PLoS Medicine by Ioannidis [p-value, a point that has been made in the medical literature for at least four decades, and in the statistical literature for decades previous. This topic has renewed importance with the advent of the massive multiple testing often seen in genomics studies.The article published in oannidis  makes thp-values, and perhaps analytic manipulation—the mathematical argument in the PLoS Medicine paper underlying the “proof” of the title's claim has a degree of circularity. As we show in detail in a separately published paper [Unfortunately, while we agree that there are more false claims than many would suspect—based both on poor study design, misinterpretation of ed paper , Dr. Ioap ≤ 0.05, instead of the actual p-value observed in a study, e.g., p = 0.001.Calculating the evidential effect only of verdicts of “significance,” i.e., Introducing a new “bias” term into the Bayesian calculations, which even at a described “minimal” level (of 10%) has the effect of very dramatically diminishing a study's evidential impact.The two assumptions that produce the above effect are:PLoS Medicine paper shows merely that the more studies published on any subject, the higher the absolute number of false positive  studies. It does not show what the papers' graphs and text claim, viz, that the number of false claims will be a higher proportion of the total number of studies published .In addition to the above problems, the paper claims to have proven something it describes as paradoxical; that the “hotter” an area is , the more likely studies in that area are to make false claims. We have shown this claim to be erroneous . The matThe paper offers useful guidance in a number of areas, calling attention to the importance of avoiding all forms of bias, of obtaining more empirical research on the prevalence of various forms of bias, and on the determinants of prior odds of hypotheses. But the claims that the model employed in this paper constitutes a “proof” that most published medical research claims are false, and that research in “hot” areas is most likely to be false, are unfounded.


The number of studies evaluating the effect of environmental exposure to genotoxic agents in children has rapidly increased in the last few years. The frequency of micronuclei (MN) in peripheral blood lymphocytes determined with the cytokinesis block assay is among the most popular biomarkers used for this purpose, although large inter- and intralaboratory variability of this end point has been observed in population studies. The availability of reference measures is therefore necessary for laboratories to validate protocols and analytical procedures, and for molecular epidemiologists, as well, to estimate the statistical power of studies and to assess the quality of data. In this article, we provide estimates of the baseline frequency of MN in children, conducting a meta-analysis of MN frequency reported by field studies in children and a pooled analysis of individual data [available from published studies and from the Human Micronucleus International Collaborative Study (HUMN) database]. Thirteen articles were selected for meta-analysis, and individual data included in the pooled analysis were retrieved from the databases of 12 laboratories. Overall means of 4.48  and 5.70  MN per 1,000 binucleated cells were estimated by the meta- and pooled analysis, respectively. A clear effect of age was detected, even within the restricted range of pediatric age considered, with significantly lower frequency values in newborns. No influence of sex was found. The study showed the advantage of using data from large collaborative studies and suggested a synergistic use of meta- and pooled analysis. DetailsDespite the large number of advantages that justify the popularity of this assay, there is an evident limitation—the large inter- and intralaboratory variability in the MN frequency. This variability may be explained partially by technical reasons, genetic variability, or sampling error. However, interscorer discrepancy and protocol differences (when different laboratories are involved) have been shown to be the most important sources of variability .The meta- and pooled analyses represent the ideal statistical tools for computing summary estimates of a biomarker frequency using data from different studies . The advIn this study, we identified the published studies reporting MN during childhood  with the aim of performing a meta-analysis of MN frequency for referent children and providing a meta-estimate of the MN baseline value. Moreover, a pooled analysis of individual data available from published studies and from the HUMN database  was perfhttp://www.ncbi.nlm.nih.gov/PubMed). The terms “micronucleus tests” existing since 1989 and “micronuclei” existing since 1990 were used as medical subject heading (MeSH) key word. To cover previous years, a free text search with terms “micronucleus” and “micronuclei” was performed. The categories “All Child: 0–18 years,” “Human,” “Language: English,” and “Publication Date: from January 1st, 1985 to September 1st, 2004” were set as search limits. This allowed retrieving 168 citations.Individuals from birth to late adolescence  were considered as children. The studies selected for inclusion in the meta-analysis were identified by systematically searching the MedLine/PubMed database  was estimated from the model and found to be highly significant . The analysis was carried out with STATA statistical software ; the random-effects model was performed with the procedure META, and the diagnostics, such as funnel plot, with the procedures METAINF and METABIAS.We computed a summary estimate of the MN frequency applying the random-effects linear model to the natural logarithm of the MN mean of each study . This moWe obtained sex-specific and age-group–specific MN mean frequencies by fitting a negative binomial random-effects model to the MN count of each subject . The negThe MN frequency for each study included in the meta-analysis is reported in p < 0.001).The layout of data selected for the pooled analysis is reported in The main purpose of this study was to provide reference values for researchers planning studies on genomic damage in children. We used two complementary approaches to compute summary measures of baseline MN frequency in children. Meta-analysis provided summary measures that, although affected by a certain degree of heterogeneity, are considered to be representative of studies published in the literature. Pooled analysis, although limited to six published studies , included 229 subjects (from six laboratories) from the HUMN database  and alloThe estimates of the baseline MN frequency in children obtained by the two approaches were by large consistent, especially considering CIs. The lower mean value estimated by meta-analysis , considering that mean ages in the laboratories contributing data to meta-analysis and in those considered for the pooled analysis were similar, is likely to be attributable to a different distribution of absolute values of MN frequency in the two sets of data. This is not surprising because studies based on cytogenetic biomarkers suffer from a certain degree of heterogeneity among laboratories, and absolute values may differ even largely.The availability of reference values is important for research teams and laboratories that need to validate protocols and analytical procedures as well as to estimate the statistical power of field studies and check the quality of data. For this purpose, baseline MN frequencies for the cytokinesis block assay have been published for an adult population .Age is the most important predictor of MN frequency, as described by cooperative studies and reported in the literature . HoweverIn the present study, the effect of age was evident even within the restricted age range considered in pooled analysis (0–19 years). The low frequency of MN detected in children < 1 year of age is noteworthy, given the growing number of studies performed on the cord blood, an easily accessible source of DNA. MN numbers were very low at birth (3.27 ‰) and increased by 66% in children 1–4 years of age (5.43 ‰), an increase that accounted for most of the age effect on MN frequency described in the literature. Besides the major challenges posed by the leaving of the protected intrauterine environment, other changes occurring in the first years of age, such as solid diet, vaccinations, and viral diseases, provide plausible explanations for this dramatic increase.No difference in MN frequency was found by sex in our pooled analysis. The effect of sex has been repeatedly reported in adult populations, and a recently published pooled analysis estimated a 19% higher MN frequency in females than in males , 2001. HIn conclusion, these results address an increasing request from researchers performing epidemiologic studies in children based on biomarkers—specifically, the availability of specific reference values in pediatric populations. The MN baseline values provided here are meant for the planning phase of a study and should not justify the conduct of future studies among children that exclude the identification of properly selected referent (control) subjects. Besides the main study findings, we showed here the advantage of using data from large collaborative research projects to improve the design of future field studies, including the efficiency of the statistical analysis.


PLoS Medicine that takes us closer to being able to adopt a pharmacogenetic approach to reduce bleeding risk from coumarin therapy.Greaves discusses a new study in  Warfarin and related coumarin-based anticoagulants are the mainstay of pharmacological management for the long-term prevention of thromboembolism. It is estimated that over 1 million residents in the United Kingdom take warfarin; in excess of 20 million prescriptions for the anticoagulant are dispensed each year in the United States of America. Despite recent advances in the development of novel, alternative, oral antithrombotics it is likely that coumarins will be used widely for at least the next decade.Coumarins inhibit the posttranslational carboxylation of glutamate residues on proteins dependent on vitamin K, including coagulation factors II, VII, IX, and X. This carboxylation process is essential for their biological activity and requires the reduced form of vitamin K. Coumarins inhibit vitamin K epoxide reductase, the enzyme responsible for the recycling of vitamin K. The vitamin K epoxide reductase complex 1 (VKORC1) gene has been identified only recently [The metabolism of warfarin depends principally upon the hepatic microsomal enzyme P450 2C9 (Cyp2C9), which catalyses degradation of the more potent S enantiomer to inactive metabolites.Coumarins have very few side effects. However, by far the most common unwanted effect, abnormal bleeding, may be lethal. Overall, the rate of life-threatening bleeding is around two per 100 patient years and more minor bleeding is common . This isIndividual patients on warfarin are within the target INR range for only 50%–70% of the time.For most clinical indications, dosing is aimed at achieving a target INR of 2.5 (range 2.0–3.0), which represents a level of anticoagulation associated with an optimal relationship between antithrombotic efficacy and bleeding risk. However, even with the best available management, individual patients on warfarin are within the target INR range for only 50%–70% of the time, on average. Although the reasons for this imprecision of dose response are often not immediately apparent, important contributing factors include co-medication with interacting drugs .In addition to this variation in dose-response within individuals, the inter-individual dose range required to achieve and maintain the target INR is exceptionally wide—for example, in the case of warfarin, a maintenance dose of 1 to >10 mg daily. Population studies suggested a genetic component to coumarin sensitivity; for example, people of Chinese origin are more sensitive, and African Americans less sensitive, than those of European ancestry ,6. This These findings raise the question of whether advances in understanding of the pharmacogenetics of oral anticoagulant therapy could beneficially influence clinical practice and patient safety by facilitating individualised coumarin dosing—hence reducing bleeding event rates. Answers are just beginning to emerge. In a retrospective study, Higashi et al. found that variant alleles of Cyp2C9 are associated with increased risk of above average INRs and longer duration to achieve stable dosing (compared to wild-type) in patients treated with warfarin . FurtherPLoS Medicine [Additional evidence of the potential for a pharmacogenetic approach to reduce bleeding risk from coumarin therapy is presented in the current issue of Medicine . ReitsmaThese intriguing findings, along with those of Higashi and colleagues, suggest that further prospective studies of the utility of a pharmacogenetic approach to improving the safety of oral anticoagulation with warfarin are justified. Such studies will of necessity be large and should include cost-effectiveness analyses. They should be implemented, but the question arises whether their importance may be undermined by the licensing for long-term use of alternative, novel oral anticoagulants that have much more predictable dose-response characteristics, such as direct thrombin inhibitors.


Extremes of temperature are associated with short-term increases in daily mortality.We set out to identify subpopulations and mortality causes with increased susceptibility to temperature extremes.We conducted a case-only analysis using daily mortality and hourly weather data from 50 U.S. cities for the period 1989–2000, covering a total of 7,789,655 deaths. We used distributions of daily minimum and maximum temperature in each city to define extremely hot days (≥ 99th percentile) and extremely cold days (≤ 1st percentile), respectively. For each (hypothesized) effect modifier, a city-specific logistic regression model was fitted and an overall estimate calculated in a subsequent meta-analysis.Older subjects , diabetics , blacks , and those dying outside a hospital  were more susceptible to extreme heat, with some differences observed between those dying from a cardiovascular disease and other decedents. Cardiovascular deaths , and especially cardiac arrest deaths , showed a greater relative increase on extremely cold days, whereas the increase in heat-related mortality was marginally higher for those with coexisting atrial fibrillation .In this study we identified several subpopulations and mortality causes particularly susceptible to temperature extremes. This knowledge may contribute to establishing health programs that would better protect the vulnerable. Extremes of temperature are associated with short-term increases in daily mortality . IdentifHere we used a case-only approach to study susceptibility to temperature extremes in 50 U.S. cities. This approach, which examines only decedents, provides important advantages compared with traditional analyses. Advantages of the approach include reduction of potential confounding by variables that are typically associated with mortality , simplification of modeling, and reduction of vulnerability to model mis-specification bias . Of partThe aim of the study was to identify factors that confer susceptibility to extreme temperatures, including sociodemographic characteristics, medical conditions, and place of death, and to explore differences in susceptibility according to the primary cause of death. We also aimed to identify specific mortality causes that experience greater relative increases on extreme temperature days, an issue that has not been addressed by previous studies.After approval of the study protocol by the Human Subjects Committee from the Harvard School of Public Health, we conducted a case-only analysis using weather and mortality data from 50 U.S. cities during the period 1989–2000. We selected all counties corresponding to the metropolitan areas of cities listed in The case-only approach was originally proposed to improve efficiency in the study of gene–environment interactions by examining the prevalence of a specific genotype only among cases. As Armstrong suggested (2003), this approach can also be used to investigate how other individual characteristics that do not vary (or vary slowly) over time  modify the effect of a time-varying exposure  on the outcome of interest  by restricting the sample to cases . In our study, we applied this approach by comparing the individual characteristics of those dying on extremely hot (or cold) days with those dying on other days. Intuitively, if an individual characteristic increases the risk of dying on extremely hot days, the proportion of decedents presenting that characteristic will be larger on extremely hot days. In other words, extremely hot days should be a predictor of the occurrence of that individual characteristic in death certificates as assessed by logistic regression . FollowiInternational Classification of Diseases, 9th revision (ICD-9) and 10th Revision (ICD-10), 1993] iFor each city, we obtained hourly weather data from the nearest National Weather Service surface station  and calculated the daily minimum and maximum temperatures. We defined extremely cold days as those with a daily maximum temperature at or less than the 1st percentile of its distribution in that city. Similarly, we defined extremely hot days as those with a daily minimum temperature at or greater than the 99th percentile. We chose the daily maximum temperature to examine extreme cold and the daily minimum to examine extreme heat because these indicate situations in which there is little relief during the day (for cold) or at night (for heat). The decision to use a city-specific definition for extreme temperatures was based on previous evidence that the relationship between mortality and temperature varies across different regions, with the largest cold effects observed in the warmer regions and vice versa . BecauseWe used data from the U.S. Environmental Protection Agency’s Aerometric Retrieval System  to estimWe conducted separate analyses for cold and heat to examine modification of the risk of dying associated to extreme temperatures. In a first stage, we fitted city-specific logistic regression models  in which the dependent variable was the hypothesized modifier . The indicator variable for extreme temperature was included as a predictor in the models. Analyses for extremely cold temperatures were conducted only for the cold months (October–April), defined as those months without any extremely hot day in any of the 42 eligible cities. Likewise, the heat analyses were performed only for the warm months (April–October). This was done to exclude hot days from the reference group for extremely cold days and vice versa, thus avoiding an over-or underestimation of the interaction in situations where the modifier of interest acts for both hot and cold days. Although seasonality per se is not a confounder in these models , if the The analyses examining subject characteristics as effect modifiers were repeated separately for deaths due to cardiovascular disease and deaths due to other primary causes. Our baseline analyses focused on extreme temperature during the day of death, because previous studies indicate the largest effect on mortality occurs then . We alsoIn a second stage of analysis, we combined the city-specific results in a random effects meta-analysis assuming that is the effect estimate in city i, νi is the variance within city i, β is the true effect of hot/cold, and τ2 is the variance of the true effect between cities, commonly referred to as the random effect. We used the restricted maximum likelihood (REML) procedure of the METAREG function in Stata  (wi = 1/( νi + τ2). For the meta-analysis of each effect modifier, cities with < 10 observations in a cell were excluded. We used the I2 statistic to assess the proportion of total variation in effect estimates that was due to between-cities heterogeneity   to calcuogeneity . We usedQ is the Q-test for heterogeneity and k is the number of cities. Here we present the overall estimates for each effect modifier obtained in the meta-analysis.where Our analyses included a total of 7,789,655 deaths across 50 U.S. cities. New York City, New York, had the highest number of deaths during the study period, 821,722, whereas smaller cities such as Boulder, Colorado; Provo, Utah; or Terra Haute, Indiana, contributed < 17,000 deaths each. Most of the subjects were > 65 years of age, white, and with a low education . A substCities in the study had a wide range of climates, with annual mean temperatures ranging from 7.8°C in Minneapolis, Minnesota, to 25.0°C in Honolulu, Hawaii. I2 < 0.30), except for dying outside a hospital, with 47% and 78% of the total variability in the cold and heat estimates, respectively, being attributable to between-cities differences. Overall, in the sensitivity analysis that used the 5th and 95th percentiles, all associations were similar but with smaller ORs and narrower CIs. For instance, the OR for low-educated subjects was 1.010 , compared with 1.016  in the baseline analyses. Results using apparent temperature were also very similar, but did not show evidence for increased heat susceptibility in diabetics . Adjustment for ozone level during the warm season resulted in similar, though somewhat lower, estimations (results not shown). The greatest change was observed for the elderly, whose estimate for heat (using the 26 cities with ozone data) changed from an OR of 1.019  to an OR of 1.014  after adjustment.In general, effect modification was modest , with thI2 of 0.55 and 0.67, respectively. When looking at lags of exposure, all the effect modifications observed in the baseline analysis occurred mainly at lag 0. Adjustment for ozone of the heat baseline models led to essentially the same results with estimates approximately 10% lower (results not shown).For some individual characteristics, the effect modification seen in In a large, multicity study we identified sub-populations with increased sensitivity to extreme temperatures. Black subjects, those ≥ 65 years of age, and diabetics were especially vulnerable to extreme heat, with some differences in susceptibility observed between those dying from a cardiovascular disease and those dying from other causes. The increase in deaths on extreme temperature days was significantly higher for out-of-hospital deaths than for inhospital deaths. Regarding specific mortality causes, cardiovascular deaths showed a higher susceptibility to extreme cold, which was particularly noticeable for cardiac arrest deaths. Another interesting finding was the marginally increased vulnerability conveyed by atrial fibrillation, which has not been previously reported.Our results suggest that advanced age increases susceptibility to temperature extremes, with a more marked effect for heat. Several studies on the effects of temperature on mortality have also found a greater susceptibility of the elderly to both cold  and hot Our results showed that blacks and low-educated subjects, which may be predictors of low socioeconomic status, were more susceptible to extreme heat. A greater susceptibility to die on hot days has been previously reported for blacks , the lowWhen examining whether some underlying chronic conditions increase the susceptibility to extreme temperatures, we confirmed the finding of An important finding of our study is that susceptibility to temperature extremes varies according to the primary cause of death. Few studies have looked at susceptibility to temperature stratifying by cause of death. A study in seven U.S. cities found that younger subjects, blacks, and those dying outside a hospital were more susceptible to extreme cold when they died from a respiratory disease, but observed no differences in susceptibility for those dying from a cardiovascular disease . In contSome evidence in the literature shows that cardiovascular deaths increase during both extremely cold days and extremely hot days . In our Given that we used a city-specific definition to determine exposure to extreme temperatures, the fact that effect modification was fairly homogeneous across cities for most of our proposed modifiers, particularly for cold effects, supports our use of a relative (rather than absolute) temperature and agrees with previous studies suggesting that populations tend to adapt to the local climate . This ada priori, this misclassification should be unrelated to ambient temperature, this may not be the case for diagnoses, such as cardiovascular disease, that have been consistently related to cold temperatures in the past. Finally, because we focused on extreme temperature days, our results cannot be extrapolated to moderately hot or cold temperatures.One limitation of our study is that, due to its case-only design, the excess risk of dying associated with extreme temperatures in each subpopulation or mortality-cause category was not estimated in absolute terms, but relative to the excess risk in other subpopulations/causes. Consequently, it was not possible to ascertain whether the negative association for myocardial infarction represented a decrease in risk on hot days or a less pronounced increase compared with other mortality causes. The most likely scenario is the latter, given previous epidemiologic evidence of an increase in the risk of myocardial infarction on hot days . Also, aIn conclusion, we confirmed in a large sample of cities that subpopulations such as the elderly, diabetics, and black subjects are especially susceptible to temperature extremes. According to our results, susceptibility of populations may vary according to the primary cause of death, suggesting that future studies on susceptibility will benefit from taking this into consideration. Finally, we found evidence that cardiovascular deaths, especially cardiac arrest deaths, show much larger increases on extremely cold days than other mortality causes.


Alternative ways to control caterpillar pests and reduce the use of pesticides in apple orchards are in the interest of the environment, farmers and the public. Great tits have already been shown to reduce damage under high caterpillar density when breeding in nest boxes in an experimental apple orchard. We tested whether this reduction also occurs under practical conditions of Integrated Pest Management (IPM), as well as Organic Farming (OF), by setting up an area with nest boxes while leaving a comparable area as a control within 12 commercial orchards. We showed that in IPM orchards, but not in OF orchards, in the areas with breeding great tits, apples had 50% of the caterpillar damage of the control areas. Offering nest boxes to attract insectivorous passerines in orchards can thus lead to more limited pesticide use, thereby adding to the natural biological diversity in an agricultural landscape, while also being economically profitable to the fruit growers. Biological control of pests is becoming increasingly important as an answer to the resistance of harmful insects to pesticides, the adverse public attitudes to pesticides and the increasing restriction of their use due to legislation Parus major L. is a common species in the Netherlands and breeds readily in nest boxes. Putting up nest boxes can easily enhance the local density of great tits [6 and personal data]. Within the breeding season great tits mainly forage for caterpillars, which is the preferred food for their nestlings Operophtera brumata L. and tortricid moths, are key pests The great tit In this study we investigated whether great tits can reduce caterpillar damage in commercially managed apple orchards, with either Integrated Pest Management (IPM) or Organic Farming (OF), by measuring damage levels of the apples in areas with and without breeding great tits.2(1) = 4.71, P = 0.03, orchard type x treatment χ2(1) = 3.88, P = 0.049; test without interaction: treatment: χ2(1) = 4.03, P = 0.045). There was no damage reduction in the OF orchards but a very substantial reduction in the IPM orchards, where the damage in the areas with breeding great tits was 50% of the damage in the control areas  = 4.18, P = 0.041; interaction with type of orchard marginally non-significant χ2(1) = 3.71, P = 0.054). The model with treatment had a higher log likelihood than the model with number of breeding pairs and was thus preferred.The effect of the number of breeding pairs of great tits on damage reduction could not be fitted when treatment also was included in the model as this number was always 0 in the control areas. Tested in the absence of treatment, breeding density had a significant effect on the amount of caterpillar damage to the apples (χ2(1) = 7.27, P = 0.007). We found earlier that in an experimental orchard, with even higher caterpillar densities, there again was a reduction in caterpillar damage when Great Tits were allowed to forage in the apple trees .Although some studies suggest that birds do not remove prey that occur at low densities Fruit growers with IPM orchards thus benefit from the presence of great tits in their orchards even without changing their normal management. Reducing caterpillar damage in the orchard by offering nest boxes to great tits is an extremely low cost measure  that can reduce damage by up to 50 %, i.e. an increase in yield of undamaged apples of 1200 kg per ha (average production is 40000 kg per ha per year).Additionally, our findings imply that the threshold caterpillar densities at which pesticides need to be applied can be raised when great tits are breeding in orchards. For example, when great tits are absent from an orchard a damage level of 3.5% is reached with an initial density of four caterpillars per tree, while when two to four pairs of great tits are foraging in an orchard the same damage occurs with eight caterpillars per tree. Offering nest boxes to attract insectivorous passerines in orchards can thus lead to more limited pesticide use, thereby adding to the natural biological diversity in an agricultural landscape, while also being economically profitable to the fruit growers.We measured caterpillar densities at the start of the growing season and damage levels to apples at harvest in two similar areas of 2 ha each, one of which was randomly assigned to the nest box treatment, in 12 Dutch apple orchards over a period of four years (1997–2000). Plots were at least 500 m apart, too far for great tits breeding in the nest box plot to forage in the control plot. In Dutch apple orchards there are two management regimes, and we used orchards under both types of regimes. We used six orchards with Integrated Pest Management (IPM) orchards where pesticides are used only if caterpillar densities exceed a control threshold Nest boxes (15 to 25) were systematically placed in the inner 1.5 ha of the nest box area to attract great tits. Six orchards were sampled in one year, five in two years and one in three years (n = 19). All nest box areas had at least one pair of breeding great tits. The laying date, clutch size and number of great tit fledglings were determined by checking nest boxes weekly from mid April until all broods had fledged.2(1) = 0.55, P = 0.36, density higher in OF than IPM orchards χ2(1) = 7.27, P = 0.007).Caterpillar densities were determined on 40 trees in each control and nest box area at the end of apple bloom by counting caterpillars on the third lowest branch of each tree. Initial densities of caterpillars did not differ between the areas with nest boxes and the control areas (repeated measures ANOVA: χIn autumn we checked 25 apples from each of 40 trees in each of the nest box and control area (thus 2000 apples per orchard) for caterpillar spring damage, as described by De Reede et al. We used a repeated measure ANOVA (to maintain the link between the area with and without great tits within an orchard) with binominal errors with caterpillar damage (number of damaged apples over number of apples scored per area) as the response variable and treatment, management type (IPM or OF) and their interaction as explanatory variables. Initial caterpillar densities (average number of caterpillars per tree) were analysed with a repeated measures ANOVA with treatment, management type (IPM or OF) and their interaction as explanatory variables. In both analyses the GenMod procedure of SAS V8 was used.Table S1Data on caterpillar damage on apples in six Integrated Pest Management (IPM) and six Organic Farming (OF) orchards: name of the orchard, year of sampling, type of management (IPM/OF), number of breeding pairs of Great Tits, number of caterpillars per tree, treatment (control/nest box area), number of apples sampled and number of damaged apples.(0.10 MB DOC)Click here for additional data file.


Protease inhibitors (PIs) are associated with hypertriglyceridemia and atherogenic dyslipidemia. Identifying HIV-1-infected individuals who are at increased risk of PI-related dyslipidemia will facilitate therapeutic choices that maintain viral suppression while reducing risk of atherosclerotic diseases. Apolipoprotein C-III (apoC-III) gene variants, which vary by race/ethnicity, have been associated with a lipid profile that resembles PI-induced dyslipidemia. However, the association of race/ethnicity, or candidate gene effects across race/ethnicity, with plasma lipid levels in HIV-1-infected individuals, has not been reported.n = 626). Race/ethnicity was a highly significant predictor of plasma lipids in fully adjusted models. Furthermore, in stratified analyses, the effect of PI exposure appeared to differ across race/ethnicity. Black/non-Hispanic, compared with White/non-Hispanics and Hispanics, had lower plasma triglyceride (TG) levels overall, but the greatest increase in TG levels when exposed to PIs. In Hispanics, current PI antiretroviral therapy (ART) exposure was associated with a significantly smaller increase in TGs among patients with variant alleles at apoC-III-482, −455, and Intron 1, or at a composite apoC-III genotype, compared with patients with the wild-type genotypes.A cross-sectional analysis of race/ethnicity, apoC-III/apoA-I genotypes, and PI exposure on plasma lipids was performed in AIDS Clinical Trial Group studies (In the first pharmacogenetic study of its kind in HIV-1 disease, we found race/ethnic-specific differences in plasma lipid levels on ART, as well as differences in the influence of the apoC-III gene on the development of PI-related hypertriglyceridemia. Given the multi-ethnic distribution of HIV-1 infection, our findings underscore the need for future studies of metabolic and cardiovascular complications of ART that specifically account for racial/ethnic heterogeneity, particularly when assessing candidate gene effects. This cross-sectional analysis of race/ethnicity, apoC-III/apoA-I genotypes, and protease inhibitor exposure on plasma lipids showed that race/ethnicity was a highly significant predictor of plasma lipids The use of potent antiretroviral therapy (ART) in patients with HIV-1 is associated with a cluster of metabolic complications, including atherogenic dyslipidemia . AnalyseThe pathophysiology of dyslipidemia in ART-treated HIV patients is multi-factorial and involves drug effects on lipid metabolism ,10, insuApoC-III is a 79-amino-acid protein whose plasma levels are directly correlated with TGs in the general population . AlthougDespite a well-described relationship of race/ethnicity with lipoproteins in the general population –33, therWe describe results of analyses from an ongoing project designed to identify candidate genes that place ART-treated HIV-1-infected individuals at higher risk of developing dyslipidemia. As a primary hypothesis, we aimed to determine whether race/ethnicity is a significant predictor of plasma lipids and ART-associated lipid abnormalities in HIV-1-infected individuals and whether apoC-III/apoA-I gene variants interacted with PI exposure to predict increased plasma TGs within race/ethnicity strata.. ACTG 372 was a rollover study for ACTG 320 to compare individuals receiving abacavir or a placebo in combination with zidovudine, lamivudine, and indinavir. Finally, A5116 was a class-sparing and regimen-simplification study for patients with advanced HIV disease. A more detailed description of the above mentioned studies can be found at http://www.clinicaltrials.gov. In our analysis, fasting lipids were recorded in all patients in A5087 prior to starting lipid-lowering therapy. Fasting lipids were recorded for patients in A5005 at least 32 wk after start of ART, and for all other studies at entry. Because lipid levels prior to HIV therapy were not consistently available, our study represents an analysis of treatment lipids across these ACTG studies.As part of New Work Concept Sheet (NWCS) 224, we performed a cross-sectional analysis of 626 HIV participants enrolled in selected AIDS Clinical Trial Group (ACTG) studies  who also had consented to collection and storage of their blood for genetic studies (A5128). ACTG studies were approved by local institutional review boards at each institution where participants were recruited and all participants gave written informed consent. These specific ACTG studies were selected because appropriate data, including fasting plasma lipids and DNA samples, were available and all participants were on ART at time of sampling. Study A5005s was the metabolic sub-study of ACTG 384, a six-arm comparison of three- or four-drug ARTs with zidovudine and lamivudine, or stavudine and didanosine, combined with efavirenz (EFV), nelfinavir (NFV), or both, in ART-naïve individuals . ACTG 50n = 440 were on stable PI ART (n = 398 on a non-RTV regimen)  or non-PI ART . Patients with short exposure to PI ART , patients not currently on PIs but who were exposed to a PI within 30 d (n = 2), and patients with unknown durations of exposure (n = 40) were excluded from analysis assessing PI effects.Among study participants, Study participants were evaluated through their respective ACTG protocols. Demographic and clinical information was provided to the NWCS224 team in a blinded, de-linked manner from the Statistical and Data Analysis Center of the Center for Biostatistics and AIDS Research at the Harvard School of Public Health. Race/ethnicity data were self-reported in these ACTG studies with race categorized as Black, Caucasian, American Indian, Alaska Native, Native Hawaiian, other Pacific Islander, or Asian; and ethnicity as Hispanic or non-Hispanic. For the purpose of our analysis, a single three-level race/ethnicity variable was defined, which represented 97.6% of the full sample: White/non-Hispanic (White), Black/non-Hispanic (Black), or Hispanic. Clinical information collected included medical histories, concomitant medications, ART history, CD4 counts, viral loads, height, weight, fasting plasma lipoproteins , and glucose. Fasting plasma lipids were measured according to the specific parent ACTG protocols. Non-HDL-c was calculated by subtracting HDL-c from total cholesterol. For the measured lipid data, >97% was confirmed to have been taken in the fasting state, the other <3% were presumed to be fasting based on study specific criteria. Body mass index was calculated as weight divided by height squared.http://www.genome.ucsc.edu]). SNPs were chosen based on prior knowledge of association with plasma lipoproteins  , Gly34Gly C/T [rs4520], and exon 4 SstI 3238(5)C/G [rs5128]), and one in apoA-I  ,28–30 an54117, −4Analyses were performed using Splus for Windows, Version 6.2 and R, version 2.0. Demographic and clinical characteristics are given overall and by specific ACTG study. Data are reported as medians and IQR for continuous variables, and as proportions for categorical variables. Raw lipoprotein data are presented by race/ethnicity, gender, and drug exposures. Log transformations were employed to normalize lipoprotein data for the purpose of modeling.As a first step, we assessed the main effects of race/ethnicity and PI exposure on plasma lipids, using multiple linear regression modeling. As a primary analysis, models were fitted treating PI exposure as a three-level factor . An analysis of PI exposure as an indicator for any current PI use was also performed. Fully adjusted multivariable models included race/ethnicity, ART exposures , age, gender, study, CD4 count, and use of lipid-lowering therapy. Maximum likelihood coefficient estimates , 95% confidence intervals (CI) for the true effects, and F-test statistics are reported. In addition, separate multivariable models were fitted for each SNP to assess their independent main effects on lipid outcomes. SNPs were coded as binary indicators for the presence of at least one variant allele  Although useful for comparison with published studies, a notable limitation of this first step is that it does not account for potential interaction between race/ethnicity, PI exposure, and genotypes.We performed all subsequent analyses stratified by race/ethnicity in order to address our primary hypothesis of an interaction between genotype and PI exposure on TGs within racial/ethnic stratum. Maximum likelihood estimates and 95% CIs for main effects of PI exposure and interaction effects between PI exposure and apoC-III/apoA-I SNPs were estimated within race/ethnicity stratum, based on fully adjusted multivariable linear regression models. In these models, PI exposure was treated as a two-level indicator for any current exposure because there is no mechanistic data to support differential interaction effects of apoC-III genotypes with distinct PIs. Estimated fold increases in TGs and corresponding 95% CIs on the un-logged scale are again reported and significance is based on a two-sided Wald test at the 0.05 level. Predicted TG levels and 95% CIs, based on these fully adjusted multivariable models, are provided for non-PI and PI groups within racial/ethnic stratum, with and without further stratification by genotype.Due to the high degree of concordance among the observed apoC-III SNPs, an additional composite genotype analysis is presented. As a primary analysis, the three apoC-III SNPs described by Tarr et al.   were conEstimation of apoC-III haplotype frequencies within race/ethnic strata and the assessment of whether these haplotypes modified the effects of PI exposure on TGs, was performed using the R functions haplo.em and haplo.glm in the haplo.stats library as described by Lake et al. . This apThe sample size provides greater than 80% power to detect moderate PI genotype interaction effect sizes of 0.23, 0.36, and 0.35 within Whites, Blacks, and Hispanics, respectively, using a two-sided level = 0.05 test, and within-cell respective assumed standard deviations of 0.7, 0.5, and 0.6 based on observed data (NCSS 2001 PASS 2000.) Notably, our analysis is not able to rule out smaller underlying interaction effects.Demographic and clinical information by study are provided in Summaries of lipid outcomes by race/ethnicity and gender are presented in Race/ethnicity was a highly statistically significant predictor of plasma lipids even after controlling for gender, study, age, CD4 count, use of lipid-lowering therapy, and ART drug exposure . Overall, current exposure to non-RTV- and RTV-containing PI regimens (compared to no current PI exposure), without consideration for genotypes, tended to be associated with higher TGs, and the strength of this association varied across race/ethnicity. Of note, non-RTV–containing PI regimens were associated with increased plasma TGs in Black/non-Hispanics . This trend was also observed in Hispanics , but not in Whites . RTV-containing PI therapy appears to be associated with even greater increases in TGs within all racial/ethnic groups, though the within-strata numbers of participants on RTV are small.As illustrated in Genotype frequencies varied markedly across race/ethnicity . For exap = 0.07), apoC-III-455 (p = 0.08), apoC-III Intron 1 (p = 0.03), and apoA-I XmnI (−2500T/C) (p = 0.06).A significant difference in the effect of PI exposure on TGs across genotypes in Whites and in Blacks was not detectable. However, predicted TG levels across genotypes did differ across the three ethnic groups , suggestn = 96 out of 104 (92.3%) Hispanics were concordant in their genotypes for −482 and −455 ; n = 99 of 104 (95.2%) were concordant for −482 and Intron 1; and n = 92 of 104 (88.5%) were concordant for all three SNPs.The tests of interaction between each SNP and PI exposure are not independent due to the high degree of concordance across genotypes: p-value = 0.04). Among Hispanic participants with no variant alleles at the three apoC-III SNPs indicated (n = 72), PI exposure (n = 57) was associated with 1.89-fold higher plasma TG levels compared to the non-PI–exposed group (n = 15); however, among Hispanic participants with a variant allele at one or more of the three apoC-III SNPs indicated (n = 32), the increase in TGs in PI-exposed participants (n = 23) versus non-PI–exposed subjects (n = 9) was only 1.06-fold  on the association of PIs with TG levels within Hispanics  Waterworth et al. [Linkage and association studies have established a relationship of apoC-III genotypes, and the apoA-I/CIII/AIV/AV gene cluster, with alterations in plasma TGs ,39,44,45h et al.  found ann = 329) [Recent work has demonstrated a potential interaction of lipoprotein genes with PI therapy in promoting lipid abnormalities in HIV-1-infected patients. Fauvel et al. studied 60 consecutive, PI-treated (mostly indinavir) White males with HIV-1, who were also taking two nucleoside reverse transcriptase inhibitors, and found that the rare apoC-III −455C, −482T, or SstI-S2(G) variants were associated with higher TGs and apoB lipoproteins as well as lower HDL-c . Tarr etn = 329) . They foBoth of these studies, predominantly of Whites, concluded that the apoC-III-482 (CT and TT), −455 (TC and CC), and 3238 (SstI) (GC and GG) genotypes may be associated with increased TG levels in HIV-1-infected patients on PIs. We did not detect significant interaction effects of these apoC-III genotypes with PIs on plasma TGs in Whites. Despite this, our findings are broadly consistent with these papers in that TG levels in White and Blacks with these genotypes on PIs in our study tended to be higher than individuals not on PI therapy; however, the magnitude of effects on lipids were more modest. These differences may reflect study design and confounding variables including age, gender, concurrent use and type of non-PI ART, the prevalence of diabetes, and use of lipid-lowering medications. Furthermore, we did not consider apoE or other candidate genes that might further modify the association of PIs with dyslipidemia. Notably, in Tarr et al. the interaction with PI therapy was only significant in those individuals with the combination of apoE and apoC-III variants .As hypothesized, we found evidence of race/ethnic differences in the association of apoC-III genotypes with PI-related dyslipidemia as well as marked race/ethnicity specific differences in SNP and estimated haplotype frequencies. Although our findings of a potential protective effect of the non-wild-type apoC-III variants on PI-induced hypertriglyceridemia in Hispanics is not consistent with studies in White samples, they are not surprising in the context of prior studies that demonstrated complex transcriptional regulation of the apoA-I/CIII/AIV/AV gene cluster , race/etRace/ethnicity is often considered a surrogate for environmental influences on lipids, but recent studies demonstrate that genetic factors also account for important differences in plasma lipids across ethnic groups ,56. For p-values, since type I errors due to multiple testing are possible with subgroup analyses. Due to the highly correlated nature of many of the reported tests, induced by the overlap in genotypes across SNPs, we included an analysis of a single composite genotype variable that is consistent with multi-locus analysis approaches in the literature [The main limitation of our study is that it was cross-sectional, involved a heterogeneous, multi-ethnic sample across ACTG studies, and had limited power to detect small interaction effects within race/ethnic strata. Our goal was to characterize trends in the data, and we caution against placing too much emphasis on the absolute terature ,15,58,59Overall, our findings of race/ethnicity-specific apoC-III-PI interaction on plasma TGs should be interpreted cautiously. Based on an examination of 43 meta-analyses of genetic association studies, Ioannidis et al. concluded that the biological impact of genetic markers on risk for common diseases is usually consistent across racial boundaries . HoweverIn summary, this study provides novel information regarding HIV-1 subgroups that may be at differential risk of developing metabolic and CV complications of ART. Approaches that account for racial/ethnic heterogeneity are mandated in pharamcogenetic studies of such complications given the multi-ethnic distribution of HIV-1 infection, concerns regarding future atherosclerotic CV disease in these populations, and the potential to use alternative strategies ,61 to reTable S1(134 KB DOC).Click here for additional data file.Table S2(37 KB DOC).Click here for additional data file.Table S3(46 KB DOC).Click here for additional data file.The EntrezGene (prior LocusLink) accession numbers for genes/proteins referred to in this paper are apoA-I (335), apoA-IV (337), apoA-V (116519), apoC-III (345), and apoE (348).One of the unfortunate side effects of protease inhibitors—one of the types of drugs used in treating HIV—is that they affect lipids in the body; for example, they cause the level of triglycerides to increase in such a way as to make it more likely that patients will develop diseases associated with abnormal lipids such as heart disease and stroke. As these drugs are taken for very long periods of time, there are concerns that there may be a future epidemic of cardiovascular disease in patients with HIV-1 who have taken such lifelong treatment.The authors wanted to see if they could identify individuals with HIV who are at increased risk of getting the lipid abnormalities caused by these drugs; in particular, they wanted to see if there were differences across different racial/ethnic groups and according to common variation in some lipid genes. Knowing about such differences might help in deciding between the various different drug treatments available.They looked at 626 people who had taken part in previous studies of HIV drugs, and who had agreed to have their blood stored for further studies such as these. They found that race/ethnicity could help predict levels of plasma lipids, and that the effect of taking protease inhibitors also appeared to differ across race/ethnicity groups. Overall, Black patients taking these drugs were less likely to have the lipid profiles that lead to cardiovascular disease, compared to Whites and Hispanics. However, individuals in all racial/ethnic groups had increases in triglyceride levels when they were given these drugs. Furthermore, Hispanics, but not Whites or Blacks, with variation in the apoC-III gene, appeared to be protected from the triglyceride-raising effect of protease inhibitorsGiven that HIV is a disease that affects all racial/ethnic groups, it will be important in the future to study carefully and in large numbers the patients of all racial/ethnic groups in studies of metabolic and cardiovascular complications of drugs used to treat HIV. It may also be necessary to look at the effect of certain genes on these lipids. In the future it may be possible to use this information to decide which patients should receive which drugs.AIDSinfo is a site run by the National Institutes of Health in the US that has many pages of information on HIV. Searching for “lipid” will bring up fact sheets on HIV drugs and the type of lipid changes discussed in this article:http://aidsinfo.nih.govTheBody.com is an HIV/AIDS resource that can be searched for information on the effects of these drugs on lipids. One page to start at is:http://www.thebody.com/treat/cardiac.html


When urine infections are missed in febrile young infants with normal urinalysis, clinicians may worry about the risk – hitherto unverified – of concomitant invasion of blood and cerebrospinal fluid by uropathogens. In this study, we determine the extent of this risk.In a retrospective cohort study of febrile 0–89 day old infants evaluated for sepsis in an urban academic pediatric emergency department (1993–1999), we estimated rates of bacteriuric sepsis (urinary tract infections complicated by sepsis) after stratifying infants by urine leukocyte counts higher, or lower than 10 cells/hpf. We compared the global accuracy of leukocytes in urine, leukocytes in peripheral blood, body temperature, and age for predicting bacteruric sepsis. The global accuracy of each test was estimated by calculating the area under its receiver operating characteristic curve (AUC). Chi-square and Fisher exact tests compared count data. Medians for data not normally distributed were compared by the Kruskal-Wallis test.Two thousand two hundred forty-nine young infants had a normal screening dipstick. None of these developed bacteremia or meningitis despite positive urine culture in 41 (1.8%). Of 1516 additional urine specimens sent for formal urinalysis, 1279 had 0–9 leukocytes/hpf. Urine pathogens were isolated less commonly (6% vs. 76%) and at lower concentrations in infants with few, compared to many urine leukocytes. Urine leukocytes (AUC: 0.94) were the most accurate predictors of bacteruric sepsis. Infants with urinary leukocytes < 10 cells/hpf were significantly less likely  than those with higher leukocyte counts  to have urinary tract infections complicated by bacteremia (N = 11) or bacterial meningitis (N = 1) – relative risk, 0 (CI:0–0.06) . Bands in peripheral blood had modest value for detecting bacteriuric sepsis (AUC: 0.78). Cases of sepsis without concomitant bacteriuria were comparatively rare (0.8%) and equally common in febrile young infants with low and high concentrations of urine leukocytes.In young infants evaluated for fever, leukocytes in urine reflect the likelihood of bacteriuric sepsis. Infants with urinary tract infections missed because of few leukocytes in urine are at relatively low risk of invasive bacterial sepsis by pathogens isolated from urine. Urinary tract infection is by far the most common serious bacterial infection in febrile young infants -8. The dFor this reason, the challenge to clinicians when utilizing screening urinalysis – heightened when relying on this test alone for decision-making  – is toWe conducted a retrospective cohort study of consecutive febrile (temperature in triage ≥ 38°C) infants aged 0 days to 89 days seen in the emergency department at Children's Hospital Boston from January 1993 to June 1999. This 6-year period immediately followed, (1) the publication in 1993 of practice guidelines for fever in young children and infants ) for each urine collection technique ,10. UrinFor young infants growing a pathogen from urine, culture results of blood and cerebrospinal fluid specimens obtained concurrently during the initial encounter were examined for evidence of concomitant infection by the same pathogen. Concomitant  bacteremia and bacterial meningitis were diagnosed if standard culture of blood or cerebrospinal fluid grew the same pathogen found in urine. Cases in which bacteremia or bacterial meningitis were diagnosed in infants with no evidence of urinary tract infection were designated non-bacteriuric sepsis.For our primary analysis, we determined the relative risk (with 95% confidence limits) of concomitant sepsis between bacteriuric infants with low as opposed to high concentrations of leukocytes in urine. Specifically, we compared the frequency of urinary tract infections associated with concomitant bacteremia or bacterial meningitis between infants that had 0–9 leukocytes/hpf versus > 9 leukocytes/hpf on confirmatory urine microscopy. For suitable parts of our analysis, we also combined infants with 0–9 leukocytes in urine with those that had negative dipstick results. We hypothesized that the risk in febrile young infants of urinary tract infections complicated by invasive sepsis would differ significantly depending on whether leukocytes in urine were few or many.The accuracy of urine leukocytes for predicting concomitant bacteremia and bacterial meningitis was determined by estimating the area under receiver operator characteristic curves (AUC) for this test. The AUC is a summative index of true to false positives for a given outcome at ordered values of a diagnostic test. An AUC value of 0.5 indicates no value of a test for diagnosis. A value of 1.0, on the other hand, shows the test is able to identify the target outcome perfectly. We adopted an AUC cut-point of 0.7 as the minimum threshold for designating a test to be useful for diagnosis ,12.In a secondary analysis, we determined the diagnostic accuracy of other variables that have the potential to be predictors of invasive sepsis, concentrating on those that are measured easily. Specifically, we estimated AUC values for body temperature and age (both recorded routinely at triage), as well as for peripheral blood leukocytes (easily obtained from blood specimens collected by finger stick). Based on previous studies, we estimated the AUC value for non-linear (J-shaped) diagnosis models of peripheral blood leukocytes ,14.Finally, among infants with positive urine culture, we gauged whether groups stratified by the leukocyte count in urine differed sufficiently in respect to other markers of infection to permit assignment to separate clinical or prognostic groups. Specifically, we compared the concentration of bacteria in urine culture, the frequency of hematuria (> 5 red blood cells/hpf) and the fraction with positive nitrites in urine at the initial visit among infants with leukocytes in urine greater versus fewer than 10 cells/hpf.Data were analyzed with the Stata statistical package . The chi-square and Fisher exact tests were used to compare count data. Medians of data that were not normally distributed were compared by the Kruskal-Wallis test.For the period of study, urine samples were obtained from 3765 febrile infants aged 0–89 days presenting to Children's Hospital Boston and sent for screening urine dipstick and bacterial culture. Of these, positive urine culture was reported in 307 (8.2%). Urine was sent for formal microscopy after dipstick analysis in 1516 (40%) of 3756 infants. Of these, 258 (17%) met study criteria for positive culture.E. coli (61), Enterococcus spp (7), Enterobacter spp (5), Klebsiella pneumoniae (4) and Citrobacter spp (2).Seventy-nine of 258 pathogens  were isolated from urine specimens with 0–9 leukocytes/hpf at the initial visit. These included: E. coli (169), Klebsiella pneumoniae (3), Pseudomonas aeruginosa (2), Enterococcus spp (2), Staphylococcus aureus (1), Citrobacter spp (1), and Enterobacter spp (1). One hundred seventy of these pathogens were cultured from urine obtained by bladder catheterization, 4 by clean void, and 5 by unknown technique.Organisms isolated from 179 cases with greater than or equal to 10 leukocytes/hpf in urine included E. coli bacteremia, 1 case of S. aureus bacteremia, and 1 case of meningitis caused by E. coli.The rate of positive urine culture increased with rising concentrations of urinary leukocytes in these infants: 6.2% (79/1279) for infants with 0–9 leukocytes/hpf, 68% (88/130) for infants with 10–50 leukocytes/hpf, and 85% (91/107) for those with more than 50 leukocytes/hpf. Twelve pathogens isolated from urine were also cultured from blood or cerebrospinal fluid. These included 10 cases of The risk of concomitant sepsis (urinary tract infections complicated by bacteremia or meningitis) varied significantly between infants with 0–9 and those with greater than 9 leukocytes/hpf in urine at the initial visit. Among 1279 infants with 0–9 leukocytes/hpf, none  had concomitant bacterial infections. In contrast, all 12 cases of invasive sepsis  occurred in 237 infants with urine leukocyte counts above 9 cells/hpf. These differences translated to a relative risk of invasive sepsis of 0 (95%CI: 0–0.06) among cases with low versus high concentrations of leukocytes in urine. All 12 cases of invasive bacteremia and meningitis occurred in infants that had bacterial concentrations in urine that exceeded 100,000 cfu/ml.In 2249 young infants, urine microscopy was not performed because they had negative urine dipstick results. Only 41 (1.8%) of these infants grew a pathogen from culture of urine. None  had concomitant bacteremia or meningitis. When these infants were combined with infants who had 0–9 leukocytes/hpf, none of 3528  had concomitant sepsis – including 0 of 41 bacteriuric infants with negative dipstick (no urine microscopy) and 0 of 258 bacteriuric infants with 0–9 leukocytes/hpf, for a rate of 0 of 299  – translating to a relative risk of sepsis (compared to infants with pyuria) of 0 (95%CI: 0–0.02). The rate of non-bacteriuric sepsis (sepsis without associated bacteriuria) was equally low (0.8%) in both groups.For identifying young infants at risk of concomitant bacteriuric sepsis at the initial encounter, the leukocyte count in urine when compared to other potential predictors was more accurate than age, temperature, peripheral bands and leukocytes in blood . Specifically, the AUC value for urine leukocytes, at 0.94, was higher than that estimated for temperature (0.52), age (0.62), leukocytes in blood (0.64), and peripheral bands (0.78).Finally, among infants subsequently reported to have a positive urine culture, there were additional differences observed – besides the risk of invasive sepsis – between those with low and high urine leukocytes, supporting categorization to separate groups, Table Screening urinalysis is only moderately sensitive and has been reported to miss approximately 20% of urinary tract infections in febrile young infants ,10,15-22Other plausible markers of bona fide urinary tract infections such as the concentration of bacteria in urine also differ significantly between bacteriuric infants with low and high leukocytes in urine, supporting categorization to separate clinical groups. Based on these differences and the relatively low risk of concomitant sepsis observed in the present study, it appears safe to manage, on an outpatient basis, febrile young infants who are well-appearing and have few leukocytes in urine, as recommended in clinical practice guidelines for fever . The risOur findings corroborate those by other investigators who have also reported differences in clinical outcomes between infants and children with urinary tract infections after stratification by the leukocyte count in urine ,22-24. OTo inform initial management decisions, therefore, it seems to us more pragmatic to identify in operational terms, as done in this study, febrile well-appearing infants at risk of bacteriuric sepsis. Risk-stratification for sepsis that is based on the urine leukocyte count undergirds expectant fever management strategies and is supported by studies that indicate not only a low risk to withholding empiric antibiotic treatment, but also the non-trivial prospect of developing acute pyelonephritis within months of antibiotic treatment in young children with few leukocytes in urine ultimately diagnosed with asymptomatic bacteriuria -28. The Our study has a number of limitations. First, being retrospective, it shares the limitations of this study design. Primarily, we cannot prove a pathophysiological link between the urine leukocyte count and the likelihood of invasive bacterial infections. It is also not possible to determine, retrospectively, whether antibiotics were administered prior to urine collection in some infants. Fortunately, at our site, antibiotic pretreatment in this age-group is highly unlikely and so would not alter our conclusions. The enhanced urinalysis technique , because of better test performance characteristics, may support more accurate diagnosis but was not evaluated by us because it is not utilized at our institution . IrrespeFinally, although not specifically evaluated in this study, we expect the short delay reported in a recent study from the first visit to a report of positive urine culture – about 16 hours  – to furIn conclusion, we show that urine leukocytes reflect well the risk of concomitant sepsis in febrile young (0–89 day old) infants with bacteriuria. Reassuringly, we find low odds of invasive sepsis by uropathogens even when urinary tract infections – the most common serious bacterial infections at this age – are missed at the initial visit on account of few leukocytes in urine. The low risk of invasive sepsis in bacteriuric infants with 'normal' screening urinalysis mitigates a major limitation of this test – a modest tendency to miss urinary tract infections – and indicates that fever management strategies reported in the literature that rely on urinalysis for screening are likely to be safe in selected febrile well-appearing young infants managed on an outpatient basis and assured of close follow-up.The author(s) declare that they have no competing interests.BKB and MBH both made substantial contributions to the conception, design, execution and completion of the study. BKB conceived the idea and designed the study in collaboration with MBH. MBH acquired the data and obtained permission from the ethical board of the institution to conduct the study. BKB performed the initial statistical analysis. Both BKB and MBH reviewed the analysis and interpretation of data. BKB drafted the manuscript, and together with MBH revised it critically for important intellectual content. Both BKB and MBH have read and approve the final manuscript.Specifications of non-linear (J-shaped) models of leukocytes (WBC) in peripheral blood for predicting the log odds of invasive sepsis:These models represent the natural log odds of invasive sepsis in febrile young infants as a function of the leukocyte count in peripheral blood. To calculate the natural log odds of this outcome in a particular patient, insert the specific peripheral blood leukocyte count into the equation. Conversion to odds is then obtained by calculating the exponent of the numerical value obtained for the equation of the natural log odds of invasive sepsis.The pre-publication history for this paper can be accessed here:


In case-control studies, elevated levels of interleukins 6 and 8 have been found to be associated with an increased risk of venous thrombosis (VT). Because of the design of these studies, it remained uncertain whether these alterations were a cause or a result of the VT. In order to distinguish between the two, we set out to measure the levels of six inflammatory markers prior to thrombosis in a population-based cohort using a nested case-cohort design.Between August 1995 and June 1997, blood was collected from 66,140 people in the second Norwegian Health Study of Nord-Trøndelag (HUNT2). We identified venous thrombotic events occurring between entry and 1 January 2002. By this date we had registered 506 cases with a first VT; an age- and sex-stratified random sample of 1,464 controls without previous VT was drawn from the original cohort. Levels of interleukins 1β, 6, 8, 10, 12p70, and tumour necrosis factor-α were measured in the baseline sample that was taken 2 d to 75 mo before the event (median 33 mo). Cut-off points for levels were the 80th, 90th, and 95th percentile in the control group. With odds ratios ranging from 0.9 (95% CI: 0.6–1.5) to 1.1 (95% CI: 0.7–1.8), we did not find evidence for a relationship between VT and an altered inflammatory profile.The results from this population sample show that an altered inflammatory profile is more likely to be a result rather than a cause of VT, although short-term effects of transiently elevated levels cannot be ruled out. An altered inflammatory profile appears more likely to be a result than a cause of venous thrombosis. Blood clots (thromboses) are a common medical problem, especially in people who have been immobilized for a variety of reasons, who have other medical or surgical conditions, or who take certain types of drugs such as oral contraceptives. As well as these factors, various genetic changes make it more likely that certain people will develop a thrombosis. The most well known of these genetic changes is in a clotting factor, Factor V. Instead of the normal clotting factor V, some people have a variant known as Factor V Leiden, which makes them more likely to develop a thrombosis.  Researchers are trying to find out if other abnormalities, particularly in levels of substances involved in inflammation, might make the chance of thrombosis more common. Identifying such changes might make it easier to predict who might be at risk of getting a thrombosis—for example, when a patient has to have an operation—and thus give them appropriate preventative measures.Previous studies have shown that the levels of inflammatory substances, known as cytokines, are raised around the time of a thrombosis. However, because of the design of these studies it was not clear whether these alterations were a cause or a result of the thrombosis. These researchers wanted to measure the levels of six cytokines before thrombosis in a large group of people and follow them to see if any particular level of cytokine made it more likely that they would go on to develop a thrombosis.During a three-year period between August 1995 and June 1997, blood was collected from 66,140 people in the second Norwegian Health Study of Nord-Trøndelag (HUNT2). Anyone who had a thrombosis between the start of the study and the beginning of 2002 was identified—506 people in all. Blood samples, taken from these people (cases) between two days and 75 months before the thrombosis happened, were used to measure levels of a number of different cytokines . These levels were then compared with those in samples also taken earlier from 1,464 people who were similar to the cases but had no thrombosis (controls). The authors found no evidence for a relationship between the chance of getting a thrombosis and a change in any of these markers of inflammation.It seems unlikely that any long-term changes in levels of cytokines make any difference as to whether or not people develop a thrombosis. Hence, any changes seen in previous studies are most likely to have been the result of the thrombosis. However, the researchers could not rule out that changes occurred in the hours or days immediately before the thrombosis, although that seems unlikely. In any case, the levels of these cytokines do not seem to be useful as a clinical tool to predict who is at risk of thrombosis.http://dx.doi.org/101371/journal.pmed.0030334.Please access these Web sites via the online version of this summary at deep venous thrombosis and pulmonary embolusMedlinePlus encyclopedia entries on venous thrombosisOmni, a health information service in the UK run by the Resource Discovery Network, has links to pages of information on HUNT studies are described in this Web siteThe  Venous thrombosis (VT) is a common and potentially lethal disorder with an estimated incidence of one to three per 1,000 individuals per year . The occAn important subgroup of risk factors comprises the levels of procoagulant proteins. Several studies have shown that elevated levels of coagulation factors VIII, IX, and XI increase the thrombotic risk –9. GenetThe association between inflammation and VT leads to the question of whether inflammation is the cause or the result of VT. There are arguments for both. In favour of a causative role is the above-mentioned possibility that inflammation may increase procoagulant protein levels and thus increase the prothrombotic state of the blood. In addition, it is well known that inflammation may promote tissue factor expression on white blood cells and endothelial cells, thus providing a trigger that may lead to thrombotic disease –22. OtheRecent case-control studies showed that raised levels of interleukins (IL) 6 and 8 are associated with a 2-fold risk for both first and recurrent thromboembolic events ,16. In tWe tested the hypothesis that a chronic inflammatory state following a proinflammatory stimulus, regardless of origin, could precede future thrombotic events. In a population-based cohort, we tested levels of cytokines  in blood samples obtained at inclusion, and examined the association with the occurrence of subsequent thrombosis.n = 94,194) in Nord-Trøndelag County (in Middle Norway) were invited to participate in a population-based health survey (HUNT2) [Between August 1995 and June 1997, all inhabitants aged 20 y or older ( (HUNT2) . HUNT2 cn = 66,140) at baseline (1995–1997), with a median age of 46 y (range 19–103 y) [The overall participation rate was 71% (9–103 y) .n = 66,140) underwent a physical examination and filled out a questionnaire at inclusion. The questionnaire contained questions about risk factors for cardiovascular disease, lifestyle, quality of life, and medication use. In addition, all participants were invited to donate 7.5 ml of blood, and acceptable serum samples were obtained from 65,291 participants (98.7%) [All consenting participants ( (98.7%) .n = 283). As a consequence, 515 cases were included, out of which 506 had blood samples available in which reliable laboratory tests could be performed.The follow-up for VT was performed as follows: the computerized diagnosis registries of all departments of the only two local hospitals  were checked for all in- and outpatient diagnoses containing ICD-9 and ICD-10 diagnostic codes for VT up until 1 January 2002. We completed case finding by searching positive diagnostic procedure codes for venography, duplex ultrasound, and Doppler ultrasound from the registries of the radiology departments in the two hospitals, and patients from Nord-Trøndelag County discharged from St. Olav University Hospital in the neighbouring county with diagnostic codes of VT. This search led to the identification of 2,136 cases with a diagnostic code of VT. Hospital records were obtained, and two physicians (I. Næss and S. Christiansen) validated each case. Cases were included only if they fulfilled the following criteria: deep venous thrombosis (DVT), an intraluminal filling defect or no venous filling on ascending contrast venography; or, no compressible venous segment or no venous flow in popliteal, femoral, or axillar veins on duplex ultrasound; or, a positive computed tomography scan or a positive autopsy; for pulmonary embolus (PE), a ventilation-perfusion scan with one or more segmental or subsegmental perfusion defects with normal ventilation; or, a contrast defect on pulmonary computed tomography scan; or, a positive autopsy. This way, 1,271 cases with a validated diagnosis of definite and probable VT were identified. These records were linked to the HUNT2 cohort, and 798 (63%) cases were identified within the cohort. We excluded all patients with previous VT, enrolment in the HUNT2 cohort after the event, or eye-vein thrombosis  and stored in a refrigerator at 4 °C. All samples were transported the same day in a cooler to the central laboratory at Levanger Hospital and stored in the HUNT biobank there the next morning at −70 °C. Samples taken on Friday were frozen the following Monday morning after being transported the same Friday evening . We haveSerum levels of IL-1β, IL-6, IL-8, IL-12p70, and TNF-α were measured using a commercial multiplex bead assay that allows accurate and reproducible measuring of multiple cytokines in a single small serum aliquot ,29. To lIn some samples, outlier levels far above the standard curves (up to 4% in cases and 3% in controls) were seen. We believe that these measurements represent artefacts of the procedure, and the results from such samples were excluded prior to the analysis. In 12 controls (0.8%) and nine cases (1.7%), the samples were missing, the volume of serum was too low to perform cytokine assays, or the cytokine measurements failed for technical reasons.We used univariate logistic regression to calculate odds ratios and their confidence intervals. Cut-off points were detectable cytokine levels at the 80th, 90th, or 95th percentile calculated in control participants. We stratified for sex, three age categories , time between blood sampling and the event, and whether the event was idiopathic or secondary. A secondary event was defined as an event that occurred after major surgery or trauma, during marked immobility , ante- or postpartum, during use of oral contraceptive pills, or in people with a history of malignancy. Linear regression was used to evaluate the relation between cytokine levels and time until the events in cases.All analyses were performed with SPSS version 11.0 .Participation was voluntary, and each participant signed a written consent. All surveys were approved by the Norwegian Data Inspectorate and by the Regional Committee for Ethics in Medical Research.General characteristics of the patients and controls are listed in Whether markers were detectable or not was similar for cases and controls. Consequently, the odds ratios calculated based on detectable versus undetectable levels were about 1.0, with narrow confidence limits . This inWe performed a sub-analysis on the samples that were taken within 1 y before the event to see if levels were higher shortly before a thrombosis occurred. They were not; if anything, they tended to be lower. In an analysis of only cases, IL-12 levels, for instance, showed a significant decrease with time closer to the event . IL-1 shUsing the 90th percentile in the control participants as a cut-off point for all markers, again no effect was found when men and women were assessed separately, or when a distinction was made between an idiopathic and a secondary event. We found no effect within the three age categories, either .We performed the current study to test the hypothesis that circulating levels of proinflammatory cytokines or chemokines are associated with a future event of VT. The data negate this hypothesis, as we found no evidence, neither in a primary analysis nor in post-hoc sub-analyses, that levels of inflammation markers were increased in individuals who later developed VT.Due to the prospective design of this case-cohort study, there was considerable variation in the time between the blood sampling and the event (this varied between 75 mo and 2 d). Therefore, we could not fully rule out the possibility of a sudden increase of inflammatory markers in hours or days before a first thrombosis. However, this possibility seems less likely, considering the results of a sub-analysis of cases developing VT in the first year after blood sampling. These patients showed no increase whatsoever in cytokine levels shortly before the event.We found a higher prevalence of detectable cytokines as compared with the prevalence recently found in the control group in the LETS, although the median levels were comparable . Both ouThe LETS found that the risk of VT increased parallel to several cytokines . This was supported by a decreased risk with increasing levels of anti-inflammatory IL-10. In the LETS, the association between cytokine levels and VT was found in samples that were collected after the event. Therefore, a model in which the thrombosis is the cause of subsequent cytokine release, and not the other way around, is the most likely explanation of the results. Such a model is also supported by the fact that C-reactive protein levels were not associated with VT in the LITE prospective study .A potential limitation of the study is that the inflammatory markers were evaluated with a method with lower sensitivity than high-sensitivity ELISAs. For example, a high-sensitivity ELISA of IL-6 may yield detectable levels in more than 90% of the control samples. On the other hand, the claims that inflammatory markers are a risk factor for VT were based on studies of similar size with assays similar to ours.In conclusion, we have investigated the role of cytokines before development of a first VT in the general population, and found no evidence of an association, not even shortly before the event. Therefore, it is unlikely that cytokine levels play an important role in determining the risk of VT.


PREP).Bipolar disorder (BD) is a major psychiatric condition that commonly requires prophylactic and episodic treatment. Lithium (Li) has been used for over 40 years now as an effective prophylactic agent. Response to Li treatment seems to be, at least in part, genetically determined. Although we ignore how Li specifically prevents mood episodes, it has previously been suggested that Li exerts an effect on the phosphoinositide pathway, and more recently, it has been proposed that Li may modulate prolyl endopeptidase , three coding SNPs and one SNP in the 5' UTR were investigated for their frequency in a BD sample of 180 excellent Li responders, 69 Li nonresponders and 126 controls. Genotyping was carried out using the SNaPshot reaction from Applied Biosystems, which is a modified fluorescent single base pair extension procedure.In this study we carried out an association study looking at the Following correction for multiple testing, no significant genotypic, allelic or estimated haplotypic differences were found between responders and nonresponders or between BD patients and controls.PREP is an interesting candidate gene to investigate in genetic studies of BD, but our findings do not support the hypothesis that genetic variation in this gene plays a major role in the etiology of BD or Li response. Lithium has been one of the most widely used and best characterized prophylactic treatments for bipolar disorder (BD). Even though the therapeutic effects of lithium have been well documented for many years, its mechanism of action remains unknown. Several plausible pathways have been hypothesized to be influenced by lithium; the phosphoinositide pathway being one . It has PREP). Prolyl endopeptidase has been shown to be highly active in the brain, especially in the frontal cortex and in the limbic system [PREP is a cytosolic serine protease that has been shown to cleave neuropeptides made up of 30 residues or less, for instance thyrotropin-releasing hormone, arginine vasopressin, and others [PREP gene and showed high levels of IP3. This increase is likely the result of the inability of Dictyostelium to dephosphorylate IP3 into IP2. Thus, these results provided experimental evidence for a link between the lack of PREP expression and increased IP3 levels. In addition, this data adds evidence to the implication of the inositol second-messenger system in BD and lithium's mode of action. These findings are in accordance with a previously reported association between abnormal PREP activity and bipolar disorder/major depression [PREP, further suggesting a possible role of prolyl endopeptidase as a mediator of these drugs' mood stabilizing action through the inositol system.One such gene is prolyl endopeptidase or prolyl oligopeptidase  and 69 were nonresponders (LiNR) to long term lithium treatment. All psychiatric assessments were carried out by experienced clinicians using the SADS-L structured interviews, and the RDC and DSM-IV criteria. Excellent lithium response was established as described previously  and summPREP gene were investigated using the ABI SNaPshot multiplex reaction and the ABI 3100 automated genetic analyzer  from venous blood samples. Nine single nucleotide polymorphisms (SNP), five intronic, three coding and one located in the 5' UTR, spaced out along the 2 test and the computer programs SigmaStat and SPSS. Haplotype and linkage disequilibrium (LD) analyses were carried out using both GENECOUNTING and its support programs [Hardy-Weinberg (HW) analysis was carried out using the Quick Chi program and those SNPs found to be in disequilibrium were not included in further analyses. Allelic and genotypic frequency distributions were compared between cases and controls, responders versus controls, nonresponders versus controls and finally responders versus nonresponders using a χprograms ,18. Our 2 values for the 8 loci investigated; the PREP3 SNP was disregarded because it was in HW disequilibrium. Upon completion of analyses several positive results were found prior to correction for multiple testing, interestingly only when the lithium responder subgroup was included in the analysis. When comparing cases and controls the PREP6 GG genotype was significantly associated with BD (p = 0.026). This genotype was also found to be significantly associated with response when LiR were compared to LiNR (p = 0.040) or to controls (p = 0.003). Furthermore, in the comparison of responders with controls, the G allele was significantly associated with LiR (p = 0.011).Table PREP8 both genotypic and allelic associations were found to be significantly associated with lithium response . PREP9 provided significant genotypic and allelic associations in the following comparisons; LiR vs. controls  and LiR vs. LiNR . However, after correcting for multiple testing (Bonferroni correction with 36 comparisons), these results became nonsignificant. We did not find any association between age of onset or sex with genotypic or allelic presentation (data not shown).For PREP4/PREP7; D' = 0.539, p = 0.0000; PREP8/PREP5; D' = 0.695, p = 0.0000; PREP8/PREP7; D' = 0.372, p = 0.00901; PREP4/PREP6; D' = 0.234, p = 0.03160).Estimated haplotype frequencies demonstrated no significant differences in any of the comparisons. Significant linkage disequilibrium was found for most marker pairings , valproate (VPA) and carbamazepine (CBZ) implicating also PREP [PREP gene [PREP in the phosphoinositide pathway and in the efficacy of these drugs [PREP in the pathophysiology of BD [PREP gene for association between lithium response and BD and found no differences when comparing genotypic or allelic distribution between bipolar patients and controls or between responders and nonresponders to lithium treatment following correction for multiple testing.The phosphoinositide pathway has garnered much interest as a potential system housing genes involved in the etiology of BD. This is mostly in part due to the inhibitory effects of lithium on key enzymes involved in the recycling and the de novo synthesis of free inositol in the cell. A novel association between this pathway and lso PREP . Their clso PREP . This grREP gene . With thse drugs . The subgy of BD -9,19 makPREP protein in patients with and without good response to lithium treatment.As it is always the case with negative association studies, it is possible that our negative results are also a consequence of methodological limitations of this study, such as reduced sample power to detect association with variants accounting for small effect sizes or limited information content of the variants investigated. In this regard, further studies using larger samples and investigating additional loci would be of interest. Moreover, expression studies could also be performed looking at the levels of the PREP gene is also housed in a region of linkage for BD, which garners additional interest to determine its role in the etiology of BD.In summary our results, albeit negative, provide an initial look into the variation of a gene involved in the phospoinositide pathway and that has been shown to play a role in the mode of action of three common BD treatments; Li, VPA and CBZ. The The author(s) declare that they have no competing interests.FM performed the molecular genetic studies, carried out analysis and drafted the manuscript. FM and GT designed the experiment. AS, GT and MA helped in drafting the manuscript. MA and PG carried out psychiatric evaluations and were responsible for sample collection. GR performed the DNA extractions. All authors read and approved the manuscript.The pre-publication history for this paper can be accessed here: