Due to their rarity and diversity, sarcomas are difficult to diagnose. Consequently, there is an urgent demand for a novel diagnostic test for these cancers. In this study, we investigated serum miRNA profiles from 1002 patients with bone and soft tissue tumors representing more than 43 histological subtypes, including sarcomas, intermediate tumors, and benign tumors, to determine whether serum miRNA profiles could be used to specifically detect sarcomas. Circulating serum miRNA profiles in sarcoma patients were clearly distinct from those in patients with other types of tumors. Using the serum levels of seven miRNAs, we developed a molecular detector, Index VI, that could distinguish sarcoma patients from benign and healthy controls with remarkably high sensitivity (90%) and specificity (95%), regardless of histological subtype. Index VI provides an approach to the early and precise detection of sarcomas, potentially leading to curative treatment and longer survival. Sarcomas are rare malignant tumours of bone and soft tissue whose diagnosis remain difficult. Here, the authors analyse serum samples from over 1000 patients and using separate discovery, training and validation cohorts, identify and validate a 7-microRNA index that distinguishes malignant sarcomas from benign disease. Benign tumors do not spread beyond the original site  and are not life-threatening. Intermediate tumors are defined as locally aggressive but nonmetastasizing or rarely (<5% of cases) metastasizing, and most cases require surgical resection for local control. On the other hand, the malignant tumors of bone and soft tissue known as sarcomas constitute an extremely rare heterogeneous group of cancers that account for 1% of adult and 15% of pediatric neoplasms; these cancers are highly metastatic and often life-threatening3.Bone and soft tissue tumors are rare diseases that arise from mesenchymal tissues and exhibit wide variation in histologic type and malignant grade. They are classified into three major categories: benign, intermediate, and malignant2. The current 5-year survival rate of patients with sarcoma is approximately 50–60%, depending on histological grade and tumor depth, size, and stage5. Due to the low degree of public and professional awareness of these malignancies, bone and soft tissue sarcomas are often diagnosed at late stages7. Moreover, due to their rarity and diversity, and despite their classification into the aforementioned malignancy categories, the diagnosis of sarcomas remains difficult8. Hence, to improve the prognosis of patients with sarcoma, early detection is essential.Despite recent advances in precision medicine and improvements in patient prognosis for several types of cancer, few advances have been made in the treatment of sarcomas. The development of molecular targeted therapy for sarcomas is hindered by the fact that these tumors are classified into more than 50 histological subtypes, and the number of cases included in studies has been limitedCurrently, histopathological examination by specialists is the only method available for the accurate diagnosis of these tumors, and no biomarkers have been identified to date. To achieve early and accurate diagnosis of sarcoma, it will be necessary to develop rapid, noninvasive, and straightforward diagnostic methods. The ideal biomarkers for this purpose should have the capacity to detect sarcomas regardless of their histological subtype. To this end, it is necessary to develop a biomarker identification strategy based on a novel principle.10. miRNA expression signatures are tumor type-specific and enable the classification of tumors by origin12. Certain subsets of miRNAs are aggressively secreted from cancer cells into the extracellular space via multiple mechanisms, including microvesicle (MV)-mediated pathways14. In light of these biological features of miRNAs, the profile of extracellular miRNAs, also known as circulating miRNAs, is expected to be cancer type-specific and reflect tumor origin16. Accordingly, the use of circulating miRNA profiles warrants further investigation as a strategy for overcoming the challenges associated with diagnosis of sarcomas.MicroRNAs (miRNAs) are a class of small noncoding RNAs that act as endogenous regulators of gene expression. The tissue-specific expression pattern of miRNAs is important for the precise regulation of cell differentiation and tissue development, and alterations in these processes are involved in the pathogenesis of cancer16; however, no consensus has emerged regarding which miRNAs are most suitable for clinical application, nor is there any standard method for collection of serum samples or preparation and detection of miRNAs. The use of circulating miRNAs in the clinic requires large comprehensive analyses of several cancer types, using standardized platforms for serum miRNA collection and detection. We recently launched a national project in Japan, entitled Development and Diagnostic Technology for Detection of miRNA in Body Fluids. This project entails the comprehensive characterization of serum miRNA profiles in more than 10,000 patients with 13 types of human cancer, including sarcomas, using the same platform and technology to evaluate each sample. The ultimate goal of this project is the development of diagnostic methods for use in clinical examinations that are capable of detecting and classifying patients according to cancer type by analyzing the circulating miRNAs in single blood samples.The use of serum miRNAs as markers for cancer diagnosis has recently been exploredHere, we describe our findings regarding the identification of promising biomarkers for the diagnosis of sarcoma. In particular, we developed Index VI, calculated from the levels of seven serum circulating miRNAs, which could discriminate among the various histological subtypes of sarcoma. Our findings provide a solution to the daunting challenges associated with the diagnosis of sarcoma.n = 414), intermediate tumors (n = 144), and benign or nontumors (n = 339).We obtained 1002 serum samples derived from patients with bone and soft tissue tumors. After excluding five patients with uterine sarcoma, 24 patients who had undergone treatment before serum collection, and 76 low-quality samples, we analyzed the miRNA profiles in material from 897 patients, including serum samples from patients with malignant bone and soft tissue tumors (sarcomas) (P > 0.01 after Bonferroni correction for multiple testing)  Fig. . Serum sg) Table . Therefo6 in more than 50% of the analyses in the malignant or benign tumor group in the discovery set were defined as abundant serum miRNAs. Unsupervised cluster analysis identified differences in circulating miRNA profiles between primary sarcomas (n = 311) and benign tumors (n = 327)  Fig. . This ef27) Fig. . Serum m27) Fig.  and PCA 27) Fig. . These rA total of 83 miRNAs exhibited high discrimination performance between sarcomas and benign tumors, based on leave-one-out cross-validation in the discovery set (Supplementary Table P < 0.05) . The results, including sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and area under the receiver operating characteristics (ROC) curve (AUC) are shown in the upper row of Table P = 0.02). A similar trend was observed in malignant soft tissue tumors  and the benign group (62.0%) Fig. . Hence, 17  and sarcoma tissues (osteosarcoma and dedifferentiated liposarcoma). Although the serum levels of all seven miRNAs were higher in malignant samples, none were significantly upregulated in sarcoma  contains a rare cancer center, and many patients with rare cancers such as sarcoma were recruited from all over Japan. Serum samples were obtained consecutively from 1002 patients with bone and soft tissue tumors who were admitted or referred to the NCCH between 2007 and 2013. All serum samples were frozen at −20 °C within 12 h of collection using serum collection tubes . Patients with uterine sarcoma and those treated with chemotherapy or radiotherapy before serum collection were excluded, as were patients with poor-quality microarray data.Control serum samples were obtained from healthy volunteers aged >35 years who were recruited from the Yokohama Minoru Clinic in 2015. The inclusion criteria for this cohort were no history of cancer and no hospitalization during the previous 3 months. Serum samples were stored at −80 °C before use.n = 30 for each) randomly selected from among patients admitted or referred to the NCCH between 2008 and 2013 and registered in the National Cancer Center Biobank. Serum samples were stored at −20 °C until use.Serum samples were obtained from patients with eight types of cancer  and the Research Ethics Committee of Medical Corporation Shintokai Yokohama Minoru Clinic (6019-18-3772). Written informed consent was obtained from each participant.http://www.mirbase.org/).Total RNA was extracted from 300 µL of serum using the 3D-Gene® RNA extraction reagent . One-half of the amount of extracted total RNA was prepared for comprehensive miRNA expression analysis using the 3D-Gene® miRNA Labeling kit, and then analyzed on the 3D-Gene® Human miRNA Oligo Chip , which was designed to detect 2555 miRNA sequences registered in miRBase release 20 or 2565 miRNA sequences registered in miRBase release 21 ] of the negative control signals, from which the top and bottom 5%, ranked by signal intensity, were removed. Once an miRNA was considered present, the mean signal of the negative controls  was subtracted from the miRNA signal. When a signal value was negative (or undetected) after background subtraction, the value was replaced by the lowest signal intensity on the microarray minus 0.1 on a base-2 logarithmic scale. To normalize the signals among microarrays, three preselected internal control miRNAs , which had been stably detected in more than 500 serum samples, were used. Each miRNA signal value was standardized using the ratio of the average signal of the three internal control miRNAs to the preset value. All microarray data of this study were in agreement with the Minimum Information About a Microarray Experiment (MIAME) guidelines.−ΔCt method was used to compare normalized expression levels. Only 28 of the 33 candidate miRNAs were analyzed because miScript PCR primers for miR-4488, miR-1908-3p, miR-1292-3p, miR-6088, and miR-4492 were not commercially available.Total RNA was extracted using the same methods as for microarray analysis and subjected to quantitative RT-PCR (qRT-PCR) analysis. Ten samples from patients with malignant bone or soft tissue sarcoma and ten samples from patients with benign bone or soft tissue tumors were selected and compared. MiRNA expression levels were investigated using miScript Primer Assays , training, and validation cohorts (between 2007/1 and 2011/6). The training and validation cohorts were randomly and blindly divided. The discovery cohort was used to select significant miRNA markers; the training cohort was used to validate miRNA markers and identify candidates for discriminant models; and the validation cohort was used to validate the discriminant models.2 scale). The representative SD was approximately 1 on a log2 scale, as shown in Supplementary Table Because the samples were chronologically divided into three cohorts, the sample size for each cohort could not be determined based on a prespecified effect size. To use serum miRNAs as discrimination biomarkers, the required minimum difference between two groups was 0.5  of each leave-out sample and dividing by the number of repeats . MiRNAs with cross-validation scores >0.6 were selected.Using the discovery cohort, only miRNAs with a signal value >2P < 0.05 by Student’s t test) were included in the subsequent analysis.In the training cohort, the levels of the selected miRNAs were subjected to cluster analysis. Thus, clusters in which the component miRNAs were upregulated or downregulated in a step-by-step manner among healthy, benign, and malignant clusters were identified. Candidate miRNAs were further narrowed down by qRT-PCR analysis of ten malignant and ten benign samples (training set 2). Serum miRNAs that were differentially expressed between two groups , compute.es package version 0.2-4, glmnet package version 2.0-3, hash package version 2.2.6, MASS package version 7.3-45, mutoss package version 0.1-10, and pROC package version 1.8. Unsupervised average linkage clustering, heatmap generation with Euclidean distances, and PCA were performed using Partek Genomics Suite 6.6 . Other statistical analyses, including logistic regression analysis, were performed using IBM SPSS Statistics version 22 . A P value < 0.01 was considered statistically significant.Univariate or age- and sex-adjusted odds ratios were calculated by logistic regression analysis. Discrimination analyses were performed using R version 3.1.2 (R Foundation for Statistical Computing, Additional details are included in Supplementary Information.Further information on experimental design is available in the Peer Review FileSupplementary InformationReporting Summary


India faces a high burden of child undernutrition. We evaluated the effects of two community strategies to reduce undernutrition among children under 3 years in rural Jharkhand and Odisha, eastern India: (1) monthly Participatory Learning and Action (PLA) meetings with women’s groups followed by home visits; (2) crèches for children aged 6 months to 3 years combined with monthly PLA meetings and home visits.We tested these strategies in a non-randomised, controlled study with baseline and endline cross-sectional surveys. We purposively selected five blocks of Jharkhand and Odisha, and divided each block into three areas. Area 1 served as control. In Area 2, trained local female workers facilitated PLA meetings and offered counselling to mothers of children under three at home. In Area 3, workers facilitated PLA meetings, did home visits, and crèches with food and growth monitoring were opened for children aged 6 months to 3 years. We did a census across all study areas and randomly sampled 4668 children under three and their mothers for interview and anthropometry at baseline and endline. The evaluation’s primary outcome was wasting among children under three in areas 2 and 3 compared with area 1, adjusted for baseline differences between areas. Other outcomes included underweight, stunting, preventive and care-seeking practices for children.We interviewed 83% (3868/4668) of mothers of children under three sampled at baseline, and 76% (3563/4668) at endline. In area 2 (PLA and home visits), wasting among children under three was reduced by 34%  and underweight by 25% , with no change in stunting . In area 3, , wasting was reduced by 27% , underweight by 40% , and stunting by 27% .Crèches, PLA meetings and home visits reduced undernutrition among children under three in rural eastern India. These interventions could be scaled up through government plans to strengthen home visits and community mobilisation with Accredited Social Health Activists, and through efforts to promote crèches.ISCRTN89911047 on 30/01/2019.The evaluation was registered retrospectively with Current Controlled Trials as The online version of this article (10.1186/s12889-019-7274-3) contains supplementary material, which is available to authorized users. Anganwadi workers) and the Ministry of Health and Family Welfare . Unfortunately, levels of child undernutrition remain high despite these workers’ efforts, especially among underserved communities. Children from Scheduled Tribe families, in particular, have the highest prevalence of stunting in the country (44%). [how to deliver nutrition interventions in such underserved communities.In India, 38% of children under five are stunted and 21% are wasted. [In this study, we report results from Action Against Malnutrition (AAM), a civil society-led, community-based initiative to supplement the efforts of frontline health and nutrition workers in seven blocks  of Jharkhand, Odisha, Bihar and Chhattisgarh. AAM included two community strategies to reduce undernutrition among children under 3 years: monthly Participatory Learning and Action (PLA) meetings with women’s groups followed by counselling through home visits; (2) crèches for children aged 6 months to 3 years combined with PLA meetings and home visits. We evaluated the effect of these two strategies on child wasting, underweight, stunting, infant and young child feeding, illness and care during illness, as well as infection control practices.Adivasi  communities disproportionately affected by undernutrition [The AAM initiative was implemented between July 2012 and March 2017. It covered seven blocks in seven districts of four states: three blocks in Jharkhand, two in Odisha, one in Chhattisgarh, and one in Bihar. These four states have high levels of chronic child undernutrition: 45% of children in Jharkhand are stunted, 34% in Odisha, 38% in Chhattisgarh and 48% in Bihar. The prevalence of acute undernutrition is also high: 29% of children in Jharkhand are wasted, 20% in Odisha, 23% in Chhattisgarh and 21% in Bihar. In addition, several districts of Jharkhand, Odisha and Chhattisgarh have a high proportion of families from AAM supported one community-based facilitator to conduct Participatory Learning and Action (PLA) meetings and home visits in a catchment area of approximately 5000 population, and two workers for crèches open to children aged 6 months to 3 years.The PLA intervention was a structured cycle of participatory women’s groups meetings convened by a local female facilitator. The cycle of meetings had four phases. In the first phase, women identified and prioritised problems related to undernutrition among children under three  using picture cards. They then identified the underlying medical and social causes for these prioritised problems through story-telling and discussions, before prioritising locally feasible strategies to address these problems. At the end of this phase, each group organised a meeting with the wider community to seek support for its chosen strategies. In the third phase, groups implemented their strategies and learned about practical actions to try at home and in the community (e.g. methods to enrich complementary foods or clearing stagnant water ponds). In the fourth phase, each group evaluated the meeting cycle. This intervention was an adaptation of an approach previously tested in Jharkhand and Odisha to improve birth outcomes. –8 SimilaAnganwadi workers; those who recently had an illness; those who lived in hamlets; and those who had recently been discharged from a Malnutrition Treatment Centre or Nutritional Rehabilitation Centre. All facilitators received a total of 12 days of training on the PLA meeting cycle and counselling. They subsequently had fortnightly review meetings with supervisors (one supervisor for 15 facilitators), during which they discussed problems and solutions to issues encountered during counselling. Each facilitator received an incentive of 3500 Indian rupees (US $ 60) per month, i.e. a little more than the 3000 INR salary paid to Anganwadi workers at the time. She was responsible for conducting 8–10 monthly PLA meetings as well as visiting 25–35 mothers of children under three in an area covering around 5000 population.The PLA facilitator also provided counselling to mothers of children under three through home visits. We chose to do home visits because a concurrent study suggested that mothers appreciated the additional conversations, demonstrations and follow-up after PLA meetings, and because home visits are recommended in World Health Organisation (WHO) tools for the Integrated Management of Childhood Illness and Infant and Young Child Feeding counselling , 12. DurAAM selected crèches as an additional, more intensive intervention to prevent undernutrition because they offered the possibility of co-locating several services: free care in a safe, smoke-free environment with clean drinking water, handwashing stations, nutritious food, growth monitoring, and psychosocial stimulation.Children in crèches received one full meal and two snacks per day, as well as two eggs per week. Meal and snack preparation were supervised to ensure caloric and protein sufficiency. The programme was available to every child in the village irrespective of their nutritional status. Within this ‘universe’ however, a special focus was given to crèche-going children who did not gain weight or whose weight decreased over two consecutive months, our operational definition of growth faltering. Children whose growth faltered and those with severe underweight were given two additional calorie-dense and protein rich meals per day. We also developed a protocol for persistent growth faltering which involved home visits and medical referrals, as described in more detail elsewhere .Anganwadi workers. Like PLA facilitators, crèche workers received 3500 INR per month. Community members participated in the selection of workers, decided on opening hours and designed the food menu. They also helped fence, repair, and decorate crèches, took part in crèche committee meetings, and sometimes contributed vegetables from their homestead gardens. In four villages, community members built the crèches themselves.Each crèche was run by two local, trained workers who were not We conducted a non-randomised, controlled  study with cross-sectional baseline and endline surveys to evaluate the effects of the two incrementally intensive strategies on child wasting, underweight, stunting, as well as infant and young child feeding, illness and infection control outcomes. The AAM consortium partners selected five blocks out of the seven participating in the programme for the evaluation: three blocks in Jharkhand  and two in Odisha (Thakurmunda and Saharpada). The block from Bihar was excluded because ensuring adequate separation between intervention and control areas proved too difficult. The block from Chhattisgarh was excluded as crèches were scattered across too large an area, making data collection highly challenging.To minimise the bias caused by geographical variability, we sought to compare the effects of interventions between areas within the same blocks. Each block was divided into three zones designated as areas 1, 2 and 3. Area 1 covered approximately 20% of each block and received regular government programmes without any intervention from AAM. For the purpose of the evaluation, area 1 was therefore designated as a control area. Area 2 covered approximately 65% of each block and received PLA meetings and counselling through home visits. Area 3 covered approximately 15% of each block and received PLA meetings, counselling through home visits, and crèches. AAM started a total of 116 crèches across the five blocks: 40 in Odisha and 76 in Jharkhand. AAM partners deliberately selected areas that had a high proportion of tribal families to open crèches in order to provide the greatest inputs to the most underserved.Anganwadi centres, and number of health sub-centres. Using this method, a total of 30 population clusters were selected from the five evaluation blocks, or six clusters per block: 10 in Area 1, 10 in Area 2 and 10 in Area 3. The estimated total population in the three evaluation areas was 144,000.Within each of the five evaluation blocks, we mapped population clusters of 8–10 contiguous villages (c.4000–5000 population each). Figure Study participants were all children under 3 years and their mothers identified during a household census in the study clusters, then selected via simple random sampling as described in the sampling section below. There were no exclusion criteria.The primary outcome for the evaluation was wasting among children under 3 years, as we expected interventions primarily focused on immediate and underlying determinants of undernutrition to have a larger effect on acute rather than chronic undernutrition. Secondary outcomes included underweight, stunting, Infant and Young Child Feeding (IYCF) practices, infection control practices, and uptake of nutrition and health services.We estimated the baseline prevalence of wasting in the study areas at 23% using district-level data from the Hungama study . We hypoWe used the 2011 Indian Census and Crude Birth Rate data from the Annual Health Survey (2011) for Jharkhand and Odisha to estimate the total number of children under three across the three study areas. The CBR for Jharkhand and Odisha were 25 and 23; we used a conservative estimate of 22 and estimated that we would find 9504 children under 3 years across all three study areas. To reach our target sample size of 4668 children, we sampled one out of two children under three in the study areas. For both the baseline and endline surveys, we first carried out a household census in all clusters to identify all children under three, then used simple random sampling to select children for anthropometric measurements.We conducted the cross-sectional baseline survey from October to March 2012–13 and the endline survey from October to April, 2015–16. Additional file We collected data on the costs of all AAM interventions from each of the implementing agencies that supported them on an annual basis through a standardised tool. All costs were adjusted for inflation, discounted at 3% per year and converted to 2017 United States Dollars (USD).Data were entered in a MS-Access database in each district, then cleaned using checks on range and frequencies. We used descriptive statistics to explore the socio-demographic characteristics of women and children and the macro z score in Stata 13.0 to generate z scores for anthropometry . In addi, to adjust all analyses for district, tribe/caste status, asset quintiles and clustering. We created these asset quintiles using Principal Component Analysis. We also selected additional variables for adjustment by identifying household, maternal and child characteristics that differed significantly (p < 0.05) between areas at baseline and endline. We checked how strongly these factors were associated with each other and dropped two of them  because they were collinear with each other and with asset quintiles. We estimated intervention effects using the difference in difference method, and effect sizes are presented as adjusted odds ratios. We adjusted for baseline differences by introducing an interaction term between area and survey wave (baseline or endline) in logistic regression models.Analyses were by intention to treat, meaning that children were included in the analyses if they participated in baseline or endline surveys, whether they took part in the interventions or not. We used logistic regression with random effects at cluster-level in Stata 13.0 to analyse data on binary outcomes (e.g. wasting), and generalised estimating equations (GEE) with an exchangeable correlation for continuous outcomes (e.g. weight-for-height z scores). We decided, a prioriWe obtained ethical approval for the study from the Institutional Ethics Committee of the Public Health Resource Network (PHRN) in Delhi on the 14th of February 2012, and from an independent ethics committee convened by Ekjut in Ranchi (Jharkhand) on the 10th of May 2013. The survey team requested consent for participation in the survey and anthropometry from mothers. This was recorded in writing or through a thumbprint impression.In the baseline survey, we found and measured 88% (1365/1556) of our target sample of children under 3 years in area 1, 80% (1248/1556) in area 2, and 68% (1255/1556) in area 3. In the endline survey, we measured 75% (1168/1556) of our target sample in area 1, 76% (1255/1556) in area 2, and 73% (1140/1556) in area 3.p-value for differences between areas < 0.001). Between 48 and 71% of families had MNREGA job cards (p < 0.001) that guaranteed 100 days of waged employment for every household whose adult members volunteered to do unskilled manual work. The proportion of mothers from Scheduled Tribes ranged from 61% in area 1 (control) to 83% in area 3 (p < 0.001). Between 54 and 60% of mothers had ever been to school (p < 0.001). We found differences in the mean ages of children who participated in the survey between areas at baseline (p = 0.017) but none at endline (p = 0.242).Table We measured exposure to the interventions: 55% (653/1177) of children in area 3 at endline had ever attended a crèche. Sixty-five percent (815/1256) of mothers of children under three had ever attended a PLA meeting in area 2, as had 72% (853/1177) of mothers in area 3.p = 0.004) and the odds of underweight by 25% , with no change in stunting  compared to control. In area 3 , the odds of wasting were reduced by 27% , the odds of underweight by 40% , and the odds of stunting by 27%  when compared to area 1 (control). Using a composite index of anthropometric failure  we found that children in Area 3 had a reduced odds of anthropometric failure , but not children in Area 2 , p = 0.617) [Table = 0.617) .Table 2EWe conducted additional sensitivity analyses on continuous z scores to check whether the interventions’ effects on anthropometry were present for weight-for-age (WAZ), weight-for-height (WHZ), and height-for-age (HAZ) z scores. These analyses are presented in Additional file Table Table Being in areas 2 or 3 was not associated with changes in the timely introduction of complementary feeding, though numbers in the 6–9 months age group were small. Figure Anganwadi workers and less likely to be weighed by them in area 3. We found a significant reduction in the proportion of children with Severe Acute Malnutrition in area 2 but no significant reduction in area 3, as consistent with our overall results for wasting.Children in areas 2 and 3 were less likely to receive food from The total and average annual costs of all interventions were USD 1,415,816 and USD 471,939 in area 3 and USD 500,931 and USD 166,977 in area 2, respectively. The estimated average annual costs of the intervention per child under three were USD 82 in area 3 and USD 7.5 in area 2. If one considers beneficiaries to be all those living in the study areas, the average annual costs of the intervention were USD 5.5 and USD 0.5 per person covered in area 3 and area 2, respectively.Our quasi-experimental study found reductions in the prevalence of wasting, underweight and stunting among children under three in areas with crèches, PLA meetings and home visits. We also found reductions in the prevalence of wasting and underweight in areas with PLA meetings and home visits only. Effects in both areas were greatest among the most marginalised children. Further analyses of continuous z scores confirmed significant effects on WAZ and HAZ in area 3, and smaller, non-significant effects on WHZ and WAZ in area 2. There were significant improvements in several but not all infant feeding and infection control practices in both intervention areas (with and without crèches).How do the effects of AAM interventions compare with those of other strategies? Global systematic reviews of breastfeeding promotion, complementary feeding education and food supplementation suggest that these can have small to moderate effects on children’s nutritional status, depending on background levels of food security –19. ThisThe existing evidence confirms that interventions to improve infant and young child feeding with no additional food or micronutrient supplementation will have only modest effects on children’s nutritional status in food insecure areas, and must be combined with interventions to control infections, enhance children’s diets, and improve women’s health and nutrition in the prenatal period . This reThe Government’s Integrated Child Development Services focus on providing food supplementation to pregnant women and children, but this remains insufficient to stem the tide of undernutrition. Crèches can provide additional supplementation and co-benefits for caregivers and children in underserved areas. Our study is one of only three experimental and quasi-experimental evaluations of crèches from India to date. A recently completed trial from rural Udaipur, Rajasthan, found a small (4%) reduction in the prevalence in child wasting 1 year after the introduction of affordable day care centres for children aged one to six years . A quasiThe effects seen on the prevalence of stunting and continuous HAZ in areas with crèches may be explained by the benefits of safe water and a smoke free environment, children’s handwashing routine, their long exposure to crèches if they attended from 6 months to 3 years, and the increased consumption of protein-rich foods, including eggs. A recent trial of egg supplementation once a day for 6 months in children aged 6–9 months in Ecuador found that eggs increased length-for-age z score by 0.63 (95 CI: 0.38–0.88) and WAZ by 0.61 (95% CI: 0.45–0.77) .How can we explain that significant effects were found on the prevalence of wasting and underweight in area 2, but not on continuous z scores? We think there are three possible reasons for this: interventions may have averted some cases of wasting without significantly changing the overall distribution of WHZ; our adjusted difference-in-difference analysis may have had different implications for binary and continuous variables; differences in wasting may have been due to chance or measurement error. We are more confident about the effects of interventions on underweight, as we found fewer WAZ values flagged as implausible in our dataset than HAZ or WHZ values. Nevertheless, the trends of increase in WAZ and WHZ in area 2 are consistent with the reductions in underweight and wasting, and effects on WAZ and HAZ in area 3 were strongly significant.The effects of AAM interventions among the most marginalised children merit further explanation. The AAM initiative selected poor districts within poor states, and provided a universal opportunity to participate voluntarily in meetings that used visual materials and stories resonating with the poorest mothers, as well as free, universal access to crèches with extra support for children with growth faltering. The objective was to shift the distribution of risk within a population rather than simply targeting high-risk individuals . The comThe cost of running crèches was higher than that of other AAM activities because of infrastructure and supplementation costs, and because crèches’ extra support for monitoring, supervision and research was included as indirect programme cost. As crèche programmes grow and run to full capacity, one might expect the costs per child to come down. Crèches may also provide several co-benefits: more mothers may be able to rest, engage in income-generation activities, and reduce their expenditure on food and care. Another substantial benefit may arise through long-term gains in children’s development. A 2012 systematic review found large, positive effects of crèches on child development outcomes, but with no studies from India . A recenOur quasi-experimental study had several strengths. It is one of only three studies that attempted to quantify the potential contribution of crèches to reducing undernutrition among children under 3 years in India. It also benefitted from a concurrent control area. The study also had limitations. First, the selection of districts, blocks and clusters in each state was purposive, and we were unable to include data from Bihar and Chhattisgarh. Although this limits the generalisability of our findings, data from Jharkhand and Odisha provide evidence for other, similar areas with high levels of child undernutrition. Second, after two attempted visits, we could interview around 75% of eligible respondents in the endline survey; we may therefore have missed more vulnerable mothers who migrated or worked for substantial periods of time outside their homes. Third, it is possible that mothers’ responses to questions about behaviours were influenced by a desire to give desirable answers following exposure to the interventions or routine ICDS activities. This would have led us to overestimate the benefit of interventions for feeding and infection control practices. Finally, we did not randomise allocation to the different interventions, and deliberately selected poorer areas with more tribal families to establish crèches. We therefore cannot rule out effects of selection bias as well as seasonal and secular changes, although our adjustments for variables that differed by area at baseline, difference in difference analyses, and the fact that baseline and endline surveys were carried out in the same season all attempted to mitigate this.What do our findings mean for policy and practice? The interventions tested in this study could be scaled up through several pathways. Since 2018, the National Health Mission recommends home visits by ASHAs to improve the care of infants and young children. ASHAs receive 3 days of training and incentives to visit caregivers at home 3, 6, 9, 12 and 15 months after birth. During these visits, they counsel caregivers on responsive feeding, immunisations, as well as play and communication for early childhood development . Our stuFuture research on integrated community interventions such as AAM should assess their cost-effectiveness, effects on children’s development, maternal mental health, and household expenditure as key potential co-benefits of these interventions.Crèches, monthly PLA meetings and counselling through home visits reduced undernutrition among children under three in rural districts of eastern India. These interventions could be scaled up through government’s existing plans for home visits and community mobilisation with ASHAs and ASHA facilitators, and through efforts to support crèches.Additional file 1:Action Against Malnutrition Survey. (PDF 308 kb)Additional file 2:Effects of interventions on children’s weight-for-height, weight-for-age and height-for-age z scores. (PDF 34 kb)Additional file 3:Dataset. (CSV 3605 kb)


Achatina fulica) is one of the most popular pet snails worldwide. This gastropod mollusk is known as the intermediate host of several parasites that cause severe diseases in pets, free-ranging vertebrates, and humans. The aim of this survey was to investigate the occurrence of parasites in giant African land snails kept as pets in southern Italy. Fresh fecal samples were collected from a total of 60 giant African land snails kept in three private collections in Campania region . In addition, microscopic analysis of mucus and histological examination of biopsy samples from the foot muscle of 30 individual snails were performed. Coprological examination revealed the presence of rhabditid nematodes identified by both morphological and molecular assessment as Rhabditella axei in two out of three examined samples, and Rhabditis terricola, Cruznema sp., and Pristionchus entomophagus in one coprological sample. No parasites were detected in the muscle biopsy samples, or in the mucus. Due to the potential harm of rhabditid nematodes, their presence in giant African land snails potentially in contact with both pets and humans should not be disregarded.Gastropods comprise nearly 60,000 species of aquatic as well as terrestrial mollusks, primarily snails and slugs. The giant African land snail ( Achatina fulica, syn. Lissachatina fulica) is native to East Africa, however it is a widespread invasive species in Asia, Oceania, and more recently in Americas, where it has been introduced accidentally or purposefully as a food source and as a pet. Its release in natural ecosystems, agricultural, and urban areas has resulted in ecological, health, and agricultural threats , were investigated for parasites. From each locality we obtained a pool of feces from 20 individual snails each. The snails included in this survey had a median age of 1.6 years ranging from 0.2 to 2 years and had not received any previous anti-parasitic treatment. Snails were fed fresh vegetables and fruits; additionally, a calcium powder supplementation  was provided twice a week. All animals were bred in Italy, privately owned and kept as pets in four indoor terrariums of 60 × 30 × 45 cm in size, in groups of 10–20 animals (2 groups of 20 snails in Pozzuoli and Caserta and 2 groups of 10 snails each in Naples). An organic peat soil , previously heat-treated (100°C for 30 min) and then free of parasites and insects, was used as substrate.Twenty grams of fresh feces were initially obtained from each of the four terrarium. When pooled feces analyzed by fresh smear, flotation and Baermann test showed positivity to rhabditid nematodes, two snails from each positive terrarium were housed individually in a sterile plastic box, and their feces were collected, immediately after defecation, in a sterile 50 ml plastic tube. Coprological examination for pooled and individual samples included fresh smear, centrifugal flotation (2 g of feces for each test) using a solution of sugar and formaldehyde (specific gravity 1.27) and Baermann test (10 g of feces for each test). Additionally, the mucus obtained during fresh smear examination was analyzed under a light microscope. Because the larval forms of several helminth species may encyst inside the foot muscle of snails, histological examination of biopsy samples harvested from the anterior and posterior areas of the foot muscle was performed in 30 snails, anesthetized with the technique described by Giannelli et al.  and GilbTemporary mounts were made by heat-killing nematodes on glass slides in a drop of water, after which a glass cover slip was applied. Nematodes were transferred into a drop of tap water on a glass slide and placed on heater (100°C) for 10 s. These specimens were used for morphological identification. An AMPLIVAL light microscope, Carl Zeiss Jena, and a Leitz Diaplan with Nomarski optics were used for observation. Morphological identification of rhabditid nematodes followed Andrassy  and Andr2O) overnight, prior to the molecular analysis, for complete removal of residuals of ethanol. Each individual nematode was transferred into a sterile Eppendorf tube (200 μl) with 20 μl of extraction buffer . Buffer and nematodes were frozen at −20°C for 20 min, and then immediately incubated at 65°C for 1 h, followed by 10 min at 95°C. The lysates were cooled on ice and then centrifuged ; 1 μl of supernatant was used for PCR.DNA was extracted from nematode specimens obtained from feces fixed in 96% ethanol collected from the three localities. Nematodes were washed in double-distilled water , 1 μl; 0.75 μl of both forward and reverse primer; polymerase, 0.1 μl; and 1 μl of DNA-extract. The PCR profiles were used as follows: for ITS 1 cycle of 94°C for 7 min followed by 35 cycles of 94°C for 60 s, 50°C for 60 s and 72°C for 60 s, and a final elongation at 72°C for 7 min (https://www.ncbi.nlm.nih.gov/genbank/).A fragment of rDNA containing the internal transcribed spacer regions  was amplified using primers 18S: 5′-TTG ATT ACG TCC CTG CCC TTT-3′ (forward), and 28S: 5′-TTT CAC TCG CCG TTA CTA AGG-3′ (reverse) . A fragmor 7 min ; for 18SRhabditella axei , Rh. terricola , P. entomophagus  and Cruznema sp. .Several morphological types of rhabditid nematodes were isolated from snail feces fixed in 96% ethanol. Obtained and edited sequences of ITS and 18S of these nematodes were compared (BLAST) with the material in GenBank and identified as Strongyloides sp., a nematode species showing close resemblance with rhabditids that has a clear parasitic zoonotic relevance , particularly in older animals, and have been identified also in chickens, dogs and pigs with incoercible diarrhea  revealed that R. axei lives in the gastrointestinal tract of its snail host where the entire nematode life cycle is completed   . In all hoptera) , 37. AltIn conclusion, our results indicate that the pet giant African land snails may serve as reservoir of several rhabditid nematodes. This snail species is among the most commonly ones kept as pets, and therefore often live in close proximity to humans. As a result, the contamination of the domestic environment through their feces is possible. Although parasitic nematodes were not isolated in this study, the giant African land snails should still be considered potential carriers of nematodes able to cause opportunistic diseases in humans. Therefore, we highlight the importance of further epidemiological research on the occurrence of free-living and parasitic nematodes in gastropod snails kept in captivity, and emphasize the need for strict control measures to reduce the risk of opportunistic infection with rhabditid nematodes in pet snail owners.Dd'O and MS conceived and planned the analysis. MS and JN carried out the analysis. CA helped shape the research and edited the manuscript. All authors provided critical feedback and contributed to the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The results revealed that otolith incorporation (Me:CaOtolith) of Sr and Ba were positively dependent on the concentrations of the elements in water (Me:CaWater). Overall, Sr was incorporated into otoliths more efficiently than was Ba, and the partition efficiency (DMe) of both elements decreased with increasing water elemental concentrations. Increasing Sr concentrations in water appeared to negatively affect the uptake of Ba into otoliths rather than facilitate it, as previously reported in fish reared in freshwater and brackish water, or showed no effect on fish in seawater. Conversely, the Ba concentration in water did not influence the otolith uptake of Sr, which agrees with the findings for other fish species. When applying otolith microchemistry to fish ecology studies, it is essential to cautiously address the interactive effects of multiple elements in the environment on otolith elemental incorporation.Although the relationship between the incorporation of an element into otoliths and the concentration of the element in water has been extensively investigated in many fish species, the interactive effects of multiple elements in water on the otolith incorporation of an element are not adequately explored or well understood. In this study, 16 treatments in triplicate using strontium (Sr; 1, 2, 3 and 4 times the ambient baseline, 6.5 mg l Fish otoliths are chemically composed of calcium (Ca) and a variety of trace elements, which are primarily incorporated from ambient water throughout their life. Once crystallized and deposited in the inner ears of fish, otoliths are physiologically stable because of metabolic inertness , 2. It i2+ within the calcium carbonate (CaCO3) matrix of otoliths, which is usually related to the ambient elemental concentration [e.g., temperature and salinity) and physiological factors  may alter otolith microchemistry directly, as well as indirectly, by altering internal metabolic and physiological processes that influence elemental incorporation [Otolith chemistry has been reported as a reliable indicator of environmental histories for many species , 12, altntration , 14, 15.poration . To dateporation –21, althporation , 22.Acanthopagrus butcheri juveniles reared in brackish water [Perca flavescens juveniles [During the formation and growth of otoliths, multiple trace elements with distinct chemical properties may be simultaneously incorporated into otoliths from ambient water. Since elemental ions may compete for space in lattice sites in the otolith matrix during crystallization, elements can potentially interact during the incorporation of a specific element in the otolith. Previous studies have commonly investigated the incorporation of an element independently and rarely considered the potential competition or facilitation by other elements. Two studies addressed this issue and demonstrated that increasing Sr concentrations in water facilitated the uptake of Ba in the otoliths of black bream sh water  and fresuveniles . HoweverParalichthys olivaceus is an important commercial fish that is widely distributed in the coastal waters of East Asia. Flounder spawns from April to June in shallow nearshore waters. After hatching, larvae are transported to coastal waters, where they finish metamorphosis and then settle. The newly settled juveniles tend to move to offshore nursery areas to forage and grow until they migrate to deep overwintering areas. In nature, all these physiological and ecological traits represent dramatic shifts in biological and environmental histories, which could eventually affect otolith microchemistry. Additionally, flounder has been bred for aquaculture and stock enhancement in East Asian countries for many years. Flounder obtain relatively stable and high survivorship once the larvae survive a “critical period” of mortality around first feeding [i.e., individual growth variation enlarged by social interaction due to size hierarchies) occurs due to aggressive attacking and cannibalism in the early juveniles [Flounder  feeding . Furtheruveniles . These aWe used flounder as an experimental fish and conducted a laboratory experiment to investigate how elemental concentration might affect otolith elemental incorporation during early life stages. In this experiment, flounder were reared from the early larval stage to juveniles for over 10 weeks. The target elements that were examined were Sr and Ba, which are the two most investigated elements because of their active incorporation into otoliths. The current study mainly tested if and how the otolith incorporation of the two elements was correlated to elemental concentration and elemental interaction.~ 40 h after fertilization (haf). At 60 haf, when hatching was supposed to finish, the hatching rate was estimated to be c. 92%.Fertilized flounder eggs were provided by the Shunyuan Fishery Station at Rizhao, Shandong Province. The eggs were acquired from the same broodstock to minimize potential genetic variability among individuals. Approximately 50,000 viable eggs were transferred into a 1000 l concrete pond that was filled with filtered seawater. The water temperature was 17.4±0.5°C throughout egg incubation. The light regime corresponded to the natural light at the laboratory (13 L at 0600–1900 h and 11 D at 1900–0600 h). The eggs started to hatch at Brachionus plicatilis as food twice per day (at 0800 and 1300 h) until they were used for the experiment. Rotifers were cultured at 21–23°C and fed Chlorella sp. Daily water changes via flowing water began on 3 dph, increasing from 30% to 150% of the total water volume with the progression of larval development. Other rearing conditions  were similar to those used for egg incubation. The incubation and rearing pond were cleaned twice a day by siphoning, and all dead larvae were counted and removed.The larvae started feeding on 3 days posthatching (dph). Following routine rearing practice and feeding management , 25, newAt 15 dph, when larvae were assumed to have survived the “critical period” of mortality and were sufficiently developed for experimental handing, they were used for the experiment.T) were randomly selected from the rearing pond with beakers, visually counted and stocked in experimental tanks (50 larvae per tank) that were randomly assigned locations, treatments and replications. The water salinity and temperature in the tanks were monitored twice a day (at 0800 and 1400 h).All experimental tanks were 50 l transparent polypropylene tanks containing 40 l filtered seawater and were provided with mild aeration under a light regime of 13L:11D. The 15 dph larvae  and Ba . A total of 16 treatments in triplicate, using Sr and Ba as categorical variables in the orthogonal design, were established to investigate the relative or interactive effects of water elemental concentrations on otolith elemental incorporation. Water elemental concentrations were manipulated by the addition of appropriate amounts of standard solutions of SrCl2·6H2O and BaCl2·2H2O in fresh seawater. The experiment was terminated when fish reached 116 dph and last for a total of 100 days.In the experiment, four water elemental concentrations were established for Sr  and approved by the Committee on the Ethics of Animal Experiments of the Institute of Oceanology, Chinese Academy of Sciences (No. 20180258). The principles of reduction, replacement and refinement were carefully followed when we developed the test. During the test, the flounder larvae were reared under routinely practiced culturing conditions to ensure their welfare. At sampling, larvae were immediately sacrificed in liquid nitrogen to alleviate the possible stress and pain. The number of embryos stocked in the tanks was controlled to satisfy the minimal requirement for this test.3 , and stored in acid-washed polypropylene bottles at 4°C until chemical analysis.Starting on the first day of the experiment, 50 ml water was randomly collected from one tank of each treatment over the course of the experiment at 10-day intervals to measure the elemental concentration in the water. After sampling, the solutions were filtered with 0.45 μm film, acidified with 2% ultrapure HNOAt the end of the experiment, three fish were randomly selected from each tank  for elemental measurements in otoliths to investigate the incorporation of Sr or Ba in relation to the elemental concentration in water or elemental interactions. After sampling, sagittal otolith pairs were extracted, cleaned with distilled water, air-dried, and then stored in microcentrifuge tubes. As sagittal otoliths are symmetrical in flounder, the right otolith of each pair was then used for chemical analysis.2O2 solution for 10 min to eliminate the remaining superficial contaminants. Thereafter, otoliths were rinsed twice with ultrapure water to remove H2O2 from the surface and dried in a laminar flow fume hood. The dried otoliths were weighed using an analytical balance , rinsed twice with ultrapure water to remove contamination during weighing, and then dried for elemental analysis. All tools for handling otolith samples were acid-washed to reduce contamination during otolith sample preparation.Otolith sample preparation and elemental measurements using solution-based inductively coupled plasma mass spectrometry  were conducted following the methods of Higgins et al.  and Bail3 (Merck) that was proportional to the otolith mass  to obtain a relatively constant Ca2+ concentration across sample solutions so that the potential matrix effects of Ca2+ were assumed to be consistent in each sample. Each sample solution was diluted with ultrapure water  to obtain a 2% HNO3 matrix solution. When the otolith samples were analyzed on an ICP-MS, instrument drift occurred due to the buildup of Ca2+ on the instrument, as well as changes in temperature, nebulizer aspiration rates and plasma, which resulted in low detection limits of the elements. All sample solutions, standards and reference materials were spiked with indium  as an internal standard to correct for instrument drift as well as potential matrix effects. Elements were quantified by an external calibration method with multi-element standards  containing the target elements.The right otoliths were fully digested in a specified volume of 10% ultrapure HNO3 solution, Merck), multi-element standards and reference materials, all with 2% HNO3 solutions as matrixes, were successively analyzed. The certified reference material  was analyzed over the course of all analysis sessions to determine the accuracy and precision of the analysis. A recovery rate of 96, 99% and 103% and an average analytical precision of 2.4, 3.1% and 4.5% relative standard deviation (RSD) were achieved for Sr, Ba and Ca, respectively. The blank was analyzed to determine the limits of detection (LODs) for each element, thus validating that the detectable concentrations of Ca, Sr and Ba in otoliths were all well above their LODs.During each analysis session, 8 otolith solutions were randomly selected and numbered from all treatments, and these samples were analyzed. The order of otolith analysis was randomized so that the analysis of any treatment could be spread over the entire analysis procedure, thus reducing potential sequence effects. Prior to each analysis session, blank  and were analyzed for Ca concentration by inductively coupled plasma atomic emission spectrometry  and for Ba and Sr concentrations by ICP-MS . The analytical procedures and methods were similar to those used for the otolith elemental measurements. In short, the internal standards and blanks were the same as those used to measure the otolith elemental concentrations. The external standards and reference material were certified standard seawater solutions. During each analysis session, 10 sample solutions randomly selected across treatments were analyzed. The precisions for the Sr, Ba and Ca concentrations in the reference solution were 1.3%, 1.3% and 1.7%, respectively. Meanwhile, the recovery rates were 98% for Sr, 102% for Ba and 96% for Ca.Water samples were diluted with 2% ultrapure HNOOtolith). The mean elemental concentrations (mean±SD) of the individuals from each tank were calculated. Elemental error, which was defined as the percentage of the absolute difference of the measured and nominal concentrations to the nominal concentration of each water sample, was used to assess how precisely the nominal water elemental concentrations were reflected throughout the experiment. The partition coefficient (DMe), which is the ratio of Me:CaOtolith to Me:CaWater, was used to assess the efficiency of elemental incorporation into otoliths from ambient water for each individual fish. Me:Cawater was the average of the elemental concentrations measured in a treatment over the course of experiment.All elemental concentration data were standardized to Ca, which was abundant and stable in all treatments, by expressing the elemental concentrations as molar ratios to Ca (Me:CaWater) on otolith elemental incorporation (Me:CaOtolith), followed by post hoc multiple comparisons (SNK test) to determine the significances between treatments. Furthermore, linear regression analyses were performed to examine the correlations between Sr:CaOtolith and Sr:CaWater at each Ba concentration or between Ba:CaOtolith and Ba:CaWater at each Sr concentration.Prior to running ANOVA, the raw data were checked for normality and homogeneity of variance using Kolmogorov–Smirnov and Levene tests, respectively. In this study, a few sets of elemental concentration data did not meet the assumptions for these statistics. Therefore, all elemental concentration data were transformed with ln(x+1). One-way ANOVAs were performed to assess whether rearing conditions  differed among treatments. Two-way ANOVA was used to evaluate the effects of elements in the environment  for Sr and 1.4% to 9.1% (average 6.0%) for Ba . The meaT) and otolith weight (WO) of fish in each treatment were 96–101 mm  and 1.94–2.15 mg . Neither LT nor WO significantly differed between treatments or replicates , although it did not differ between treatments with the same Sr concentration , yet they did not differ between treatments of the same Ba concentration , 1.70–5.48 (2Ba), 1.62–6.11 (4Ba) and 1.66–5.83 (6Ba) mmol mol-1 . At each Ba concentration, Sr:CaOtolith significantly increased as Sr:CaWater increased . At the same Sr concentration, Sr:CaOtolith did not differ among the Ba concentrations (P>0.05). Sr:CaOtolith and Sr:CaWater were positively correlated at each Ba concentration , 8.00–16.44 (2Sr), 5.32–15.39 (3Sr) and 5.61–15.96 (4Sr) μmol mol-1 . At each Sr concentration, Ba:CaOtolith significantly increased as Ba:CaWater increased (P<0.05). Ba:CaOtolith significantly differed between most Sr concentrations at each Ba concentration (P<0.05). A significant but weak negative correlation was detected between Ba:CaOtolith and Sr:CaWater at each Ba concentration . Ba:CaOtolith and Ba:CaWater showed a positive linear relationship at each Sr concentration , but remained relatively constant among Sr concentrations at high concentrations (4Ba and 6Ba).Dectively . DSr tenBa values were 0.08–0.19, 0.08–0.25, 0.07–0.17 and 0.08–0.17 among the Ba concentrations at 1Sr, 2Sr, 3Sr and 4Sr concentrations, respectively  measured in previous experimental studies except for the high Ba value (4610–6320 μmol mol-1) observed in Collingsworth et al. [Otolith and Ba:CaOtolith of each treatment fell within the ranges  is a predictable indicator for assessing the efficiency of elemental incorporation into otoliths from ambient water. DSr appears to have a relatively consistent range in experimental studies, even though the studies differed in experimental design  reported in marine fish in experimental studies  except for yellow perch Click here for additional data file.


Since these conditions often influence coral community structure and introduce physiological challenges to its resident organisms, changes in reef-associated fishes are expected. We present critical baseline data on fish biodiversity and benthic composition for the Al Wajh Bank. Underwater visual census of conspicuous fishes and standardized collections of cryptobenthic fishes were combined to provide a comprehensive assessment of these fish communities. We documented 153 fish species and operational taxonomic units, including undescribed species, within 24 families on reefs largely dominated by hard coral and soft sediment (39% and 32% respectively). The families Pomacentridae and Gobiidae contributed the most towards fish diversity and abundance. Bray-Curtis dissimilarity distances among sampled sites suggest a distinctive fish community within the lagoon, and coefficients of variation for each species show high variation in their distribution across the lagoon. Species accumulation curves predict that additional sampling would document many more species throughout Al Wajh. Our findings provide the most extensive biodiversity survey of fishes from this region to date and record the condition of the reef prior to major coastal development planned to occur in the near future.Al Wajh Bank in the northern Red Sea contains an extensive coral reef system that potentially supports a novel fish community. The large (1500km Coral reefs contain some of the most species-rich communities of marine fishes , 2, yet Studies documenting reef fish communities in the Red Sea are limited when compared to the relatively accessible Great Barrier Reef and Caribbean reefs . Coral r2 region contains roughly 50 islands scattered across a sandy basin with a maximum depth of ~30m. The extensive barrier reef nearly encloses the entire lagoon, with only three narrow channels deeper than 5m, between 100m to 900m wide, connecting to outside the lagoon. The reef’s bathymetry is unique among other Red Sea coastal systems, which are typically composed of isolated pinnacles extending vertically from the continental shelf up to 200m deep and therefore exposed to unrestricted water flow. There is little documentation on the hydrology within Al Wajh Bank, but its contained and shallow bathymetry suggests there is low wave action and water circulation. These properties interact with evaporation and aeolian dust resulting in an environment with high salinity and turbidity, and extreme annual temperature fluctuations. The annual change in sea surface temperature (SST) inside the lagoon is nearly double that from outside  near the towns of Al Wajh and Umluj. The 1500km outside . Lagoon e region , 23, pose region . In the Plans to transform Al Wajh Bank into a major tourist destination are expected to commence in 2019 as a part of Saudi Arabia’s Vision 2030 , 26. Wit2 PVC quadrate and were established near the start of the transects on the first relatively flat section of the reef matrix observed.Surveys were conducted over three days, from January 31st to February 2nd, 2016. Fish communities and benthic cover were surveyed at eight sites across the region  between Benthic surveys were conducted along the transects. At fixed points every 0.5m  along each transect, the type of benthic organism or substrate directly beneath the transect was recorded. Benthic types were classified into seven major categories: hard coral, soft coral, dead coral, sponges, turf algae, rubble & pavement, and sand. Hard corals were further identified to family or genus. Reef rugosity and height were observed at the collection stations as a proxy for habitat complexity. Rugosity was calculated as the linear distance between the ends of a 2m chain laid across the contour of the reef divided by the length of the chain, replicated three times. Height was measured as the vertical distance from the substrate to the three highest points of the reef structure within the quadrate.Underwater visual surveys (UVS) are a commonly employed survey method in reef fish studies  as they Ichthyocide surveys and other standardized methods for targeting cryptobenthic fishes are more laborious than UVSs, but they have been shown to significantly increase observed species richness , 30. At Due to the inherent difficulties associated with identifying cryptobenthic reef fishes, sequence data were obtained from specimens collected during the rotenone survey and were used in conjunction with visual identification to assign species names where possible. Reliable genetic data could not be obtained from all specimens, so a number were simply identified visually. Total genomic DNA was extracted from fin tissue samples using the HotSHOT protocol  and then5’-TGT AAA ACG ACG GCC AGT CGA CTA ATC ATA AAG ATA TCG GCA C-3’ and FishR2 5’-CAG GAA ACA GCT ATG ACA CTT CAG GGT GAC CGA AGA ATC AGA A-3’. For each sample, polymerase chain reaction (PCR) was performed on a 12.5μL mixture of 1μL extracted DNA, 6.25μL Qiagen MasterMix, 4.25μL H2O, and 0.5μL of each primer (10 μM), using the following cycling parameters: 95°C for 15min; 35 cycles at 94°C for 60s, 50°C for 60s, 72°C for 45s; 72°C for 10min; store at 4°C. PCR products were visually verified using a 1% agarose gel with SYBR® Green dye, then cleaned using Qiagen ExoSAP , and then sequenced using the Sanger method [The mitochondrial cytochrome c oxidase-I (COI) gene has been shown to effectively discriminate between fish species . This str method  at the Kr method , which ir method .Caesio sp. was detected via UVS and Caesio caerulaurea was detected in the ichthyocide survey. However, the former was removed from analysis, as it has potential to be C. caerulaurea. To analyze community structure and variation across Al Wajh, we generated a non-metric multidimensional scaling (nMDS) plot with Bray-Curtis dissimilarity distances between sites [Abundance data for UVS and ichthyocide surveys were scaled to fish-per-square-meter, then merged for all sites. Fishes described to the genus level were discarded from analysis if they could not be distinguished from another species within the genus to avoid double-counting species. For example, en sites , 36 and We compared the coefficient of variation (CV) for the species contributing 80% of the total abundance to better understand species-level distributions. The CV was calculated by dividing the standard deviation by mean abundance, which allowed us to compare how localized (larger CV) or wide-spread  a species is. A species found at a single site will have a CV of 2.83 .SIMPER analyses were used to compare fish communities from UVS and ichthyocide surveys to data collected from published surveys along the Saudi Arabian coastline of the Red Sea , 29. RobSampling was assessed using rarefaction curves . Curves This research was undertaken in accordance with the policies and procedures of the King Abdullah University of Science and Technology (KAUST). Permits for sampling in Saudi Arabian waters using the ichthyocide rotenone were obtained from the Saudi Arabian coastguard. No specific permissions were required, as the study did not involve endangered or protected species. We were unable to obtain ethics approval or a waiver because no ethics board or committee for working with animals existed within KAUST or the Kingdom of Saudi Arabia at the time of collection.This study documented 6427 individual fishes consisting of 153 distinct species and operational taxonomic units (OTUs), within 24 families . OTUs weTaeniamia bilineata (Apogonidae), but 97% of the individuals were observed at site 3 alone. Similarly, the most abundant species observed in UVS transects was Chromis viridis (Pomacentridae), which was almost exclusively found at site 7. The following species were abundant but restricted to one site only: Cheilodipterus novemstriatus (Site 3), Nectamia annularis (Site 3), T. bilineata (Site 3), Pseudocheilinus hexataenia (Site 6), Eviota prasina (Site 6), C. viridis (Site 7), Pristotis cyanostigma (Site 7). Gnatholepis sp. 2 (Site 8) and Pseudocheilinus evanidus (Site 8). The most abundant widely-spread species included Pomacentrus albicaudatus, Trimma avidori, Asterropteryx semipunctata, Eviota zebrina, Eviota sp. “Red Sea 3,” Neopomacentrus cyanomos, Eviota distigma, Pomacentrus trilineatus, and Callogobius bifasciatus.After combining data from both survey methods, the composition of fishes in Al Wajh Bank was highly skewed towards 22 species (14% of the species observed) comprising 80% of the total abundance , of whicP. trilineatus and N. cyanomos. Richness and abundance at site 8 was greater than at site 6, but site 6 was more diverse  . The sam diverse .Rarefaction curves do not appear to approach a maximum, particularly in the site-based curves, suggesting that there were additional (undetected) species in Al Wajh Bank . The curCaesio striata, P. albicaudatus, P. trilineatus, P. cyanostigma, N. cyanomos, Neopomacentrus miryae, and Neopomacentrus xanthurus, cryptobenthic species including E. zebrina, Asterropteryx semipunctata, and T. avidori, and the sand associated Parupeneus forsskali, Taeniura lymma, and Torpedo sinuspersici . Soft corals comprised 5% of the benthic habitat, 72% of which were within Xeniidae. About 3% of the benthos was occupied by sponges. Rubble & pavement was most abundant at site 8, whereas turf algae was most abundant at site 6. Dead coral consistently comprised about 3% of the benthos. We expected to observe a higher frequency of dead coral following a major bleaching event in 2015 that had occurred only a few months before sampling and impacted other Red Sea reefs [Reef habitats across Al Wajh Bank were dominated by two major categories–hard coral and sand . 95% of ea reefs .Al Wajh Bank is isolated and environmentally distinct from other Red Sea reefs, however its remote location has limited field studies within the area. Moreover, potential changes in the reef’s undocumented communities caused by development necessitates the need for baseline ecological data. Most of the biodiversity surveys in the northern Red Sea have been conducted in the Gulf of Aqaba –42, 350kRoberts et al.  reportedN = 12) from the family Gobiidae. It is possible these names represent described species that our team was unable to identify, but there exists potential for some to be undescribed. The OTUs E. sp. “Red Sea 1” and E. sp. “Red Sea 3” were first documented in 2013 [The most complete checklist of fishes in the Red Sea lists 1207 species , but thi in 2013 , but reqBased on the rarefaction curves  and specThe extreme physical properties of Al Wajh Bank provide a unique opportunity to study how corals live in stressful conditions . Under sIn 2017, Saudi Arabia announced the launch of The Red Sea tourism project as a part of Saudi Vision 2030 , 26. TheFishing activity at Al Wajh Bank is also present, but its broader impact is largely unknown. Fishermen likely arrive from ports in the nearby towns of Al Wajh and Yanbu. Low accessibility to Al Wajh’s remote reefs may likely result in low fishing pressure, but fishing may also be encouraged by infrequent coastguard patrols . Along tAl Wajh Bank is unique in its physical properties and supports a fish community distinct from other Red Sea reefs. We recognize the region as a new type of reef habitat in the Red Sea, characterized by its unique bathymetry and water conditions. The ecological role of Al Wajh in the greater Red Sea is largely unknown, however habitat diversity positively correlates to species diversity , and botThe body of research on coral reefs in the Red Sea is small in comparison to other reef systems, and few studies consider local variation along the Red Sea’s 12° of latitude, but existing work depicts the region as high in diversity and endemism. The Kingdom of Saudi Arabia is undergoing a period of rapid social and economic change, and marine life in the Red Sea is becoming increasingly intertwined in these changes. The lack of historical data, however, makes it difficult to document anthropogenic changes and apply appropriate management strategies. Al Wajh Bank is expected to be transformed into a tourist hotspot in the coming years, and the health and vibrancy of its ecosystem is vital to research on extreme conditions found in the Red Sea and the long-term economic success of the region. Here, we provide baseline data on the fish community and other characteristics of Al Wajh Bank to inform future research and decisions that may affect the reef. The authors recommend that future surveys in this area continue to use combined UVS-ichthyocide surveys to capture taxonomic breadth and for comparability with this study. It is also advised that additional surveys be conducted during summer months to detect potential seasonal differences in the reef community.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 TableMatches were determined using COI sequences with a > 96% match.(XLSX)Click here for additional data file.


Preparing an antibiotic stewardship program requires detailed information on overall antibiotic use, prescription indication and ecology. However, longitudinal data of this kind are scarce. Computerization of the patient chart has offered the potential to collect complete data of high resolution. To gain insight in our global antibiotic use, we aimed to explore antibiotic prescription in our intensive care unit (ICU) from various angles over a prolonged time period.We studied all adult patients admitted to Ghent University Hospital ICU from 1 January 2013 until 31 December 2016. Antibiotic prescription data were prospectively merged with diagnostic  and microbiology data by ICU physicians during daily workflow through dedicated software. Definite focus of infection and probability of infection (classified as high/moderate/low) were reassessed by dedicated ICU physicians at patient discharge.During the study period, 8763 patients were admitted and overall antibiotic consumption amounted to 1232 days of therapy (DOT)/1000 patient days. Antibacterial DOT  were linked with infection in 80%; the predominant foci were the respiratory tract (49%) and the abdomen (19%). A microbial cause was identified in 56% (3169/5686). Moderate/low probability infections accounted for 42% of antibacterial DOT prescribed for respiratory tract infections; for abdominal infections, this figure was 15%. The median treatment duration of moderate/low probability respiratory infections was 4 days (IQR 3–7). Antifungal DOT  were linked with infection in 47% of total antifungal DOT. Antifungal prophylaxis was primarily administered in the surgical ICU (76%), with a median duration of 4 DOT (IQR 2–9).By prospectively combining antibiotic, microbiology and clinical data we were able to construct a longitudinal, multifaceted dataset on antibiotic use and infection diagnosis. A complete overview of this kind may allow the identification of antibiotic prescription patterns that require future antibiotic stewardship attention.The online version of this article (10.1186/s13054-018-2178-7) contains supplementary material, which is available to authorized users. Antibiotics are among the most prescribed drugs in the intensive care unit (ICU) , 2. The Surveillance of antibiotic prescription is a first and essential step to measure antibiotic expenditure, to document physicians’ incentives to prescribe antibiotics and to identify areas of potential overuse or misuse which could then be a target for antimicrobial stewardship interventions , 13–15. In this manuscript, we present a complete and in-depth analysis of global antibiotic prescription and infection diagnosis in a university hospital ICU over a 4-year period. These data were collected with the help of a locally developed software program, which has been designed to link pharmaceutical, clinical and microbiological data together with diagnostic interpretation while performing daily bedside clinical work. As such, we were able to get a bird’s eye view of our local antibiotic prescribing practices, which can then serve as a starting point for the future construction of an antibiotic stewardship program (ASP).This study was conducted from 1 January 2013 until 31 December 2016 at the medical (14 beds) and surgical (22 beds) ICU of Ghent University Hospital (1054 beds). The Ghent University Hospital Ethics Committee approved the study (registration number B670201628197) and waived informed consent based on the non-interventional nature of this study and the complete anonymization of patient data. Patients aged 16 years or older were included.An Intensive Care Information System  integrating computerized physician order entry for medication prescriptions, computerized medication administration recording and clinical patient monitoring data has been available at the bedside since 2003. Patients are managed in a closed ICU model. Antibiotic prescriptions are at the discretion of the attending senior ICU physician, without the use of stringent protocols or antibiotic restrictions. As a rule, postoperative prophylactic treatment is not prolonged for more than 24 h following the procedure. Empirical antibiotic choices are guided by systematically collected surveillance cultures whenever available. Direct microscopic examination is performed on all diagnostic respiratory and per-operative samples. Pathogen identification is routinely performed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Microbiology results are reported electronically. Interdisciplinary staff meetings with medical microbiologists reviewing all antibiotic prescriptions take place once weekly in the medical ICU and three times weekly in the surgical ICU; these staff also include the presence of infectious diseases specialists in the surgical ICU. In addition, daily advice and follow up by these specialties is possible on a demand basis. Treatment duration and opportunities for antibiotic de-escalation are evaluated daily by the attending ICU physician and during the interdisciplinary discussions.2O), a semi-recumbent position (30–45°) and daily assessment of sedation.A ventilation-associated pneumonia (VAP) prevention bundle was used during the entire study period and involved the use of strict hand hygiene, oral care with chlorhexidine, endotracheal tube cuff pressure control  was developed by a consortium of the Ghent University Hospital ICU and the Department of Information Technology (INTEC) of the Faculty of Engineering of Ghent University . The proPrescription indication and antimicrobial utilization were described for all patients that were admitted during this study period. In microbiologically confirmed infections, the timing of appropriate antibiotic therapy was defined as the point in time at which all pathogens involved in the infection were covered by at least one component of the treatment.Antimicrobial days of therapy (DOT) per admission and per patient days is recommended as utilization metric by the STEWARDS panel and others , 14, 22.Subgroup analyses were performed in the patient populations with an ICU length of stay (LOS) equal to or more than 48 h versus less than 48 h, respectively, as this latter subgroup consists mainly of the less severely ill patients in our setting . In addition, patients with an ICU LOS of 48 h or more constitute the population at risk of developing ICU-acquired infection, which was defined as an infection emerging after more than 48 h of ICU admission.p < 0.05. For outcome analysis, we only included the last ICU episode of patients with consecutive ICU admissions. Statistical analysis was performed using R Statistical Software (version 3.4.2).Categorical variables were expressed as frequencies (percentages) and continuous variables were described as medians with the interquartile range . Differences in categorical variables were calculated using the Pearson chi-square test. The Mann–Whitney U test was used to compare continuous variables. Statistical significance was defined as A total of 10,743 ICU admissions were recorded in 8763 patients, resulting in a total of 47,403 patient days from 1 January 2013 until 31 December 2016. ICU and hospital mortality were 10.7% and 15%, respectively. Median Acute Physiology and Chronic Health Evaluation (APACHE) II score at admission was 18 (IQR 13–25). Mechanical ventilation was provided in 3958 admissions (36.8%) with a median duration of 2 days (IQR 1–6), resulting in 20,897 ventilation days. Vasopressor therapy was administered in 3639 admissions (33.9%) with a median duration of 2 days (IQR 2–4). Detailed information on patient characteristics is presented in Additional file Staphylococcus aureus isolates in our ICU population. Vancomycin resistance was present in 1.9% of the Enterococcus species isolates. Extended spectrum beta-lactamase production (ESBL) was present in 33% of Enterobacteriaceae isolates, whereas carbapenemase production was present in 1.2%.Methicillin resistance was present in 23% of the Patients were exposed to at least one antibiotic class in 66% 7051/10743) of ICU admissions. An infection was present within the first 48 h of ICU admission in 35% (3804/10743) of admissions. An ICU-acquired infection was diagnosed in 23% (1096/4851) of admissions with an ICU length of stay of more than 48 h. Detailed information on antibiotic exposure per ICU episode is provided in Fig. /10743 ofA total number of 10,731 treatment courses (infection bars) was recorded during the study period. Respectively 4525 (42.2%) and 6206 (57.8%) of these courses were prescribed for prophylaxis and for infection. Fungal infections represented 8% (520/6206) of the infectious episodes. Infections were microbiologically confirmed in 56% (3169/5686) and 63% (327/520) of bacterial and fungal infections, respectively. Antibiotic treatment initiated in the ICU was considered appropriate within 12 h and 24 h, respectively, following treatment initiation in 83% and 87% of microbiologically confirmed infections. Infections were ICU-acquired in 28% of cases (1767/6206). The focus of the bacterial infections was predominantly respiratory and abdominal . The crude ICU mortality rate in patients with respiratory and abdominal infections was 22.1% and 19.2%, respectively. Infection probability was classified as high, moderate or low in 50%, 34% and 16%, respectively, of the respiratory infections compared to 76%, 16% and 8%, respectively, of the abdominal infections. The median treatment duration of high, moderate and low probability respiratory infections for which the course was completed on the ICU was 7 days (IQR 5–10), 6 days (IQR 4–8)] and 4 days (IQR 2–6), respectively. Only 19% of the respiratory infections were classified as ventilator-associated. A total of 345 cases of VAP and 182 cases of ventilator-associated tracheobronchitis (VAT) were diagnosed, resulting in VAP and VAT incidences of 16.5/1000 ventilation days and 8.7/1000 ventilation days, respectively. Respectively, 52%, 37% and 11% of VAP were classified as having a high, moderate or low probability of infection, translating into VAP incidences of 8.6, 6.1 and 1.8/1000 ventilation days, respectively. VAP incidence was 17.0/1000 ventilation days in the medical ICU and 16.1/1000 ventilation days in the surgical ICU. The median treatment duration of VAP and VAT episodes in the ICU was 7 days (IQR 5–9) and 6 days (IQR 4–7), respectively. The crude ICU mortality rate in patients with VAP and VAT was 32.6% and 21.1%, respectively  were administered, resulting in 58,413 DOT (1232 DOT/1000 patient days). Detailed utilization analysis per antibiotic agent and infection probability is presented in Table Prophylactic therapy accounted for 25% of the total antimicrobial DOT; first-generation cephalosporins, folate pathway inhibitors, monobactams and azoles were predominantly used in this setting. Antibacterial prophylaxis mainly comprised perioperative prophylaxis (46%) and prolonged prophylaxis in the immunosuppressed patient (19%). The median duration of perioperative prophylaxis was 8 h (IQR 8–17). In addition, 9% and 11%, respectively, of antibacterial prophylaxis was initiated in the setting of trauma and following aspiration with respective durations of 3 DOT (IQR 2–4) in trauma patients and 3 DOT (IQR 2–5) in the setting of aspiration. Antifungal prophylaxis accounted for 53% of the total antifungal DOT and was primarily administered in the surgical ICU (76%), with a median duration of 4 DOT (IQR 2–9).Seventy-five percent of the total antimicrobial DOT was used to treat infections. Of the total amount of DOT used to treat bacterial respiratory infections, 42% was used to treat infections with a moderate and low probability; for bacterial abdominal infections, this figure was 15%.Enterobacteriaceae were the predominant bacterial species that were designated as causative pathogens in both respiratory and abdominal infections . Amoxicillin/clavulanic acid resistance and cefuroxime resistance was present in 48% and 39% of the Enterobacteriaceae isolates, respectively. Non-fermenting Gram-negative bacilli were the second most prevalent pathogens associated with respiratory infections (15%), and mainly consisted of Pseudomonas aeruginosa. Piperacillin-tazobactam, ceftazidime and meropenem resistance was present in 27%, 20% and 20%, respectively, of the Pseudomonas isolates. Enterococci were the second most prevalent pathogens that were linked as causative in the abdominal infections (20%) and ampicillin resistance was present in 44% of the isolates  and timespan covered (4 years).Our study confirms that the antibiotic burden in the ICU is very high. Respectively, 66% of all admitted patients and 84% of patients with an ICU stay of more than 48 h were exposed to at least one class of antibiotic. These figures are consistent with the results of the one-year prospective surveillance study of Bergmans et al. and with the EPIC II point-prevalence study, which reported antibiotic prescription in 61% and 71% of admitted patients, respectively .Clostridium difficile infection, rates of antibiotic-resistant pathogens, days of antibiotic therapy/number of admissions, days of antibiotic therapy/patient days and redundant therapy events. All of these metrics may be derived from separate electronic data sources  and a connection between the different elements is not mandatory; however, these metrics are crude and unable to provide insight into antibiotic prescription. By linking these sources, a deeper understanding of the different factors driving antimicrobial use can be obtained, as illustrated by this study.The need for detailed antibiotic prescription surveillance and feedback to the clinician was already acknowledged in the very early stages of antimicrobial stewardship, but there is disagreement about which appropriate measures to select , 23, 24.For example, the respiratory system accounted for half of identified sources of bacterial infection and more than one third of the total antibacterial DOT. Compared to abdominal infections, which represented the second largest group of bacterial infections and destination for antibiotic consumption, respiratory infections were less frequently categorized as highly probable , which also reflected the amount of DOT that was designated to treat these highly probable infections . One quarter of the infections that were diagnosed in our ICU were ICU-acquired, which is in contrast with the study of Bergmans et al. where half of the infections were ICU-acquired and almost exclusively occurred in ventilated patients. Whereas the authors of the previous study concluded that stewardship should be focused on the prevention of ventilator-associated respiratory infections, this statement may apply less to our ICU population. In addition, restricting duration of antibiotic therapy in VAP of high probability will offer little gain, as the median treatment duration was only 7 days. In contrast, more restrictive use of antibiotics in suspected respiratory tract infections with cultures remaining negative and/or a swift clinical resolution, could result in a more profound reduction of antibiotic consumption. In addition, we observed that prophylactic treatment accounted for one fourth of the total DOT. Although this figure seems extremely high, more in-depth evaluation showed that two thirds of the antibacterial prophylactic treatment consisted of perioperative prophylaxis and prophylaxis in the immunocompromised patient, which conformed to guidelines with regard to treatment duration in the ICU. Whether aspiration and trauma justify antibiotic prophylaxis, and for what duration, is still a matter of debate; more recent studies promote restrictive use of antibiotics in this setting –29. We bDespite the recognition of the importance of high-quality surveillance data to support antibiotic stewardship, few studies have provided detailed data on ICU global antibiotic consumption and infection diagnosis over an extended period of time , 19, 31.Our study has limitations. First, building the database starting from computerized physician order entry depends on adequate filling in of the “pop-up” questions that are triggered by it, and by the persistent commitment of attending physicians or infection control personnel in linking the various information sources and finalizing infection diagnosis. Second, as stated before, the lack of adherence to strict criteria in labeling infection diagnosis in the current design hampers multicenter application . ProbablWe were able to obtain a unique bird’s eye view on global antibiotic use and infection diagnosis in our ICU over a 4-year time period by analysis of a multifaceted dataset, which was collected during the daily clinical workflow of ICU physicians with the help of information technology. In doing so, we revealed antibiotic prescription patterns that merit the attention of antibiotic stewardship.Additional file 1:2)/fractional inspired oxygen (FiO2) ratio, sequential organ failure assessment score (SOFA)). For every antibiotic prescription that is entered via computerized physician order entry, a horizontal bar is created that runs just above the timeline and lengthens upon duration of the prescription (antibiotic bar). This bar is accompanied by a second bar running in parallel below the timeline and describing the indication for this antibiotic . The infection bar is created in a two-step fashion. A preliminary version is fed by data from a short questionnaire that “pops up” in real time after any antibiotic prescription and inquires the prescriber about indication, likely focus, severity and probability of infection and presence of microbiological data guiding antibiotic choice. This preliminary bar can be altered manually when more data on the origin and clinical evolution of the infection become available. Multiple antibiotic prescriptions can thus be linked with the same infection bar; in addition, the same antibiotic bar can be linked with multiple infection bars . For each infectious episode, focus, severity and probability of infection is selected from a drop-down menu. More detailed information on prescription and infection are revealed on the base of the screen by hovering over the bars. All positive microbial culture results and matching susceptibility patterns are automatically indicated above the timeline using small symbols, whereas selected microbiological isolates that are linked to an infection are displayed underneath the timeline. (JPG 479 kb)Screenshot of COSARA central infection dashboard view. Time-graphs at the top of the page show the evolution of selected clinical (e.g. fever) and laboratory ) variables and indicators of severity-of-illness Additional file 3:Staphylococcus aureus promoting glycopeptide prescription in suspected pneumonia with negative sputum cultures). (JPG 298 kb)Screenshot of COSARA antibiotic-infection combinations linked to microbiology data overview. The coupled antibiotic-infection bars can be linked to microbiological culture results; pathogens may be designated as causative pathogens or as non-causative pathogens influencing antibiotic prescription Additional file 5:Patient characteristics. (DOC 36 kb)Additional file 6:Treatment duration per focus of infection. (DOC 41 kb)Additional file 7:Antimicrobial use per antimicrobial class and per year. (DOC 61 kb)