59% had an annual household income above $50,000; 61% had attended college; and 44.1% had never smoked cigarettes. At baseline, 36.8% ate at least 35 servings of fruits and vegetables per week, and 23.7% exercised at least 2.7 hours a week .Both before and after diagnosis, 55.5% of women used multivitamins; 57.3% used single vitamins; 35.5% used prayer, 29.2% botanicals, 21.6% special diets, and 16.6% mind-body practices. 25.4% of women used at least one practitioner-based modality both before and after diagnosis . 67.2% of women started some form of self-care after diagnosis. The modalities most commonly started after diagnosis were multivitamins (20.5%), single vitamins (19.1%), support groups (17.4%), other dietary supplements (12.3%), and mind-body practices (11.6%). 14.8% of women began using a practitioner-based modality after diagnosis. The particular modalities most commonly initiated after breast cancer diagnosis were calcium (25.3%), vitamin E (22.6%), green tea (20.9%), support groups (17.4%), vitamin C (16.5%), low-fat diet (11.0%), and glucosamine (10.3%) .10.6% of women stopped using multivitamins after diagnosis. The most common modalities stopped after diagnosis were iron (11.9%) and vitamin C (9.3%). A small proportion of women (7.2%) stopped using all practitioner-based modalities. Chiropractic, the most common ever-used practitioner-based modality (210 users or 27.5%), was the modality most commonly discontinued after diagnosis (14.5%) . As previously reported , all 764Higher education was a strong predictor of both starting mind-body practices after diagnosis and using such practices before and after diagnosis (continuous use); education was also associated with continuous use of botanicals and dietary supplements. Income was associated directly with continuous single vitamin use and inversely with continuous use of other dietary supplements ; income Healthy behaviors reported at the baseline interview were more strongly associated with continuously using self-care than with starting self-care after a diagnosis with first primary breast cancer. Those with the highest level of physical activity (>2.7 hr/week) were 2.4 times as likely to use mind-body modalities before and after diagnosis as those who reported no physical activity. Women who had a mammogram within 5 years before diagnosis were 2 times as likely to be continuous users of single vitamins as women who had not had a recent mammogram. Women whose intake of fruits and vegetables was high were 50% more likely to use multivitamins and/or botanicals continuously than those whose intake was low .Current smokers had 60% lower odds of following a special diet before and after diagnosis than never smokers . Women wHigh BMI was also associated with continuous use of practitioner-based modalities (OR = 2.5); high fruit and vegetable intake was associated with starting practitioner-based CAM after diagnosis (OR = 1.8). In our multivariable logistic regression models, among women with a diagnosis of first primary breast cancer, the strongest and most common predictors of continuous use of one type of self-care were continuous use of other types of self-care. In particular, users of one type of ingestible self-care were more likely to use other ingestible agents. Women who continuously used single vitamins were nearly 4 times as likely to use multivitamins and/or use other dietary supplements and 1.6 times as likely to use botanicals before and after diagnosis as other women. Similarly, women who used botanicals before and after diagnosis were nearly twice as likely to use multivitamins and single vitamins before and after diagnosis as never users of botanicals .Women who used ingestible forms of self-care were also more likely to begin using new categories of ingestible self-care after diagnosis with first primary breast cancer. Women who used single vitamins before and after diagnosis were 10 times as likely to start use of botanicals after diagnosis as never users; women who used other dietary supplements before and after diagnosis were 3 times more likely to start using botanicals after diagnosis than never users .Continuous users of mind-body modalities were more than 7 times as likely to use prayer, 5 times as likely to use support groups, and more than twice as likely to use practitioner-based modalities continuously as non-users. Neither stage of disease nor hormone receptor status was associated with continuous or new use of self-care categories. The only clinical variable associated with self-care was treatment type: women who received tamoxifen treatment had 1.7 times greater odds of starting use of single vitamins after diagnosis than women who were not treated with tamoxifen . We present a novel method to describe changes in CAM use before and after cancer diagnosis by comparing those who started using, stopped using, continued using, or never used a category of CAM following diagnosis. Cancer survivors access multiple forms of CAM and we specifically examined differences in patterns of CAM that involved “self-care” as compared to those that were based on “practitioner-care.” Among women diagnosed with a first primary breast cancer who self-reported their use of complementary and alternative medicine modalities in the follow-up components of the Long Island Breast Cancer Study Project, the factors most commonly and strongly associated with using self-care and CAM modalities were use of other categories of self-care. In particular, women who used one form of ingestible self-care  were more likely than other women either to continue or to start using other ingestible self-care. Healthy behaviors were associated with self-care practices that were used before diagnosis and continued afterwards. Behavioral factors were more predictive of self-care use after breast cancer diagnosis than were indicators of disease prognosis.In a nationally representative survey conducted in 1997, which is close to the period when the LIBCSP cases were diagnosed with breast cancer, 42% of adults reported using at least 1 of 16 CAM modalities (these data did not include daily vitamins) . 95% of A recent cross-sectional study reported that 75% of adult cancer patients used CAM and of those, close to 60% began using CAM after their cancer diagnosis . It has It is not surprising that the strongest predictor overall of using a self-care category before and after diagnosis with a first primary breast cancer, and of starting a self-care category after diagnosis, was using other forms of self-care. Women who already use some form of CAM or a self-care practice are likely to be aware of other CAM modalities. It is also not surprising that ingestible forms of CAM were commonly used together. Women who already take one type of supplement do not need to make much additional effort or to undergo a significant change in behavior to try new supplements. Continuous use of botanicals was the factor most commonly associated with starting new forms of self-care, including single vitamins, mind-body, other dietary supplements, and special diets. However, starting practitioner-based CAM was not associated with prior use of self-care modalities. High intake of fruits and vegetables at the baseline interview was associated with the use of multivitamins before and after diagnosis. High recreational physical activity level reported at the baseline interview was positively associated with continuous use of mind-body practices, and high BMI at baseline was negatively associated with continuous use of botanicals, other dietary supplements, and mind-body modalities. However, healthy behaviors were not strongly associated with starting self-care practices after diagnosis; most women who engaged in healthy behaviors were already using self-care. Neither stage of disease nor hormone receptor status was associated with starting either self-care or practitioner-based modalities. Most self-care and practitioner-based CAM use occurred during tamoxifen treatment, perhaps because tamoxifen is an oral agent that patients use for 5 years, whereas chemotherapy and radiation treatments have a usual duration of several months or weeks. In addition, women who used tamoxifen may have been more willing to use other oral or ingestible agents than women who did not use tamoxifen. Mind-body practices were also common during adjuvant treatment, perhaps because women who were told not to take dietary supplements during those treatments used noningestible forms of self-care instead. The modalities that were most commonly initiated after diagnosis with breast cancer among women in our study were also the modalities most frequently used during adjuvant treatment. Although our study participants were not asked why they used the modalities they chose, data from other studies suggest that patients initiate self-care after breast cancer diagnosis due to fear or experience of treatment-related side effects –27. A reAmong the limitations of our study, the follow-up data were collected nearly a decade ago; CAM and self-care practices may be different now. And because the data were collected more than 5 years after diagnosis and treatment, patients may not have accurately recalled their prior CAM use. Although minority race/ethnicity was associated with beginning practitioner-based CAM, only 6% of the sample were non-White, and thus the associations observed are unstable and must be interpreted with caution. In addition, although two-thirds of the case women who participated in the original LIBCSP case-control study provided some follow-up data, only 55.4% personally completed the full follow-up questionnaire, including the instrument developed by our team to assess use of CAM. We previously reported that nonrespondents to the full follow-up were of lower socioeconomic status than respondents (response bias) . The welGrowing evidence suggests that CAM and other self-care modalities may be effective in reducing treatment-related side effects, including nausea and vomiting, as well as in reducing stress and easing pain , 29. FurWe found that a diagnosis of breast cancer was associated with subsequent behavioral changes and that more patients undertook new forms of self-care than abandoned the forms they had been using. The most popular modalities were those that are ingestible and readily available without a gatekeeper, and they were commonly used in combination. These findings are important because they identify patterns of CAM use in a well-characterized population of cancer patients, including how CAM use changed after diagnosis and during treatment. Based on research conducted to date, evidence-based guidelines are available for providers and patients about the safety and effects of various CAM modalities in the oncology setting –36 and c


To bring down the per-sample cost, one approach is to pool barcoded NGS libraries before sample enrichment. Still, we lack a complete understanding of how this multiplexed tNGS approach and the varying performance of the ever-evolving analytical tools can affect the quality of variant discovery. Therefore, we evaluated the impact of different software tools and analytical approaches on the discovery of single nucleotide polymorphisms (SNPs) in multiplexed tNGS data. To generate our own test model, we combined a sequence capture method with NGS in three experimental stages of increasing complexity .Compared to classical genotyping, targeted next-generation sequencing  across multiplexed samples, with <10% strand bias. However, the SNP calling quality was substantially affected by the choice of tools and mapping strategy. With the aim of reducing computational requirements, we compared conventional whole-genome mapping and SNP-calling with a new faster approach: target-region mapping with subsequent ‘read-backmapping’ to the whole genome to reduce the false detection rate. Consequently, we developed a combined mapping pipeline, which includes standard tools , and tested it on public HiSeq2000 exome data from the 1000 Genomes Project. Our pipeline saved 12 hours of run time per Hiseq2000 exome sample and detected ~5% more SNPs than the conventional whole genome approach. This suggests that more potential novel SNPs may be discovered using both approaches than with just the conventional approach.tNGS. Our study has also shown the benefit of computing inter-sample SNP-concordances and inspecting read alignments in order to attain more confident results.We recommend applying our general ‘two-step’ mapping approach for more efficient SNP discovery in  Correlations between genotype and phenotype variations have traditionally been studied by determining the genotype of known markers. For example, genome-wide association studies (GWAS) have revealed associations of known common variants with several of the common diseases. But these associations typically explain less than 25% of the heritable risk estimated for each of those diseases ,2. This tNGS) data? What is the effect of the varying performance of the rapidly evolving alignment and analytical tools? Can a sample multiplexing approach reduce the analysis and study costs? To address these questions, as a test model, we combined a hybridization-based sample enrichment method  with an NGS platform . With sample mix-up control and enrichment cost reduction in mind, we evaluated the enrichment of barcoded SOLiD libraries , rather than preparing NGS libraries after enrichment [tNGS method on E. coli, as a simple model to optimize the process, and human genomic samples in three experimental stages of increasing scope and complexity, culminating in a SNP concordance evaluation for the BRCA1 and BRCA2 cancer genes.Target enrichment methods rely either on PCR or sequence-specific nucleic acid hybridization, and each method has unique advantages and disadvantages -7. The crichment -10. Indirichment ,8,11-14.tNGS data, we carefully analyzed the data using different software and tools. We found that SNP detection depends strongly on the chosen analytical tools and settings, rather than on key enrichment measures such as high and uniform coverage, the percentage of reads mapped on target, or an adequate enrichment fold. Unexpectedly, all evaluated tools failed to identify a large proportion of true sequence variations . Whole-genome (WG) mapping/SNP-calling was time-consuming, but with the benefit of few false positive SNPs. Target region (TR) mapping/SNP-calling was faster and yielded more true SNPs than WG mapping, but led to a high proportion (up to 50%) of likely false positive SNPs. Target region mapping can force-map reads from other genomic loci into the target, leading to false positive SNPs and requiring a postprocessing cleanup. To benefit from both methods, we here report a novel ‘two-step’ mapping approach that starts with TR mapping/SNP-calling, followed by backmapping only the SNP-supporting reads to the WG. Because of the recent wide interest in whole exome sequencing, we also applied our approach to public human exome enrichment data generated by Illumina HiSeq instruments. Moreover, our report includes detailed practical instructions, such as validating SNPs by computing inter-sample SNP-concordances between multiplexed technical replicates, filtering for novel SNPs, or establishing and evaluating a tNGS method.To identify a suitable analytical approach in terms of computational time and accuracy of sequence variant detection in tNGS model) and a fourth, purely analytical stage using human exome data. The study design, the established wet-lab workflow, and the bioinformatics workflows for our test tNGS model are presented in Figure E. coli for 68 genes and sequenced them on a SOLiD NGS platform. In the second stage, we enriched three pools of barcoded libraries of E. coli for the same 68 genes and sequenced these on the same NGS platform. The results of this stage showed reproducible uniform coverages and enrichment folds for most barcodes and multiplexes tested. Full details on the target enrichment experiments and results, especially the wet-lab procedure, can be found in the Additional file 1. In the following results section we concentrate on the respective data analysis and tool development.We developed a novel, fast two-stage backmapping method in the course of three experimental and analytical stages (our test BRCA1 and BRCA2). We enriched these genomic regions at different multiplexing levels . We also included two non-barcoded controls. The sequencing results and enrichment measures are summarized in Additional file In the third stage, we focused on enriching pools of barcoded libraries of HapMap individuals for non-repeat-masked regions of two clinically relevant human cancer genes  is a typical objective of resequencing. To test how well variants are discovered using our multiplexed For the Yoruban sample we observed a good SNP overlap (15/15) and reached a concordance of 14/15 through visual inspection in IGV. Bioscope called only 13/15 SNPs with the chosen settings and also with many alternative settings (Bioscope_settings.xls at ). Our maFigure BRCA1/2 experiment  to two human exome Illumina HiSeq2000 data sets: a female CEU HapMap individual and her father  SOLiD libraries instead of gDNA to one selected microarray-based sequence capture method. We tested this pre-enrichment sample multiplex approach by sequencing 68 E. coli genes as well as two human cancer genes (BRCA1 and BRCA2) in three independent stages and different multiplexing folds on the SOLiD system. We achieved good and reproducible coverage profiles for the TRs across most of the different multiplexed samples, enriching human exonic as well as intronic regions with less than 10% strand bias . We further recommend, if cost allows, to duplicate samples (which is easily performed in a multiplexed experiment) and validate computed SNPs by inter-sample concordance checks between these technical replicates.SNP-callers were originally optimised for low or moderate variations in coverage. In other words, extremely high coverages or high coverage gradients are currently challenging from the bioinformatics point of view. Even for non-enriched samples, some degree of automatic or manual postprocessing is required specifically to distinguish SNPs from misalignments . A previBRCA1/2 regions, or 61 MB of exome regions, compared to 3 Gb of human genome) and cleaning up force-mapped reads. For exomes, we cut run time by more than half, enabling overnight runs on an 8-core node, or turnaround within a working day (4–5 hours) on a 16-core node. This run time cut is necessary to match the speed of new high-throughput platforms such as the Illumina HiSeq2500, which takes 27 hours for producing 120 Gb , the genome-sized data traffic may overload the compute cluster when too many samples are processed in parallel. Compared to BioScope, other pipelines such as BWA alleviate this problem, but a complete exome run using BWA, SAMtools and Picard nevertheless takes about a full day. Our aim was to cut the computational time significantly by mapping to the small TR  for a reliable and efficient analysis of exome and targeted enrichment data. To attain confident results, we specifically recommend the SNP-validation between duplicated samples and a final tNGS test model is shown in Figure The study design for our We performed read mapping and SNP calling with SOLiD Corona Lite and later switched to Bioscope (a pre-release version of v1.2), after the manufacturer advised that the Corona Lite SNP-caller (pre-release diBayes) was not designed for enriched samples . Our Bioscope mapping and SNP-calling settings (Bioscope plan and ini files) are fully documented on the project homepage . For SNPE. coli WG mapping we used the E. coli K-12 M1655 genome  as our mapping reference. For human WG mapping we used the NCBI36/hg18 assembly as our mapping reference. For TR mapping we constructed a single FASTA reference sequence consisting of greater TRs, each separated by a block of 50 Ns (i.e. taking into account the read length of 50). Greater TRs consisted of the actual TRs, extended by 49 bases and merged with adjacent TRs if an overlap occurred. The reason for defining greater TRs was to map reads which partially overlap actual TRs. For E. coli we targeted 68 genes with a total length of 90 kb. For the human samples we targeted 198 small regions with a total length of 90 kb in two genes (a reduction of the size of the intended TRs from 165 kb to 90 kb because of the repeat-masker to enable designing efficient capture probes).We mapped the 50-mer reads using two separate mapping strategies for each sample: WG mapping  and TR mapping . For We wrote R-scripts  for the As validated references for our own SNP-calling, we created consensus SNP lists for the Yoruban HapMap individual NA18507. We created our ‘gold’ consensus from the HapMap3 data , WG IllTo clean up SNPs obtained after our fast TR mapping approach, we initially tested a ‘SNP-backmapping’ approach . This did not eliminate all potential false positive SNPs Figure . We thenTo evaluate the performance of our backmapping pipeline we used publicly available sequence data of a HapMap CEU family trio (ID 1463) who had been repeatedly sequenced within the 1000 Genomes Project . As reprWe aligned the reads to the human genome reference NCBI GRCh37  using BWThe exome-based reference sequence for the TR mapping was created as follows: We downloaded a file of exonic regions, represented by the compatible Consensus Coding Sequence (CCDS) file ‘CCDS.20110907.txt’ at . We thenWe uniquely extracted reads covering heterozygous SNPs, from the BAM-file. As the reads had been sequenced from paired-end libraries, we also extracted the according mate from the BAM-file and created new FASTQ files containing these reads and their (possibly unmapped) mates. We implemented the read-extraction process in a self-written shell script which used VCF-Tools , SAMtoolUsing a self-written Python script and the coordinate transformation file (see above), we transformed the coordinates in the SNP-list from the exome reference coordinate system to the genomic coordinate system. We divided the SNP-list into heterozygous SNPs and homozygous SNPs. Because the heterozygous SNPs can be artifacts resulting from the alignment of non-exonic reads to the exome reference, we extracted the corrected genotype at the SNP-coordinates from the backmapped WG BAM file using the pibase software.None of the co-authors provided competing of interests.AE, AF designed the study; NS, AK, PFS performed the sample enrichment; AE designed the next-generation sequencing experiments; MF, AK, IT, B-SP, BS, NS performed data analysis; IT, B-SP, MF created the tNGS Backmapping pipeline; AE, MF interpreted the data and wrote the manuscript; NS, PR, IT, SS, AF proof-read and edited the manuscript; AE, MF, IT, B-SP, PR, AF revised the manuscript; AE, PFS, PR, AF coordinated the project. All authors read and approved the final manuscript. Supporting Methods and Results. It includes additional details of the Methods, Results and Discussion. - It lists all the Supplementary Tables and Figures .- It also includes Tables A1-A3 and Figures A1-A5.Click here for file Supporting Tables. Tables S1-S26.Click here for file Supporting Figures. Figures S1-S9.Click here for file


Current knowledge on morbidity and mortality in HIV-infected children comes from data collected in specific research programmes, which may offer a different standard of care compared to routine care. We described hospitalization data within a large observational cohort of HIV-infected children in West Africa (IeDEA West Africa collaboration).We performed a six-month prospective multicentre survey from April to October 2010 in five HIV-specialized paediatric hospital wards in Ouagadougou, Accra, Cotonou, Dakar and Bamako. Baseline and follow-up data during hospitalization were recorded using a standardized clinical form, and extracted from hospitalization files and local databases. Event validation committees reviewed diagnoses within each centre. HIV-related events were defined according to the WHO definitions.From April to October 2010, 155 HIV-infected children were hospitalized; median age was 3 years [1–8]. Among them, 90 (58%) were confirmed for HIV infection during their stay; 138 (89%) were already receiving cotrimoxazole prophylaxis and 64 children (40%) had initiated antiretroviral therapy (ART). The median length of stay was 13 days (IQR: 7–23); 25 children (16%) died during hospitalization and four (3%) were transferred out. The leading causes of hospitalization were WHO stage 3 opportunistic infections (37%), non-AIDS-defining events (28%), cachexia and other WHO stage 4 events (25%).Overall, most causes of hospitalizations were HIV related but one hospitalization in three was caused by a non-AIDS-defining event, mostly in children on ART. HIV-related fatality is also high despite the scaling-up of access to ART in resource-limited settings. In 2010, the HIV/AIDS epidemic led to 1.8 million deaths worldwide, 14% of which occurred in children below 15 years of age . Since 2Current knowledge on morbidity and mortality comes mainly from data collected in specific research programmes, which may offer a different standard of care than that offered outside of a research context, where means for diagnosis and treatment are more limited. Hospitalization represents an important outcome measure, as a reliable indicator for morbidity and mortality, as well as healthcare utilisation.In this study, we described hospitalization in tertiary hospital wards from a large prospective observational cohort of HIV-infected and HIV-exposed children in the paediatric West African International epidemiological Database to Evaluate AIDS (IeDEA) Collaboration.http://www.iedea.org/) was initiated in July 2006 to address evolving questions in HIV/AIDS research, unanswerable by single cohorts, by collecting and harmonizing data from multiple HIV/AIDS cohorts and programmes. In 2012, the paediatric IeDEA West Africa collaboration (pWADA) involved 11 paediatric HIV/AIDS clinics spread out over seven countries in West Africa.The IeDEA network  were supposed to routinely receive cotrimoxazole prophylaxis from six weeks of age, then an EID based on virological methods, which needs to be confirmed by serology from 15 months after the end of breastfeeding in breastfed children. HIV-infected children are typically seen at least every three months, and CD4 counts are measured every six months; ART, cotrimoxazole prophylaxis and blood analyses are free of charge. Children initiated ART according to the 2008 WHO recommendations, contemporaneous with the practices at time of ART initiation : all HIVWe performed a six-month cross-sectional multicentre survey from April to October 2010 in five HIV-specialized paediatric hospital wards involved in the pWADA IeDEA collaboration: the Gabriel Touré Teaching Hospital , the National Teaching Hospital , the Albert Royer Children's Hospital , the Général de Gaulle Teaching Hospital  and the Korle Bu Teaching Hospital . All newly hospitalized HIV-infected children≤18 years, previously known as HIV-infected patients  or diagnosed as HIV-infected during hospitalization were eligible, regardless of ART.Baseline and follow-up information during hospitalization were recorded using a standardized clinical form. Data were extracted from hospitalization medical records and local databases. The following variables were recorded: social-demographic data ; data of HIV diagnosis, type and circumstances of diagnosis, tuberculosis history; ART history ; current prophylaxis; co-morbidity history, immunization status; date of hospitalization, cause, date of first symptoms, weight, height, WHO stage, CD4 count and viral load – when available – complete blood counts; diagnosis and type of diagnosis , degree of severity; date of end of hospitalization, vital status at exit, underlying cause of death in that event. In case of hospitalization, the available diagnosis tools were provided to children's family. Unfortunately there were no data on prior PMTCT services on care as HIV-exposed infants. However, a proxy for identified HIV-exposed infants could be assumed from the children who were already on cotrimoxazole among those newly diagnosed.Ad-hoc event validation committees reviewed diagnoses within each centre. HIV-related events were defined according to the WHO definitions . In thisImmunodeficiency was defined according to age at time of CD4 measurement for those with available data, according to WHO definitions . Immune In order to determine one underlying cause for each hospitalization, the following priorities were applied if a patient had multiple diagnoses: (1) WHO stage 4 opportunistic diseases, (2) WHO stage 3 opportunistic diseases, (3) other non-AIDS-defining infections, (4) other cancerous events, (5) WHO stage 4 cachexia, (6) other diseases and (7) non-specific events including non-specific WHO stage 3 events .When a medical etiological diagnosis for hospitalization could not be identified, we reviewed diagnoses that comprised symptoms or clinical signs and attributed the hospitalization to those. Finally, we grouped the prioritized diagnoses associated with hospitalization into the following four categories: WHO stage 4 events (including cachexia); WHO stage 3 opportunistic infections; other non-AIDS-related infections; and other diseases or non-specific diseases . To determine the underlying cause of death, we used the International Classification of Diseases, Tenth Revision rules  and defiWe described the patterns and causes of hospitalization in children according to the WHO staging, knowledge of HIV infection at time of hospitalization, cotrimoxazole prophylaxis and ongoing ART at the time of hospitalization. Categorical data are presented as frequencies (percentage) and continuous variables as median [interquartile range (IQR)]. Proportions were compared by Fisher's exact tests.n=90, 58%); these children were aged in median 2 years (IQR: 1–6 years), 92% had reached WHO stage 3 or 4 of the disease, and 88% were already receiving cotrimoxazole prophylaxis (n=80), a proxy of HIV-exposure.In 2010, 1486 children were known to be HIV-infected and were followed up in these five clinics. From April to October 2010, 155 HIV-infected children were hospitalized once in one of the five participating tertiary hospitals in West Africa. Their main characteristics at entry to the hospital are presented in Overall, 64 children were receiving ART of which 36 (56%) had already initiated treatment prior to hospitalization, 64% of which were aged>5 years; the median time on ART among those children was 9.8 months (IQR: 1.6–32). Among those who were not on ART at time of hospitalization, all (except two for whom eligibility could not be ascertained) were eligible according to 2008 WHO guidelines.p<0.001).However, only 30% initiated treatment during hospitalization. These children were similar to those who did not initiate ART in terms of age, stage of the disease and cause of hospitalization. However, the median length of stay was significantly higher in those initiating ART  compared to those who did not . Baseline characteristics were similar between both groups as was the distribution of events  died during hospitalization. These children were hospitalized for a significantly shorter period of time  compared to those who remained alive ; 25 children (16%) died during hospitalization, and four (3%) were transferred out to another ward or to another hospital. The most frequent causes of hospitalizations were: WHO stage 3 opportunistic infections (37%), other non-AIDS-defining infections (28%), WHO stage 4 events including cachexia (25%) and other diseases including WHO stage 3 non-specific diseases (10%). Among the non-AIDS-defining infections, the most frequent diagnoses were the following: infectious diarrhoea (31%), malaria (24%) and pneumonia (15%). Two hospitalizations were related to suspected adverse events to ART: one child presented severe anaemia and another, a suspected muco-cutaneous disease.Many children were hospitalized with a second or third diagnosis associated with the main cause for hospitalization, yielding a total of 305 diagnoses, as described in The majority of the diagnoses were definite (81.6%). Of the 50 presumptive diagnoses, 40% were suspected cases of tuberculosis , and 6% were suspected HIV encephalopathy (one case out of four was definite).p=0.0383 and p<0.0010 for cachexia and weight loss, respectively).p=0.0321) . We also note that hospitalizations for cachexia were highest among children receiving no treatment at all (61.5%) compared to those on cotrimoxazole only (26.4%) and those on both ART and cotrimoxazole (6.3%) (p<0.001).When comparing the overall causes by treatment groups at time of hospitalization , we observed a significant difference in the distribution of events with a higher proportions of non-AIDS-defining events in those who were on both cotrimoxazole and ART compared to those receiving cotrimoxazole only or no treatment at all (=0.0321) . When stn=6), acute respiratory disease (n=6) and chronic diarrhoea (n=3)  .This cross-sectional study documents the causes of hospitalization in HIV-infected children in West Africa. We make several observations. First, of the 155 hospitalized children, 58% were unaware of their HIV status and were newly diagnosed during hospitalization; of these children, all were eligible for ART initiation and less than a third initiated treatment. Second, 62% of causes were AIDS-related, highlighting the advanced stage of HIV disease in these children at the time of hospitalization. Furthermore, we showed that among all of the events, 53% were in relation to an infection, underlining a context of residual infectious morbidity, despite cotrimoxazole prophylaxis and ART initiation. Indeed, the proportion of AIDS-defining causes of hospitalization was lower in children who were on both ART and cotrimoxazole. However, these children evidently still experience a substantial amount of non-AIDS-defining events serious enough to cause hospitalization.The median age at hospitalization was 3 years, and we showed that hospitalization was an opportunity to diagnose 58% of HIV cases in this study. The fact that most of these newly HIV diagnosed children were already receiving cotrimoxazole prophylaxis at the time of their hospitalization indicated that they were already identified as HIV-exposed infants, but they had not yet been tested for HIV or had not been informed of their status. This reflects the flaws in the prevention of mother-to-child transmission of HIV (PMTCT) services in resource-limited settings and many missed opportunities for early HIV postnatal diagnosis and care. Indeed, a large proportion of HIV-infected infants never enters the pathway to early HIV care and is diagnosed at a late age, once symptomatic, initiating ART too late. This leads to severe morbidity and mortality ,15,24,25Care interventions aiming at identifying the earliest possible HIV-infected infants and treating them according to current guidelines would drastically reduce costs of HIV care. Indeed, previous studies suggest that hospitalization constitutes 15–49% of total HIV care, particularly in children at the most advanced disease stage ,30. UnfoMany studies in resource-limited settings have described hospitalizations in HIV-infected children, but tend to be restricted to cause-specific hospitalizations or focused on HIV-exposed rather than HIV-infected children –38. In trecurrent pneumonia constituted a large proportion of the causes of hospitalizations, significantly larger in those who were not on ART. The burden of respiratory manifestations in HIV-infected children is well documented, and our results are consistent with those of previous studies, in both high-resource and limited-resource settings [Overall, 72.5% of events were AIDS and 27.5% non-AIDS defining. Among AIDS-defining events, respiratory diseases, particularly settings ,44, inclsettings . In addisettings ; in our settings , we obseAlmost half of the hospitalizations were caused by cachexia or underweight. This is consistent with previously published data on HIV/AIDS and malnutrition. A cross-sectional study led in Kenya reported the prevalence of malnutrition in HIV-infected children to reach 40% in 2008 . AnotherAlthough ART-treated children were less affected by cachexia and underweight, 15% of ART-treated children were still hospitalized for those causes. Similar observations have been reported elsewhere, in the ARROW open label randomized trial led in Uganda and Zimbabwe, authors reported that one in nine children with advanced HIV required hospitalization for severe malnutrition after ART initiation . PossiblThe third leading cause of hospitalization in this study is malaria. Previous studies have reported protective effects of cotrimoxazole when studying HIV and malaria co-infection ; a recenOur study has several drawbacks. As we have discussed previously, this study was conducted in tertiary hospitals where the distribution of severe morbidity may differ from that in more rural settings. Furthermore, social barriers and stigma associated with HIV/AIDS often lead to delays in accessing care and receipt of HIV diagnosis. In the absence of ART, mortality is severe among infants<2 years , which cOverall, over half the children were newly diagnosed with HIV underlining the urgent need to improve access to early HIV diagnosis and treatment. Furthermore, one hospitalization in two is related to infectious diseases and one hospitalization in three was caused by a non-AIDS-related event; this proportion was higher in children on ART, reflecting a context of prevalent severe morbidity that may be beyond the threshold of ART effectiveness. In addition to ART, efforts must continue to focus on increasing the availability of cotrimoxazole prophylaxis for all in order to reduce effectively clinical morbidity in HIV-exposed and HIV-infected children.


Patient recall or the application of population norms are commonly used methods to estimate (unobservable) health status prior to acute-onset illness or injury; however, both measures are potentially subject to bias. This article reports tests of the validity of both approaches, and discusses the implications for reporting changes in health-related quality of life following acute-onset illness or injury.Recalled pre-injury health status and health status at 5- and 12-months post-injury were collected from participants in a prospective cohort study of people injured in New Zealand. Reported post-injury health status was compared with recalled pre-injury status and New Zealand norms for two groups: those who reported having fully recovered, and those who had not.There was a small but statistically significant difference between pre- and post-injury health state valuations for people who had fully recovered, with recalled pre-injury health status being higher than reported post-injury health. Perceived health status for those who had fully recovered was significantly higher than the population norm.Retrospective evaluation of health status is more appropriate than the application of population norms to estimate health status prior to acute-onset injury or illness, although there may be a small upward bias in such measurements. Generic measures of health status are designed to gauge changes in people’s health status over time such as their recovery from illness or injury. Instruments such as the Health Utilities Index, SF-6D and EQ-5D are used for deriving health state preference values for calculating Quality-Adjusted Life Years (QALYs) for use in economic cost-effectiveness analyses . This arThe EQ-5D has been included in national population health surveys in the United Kingdom, Canada, China, Finland, Spain, Denmark, the United States and New Zealand ,4. The Ne.g. cancer, stroke, injury), participants are usually recruited only after the health event has occurred. In such cases, researchers tend to adopt one of two approaches. Either they apply population norms to estimate pre-onset health, or they ask participants to ‘recall’ their pre-onset health status. Both approaches have limitations.To measure change in health status, information about pre- and post-intervention health is required. Similarly, if the focus is determining health burden borne by groups affected by particular conditions, information about health before and after the onset of the condition is required. However, in studies looking at acute-onset conditions  underway in New Zealand provides an opportunity to investigate the validity of using retrospective evaluation or population norms as proxies for pre-onset health valuation using the EQ-5D. This article reports the results of this analysis and discusses the implications for reporting changes in health-related quality of life (HRQoL) following illness or injury.The Accident Compensation Corporation (ACC) provides universal no-fault insurance for people injured in New Zealand. This article uses data from POIS, a prospective cohort study of individuals, aged between 18 and 64 years, recruited from the ACC entitlement claims register between December 2007 and June 2009 . Particii.e. reported having “completely recovered” and having attained at least their pre-injury functioning on all 12 WHODAS items.Two components of the POIS interview were used to identify “fully recovered” participants. First, participants were asked at the start of the 5-month and 12-month interviews whether they had completely recovered or were still affected by their injury. Second, they were assessed on the 12-item World Health Organization Disability Assessment Schedule (WHODAS 2.0), an instrument developed by the World Health Organization (WHO) to measure disability . The WHOAt the first interview, participants were asked to complete the EQ-5D with respect to their pre-injury health status. They also did the same with respect to their (current) health status at 5- and 12-months after injury. The New Zealand EQ-5D valuation set [To test the validity of participants’ retrospective evaluations of their health status, we compared their recalled pre-injury health with their reported health at 5- and 12-months after injury. These comparisons were performed for two groups: participants who reported having fully recovered, and others who reported having not. We hypothesised that for fully-recovered participants if their recalled pre-injury health status is unbiased then it would be the same as their post-injury status.We also compared participants’ health valuations with population norms from the survey of the New Zealand general population undertaken in 1999 from which the above-mentioned EQ-5D valuation set was derived . RespondVia the survey of the general population, 1250 respondents described their health status on the EQ-5D, of whom 964 were aged 18–64 and identified their sex, allowing for construction of adjusted population norms.As represented on the EQ-5D’s five dimensions, 2842 POIS participants recalled their pre-injury health status at the first interview, and 1475 and 2262 respectively also reported their current health status at the 5- and 12- month post-injury interviews. Fewer participants completed the second (5-month) interview, as many had completed the first interview at the time the second interview was scheduled for, due to unanticipated delays in recruitment and interviewing . Via theTable i.e. participants’ reports of having “completely recovered” and having attained at least the same WHODAS level – as each measure on its own may miss some important aspects of recovery. For example, at the 12-month interview, 970 participants reported that they had “completely recovered” and 1025 reported they had attained at least the same level of functioning as pre-injury on all WHODAS dimensions, but only 706 passed both these tests.Recovery status was reported by 1248 participants at the 5-month interview, of whom 287 (23%) were fully recovered, and by 1937 participants at the 12-month interview, of whom 706 (36%) were fully recovered. We used two measures of recovery – The most common injury types were: spine dislocation, sprain, or strain; upper extremity fracture; upper extremity dislocation, sprain, or strain; lower extremity fracture; and lower extremity dislocation, sprain, or strain. Note that the injury data record more than one injury type for many participants, so the percentages do not add to 100.The mean (unadjusted) EQ-5D health state value for the general population was 0.82. For the POIS cohort, their mean recalled pre-injury value was 0.94, falling to 0.75 and 0.78 5 and 12 months after injury respectively, where all three estimates are statistically significantly different than the general population mean (p < 0.001).If recalled pre-injury health valuation is unbiased, we would expect that: (1) pre-injury health state values are statistically the same as post-injury values for fully recovered participants, and 2) pre-injury health state values are significantly higher than post-injury values for non-recovered participants. We found a small but statistically significant positive difference for participants who had fully recovered, and a large positive difference for participants who had not fully recovered . However, there is also a possibility that recall bias may have been more pronounced in our study due to our 3-month delay between the injury event and measurement of recalled pre-injury health status.These results contrast with those of Watson and colleagues , who fouSeveral studies have found that the general population may not be representative of populations of ill or injured individuals in terms of pre-onset health status -15. Somei.e. experience with the new health state). This argument also implies that recalled pre-injury evaluation should be used instead of population norms to assess changes in health status, as the general population has not had the same injury experience as the study population.An alternative explanation for the observed difference between self-reported health and the general population norm is ‘response shift’. This is the theory that individuals’ reference points for health status valuations change as their health changes . Study pImplicit theories of memory may help to explain the small bias found in recalled health status . One impRetrospective evaluation of pre-onset health status is likely to be more appropriate than applying population norms to measure the effects of acute-onset illness or injury on HRQoL, although users of this approach should be aware of the potential for a small upward bias in such measurements.POIS: Potential Outcomes of Injury Study; SF-6D: Short Form 6-dimension health status instrument; EQ-5D: EuroQol 5-dimension health status instrument; AQoL: Assessment of Quality of Life health status instrument; QALY: Quality-adjusted life year; NICE: National Institute for Health and Clinical Excellence; PROMS: Patient Reported Outcomes; HRQoL: Health-related quality of life; ACC: Accident Compensation Corporation; WHO: World Health Organization; WHODAS 2.0: World Health Organization Disability Assessment Schedule.All authors declare that they have no competing interests that may be relevant to the submitted work.RW led the preparation of this article with support from SD, and conducted the statistical analysis. SD leads the POIS research team. PH and JL are POIS co-investigators. All authors contributed to the writing and editing of the manuscript. RW is guarantor. All authors read and approved the final manuscript.


Staphylococcus aureus (MRSA) infections during single hospitalizations and subsequent readmissions to the same institution. None have assessed the comprehensive burden of MRSA infection in the period after hospital discharge while accounting for healthcare utilization across institutions.Many studies have evaluated methicillin-resistant We conducted a retrospective cohort study of adult patients insured by Harvard Pilgrim Health Care who were newly-detected to harbor MRSA between January 1991 and December 2003 at a tertiary care medical center. We evaluated all MRSA-attributable infections associated with hospitalization in the year following new detection, regardless of hospital location. Data were collected on comorbidities, healthcare utilization, mortality and MRSA outcomes. Of 591 newly-detected MRSA carriers, 23% were colonized and 77% were infected upon detection. In the year following detection, 196 (33%) patients developed 317 discrete and unrelated MRSA infections. The most common infections were pneumonia (34%), soft tissue (27%), and primary bloodstream (18%) infections. Infections occurred a median of 56 days post-detection. Of all infections, 26% involved bacteremia, and 17% caused MRSA-attributable death. During the admission where MRSA was newly-detected, 14% (82/576) developed subsequent infection. Of those surviving to discharge, 24% (114/482) developed post-discharge infections in the year following detection. Half  of post-discharge infections caused readmission, and most  occurred over 90 days post-discharge.In high-risk tertiary care patients, newly-detected MRSA carriage confers large risks of infection and substantial attributable mortality in the year following acquisition. Most infections occur post-discharge, and 18% of infections associated with readmission occurred in hospitals other than the one where MRSA was newly-detected. Despite gains in reducing MRSA infections during hospitalization, the risk of MRSA infection among critically and chronically ill carriers persists after discharge and warrants targeted prevention strategies. It is well known that MRSA acquisition confers a high risk of infection during the same hospitalization. Studies performed in the past twenty years indicate that approximately 11–20% of patients who newly acquire MRSA develop subsequent infection during hospitalization However, less is known about the long-term risks of MRSA acquisition. This is important because post-discharge medical care is often provided through a variety of methods, such as nursing homes, rehabilitation centers, and home health care services, and hospital length of stay continues to shorten. Recent data indicate that over half of hospitalizations in the United States are 3 days or less The need to understand post-acquisition MRSA risks is magnified by widespread initiatives to identify MRSA carriers. As of 2011, fifteen states have enacted legislation involving screening, reporting, or other laws targeting MRSA carriers We sought to assess the risk of subsequent MRSA infection, hospitalization and death among high-risk inpatients newly-detected to harbor MRSA at a tertiary hospital to understand the magnitude of MRSA burden in a sick cohort. To reduce the complexity of potential differential virulence due to community-acquired versus hospital-associated MRSA strains, we studied a cohort during a time period when healthcare-associated MRSA was the dominant clone at a single institution.We conducted a retrospective cohort study to evaluate the frequency of subsequent MRSA infection and death among patients newly-detected to harbor MRSA. We identified a cohort of adult patients insured by Harvard Pilgrim Health Care who were newly-detected to have a MRSA-positive culture between January 1, 1991 and December 31, 2003 at Brigham and Women's Hospital, a 750-bed tertiary care academic medical center in Boston, MA. This study period corresponds to a time window when genetic testing (pulse field gel electrophoresis) was being performed on a representative sample of hospital MRSA strains, and nearly all strains were confirmed to be healthcare-associated clones . In addition, nares surveillance testing for MRSA was not routinely performed during the study period.Harvard Pilgrim Health Care is a full-service health benefits company serving patients throughout New England with a network of over 135 hospitals and 28,000 physicians For outcomes, all inpatient admissions in the year following MRSA detection were identified by date and hospital name, and discharge summary and microbiologic culture data were requested. If evidence of MRSA-positive cultures was found during inpatient hospitalizations, full medical records were obtained. Outpatient MRSA cultures were not assessed. Medical records were reviewed to identify the source of the MRSA-positive culture at the time of new detection and determine whether the positive culture represented colonization or infection based on Centers for Disease Control and Prevention criteria International Classification of Diseases, Ninth Revision codes from healthcare claims data in the year prior to MRSA detection. Identified comorbidities were verified through medical record review. For all patients newly-detected with MRSA during an inpatient stay, we also determined their preadmission location, hospital discharge disposition, intensive care unit admission, and length of hospital stay.For descriptive purposes, we obtained demographic, comorbidity and healthcare utilization data for all patients from Harvard Pilgrim Health Care insurance records. Healthcare utilization included acute hospital admissions, sub-acute hospital admissions, admissions to an intensive care unit, emergency room visits, outpatient visits, surgical and non-surgical procedures in the year before and after MRSA detection. Comorbidities were identified using diagnoses of heart disease, asthma, diabetes mellitus, end-stage renal disease, end-stage liver disease, and noncancer immunocompromised states based on We assessed patient characteristics as the proportion of total patients with the specified attribute. We determined the proportion of patients who were colonized or infected at the time of detection and the proportion of patients who developed any subsequent infection in the year following MRSA detection. We evaluated all-cause mortality among patients newly-detected with MRSA using both Harvard Pilgrim Health Care insurer records and the Massachusetts Mortality File. We also assessed whether deaths were associated with MRSA infection at the time of detection or during the year following detection. If deaths were associated with MRSA, we further determined whether deaths were attributable to MRSA. Death was deemed to be associated with MRSA if MRSA bacteremia was found within 7 days of death or if there was active MRSA infection at the time of death as previously described 2 tests. Variables significant in bivariate testing at a level of α<0.2 were entered into a logistic regression model. Final model variables were retained at α = 0.05.Potential predictors of subsequent MRSA infection among patients newly-detected with MRSA were assessed using χWe identified 591 patients insured by Harvard Pilgrim Health Care with an MRSA-positive culture between January 1, 1991 and December 31, 2003 at the Brigham and Women's Hospital who had no prior evidence of MRSA-positivity. In keeping with rising MRSA prevalence across the U.S., 22% (N = 123) of the cohort was identified from 1991–1995 (N = 189), 31% from 1996–1999, and 47% (N = 279) from 2000–2003. Patient characteristics are summarized in Of the 591 patients who were newly-detected to harbor MRSA, 138 (23%) were colonized and 453 (77%) were infected at the time of entry into the cohort. Among those patients who were colonized, common sources of MRSA colonization included sputum (53% of patients), nares (24%), and wounds (9%). Infections at the time of MRSA detection most often included pneumonia (41% of patients) and skin and soft tissue (19%) infections, with 18% of detection infections associated with bacteremia.2 = 7.48, p = 0.82) of cohort entry. There was also no difference in infection risk according to whether patients were colonized  or infected  at the time of new detection . Among inpatients, the risk of subsequent infection differed significantly according to whether patients were admitted from another hospital , home , or a rehabilitation or skilled nursing facility  .Overall, 196 patients (33%) developed 317 discrete and unrelated infections in the year after detection of MRSA carriage . The meaThe risk of subsequent infection during the admission in which MRSA was newly-detected was 14% (82/576). Among those surviving to discharge, the risk of post-discharge MRSA infection was 24% (114/482). These 114 patients had a total of 185 post-discharge infections. The mean time to first post-discharge MRSA infection from the time of new detection was 97 days . Over half  of post-discharge MRSA infections occurred more than 90 days after discharge, and 45 (25%) occurred more than 180 days after discharge. Overall, 21 (11%) post-discharge MRSA infections would have been missed if surveillance was not extended to hospitals other than the one at which MRSA detection was first noted. Furthermore, 99 (54%) were the cause of hospital readmission and an additional 19 (10%) were present upon hospital readmission. Among post-discharge MRSA infections associated with hospital readmission, 97 (82%) were to the same institution in which MRSA was newly-detected.2 = 0.01, p = 0.92). There was also no difference in the risk of post-discharge infection according to whether patients were discharged home , to a rehabilitation or skilled nursing facility , or to another hospital  .The distribution of pre- and post-discharge infections among patients newly-detected to harbor MRSA is shown in The vast majority of patients (85%) were re-hospitalized in the year following detection with MRSA . Among p2 = 0.14, p = 0.93). In comparison, overall annual mortality from Brigham and Women's Hospital excluding obstetric patients ranged from 1.2–2.0% during the study period.Within one year of MRSA acquisition, death occurred in 46% (269/591) of patients. Over one-third of all deaths  were associated with MRSA, and 20% (54/269) were due to MRSA. This translated into a 9.1% risk of death due to MRSA among newly-detected carriers in this cohort. Among those developing MRSA infection, 48% (94/196) died with MRSA infection, and 28% (54/196) died due to MRSA infection. The time to death from the time of detection  and discIn logistic regression modeling, male gender and age <35 and ≥85 years of age were predictive of subsequent infection with MRSA . The onlTo our knowledge, this work represents the first comprehensive assessment of MRSA infection, hospitalization and death in the year following acquisition in a high-risk tertiary care population. In this cohort, we found that one-third of patients developed subsequent MRSA infection in the year following acquisition. This risk of MRSA infection is substantially higher than those reported in prior studies While advances in infection prevention Notably, the risk of MRSA infection was not constant over the year following new detection. These data found that infection risk was highest in the 3 months following detection, remained high but to a lesser degree in the next 3 months and then plateaued in the final 6 months of the one-year follow-up. This is consistent with an elevated risk during peri-hospitalization periods, similar to what we previously reported in long-term MRSA carriers We recognize that it is possible, and even likely, that many patients in our cohort acquired MRSA long before detection. Indeed, 40% of patients had an MRSA-positive culture within 2 days of hospital admission, and 81% were hospitalized in the year prior to detection. However, as described above, we believe that MRSA infection risk among carriers may be more strongly linked to hospitalization than time from acquisition. This is supported by the fact that patients do not develop immunity to prior infecting strains of MRSA Clostridium difficile in predominantly tertiary care hospitals demonstrated a one-month all-cause mortality of 25% and attributable mortality of 7% The patients in this study reflect the case mix of a tertiary care academic medical center. It further reflects the case mix of patients who are at risk of MRSA acquisition. The combination of these two is reflected by the very high all-cause mortality one year after MRSA detection. While the risks of subsequent MRSA infection are unlikely to reflect the experience of patients who harbor MRSA in outpatient settings or community hospitals, it highlights the remarkably elevated risk in those who are critically and chronically ill. We postulate that this may reflect the risk of MRSA sequelae not only among tertiary care patient populations, but also among MRSA-positive inpatients with the identified risk factors of advanced age, diabetes, renal insufficiency, or decreased albumin, regardless of hospital type or location. The 33% one-year risk of MRSA infection and the 9% attributable risk of death due to MRSA is concerning in an aging population with rising comorbidities. It is also consistent with the invasiveness of this pathogen. This is not dissimilar to other healthcare-associated pathogens This study has several limitations. As mentioned above, it is generalizable only to high-risk MRSA carriers or institutions that care for high-risk patients. In that context, these data may still underestimate infection risk since outpatient MRSA infections were not assessed. In addition, this study describes the risk of subsequent MRSA infections among hospitalized carriers of healthcare-associated strains . Results may not apply to U.S. hospitals reporting an increasing prevalence of community-associated MRSA strains during this period In summary, we show that commercially-insured patients from a tertiary care medical center incur substantial risks of MRSA morbidity and mortality in the year following detection of carriage. One in three MRSA carriers developed MRSA infection, and one in eleven died because of MRSA infection. Nearly 60% of MRSA sequelae occurred after discharge from the hospitalization in which MRSA was newly-detected, and readmission due to MRSA infection was common. These results suggest that preventative interventions seeking to reduce MRSA morbidity and mortality should target patients newly-detected with MRSA and perform post-discharge follow-up across healthcare facilities to account for the substantial risks observed in the period after hospital discharge.


Out-of-hospital cardiac arrest (OHCA) is a common medical emergency with significant mortality and significant neurological morbidity. Helicopter emergency medical services (HEMS) may be tasked to OHCA. We sought to assess the impact of tasking a HEMS service to OHCA and characterise the nature of these calls.Retrospective case review of all HEMS calls to Surrey and Sussex Air Ambulance, United Kingdom, over a 1-year period (1/9/2010-1/9/2011). All missions to cases of suspected OHCA, of presumed medical origin, were reviewed systematically.HEMS was activated 89 times to suspected OHCA. This represented 11% of the total HEMS missions. In 23 cases HEMS was stood-down en-route and in 2 cases the patient had not suffered an OHCA on arrival of HEMS. 25 patients achieved return-of-spontaneous circulation (ROSC), 13 (52%) prior to HEMS arrival. The HEMS team were never first on-scene. The median time from first collapse to HEMS arrival was 31 minutes (IQR 22–40). The median time from HEMS activation to arrival on scene was 17 minutes (IQR 11.5-21). 19 patients underwent pre-hospital anaesthesia, 5 patients had electrical or chemical cardioversion and 19 patients had therapeutic hypothermia initiated by HEMS. Only 1 post-OHCA patient was transported to hospital by air. The survival to discharge rate was 6.3%.OHCA represents a significant proportion of HEMS call outs. HEMS most commonly attend post-ROSC OHCA patients and interventions, including pre-hospital anaesthesia and therapeutic hypothermia should be targeted to this phase. HEMS are rarely first on-scene and should only be tasked as a first response to OHCA in remote locations. HEMS may be most appropriately utilised in OHCA by only attending the scene if a patient achieves ROSC. Out-of-hospital cardiac arrest (OHCA) is a common cause of mortality and serious neurological morbidity . EffortsSurvival from OHCA is largely dependent on pre-hospital events. Survival with good neurological outcome is influenced by good quality cardio-pulmonary resuscitation (CPR)  and earlIn several countries, helicopter emergency medical services (HEMS) are tasked to attend OHCA incidents. HEMS can provide a rapid response to the scene, particularly in rural or difficult to access locations. HEMS often carry a pre-hospital doctor as part of the team who is frequently capable of administering pre-hospital anaesthesia, advanced airway interventions, inotropic support and initiating therapeutic hypothermia. Advanced post-ROSC care can be initiated pre-hospital by HEMS and the patient transported rapidly, possibly for early percutaneous coronary intervention, to hospital, particularly in cases of ST-elevation myocardial infarction . The impSurrey-Sussex HEMS provides advanced pre-hospital care to the southeast of England. Two helicopters, located at two separate bases provide pre-hospital medical and trauma care to three counties (population approximately 2.5 million) and undertakes approximately 600 missions per year. Approximately 60% of missions are for trauma and 40% for acute medical illness. The incidence of OHCA is approximately 40 per 100,000 annually. The HEMS team consists of a pilot, a pre-hospital doctor, from an anaesthesia or emergency medicine background, and a critical care paramedic. Cold intravenous saline is carried in a bespoke cool box to initiate therapeutic hypothermia in all patients who achieve return of spontaneous circulation, in whom there is no contraindication to cooling. The HEMS team can reach any point in the counties within 20 minutes of dispatch. HEMS can currently be activated to all cases of OHCA, although were death is suspected  the dispatcher may wait for further information from a land ambulance crew prior to activating HEMS. We sought to assess the impact of tasking a HEMS service to OHCA and characterise the nature of these calls.A retrospective case review of all missions conducted by Surrey-Sussex HEMS over a one-year period (1/9/2010-1/9/2011) was conducted. All missions were reviewed and those with medical OHCA as the initial dispatch were selected. Inclusion criteria were cases were HEMS attended an OHCA of non-traumatic aetiology. Exclusion criteria were HEMS stand-downs and case where the patient had not suffered a cardiac arrest or the OHCA was felt to be traumatic in origin.HEMS is activated by a designated HEMS paramedic in the ambulance dispatch centre. The HEMS paramedic was the ability to screen all calls in real time and also listen to select calls if needed. Specific dispatch criteria exist for tasking HEMS to trauma incidents but tasking to medical incidents is at the discretion of the HEMS paramedic.The ambulance dispatch log, HEMS patient record sheet and patient vital signs summary were all reviewed. Utstein data was reviewed for each patient and collated on a Microsoft Excel (Microsoft Inc) database. Descriptive statistics were used to describe the study cohort with median and interquartile range used for continuous variables. The study was registered as a service evaluation and formal ethical approval waived. Patients were followed up to hospital discharge.During the one-year research period, Surrey-Sussex HEMS attended 636 missions. Of these, 89 (14%) were to suspected OHCA. In 23 (26%) cases HEMS was stood down en-route to scene by the land ambulance crew. In these cases the patient was either not in cardiac arrest on arrival of the ambulance or pronounced dead by the land ambulance crew. In two cases (2%) the HEMS crew attended a scene and found a patient who had not suffered a cardiac arrest.Overall, 64 OHCA HEMS missions were included. The patient was male in 49 (77%) cases. Patient age ranged from 3 months to 87 years (mean= 54 years). There were 3 cases of paediatric (<16 years) OHCA.The location of OHCA was a private residence in 26 (41%) cases, public area in 34 (53%) cases and not documented in 4 (6%) cases. 39 (61%) cases were witnessed and 25 (39%) cases unwitnessed. Bystander CPR was performed in 45 (70%) cases, not performed in 11 (17%) cases and not documented in 9 (14%) cases.The initial cardiac rhythm was ventricular fibrillation in 36 (56%) cases, pulseless electrical activity in 14 (22%) cases and asystole in 14 (22%) cases.The average response time to suspected OHCA calls by land ambulance in Surrey and Sussex is less than 8 minutes with the first ambulance dispatched within 2 minutes of the initial emergency call in the majority of cases. The median time from initial emergency call to HEMS activation was 11 minutes (IQR 6–22 mins). The median response time from HEMS activation to HEMS arriving with the patient was 17 minutes (IQR 11–21 mins). The median time from initial emergency call to HEMS arriving with the patient was 31 minutes (IQR 22–40 mins). The median time from emergency call to arrival at hospital was 90 minutes (IQR 79–101 mins).In total, 25 (39%) patients achieved ROSC at-scene, 13 (20%) achieved ROSC prior to HEMS arrival and 12 (19%) post-HEMS arrival. Of the paediatric cases, 2 patients achieved ROSC, 1 did not and all were transported to hospital.All patients who achieved ROSC were sedated, paralysed and ventilated following HEMS arrival. 4 patients who achieved ROSC required drug-assisted rapid sequence induction (RSI).°C) was achieved prior to hospital arrival in 15 patients. A summary of the OHCA call and interventions performed by HEMS is shown in Figure HEMS thrombolysed two patients pre-hospital, both for suspected massive pulmonary embolism. HEMS performed chemical/electrical cardioversion in 5 patients for unstable broad complex tachycardias. Therapeutic hypothermia was initiated in 19 post-ROSC patients using cold intravenous saline and target temperature (<34All patients who achieved ROSC (n=25) were transported to hospital, 24 by land ambulance and 1 by air ambulance. Only 2 (3%) patients re-arrested during transport to hospital and both were being transported by land. Of the post-ROSC patients transported, 9 were taken directly for percutaneous coronary intervention following a post-ROSC 12-lead ECG suggestive of a primary coronary occlusive origin. Of the patients seen by HEMS, 4 (6.3%) survived to discharge from hospital.We found the HEMS response to OHCA to be predominantly for post-ROSC patients. In our setting, the interventions (CPR and defibrillation) were delivered by ambulance ground units in all cases of OHCA prior to HEMS arrival. In the majority of cases in our region, OHCA are attended by paramedics capable of performing Advanced Life Support, including intubation. The paramedics are not currently permitted to administer anaesthetic agents, muscle paralysis or initiate cooling. In geographical settings such as south east England, any delay in activating HEMS will make ground units more likely to be first on-scene, as shown in this case series. Except where geographical location may present a challenge or delay emergency medical response, HEMS activation to OHCA should be to deliver advanced post-ROSC care. Provided there is early, competent land-based emergency medical intervention, activation or landing at the scene of an OHCA, with the associated risks to medical crew, public and aircraft, may be best reserved for OHCA patients who have achieved ROSC. In our study HEMS were never the first emergency medical response to reach an OHCA patient. There are theoretical benefits of HEMS providing early critical care intervention, such as pre-hospital anaesthesia and initiating prehospital therapeutic hypothermia but further research is warranted to evaluate the impact of these interventions on outcome.Landing a helicopter at the scene of OHCA is not without risk. In systems were ground and air-based emergency medical services operate together, it may be appropriate for direct communication to occur between the ground and air teams, with HEMS only attending the scene in cases of ROSC, unless a specific HEMS intervention is required.Previous studies examining the role of HEMS in OHCA have come to similar conclusions. Skogvoll et al. suggested HEMS may contribute to improved survival through enhanced post-ROSC care . HospitaThere is little evidence to support transporting OHCA patients by air who remain in cardiac arrest, except in special circumstances, for example hypothermic patients. Quality of cardiopulmonary resuscitation has been shown to be poor during aeromedical transport , but thePost-ROSC, HEMS can facilitate rapid transport for PCI, particularly if the ground transport time is long. HEMS transport of post-ROSC OHCA patients has previously been suggested to improve outcome  but thisThis study has several limitations, namely the retrospective nature of the case review and the relatively small number of patients included. It was not possible to quantify the impact of specific HEMS interventions on patient outcome. However, the results suggest a clear outcome that HEMS is likely to only be beneficial to post-ROSC patients, except in a few select cases. In the absence of any formal OHCA dispatch criteria, HEMS are also likely to be dispatched to a select subgroup of OHCA. These are likely to include witnessed, young age, high prevalence of VF as the presenting rhythm or OHCA occurring in remote locations.OHCA represents a significant proportion of HEMS call outs. HEMS most commonly attend post-ROSC OHCA patients and interventions, including pre-hospital anaesthesia and therapeutic hypothermia should be targeted to this phase. HEMS are rarely first on-scene and should only be tasked as a first response to OHCA in remote locations. OHCA patients in this study rarely re-arrested and transport to hospital by air should not be precluded by fear of re-arrest. HEMS may be most appropriately utilised in OHCA by only attending the scene if a patient achieves ROSC.HEMS: Helicopter emergency medical services; IQR: Inter-quartile range; OHCA: Out-of-hospital cardiac arrest; PCI: Percutaneous coronary intervention; ROSC: Return-of-spontaneous circulation; RSI: Rapid sequence induction.The authors declare that they have no competing interests.Both authors contributed to data collection, analysis and manuscript preparation. Both authors read and approved the final manuscript.