Infectious travelers’ diarrhea (TD) is a well-appreciated problem among service members serving abroad, particularly where infrastructure is limited due to ongoing combat operations, and efforts at sanitation and hygiene may not be considered an immediate priority. Bacterial and viral causes of travelers’ diarrhea are well-described among deployed service members, however, gastrointestinal protozoan infections among deployed service members are less well documented. This study’s purpose was to identify potential risk factors for, and clinical presentations of, enteric protozoan infections in an active duty military population deployed to combat operations in the Southwest Asia.A cross-sectional study of enteric protozoan infections among US service members deployed in Al-Asad Air Base, Iraq in support of Operation Iraqi Freedom (OIF) was conducted in summer 2004. Subjects were obtained through a randomized sector sampling scheme, and through presentations for care at the air base medical facilities. All study participants provided a stool sample, either diarrhea or solid, upon study entry and completed a questionnaire documenting demographic information, clinical symptoms of any prior diarrheal episodes, and health risk behaviors. Basic diagnostic microscopy for protozoa was conducted to include acid-fast and modified trichrome staining.Blastocystis hominis (n = 36), Entamoeba coli (n = 25), Endolimax nana (n = 20), and Entamoeba histolytica (n = 5) were the predominant organisms isolated. Crude incidence of prior episodes of diarrhea was greater among subjects from whom enteric protozoa were isolated compared to those without . Bivariate analysis of health risk and hygiene behaviors found increased odds for presence of Blastocystis hominis among those service members who reported off base ice  and raw vegetable consumption .Four hundred thirty-seven subjects were included in the analysis, and 75 (17.1 %) subjects were found to have enteric protozoan infections as identified by diagnostic stool microscopy. This study suggests that US service members deployed to the early stages of OIF were at greater risk of acquiring enteric protozoa than previously understood. The noted prevalence of enteric protozoa among US service members in this study is higher than in prior reports, approaching prevalence expected in the general host nation population, suggesting that US service members operating at Al-Asad Air Base in early OIF were exposed to greater degrees of fecally contaminated food and water, and poor hygienic and sanitation practices. Consumption of food and water prepared by host nation parties in Southwest Asia may place US service members at risk for acquiring intestinal protozoa. Shigella, enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and norovirus. However, descriptions of gastrointestinal protozoan infections among deployed service members are less well documented. This study’s purpose was to identify potential risk factors for and clinical presentations of enteric protozoan infections in an active duty military population deployed to combat operations in the Southwest Asia, specifically Iraq. This study had two objectives: (1) to characterize the burden of intestinal protozoan infection within an active duty military population performing combat operations in Southwest Asia, and (2) to examine the potential risk factors and health risk behaviors associated with the observed prevalence of intestinal protozoa within this population. An improved understanding of protozoan associations with infectious diarrhea may lead to understanding the risk, optimization of empiric therapy, and identification of potential methods for prevention.Infectious diarrhea has remained a problem for service members serving abroad for centuries, particularly in those settings where infrastructure is still limited due to ongoing combat operations, and efforts at sanitation and hygiene may not be considered an immediate priority . OutbreaThis study was conducted at Al-Asad Airbase, Iraq in summer 2004. Al-Asad Airbase is an Iraqi Air Force base located in Al Anbar province of western Iraq. At the time of this study, the base was home to approximately 8,000 U.S. service members, predominantly U.S. Marines from marine expeditionary and aviation units. A small hospital and several smaller clinics were established within fixed facilities on the base. Primary food service consisted of two large cafeterias which provided three hot meals per day. The cafeterias were staffed with a significant number of foreign contractors, which is a common practice among military contractors. These facilities were regularly inspected by Navy Preventive Medicine Unit personnel. Additional host nation food vendors were available on base at times, but these sources were not inspected by preventive medicine personnel. Military personnel may have been exposed to off base sources of food and drink during patrols or engagements among the Iraqi populace. Bottled water was available for consumption. Toileting was limited to chemical toilets.Individuals were considered eligible for the study if they were active duty military or a military beneficiary, 18 years or older, had received no antibiotic therapy within the 72 h prior to enrollment , and had been in Iraq for more than 48 h. Only those subjects for whom stool microscopy results and survey data were available were included in the final analysis.This study was conducted over a three week period in summer 2004 as part of larger cross-sectional, case finding study that sought to identify cases of infectious diarrhea and asymptomatic controls. A total of 552 subjects were recruited to the study including acute diarrheal cases presenting for medical care, acute diarrheal cases not presenting for medical care (identified during a randomized sector convenience sampling scheme encompassing non-restricted areas of the airbase), and asymptomatic participants (similarly located through the same randomized sector sampling scheme). The random sector sampling scheme was created by dividing the base into 12 sectors, sans restricted areas. Investigators initially randomly selected four sectors a day for three days (via the use of a multi-sided die) for selection of “cases not seeking care” and asymptomatic patients. One of four study teams were sent to each selected sector each day to recruit and enroll subjects through a convenience sampling technique. The rotation of sectors for sampling was continued throughout the study period based on the original three days randomization. Subjects included in the final analysis for this study were those subjects identified in the larger study for which results of stool diagnostic studies for protozoa were available. In total, 437 subjects were included for analysis, of which 111 were symptomatic diarrheal cases at the time of enrollment.All study participants provided a blood sample and a stool sample, either diarrhea or solid, upon study entry and completed a questionnaire documenting demographic information, clinical symptoms of any prior diarrheal episodes, and health risk behaviors. Study questionnaires were collected at the time of stool sample submission. Stool specimens and study questionnaires were placed into a cooler and transported to the laboratory twice each day. All stool samples underwent microbiologic analysis for bacterial, viral, and protozoan causes as part of the larger cross-sectional study. Basic diagnostic microscopy for protozoa was conducted by NAMRU-3 staff to include acid-fast and modified trichrome staining.A case of enteric protozoa infection was defined by the direct visualization on diagnostic stool microscopy of protozoal cysts or trophozoites from the stool specimen submitted. This case definition was utilized both for those subjects with acute diarrhea at the time of enrollment, and for those subjects that were asymptomatic on enrollment.Study stool samples and questionnaires were linked by a unique identification number (UID) assigned to each subject. This was done to ensure and maintain the confidentiality of study subjects. The UID was included on all questionnaires, clinical forms, stool samples, and laboratory forms. All data was entered and verified using Microsoft Access database.Blastocystis hominis was isolated to those subjects with no enteric protozoa isolated from stool. Univariate analysis was conducted using standard statistical tests. Comparisons of median age, median time in theater, and median time at Al-Asad were conducted with Wilcoxon Rank Sum tests. Testing of independent categorical variables was conducted with either Pearson’s chi-square test or Fisher’s Exact test. Crude incidence rates of prior episodes of diarrhea were calculated using the number of self-reported prior episodes of diarrhea during the deployment, and the self-reported days in theater. Poisson regression was used to estimate incidence rates and confidence intervals, and crude incidence rate ratios. Health risk behavior survey responses in some cases utilized Likert scales. For analysis, these responses were dichotomized so that responses of “always” or “frequently” were treated as “yes”, and those responses indicating “rarely” or “never” were treated as “no.” Likert responses of “sometimes” were removed from analysis. Unadjusted odds ratios were obtained through bivariate logistic regression analysis to assess the strength of association between individual health risk behaviors and the presence of enteric protozoa. All data analysis was conducted using Stata/IC Version 12.1,  and statistical significance was set at p < 0.05.Subjects from whom enteric protozoa were identified were compared to those with no enteric protozoa identified from stool samples. Due to the frequency in which it was identified, additional analysis was conducted comparing those subjects from whom only This study (DOD# NAMRU3.2004.0011) was reviewed and approved by the Institutional Review Board of the US Naval Medical Research Unit No. 3 in compliance with all Department of Navy Federal regulations governing the protection of human subjects. Informed consent was obtained and documented from all adult participants.This study determined there were a total of 437 observations available for analysis. There were no significant differences found by median age, median days in theater, or median days at Al-Asad Airbase when comparing subjects with enteric protozoa to those without protozoa isolated Table . No diffBlastocystis hominis (n = 36), Entamoeba coli (n = 25), and Endolimax nana (n = 20), made up 90 % of all identified protozoa with multi- protozoan co-infections included  and Giardia lamblia (n = 1).Enteric protozoa were isolated from 75 (17.1 %) stool samples. coli n = 5, and Eninis n = 6, Entamop = 0.08). There were no differences noted in prevalence of reported current fever. Among those who reported diarrhea previously during the deployment, there were no significant differences in median number of prior episodes (3), and no significant differences in diarrhea associated symptoms. A near significant (p = 0.07) greater proportion of subjects with protozoa identified reported having previous episodes of diarrhea of such severity that back-up personnel were required to come in to cover the subject’s work shift. Additionally, near significant differences were noted in reporting off base travel in the prior 5 days. Subjects without protozoa identified tended to more frequently report off base travel in the last 5 days (p = 0.06)  compared to those subjects with protozoa identified  Table .Table 4CBivariate analysis suggested that those who reported having drinks with ice or eating raw vegetables with meals while off base were at increased odds of having protozoa identified in their stool samples, although results were not significant Table . Point eBlastocystis hominis was the only protozoa identified in the stool samples of 27 subjects , abdominal cramps in nine (60 %), headache in nine (60 %), fever in five (33 %), blood in stool in two (13.3 %), and vomiting in one (6.7 %). These proportions are not significantly different from those seen in subjects without protozoa identified. However, a greater proportion of subjects with Blastocystis hominis reported needing to have a backup called to cover their shift due to gastrointestinal upset compared to those subjects without protozoa identified .Of the 75 subjects from which protozoa were identified, ts Table . FifteenBlastocystis hominis identified to those subjects without protozoa identified found that subjects with Blastocystis hominis were at about 70 % lesser odds of reporting diarrhea on a prior deployment . Twenty-three (85 %) of 27 subjects with Blastocystis hominis only identified denied having had diarrhea on prior deployments. Point estimates also suggested a trend toward lesser odds of having traveled off base in the last 5 days for those subjects with Blastocystis hominis only identified . Most notably, subjects with Blastocystis hominis only identified reported over three times greater odds of using ice in drinks off base , and over eight times greater odds of having eaten raw vegetables off base .Bivariate analysis Table  comparinet al.’s report on gastroenteritis in U.S. Marines during the initial invasion of Iraq mentions only a single case of diarrhea of protozoan etiology (Cryptosporidium) in 129 stool samples obtained within southern Iraq [et al.’s analysis of service members arriving in Qatar for “rest and relaxation” leave from Iraq or Afghanistan in 2006 found enteric protozoa in as many as 20 % of those service members with diarrhea [Blastocystis hominis, Entamoeba coli, and Endolimax nana. The Monteville et al. study did include members with longer deployments which may have contributed to the greater prevalence of intestinal protozoa. This suggested perhaps a greater role of gastrointestinal protozoa in diarrhea among deployed service members than previously considered, particularly on extended deployments. Historically, Hyams and colleagues collected over 400 stool samples from service members with gastroenteritis during deployment to Saudi Arabia in preparation for the First Gulf War with Iraq. Their analysis of these samples found a bacterial pathogen in about half of all submitted samples, but found no evidence of intestinal protozoa in any of the over 400 samples [Giardia cysts in only 9 specimens with no evidence of other intestinal protozoa [Multiple prior reports have addressed the impact of infectious diarrhea on combat operations during Operation Iraqi Freedom (OIF). However, limited references have been made to identified enteric protozoa during either OIF or the prior war in Iraq. Thornton diarrhea . Enteric samples . Malone Blastocystis hominis, Entamoeba coli, and Endolimax nana as the most commonly identified enteric protozoa. This study also appears to confirm that the most frequently isolated protozoa among US service members deployed to the Middle East are Blastocystis hominis, Entamoeba coli, and Endolimax nana as similarly noted in two prior studies [Entamoeba coli, and Endolimax nana. The pathogenicity of Blastocystis hominis remains controversial up to the present, and would have been considered a commensal at the time of the study [Looking at deployments to the Middle East outside the Iraq theater, a 1991 report on almost 200 stool samples collected from service members with acute diarrhea during a deployment to Egypt also did not find evidence of intestinal protozoa in these samples . A study studies , 10. It he study .e.g., social desirability bias), and compliance with these and other proper hygiene practices may well have been much lower. The consumption of off base ice which presumably would have been provided in an off base restaurant or a setting where prepared food was provided indeed had the greatest associations with the presence of Blastocystis hominis in stool.The prevalence of enteric protozoa in this study is high and suggests that sanitary conditions for US service members at the time of this study were less than optimal. The reported compliance with appropriate hygiene measures of washing hands before eating was quite high , as well as washing hands after toileting , although, these types of questions may introduce a bias,  in US service members has not been previously reported. Results in this study would at least suggest a greater than expected prevalence of Blastocystis hominis among a group of generally healthy deployed US service members. Those subjects with only Blastocystis hominis identified in their stool did not report significant differences in gastrointestinal symptoms compared to those subjects with no enteric protozoa identified. However, the small number of Blastocystis hominis cases that reported associated symptoms of prior diarrhea (only 15 of 27 answered survey questions regarding prior episodes of diarrhea) limits the ability to detect any real differences. Significant controversy remains in the current literature as to the true pathogenicity of Blastocystis hominis, and the need to treat when the organism is identified in stool [Blastocystis hominis infection did not differ significantly from those subjects without enteric protozoa, the finding that subjects with only Blastocystis hominis isolated more frequently reported having had a back-up called to cover their shift suggests that whatever symptoms were present were possibly more severe among those with Blastocystis hominis. This may be another indication of the potential pathogenicity of Blastocystis hominis, at least among service members who were presumed to have acquired the protozoa upon arrival into theater. More likely, however, is that the presence of Blastocystis hominis among those requesting back-up may simply be indicative of a prior ingestion of food or drink with heavy fecal and multi-pathogen contamination, to include viral and bacterial causes of acute diarrhea.This significant proportion of in stool –13. WhilThe finding of a trend toward a greater proportion of subjects with protozoa identified reporting no off base travel in the prior five days may be the result of gastrointestinal infection or illness acquired during previous off base travel. Perhaps, these symptoms of these illnesses prevented service members from participating in off base operations in the days immediately prior to study entry. The finding of greater incidence of episodes of diarrhea among those subjects with protozoa identified suggests that those subjects may have had ongoing or recurrent episodes of diarrhea rather than acute episodes which subsequently resolved as may be expected with an acute bacterial diarrheal infection. Again, these recurrent episodes may have contributed to service members having remained on base in the five days prior to study entry. This may be further evidenced by the finding of a trend toward greater odds of having required a back-up to be called in to cover a service member’s shift among those with protozoa identified.Entamoeba coli, Endolimax nana, and Entamoeba histolytica by diagnostic microscopy, but recognize that some may note the possibility of misidentifying one species for another. Regardless of this possibility for misidentification, the presence of any of these organisms would indicate fecal contamination of food and/or water sources, and may indicate risk for other pathogenic enteric protozoa. For this study, we were not able to consider the possibility of bacterial or viral co-pathogens that may have contributed to the clinical presentation of study participants. Furthermore, this study is not a comprehensive assessment for enteric protozoa as only direct microscopy was used for identification. More sensitive protozoan identification methods are now available and FDA-approved, such as enzyme immunoassays for Entamoeba histolytica and Giardia, and polymerase chain reaction for Cryptosporidium and Giardia [This study has a number of notable limitations. It is a cross-sectional study and therefore subject to the limitations of all cross-sectional studies. Most notably, we are unable to clearly establish a causal association between reported health risk behaviors and presence of enteric protozoa as all information was collected simultaneously. We have limited information regarding where study subjects may have traveled while en route to Iraq, or how long they may have remained at those locations. Some service members did report prior deployments to a variety of locations, and it is likely that some service members may have been on shipboard deployments with en route ports of call prior to their arrival in Iraq. We are unable to determine which, if any, subjects may have arrived to theater already carrying enteric protozoa, though this is unlikely. We are confident in our ability to discern  Giardia . This stBlastocystis hominis. Our results suggest that the presence of Blastocystis hominis is associated with greater lost work days, but we are unable to conclude at this time whether these lost work days are attributable to Blastocystis hominis directly, or if its presence is simply indicative of exposure to a heavy, and potentially diverse, pathogen load previously.This study also has several strengths. To our knowledge, this is the only study to date specifically exploring the prevalence of enteric protozoa among service members deployed to Operation Enduring Freedom or Operation Iraqi Freedom. The prior reports that have included protozoa etiologies of acute diarrhea among service members deployed to Southwest Asia have not attempted to link diarrheal associated symptoms with enteric protozoa presence. Our study was able to combine information on health risk behaviors, associated clinical symptoms, and stool diagnostic microscopy. Additionally, unlike any prior study among deployed US service members, this study was also able to associate health risk behavior information with stool samples positive for Blastocystis hominis presence in stool samples and prior reported use of ice in off base drinks, and consumption of raw vegetables when eating off base during the deployment. Similar, but not statistically significant, results were noted when all protozoa were pooled for analysis. This study reiterates the need for deployed US service members to consume food and water only from sources that have been approved by US preventive medicine and public health officials. Consumption of food and water prepared by host nation parties will likely place US service members at risk for acquiring intestinal protozoa. The noted prevalence of enteric protozoa among US service members in this study is higher than in prior reports, approaching prevalence expected in the general host nation population, which suggests that US service members operating at Al-Asad Air Base in early stages of Operation Iraqi Freedom were exposed to greater degrees of fecally contaminated food and water, and poor hygienic and sanitation practices. Future studies should consider the impact that intestinal protozoa have on unit readiness, and the clinical effects of Blastocystis hominis infections on deployed US service members. Study subjects were not followed beyond the study window; therefore, we are unable to comment on the potential long term gastrointestinal consequences of these protozoan exposures. Future work should also consider the possibility of extended alteration of gut flora following exposure to enteric protozoa.This is the only study to date that attempts to determine health risk behavior associations specifically with the presence of enteric protozoa among deployed US service members. This study found strong associations with


Performance at identification lineup was assessed in eighty‐five 6‐ to 11‐year‐old typically developing children. Children viewed a live staged event involving 2 male actors, and were asked to identify the perpetrators from 2 separate lineups (one perpetrator‐present lineup and one perpetrator‐absent lineup). Half the children took part in lineups adapted by a registered intermediary , and half took part in “best‐practice” lineups, according to the current guidance for eyewitness identification in England and Wales. Children receiving assistance from a registered intermediary (relative to children who received best‐practice lineups) were more accurate in their identifications for perpetrator‐present lineups, and there was some evidence that they were also more accurate for perpetrator‐absent lineups. This provides the first empirical evidence for the effectiveness of registered intermediary support during identification lineups. Further, these component skills improve markedly during childhood  have been suggested to make them more appropriate for child witnesses  are entitled to a registered intermediary (RI) at all stages of an investigation . An RI is an impartial, trained professional who facilitates understanding and communication between vulnerable witnesses and members of the justice system; ensuring that communication is complete, coherent, and accurate ; preparing reports detailing recommendations at different stages of the justice process; and advising more widely on how to enable the vulnerable individual to communicate their best evidence , suggest that the use of RIs is associated with increased access to justice, at both investigative and trial stages of cases  and, 1 week later, took part in a full evidential interview and identification lineup (at which some of the children received the support of an RI). The RI intervention at lineup included recommended adaptations to Police and Criminal Evidence Act (1984) Code D practice: showing the sequential lineup presentation once, opposed to twice, which could reduce fatigue and memory decay by reducing the presentation period; showing a simultaneous matrix of faces from the lineup, which could decrease the working memory load Cowan,  and lead22.1Participants were English‐speaking 6‐ to 11‐year‐old typically developing children attending one of four mainstream primary schools based in low/mid SES areas in a large, multi‐ethnic city . The sample comprised 85 children  between the ages of 6 years 6 months and 11 years 2 months (mean = 8 years 6 months), none of whom had diagnosed developmental disorders or special educational needs. The age range was selected as it encompassed a range of ages utilised in previous research, but was restricted enough to ensure that the staged event was suitable for all participating children. Participants were semirandomly allocated to the RI or best‐practice condition; strict random allocation was impossible due to practical issues, schools, and the need to test all children in the RI condition last , medium (w = .30), and large  on lineup accuracy (correct/incorrect), a post hoc power analysis on the sample of 85 was conducted using the software package, GPower  = 1.07, p = .30, or Perpetrator 2, χ2 = .16, p = .69. Data for the two events were, therefore, combined.Children watched a live event during school assembly of two male actors giving a talk about what school was like a long time ago. Towards the end of the talk, a minor crime (the theft of keys/phone) took place . They had completed necessary training provided by the Ministry of Justice, and also contributed to this training course for several cohorts of new RIs.2.2.3n = 42) saw Perpetrator 1 in the first lineup and other children (n = 43) saw Perpetrator 1 in the second lineup, and vice versa. There was no effect of lineup order for Perpetrator 1 accuracy, χ2 = .17, p = .68, or Perpetrator 2 accuracy, χ2 = .30, p = .59, so data were combined across the lineups.Immediately following the investigative interviews, children viewed two video lineups; one for each actor in the staged event. Each child viewed one “perpetrator present” lineup (in which one of the actors was present) and one “perpetrator absent” lineup (in which neither actor was present). Some children  Code D (2011), which gives provision for identification procedures in England and Wales. Each lineup contained nine (colour) video images of head and shoulders, facing front. Heads turned to the left and to the right profile, and then back to the front. PACE Code D specifies that each video “lineup” consists of a minimum of nine images, including one suspect; the witness must be advised that the culprit may not be present; the witness must view the entire sequential lineup twice before making any identification; and if the witness is unable to make a positive identification they should say so. The foils contained within each lineup were chosen by experienced police employees using their national database, PROMAT™. The lineups for the children in the best‐practice condition were run in accordance with PACE Code D (as described below). For logistical reasons, lineup administrators were not blind to the identity of the perpetrator, but they sat behind each child out of his/her eye line to avoid inadvertently influencing the lineup result.The possible lineup responses were correct hits of the perpetrator, foil identification, or incorrect rejection (for perpetrator present lineups); or correct rejections, false identification of innocent suspect, or foil identification (for perpetrator absent lineups). To ensure the lineup was not biased towards the suspect, a measure of lineup bias was calculated  = 5.76, p = .02. For Perpetrator 2, there was no significant effect of RI on lineup accuracy, χ2 = .00, p = 1.00. Inspection of the data collected from the children seen by each RI (n = 19 each) revealed that the children were comparable across all control measures (discussed next), although there was a trend towards the children seen by RI2 having higher facial memory scaled scores  on a subtest of a standardised memory battery , t(36) = −1.92, p = .06. This may account for the better performance of the children seen by RI2, relative to RI1, on one of the lineups.Importantly, although the RIs were present throughout the lineups (seated next to the child), their role did not extend beyond giving advice to the lineup administrator regarding instructions and procedure, as listed above. In addition, RIs had no knowledge of the identity of the perpetrators. There were two RIs involved in the study. For Perpetrator 1, one RI (RI2) elicited greater lineup accuracy than the other (RI1), χ2.2.4An extensive range of standardised cognitive measures  were administered to ensure that cognitive skills that might affect identification lineup performance were controlled between groups  from the second edition of the Wechsler Abbreviated Scale of Intelligence  on all cognitive variables. Age differed between the two groups, as did facial memory. Therefore, these variables were controlled in subsequent analyses.First, potential group differences in cognitive variables that might impact on witness performance were assessed . Table To examine the effect of condition (RI vs. best‐practice) on lineup accuracy and perpetrator presence or absence, whilst controlling for variables that differed between groups , two logistic regressions were conducted (one for each perpetrator). For both perpetrators, logistic regression analyses were performed with lineup accuracy (correct or incorrect) as the dependent variable. Predictor variables were condition, lineup presence, facial memory, and age in months.n = 85) were analysed and the full model significantly predicted lineup accuracy . The model accounted for between 15.1% and 21.6% of the variance, with 82% of correct performance (but only 62.5% of incorrect performance) successfully predicted. Table 2 = 7.40, p = .007, than the best‐practice condition  were analysed and the full model significantly predicted lineup accuracy . The model accounted for between 19.7% and 27.9% of the variance, with 84.7% of correct performance (but only 42.3% of incorrect performance) successfully predicted. Table p = .003) compared with the best‐practice condition. The odds ratio of an accurate lineup response occurring on the best‐practice perpetrator present lineup was 1.5, whereas on the RI perpetrator present lineup, all 19 participants were correct. RI presence led to a greater number of correct rejections on the perpetrator absent lineup, χ2 = 4.44, p = .04, compared with the best‐practice condition  = −1.08, p = .29. The results remained the same: for both perpetrators, there was a significant effect of condition on lineup accuracy: Perpetrator 1, χ2 = 4.46, p = .04; and Perpetrator 2, χ2 = 13.22, p = .001. For Perpetrator 1, the odds ratio of an accurate lineup response occurring on the best‐practice lineup was 1.47 and on the RI lineup was 4.8. For Perpetrator 2, the odds ratio of an accurate lineup response occurring on the best‐practice lineup was 1.14 and on the RI lineup was 13.5; thus, participants were more likely to be correct when in the RI condition for both perpetrators.As previously noted, scores on the facial memory subset of the Test of Memory and Learning differed between children in the two conditions, with children in the RI condition performing better on this subtest than the children in the best‐practice condition. Because facial memory is likely to be closely linked to identification performance, further analyses were conducted after removing nine children with the highest facial memory scores from the RI group, in order to check whether the significant effect of condition remained when facial memory scores were equivalent, 4Although the use of RIs in England and Wales is steadily increasing, there has been little empirical work exploring their efficacy, and none concerning their potential impact on identification lineups. This study was a novel evaluation of the effectiveness of RI assistance during an identification lineup in 6‐ to 11‐year‐old typically developing children. Results demonstrated a beneficial effect of RI intervention on lineup identification accuracy: adaptations to the lineup protocol suggested by RIs resulted in a higher number of correct identifications for perpetrator present lineups, with some evidence that these adaptations were also effective for perpetrator absent lineups.The current findings have forensic relevance in jurisdictions worldwide, given the international interest in RI schemes . This could have been effective via reducing fatigue (which may be likely in child witnesses). Additional analyses were conducted to explore this explanation . In PACE Code D, there is no protocol for adapting the lineup for vulnerable child witnesses, however, in this study, RIs highlighted the different lineup response options  and checked the child's understanding of their response . Previous research has shown that using visual prompts during a lineup  can be effective at improving lineup performance  lineup presentation, plus the simplification of the instructions and procedures , could have been key features that improved performance for children supported by an RI. Further research is required to replicate these findings and to tease out which RI adjustments are helpful to child witnesses.In this study, the presence of an RI improved performance on both perpetrator present lineups, but only had a positive effect on one of the two perpetrator absent lineups. Previous research has shown that children find perpetrator absent lineups more problematic than perpetrator present lineups. This could be because the mere presentation of photos/videos could imply to the child that the perpetrator features in the lineup, and that they are required to make a selection  and there was a realistic 1‐week delay between the presentation of the staged event and the subsequent lineup. As such, the research was more ecologically valid than many other studies on the performance of child witnesses during lineups, giving confidence in the results.In conclusion, this study highlights beneficial effects of RIs during identification lineups with 6‐ to 11‐year‐old typically developing children. However, one limitation is that this was an experimental study involving a mild and nonthreatening staged event. In addition, by the time the children were engaged in the lineup process (which was conducted in a familiar environment), they were comfortable with the RI and the interviewer/lineup administrator . In real‐life, children may be taking part in a lineup identification whilst experiencing high levels of stress and anxiety, which may (negatively) impact on their performance. One crucial aspect of the RI role is to assist in the management of such issues: to enable any trauma to be managed appropriately and to ensure the witness can engage and communicate with justice professionals (The Advocate's Gateway,


II). In the present work, four binding modes of TXB on LII were identified by a contact-map based clustering method. The highly flexible binary complex ensemble was generated by parallel tempering metadynamics simulation in a well-tempered ensemble (PTMetaD-WTE). In agreement with experimental findings, the pyrophosphate group and the attached first sugar subunit of LII are found to be the minimal motif for stable TXB binding. Three of the four binding modes involve the ring structure of TXB and have relatively higher binding affinities, indicating the importance of the ring motif of TXB in LII recognition. TXB-LII complexes with a ratio of 2:1 are also predicted with configurations such that the ring motif of two TXB molecules bound to the pyrophosphate-MurNAc moiety and the glutamic acid residue of one LII, respectively. Our findings disclose that the ring motif of TXB is critical to LII binding and novel antibiotics can be designed based on its mimetics.Teixobactin (TXB) is a newly discovered antibiotic targeting the bacterial cell wall precursor Lipid II (L II), one of the most essential intermediate products for the biosynthesis of bacterial cell walls, was recognized as an antibiotic target for decades1. Structurally, one LII molecule is composed of one bactoprenol hydrocarbon chain (C55), which is embedded in the cell membrane, a disaccharide of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), a penta-peptide attached to the MurNAc , and a pyrophosphate group (PP) linking the bactoprenol anchor and MurNAc3. The disaccharide and penta-peptide form the peptidoglycan subunit is further cross-linked through the penta-peptide to provide mechanical strength to the cell wall. LII is critical to the cell wall synthetic pathway4: the molecule is first assembled to the C55 anchor in the cytoplasmic side of the plasma membrane, and then translocated to the periplasmic side where the pentapeptide-linked disaccharide is released from the anchor to build the cell wall. The remaining C55-PP is translocated back to the cytoplasmic side of the membrane by a not well characterized recycling pathway5. Thus, preventing the translocation of LII is bona fide strategy to kill bacteria by interfering with a critical step in the cell wall synthetic pathway. Some antibiotics, such as vancomycin, nisin, ramoplanin and mannopeptimycin2 exert their antimicrobial activity by interacting with LII synthesis.Lipid II  with the D-Ala-D-Ala segment of the penta-peptide of LII7. Several of these complexes can even form a super-complex through the D-Ala-D-Ala segment9. However, bacteria develop resistance to vancomycin by mutating the penta-peptide terminus from D-Ala-D-Ala to D-Ala-D-Lac. The binding affinity of vancomycin to the mutated LII is 1000-fold lower mainly due to the introduction of a lone pair repulsion and the loss of central H bonds10. To counteract such effect, a vancomycin derivative was developed with similar binding affinity for both normal LII and mutated LII.The interaction between vancomycin and Let al. discovered a new LII-targeted antibiotic named teixobactin (TXB) by screening the uncultured bacteria with the help of iChip technology11. TXB is produced by the Gram-negative bacterium Eleftheria terrae and can effectively kill Gram-positive pathogens without any drug-resistance developed. Structurally, this compound contains 11 amino acids  and interaction of TXB with pyrophosphate and MurNAc group of LII is critical for binding.Recently, Ling ids Fig. , four ofet al. synthesized a TXB analogue by replacing the End-10 residue with an arginine residue12, and found this analogue only had a slight increase of the minimum inhibitory concentration (MIC) against Gram-positive bacteria when compared to TXB. Structure-function relationships of TXB and several new analogues indicated that the D-amino acids were important for activity, as almost a total loss of activity was observed when they were replaced by L-amino acids14. Further, modification of NmPhe-1 by the substitution of a methyl group with an acetyl group13, or substitution of the NH proton with one more methyl group, or introduction of a hydroxyl group to aromatic ring15 reduced the antimicrobial activity. Nowick’s group reported the importance of the ring motif and suggested the TXB-LII binding was through H bond interaction between amide NH groups on the ring motif of TXB and pyrophosphate group of LII16, which was similar to nisin-LII binding17. The importance of hydrophobicity from the N-terminal tail was also discussed in their work: the TXB analogue still remained bactericidal after replacing the residues NmPhe-1 to Ile-5 with a dodecanoyl group.The finding of such novel antibiotics stimulated follow-up studies to establish the TXB structure-function relationships. However, the great difficulties in the synthesis of the residue enduracididine (End-10) are the main obstacles in the synthesis of TXB. Jad II. However, a detailed structural model of the TXB-LII complex is not available. In the present work, molecular dynamics (MD) simulations with an enhanced sampling technique were performed to explore the binding phase space of TXB-LII complex with a ratio of 1:1, to figure out the specific binding modes of TXB and LII. Four different binding modes are discovered in our simulations, and the results indicate the ring motif of TXB plays an important role in recognizing LII. Structural models of the TXB-LII complex with a ratio of 2:1 are suggested, which are also found stable in the lipid bilayer environment. These discoveries provide new insights to understand the binding mechanism of TXB to LII.These previous studies provided clues on the important function groups of TXB interacting with LII increases the diversity of binding gestures. With so many polar residues, such as charged residues PP-4, Glu-7, Lys-8, Ala-10  in LII, and the polar ring motif in TXB, a lot of local minima could be foreseen on the free energy surface (FES) of TXB- LII binding. Three repeats of the simulation were run for 200 ns to investigate the binding of one TXB and one LII. The number of contacts between the two molecules i,j = 1, when the distance between atoms i and j is no larger than 0.4 nm, and P(t)i,j = 0, when their distance is larger than 0.4 nm. Compared with the residue-based contact map, this atom-based contact map avoids the accumulative effects, i.e. several atom pairs with low frequency ends up to one residue pair with high frequency, thus provides contact information with higher accuracy.The number of contacts in our PTMetaD-WTE simulation of 1 TXB and 1 L0 K Fig. , the PTM0 K Fig. . However0 K Fig.  was utilAccording to the contact map, 25 residue-pairs with high occupancy were selected and labelled with the letters from a to y, respectively. The 25 contacts are statistically meaningful, but for a specific binding mode, usually not all the 25 contacts are involved. The next step was to re-label each frame obtained from the simulation by identifying which contacts existed. For example, if a frame is labeled as “opqrst”, it means the contacts of o, p, q, r, s and t exist in this frame. In this way, all the frames could be re-grouped by their labels. The label, which is a combination of the contact letter a to y, functions as a natural metrics for binding mode identification. The 10 most popular binding modes are shown in Fig. 18 method was used to classify the frames of each binding mode, based on RMSD with respect only to the contacted residues with a cutoff of 0.25 nm. Concretely, that is RMSD of residues Ala-6, Glu-7 (LII) and Ser-7, Thr-8, Ala-9, End-10, Ile-11 (TXB) for binding mode 1 (BM1); RMSD of residues GlcNAc-1, PP-4 (LII) and Ser-7, Thr-8, Ala-9, End-10 (TXB) for binding mode 2 (BM2); RMSD of residues Glu-7, Lys-8, Ala-9, Ala-10 (LII) and Ile-2, Ser-3, Gln-4 (TXB) for binding mode 3 (BM3); and RMSD of residues Ala-10 (LII) and Ser-7, Thr-8, Ala-9, End-10, Ile-11 (TXB) for binding mode 4 (BM4). The representative conformations of each binding mode are presented in Fig. The gromosII. BM2 was characterized by the H bonds between four amide groups on TXB ring motif and PP-4 of LII, additionally, one H bond was also found between the hydroxyl group of Ser-7 of TXB and PP-4 of LII, as well as two H bonds between the sidechain-ring of End-10 of TXB and GlcNAc-1 of LII. BM3 was stabilized by three H bonds one of which is formed between the backbone carbonyl group of Gln-4 and the amide group of Ala-10, one is between the backbone amide group of Gln-4 and the carbonyl group of Lys-8, and another one is formed between the hydroxyl group of Ser-3 and the carboxyl group of Glu-7. And BM4 was similar to BM1, the difference is the carboxyl group was from Ala-10 instead of Glu-7. Occupancy for each of these H bonds during 600 ns simulation (3 repeats with 200 ns each) is listed in Table The featured contacts of BM1 were mainly maintained by the H bonds interaction between the four amide groups on the TXB ring motif (from residue Thr-8 to residue Ile-11) and the carboxyl group of Glu-7 on LFurther analysis of the featured contacts of BM1 and BM4 finds that the carboxyl groups of Glu-7 and Ala-10 come from the backbone: Glu-7 covalently bonds to Lys-8 via the side chain carboxylate instead of the backbone carboxyl and Ala-10 is located at the C-terminus. These two carboxyl groups cannot be changed by side-chain mutations. Both carboxyl groups could rotate during the binding. Therefore, both oxygen atoms in each carboxyl have the opportunity to form H bonds with the four amide groups in the ring motif of TXB  are shown in Fig. II system. The inter-group distance in TXB-C15PP system showed larger fluctuation than that in TXB-LII system at the end of simulations, and unbinding happened in repeat 1 of TXB-C15PP system, indicating that the introduction of GlcNAc-1 in LII further stabilized the binding. Our results agree with the experimental finding11 that the minimal motif of LII required for stable binding with TXB is the pyrophosphate (PP-4) and the first sugar (GlcNAc-1) moieties, both of which are highly conserved in bacteria2.Since the ring motif of TXB makes polar-contacts only with one phosphate group of the pyrophosphate residue, we examined whether one phosphate group is enough for a stable binding. Two new simulation systems were designed to verify the importance of the PP-4 motif. One system contained one TXB molecule and one C15P molecule, which was composed of a bactoprenol carbon chain covalently bonded to a phosphate group. The other system contained one TXB and one C15PP molecule, which was made of a carbon chain and a pyrophosphate group. Force field parameters for both C15P and C15PP molecules were taken from GAFF. Each system was run by triplicate and simulations started from the bound conformation. Distances between the ring motif and the phosphate (TXB & C15P system) or the pyrophosphate  utilize the ring motif of TXB to attract the charged residues of Lion Fig.  embeddedion Fig. . The RMSII binders show some differences and similarities. For instance, vancomycin binds to the negatively charged residue (the D-Ala-D-Ala segment of LII)7 through the formation of a “carboxylate anion binding pocket”24. Tridecaptin A1, a Gram-negative LII binder (Gram-negative LII has a meso-diaminopimelic acid instead of a lysine on the penta-peptide motif), forms H bonds with the carboxylate of meso-diaminopimelic acid, and the pyrophosphate moiety is not involved in the binding25. Mersacidin has an electrostatic-interaction dominated binding to LII, and the terminal GlcNAc sugar is included in the binding26. In contrast, Nisin binds to LII through the H bonds between the backbone amides on the ring structure and the pyrophosphate moiety17, and shares features with TXB  to test the reliability of our force field parameters. The topology file of nisin was developed with the same strategy as we described in Methods. The nisin-LII binding mode was well maintained during the 100 ns . However, considering the relatively higher binding affinity of BM1 and BM2, we believe that the model BM1&BM2 is the most probable model.The contact map of the last 50 ns is presented in Fig. II molecule. Such binding mode was found for ramoplanin, which dimerizes under membrane environment before binding to LII27. Therefore, another 400 ns PTMetaD-WTE simulation was carried out to simulate the interaction between two TXB molecules in solution. Based on the contact map and the subsequent unbiased MD simulation  were discovered. These structural models explain several experimental observations by confirming that: 1) the pyrophosphate-MurNAc moiety of LII is the minimal motif for a stable binding in BM1; 2) The ring motif of TXB is critical for its bactericide function; and 3) Binding sites in LII, the pyrophosphate-MurNAc moiety, the carboxyl group on the backbone of glutamic acid residue and the carboxyl group on the backbone of C-terminus alanine residue play essential roles in the synthesis of the bacterial cell wall. Thus, TXB resistance mutants of LII are unlikely to develop. To further validate such binding modes, X-ray and NMR approaches may be needed. Based on the flexible nature of such binding it is anticipated that direct structure characterization with the crystallography or NMR may be difficult. With our predicted complex models as a working hypothesis, experiments can be designed to explore the recognition mechanism between TXB and LII.Using PTMetaD-WTE simulations, we explored the binding phase space between the two molecules of TXB and LII molecules are described by AMBER99SB force field28, except End-10 in TXB, which is not defined in AMBER99SB. We chose GLYCAM_06j-1 force field29 to describe the sugar residues GlcNAc-2 in LII. However, the other sugar residue MurNAc was not available in the force field. MurNAc could be considered as the ether of lactic acid and GlcNAc, so it was divided into two residues GlcNAc-1 and Lac-5. Same as End-10 in TXB, Lac-5 is not a standard residue either, therefore, we generated their topologies, as well as the topologies of other undefined residues like C15-3 and PP-4 in LII, based on generalized amber force field (GAFF)30. Partial charges for the undefined residues  were fitted based on Restrained Electrostatic Potential (RESP) method in which the electric potential was calculated with Gaussian 09 at the level of HF/6-31 G*. The partial charges of some residues on the ring motif of TXB and Glu-7 of LII bonded with the neighboring residues in a non-standard way were recalculated using the same strategy.All the amino acid residues in TXB and L31. We mainly focused on the interaction between TXB and LII; therefore, the membrane was not included in the exploration of the binding phase space of TXB-LII complex to save the computational resources. This is reasonable according to the original study11, which claimed that purified LII could form complex with TXB in vitro, indicating the membrane is not involved in TXB-LII binding. The simulation systems were composed of TXB, LII (ratio of them would be mentioned in results and discussion part), TIP3P water32, and counter ions NaCl. Counter ions were modified by Joung et al.33 and added to a concentration of 0.17 M while neutralizing charges of the simulation systems. A leap-frog algorithm was used to integrate Newton’s equation of motion, and the time step was set to 2 fs along with the application of LINCS algorithm34, by whom all the covalent bonds between hydrogen atoms and heavy atoms were constrained. The cutoff for both van der Walls (VDW) interaction and short-range electrostatic interaction were set to 1.0 nm, and a Particle mesh Ewald (PME)35 method was employed to deal with the long-range electrostatic interaction. After the energy minimization, two short simulations with position restrains of all atoms of TXB and LII were performed to equilibrate the simulation system: 100 ps simulation in NVT ensemble to heat up the system temperature to 300 K and another 100 ps simulation in NPT ensemble to adjust the system pressure to 1 bar. After that, the product simulations were run 200 ns in NPT ensemble with all position restrains removed. The temperature and pressure of the system were maintained by V-rescale temperature coupling method and Parrinello-Rahman pressure coupling method respectively. Each kind of simulation was repeated three times by random assigning initial velocities.MD simulations were performed with GROMACS package, version 4.6.7II complex was stabilized in the aqueous simulations, we checked the stability of the complex in a membrane environment. The membrane model was built utilizing a web server MemBuilder II and parameterized with a Slipids force field published by Jambeck and Lyubertsev38, which is compatible with AMBER force field. In the prepared lipid bilayer, each leaflet of the membrane contained 48 negatively charged POPG and 16 zwitterionic POPE. Then one POPG was removed from the upper leaflet and our lipid II model was embedded. The membrane model was equilibrated for 50 ns at 300 K and 1 bar, and its area per lipid (APL) reached a value of 0.60 nm2, which was comparable with experimental results . After that, two TXB molecules were added onto the LII molecule in the predicted 2:1 complex configuration in the simulation box and 100 ns production MD simulation was performed. The distances between the ring motifs of the two TXBs and their respective targeting residues of LII were measured to illustrate the stability of the TXB-LII complex.After the TXB-LIn most cases, MD simulation is useful in exploring interesting events only when the system is ergodic in the simulated timescale. However, practically, simulation system usually presents metastability, and sampling would be trapped by some local minima of free-energy, making sufficient sampling unaffordable. Therefore, it is always a challenge for MD simulation to efficiently explore the large regions of phase space due to the limited time scale that the computational resources could reach.42 is one of the most popular sampling-enhancing methods, especially to validate and efficiently probe rare events, and to reconstruct free energy surfaces (FES)46. In metadynamics, a small repulsive Gaussian shaped potential, which is the function of the selected collective variables (CVs), is added to internal potential of the system every specified number of MD steps. At time t, the accumulated Gaussian potential VG can be written as,R is the microscopic coordinates and R. σi is the Gaussian width and W and deposition stride τG: Metadynamicset al. published a well-tempered metadynamics in 200847 to improve the convergence of metadynamics by utilizing a modified external bias,T has a temperature dimension.Barducci δs,s(t) is replaced by Gaussian, the difference between equation  to underlying free energy rather than oscillating around it and greatly increasing the availability of metadynamics. However, some hidden degrees of freedom that could not be described by the chosen CVs, would still limit exploration of the phase space, especially for those systems with high conformational complexity.Time derivative of this bias potential could easily be obtained,mentclass2pt{minim48, which is realized by well-tempered metadynamics when the system energy is used as CV, fluctuations of energy are enlarged, making it possible to have an efficient exchange with less replicas. Sampling efficiency of PTMetaD-WTE was used for protein folding50, peptide adsorption onto surfaces51, and protein oligomerization52.Another commonly used method to enhance sampling is Replica Exchange MD (REMD), such as parallel tempering (PT). In PT, non-interacting replicas of the system are simulated with different temperatures and exchange with adjacent replicas. Energy barriers might be overcome when higher temperatures are accessed by replicas, allowing for the exploration of new conformational space. Metadynamics can be easily combined with PT (PTmetaD), and will dramatically enhance the sampling efficiency of each replica. Although PTmetaD compensates the weakness of PT or Metadynamics in some degree, a sufficient overlap between the potential energy distributions of neighboring temperatures is necessary to reach an efficient exchange, which usually means the temperature gap cannot be too large and lots of replicas are required to achieve the sampling at a high temperature. However, in well-tempered ensemble (WTE)53. 16 replicas were simulated in NVT ensemble to avoid the instability of simulation box in NPT ensemble at high temperature. Temperatures varied from 300 K to 546.6 K and followed an exponential distribution: Ti was the temperature of replica i, and k = 0.04. Each replica contained 1 TXB, 1 LII, 6506 TIP3P water molecules as well as 24 sodium ions and 21 chlorine ions. 16 starting conformations of the TXB-LII complex with different contacts were generated from a standard MD simulation at high temperature (500 K). Before the PTMetaD-WTE simulations, 500 ps simulation was performed to equilibrate each replica.Our PTMetaD-WTE simulation was performed using Gromacs 4.6.7 package with the help of plumed 2.1.3 plug-inII heavy atoms was used as the new biased CV. In metadynamics simulation, CV should be wisely chosen to describe the degrees of freedom of system to achieve convergence. However, with the benefit from WTE, such limitation is largely reduced. The reason we chose the number of contacts as CV, instead of other generic CV such as inter-molecular distance, is that the contacts naturally include the information of interacting pairs between the two molecules, which is closely related to the binding modes. Number of contacts i and j were labels of heavy atoms from molecule A and B respectively, PTMetaD-WTE simulations were separated into two steps. First, 20 ns PTMetaD simulation was run with biases only on the potential energy. During this step, Gaussian potential was added every 500 calculation steps (1 ps), and a bias factor equal to 60 was adopted. Gaussian height was set to 2 kJ/mol and Gaussian width was set to 500 kJ/mol. Probabilities of neighboring-replica-exchange could reach 45%. In the second step, the accumulated biases on the potential energy CV in the first step were kept as a static additional bias potential, but no more bias were added to the potential energy. Number of contacts between TXB and L54. The instantaneous biased frequency U (R) is internal potential, and β is the inverse of product of temperature and Boltzmann constant. Equation (55:F(s) is the free energy as a function of CV In metadynamics simulation, weight of each sampled frame is biased by the additional Gaussians. The unbiased weight of each frame can be obtained from biased one based on the script published along with this workTherefore, the unbiased weight of each sampled frame has a value of The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Supplementary information


Mycobacterium tuberculosis (M. tb) H37Rv, is absent in the closely related Mycobacterium bovis and M. bovis Bacille Calmette-Guérin (BCG). On the other hand, we previously found that Mycobacterium marinum has an extended RD4 which includes a number of genes involved in the biosynthesis of lipooligosaccharides (LOSs). As such, there appears to be a gradual decay of RD4 in mycobacterial genomes in the order of M. marinum, M. tb, and M. bovis (including BCG). To understand the potential effect of RD4 on mycobacterial virulence, in this study, we cloned the entire (Rv1501–1516c) and partial (Rv1501–1508c) RD4 into an integrating vector. These constructs were introduced to M. bovis BCG and M. marinum and the virulence of the RD4 knock-in strains were evaluated in the SCID mice and zebrafish infection models, respectively. BCG containing the entire RD4 exhibited similar levels of virulence to the parental strain but BCG containing partial RD4 (Rv1501–Rv1508c) was more attenuated. Similarly, zebrafish infection experiments showed that addition of partial RD4 also appeared to attenuate the virulence of M. marinum. However, M. marinum containing entire RD4 was more virulent than the wild type strain. Interestingly, BCG strains containing the entire or partial RD4 exhibited better protection of zebrafish against M. marinum challenge than the parental BCG. Taken together, our data suggest that RD4 plays a role in mycobacterial virulence and that RD4 knock-in BCG strains confer improved protection. Our study has provided new insights into the biological function of RD4 and evolution of mycobacterial genomes.Comparative genome analyses have revealed a number of regions of difference (RD) among mycobacterial species. The functional consequences of most of these genome variations have not been studied. RD4, which encompasses Rv1506c-Rv1516c of  Mycobacterium tuberculosis (M. tb) complex (MTBC) comprises a group of closely related subspecies that shares 99.9% identical genome sequences but differs widely in terms of host tropisms and pathogenicity. These include the human-adapted M. tb, Mycobacterium africanum, and Mycobacterium canettii and the animal-adapted M. bovis, Mycobacterium caprae, Mycobacterium microti, Mycobacterium pinnipedii, Mycobacterium orygis, and Mycobacterium mungi  vaccine , ranging in size from 2 to 12.7 kb, are absent in the genome of M. bovis BCG-Pasteur   of Mycobacterium bovis BCG strains were grown at 37°C in Middlebrook 7H9 broth (Difco™) supplemented with 0.2% glycerol, 10% albumin-dextrose-catalase , and 0.05% Tween 80 or on Middlebrook 7H11 agar (Difco™) supplemented with 0.5% glycerol and 10% oleic acid-albumin-dextrose-catalase . Mycobacterium marinum strains were grown in the same media except the growth temperature was at 30°C. Escherichia coli strain DH5α was used for routine manipulation and propagation of plasmid DNA. E. coli strains were grown in LB broth or agar. Antibiotics were added as required: kanamycin, 50 μg/mL for E. coli and 25 μg/mL for BCG or M. marinum strains.M. tb genomic DNA as template. The PCR fragment was digested with SalI and HindIII and ligated into pMV306 predigested with these two enzymes. To construct pMV306::Rv1501-1508c, the forward primer 5′- CCTCGAAGCTTTCATGATACCGGTTCCATAGGTCCAATC-3′ and reverse primer 5′- TTGGCTAGCAACCGCGCGAGGTCCTC-3′ were used for PCR amplification using M. tb genomic DNA as template. The PCR fragment was digested with HindIII and NheI and ligated into pMV306::Rv1501-1502 predigested with HindIII and XbaI. To construct pMV306::Rv1501-1516c, the bacterial artificial chromosome Rv264 .The integrating vector pMV306  were pelleted and resuspended in 800 μL Trizol. Cells were disrupted by bead beating. The supernatant was then extracted with chloroform:isoamyl alcohol (24:1) and precipitated with isopropanol. Crude RNA samples were treated with gDNA Eraser and reversely transcribed using the PrimeScript™RT reagent Kit (Takara) according to the manufacturer's protocol. The resulting cDNA was used as the template for PCR amplification using primers specific to Rv1501 , Rv1507c , and Rv1516c .Mycobacterial cultures , and Four C57BL/6 mice were immunized subcutaneously with a mixture containing 100 μl purified Rv1505c protein (10 μg) and 100 μl of Freund's incomplete adjuvant (Sigma). The immunization procedure was repeated two more times (2 weeks apart). Two weeks after the last immunization, mice were sacrificed and their sera were collected.M. marinum strains, cell lysates were prepared and subjected to standard Western blot analysis. The primary antibody was the antisera against Rv1505c protein prepared above (1:100 dilution).For analysis of Rv1505c expression in recombinant BCG and M. bovis BCG-China and M. marinum 535 was performed as previously described  (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco). Cells were seeded at a density of 106 cells per well in DMEM medium supplemented with 10% FBS for 16 h. Macrophage cells were then infected with BCG or M. marinum at multiplicity of infection (MOI) of 10 and 1, respectively, for 4 h. Cells were washed three times with PBS and subsequently, incubated at 37°C (for BCG) or 32°C (for M. marinum) in fresh media containing 20 μg/mL gentamycin. At various time points post-infection, the infected macrophage monolayers (three wells per strain) were washed twice with fresh media and then lysed with 0.1 ml of 1% Triton X-100 (Sigma) to release intracellular mycobacteria. The number of intracellular mycobacteria was enumerated by plating appropriate dilutions on Middlebrook 7H10 agar plates containing appropriate antibiotics.Infection of murine macrophage cell line J774A.1 with recombinant strains of All of the animal procedures were approved by the local animal care committees at Fudan University. All methods were performed in accordance with the relevant guidelines and regulations.7 CFU of the different BCG strains in 0.1 ml PBS/0.01% Tween-80 and were monitored for survival. At day 1 post-infection, 2 mice from each group were sacrificed and the lungs and spleens were harvested, homogenized in PBS, and plated on 7H11 agar to enumerate bacterial burden. This was performed to confirm the actual infection dosage: 5.92 ± 0.10 log10, 6.16 ± 0.14 log10, and 6.15 ± 0.09 log10 CFUs in the spleens and 4.43 ± 0.11, 4.42 ± 0.10 log10, and 4.45 ± 0.12 log10 CFUs in the lungs for BCG-China::pMV306, BCG-China::Rv1501-1508c, and BCG-China::Rv1501-1516c groups, respectively. There was no significant difference on the infection dosage among the three groups.SCID mice infection with BCG was performed as described previously  were vaccinated intraperitoneally with 104 CFU of recombinant BCG or PBS as the negative control. At day 30 post-infection, six live fish from each group were sacrificed and homogenized. The homogenates were plated by serial dilution on Middlebrook 7H11 agar and incubated at 30°C for 7 days to determine the CFU of M. marinum.Groups of adult zebrafish (20 per group) were vaccinated intraperitoneally with 10M. bovis BCG strains (BCG-China and BCG-Japan) and two M. marinum strains (535 and 1218R). PCR analysis using primers specific to Rv1507A (for Rv1501-1508c) and Rv1515c (for Rv1501-1516c) confirmed the successful integration of these two fragments into the mycobacterial genomes  and entire (Rv1501-1516c) regions of RD4, respectively, into a mycobacterial integrating vector pMV306. These constructs were introduced to two s Figure . To detes Figure . Resultss Figure .M. marinum 535 knock-in strains as the representatives and examined their growth in 7H9 broth and in J774 macrophages. There was no difference in growth under these conditions between the knock-in strains and the respective control strains  were intravenously infected with 107 CFU of the BCG strains and their survivals were monitored. The survival curves were plotted using a Kaplan-Meier analysis.We first determined the virulence of the BCG-China knock-in strains in SCID mice. Since BCG is highly attenuated, the virulence of BCG strains is routinely evaluated in immunocompromised SCID mice lacking T- and B-lymphocytes. Groups of SCID mice (p < 0.01) and the BCG-China::Rv1501-1516c group (p < 0.01). There was no significant difference between the parental and the BCG-China::Rv1501-1516c groups.The median survival time for the parental, the BCG-China::Rv1501-1508c and BCG-China::Rv1501-1516c groups were 63, 77, and 67.5 days, respectively Figure . Log-ranM. marinum 535 knock-in strains in zebrafish. Zebrafish have been widely used as a laboratory model for studying M. marinum infection, which manifests both acute disseminated disease and chronic persistent infection  were intraperitoneally infected with 100 CFU of WT M. marinum 535 and the two RD4 knock-in strains, and were monitored for survival. The median survival time for the WT, M. marinum:: Rv1501-1508c, and M. marinum::Rv1501-1516c were 19, 23, and 15 days, respectively  and the WT M. marinum (p < 0.05). The M. marinum::Rv1501-1508c group appeared to survive longer than the WT group with the difference approaching significance (p = 0.08).We next determined the virulence of y Figure . ZebrafiM. marinum infection model. Oksanen et al. previously showed that vaccination of with BCG or DNA vaccines expressing known antigens  protects adult zebrafish against M. marinum infection, suggesting that zebrafish is a promising new model for preclinical TB vaccine research  were vaccinated intraperitoneally with 104 CFU of BCG strains or PBS as the negative control. Thirty days after BCG vaccination, zebrafish were infected intraperitoneally with 10 CFU of M. marinum 535 and were monitored for survival. Log-rank analysis of the survival curves revealed that the median survival time of the unvaccinated group (PBS), the parental BCG, the BCG-China::Rv1501-1508c and BCG-China::Rv1501-1516c groups were 27.5, 30, 45.5, and 54 days, respectively .Groups of adult zebrafish  were vaccinated by the BCG strains and infected with M. marinum as described above, and six live fish from each group were sacrificed at 30 days post-infection to determine the bacterial burdens. Consistent with the survival curve data, the M. marinum burdens in the BCG-China::Rv1501-1508c and BCG-China::Rv1501-1516c groups were 1.73 and 2.25 log10 lower than that in the BCG-China group, respectively  and the bacterial burdens in the two knock-in BCG groups were also significantly lower than the unvaccinated group  in M. marinum increased its virulence, whereas this effect was not observed in M. bovis BCG  in BCG and M. marinum, the biosynthesis of LOSs was found to be linked to the secretion of PE-PGRS proteins  in animal models will help to address this question. While further studies will be required to test our hypotheses, the RD4 knock-in BCG could be a promising candidate for the development of more effective TB vaccines to replace current BCG.The lack of RD4 in BCG strains may also be a disadvantage in terms of providing protection against JL conceived and supervised the project, wrote the paper. LZ supervised the project. HR, XL, CL, JY, FC, and RS performed the experiments.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.


This study aimed to determine the trends in prevalence of childhood overweight and malnutrition in a large Chinese ethnic minority population from 1991 to 2010. In the Chinese National Survey on Students’ Constitution and Health from 1991 to 2010, multistage stratified sampling was conducted in the series of cross-sectional studies. Participants were 7–18-year-old students randomly selected by sex and region, and included Han and 26 ethnic minorities. During the survey period, the overall prevalence of overweight increased from 5.8% to 13.5%, and malnutrition trend increased from 3.6% to 4.1% in ethnic minority children and adolescents. Moreover, Korean and Mongol children were more likely than Han children to be obese . Among these minorities, the Dongxiang and Li children were more likely to be malnourished . Shui, Khalkhas, Lisu, and Monguor children were less likely to be overweight and malnourished compared with the Hans. The prevalence of overweight among ethnicities increased yearly while that for malnutrition has fluctuated over the past few decades. Overweight and malnutrition are major health concerns in many low- and middle-income countries, including China136Many studies have reported the prevalence of overweight and obesity among children and adolescents in China, but those on malnutrition remain sparse. Concerns are increasing regarding epidemic trends in overweight and obesity, especially among the Hans. However, the prevalence rates of overweight and malnutrition among minorities remain unknown, with the exception of some studies on Tibetan, Mongolian and Uygur children1112Thus, this study aims to describe the prevalence rates of overweight and malnutrition among school-aged children and adolescents from different ethnic groups.The Chinese National Surveys on Students’ Constitution and Health (CNSSCH) is a series of complex multistage, cross-sectional, nationwide surveys on the physical fitness and health status of students in China. Becoming a continuous survey in 1985, the CNSSCH has released data every 5 years since, with currently six completed surveys.In this study, Han participants were school students aged 7–18 years, randomly selected from 31 mainland provinces. The participants were classified by gender and region  within each province, and divided into four groups with equal sample sizes from three socioeconomic classes . The prevalence of overweight among Han children and adolescents has been reported elsewhereParticipants of ethnic minorities were primary and secondary school students aged 7–18 years, randomly selected from 13 provinces. Mongolian, Hui, Uyghur, Zhuang and Korean student participants were classified by gender and region . Other ethnic minorities were classified by gender only, not by region , and werOur study was approved by the ethics committee of the Medical College of Soochow University in Suzhou, Jiangsu, China, and followed the tenets of the Declaration of Helsinki. Verbal informed consent was obtained from all students aged 7–18 years and from their parents after the nature of the study had been explained.th percentile weight to the same height population. The standards were created from 1985 CNSSCH data. Overweight was defined as >110% of the standard weight-for-height, after adjusting for age and sexData were obtained from normal students who were well-developed, physically and psychologically healthy, and able to participate in various sports activities. Students who had multiple organ dysfunction , physical disability and deformation, acute diseases , and females who were menstruating were excluded from participating in the fitness test of the survey. Height (cm) and weight (kg) were measured by the same trained technicians. Weight was measured to the nearest 0.1 kg using a balance-beam scale while the participants were wearing lightweight clothing. Height was measured to the nearest of 0.1 cm with a portable stadiometer while the participants were barefoot . Weight We estimated distributions of overweight and malnutrition by gender and residence . P < 0.05 was considered statistically significant. To test for trends in the prevalence rates of overweight and malnutrition among children and adolescents during 1991–2010, we used generalized linear models (GLM) with a log function based on binomial distribution. The GLM was also used to generate rate ratios  between the Han and ethnic minority groups, after adjusting for age, group, sex, and region. The overall prevalence rate of ethnic minority groups was normalized by means of weighting by the population of ethnicities from China’s 2010 census data. Age-adjusted prevalence of every ethnic minority was also directly adjusted according to China’s 2010 census population age structure. Data were analysed using SAS  statistical software.From 1991 to 2010, the overall prevalence of overweight significantly increased among ethnic minority children and adolescents from 5.8% to 13.5%  . HoweverA lower rate of prevalence for overweight and malnutrition was found among children living in rural regions compared with those living in urban regions . Likewise, a lower rate of prevalence in other regions was found when compared with that in urban regions  . The preIn 2010, the Koreans had the highest overall prevalence of overweight at 30.6% , followed by the Mongolians at 22.3%  . Among eFrom 1991 to 2010, the Koreans and Mongolians were more likely than the Hans to be obese . The overall RR for the minority/Han children for overweight was 0.53 . Among these minorities, the Dai, Salar, Zhuang, Zang, Dongxiang, and Li were more likely to be malnourished than other minorities. The overall RR for minority/Han children for malnutrition was 0.81  .Further analysis of the trend among the outliers in In this nationwide health survey of Chinese children, we observed that the prevalence of overweight in ethnic minorities had increased over the past few decades. Compared with the Hans and excluding Korean and Mongolian children, most ethnic minorities had a lower risk for the prevalence of overweight. The results also indicated that the children living in urban areas had a higher prevalence of overweight than their rural counterparts. Girls had a higher prevalence of malnutrition than boys. Moreover, most minorities had a lower risk for the prevalence of malnutrition among children and adolescents compared with the Hans from 1991 to 2010. Although the overweight prevalence among ethnic minorities was increasing yearly, the trend for malnutrition fluctuated.Ethnic minorities had a lower prevalence of overweight overall compared with the Hans. A main reason for this may be due to genetic differences between the Hans and some ethnic minorities. For example, the Q27E genotype and allele frequency distribution differs among the Hans and Uyghurs. ADRB2 is associated with fat mobilization and stimulus generated by heat, and its genetic polymorphisms are significantly susceptible to obesity in the human body. Among these polymorphisms, Q27E is most common and is related to diseases such as obesity, primary hypertension, and type 2 diabetesHowever, due to the development of local tourism, frequent cultural exchanges, and economic exchanges between the Han and ethnic minorities, the recent prevalence of overweight among ethnic minorities was found to rise. The Mongol and Korean children had a higher overall prevalence of overweight than the Han children, which could be due to different factors. Mongolians living in the plateau region consume a large amount of red meat in order to adapt to the high altitude and frigid weatherChildren and adolescents in most ethnic minorities were at a lower risk for malnutrition compared with the Han children from 1991 to 2010. China has adhered to the policy of national equality and unity for an extended period. Furthermore, the Chinese government greatly supports the development of ethnic minorities and has intensified reform and opening-up among minorities. For example, the government gives preferential policies and several subsidies to ethnic minorities. The government also engages in the construction of infrastructures among ethnic minorities, such as hospitals, and trade development, which helps decrease poverty. However, national finance cannot support the entire Han population that lies below the poverty line. Thus, more parents than in the past work far from their hometowns and consequently cannot care for their children. This is a possible reason why the Hans were found to have a relatively high rate of malnutrition compared with ethnic minorities from 1991 to 2010.The differences among age groups, similar to that of other nations, are more likely caused by children’s growth and sexual maturity than environmental and behavioural factors. Furthermore, we used a statistical definition of obesity and malnutrition that was based on a comparison with the 1985 reference population represented in the CNSSCH growth charts. The charts were created for comparing within specific sex and age groups and not for comparing across sex and age groups. Age differences in prevalence reflect differences from the original reference population. Therefore, we did not focus on the variation from age, and examined the trend of overweight and malnutrition among ethnic minority children, after adjusting for age group as a confounder.In 1995, the prevalence of malnutrition was high among minorities in the 13–15- and 16–18-year age groups. Mongolian, Shui, Tujia, Bai, Dai, and Yi children were not included in the 1995 survey. The prevalence of malnutrition among the Zang, Uyghur, and Zhuang children in 1995 was higher than that in previous years. Moreover, the Mongolians, Yis, Uyghurs, and Zhuangs had relatively heavier weight. These data may contribute to findings that the prevalence of malnutrition was high among minorities in certain age groups in 1995.In general, minorities with greater overweight prevalences were less likely to be affected by malnutrition prevalence. Interestingly, results from the Shui, Khalkhas, Lisu, and Monguor children were inconsistent with this finding. The Khalkhas and Monguors are nomadic ethnicities, whereas the Shuis and Lisus livelihoods are based on agriculture, which requires physical strength. This may be the reason why these four ethnicities had a higher prevalence of malnutrition than other minorities.In China, the overweight prevalence among ethnic minorities increased yearly and the trend of malnutrition fluctuated. However, in developed countries, a stable prevalence of childhood overweight and obesity over the past 15 years has been reported, while the prevalence of malnutrition has been found to be increasing, especially in girlsFrom a public health perspective, it is important to monitor and mitigate trends in overweight and malnutrition among children and adolescents. Non-communicable diseases (NCD) killed 38 million people globally in 2012, accounting for 68% of total annual death worldwideWe acknowledge study limitations. First, this study analysed the Han and 26 other ethnic minority groups, but did not include another 29 ethnic minority groups. However, the remaining ethnic minorities account for <15% of the entire ethnic group population. Second, all Han participants in the 1991 survey were selected from a high socioeconomic population. Therefore, the prevalence of malnutrition is likely underestimated, and the trend of overweight likely overrated. Third, the definitions used for overweight and malnutrition in this article were not global criterions. Although different definitions of overweight and malnutrition in children and adolescents are used worldwide, they may be unsuitable for China’s children and adolescents. However, the standard weight-for-height references were created from the 1985 CNSSCH data , which had the largest sample size among the six existing surveys. The World Health Organization further recommends using these weight-for-height references. Since 1985, the standard weight-for-height references have been widely used for evaluating children’s nutritional status, and overweight and malnutrition in China.We observed that the overweight prevalence among ethnic minorities was increasing yearly and that the trend of malnutrition fluctuated. We also examined the relationship between overweight and malnutrition among ethnic minorities. The results of this study could help the Chinese government to better understand the prevalence of overweight and malnutrition among ethnic minorities and take measures to improve the present situation.How to cite this article: Guo, S. et al. Prevalence of overweight and malnutrition among ethnic minority children and adolescents in China, 1991–2010. Sci. Rep.6, 37491; doi: 10.1038/srep37491 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Alcohol use is a global health issue and may influence activity performance in a variety of domains, including the occupational and domestic spheres. The aim of the study was to examine the influence of annual drinking frequency and binge drinking (≥6 units at one occasion) on activity impairments both at work (sickness presenteeism) and outside the workplace.Employees (n = 3278), recruited from 14 Norwegian private and public companies, responded to a questionnaire containing questions from the Alcohol Use Disorders Identification Test (AUDIT) and the Workplace Productivity and Activity Impairment questionnaire (WPAI).Multiple hierarchical regression analyses revealed that binge drinking was associated with both sickness presenteeism and impaired daily activities, even after controlling for gender, age, educational level, living status and employment sector. Annual drinking frequency was associated with impaired daily activities, but not sickness presenteeism.Binge drinking seems to have a stronger influence on activity performance both at work and outside the workplace than drinking frequency. Interventions targeting alcohol consumption should benefit from focusing on binge drinking behavior. Alcohol use constitutes a global health issue. Harmful use of alcohol has been found to be involved in more than 200 different injury and disease conditions . The WorAlcohol consumption may influence activity performance in a variety of domains, including the occupational and domestic spheres. In his general model of employee substance use and productivity outcomes, Frone  proposesAlcohol consumption are often associated with impaired daily activities, such as difficulties in carrying out daily routines  and mobiDifferent drinking patterns can have dissimilar effects on outcome measures. One may distinguish between (a) drinking frequency, i.e., the typical frequency of drinking in a given period of time, and (b) episodic heavy drinking (binge drinking). Binge drinking is often operationalized as consuming five drinks or more on one occasion , 15. HowIn line with Bacharach and colleagues , it may The present study was conducted in Norway, a country in which alcohol is a legal and widely used drug. Traditionally, Norway has been characterized as a spirit-drinking country with binge drinking during the weekends and abstinence during weekdays, i.e., a dry drinking culture . HoweverBased on a public health perspective and justified by the total consumption model , Norway Knowledge on the relationship between alcohol consumption on one hand and sickness presenteeism and impaired daily activities on the other, is limited within working populations that are not in clinical treatment for alcohol abuse or -dependence. To be able to provide early identification and public health programs targeting risky drinking, such knowledge might be crucial. Moreover, there seems to be a shortage of studies that have explored and compared activity restrictions both within and outside the workplace.The aim of the present study was therefore to explore the influence of annual drinking frequency and binge drinking on sickness presenteeism and impaired daily activities in a sample of Norwegian employees.This study is part of the Norwegian national WIRUS project , where one of the studies are the WIRUS-Screening study. Other results from WIRUS are published elsewhere . The stuThe employees were invited to participate in a web-based alcohol screening study, where they answered questionnaires designed to measure alcohol consumption, sickness presenteeism and impaired daily activities. A total of 4,275 employees (29.8%) responded to the questionnaire. However, 997 employees were excluded because of missing values on key variables or as a result of being abstainers, leaving a final sample of 3,278 individuals. Characteristics of the study sample, the invited sample and the Norwegian workforce are presented in The study sample consisted of 32.6% males and 67.4% females. 68.5% of employees were aged ≥40 and 75.3% had completed a university or college education. 10.0% of the respondents were employed within the five private sector companies , while 90.0% were employed within the nine public sector companies .Two questions were used to measure alcohol consumption. Both items were taken from the Norwegian translation of the Alcohol Use Disorders Identification Test (AUDIT), developed by the World Health Organization . Annual Sickness presenteeism and impaired daily activities were measured by one item each taken from a Norwegian translation of the Work Productivity and Activity Impairment questionnaire (WPAI). Sickness presenteeism was measured on a visual analogue scale ranging from zero (no influence on productivity) to ten (obstructed productivity completely), where respondents answered the following question: "During the past seven days, how much did alcohol consumption affect your productivity while you were working?". The WPAI has demonstrated satisfactory psychometric properties  and measSimilarly, impaired daily activities were measured by asking respondents: "During the past seven days, how much did alcohol consumption affect your ability to do regular daily activities, other than work at a job?". Responses were given on a visual analogue scale from zero (no influence on activities) to ten (obstructed activities completely).Sickness presenteeism and impaired daily activities were entered as continuous variables in correlation and regression analyses, and collapsed into two categories (impairment/no impairment) for utilization in crosstabulation. No impairment reflected a score of zero, while impairment included scores ranging from one to ten on the visual analogue scale.Earlier studies have found variables such as gender, age, educational level and family life to be associated with activity performance in working populations , 30. Ther) were performed to explore the strength and direction of the unadjusted relationships between the variables. Contingency tables were constructed to estimate the odds and risks of impairment given low or high levels of annual drinking frequency and binge drinking, respectively. Adjusted multiple hierarchical regression analyses were applied to investigate the influence of annual drinking frequency and binge drinking episodes on sickness presenteeism and impaired daily activities. Control measures were entered at stage 1 and alcohol measures were entered in stage 2 to evaluate the model as a whole, as well as the influence of each independent variable. Significant results were defined as p < .05.All statistical analyses were performed with IBM SPSS version 24. Bivariate correlation analyses . Respondents were informed about the study's aim and confidentiality, assured that participation was voluntary and provided written informed consent.As seen in Almost two out of ten (19.7%) employees reported “frequent drinking” during last year, i.e., consumption on a weekly or almost daily basis, while the majority (80.3%) reported “infrequent drinking” (maximum four times a month). Approximately one out of ten (11.0%) employees reported “recurrent binge drinking” during the last year , while 89.0% reported “never or rarely binge drinking”.As seen in As shown in F , ΔR2 = .005, p < .001. In the fully adjusted model, binge drinking was the only independent predictor associated with sickness presenteeism . Annual drinking frequency did not display a statistically significant contribution to the model .The sickness presenteeism hierarchical regression model is presented in F , ΔR2 = .025, p < .001. After controlling for gender, age, educational level, employment sector and living status, both annual drinking frequency and binge drinking were significantly associated with impaired daily activities. Binge drinking  displayed a stronger influence on daily activity impairment than annual drinking frequency .The impaired daily activities hierarchical regression model is presented in The aim of the present study was to explore the influence of annual drinking frequency and binge drinking on activity impairments both at work (sickness presenteeism) and outside the workplace. Results showed that (a) binge drinking was associated with higher levels of sickness presenteeism and impaired daily activities, (b) binge drinking had a stronger influence on daily activities than on sickness presenteeism, and (c) annual drinking frequency significantly influenced the employees’ daily activities but it did not affect sickness presenteeism.Binge drinking was associated with both higher levels of sickness presenteeism and impaired daily activities outside the workplace. Binge drinking is known to have several short-term effects such as hangovers, decreased attention and reduced concentration, as well as other temporary physical, cognitive and psychological disturbances . These cSomewhat surprisingly, the association between binge drinking and impaired daily activities was stronger the association between binge drinking and sickness presenteeism. Similarly, annual drinking frequency displayed an influence on impaired daily activities but not on sickness presenteeism. An explanation for these findings could be that (heavy) drinking usually occurs on days preceding weekends and holidays, when the employees have a day off from work .Studies on drinking patterns have found that people drink less before conducting "serious" activities that require long-term commitment and focus, such as work activities, due to the impact heavy drinking can have on performance . AnotherBy comparing standardized regression coefficients and probability values, the present study found that annual drinking frequency had less influence on both activity performance measures compared to binge drinking. It is possible that, whereas binge drinking episodes result in more short-term disability and impairments, a pattern of frequent consumption can have more long-term consequences which do not immediately influence employees' activity performance in a short-term perspective . IndividFindings from the present study might indicate that binge drinking has a stronger influence on activity performance than annual drinking frequency, both at work and outside the workplace. Hence, individual and collective interventions aimed at preventing the development of alcohol-related problems may benefit from specifically targeting alcohol consumption behavior characterized by high levels of binge drinking. The findings from this study may in particular have implications for public sector employees, as a result of well educated female employees above age 40 and employed within public administration constituting a large proportion of the study sample.The present study has some limitations. It was based on a cross-sectional design and, hence, it is not possible to draw causal inferences from the associations identified. The relationship between alcohol consumption and activity performance may, as emphasized by Frone , be modep < .001), with a 4.0% underrepresentation of employees younger than 40 years old. Although non-response is a less prominent threat to associations between variables than to prevalence estimates [This study was based on a relatively large sample . The final response rate, however, was low (22.8%). Moreover, comparisons between our study sample and characteristics of the entire Norwegian workforce did reveal that older, highly educated and female employees were somewhat overrepresented in this study. On the other hand, our study sample was to a much lesser degree different from our invited sample regarding gender and age distributions. Gender distribution in the study was not significantly different from the invited sample. Age distribution, however, was significantly different  and outside the workplace. Binge drinking was stronger associated with activity impairments than annual drinking frequency, and binge drinking was stronger associated with daily activities than with workplace performance. Although further longitudinal research is needed, the findings of the present study implicate that interventions targeting alcohol consumption should place large emphasis on binge drinking behavior.