With the increasing use of anti-TNF therapy in inflammatory bowel disease (IBD), a shift of costs has been observed with medication costs replacing hospitalization and surgery as major cost driver. We aimed to explore the evolution of IBD-related costs over two years of follow-up.In total 1,307 Crohn's disease (CD) patients and 915 ulcerative colitis (UC) patients were prospectively followed for two years by three-monthly web-based questionnaires. Changes of healthcare costs, productivity costs and out-of-pocket costs over time were assessed using mixed model analysis. Multivariable logistic regression analysis was used to identify costs drivers. In total 737 CD patients and 566 UC were included. Total costs were stable over two years of follow-up, with annual total costs of €7,835 in CD and €3,600 in UC. However, within healthcare costs, the proportion of anti-TNF therapy-related costs increased from 64% to 72% in CD (p<0.01) and from 31% to 39% in UC (p < 0.01). In contrast, the proportion of hospitalization costs decreased from 19% to 13% in CD (p<0.01), and 22% to 15% in UC (p < 0.01). Penetrating disease course predicted an increase of healthcare costs (adjusted odds ratio (adj. OR) 1.95 (95% CI 1.02–3.37) in CD and age <40 years in UC (adj. OR 4.72 (95% CI 1.61–13.86)).BD-related costs remained stable over two years. However, the proportion of anti-TNF-related healthcare costs increased, while hospitalization costs decreased. Factors associated with increased costs were penetrating disease course in CD and age <40 in UC. Crohn’s disease (CD) and ulcerative colitis (UC), collectively known as inflammatory bowel diseases (IBD), are characterized by chronic relapsing intestinal inflammation that may lead to severe complications and disability. Therefore, IBD represent a high economic burden to society.–8 The eaWith the introduction and increasing use of anti-TNF therapy in IBD, a major shift of costs has been observed with medication costs replacing in-patient care, such as hospitalization and surgery, as the greatest source of healthcare expenditure. Most preThe ‘Costs Of Inflammatory bowel disease in the Netherlands’ or COIN-study has been initiated to generate longitudinal cost data in order to assess the impact of anti-TNF therapy on IBD-related costs. In the present study we aimed 1) to assess the evolution of costs of IBD over a period of two years, 2) to explore the contribution of healthcare, productivity and out-of-pocket costs on IBD-related costs; and 3) to identify predictors for high costs over two years of follow-up.From October 2010 to October 2011 we invited all IBD patients aged 18 years or older from seven university hospitals and seven district hospitals to participate in the COIN-study by letter .A secure web-based questionnaire was developed to obtain baseline characteristics and collect cost data on a three-month basis during two years of follow-up. The cohort organisation and study follow-up protocol have been described in detail elsewhere. The studDemographic characteristics included gender, age, age at diagnosis, education level, work status, family history, and smoking status. Clinical characteristics included subtype of IBD, disease duration and localization, disease behaviour, stoma or pouch surgery, and clinical disease activity.In accordance with Drummond et al., we distData analysis was performed using SPSS version 18.0. Descriptive statistics were used to characterize patients with CD and UC. We report means with a standard deviation (SD) and medians with an interquartile range (IQR). Comparisons between CD and UC patients were analysed with Student’s t-test for continuous variables and χ2 for dichotomous variables. To compare medians, the Mann-Whitney U test was used. Costs were reported as mean cost/patient with a 95% confidence interval.To control equality between the study population (i.e. responders) and the patients who were lost to follow-up over time (i.e. non-responders) we performed a non-responder study. To account for missing data and repeated measurements, we used a generalized mixed model to compare costs between different subgroups.We performed a multivariate logistic regression analysis to identify factors predicting increase of healthcare costs over two-years of follow-up. As a dependent variable we used the 10 percent of patients who displayed the highest increase in healthcare costs over two years of follow-up. Variables that reached borderline significance (p<0.1) in the univariate analysis were considered for inclusion into the multivariate models. We fitted separate models for UC and CD. P-values <0.05 were considered statistically significant.At baseline, 1,307 CD patients and 915 UC patients were included. The two-year follow-up questionnaire was filled-out by 736 CD patients and 566 UC patients . Additional response rates per time point are provided in The baseline characteristics of the study population completing the two-year follow-up are described in Over the two-year follow-up period, IBD-related costs did not change . The meaIn In The widespread use of anti-TNF in the treatment of patients with IBD has changed the healthcare landscape radically and has led to a major shift in cost profiles. For the Most of the IBD-related costs were incurred by anti-TNF therapy, both in CD and UC patients. The present data underscore our previous observations that healthcare expenditures in IBD shift from costs related to hospitalization and surgery to costs driven by medication use. Due to tAn important observation is the ongoing rise of anti-TNF therapy-related costs, with a concurrent reduction of hospitalization costs. A similar trend in increase of anti-TNF therapy-related costs has been found in rheumatoid arthritis.;15 In twEven though healthcare cost differ to a large extend between Western countries, comparable trends in treatment paradigms should have induced the same alterations in cost profiles as observed in our study. For example, Kappelman et al. studied healthcare costs using medical and pharmacy claims from an administrative database between 2003 and 2004, in which 10% of all CD patients had at least two claims of infliximab infusions. In this The large sample size and longitudinal data enabled us to study predictors of healthcare costs over time. In CD, penetrating disease was found to be associated with an increase of costs over two years of follow-up. This can be attributed to the fact that a penetrating disease is a predictor of poor outcome in CD, resulting in frequent surgery and hospitalizations–21. FurtIn UC patients younger than 40 years of age, an increase of healthcare costs was encountered as well. We found a 100% increase of anti-TNF use among young UC patients during two years of follow-up. This finding is in line with previous studies in which younger age in UC was found to be associated with a more severe disease course and an increased risk of relapses.–24 FurthOur study has several limitations. First, an inherent limitation of a longitudinal study using a web-based questionnaire design is the high rate of loss to follow-up. We tried to reduce the impact of this problem by using mixed models to correct for missing values. Furthermore, we performed a non-responder study, which showed that responders  were older and had a longer disease duration. Since costs in elderly IBD patients are lower than in younger patients, we may hIn conclusion, there is an apparent shift in cost profiles from surgery and hospitalization towards anti-TNF therapy. However, total IBD costs remain remarkably stable over time, suggesting that the anti-TNF-related costs are compensated by a reduction of hospitalization costs. This may corroborate the notion that investment in expensive medical therapy might be cost-effective from a pharmaco-economical point of view,presuming that a reduction in hospital admission is equal with an improvement in quality-of-life. Whether long-term anti-TNF therapy is truly cost-effective in IBD has yet to be determined. Further careful monitoring of changes in the costs of care for IBD patients will aid timely, sensible economic decision-making.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 TableA. Average healthcare costs/patients per 3 months in CD patients (€). B. Average healthcare costs/patients per 3 months in UC patients (€). C. Proportion of healthcare costs in CD (%). D. Proportion of healthcare costs in UC (%).(DOCX)Click here for additional data file.


The 2013-2015 Ebola Virus Disease outbreak in West Africa had similar nuances with the 1976 outbreaks in Central Africa; both were caused by the Zaire Ebola Virus strain and originated from rural forested communities. The definitive reservoir host of Ebola virus still remains unknown till date. However, from ecological perspective, it is known that the virus first emerged from forest ecotypes interfacing with human activities. As at March 2015, the outbreak has claimed over 9000 lives, which is unprecedented. Though it remains unproved, the primary sources of infection for past and present outbreaks are forest dwelling, human-hunted fauna. Understanding the ecological factors at play in these forest ecotypes where wild fauna interface with human and causing pathogen spill over is important. A broad based One Health approach incorporating these ecological concepts in the control of Ebola Virus Disease can effectively ameliorate or forestall infection now and in the future. In 1976, a novel virus that emerged from the Congo basin and was later identified as a new filovirus following virological and immunologic investigation . The WHOThe biodiversity of the forest ecotype intimately related to EBOV spill-overs and subsequent EVD outbreaks provides habitats to a wide range of fauna and their supporting flora: humans continuously interact with the biotic and abiotic components of this ecotype in complex ways that drive and engender spill-over events. Many of these animals are equally susceptible to zoonotic infectious diseases like Ebola. Hence the combination of susceptibility to agents of infectious disease that can also be transmitted to humans makes wildlife a potentially dangerous reservoir of pathogens. Though many of such wildlife associated zoonotic infections like SIV-HIV, rabies, influenza and corona viruses have been reported to cause morbidity and mortality . HoweverThe one health paradigm provides a platform for public education and control of incessant EVD scourge in West and Central Africa. This entails coming to term with the reality that EVD is first animal virus that is transmitted to human (zoonoses). Globally, zoonoses account for over 60% of all infectious diseases  and hund


In contract, serum IL-10 levels in non-carrier cattle (n = 21) were very low or undetectable during the same period.The factors leading to persistent infection of foot-and-mouth disease (FMD) virus in ruminants are not well defined. This paper provides evidence of the presence of interleukin-10 (IL-10) early in the course of infection (1–4 days) as a factor in the development of persistence of FMD virus in cattle. Results showed that serum IL-10 in carrier cattle infected with FMD virus type O ( Foot-and-mouth disease (FMD) virus is a small, non-enveloped single stranded, positive sense RNA virus (Picornaviridae family) and has seven serotypes: O, A, C, Asia 1, and Southern African Territories (SAT) 1, 2 and 3, all of which cause a highly contagious vesicular disease in cloven-hoofed animals  and the P < 0.05 was considered statistically significant.To address this, serum samples collected at 0, 1, 2, 3, and 4 days post-infection (dpi) from 15 cattle of varying ages infected with the type O UKG/34/2001 virus and similarly from ten cattle infected with the type O SKR/1/2000 virus were used for detection of IL-10 and IFN protein. Detailed results regarding the clinical profiles are presented elsewhere . Brieflyn = 4) and non-carriers (n = 21), IgG antibodies to the structural proteins of FMD virus were quantified in sera using liquid phase blocking ELISA (LPBE) as described previously [n = 15) infected with the type O UKG/34/2001 had seroconverted to be FMD positive. Type-specific antibodies were detected in all inoculated animals by 4 dpi . Similar results were also observed in sera collected from the cohort infected with the type O SKR isolate (data not shown). The results obtained suggest that the IgG antibody response to structural proteins of FMD virus is not associated with the development of persistence in cattle, which is consistent with previous reports [To analyze differences in humoral responses between carriers . In the carrier group, serum IL-10 was detected and peaked at 1 or 2 dpi and rapidly declined afterwards. In contrast, it was evident that levels of IL-10 in sera from the non-carrier group (n = 21) were much lower or undetectable. Comparison of serum IL-10 levels between carrier and non-carrier cattle showed that levels from carrier cattle during early infection were significantly higher than in non-carrier animals , suggesting an association of IL-10 response during the acute phase of infection with a common outcome of FMD disease (i.e. persistence) in cattle. Age at infection is known to influence the establishment of viral persistence [n = 12) became carrier (Table To investigate if elevation of serum IL-10 during the acute infection correlates with development of persistent infection in cattle, IL-10 was measured basically following the method of Kwong et al. . Brieflyroup n =  were mucsistence  and bactsistence . It has sistence  as obserThe results obtained in this study suggest that the humoral IgG response to structural proteins of FMD virus is not associated with the development of persistence in cattle, however, the previous study showed that persistence with FMD virus was less likely to be established if effective immune response to vaccination had developed . IL-10 i


The aim of this study was to identify costs in patients diagnosed with Celiac disease.This retrospective case control study covered the period 2003–2006 and was conducted in a large Israeli Health Maintenance Organization insuring over two million members. Our cohort comprised 1,754 patients with Celiac disease with a control group of 15,040. Costs were aggregated according to main cost-branches and computed individually for each member. A linear step wise regression was performed with costs being the dependent variable and the independent variables; age, gender and the presence of celiac disease. Costs were compared with patients suffering from other chronic diseases.The total costs of the patients with celiac disease were significantly higher than that of the control group for hospital admission, medications, laboratory and imaging. Hospital admission rate was 7.98% as opposed to 7.1% for the control group (p = 0.06). When compared with other chronic illnesses, the costs of patients with celiac disease were similar to those of patients with diabetes and hypertension.Patients with Celiac disease utilize medical services more than the general population. This research suggests that the use of medical resources by patients with Celiac disease may be higher than previously thought. Celiac disease (CD) is an inherited autoimmune disorder that is triggered by the ingestion of gluten  with a pSettings & participants: All residents of Israel are insured by one of four non-profit, integrated Preferred Provider Organizations (PPO). This study was conducted in Maccabi Healthcare Services (MHS), the second largest PPO in Israel insuring over two million members. According to Israeli National Health Insurance Act legislated in 1994, MHS is obliged to insure every citizen who wishes to join it, irrespectively to age, gender, physical condition or any other criterion. Therefore, every section in the Israeli population is well represented in MHS.Sources of data: The MHS central data repository retains complete historical records of patient demographic data, physician data, laboratory results, and costs using the patient's unique national identification number. A computerized medical record (CMR) is used by all 3780 physicians caring for a population of two million individuals in over 4000 community clinics/surgeries. Patients have blood samples taken at one of the 260 collection points throughout the country and all laboratory testing is undertaken in one central location. There are no patient copayments for laboratory testing. The CMR runs locally on a personal computer located in the physician’s office and is used exclusively for the physicians' patients insured by MHS. It exchanges data with the central data repository using proprietary transactions sent by modem over telephone lines or through a wide area network. Laboratory test results, among other data, are downloaded on a daily basis and are reviewed by physicians at their leisure either on-screen or in printed form. After being accepted by the doctor, these lab reports become an integral part of the patient’s medical record. This study was approved by the PPO’s Institutional Review Board on 23.7.2006.Cohort definition: This retrospective case control study covered the period 2003–2006. All patients that were identified with CD from this period were included in our cohort. We compared costs between patients with and without CD. The cohort with CD was defined by an ICD9 diagnosis in their CRM and a positive blood test for CD serology . The control group from the rest of the MHS population was matched in clusters for gender and age on an 8:1 basis.We compared costs by admission, medication purchase, laboratory, imaging  and overall costs. The number of laboratory test were broken down by Hb, WBC, Bilirubin, Creatinine, Cholesterol, HDL, LDL, Triglyceride, AST, Urea, Uric Acid, Total T4, Free T4, TSH, IgA, IgG, IgM, IgE. In addition we compared the number of visits to primary and secondary care physicians as well as visits to dieticians.We also examined the comorbidities of the two groups as follows: Oncology patients within five years of diagnosis or active treatment. Diabetes mellitus (DM), Ischemic heart disease (IHD), Myocardial infarction (MI), Atrial Fibrillation (AF), Congestive cardiac disease (CHF) Hypertension (HTN), and those patients defined by the health ministry as having selected severe illness which includes dialysis, AIDS, thalassemia major. We also compared costs after a logarithmic transformation.Cost assessment: In the MHS accounting system every treatment is automatically converted into money terms according to real market prices. The databases allow all costs to be aggregated according to main cost-branches  and computed individually for each member.CD-related cost assessment: The actual direct cost of a CD patient was assessed by comparing his/her cost during study period with the mean cost of all other MHS members, matched for age group and gender. No externalities or direct non-medical costs were included. Cost of gluten free nutrition was not included. All costs were computed in New Israeli Shekel (NIS).Statistical analysis: Confidence intervals for patient and disease-related costs were computed by the independent sample T-test and Mann–Whitney U-test for continuous variables and Chi test or Fisher’s exact test for discrete variables. The analysis was done independently for each of the three years and as an average for the period 2003–6 for each index case and their controls. A linear step wise regression was performed for the independent variables such as age, gender and the presence of CD. The dependent variable was the log of costs rather than the actual costs because the costs were not distributed normally. The significance level for all comparisons was corrected by the false discovery rate (FDR) . The anaOur cohort of patients with celiac disease comprised 1,754 patients aged 25.7 ± 19.2 years with a control group of 15,040 patients aged 28.8 ± 26.4 years. The proportion of males in these two groups was 34% and 42% respectively (p < 0.001). The difference in age between the two groups was not significant (p = 0.15).The total costs of the patients with CD were significantly higher than that of the control group. The data is presented as a log value in order to approximate a standard distribution Figure . Table 1When examined for use of imaging, a significant difference between groups was found only for ultrasound. 41% for the celiac patients compared to 35.7% for the rest (p < 0.001). Utilization of primary and secondary care showed a statistical difference between groups for visits to gastroenterologists 1.06 and 0.23 visits per year (p < 0.001) respectively and medical nutritionists 0.5 and 0.24 visits per year (p < 0.001) respectively. Endoscopy costs fall under two separate categories in Table When we examined for comorbidity between the groups by performing logarithmic regression we found that the celiac patients had Odds Ratios of 1.073 (95% CI 1.014-1.136) and 1.055 (95% CI 0.998-1.116) for Cardiac disease and Diabetes Mellitus respectively. When we examined the costs between groups using stepwise logarithmic regression and the log of total costs as the dependent variable, Table  we see tThe clear finding from our study is that patients with CD utilize medical services more than general population in most categories of expenditure examined. This is somewhat surprising in view of the fact that many of these patients will be asymptomatic and observing their gluten free diet. Our null hypothesis was that diagnosed patients would have costs that were similar to the control group. Previous studies have shown a decrease in medical expenditure after the initiation of treatment. A 30% reduction in direct medical costs after diagnosis of CD was reported by Green ; the meaBy our study design, it is impossible to prove or refute previous findings suggesting a decline in medical costs after diagnosis. However, in our study, where all patients already had a diagnosis of CD at baseline, there was no decrease in medical costs over the years. However it should be remembered that that most patients who have CD and adhere to their gluten free diets convert to negative serology. Thus this group would not be represented in our cohort and it may be that our cohort is a sicker population of patients.When compared to the costs of other chronic disease states our sample shows similar costs to that of diabetes and hypertension. It should be remembered that the average age of our sample was under thirty years of age. This can be compared to an average age of 61.6 years for MHS patients with diabetes . If our Our study has a few limitations: the case definition of our CD group was made using the diagnosis and supporting laboratory data but was not validated by biopsy results. The lack of information in our study relating to time of diagnosis is a deficiency as we know that CD costs peak in proximity to the time of diagnosis and if for some reason there were a high number of diagnoses of new CD in close proximity to the years 2003–6 this could create bias. In addition, we do not know how many of the CD patients were adhering to their gluten free diets, and it could be that the increased medical costs were due to non-adherence and complications of CD. Also, costs of gluten free products were not included in our analysis, though this would increase the medical costs of CD, strengthening the refutation of our null hypothesis. Although all sections of the population are well represented in MHS, MHS has higher market penetration in the centre of the country which is both wealthier and with greater access to secondary care than the periphery. This may result in more costs than in the country’s periphery. Finally, we do not know the length of time from diagnosis of each patient, whether they were symptomatic or not and what was the impact of their disease on quality of life, information that would enable us to explore the factors related to the increased costs. The strength of our study is that our results are based on analysis of complete medical utilization costs and admission data with the exception of private consultations in a whole population depicting the medical costs in real life.As the incidence of CD is increasing in all countries, it becomes more important to assess the financial consequences of diagnosis. Our study suggests that the patients with CD require increased medical resources similar to patients who suffer from diabetes or hypertension, unrelated to the time of diagnosis.The authors declare that they have no conflict of interest. There has been no external funding for this study.ADH and RS planned the study. RE and ADH drafted and reviewed the manuscript. ML was responsible for the statistical analysis. Each author has approved the final draft submitted.


We summarize what is known about this virus and its global expansion as of mid-February 2016. Zika virus is a mosquitoborne flavivirus that is the focus of an ongoing pandemic and public health emergency. Previously limited to sporadic cases in Africa and Asia, the emergence of Zika virus in Brazil in 2015 heralded rapid spread throughout the Americas. Although most Zika virus infections are characterized by subclinical or mild influenza-like illness, severe manifestations have been described, including Guillain-Barre syndrome in adults and microcephaly in babies born to infected mothers. Neither an effective treatment nor a vaccine is available for Zika virus; therefore, the public health response primarily focuses on preventing infection, particularly in pregnant women. Despite growing knowledge about this virus, questions remain regarding the virus’s vectors and reservoirs, pathogenesis, genetic diversity, and potential synergistic effects of co-infection with other circulating viruses. These questions highlight the need for research to optimize surveillance, patient management, and public health intervention in the current Zika virus epidemic. Aedes africanus mosquitoes from the same forest . Furthermore, 29 other countries in the Americas have reported autochthonous Zika virus transmission, including Puerto Rico and US Virgin Islands  Table 2 Given the wealth of new information about Zika virus, we conducted a literature review to summarize the published findings. This review contextualizes the ongoing Zika virus epidemic in the Americas and identifies knowledge gaps that must be addressed to combat Zika virus successfully.Using the keywords “Zika,” “ZIKV,” “ZIKAV,” and “Zika virus,” we searched Google, PubMed, Web of Science, Scopus, and ProMed Mail. We reviewed all literature published through February 16, 2016, including peer-reviewed journal articles, infectious disease reporting system broadcasts, and public health agency information . To ensure the capture of all information, we cross-referenced the bibliographies of reviewed articles. The search included English-language and foreign-language articles, which were computer translated.Flaviviridae, which includes several other mosquitoborne viruses of clinical importance   genus. Several Aedes spp. have been implicated, including Ae. aegypti, Ae. africanus, Ae. hensilli, and Ae. albopictus ; others have a broad geographic span   has been described; however, confounding by contemporaneous vectorborne transmission in these instances cannot be excluded. For example, the patient who became infected with Zika virus after a monkey bite had concomitant exposure to mosquitoes, a more plausible route of acquisition (41) reported recent exposure to mosquitoes . Probable intrapartum transmission has also been described: 2 newborns were found to be viremic with Zika virus <4 days after being born to infected mothers (Intrauterine transmission is supported by the finding of Zika virus RNA by reverse transcription PCR (RT-PCR) in amniotic fluid of 2 mothers with symptoms of Zika virus infection during pregnancy; both delivered babies with microcephaly . Commonly reported symptoms include rash, fever, arthralgia, myalgia, fatigue, headache, and conjunctivitis (°C –38.0°C) .In humans, the incubation period from mosquito bite to symptom onset is ≈3–12 days. Infection is likely asymptomatic in ≈80% of cases , Table 3.ctivitis  Table 3.48). However, concern exists that these findings may in part be artifactual, resulting from previous underreporting of cases and confounding by other risk factors for microcephaly (49). Because systematic surveillance for microcephaly was not previously undertaken, the baseline rate of microcephaly in Brazil is unknown, and subsequent reports suggest that a substantial proportion of infants that reportedly have microcephaly do not actually have the condition (50).More severe clinical sequelae have increasingly been associated with Zika virus. During the ongoing outbreak in Brazil, reports of infants born with microcephaly have markedly increased  (51). However, this finding should be cautiously interpreted; reports included only 17 cases, and none were laboratory-confirmed Zika virus cases. In addition, the true baseline rate of such malformations before the outbreak is unknown (51).Health officials in French Polynesia have reported an apparent increase in congenital central nervous system (CNS) malformations, coinciding with the outbreak occurring during 2013–2014 (52) and progression of disease in directly infected mouse brains (53). One hypothesis for Zika virus’s role in CNS malformations pertains to the virus’s hijacking of autophagy during viral replication (54). Some cellular proteins have a dual role in autophagy and centrosome stability; a normal number of centrosomes is important for brain development (54). An increase in centrosomes in mice has been shown to result in microcephaly (54). Therefore, Zika virus’s interference in autophagy has been hypothesized to lead to an increase in centrosome number and microcephaly; this potential role in malformations merits further investigation.A plausible neuropathologic link between Zika virus and CNS anomalies is supported by research showing viral neurotropism in intraperitoneally infected mice (55). A surge in Guillain-Barre syndrome cases has been observed in Brazil, Colombia, El Salvador, Suriname, Venezuela, and French Polynesia during outbreaks; however, Zika virus has been laboratory confirmed in only some of these cases (55).Severe neurologic sequelae have also been described in adults, including meningitis, meningoencephalitis, and Guillain-Barre syndrome . Hematospermia was reported in 2 cases .Nonneurologic sequelae include transient hearing loss, hypotension, and genitourinary symptoms (44). Besides 1 infant death, 3 other fatalities were reported (2 from Brazil and 1 from Colombia): 1 man with lupus erythematosus, chronic corticosteroid use, rheumatoid arthritis, and alcoholism; and 2 girls 16 years of age, 1 with sickle cell disease (59). Rare deaths have been described in patients infected with Zika virus . Mild elevations in inflammatory markers , serum lactate dehydrogenase, or liver enzymes have been described . These findings are observed in many other viral infections, including the co-circulating viruses DENV and CHIKV, so none of these laboratory alterations reliably distinguish among these infections.Information on laboratory findings for Zika virus infection is limited. Complete blood count is often normal; even if blood count is abnormal, changes may be nonspecific  (63). Commercial assays have been developed, including a PCR-based assay that has been approved by the Communauté Européenne  and a serologic assay that has been approved by the US Food and Drug Administration for restricted use in emergency situations (64). Testing has typically been performed by large reference laboratories  and universities. CDC’s typical turnaround time is 4–14 days. Appropriate tests are selected by the laboratory on the basis of clinical information provided by the requesting healthcare provider (65). To coordinate sample collection, providers should contact local public health agencies before testing.Clinical evaluation alone is unreliable for a diagnosis of Zika virus infection. Because of clinical overlap with other arboviruses, diagnosis relies on laboratory testing. Evaluation for Zika virus, CHIKV, and DENV should be undertaken concurrently for all patients who have acute fever, rash, myalgia, or arthralgia after recent (previous 2 weeks) travel to an area of ongoing Zika virus transmission (63). In contrast, serologic testing is not recommended during the acute phase, when Zika virus IgM may be undetectable . Consequently, testing algorithms are based on sampling relative to symptom onset, and serologic testing should be considered if samples are negative for Zika virus by RT-PCR (63).Molecular amplification  on serum samples remains the most specific diagnostic approach and is the preferred testing method for Zika virus during the acute phase of illness (<7 days from symptom onset) . Therefore, positive serologic test results should be confirmed with testing that uses an alternative platform such as a seroneutralization assay  (63).Serologic testing has limitations. Zika virus IgM and IgG are notoriously cross-reactive with those against other flaviviruses (particularly DENV), limiting specificity (63). Urine and saliva may offer alternatives, particularly when blood collection is difficult . Viruria may persist longer than viremia. One study reported that Zika virus RNA was detected in urine up to 20 days after viremia had become undetectable (62); therefore, RT-PCR testing of urine should be considered when Zika virus is clinically suspected, despite negative serum testing . Similarly, RT-PCR conducted with saliva has been shown to increase the detection rate during the acute phase of infection but does not extend the window of detection of Zika virus RNA; consequently, blood remains the preferred sample (66).The type of sample can also affect the probability of detection. Although diagnostic testing is performed primarily on serum or cerebrospinal fluid, the diagnostic utility of other specimen types  is being evaluated (67).No specific treatment or vaccine is available for Zika virus infection. Management is supportive and includes rest, fluids, antipyretics, and analgesics. Aspirin and other nonsteroidal antiinflammatory drugs should be avoided until dengue is excluded because of the risk for hemorrhage among dengue patients (Ae. aegypti peak biting times (early morning and late afternoon) (68). Community-level strategies target mosquito breeding through elimination of potential egg-laying sites  by drying wet environments or using insecticide treatment (68). Pregnant women residing in countries that are not Zika virus–endemic are advised against travel to affected countries (69). Testing should be offered to all pregnant women who have traveled to areas with ongoing Zika virus transmission (70). Serial fetal ultrasounds should be considered to monitor fetal anatomy and growth every 3–4 weeks in pregnant women with positive or inconclusive Zika virus test results, and the infant should be tested at birth (70). Men who reside in or have traveled to an area of active Zika virus transmission and who have a pregnant partner should abstain from sexual activity or use condoms during sex; similar guidelines apply for men with a nonpregnant female sex partner who is concerned about sexual transmission of Zika virus (58).Other general measures focus on prevention of mosquito bites, including individual protection , particularly during known Zika virus has been declared a public health emergency. As many as 1.3 million persons have been affected in Brazil alone  Table 2,Foremost, diagnosis remains suboptimal. Diagnostic guidelines are contingent on laboratory testing that is not widely available. Although commercial tests for Zika virus are limited in number and availability, more are in development, including prototype multiplex molecular assays that concurrently test for Zika virus, CHIKV, and DENV . However, although not unique to Zika virus, laboratory infrastructure and testing capability is lacking in resource-constrained settings where Zika virus is most prevalent.44). In the United States, multiple factors guard against the explosive epidemic occurring throughout Central and South America. Specifically, lower rates of human crowding in urban areas, wider access to air conditioning and mosquito repellants, and waste management limit mosquitoborne transmission, which has been the case for DENV (71). Nonetheless, further entomologic research is needed to define the range of Zika virus vectors and identify new areas where autochthonous transmission could take place to enable early intervention. Investment is also needed in durable control measures such as adaptable vaccine platforms for arboviruses; currently, no Zika virus vaccines are in advanced development  are a current priority, pending advances in diagnostics; the World Health Organization and the Pan American Health Organization have issued recommendations ; however, absence of a licensed pathogen reduction technology for use in red cells, high incremental cost, and technical barriers render such technology an unlikely short-term solution.Given reports of possible transfusion-transmitted Zika virus, the pandemic also has implications for the blood supply within Zika virus–endemic and nonendemic regions. The US Food and Drug Administration recommends 28-day deferral for blood donors with confirmed or suspected Zika virus infection (73). Although autochthonous transmission in the United States is unlikely to match the scale of the epidemic in Central and South America, much about Zika virus and the way the pandemic will evolve are unknown. Continued vigilance is warranted, along with a concerted effort toward improving our understanding, management, and prevention of this emerging pathogen.Zika virus has the propensity to infect large numbers of persons with severe consequences in some cases. The epidemic has serious medical, ethical, and economic ramifications, particularly in countries where the resources for early diagnosis are lacking and potential intervention measures  are discouraged or illegal (Technical Appendix. Additional references and tables summarizing findings of literature search regarding current knowledge of Zika virus.


Vaccinia virus A33 is an extracellular enveloped virus (EEV)-specific type II membrane glycoprotein that is essential for efficient EEV formation and long-range viral spread within the host. A33 is a target for neutralizing antibody responses against EEV. In this study, we produced seven murine anti-A33 monoclonal antibodies (MAbs) by immunizing mice with live VACV, followed by boosting with the soluble A33 homodimeric ectodomain. Five A33 specific MAbs were capable of neutralizing EEV in the presence of complement. All MAbs bind to conformational epitopes on A33 but not to linear peptides. To identify the epitopes, we have adetermined the crystal structures of three representative neutralizing MAbs in complex with A33. We have further determined the binding kinetics for each of the three antibodies to wild-type A33, as well as to engineered A33 that contained single alanine substitutions within the epitopes of the three crystallized antibodies. While the Fab of both MAbs A2C7 and A20G2 binds to a single A33 subunit, the Fab from MAb A27D7 binds to both A33 subunits simultaneously. A27D7 binding is resistant to single alanine substitutions within the A33 epitope. A27D7 also demonstrated high-affinity binding with recombinant A33 protein that mimics other orthopoxvirus strains in the A27D7 epitope, such as ectromelia, monkeypox, and cowpox virus, suggesting that A27D7 is a potent cross-neutralizer. Finally, we confirmed that A27D7 protects mice against a lethal challenge with ectromelia virus. Before the eradication of smallpox (variola virus) from nature, hundreds of million of people succumbed to the infection. The discovery of vaccinia virus (VACV), the active ingredient of the smallpox vaccine, ultimately led to the eradiation of smallpox from the human population. Vaccination with VACV leads to a strong antibody response that protects against variola virus. As the protective antibodies recognize viral proteins that are highly similar in sequence between the different orthopox strains, such as A33 used in this study, several antibodies have the capacity to neutralize a larger breath of orthopx viruses. In this study we have identified an anti-A33 antibody from a larger panel that exhibits a unique binding mode to A33. This antibody, A27D7, is also resistant to single amino acid changes throughout the protein and binds to engineered A33 variants that mimic ectromelia and orthopox A33 in the antibody-binding site. As the antibody further protects against ectromelia infection of mice, this antibody appears to be a potent orthopox cross-species protective antibody with therapeutic potential. Inoculation with vaccinia virus (VACV) protected against smallpox, a deadly disease caused by the related orthopoxvirus, variola (VARV) . Its sucst century. More recently a vaccinia virus clonal isolate, ACAM2000 has been used, produced using modern cell cultures . Cl. Cl50]. escribed  [50].5 cells/well into 24-well Costar plates  and EEV VACVWR neutralization was performed the following day using Mabs or pAbs samples at 10μg/ml  or 1/100  respectively in the absence or the presence of baby rabbit complement 10%   as described previously . , and the liquid was rapidly inhaled by the mouse. Mice were weighed daily to assess disease progression. Mice were euthanized if and when 25% weight loss occurred. A dose of 1x105 PFU of VACVWR was the standard dose given to 7-week-old BALB/c females. For A33 mAb protection studies, mice were treated by i.p. injection with 100 μg of antibodies 1 day before infection. Control mice received anti-A10 BG3.1 antibody, which is known to provide no protection. An additional group received anti-B5 B126 as positive control. A third group received anti-L1 M12B9, which was expected to protect against death but not weight loss , was selected based on the highest identity score in a PDB-BLAST. MR was performed using Phaser MR  and a si.3/37.4% . Refinem.3/37.4%  and rest.3/37.4% . NCS ref.4/28.2% .1 (R = 50.71%). The SEC profile of the complex suggested one Fab molecule bound to two A33 molecules , for a total MW of ~72 kDa resulting in a Matthews coefficient of 2.30 Å3/Da and 46.5% solvent. On this basis, MR was carried out with three independent components as starting models: the variable domain of the anti-tetrahydrocannabinol Lκ/HigG1 Fab fragment [pdb code 3LS4], its associated conserved domain, and one A33 monomer extracted from the previously deposited A33 dimer structure [pdb code 3K7B]. This Fab template shares the highest light chain identity (97%) with A27D7, among reported X-ray structures. Alternating cycles of manual refinement with COOT [free = 20.1/22.6%), which ranked in the 99th percentile for clashes and in the 100th percentile for geometry, compared to structures of similar resolution (Molprobity) [Initial crystals grew in 0.1 M sodium cacodylate pH 6.5, 0.2 M NaCl, 2.0 M ammonium sulfate with a protein concentration of 5.1 mg/ml . Data from a single crystal was collected at SSRL BL11-1. MR was performed with Phaser MR  and a siith COOT  and restith COOT  led to tprobity) .To assess binding kinetics of MAbs A2C7, A20G2, and A27D7 to WT-A33, we performed BLI assays using the OctetRED instrument . In a first experiment we immobilized MAbs on Anti-mouse Fc Capture (AMC) biosensors and tested binding to WT-A33 in solution. Biosensors were loaded with 10 μg/mL MAb diluted in 1X kinetics buffer  over 5 mins. MAb-loaded tips were tested against two-fold serial dilutions of WT-A33 analytes (0.78–50 nM). Binding kinetics were calculated using Octet Data Analysis 7.1 software  with curve-fitting statistics. The association steps were performed over 10 mins and dissociation steps performed over 20 mins. A negative control antibody (EE11) that targets the VACV antigen D8 was run in parallel to all assays for subtraction of background binding signal. Curves were aligned to baseline.D, kon, and koff were determined by global fitting of association and dissociation steps for all dilutions assuming a 1:1 binding model.To test the effect of the A33 mutations on antibody binding, we immobilized biotinylated A33 (BtA33) using streptavidin (SA) biosensors. SA biosensors were loaded with 1.5 μg/mL BtA33 protein in 1X kinetics buffer  over 5 mins. A33-loaded tips were tested against three-fold serial dilutions of IgG analytes (27.4 pM-20 nM). The association steps were performed over 10 mins and dissociation steps performed over 20 mins. Buffer control against biosensors loaded with WT-A33 was subtracted from raw data and curves were aligned to baseline. K2+ and Mg2+ was slowly loaded into nares (5 μl/nare). Mice subsequently were left in situ for 2 to 3 min before being returned to their cages. Mice were monitored daily for mortality and morbidity, as measured by weight change.Four- to six-week-old female C57BL/6 mice were obtained from Harlan Laboratories , housed in filter-top microisolator cages, and fed commercial mouse chow and water ad libitum. The mice were housed in an animal biosafety level 3 containment area. The day before challenge, mice were treated i.p. with 100 μg of the specified antibody. Immediately before challenge, mice were anesthetized with 0.1 ml/10 g body weight of ketamine HCl (6 mg/ml) and xylazine (0.5 mg/ml) by i.p. injections. One thousand PFU of ECTV (Moscow strain) in PBS without CaAnimal husbandry and experimental procedures were approved by the Institutional Animal Care and Use Committee of Saint Louis University  and by the Department of Laboratory Animal Care and the Animal Care Committee of the La Jolla Institute . We follow PHS Policy on humane care and use of laboratory animals.www.rcsb.org) with codes 4LQF, 4LU5, and 4M1G. The nucleotide sequences of the anti-A33 antibodies have been deposited in GenBank, and accession numbers are listed in The coordinates and structure factors of the A33:Fab complexes have been deposited in the Protein Data Bank (S1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S1 FigPurified anti-A33 MAbs , or no primary antibody (-1’) were tested for binding to overlapping A33 biotinylated 20mer peptides. All seven A33 anti-A33 MAbs bind recombinant A33 protein (A33) but failed to bind any linear peptide, indicating the conformational nature of A33 epitopes.(TIF)Click here for additional data file.S2 FigLegend is the same as for (TIF)Click here for additional data file.S3 Figon), dissociation rate (koff), affinity constant (KD), and fit quality scores are reported in Real-time binding curves of MAbs A2C7, A20G2 and A27D7 to immobilized wild-type A33 and indicated A33 mutants are shown. Association (10 min) and dissociation (20 min) steps are represented. Curves are colored according to their specific antigen concentration . Association rate (k(TIF)Click here for additional data file.S4 FigVACV orthologs . VACV: vaccinia virus; VARV: variola virus; CMLV: camelpox virus; MPXV: monkeypox virus; MYXV: myxoma virus; CPXV: cowpox virus; ECTV: ectromelia virus; RFV: rabbit fibroma virus; TATV: taterapox virus; LSDV: lumpy skin disease virus; GTPV: goatpox virus; SPPV: sheeppox virus; SWPV: swinepox virus; DPV: mule deer pox virus; YMTV: Yaba monkey tumor virus; TANV: tanapox virus; YLDV: Yaba-like disease virus; MOCV: Molluscum contagiosum; ORFV: Orf virus; BSPV: bovine papular stomatitis virus. (B) GENIO/logo representation of the alignment performed with the closest A33VACV orthologs. (C) The model of A33VACV as extracted from A20G2-Fab/A33 complex structure is represented as a blue surface for conserved residues and in red and green for residues that vary among the orthopox species, as defined in (A). As expected, all variable residues are located on the surface. There are a total of 37 potential variations over the whole A33 sequence, among which eighteen can be visualized on the A33 model (residues 98–185). Amino acid positions in C can differ with alignment in B by up to 3, due to different lengths of the sequences.(A) Phylogenic cladogram of A33 orthologs. A33 sequence is overall highly conserved. All 106 sequences of A33 orthologs were downloaded from the Poxvirus Bioinformatics Resource Center server (poxvirus.org). Sequence alignment was performed with MEGA5 software using ClustalW with the GONNET similarity matrix and manual refinement. Phylogenic analysis was performed with a global gap removal to exclude gaps from alignments. Maximum Likelihood trees were constructed using the Jones-Taylor-Thornton amino acid substitution model with rates among sites Gamma distributed with Invariant sites. The robustness of trees was evaluated by bootstrap analysis with 1,000 rounds of replication. Non-redundant sequences of the clade framed in red are representative of A33 variation profile for the closest A33(TIF)Click here for additional data file.


Physical inactivity in children is an important risk factor for the development of various morbidities and mortality in adulthood, physical activity already has preventive effects during childhood. The objective of this study is to estimate the association between physical activity, healthcare utilization and costs in children.Cross-sectional data of 3356 children aged 9 to 12 years were taken from the 10-year follow-up of the birth cohort studies GINIplus and LISAplus, including information on healthcare utilization and physical activity given by parents via self-administered questionnaires. Using a bottom-up approach, direct costs due to healthcare utilization and indirect costs resulting from parental work absence were estimated for the base year 2007. A two-step regression model compared effects on healthcare utilization and costs for a higher (≥7 h/week) versus a lower (<7 h/week) level of moderate-to-vigorous physical activity (MVPA) adjusted for age, gender, BMI, education and income of parents, single parenthood and study region. Recycled predictions estimated adjusted mean costs per child and activity group.The analyses for the association between physical activity, healthcare utilization and costs showed no statistically significant results. Different directions of estimates were noticeable throughout cost components in the first step as well as the second step of the regression model. For higher MVPA (≥7 h/week) compared with lower MVPA (<7 h/week) total direct costs accounted for 392 EUR (95% CI: 342–449 EUR) versus 398 EUR (95% CI: 309–480 EUR) and indirect costs accounted for 138 EUR (95% CI: 124–153 EUR) versus 127 EUR (95% CI: 111–146 EUR).The results indicate that childhood might be too early in life, to detect significant preventive effects of physical activity on healthcare utilization and costs, as diseases attributable to lacking physical activity might first occur later in life. This underpins the importance of clarifying the long-term effects of physical activity as it may strengthen the promotion of physical activity in children from a health economic perspective. Physical activity (PA) has several preventive effects on physical and mental health -3. PhysiData from the German Interview and Examination Survey for Children and Adolescents (KiGGS) show a pattern of insufficient PA for children in Germany: only 15.3% of children and adolescents between 4 and 17 years of age fulfill World Health Organization (WHO) recommendations regarding PA (moderate to vigorous intensity) of at least 60 minutes a day .A lack of PA influences the development of obesity and a growing prevalence of obesity can be observed in children ,10. In GWhile there has been some research on the impact of childhood obesity on healthcare utilization and costs, the association between PA, healthcare utilization and costs has barely been explored for children. A prospective Dutch study with 996 primary schoolchildren describes the economic burden of injuries that occur during physical education class, leisure time or organized sports . Only onUntil now, there are no studies in Germany analyzing the relationship between PA, healthcare utilization and costs for children. Assuming preventive effects of PA on healthcare utilization and costs on the one hand and economic burden resulting from PA-related injuries on the other, it is still uncertain whether savings or additional costs predominate in physically active children. The aim of this cross-sectional study is to analyze the correlation between different levels of PA and healthcare utilization as well as costs for children aged 9 to 12 years based on data from the GINIplus- and LISAplus studies.The cross-sectional data were taken from the 10-year follow-up of two prospective population-based birth cohort studies: The GINIplus study (The German Infant Study on the Influence of Nutrition Intervention plus Air Pollution and Genetics on Allergy Development) and the LISAplus study (Influence of Life-style Factors on Development of the Immune System and Allergies in East and West Germany plus Air Pollution and Genetics on Allergy Development). Included in both studies were healthy, fullterm newborns with a birth weight >2500 g who are of German descent and live in the proximity of study centers in Munich, Leipzig, Bad Honnef and Wesel. In these areas, newborns were recruited from obstetric clinics between the mid- to the late 1990s . FurtherBoth study protocols were approved by the local ethic committees  and written informed consent was obtained from all participating families.Starting with 5991 newborns in GINIplus and 3097 newborns in LISAplus at baseline, after 10 years about 55% of all individuals were left for data collection. The cross-sectional data for the 10-year follow-up  were available between 2005 and 2009 depending on the birth date of the individuals . The 10-Data on the exposure PA are missing for 286 of those children. To avoid unnecessary further loss of data, we included children in the analyses for whom only data on covariates were missing but data for the exposure PA were available. Thus, the analyses are based on data for 3356 individuals.PA is a generic term for any movement of the body which is produced by the skeletal muscles and increases energy use above the metabolic rate at rest . In thisThe WHO and various other guidelines advise children to be physically active on a moderate-to-vigorous intensity to maintain a basic level of health ,20,21. WTherefore, hours of moderate PA and vigorous PA that were reported by the parents of participating children were added up to build a sum variable of MVPA weighting moderate and vigorous PA equally and combining the information on intensity, quantity and frequency of PA in h/week MVPA.For the primary analysis, the sum variable MVPA was dichotomized into two levels : <7 h/week MVPA (WHO-recommendations not met) and ≥7 h/week MVPA (WHO-recommendations met). A four-level MVPA-variable was considered in a sensitivity analysis model categorized in: <3.5 h/week MVPA, ≥3.5 h/week and <7 h/week MVPA, ≥7 h/week and <10.5 h/week MVPA, ≥10.5 h/week MVPA.Covariates used in the analysis are age, gender, body mass index (BMI) of children, highest education level of parents, relative income position of the household , single parenthood and study region. Socioeconomic information was obtained from parents via self-administered questionnaires.As measures of children’s socio-economic background information on the education level and income of parents were used. Education level of parents was given by the maximum completed school years of either of the parents: “low” (<10 years), “medium” (=10 years), “high” (>10 years). In cases of missing information  single imputation was conducted, applying the Markov Chain Monte Carlo method (PROC MI in SAS) . Completa [Information on parents’ net household income was converted into equivalence income according to the modified Organization for Economic Cooperation and Development (OECD) scale ,24. Equia , the relAnthropometric data of weight and height were recorded at the physical examination of the children by trained medical staff and BMI was calculated as weight in kilograms divided by height in meters squared. BMI data were classified according to German age- and sex-specific percentile cut-off points for children, resulting in the categories “severely underweight” (<P3), “underweight” (P3 to < P10), “normal weight” (P10 to P90), “overweight but not obese” (>P90 to P97) and “obese” (>P97) ,27.In the healthcare utilization questionnaire parents reported whether their child had used healthcare services  as well as the number of physician visits , dermatologist, pulmonologist, emergency doctor and other specialist), therapist visits , the number of hospital days and inpatient rehabilitation days of their children. In addition parents provided information on their work absence days  required due to health problems of their children. All questions referred to the previous 12 months.On the basis of this individual level data direct medical costs were assessed applying a bottom-up approach and unit prices. Physician costs were calculated using prices per physician visit for each medical specialty, taken from a national costing guideline from the Working Group Methods in Health Economic Evaluation (AG MEG) ,28. For Direct medical costs were calculated as total costs  as well as in the separate subcategories: physician, therapist, hospital and inpatient rehabilitation costs.Indirect costs resulting from parental work absence were calculated as age- and gender-specific mean costs per day of lost work for employees multiplied by the number of lost work days caused by the child . The humAll costs were denominated in Euros and referred to the year 2007 . More deDifferences of variable distributions for both cohorts (GINIplus and LISAplus) have already been analyzed elsewhere and no noticeable differences were found . TherefoBivariate analyses were conducted for the MVPA variable of primary analysis and each covariate separately, for the variables education level and relative income position as well as for the MVPA variable and each cost category.The primary regression model analyzes the association between MVPA and direct medical costs  as well as the association between MVPA and indirect costs . The regression model was adjusted for the covariates age, gender, BMI of children, education level of parents, relative income position of the household , single parenthood and study region.Descriptive analysis of the outcome total costs showed a positively skewed distribution. To account for positively skewed cost data and for a high number of zero-costs , a two-step regression approach was applied to model the costs. The first step consists of a logistic regression model (PROC LOGISTIC in SAS). Handling a binary response variable regarding utilization (yes/no) it estimates the association between MVPA, covariates and the odds of generating costs in a particular cost category. In the second step, a generalized linear regression model (PROC GENMOD in SAS) was used to assess the association between MVPA, covariates and the extent of costs caused by the healthcare utilization of the children in a particular cost category. A gamma distribution with log-link function was assumed .Four types of sensitivity analyses were conducted on each of the cost components and for both steps of the model. Sensitivity analysis model 1 (SAM 1) reperformed the primary analysis excluding two individuals due to high utilization of healthcare services . In SAM 2 the categorization of the MVPA variable was changed from dichotomous into four-level: <3.5 h/week MVPA, ≥3.5 and <7 h/week MVPA, ≥7 and <10.5 h/week MVPA and ≥10.5 h/week MVPA. Two further models were adjusted for the same covariates as the primary analysis, but in each instance one extra variable was included in the calculation: SAM 3) an interaction term of MVPA and gender, SAM 4) an interaction term of MVPA and BMI. All sensitivity analyses were considered as explorative, therefore no adjustment for multiple testing was made.Additionally, recycled predictions were applied combining both steps of the regression model to assess the overall association between PA and costs. Therefore, adjusted mean costs and the cost differences were estimated for the two MVPA groups (WHO recommendations met vs. WHO recommendations not met). 1000 bootstrap replications and the percentile method were used to estimate 95%-bootstrap-percentile-intervals ,35. For Table t-test for independent samples. There is no significant difference in mean values for age between MVPA groups. Significant associations were observed for MVPA and single parenthood as well as for MVPA and study region (Pearson chi-square tests).Bivariate analyses for the dichotomous MVPA variable and covariates show that there is no significant association between MVPA and gender, MVPA and BMI, MVPA and education level of parents or MVPA and relative income position of the household (Pearson chi-square tests). Bivariate analysis for MVPA and age was conducted using the In Table For bivariate analysis of MPVA and cost data the non-parametric Wilcoxon-Mann-Whitney test was applied. There is no significant difference in mean costs between MVPA groups for total direct costs, physician costs, therapist costs, hospital costs, inpatient rehabilitation costs or parental work absence costs.Table The analysis showed no statistically significant results. Comparing MVPA ≥ 7 h/week and MVPA < 7 h/week, the odds ratio estimates  showed different directions throughout cost components: the probabilities of total direct costs and inpatient rehabilitation costs were 0.95fold (95% CI: 0.76-1.19) and 0.97fold (95% CI: 0.52-1.84), while probabilities of physician, therapist, hospital or indirect costs were 1.06fold (95% CI: 0.86-1.30), 1.05fold (95% CI: 0.88-1.25), 1.20fold (95% CI: 0.85-1.68) and 1.04fold (95% CI: 0.87-1.26).Regarding further parameters of the regression model, there are single significant odds ratio estimates: A one unit increase in age is associated with a twofold higher probability of hospital costs. For boys, the odds ratio estimate for therapist costs is higher compared with girls. Regarding BMI, the probabilities of inpatient rehabilitation costs for severely underweight and for obese children are higher compared with normal weight children .More detailed analyses were done regarding the probability of costs for further specialists as well as for further therapists. The analyses did not reveal systematic changes in comparison with the reported overall analysis on the probability of physician and therapist costs.The results for the second step of the regression model  are presented in Table The analysis showed no statistically significant results. Different directions of estimates (extent of costs where costs occurred) were noticeable throughout cost components in the second step of the regression model: For higher MVPA (≥7 h/week) compared with lower MVPA (<7 h/week) the extent of total, physician, hospital and rehabilitation costs was 0.99fold (95% CI: 0.88-1.12), 0.96fold (95% CI: 0.90-1.03), 0.79fold (95% CI: 0.57-1.09) and 0.91fold (95% CI: 0.42-1.98), while therapist and indirect costs were 1.11fold (95% CI: 0.93-1.32) and 1.06fold (95% CI: 0.95-1.18).Regarding further parameters of the regression model, there are single significant estimates: For a one unit increase in age, total costs are higher . Boys showed increased total and therapist costs compared with girls. Regarding BMI, total direct costs are higher for all weight categories compared with normal weight. The highest total costs can be observed for obese children .More detailed analyses were done regarding the costs for further specialists as well as for further therapists. The analyses did not reveal systematic changes in comparison with the reported overall analyses on the extent of physicians and therapists costs.Sensitivity analyses were conducted on all cost components and on both steps of the regression model. They did not systematically change the results in comparison with the primary analysis. In Table The information that can be added through sensitivity analyses is related to the second step of the regression model. In SAM 3 and SAM 4 there are some significant values: If costs occur, boys show a lower extent of total costs , while girls show an increased extent of total costs  for higher compared with lower MVPA. If costs occur, for underweight children total costs double for higher compared with lower MVPA.Combining the two steps of the regression model, the results for the recycled predictions estimate adjusted mean costs and 95% bootstrap-percentile-intervals for both MVPA groups  compared with lower MVPA (<7 h/week) physician costs accounted for 95 EUR (95% CI: 91–100 EUR) versus 99 EUR (95% CI: 91–107 EUR), therapist costs for 114 EUR (95% CI: 98–131 EUR) versus 101 EUR (95% CI: 82–121 EUR), hospital costs for 149 EUR (95% CI: 113–201 EUR) versus 161 EUR (95% CI: 92–249 EUR) and inpatient rehabilitation costs for 38 EUR (95% CI: 26–270 EUR) versus 43 EUR (95% CI: 17–360 EUR). All effects, however, are not significant.This study analyzed the association between PA, healthcare utilization and costs for children based on cross-sectional data of the 10-year follow-up from two German birth cohort studies. The results show that a majority of children fulfill WHO-recommendations of ≥ 7 h/week MVPA and seem to be quite active. No statistically significant association between PA and healthcare utilization and costs was observed. The results show different directions of association. Basically, physically active children are healthier in terms of fitness . Having This study has strengths and limitations. It is the first study analyzing the association between PA, healthcare utilization and costs for children using a bottom-up approach which needs fewer assumptions regarding individual utilization compared with top-down approaches. A broad spectrum of healthcare services is captured  and as one of the first studies it even takes into account an aspect of indirect costs . Using an excess cost approach, the present study is able to capture all costs related to MVPA or its health consequences and to compare costs between groups of different MVPA level.A Canadian study analyzed similar associations: Kirk et al. explored the association between health behaviors (including PA) and healthcare utilization in Canadian schoolchildren using a top-down approach and a cross-sectional study design. They linked survey data from the Children’s Lifestyle and School Performance Study (CLASS) with Nova Scotia administrative health data. To measure healthcare utilization and costs they only use physician visits and physician costs as outcome. As in the present study, Kirk et al. found no statistically significant association between PA and healthcare utilization , but forThe present study is subject to some limitations. As a cross-sectional design was used, statements about causal relationships, accumulative or long-term effects of PA on healthcare utilization and costs cannot be made. In the long run, for physically active people, savings potential is assumed . Rütten Preventive effects of PA on healthcare utilization and costs were assumed but even an inverse causation is conceivable. Children being less physically active might nevertheless show a higher probability of healthcare utilization and higher costs. This can be the case if children have serious (chronic) diseases that might restrain them from being physically active. The higher probability of healthcare utilization and higher costs might then not be associated with PA, but with the disease itself.There are limitations regarding the estimation of cost data in the present study. This study was not able to account for actual expenditures, but applied updated contact prices based on mean values suggested by the AG MEG . Costs cThe estimation of indirect costs is limited to one aspect of indirect costs  focusing on production losses in paid work. But this can only be regarded as an approximation of indirect costs because it only takes into account employed people and disregards unpaid work. Further indirect costs concerning children individually are also conceivable as for example negative effects on their education or career opportunities. Regarding utilization data, on which cost estimations are based, the study cannot exclude recall bias, because parents of participating children provide information about the previous 12 months. The authors do not assume an effect on the validity of their study .As a tendency of “overreporting” PA is known from other surveys , an overFor the construction of the exposure variable MVPA it has been assumed that the WHO recommendation of 60 minutes/day MVPA can be extrapolated to 7 h/week MVPA. This was necessary as PA in this study was recorded in h/week. It does, however, not ensure that children are physically active daily which might have an influence on possible health effects of MVPA. Further, other health behaviors like food habits may possibly confound the association between MVPA, healthcare utilization and costs and could not be taken into account.Furthermore it has to be noted that the study sample is not representative for German children .As in the 10-year follow-up only about 55% of the baseline individuals are included, non-response bias cannot be ruled out either.This study may be regarded as one of the first steps in investigating the association between PA, healthcare utilization and direct as well as indirect costs in children. Even if the study did not show significant results, it is important because it examined possible short-term effects in this association. Setting the focus on the association between PA and healthcare costs rather than on the association between a disease and healthcare costs, the study makes a contribution to the exploration of health behaviors and protective factors in primary prevention. Long-term effects remain to be analyzed to clarify the public health importance of PA. Therefore, further studies which apply a lifetime perspective and observe the participants from childhood into adulthood are needed. As it is scientifically known that positive health effects of PA in children are possible, the focus of further studies should be on these aspects: a better understanding of the PA types and the sport forms that generate health effects in children (paying particular attention to their growth process) and calculating its subsequent economic impact more exactly. This might strengthen and underpin the promotion of PA in children from a health economic perspective.aWe chose the reference year 2007 for reported numbers, because it was the year of data collection in the 10-year birth cohort.


The Member States of European Union are encouraged to improve the general conditions for the production and marketing of apicultural products. In Belgium, programmes on the restocking of honey bee hives have run for many years. Overall, the success ratio of this queen breeding programme has been only around 50%. To tackle this low efficacy, we organized sanitary controls of the breeding queens in 2012 and 2014.Varroa destructor Macula-like Virus occurred at very low prevalences (≤5%). Remarkably, we found Apis mellifera carnica bees to be less infected with Deformed Wing Virus than Buckfast bees (p < 0.01), and also found them to have a lower average total number of infecting viruses (p < 0.001). This is a significant finding, given that Deformed Wing Virus has earlier been shown to be a contributory factor to winter mortality and Colony Collapse Disorder. Moreover, negative-strand detection of Sacbrood Virus in eggs was demonstrated for the first time.We found a high quantity of viruses, with more than 75% of the egg samples being infected with at least one virus. The most abundant viruses were Deformed Wing Virus and Sacbrood Virus (≥40%), although Lake Sinai Virus and Acute Bee Paralysis Virus were also occasionally detected (between 10-30%). In addition, Aphid Lethal Paralysis Virus strain Brookings, Black Queen Cell Virus, Chronic Bee Paralysis Virus and High pathogen loads were observed in this sanitary control program. We documented for the first time vertical transmission of some viruses, as well as significant differences between two honey bee races in being affected by Deformed Wing Virus. Nevertheless, we could not demonstrate a correlation between the presence of viruses and queen breeding efficacies.The online version of this article (doi:10.1186/s12917-015-0386-9) contains supplementary material, which is available to authorized users. In view of the spread of varroasis – a mite infestation of the honey bee – over Europe and the problems which this disease has brought about in the beekeeping sector, the Member States of the European Union have been encouraged to set up national programmes aimed at improving the general conditions for the production and marketing of apicultural products. In Belgium, such apicultural programmes now exist for many years and particularly in the Flemish region, a lot of effort has been put in the restocking of hives. Within this programme, a limited number of recognized breeders are provided with the possibility to travel to a land mating yard in Belgium (Kreverhille) and island mating yards in Germany  and the Netherlands  with selected virgin queens. When these fertilized queens perform well they become the new breeding queens two years later, and are distributed on a large scale among the other beekeepers. Overall, this programme enjoyed a high participation rate amongst the beekeepers, but failed to a certain extent in terms of the efficacy of the queen breeding programme. This is evident from the fact that in the past four years, between 5,948 and 6,195 larvae were grafted, but only 61.4-70.8% could be raised to newborn queens and from these only 75.0-79.9% became egg-laying. Thus overall, the success ratio of the queen breeding programme has been only 49.1-53.1%, a fairly low number   vs. an average of 2.3 infecting viruses in Buckfast ]. No significant differences were found in the incidence of the other viruses screened .The most abundantly detected viruses were DWV  and SBV 42%, 33/78). LSV and ABPV were moderately detected in 28% (22/78) and 14% (11/78) of the samples. The other viruses ALPV, BQCV, CBPV and VdMLV had only low prevalences, respectively 5% (4/78), 5% (4/78), 1% (1/78) and 3% (2/78). Remarkably, %, 33/78.Our results, however, did not indicate a correlation between the virus burden  and queen breeding efficacy  and Asian honey bees (Apis cerana) [A. cerana [Furthermore, we have detected the negative-strand of SBV. Although this might indicate that SBV replicates in eggs, it is also possible that this originates from transovum transmission, such as surface contamination with sperm containing negative-strand RNAs. Replication of SBV was previously reported in adults and larvae of European ( cerana) -32. This cerana) , but thi cerana) ,33,34. L. cerana ,36. Althet al. [A broad virus screening of honey bee eggs was not yet performed. Nevertheless, few studies reported the presence of viruses -8,19,37 et al. ).Apis mellifera carnica bees to be less infected with Deformed Wing Virus (p < 0.01) than Buckfast bees, and also found them to have a lower average total number of infecting viruses (p < 0.001). This is a significant finding, given that Deformed Wing Virus has earlier been shown to be a contributory factor to winter mortality, and offers interesting perspectives for breeding virus-resistant bees. However, we cannot make general conclusions about the virus-resistant state of carnica race compared to Buckfast race solely based on our data. Concluding, further sanitary screenings in the context of queen breeding seems advisory, especially because BQCV infection is a common cause of queen larval death [A survey of viruses in honey bee eggs in the context of a queen breeding program demonstrated high incidences of two viruses (DWV and SBV) and moderate to low incidences of a further six viruses . Vertical transmission (transovum or transovarian) of some viruses  was demonstrated for the first time as well as negative-strand detection of SBV. We could not demonstrate a correlation between the presence of viruses and the low queen breeding efficacies. Remarkably, we found al death .Apis mellifera), which is neither an endangered nor a protected species.The study involved the European honey bee (


Traumatic Brain Injury (TBI) has gained attention in the past decade as a “signature injury” in the conflicts in Iraq and Afghanistan. TBI is a major burden for both the military and civilian population in the US and worldwide. It is a leading cause of death and disability in the US and a major health services resource burden. We seek to answer two questions. What is the evidence regarding the association of TBI with health services utilization and costs in the US and worldwide? What is the evidence regarding racial/ethnic, gender, geographic, socio-economic and other disparities in health services utilization and cost in the US and worldwide? To attain this goal we searched several databases using key words to perform a systematic review of the literature since 2000. We found 36 articles to be eligible for inclusion in the review. The evidence demonstrates a wide variation in health services utilization and costs depending on population of study and severity of TBI. The evidence also supports the existence of racial/ethnic, gender, insurance, geographic disparities in the US as well as other unique disparities worldwide. Traumatic Brain Injury (TBI) has gained attention in the past decade as a “signature injury” in the conflicts in Iraq and Afghanistan . TBI is TBI is a major health services resource burden with approximately 1,365,000 (80.7%) emergency department (ED) visits and 275,000 (16.3%) hospitalizations annually  were searched for articles published between January 2000 through June 2013 using a reproducible strategy. Four searches with broad search terms were performed in each database using MeSH headings search. The first search used the terms The following inclusion criteria were used to determine eligible study characteristics: (1) must be published in English, (2) must include health services cost or utilization related to TBI (3) must include adults. Exclusion criteria included: (1) did not focus on clinical efficacy, (2) was not limited to describing cost-effectiveness of an intervention, and (3) did not focus on pediatric TBI.The process used to screen the citations is shown in Data collected from the eligible articles is shown in Tables Of the 858 articles found through searching three databases, 36 were eligible for inclusion in this review. See Type of cost and utilization are included in Tables Costs associated with TBI vary widely depending on the population studied, severity of injury and time period. In an early review of mild TBI without surgical intervention, indirect costs were much higher than direct costs, accounting for 92% of total costs in 1981 dollars Since 2000, there have been a number of studies in the US and world wide of health services costs associated with TBI in various populations with costs ranging from $9,000 to $103,667. . As a comparison, total health services costs for non-TBI matched controls were $7,377 . Mean costs were 76% higher in the three years after injury for the mild TBI group and 5.75 times greater for the moderate/severe group compared to controls . Another case control study for that for definite and probable TBI, most incremental costs occurred within the first six months while significant long-term incremental medical costs were not apparent among one-year survivors  veterans treated in the VA found that OEF/OIF veterans with TBI had 4 times higher median health services costs relative to OEF/OIF veterans without TBI  In one study, private insurance was associated with a shorter length of stay , job skills (46.3%), and increasing income (50.5%) (Among 79 patients with moderate/severe TBI in Netherlands, those with a high locus of control were more likely to visit medical specialists and use supportive care than those with lower levels of internal locus of control. . In AustWe reviewed a decade of literature regarding the utilization and cost of health services for TBI in civilian and military adults in the US as well as other countries. We found that cost estimates vary depending on the study population, TBI severity and presence of other mental health co-morbidities. We also found there was evidence of significant racial/ethnic disparities in utilization. However, there is need for further research on cost of TBI over time and better understanding of racial/ethnic, geographic and socioeconomic variations in cost and utilization among individuals with TBI in both civilian and military/Veteran populations in the US and worldwide.Our study has a number of limitations. We only searched literature since the year 2000 and included studies only published in the English language. We also only reviewed published studies, which may reflect publication bias, as some analyses are not published.We can conclude, however, that the cost and utilization of TBI is an important area of research for the next 5 years, especially considering the growing recognition of the impact of mental health on health costs and outcomes. A potential concern, which has been addressed only marginally in the literature, is racial/ethnic, geographic, and socio-economic disparities in utilization of health services for TBI and its associated co-morbid conditions. There is also a need to control for severity and follow patients over time to truly compare costs and utilization. Future research should investigate the change in cost during the first year vs. over the lifetime, and costs for those who receive mental health services vs. those who do not.


To the Editor: Molecular epidemiologic investigations and virologic surveillance contribute notably to the control and prevention of measles  for isolation of measles virus from throat and nasopharyngeal swab samples. For serologic confirmation of cases, we used a commercial measles IgM ELISA kit . Virus genotyping was based on the 450-nt coding sequence for the carboxyl terminus of nucleoprotein (N) of measles virus, as recommended by the World Health Organization  and previously described primers (We used the Vero/human-SLAM cell line (PCR products could be amplified from 16 of the 24 samples analyzed. Ten samples provided high quality sequence reads for the N gene coding region, which were used for further analysis. Clinical and demographic data for these 10 cases, virus isolation status, and GenBank accession numbers of the sequences are summarized in the Phylogenetic analysis revealed 1 of the 10 measles virus strains to be of genotype D8 Figure 1,The B3 genotype identified in this study could be a previously undetected genotype endemic to India or a recent importation. B3 is endemic to many countries in Africa, and its importation into Europe and North America and elsewhere has been described (This study was approved by the Institutional Human Ethics Committee of the Rajiv Gandhi Centre for Biotechnology. It was carried out as part of the National Virology Network Laboratory Program of the Indian Council of Medical Research, which provided financial support. We used measles virus genotype B3 sequences detected globally to analyze origins of strains identified in India.


There are different viruses that cause respiratory infections. Most of the time it is very difficult to know the causative agent from the respiratory symptoms. In order to identify the possibility of a virus being the causative agent of the respiratory infection and to identify the type of virus that is causing the respiratory symptoms, nasal swabs were collected from patients with symptoms of acute respiratory infection.During a 6-month period (1 February-31 July 2014) 365 nasal swabs were collected from patients with symptomatic respiratory infections admitted to Hospital Israelita Albert Einstein, São Paulo, Brazil for polymerase chain reaction virus detection (CLART Pneumovir) capable of detecting 14 respiratory viruses.Almost half of the samples collected tested positive. Specifically, 48.76 % (178/365) of samples were positive for respiratory virus. Syncycial respiratory virus accounted for 19.66 % (35/178), influenza H3N2 for 14.61 % (26/178), parainfluenza virus for 7.3 % (13/178), influenza B virus for 6.18 % (11/178), influenza H1N1 for 1.69 % (3/178) and influenza C for 1.69 % (3/178).Respiratory virus is a common cause of respiratory infection requiring hospital admission, rhinovirus being the most prevalent. The identification of the type of respiratory virosis is crucial for virus isolation precautions.