We also examine the diagnostic value of individual symptoms and signs combined with dipstick test results in terms of clinical decision making.Acute urinary tract infections (UTI) are one of the most common bacterial infections among women presenting to primary care. However, there is a lack of consensus regarding the optimal reference standard threshold for diagnosing UTI. The objective of this systematic review is to determine the diagnostic accuracy of symptoms and signs in women presenting with suspected UTI, across three different reference standards (10Searches were performed through PubMed (1966 to April 2010), EMBASE (1973 to April 2010), Cochrane library (1973 to April 2010), Google scholar and reference checking.2 CFU/ml were included. Synthesised data from a high quality systematic review were used regarding dipstick results. Studies were combined using a bivariate random effects model.Studies that assessed the diagnostic accuracy of symptoms and signs of an uncomplicated UTI using a urine culture from a clean-catch or catherised urine specimen as the reference standard, with a reference standard of at least ≥ 102 CFU/ml; 55.4% at ≥ 103 CFU/ml and 44.8% at ≥ 102 CFU/ml ≥ 105 CFU/ml. Six symptoms are identified as useful diagnostic symptoms when a threshold of ≥ 102 CFU/ml is the reference standard. Presence of dysuria (+LR 1.30 95% CI 1.20-1.41), frequency (+LR 1.10 95% CI 1.04-1.16), hematuria (+LR 1.72 95%CI 1.30-2.27), nocturia (+LR 1.30 95% CI 1.08-1.56) and urgency (+LR 1.22 95% CI 1.11-1.34) all increase the probability of UTI. The presence of vaginal discharge (+LR 0.65 95% CI 0.51-0.83) decreases the probability of UTI. Presence of hematuria has the highest diagnostic utility, raising the post-test probability of UTI to 75.8% at ≥ 102 CFU/ml and 67.4% at ≥ 103 CFU/ml. Probability of UTI increases to 93.3% and 90.1% at ≥ 102 CFU/ml and ≥ 103 CFU/ml respectively when presence of hematuria is combined with a positive dipstick result for nitrites. Subgroup analysis shows improved diagnostic accuracy using lower reference standards ≥ 102 CFU/ml and ≥ 103 CFU/ml.Sixteen studies incorporating 3,711 patients are included. The weighted prior probability of UTI varies across diagnostic threshold, 65.1% at ≥ 10Individual symptoms and signs have a modest ability to raise the pretest-risk of UTI. Diagnostic accuracy improves considerably when combined with dipstick tests particularly tests for nitrites. Acute uncomplicated urinary tract infections (UTI) are one of the most common bacterial infections among women presenting to primary care, with an annual incidence of 7% for all ages of women peaking at 15-24 years and women older than 65 [5 colony-forming units (CFU) of a single uropathogen, in a clean catch or catherised urine specimen [2 [3 [The original reference standard for diagnosing UTI was the presence of significant bacteriuria, defined as the isolation of at least 10specimen . Howevercimen [2 -7 and 10en [2 [3 -11.The pre-test probability of asymptomatic bacteriuria in women of reproductive age is approximately 5% ,13. HoweAlthough empirical treatment of UTI is most cost-effective ,16, pres2, 103 and 105 CFU/ml. In addition, we aim to determine the diagnostic accuracy of symptoms and signs combined with dipstick test results.A previous systematic review established the diagnostic accuracy of symptoms and signs for UTI , howeverThe PRISMA guidelines for reporting on systematic reviews and meta-analysis were followed to conduct this review , Embase (1973 to April 2010) and the Cochrane Library (1973 to April 2010). A combination of MeSH terms and text words were used including: 'urinary tract infection/pyelonephritis/cystitis/urethritis', 'physical examination/medical history taking/professional competence', 'sensitivity and specificity', ' reproducibility of results/diagnostic tests, routine/decision support techniques/bayes theorem/predictive value of tests'. All combinations were restricted to 'women and female'. This search was supplemented by checking references of filtered papers and searching Google Scholar . No restTo be eligible for inclusion, the studies had to fulfil the following criteria:1) Have a study population of adult symptomatic women with suspected uncomplicated UTI presenting to a primary care setting.2) Use a cohort or cross-sectional study design. Case control studies were excluded.2 CFU/ml.3) Investigate the diagnostic accuracy of symptoms and signs of UTI using a urine culture from a clean-catch or catherised urine specimen as the reference test, with a diagnostic threshold of at least ≥ 104) Include sufficient data to allow for the calculation of sensitivity, specificity, negative and positive predictive values and the prevalence of uncomplicated UTI.The number of true positives, false positives, true negatives and false negatives for each sign and symptom were extracted from each of the studies and a 2 × 2 table was constructed. Discrepancies were resolved by discussion between the two reviewers (LG and GC). Authors were contacted to provide further information when there was insufficient detail in an article to construct a 2 × 2 table.The methodological quality of the selected studies was evaluated independently by two reviewers (LG and GC) using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS) tool, a validated tool for the quality assessment of diagnostic accuracy studies . This to2 CFU/ml, ≥ 103 CFU/ml and ≥ 105 CFU/ml. However, pooled estimates cannot be calculated using the bivariate model with less than 4 studies.We used the bivariate random effects model to estimate summary estimates of sensitivity and specificity and their corresponding 95% confidence intervals. This approach was used as it preserves the two-dimensional nature of the original data and takes into account both study size and heterogeneity beyond chance between studies . In addiWe plotted the individual and summary estimates of sensitivity and specificity for each symptom and sign at the different threshold levels in a receiver operating characteristic graph, plotting a symptom's sensitivity (true positive) on the y axis against 1-specificity  on the x axis. We also plotted the 95% confidence region and 95% prediction region around the pooled estimates to illustrate the precision with which the pooled values were estimated  and to illustrate the amount of between study variation (prediction ellipse). We assessed heterogeneity visually using the summary ROC plots and statistically by using the variance of logit transformed sensitivity and specificity, with smaller values indicating less heterogeneity among studies.2 CFU/ml, ≥ 103 CFU/ml and ≥ 105 CFU/ml. Using Bayes theorem the post-test odds of a UTI were estimated by multiplying the pretest odds by the likelihood ratio, where pre-test odds is calculated by dividing the pre-test probability by (1-pre-test probability) and the post-test probability equals post-test odds divided by (1 + post-test odds) [To examine the influence of threshold effects when considering alternative reference standards we conducted subgroup analysis across the three different thresholds: ≥ 10st odds) . Finallyst odds) .We used Stata version 10.1, particularly the metandi commands, for all statistical analysesTwo researchers  screened all potential articles and agreed that the full text of 51 articles should be examined. Nineteen relevant studies were identified by our search strategy -10,24-362 CFU/ml. The mean weighted prior probability using a reference test of ≥ 103 CFU/ml and ≥ 105 CFU/ml is 55.4% and 44.8% respectively. Summary characteristics of each included study are presented in Table The sixteen studies included 3,711 patients and were carried out in a primary care setting. One study was based in the USA , two in The summary diagram of the quality assessment is shown in Figure 2 CFU/ml, as their 95% confidence interval values do not cross the line of no effect. Presence of dysuria, frequency, hematuria, nocturia and urgency are found to increase the probability of UTI. Presence of vaginal discharge decreases the probability of UTI. Presence of hematuria in urine has the highest diagnostic utility (LR+ 1.72), with a specificity of 0.85 and a sensitivity of 0.25, thus hematuria when present is more useful in 'ruling in' UTI. In contrast, all other significant symptoms are identified as being more useful in 'ruling out'. A similar pattern of results emerge using a higher reference standard threshold ≥ 103 CFU/ml. Consistent with lower threshold effects dysuria, frequency and urgency remain significant symptoms for ruling out a urinary tract infection at ≥ 105 CFU/ml.Sixteen studies examined the accuracy of ten different symptoms and signs of UTI. The pooled sensitivities, specificities and the respective variance of the logit-transformed sensitivity and specificity, for individual symptoms and signs at each of the three reference standard threshold levels are presented in Tables The individual and summary estimates of sensitivity and specificity, the 95% confidence region and 95% prediction region for each symptom and sign at each of the threshold levels are presented in a receiver operating characteristic graph in figures 2 CFU/ml and to 67.4% (95% CI 60.6 - 73.6) using ≥ 103 CFU/ml. Presence of vaginal discharge decreases the pre-test probability from 65.1% to 54.1% (95% CI 48.3 - 59.9). The probability of a UTI increases to 93.3% (≥ 102 CFU/ml) and 90.1% (≥ 103 CFU/ml) when the presence of hematuria is combined with a positive dipstick test for nitrites  where the prevalence of symptoms may differ and increase the chance of spectrum bias.2 CFU/ml, ≥ 103 CFU/ml and ≥ 105 CFU/ml. Lastly, this study highlights the additional importance of using dipstick test, particularly tests for nitrites, as an additional diagnostic tool when ruling in a UTI diagnosis based on particular symptomatology.The systematic search, the conservative inclusion criteria, the inclusion of additional data from authors, and the quality assessment of the included studies can be seen as strengths of this study. In addition, given the lack of consensus regarding reference standard thresholds for UTI, the current study is the first study to determine the diagnostic accuracy of symptoms and signs across the three thresholds ≥ 105 CFU/ml. Finally, while the probability of UTI increases when the presence of certain symptoms are combined with positive dipstick test results, it is important to acknowledge that the predictive value of the dipstick test result, was taken from a meta-analysis which included men and pregnant women [We acknowledge that this review has limitations. Variability of diagnostic accuracy estimates across studies is high. This may be due to the fact that we did not restrict the age of women included in the meta-analysis. It is known that the prevalence of UTI differs across age groups, peaking at 15-24 years and greater than 65 years . In addint women .2 CFU/ml and of ≥ 103 CFU/ml. In addition, combining nitrite dipstick test results with clinical symptoms and signs is useful for ruling in a UTI diagnosis and deciding on the optimal patient management strategy. More recently, formal clinical prediction rules for UTI that incorporate the independent effects of symptoms and signs into a "risk score" have been proposed as an alternative management strategy when considering antibiotic treatment [Individual symptoms and signs will modestly increase the post-test probability and cannot accurately 'rule in' or 'rule out' the diagnosis of a UTI. Subgroup analysis shows improved diagnostic accuracy using lower reference standards of ≥ 10reatment . This apreatment .The current approach of evaluating symptoms and signs as a diagnostic test is in general two-dimensional, and ignores symptom severity ,9,28 In Individual symptoms and signs, independent of reference standard threshold have a modest ability to 'rule in' or 'rule out' the diagnosis of UTI. Use of a dipstick test enhances diagnostic utility and reduces the chance of prescribing unnecessary antibiotics. Future studies should focus on the refinement of diagnosis utilising information on severity and duration of symptoms, alone, in combination and alongside dipstick testing.The authors declare that they have no competing interests.All authors were involved in the study conception and design. LG performed a systematic search of the literature. Both LG and GC screened potential articles, evaluated the methodological quality of studies, acquired data for analysis, performed statistical analysis and interpretation of data and drafted the paper. TF, FVL and BDD critically revised the draft manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2296/11/78/prepubPRISMA checklist. Guidelines for the reporting on systematic reviews and meta-analysesClick here for file


BRCA2 and, at lower frequency, of BRCA1 are implicated in a relatively small proportion of MBC cases. Common polymorphic variants in BRCA1 and BRCA2 genes may represent breast cancer (BC) susceptibility alleles and could be associated with a modestly increased risk of MBC at population level. Considering the relevant role of BRCA2 in MBC, we investigated whether the BRCA2 N372H variant, representing the only common non-synonymous polymorphism in BRCA2, might modulate the risk of BC in male populations.Male breast cancer (MBC) is a rare disease and little is known about its aetiology. Germ-line mutations of BRCA1 and BRCA2 mutations, with 261 male population controls, all residing in Tuscany, Central Italy. All MBC cases and controls were genotyped for the BRCA2 N372H allele by TaqMan allelic discrimination assays. To evaluate the genotype specific risk of the BRCA2 N372H variant, MBC carriers of germ-line BRCA1/2 mutations were excluded from the analyses.A case-control study was performed comparing a population-based series of 99 MBC cases, characterized for No association emerged in univariate and age-adjusted analyses. Age-specific analyses suggested an increased risk for the HH homozygous genotype in subjects younger than 60 years. A statistically significant interaction emerged between this genotype and age (p = 0.032). When analyses were restricted to MBC cases enrolled in the first 4 years following diagnosis, a recessive model showed a significantly increased risk of MBC in HH subjects younger than 60 years .BRCA2 HH homozygous genotype might be positively associated with an increased risk of MBC in men younger than 60 years.Overall, our findings, although based on a relatively small series, suggest that the  Male breast cancer (MBC) is a rare disease and little is known about its aetiology compared with female breast cancer (FBC). MBC represents less than 1% of all cancers in men and its incidence is increasing in younger men .BRCA1 (OMIM #113705) and, to a larger extent, BRCA2 (OMIM # 600185), are implicated in MBC. Both genes are estimated to be responsible respectively for up to 16% and 76% of the MBCs in high-risk breast/ovarian cancer families [BRCA1/BRCA2 mutations are sharply different in ethnically diverse population- and clinic-based series ranging from 0% to 4% for BRCA1 and from 4% to 40% for BRCA2 [BRCA1/BRCA2 genes.Similar to FBC, a positive family history (FH) of breast cancer (BC) is associated with increased risk of MBC and approximately 15% to 20% of male patients with BC have a positive FH . The two OMIM #11705 and, families . The fre2 OMIM # 00185, arBRCA1/BRCA2 genes may represent BC susceptibility alleles and could be associated with a modest risk of MBC that would explain, on population basis, a large proportion of the disease.Low-penetrance polymorphisms in BC susceptibility genes are present in a high percentage of individuals and might account for BC risk at population level. In this respect, common polymorphic variants in BRCA2 gene that results in an amino acid change and has a rare allele frequency greater than 10%. Little is known on the functional effect of this polymorphism, although the substitution of asparagine  by histidine (a basic amino acid) may be expected to affect BRCA2 structure and function as it falls in a region that has been shown to interact with the histone acetyltransferase P/CAF prior to transcriptional activation of other genes [The N372H rs144848) polymorphism is the unique variant in 48 polymoBRCA2 N372Hpolymorphism on the risk of breast cancer in female populations with inconsistent findings. The HH homozygous genotype was reported to be associated with a 1.3- to 1.5-fold increased risk of breast and ovarian cancer [BRCA2 N372H polymorphism on BC risk has not yet been investigated in male populations.Several studies have analysed the effect of the n cancer -11. In cn cancer -15. A ren cancer . To our BRCA2 in MBC, we have performed a population-based case-control study to investigate the role of BRCA2 N372H variant on BC risk in men, comparing a series of MBC, characterized for BRCA1 and BRCA2 mutation status, with a control group from the same area of Central Italy .Considering the relevance of In a previous study , we had Overall, after exclusion of deceased and migrated patients, 85 additional unrelated MBC were traced and invited to participate into the study. Eleven cases refused to participate, mostly because of advanced age or severe illness. Confirming our previous response rate of about 80%, 74 of these 85 MBC cases (87.1%) agreed to participate. According to the original study protocol, each MBC patient provided: 1) informed consent; 2) blood samples; 3) detailed information on his personal and family history of cancer at any site, including all first- and second-degree relatives of both genders; 4) a detailed smoking and working history up to the date of BC diagnosis. Information has been validated by available sources . Procedures to maintain confidentiality for all the information collected have been developed and strictly applied. The study was approved by the local Ethical Research Committee .In the frame of a multi-site epidemiological project, two series of healthy adults of both sexes have been randomly selected from the municipality lists of two areas in Tuscany: the city of Florence and one rural area ,19. All BRCA1 and BRCA2 coding sequences, including intron-exon boundaries, were analysed by using single strand conformation polymorphisms (SSCP) combined with protein truncation test (PTT) and automatic sequencing analysis [BRCA1: Genbank, U14680; reference sequence for BRCA2: Genbank, NM_000059).Buffy coat aliquots from MBC cases and controls were anonymously shipped to the research laboratory  where genomic DNA was extracted using QIAamp DNA Mini Kit . The entire analysis ,20. MutaBRCA2 N372H (rs144848) polymorphism was analysed by TaqMan allelic discrimination using Real-Time PCR. Fluorescent hybridisation probes, labelled with 6-carboxyfluorescin (FAM) to detect the 1342C (372H) allele and with Texas Red to detect the 1342A (372N) allele were specifically designed. TaqMan assays were performed in a reaction volume of 50 μl, comprising 100 ng of DNA, 400 nM each probe, 400 nM each primer and 1 × Universal Master Mix (Biorad). The following conditions were used for amplification: 3 minutes at 95°C and 55 cycles at 95°C for 10" and 63°C for 45". The Real-Time PCR was accomplished on iCycler thermal cycler (Biorad) with 96 × 0.2 ml samples. The fluorescence was visualised through iCycler Optical System (Biorad) during Real-Time PCR and analysed by using the iCycler IQ Real-Time Detection System (Biorad) 3.0 software. About 15% of the genotyping results were confirmed by sequencing analysis. The genotype controls for the three possible genotypes plus "no template" controls were always included in each analysis.The 2 test with one degree of freedom comparing expected genotype frequencies (based on observed qs) to observed genotype frequencies. The association between MBC risk and BRCA2 N372H was measured by the odds ratio (OR) and its corresponding 95% confidence interval and was estimated using unconditional logistic regression after adjustment for age considered as dichotomous variable . The analysis were performed with four logistic regression models based on a co-dominant, dominant, recessive and multiplicative codominant effect (inheritance model). In the dominant model, both the heterozygous variant and rare homozygous variant were combined in a dummy variable. In the recessive model, the variant was defined in a dummy variable as only the rare homozygous genotype. In the co-dominant model both rare homozygous and heterozygous variant effects were estimated using two dummy variables while in the multiplicative codominant model a dose-response effect were tested on the variable counting the number of copies of the H allele. Analyses have also been carried out according to stratification by age at diagnosis for cases and age at interview for controls (below/above 60 years) and restricted to MBC cases with a time interval after diagnosis shorter than the median value of the series (4 years). Interactions between age, modelled as dichotomous variable , and genotype under co-dominant, dominant, recessive and multiplicative codominant models, were assessed using Likelihood Ratio test (LR) comparing logistic regression models with and without interaction term.Allele frequencies have been calculated as the number of alleles divided by the number of chromosomes. Genotype frequencies have been calculated as the number of participants with a particular genotype divided by the number of participants. Tests for Hardy-Weinberg equilibrium among cases and controls have been assessed using Pearson's χBRCA2 N372H polymorphism on MBC risk, we carried out a population-based case-control study based on a total of 99 MBC cases and 261 adult male controls from the same area of Central Italy . The mean age at interview was 67.9 (SD 11.8) in cases and 55.9 (SD 6.9) in controls (p < 0.0001). The mean age at diagnosis in the MBC series was 63.4  and the time interval between diagnosis and interview ranged from 0 to 26 years (median 4.0). A detailed FH for breast and ovarian cancers diagnosed in first-degree relatives was collected for all MBC cases. Overall, 25.3% (25/99) of MBC patients reported a first degree FH positive for breast/ovarian cancer.In order to evaluate the putative influence of the BRCA1 and BRCA2 mutations. Overall, 8/99 (8.1%) MBC cases resulted to be mutated in BRCA1/BRCA2 genes: 6 cases (6.1%) carried BRCA2 mutations and 2 cases (2.0%) carried BRCA1 mutations. All 99 MBC cases and 261 controls were analysed for the BRCA2 N372H allele and genotype frequencies. Genotype distribution was consistent with Hardy-Weinberg equilibrium (p = 0.529) among our population controls and cases (p = 0.067). In order to evaluate the genotype specific risk of the BRCA2 N372H polymorphism, MBC carriers of germ-line BRCA1/2 mutations (8 cases) were excluded from the analyses.MBC cases were characterized for BRCA2 N372H genotypes was observed between MBC cases and controls (p = 0.46). The age-adjusted analysis of the genotype-specific risks showed that individuals with NH heterozygous and HH homozygous genotypes were not at increased MBC risk. These results were confirmed on dominant, recessive and multiplicative codominant transmission models (data not shown). Separate analyses stratified according to age (below/above 60 years) showed a tendency towards an inverse association between the H allele and MBC risk in the older sub-group (age > 60 years), although far from being statistically significant. On the contrary, an increased MBC risk  was observed in younger subjects (age ≤ 60 years) on the basis of a recessive model .As shown in Table interaction = 0.17 (95%CI = 0.04–0.83). Sensitivity analyses were carried out based on cut-off points for both age at diagnosis and time interval after diagnosis different from those identified a priori. These analyses confirmed the association, when older individuals (up to 65 years of age) or cases with a longer time interval (up to 6 years) were included.We carried out additional analyses taking into account the length of the interval between diagnosis and blood donation among cases. Overall, no association between the H allele and MBC risk was observed, when analyses were restricted to a comparison between the group of 53 MBC cases with an interval shorter than the median value of the whole series ≤4 years) and the series of controls (Table  years anBRCA2 N372H (1342A > C) polymorphism on MBC risk in a population based case-control study. Overall, a positive association between the HH genotype and an increased risk of MBC was suggested in men younger than 60 years; this association was particularly strong (with a five-fold increased risk) when analyses were restricted to 53 MBC cases enrolled in the study with a blood donation not later than 4 years after their diagnosis. To our knowledge, the present study is the first reporting an analysis of the association between BC risk and the BRCA2 N372H variant in men.We evaluated the effect of the BRCA2 N372H variant on BC risk has been investigated by several studies in women from different populations with inconsistent findings that might reflect genetic differences across populations. In particular the HH genotype was associated with a 1.3- to 1.5-fold increased risk of BC in series from Northern Europe [The role of the n Europe , Australn Europe  and in an Europe . This asn Europe , Japan [n Europe  and in an Europe .BRCA2 N372H variant and increased MBC risk in men younger than 60 years. This effect on MBC risk was particularly evident when analyses were restricted to the sub-group of MBC cases with an interval between diagnosis and blood donation ≤ 4 years, in order to avoid a possible selection bias related to the enrolment of MBC cases with a longer survival in the attempt of expanding the series of patients affected with this rare disease. Overall, we had found a statistically significant interaction between N372H genotype and age: the effect on MBC risk of the HH homozygous genotype was significantly modified by age. In this respect, it is noteworthy that a recent pooled analysis with over 15,000 BC cases and 15,000 controls provided some evidence of an interaction of BRCA2 N372H with age, suggesting a possibly increased BC risk in women younger than 40 years of age [Here, we performed a population-based case control study on male subjects all residing in the same area of Central Italy (Tuscany). Our analyses based on a recessive transmission model suggested an association between the s of age .BRCA2 N372H variant on BC risk could be modulated by the interactions with hormonal factors. In males, the hormonal background is not influenced by reproductive factors as in females and sex steroid levels decline with age [BRCA2 N372H variant with idiopathic male infertility, a condition possibly related to increased sensitivity to estrogens, has been recently suggested [BRCA2 N372H variant increased MBC risk in younger men that may show higher steroid hormones levels.It could be speculated that the effect of the with age . Intriguuggested . Here, wBRCA1/2 germ-line mutations [On the other hand, MBC risk could be influenced by interactions between genetic and environmental factors. Recently, we reported a possible modifying effect on MBC risk of an occupational exposure to chemicals, as polycyclic aromatic hydrocarbon, in subjects carrying utations .BRCA2 N372H variant might affect fetal survival in a sex-dependent manner. In fact, an excess of NH heterozygotes in newborn females, compared to newborn males, has been observed and the BRCA2 N372H genotype distribution showed a significant deviation from the Hardy-Weinberg equilibrium in adult female controls with an excess of heterozygotes and a deficit of both homozygotes [It has been also suggested that the ozygotes . In our ozygotes  and for ozygotes ,14.Overall, our findings, although based on a relatively small series, suggest that the HH homozygous genotype might be positively associated with an increased risk of MBC in men younger than 60 years. This effect appears to be modified by age, with a statistically significant interaction. Due to the rarity of MBC, larger collaborative studies are needed to confirm our results in different male populations.The author(s) declare that they have no competing interests.BRCA1 and BRCA2 mutational analysis. FS coordinated statistical analyses. LO participated in study design and supervision of experimental conduct and analysis, interpretation of results, drafting and revision of the manuscript, and approved the final version.DP participated in study design, coordination of field work, analysis and interpretation of data and critical review of the manuscript. MF performed SNP genotyping analyses and drafted the manuscript. GM and CS contributed to study design and statistical analyses. MC, MAC and IZ collected samples, clinical data and performed statistical analyses. RL, CDA, PR performed All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Relative little attention has been devoted until now to the combined effects of gene polymorphisms of the hemostatic pathway as risk factors for Myocardial Infarction (MI), the main thrombotic complication of Coronary Artery Disease (CAD). The aim of this study was to evaluate the combined effect of ten common prothrombotic polymorphisms as a determinant of MI.n = 307; n = 182; respectively). An additive model considering ten common polymorphisms  was tested. The prevalence of MI increased linearly with an increasing number of unfavorable alleles . In a multiple logistic regression model, the number of unfavorable alleles remained significantly associated with MI after adjustment for classical risk factors. As compared to subjects with 3-7 alleles, those with few (≤2) alleles had a decreased MI risk , while those with more (≥8) alleles had an increased MI risk . The number of procoagulant alleles correlated directly  with endogenous thrombin potential.We studied a total of 804 subjects, 489 of whom with angiographically proven severe CAD, with or without MI (The combination of prothrombotic polymorphisms may help to predict MI in patients with advanced CAD. Myocardial Infarction (MI), the leading complication of coronary atherosclerotic disease (CAD), generally occurs in the late stages of disease because of coronary thrombosis superimposed on a ruptured/unstable plaque In this study we evaluated the combined effect of ten common genetic variants, with known modest effects on the hemostatic balance (listed in 2-test).Supplemental Supplemental 2 for linear trend = 10.68; P = 0.001). In a multiple logistic regression model the prothrombotic score remained significantly associated with MI after adjustment for sex, age, degree of CAD, smoke, BMI, LDL- and HDL-cholesterol . Using the median of PS as cut-off, CAD patients with >5 alleles had a significantly increased risk of MI as compared to subjects with ≤5 alleles . Using approximately the 5th and the 95th percentiles of PS distribution , the study population could be classified into in 3 subgroups: a low-risk group with less than 3 unfavourable alleles (n = 26), an intermediate-risk group with 3 to 7 unfavourable alleles (n = 417), and a high-risk group with more than 7 unfavourable alleles (n = 46). The prevalence of MI among these groups increased progressively , while they were similar for the other clinical and laboratory variables (data not shown). Considering the intermediate-risk group as the reference group, carriers of <3 alleles had a lower risk of MI, while carriers of >7 alleles had an increased risk . As shown in sed risk . Comparised risk . The AUCTo get insights on the pathophysiological effect of combined hemostatic alleles, we assessed the characteristics of thrombin generation activity curves as a function of the number of procoagulant alleles . Since this assay pertains only to the coagulation pathway, the three platelet-related polymorphisms were not considered for this analysis. This assay was performed in a subset of 29 CAD patients , selected among those without possible confounders , in order to form three groups matched for age and sex representing the previously defined risk groups . The number of procoagulant alleles was significantly associated with ETP and with Start Tail, but not with Lag Time, Peak or Time to Peak . SimilarEvidence that a hypercoagulable state is associated with increased mortality has been provided by some recent studies P values, we found no consistent association when each polymorphism was considered individually. Indeed, CAD and MI are paradigms of complex disease, in which the effect of individual genes on the risk is anticipated to be weak This study focused on relatively few genetic variants associated with defined biochemical alterations. While some of them (i.e. Factor V Leiden and prothrombin 20210 G>A) are established risk factors for venous thromboembolism, their association with arterial thrombosis is much less convincing in vitro functional studies showed an association between the number of procoagulant alleles and thrombin generation. The latter is known to be a highly variable and complex phenomenon modulated by the interplay of several factors, none of them with predominant influence, many of them under genetic control Recently, a polygenic approach has been demonstrated to be a valid tool to identify subjects at risk for another complex trait such as type 2 diabetes One strength of our study is the clear definition of phenotypes, allowing comparison of patients with angiographically proven, advanced CAD, with or without MI. The CAD population had a substantial burden of traditional risk factors and thus represented a typical patient population seen in clinical practice.Our study has several limitations, including the relatively low number of subjects and polymorphisms and a retrospective case-control design. In this setting the possible confounding of the survivor effect should also be taken into account. The prothrombotic score, calculated as the sum of prothrombotic alleles, is likely an oversimplification, since it standardized the contribution of each gene variant and does not allow distinguishing the possible different transmission models, as well as the different biological weight of the polymorphisms. Nevertheless, for complex traits the presence of additive effects of many genes is considered more likely than interactive effects This study can be viewed as hypothesis-generating, shedding light into the potential usefulness of a polygenic approach in appropriate clinical contexts. Indeed, the predictive power of our additive genetic model was relatively low, yielding an area under the ROC curve (AUC) of 0.58. The AUC is a measure of the discriminatory power of a test, ranging from 0.5 for no discriminatory power to 1 for a perfect test in vitro measurements of thrombin generation. In the specific context of advanced CAD, similar approaches may be useful as surrogate markers of the propensity to form blood clots leading to MI. Further studies on larger samples are needed to confirm this intriguing working-hypothesis, as well as to improve predictive modelling.Our data support the idea that, while individual genetic susceptibility variants are of limited clinical use, the combined information from a number of these variants can permit the identification of groups of people at high and low risk of developing a complex trait such as MI This study was performed within the framework of the Verona Heart Project, a regional survey aimed to search for new risk factors for CAD and MI in subjects with objective angiographic documentation of their coronary vessels. Details about enrolment criteria have been described in detail elsewhere All participants came from the same geographical area , with a similar socio-economic background. At enrolment, a complete clinical history was collected, including the assessment of cardiovascular risk factors such as obesity, smoking, hypertension and diabetes. The study was approved by the Ethic Committee of our Institution . A written informed consent was obtained from all the participants after a full explanation of the study.Samples of venous blood were drawn from each subject at enrolment, before coronary angiography and after an overnight fast. Serum lipids, as well as other CAD risk factors, including high-sensitivity C-reactive protein (hs-CRP) were determined as previously described Genomic DNA was extracted from whole blood samples by a phenol-chloroform procedure using the Puregene kit (Gentra Systems) according to the manufacturer's protocol. The 10 genetic polymorphisms, selected on the basis of prior evidence of potential functionality in modulating the hemostatic pathway, are listed in This assay was performed in a subset of CAD patients on samples drawn at enrolment, in order to evaluate a possible functional counterpart of an increasing number of prothrombotic alleles in terms of propensity to form blood clot. Plasma sample were centrifuged at 23,000 g at 4°C for 1 hour before testing. Calibrated automated thrombin activity measurement was conducted according to Hemker et al. 2-test or the Fisher exact-test when indicated. Hardy-Weinberg equilibrium was tested for each genotype within each group by means of χ2-test. A value of P<0.05 was considered statistically significant.Calculations were performed mainly with SPSS 13.0 statistical package . Distributions of continuous variables in groups were expressed as means±standard deviation. Logarithmic transformation was performed on skewed variables, for whom geometric mean with 95% confidence interval (CI) are given. Quantitative data were assessed using the Student's t-test or by ANOVA with Tukey's post-hoc comparison of the means. Correlations between quantitative variables were assessed using Pearson's correlation test. Qualitative data were analyzed with the χ2-test, with the values predicted on the basis of the Hardy-Weinberg equilibrium. To assess the extent to which gene polymorphisms were associated with MI, odds ratios with 95% CIs were estimated by univariate logistic regression analysis. Adjustment for other variables  was performed by adding those covariates in a set of multiple logistic-regression models.Within each group examined, the frequencies of the genotypes associated with each of the polymorphisms were compared by the χ2 for linear trend analysis. The prothrombotic score was analysed by logistic regression both as continuous variable and as categorised variable. Odds ratios with 95% CIs were estimated by univariate logistic regression analysis and then by multiple logistic regression with adjustment for number of affected vessels, age, sex, smoking, BMI, LDL- and HDL-cholesterol. The predictivity of our models was then evaluated by the receiver-operating-characteristics (ROC) curve, estmating the area under the curve (AUC).The existence of gene-gene interactions was first explored by a data mining technique similar to the Adaboost algorithm, and based on classification and regression trees (CART): the gradient boosting machine Table S1Genotypes frequencies (%) in CAD-free and in CAD subjects.(0.08 MB DOC)Click here for additional data file.Table S2Characteristics of the CAD population, with or without MI.(0.06 MB DOC)Click here for additional data file.Figure S1ROC for the information provided by our polygenic model of prothrombotic alleles after fitting a logistic regression model.(0.05 MB DOC)Click here for additional data file.


Preeclampsia is a leading cause of maternal and fetal/neonatal mortality and morbidity worldwide. The early identification of patients with an increased risk for preeclampsia is therefore one of the most important goals in obstetrics. The availability of highly sensitive and specific physiologic and biochemical markers would allow not only the detection of patients at risk but also permit a close surveillance, an exact diagnosis, timely intervention (e.g. lung maturation), as well as simplified recruitment for future studies looking at therapeutic medications and additional prospective markers. Today, several markers may offer the potential to be used, most likely in a combinatory analysis, as predictors or diagnostic tools. We present here the current knowledge on the biology of preeclampsia and review several biochemical markers which may be used to monitor preeclampsia in a future, that, we hope, is not to distant from today. Preeclampsia occurs in 2–5% of pregnancies in the Occident, but it complicates up to 10% of pregnancies in the developing countries, where emergency care is often inadequate or lacking. Therefore we are in need of a widely applicable and affordable test that could permit presymptomatic diagnosis in order to identify and monitor the patients at risk and thus provide the best prenatal care for these women and their child. Such a test would also be of benefit to confirm a confounding clinical diagnosis and for future studies investigating prophylactic treatments or temporizing therapies.To be effective a screening test need to be sufficiently sensitive and specific and must provide an adequate postive predictive value . Today, Furthermore, when evaluating new screening strategies, not only sensitivity, specificity and predictive values should be taken into account, but also costs, patient's acceptability and quality control . Thus, tPreeclampsia is a multi-system disorder of pregnancy, which is characterized by new onset hypertension  and proteinuria , that develop after 20 weeks of gestation in previously normotensive women [The disorder has a higher incidence among nulliparous women, in women who conceive with assisted reproduction techniques, and in women affected by autoimmune disorders, reflecting the probable influence of an "inexperienced" or dysregulated maternal immune system in its emergence ,6. On thDespite extensive clinical trials, up to date, no therapeutic approaches are available for either treatment or prevention of preeclampsia. Anti-hypertensive drugs, corticosteroids for lung maturation or magnesium sulfate to prevent from eclampsia (RCOG Guideline No. 10(A)) are given to handle (or prevent the worsening of) the symptoms and can thus temporize over the short term to allow for safe delivery with a more mature fetus. However, the maternal risks must be carefully weighted against the possible fetal benefits in temporizing management, as the risk of fatal deterioration of the maternal and/or fetal health condition is high. Several prophylactic therapies  have so far failed to prove efficacious in the prevention of preeclampsia in healthy, nulliparous subjects, although some benefit has been shown in high risk groups  but also on the health costs associated with this poor medical condition. Since many years, different biophysical and biochemical markers have been investigated, based on pathophysiological observations that have been noted in case of preeclampsia, such as placental dysfunction, a generalized inflammatory response, endothelial dysfunction and activation of the coagulation system.- Miss-diagnosis is still an issue in hospital- or community midwifery care owing to the multiple clinical symptoms associated with the syndrome . The ava- Biochemical markers might allow the stratification of preeclamptic patients in different categories according to symptoms severity and/or pregnancy outcome and thus improve its clinical management .- Very importantly, biomarkers might ensure a reliable early disease assessment in asymptomatic pregnant women, in particular among target groups at increased risk based on their clinical history (preeclampsia or hypertension in a previous pregnancy) or pre-pregnancy state .On account of the current understanding of the etiology of this life-threatening pregnancy disorder, a major focus of research has recently been the identification of placental factors showing abnormal expression in preeclamptic placentas and the assessment of their potential use for non-invasive early prediction or early detection. On the other hand, it appears that maternally-expressed proteins may also serve this purpose. We review below a selected choice of the most promising markers that have been identified up to now Table , with anAngiogenesis requires the complex interplay between the pro-angiogenic factors vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) with their cognate receptors VEGF receptor-1 -1) and VEGFR-2 (for a review on the function of these factors: ). InterePlacental cells also secrete a soluble isoform of flt-1, which is generated through alternative splicing of the messenger RNA and acts as an anti-angiogenic factor by interacting with, and thereby neutralizing, PlGF and VEGF . There iMaternal blood levels of sflt-1 were shown to correlate with the severity of preeclampsia, whereas, in an opposite manner, the quantities of bioactive VEGF and PlGF were further decreased in patients with severe symptoms compared to normal pregnant women or preeclamptic patients with mild symptoms ,33,38. ARemarkably, when introduced into pregnant rats, exogenous sflt-1 triggers hypertension and proteinuria, symptoms akin to those in preeclampsia . BesidesAccording to some studies, the presymptomatic alterations in sflt-1 levels appeared to be specific for preeclampsia as no changes are detected in women who later deliver SGA neonate or whose pregnancies are complicated by IUGR, compared to women with normal pregnancy outcome ,52. HoweIt has recently been reported that patients with preeclampsia have lower plasma concentrations of soluble VEGF-R2 . HoweverEndoglin (Eng) is a co-receptor for transforming growth factor (TGF)-β1 and TGF-β3 that is highly expressed on cellular membranes of the vascular endothelium and on the syncytiotrophoblast ,56. It fSoluble Eng is present in substantial excess in preeclamptic patients compared to normotensive controls, and its concentrations appear to increase with the severity of the symptoms and are the highest in preeclampsia complicated by the HELLP symptom ,59,60. PLike sflt-1, sEng concentrations raise during the last 2 months of normal pregnancy. In pregnancies ending with preeclampsia, this increase occurs earlier and is steeper ,62-65. TSeveral longitudinal case-control studies have therefore evaluated the potential of sEng in combination with the pro- and anti-angiogenic factors PlGF and sflt-1 for the prediction of preeclampsia ,62,63. TP-selectin is a member of the selectin family of cell surface adhesion molecules. It is expressed by platelets and endothelial cells upon activation and plays crucial roles in inflammatory reactions by supporting the recruitment and activation of circulating leucocytes, and in coagulation through the generation of leukocyte-derived "blood-borne" tissue factor ,68. P-sePreeclampsia is associated with extensive platelet activation -72. P-seInterestingly, it has recently been shown that alterations in the levels of soluble P-selectin before 20 weeks of gestation antedate the symptoms -81. ThisSRY (sex determining region Y) and DYS [maspin gene, which is hypomethylated in fetal tissue [RASSF1A [RASFF1A approach in 10 women with preeclampsia and 20 controls has been published [Since its detection in maternal plasma many approaches have been tested to use cell free fetal DNA for non-invasive diagnostic approaches. These include qualitative analyses like fetal sex analysis , determi and DYS , alterna and DYS -98 and bl tissue  or the h[RASSF1A . Althougublished . cffDNA ublished . Currenta disintegrin and metalloprotease 12) is a membrane bound zinc dependant protease and belongs to the ADAM protein family, a group of proteins involved in cell-cell and cell-matrix interactions in fertilization, muscle development and neurogenesis [ADAM12  was first isolated in 1983 by Bohn et al. ,116. It Here again the combination of several diagnostic tools results in improved predictive power as was shown by combined measuring of first trimester serum PP-13 levels and median uterine artery pulsatility index by ultrasound. This combination achieved a detection rate for preeclampsia of 90% with a false positive rate of 6% . HoweverPentraxin 3  belongsPAPP-A  is a disulfide bond linked homodimeric peptidase of 1628 amino acids and a mass of 400 kDa . It can Visfatin (nicotinamide phosphoribosyltransferase (Nampt) enzyme, EC 2.4.2.12) is an adipokine secreted by adipose tissue and involved in the biosynthesis of nicotinamide adenine dinucleotide as it catalyzes the condensation of nicotinamide with 5-phosphoribosyl-1-pyrophosphate to yield nicotinamide mononucleotide ,153. SevAdrenomedullin is a peptide consisting of 52 amino acids that exhibits a long lasting hypotensive effect and was first isolated from pheochromocytoma by Kitamura et al. in 1993 . It is eThis is an old actor whose role in the pathogenesis of preeclampsia is currently being reevaluated. There is long time evidence for a role for the renin-angiotensin system in pregnancy. Exactly a decade ago, it was discovered that patients with preeclampsia develop agonistic autoantibodies against the second extracellular loop of the AT1 receptor . SeveralThe inadequate placental perfusion has lead to the use of Doppler ultrasonography to assess the velocity of the blood flow in the uterine arteries. A persistence of an early diastolic notch after 24 weeks of gestation or abnormal flow velocity ratio's has been associated with an inadequate trophoblast invasion. Pregnancies associated with an abnormal uterine Doppler after 24 weeks of gestation (high pulsatility index and/or presence of an early diastolic notch) are associated with a more than six fold increase in the rate of preeclampsia .Among high-risk patients with a previous preeclampsia, doppler ultrasound of the uterine arteries has an excellent negative predictive value, thus it is an important tool in patient management and care which is of paramount benefit for patients with PE in a previous pregnancy. However effort must be made so that this sophisticated technology becomes available in centers throughout underdeveloped countries.A recently published systematic review assessed the use of Doppler ultrasonography in case of preeclampsia . A totalCurrent data do not support the use of Doppler ultrasonography for routine screening of patients for preeclampsia . HoweverAlthough a panel of promising biomarkers already exists, a lot of effort is made to find novel candidates that bear a greater potential to identify women at risk for preeclampsia, in order to provide the best possible care for these mothers and children. Several approaches for the identification of novel potential biomarkers can be applied. Microarrays offer the possibility to rapidly screen the placental transcriptome for up- and down-regulated transcripts in preeclamptic samples compared to healthy controls hoping that the resulting proteins are excreted and detectable in maternal plasma. Comparative transcription analyses have been performed to some extent by several groups ,176-183,® reagent by Applied Biosystems [A more direct approach is to compare the proteome in the maternal circulation as transcriptome and proteome do not comply with each other. To cope with the complexity of human plasma, high throughput methods need to be employed. The classic approach is comparative 2D-gelelectrophoresis of albumin depleted plasma and subsequent mass spectrometric analysis of the remaining proteins that show different quantities -191. Thiosystems . Up to eosystems -195.A new direction of research in the field of complex diseases is metabolomics, which consists in the global analysis of endogenous and secreted metabolites in a biological system. The first reports investigating the placental metabolome under varying oxygen tensions or in plasma of preeclamptic patients revealed novel redox biomarkers and suggest that this technology might bring new insights into placental function ,197.However, the different -omics profiling techniques are not suited for simple, low-cost, and rapid routine clinical screenings. Their use is primarily for the identification of novel biomarkers within global analyte groups. The next step is then to develop straightforward methods specifically designed for use in a hospital environment.Despite there exists many different potential markers for preeclampsia, the reliability of these markers in predicting preeclampsia has been inconsistent between different studies. Furthermore, preeclampsia is a multifaceted disorder, certain say it is not one but several diseases. Therefore, there is a need for high quality, large scale multi-center trials which enroll patients with different risks of developing the syndrome and throughout multi-ethnical background, in order to assess the predictive value of different markers and finally propose the best marker combination for a routine use in clinical settings.The authors declare that they have no competing interests.SG, CR and OL drafted and wrote the manuscript, RZD, ST, WH and SH: helped to draft the manuscript and gave important inputs. All authors read and approved the final manuscript.


Current 2005 guidelines for advanced cardiac life support strongly recommend immediate defibrillation for out-of-hospital cardiac arrest. However, findings from experimental and clinical studies have indicated a potential advantage of pretreatment with chest compression-only cardiopulmonary resuscitation (CPR) prior to defibrillation in improving outcomes. The aim of this meta-analysis is to evaluate the beneficial effect of chest compression-first versus defibrillation-first on survival in patients with out-of-hospital cardiac arrest.Main outcome measures were survival to hospital discharge (primary endpoint), return of spontaneous circulation (ROSC), neurologic outcome and long-term survival.Randomized, controlled clinical trials that were published between January 1, 1950, and June 19, 2010, were identified by a computerized search using SCOPUS, MEDLINE, BIOS, EMBASE, the Cochrane Central Register of Controlled Trials, International Pharmaceutical Abstracts database, and Web of Science and supplemented by conference proceedings. Random effects models were used to calculate pooled odds ratios (ORs). A subgroup analysis was conducted to explore the effects of response interval greater than 5 min on outcomes.P = 0.979), survival to hospital discharge  or favorable neurologic outcomes . For 1-year survival, however, the OR point estimates favored chest compression first  but the 95% CI crossed 1.0, suggesting insufficient estimate precision. Similarly, for cases with prolonged response times (> 5 min) point estimates pointed toward superiority of chest compression first , but the 95% CI again crossed 1.0.A total of four trials enrolling 1503 subjects were integrated into this analysis. No difference was found between chest compression-first versus defibrillation-first in the rate of return of spontaneous circulation (OR 1.01 [0.82-1.26]; Current evidence does not support the notion that chest compression first prior to defibrillation improves the outcome of patients in out-of-hospital cardiac arrest. It appears that both treatments are equivalent. However, subgroup analyses indicate that chest compression first may be beneficial for cardiac arrests with a prolonged response time. There are an estimated 294,851 emergency medical services (EMS)-assessed out-of-hospital cardiac arrests (OHCA) in the United States each year . DespiteCurrent guidelines of the European Resuscitation Council (ERC) and the American Heart Association (AHA) were last updated in 2005 and emphasize the importance of early defibrillation. The International Liaison Committee on Resuscitation (ILCOR), ERC and AHA clearly prioritize early defibrillation ,8. HowevThere is, however, clinical equipoise whether professional chest compression only promptly followed by defibrillation could increase myocardial "readiness" for defibrillation. Data from the first randomized clinical trials (RCT) have shown conflicting results, but most studies were limited in size and underpowered to allow definite conclusions. A recent large-scale observational study indicated potential benefit for preshock chest compressions .This is the first meta-analysis to systematically review the current research on chest compression first as compared to defibrillation first on outcomes in patients with OHCA.The study was performed according to PRISMA guidelines . For details on search strategy for MEDLINE, see Additional file A search was conducted of SCOPUS, MEDLINE (via PubMed), BIOS, EMBASE, the Cochrane Central Register of Controlled Trials, International Pharmaceutical Abstracts database, and Web of Science from January 1, 1950, to June 19, 2010, supplemented by the conference proceedings of the American Heart Association (2006-2009), the American College of Cardiology (2006-2010), the European Society of Cardiology (2001-2009), the symposium on Transcatheter Cardiovascular Therapeutics (2006-2009), the World Congress of Cardiology (2006-2009) and the European Resuscitation Council Scientific Symposium (2006-2009). We also considered published review articles, editorials, and Internet-based sources of information  independently reviewed the titles and abstracts of all citations to identify potentially relevant studies and to exclude duplicates. The corresponding publications were reviewed in full text by three investigators  to assess whether studies met the following inclusion criteria: 1) randomized treatment assignment to chest compression first versus defibrillation first, 2) human study and 3) included outcome data on one of the four following clinical outcomes: return of spontaneous circulation, survival to hospital discharge, neurological outcome at discharge or survival at 1 year  using the prepared standardized extraction database (MS Excel); data on outcome (see endpoint definition below), total patient numbers per group, and covariables of interest  were extracted. The quality of each trial was assessed using the Jadad scale to ensure sufficient quality but was not implemented in the analysis due to relevant limitations of such approaches ,14. AbsoThe primary endpoint of this analysis was survival to hospital discharge. However, the endpoints are presented in a chronologic order as follows:1. Return of spontaneous circulation (ROSC)2. Survival to hospital discharge3. Favorable neurologic outcome at discharge  score 1 or 2)4. Long-term outcome "Favorable neurological outcome" was defined as a CPC score of 1 or 2 .We regarded a relative change of at least 20-25% as clinically relevant. Power analyses of prospective randomized trials evaluating interventions for OHCA  used variable definitions for "clinically relevant" differences in survival, ranging from 32-550% -19. Ther2I. I2 can be interpreted as the percentage of variability due to heterogeneity between studies rather than sampling error. On the basis of findings in a previous observational study, an a priori subgroup analysis of response time from event to EMS arrival (≤5 min versus >5 min) was also conducted  for the chest compression-first group as compared to 11.4% [7.1-16.6%] for the defibrillation-first group.As summarized for all response times in Figure P = 0.979; heterogeneity: I2 = 74.9%, P = 0.05).The average weighted proportion of patients with favorable neurological status was 13.7% [4.9-25.9%] after chest compression first and 13.3% [9.0-18.3%] after defibrillation first. As seen in Figure P = 0.092; heterogeneity: I2 = 0%, P = 0.647). However, the 95% confidence intervals crossed 1.0, indicating insufficient precision of the effect size estimation and resulting in statistical nonsignificance. The average weighted proportion of patients able to leave the hospital after cardiac arrest with chest compression first it was 11.0% [4.8-19.5%] as compared to 8.6% [4.8-13.4%] for patients treated with defibrillation first.As shown in Figure Figure In Figure P = 0.872; heterogeneity: I2 = 0%, P = 0.73).As shown in Figure P = 0.263; heterogeneity: I2 = 0%, P = 0.954)  Figure . The 95%P = 0.300 . Again, the 95% confidence interval crossed 1.0, and the difference was therefore not statistically significant.As Figure P = 0.705 .No relevant differences were found for patients with a response time >5 minutes in ROSC Figure , the OR P = 0.353; heterogeneity: 59.1%; P = 0.062)  Figure .P = 0.879; heterogeneity: I2 = 84.2%; P = 0.012).As Figure P = 0.033. That is, for every absolute increase of 1 time unit (1 second) in the response time, the log odds ratio increased by 0.0051 (in direction to superiority of a chest compression-first approach). At around 600 seconds (10 min) response time, the regression line crosses OR 1.0 (equipoise between the two interventions). Additional file This analysis showed a significant effect of the mean response interval of each study in the control arm on the effect of predefibrillation chest compression; the point estimates of the OR pointed toward inferiority of predefibrillation chest compression for studies with short mean response intervals but toward superiority for studies with longer mean response intervals  and to avoid RV distension during VF, which helps to reduce the risk of occurrence of "nonperfusing" postdefibrillation rhythms  ,31. Two a priori to include only randomized, controlled trials in our meta-analysis, our results may differ from these large observational studies. Finally, it may be that the treatment effect of chest compression first may be dependent on the response interval from the time of call to EMS response. Further research, with patient-level data, will need to be conducted to assess whether this finding is consistent.Several aspects could explain this controversy. First, findings from experimental animal studies may not apply to humans, especially since most models use electrical induction of ventricular fibrillation, which may not appropriately reflect the majority of cardiac arrests in humans . In a moThe possible difference in treatment effect for longer-lasting  makes plausible sense from a pathophysiological standpoint. Cardiac arrest (due to ventricular tachycardia/fibrillation (VT/VF)) is definitively not a static event. Rather, it is a dynamic process with sometimes continuous transitions starting with VT, transforming into coarse and then into fine amplitude VF and finally into asystole; these different electrocardiogram morphologies are obviously associated with different degrees of defibrillation success . During Niemann et al. demonstrated the superiority chest compression first in a dog model , but fouIn 2002, Weisfeldt et al. proposed a three-phase time-sensitive model for treatment of sudden cardiac arrest: the electrical phase  where predefibrillation chest compressions could be meaningful, and the metabolic phase (> 10 min), where survival rates are poor in general . The autIt has to be considered that nonstratified overall results showed odds ratios very close to 1.0; that is, no treatment effect with fairly narrow confidence (precision) intervals and with very little heterogeneity. In contrast, OR point estimates pointed toward superiority of predefibrillation chest compressions for those cardiac arrests with prolonged EMS response, while in patients with shorter EMS intervals these OR estimates pointed toward superiority of a defibrillation-first approach Figures  and 6. ORCT data are considered the "golden standard" and superior to observational studies. Clearly, the latter are more prone to be biased by confounding, and, accordingly, we considered RCT exclusively in this meta-analysis. Nevertheless, there are caveats for RCT also ; this isA further limitation of this study is the heterogeneity of the study protocols. Three of the four included trials use the 2000 guidelines with a "three-shock protocol" -18,while one study utilized a single shock application (as advocated in the current 2005 guidelines) in the chest compression first group . All fouP = 0.45).Finally, we did not have the complete set of individual patient data, and our analyses are thus based on study-level data. Therefore, we could not adjust the analysis for covariables. For example, the 1-year survival data for the study by Jost et al.  are baseThe results of this meta-analysis demonstrate that survival is equivocal for the chest compression-first group as compared to the defibrillation-first group. Thus, current guidelines emphasizing early defibrillation still appear appropriate. However, the study revealed signals toward possible superiority of predefibrillation chest compressions for patients with a response interval of >5 min; the statistical power of this study was insufficient for such subgroup analyses, and none reached statistical significance. These signals suggest that the optimal treatment of cardiac arrest patients may depend on the duration of the event and the timeliness of the response. Future research will need to be conducted to assess whether this differential effect is seen in patients treated for out-of-hospital cardiac arrest. This may lead to different treatment guidelines based on the duration of the arrest and the interval of the response.AHA: American Heart Association; CPR: Cardiopulmonary resuscitation; ERC: European Resuscitation Council; EMS: Emergency medical services; ILCOR: International Liaison Committee on Resuscitation; OHCA: Out-of-hospital cardiac arrests; OR: Odds ratio; RCT: Randomized clinical trials; ROSC: Return of spontaneous circulation.The authors declare that they have no competing interests to disclose. PM is supported by a postdoctoral fellowship grant from the Swiss National Research Foundation, Switzerland. The funding organizations had no role in the design and conduct of the study; the collection, management, analysis, and interpretation of the data; or the preparation, review, or approval of the manuscript.PM, CS conceptualized and designed this meta-analysis. PM, BH, CS were substantially involved in data acquisition . PM, GK, CS performed the analyses and were substantially involved in data interpretation. PB, DJ, IJ provided data used for the analysis and relevantly contributed to the interpretation and intellectual content of the manuscript. PM and CS drafted the manuscript. All authors revised the manuscript critically for important intellectual content. All authors approved the final version.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/8/52/prepubSupplementary table 1. PRISMA statement checklist.Click here for fileSupplementary table 2. Detailed literature search strategy with search terms used for Medline.Click here for fileSupplementary figure 1. Meta-regression plot. Meta-regression plot of odds ratios (OR) versus response interval (seconds). Size of circles indicate study weights in a mixed-effects model.Click here for fileSupplementary table 6. Predicted odds ratios for variable response intervals.Click here for fileSupplementary tables 3 - 5. Sensitivity analyses with different meta-analytical approaches.Click here for file


Toluene diisocyanate (TDI) is a highly reactive compound used in the production of, e.g., polyurethane foams and paints. TDI is known to cause respiratory symptoms and diseases. Because TDI causes symptoms in only a fraction of exposed workers, genetic factors may play a key role in disease susceptibility.CYP1A1*2A, CYP1A1*2B, GSTM1*O, GSTM3*B, GSTP1 I105V, GSTP1 A114V, GSTT1*O, MPO -463, NAT1*3, *4, *10, *11, *14, *15, NAT2*5, *6, *7, SULT1A1 R213H; immune-related genes: CCL5 -403, HLA-DQB1*05, TNF -308, TNF -863) and symptoms of the eyes, upper and lower airways (based on structured interviews).Workers (N = 132) exposed to TDI and a non-exposed group (N = 114) were analyzed for genotype . TDI-exposed individuals with TNF-308 A were protected against the majority of symptoms, but it did not reach statistical significance. In the non-exposed group, however, TNF -308 A carriers showed higher risk of the majority of symptoms . Individuals with SULT1A1 213H had reduced risk both in the exposed and non-exposed groups. Other polymorphisms, showed associations to certain symptoms: among TDI-exposed,NAT1*10 carriers had a higher risk of eye symptoms and CCL5 -403 AG+AA as well as HLA-DQB1 *05 carriers displayed increased risk of symptoms of the lower airways. GSTM1, GSTM3 and GSTP1 only displayed effects on symptoms of the lower airways in the non-exposed group.For three polymorphisms: Specific gene-TDI interactions for symptoms of the eyes and lower airways appear to exist. The results suggest different mechanisms for TDI- and non-TDI-related symptoms of the eyes and lower airways. Exposure to diisocyanates is well known to cause asthma and symptoms such as cough, wheezing, and dyspnoea . MoreoveBecause diisocyanates cause asthma in only a minor fraction of exposed workers, genetic factors probably modify disease susceptibility . The metNon-enzymatic hydrolysis converts isocyanates to their corresponding amines. There is ample proof that aromatic and heterocyclic amines are activated by oxidation of the primary amine by cytochrome P450 enzymes to aryl hydroxylamines. The most important oxygenase is CYP1A2, but also CYP1A1 and CYP1B1 can activate a range of arylamines to reactive intermediates . AnotherDQB1*0503 allele among subjects with TDI-induced asthma [There are also several immune-related genes of relevance for the susceptibility to respiratory diseases. The chemokine CCL5  is responsible for the recruitment of inflammatory cells, such as eosinophils and T-lymphocytes. The HLA class II molecules are involved in the immune responses as they bind antigen-derived peptides and present them to lymphocytes via the T-cell receptor. Mapp et al. have shown overrepresentation of the d asthma . Tumor nd asthma  and a std asthma .The objective of the present study was to investigate whether the associations of TDI and symptoms of the eyes and airways were modified by relevant functional polymorphisms in metabolizing enzymes and/or in proteins involved in airway inflammation. Thus, a group of workers exposed to isocyanates, mainly TDI, and a non-exposed group, not exposed to any isocyanates, were analyzed for genotype and health effects.The study encompassed eleven plants in the southern part of Sweden. These plants are described in detail in Sennbro et al. . Plants There were in total 184 occupationally TDI-exposed workers employed at the plants and 132 workers participated in the study. Out of a total 198 employees from five different facilities in southern Sweden, a group consisting of 114 workers, not occupationally exposed to isocyanates or performing any plastic or rubber work, was included as well. The pre-requisites to be included in the study were that participants had agreed to take part in the genetic analysis and that they were present at work/at the work shift when the medical examinations took place. The main limiting factor for inclusion in the study has been absence at work shift on the day that the medical examination took place.The characteristics of exposed and non-exposed workers are presented in Table The median age in the exposed group was 36 years (range 18–64), and in the non-exposed group 39 (18–61). The non-exposed group has been described in more detail in Sennbro et al. ; they diThe health examinations are described in detail in Littorin et al. . In the Personal exposure was monitored by air measurements and by analysis of biomarkers of exposure in urine and plasma ,22. The DNA for genotyping was prepared from white blood cells. Due to variations in success rate of the genotype analyses, the number of analyzed individuals differs to some extent between the different polymorphisms. Moreover, all 246 individuals approved analysis of metabolizing genes, whereas 239 individuals approved analysis of immune-related genes.CYP1A1 gene were analyzed by PCR, as previously described [MspI restriction fragment length polymorphism in the 3'end of CYP1A1  is due to a C>T base substitution 264 base pairs downstream the poly(A) signal. The CYP1A1*2B allele (A>G) contains an additional replacement of isoleucine by valine at residue 462 in the heme binding region of the enzyme.Two closely linked single nucleotide polymorphisms (SNPs) in the escribed . The MspGSTM1 and GSTT1 null, and GSTP1 I105V (A>G) polymorphisms was performed as previously described [GSTP1 114 Ala (A) to Val (V) substitution was analyzed by PCR and Dynamic Allele Specific Hybridization (DASH) [GSTM3 was analyzed by PCR/RFLP using the restriction enzyme MnlI according to Inskip et al [Genotyping for the escribed . The GSTn (DASH) , using 5ip et al .NAT1 gene, 559 C>T, 560 G>A, 640 T>G, 1088 T>A and 1095 C>A, were analyzed to determine the alleles *3 (1095 C>A), *4 (wild type), *10 (1088 T>A and 1095 C>A), *11 (640 T>G), *14 (560 G>A) and *15 (559 C>T), essentially as described by Deitz et al [NAT1*10 allele and not carrying the *14 or *15 alleles were considered to have the rapid acetylator phenotype (R). Individuals homozygous for NAT1*3, *4 or *11 alleles were considered to have the intermediate phenotype (IM). The slow acetylator alleles NAT1*14 and *15 were rare in the study population: there were two individuals carrying *10*14 and *10*15 and four individuals carrying *4*14 and all were considered as slow acetylators (S). NAT2 polymorphisms  were analyzed as previously described [NAT2 phenotype was deduced from the genotype and subjects were classified as slow (S = carriers of two mutations), intermediate (IM = carriers of one mutation) or rapid  acetylators.Five different SNPs in the tz et al . Howeverescribed . The NATMPO G-463A polymorphism, a PCR/RLFP method with AciI was used [SULT1A1, Arg213His (R213H) was analyzed by PCR/RLFP using the restriction enzyme Bsp143II to distinguish the G and A alleles according to Engelke et al [For the was used . The SULke et al .CCL5 -403G/A was performed with a Taqman-based assay ), forward (5'-CACTTTATAAATAACATCCTTCCATGGAT) and reverse primers (5'-TCAAAGTTCCTGCTTATTCATTACAGA), MGB-probes , as well as 20 ng of template. The two polymorphisms in TNF (-308 G/A and -863 C/A) were analyzed with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry  at the SWEGENE Resource Center for Profiling Polygenic Disease, Lund University, Malmö. HLA-DQB1*05-genotype was assessed with sequence-specific primers and amplification control primers as previously reported [Analysis of In all types of genetic assays, positive controls for each genotype as well as a negative control (no template) were included. Furthermore, for all assays at least 5% of the samples were reanalyzed and the agreement between these analyses was always 100%.GSTM1, GSTT1, NAT1 and NAT2 were dichotomized according to the inferred phenotype: present vs. null for GSTM1 and GSTT1, slow+intermediate vs rapid for NAT1 or intermediate+rapid vs slow, and slow vs. rapid+intermediate acetylation for NAT2. The main effects of the remaining genes were first assessed separately for each genotype; genotypes were subsequently grouped based on prior biological knowledge of the allele function or the frequency distribution in our material. For analysis of gene-gene interactions, the analysis was limited to genotypes that were significantly associated with symptoms in the single gene analysis. For example for symptoms of the eyes, the combinations of NAT1 (categorized as IM+S/R)+SULT1A1 (GG/AG+AA), NAT1+TNF-308 (GG/AG+AA), and SULT1A1+TNF-308 were evaluated. All gene-gene combination analyses were adjusted for sex, age, atopy and smoking.The Hardy Weinberg equilibrium, which is the principle for the relation between frequencies of alleles and the genotype of a population was here used for indication of genotyping errors and was tested for all genes with the Chi-square test. The difference in characteristics for the study subjects was evaluated with non-parametric tests . The genetic influence on the symptoms was evaluated by comparing different genotypes among exposed and non-exposed separately in logistic regression analysis. In a further analysis, interaction between genotype and exposure status (exposed/non-exposed) was evaluated using logistic regression with an interaction term (genotype)*(exposure). Effects were estimated using odd ratios (ORs) together with 95% confidence intervals (CIs). Statistical significance refers to P < 0.05 (two-tailed) or, equivalently, to 95% CI for an OR that excludes 1.0. Unadjusted as well as adjusted analyses were performed. Based on the general knowledge of effects on symptoms of the airways and the eyes, we adjusted for age, sex, smoking (nonsmoker + former smoker vs. smoker), and atopy. Due to missing information, 112 non-exposed and 128 exposed workers could be included in the adjusted analysis compared to 114 non-exposed and 132 exposed workers in the unadjusted analyses. The genotypes of The data were analysed for patterns of associations between genetic markers and different symptoms and such patterns were presented, although some effect estimates/interaction terms did not reach statistical significance. For genetic markers with no obvious pattern of associations, only those resulting in significant effect estimates/interaction terms were presented.MPO (p = 0.008) and HLADQB1*0501 (p = 0.038) between exposed and non-exposed groups , genotype distributions for 1 p = 0.08 betweenIn Table CYP1A1*2A, CYP1A1*2B, and TNF -308 there was a pattern consistent with interaction between genotype and exposure status for the majority of symptoms investigated  and carriers of the CYP1A1*2B variant, had an increased risk for symptoms as compared to the non-exposed subjects. For TNF-308, the variant A allele was associated with a higher risk of symptoms among the non-exposed group, whereas the opposite was found to be true for exposed subjects. Among exposed subjects, the A variant allele was found to protect particularly against wheeze etc., but also against dry cough and cough with mucus  among exposed workers. There were too few individuals with DQB1*0503 or *0504 for any association analysis.For other polymorphisms, associations were restricted to certain symptoms Table . The rapGSTM3 AB+BB were protected from cough with mucus among non-exposed subjects, with statistical significance for interaction. In the non-exposed group,GSTM1 null as well as GSTP1*105I/V+V/V individuals displayed significantly higher risk of cough with mucus. For GSTP1, evidence of genotype-exposure interaction was found as well. Among exposed subjects, TNF-863 CA+AA carriers had higher risk of cough with mucus than CC . Variant allele carriers of MPO were protected against nose bleed among exposed workers.Individuals with NAT1+SULT1A1 and symptoms of the eyes, carriers of NAT1 IM+S and SULT1A1 AG+AA had an OR = 0.19 95% CI 0.066–0.53; for the combination of TNF-308 and SULT1A1 and wheeze etc., carriers of TNF-308 AG+AA and SULT1A1 AG+AA displayed an OR = 0.27 95% CI 0.081–0.91. There was no strong evidence for interaction apart from the combination of GSTM1 and GSTP1 for cough with mucus among non-exposed workers, where carriers of GSTM1 null and GSTP1*105 Val- displayed OR = 22, 95% CI 2.5–200, compared to GSTM1 present and GSTP1*105 Ile/Ile.The gene-gene combinations analysis resulted in statistically significant associations between gene-gene combinations and symptoms. However, the results did not differ much from the results in the single gene analysis: e.g. for the combination CYP1A1 genotypes appeared to modify the effect of TDI. Similar pattern for the MspI (*2A) variant as the Val (*2B) variant was found, which probably is due to linkage between these two polymorphisms [MspI variant alone on the CYP1A1 activity [Gene-exposure interaction was observed for symptoms of the eyes and lower airways, whereas there were no strong indications of interaction for nose bleed. The orphisms . Howeverorphisms ,33. Theractivity . The rolactivity . HoweverTNF -308 and non-occupational asthma [TNF -308 was associated with reduced risk among the exposed whereas the variant of -863 was associated with increased risk. Thus, our results indicate that genotypes associated with low TNF circulating levels (-308 G and -863 A) may be linked to symptoms in the TDI-exposed. The impact of TNF polymorphism (-308) on TDI-asthma has been analyzed in a recent study by Beghé et al [Elevated levels of TNF are frequently observed in bronchoalveolar fluid of asthmatic subjects . The -30l asthma . Howeverhé et al , but no SULT1A1, the variant allele of R213H (G/A), carries in vitro only 10–25% of the activity of the more common allele [SULT1A1 showed significant effect on symptoms from the eyes and on wheezing, and a similar non-significant pattern was observed for all symptoms analyzed. With regards to eye symptoms, homozygotes for the more common allele G had up to three times higher risk than individuals heterozygous or homozygous for the low-activity A allele. However, the data did not suggest a gene-exposure interaction due to the fact that a protective effect was seen for exposed as well as for referents.In n allele ,42. In oNAT1 and NAT2 activity could hypothetically contribute to the formation of reactive damaging metabolites, by O-acetylation, in the eyes and airways. Hence, the rapid NAT1 and NAT2 genotypes could increase an individual's risk for TDI-associated symptoms. Gene-TDI interaction was suggested for NAT1 only for eye symptoms, where the rapid genotype showed approximately two times higher risk for symptoms among exposed workers. For NAT2 rapid acetylators, an increased risk was suggested for this symptom as well . For the lower airways, an association between NAT1 slow genotype and increased risk for dry cough was found (data not shown), but it was only among non-exposed and there were no symptomatic exposed individuals with slow genotype, making it very difficult to evaluate the effect of this genotype. Thus, in contrast to Wikman and colleagues (14), we did not find any reliable associations with neither NAT1 nor NAT2 with the symptoms of the lower airways. The difference between the studies on isocyanate-exposed workers could be due to differences in selection of study subjects: in contrast to the study by Wikman et al., this is a cross-sectional study and the selection criterion was exposure and not disease. Moreover, we studied predominantly TDI-exposed individuals and did not include study subjects with a mixed diisocyanate exposure. Furthermore, in contrast to Wikman, we did not select asthma cases but rather analyzed symptoms which may be early indicators of asthma-related changes.Higher CCL5 results in a new consensus element for the GATA transcription factor family [CCL5 genotype appeared to have an effect on the risk for TDI-associated airway symptoms such as wheeze etc. and cough with mucus: the variant allele was associated with approximately three times higher risk among exposed, suggesting a CCL5-TDI interaction. An association for CCL5 in TDI-associated airway symptoms is also suggested from a recent study showing increased expression of CCL5 in vitro in bronchial epithelial cells when stimulated with TDI [The -403G/A variant of r family , which swith TDI .HLA-DQB1*05, in particular the *0502 allele, and isocyanate-related symptoms of the lower airways. Compared to the *0501 allele, the *0502 has an exchange of a valine to a serine at amino acid position 57 in the beta chain of HLA class II molecules. Mapp et al. [There was also evidence for association of p et al.  have foup et al. .GSTM1*O subjects. We found only a non-significant higher risk of cough with mucus among the exposed subjects, and the strongest effect was found among non-exposed subjects. The differences in study design as discussed above for NAT1 and NAT2 could also explain differences in results for GSTM1 compared to Piirilä et al., since both Wikman's and Piirilä's reports are based on the same study population. Other studies have reported higher risk of non-occupational asthma for GSTM1*O subjects [GSTM3 in relation to diisocyanate-asthma and found later reactions in the bronchial provocation test among GSTM3*AA individuals. We also found that GSTM3 was associated with airway symptom: there was a protective effect of GSTM3*B on cough with mucus. However, this was only evident among the non-exposed individuals. For GSTP1, the exposed I/V+V/V displayed a tendency towards a protective effect, which is in line with the study by Mapp et al. (13) on TDI-asthma and GSTP1. On the other hand, GSTP1 I/V+V/V subjects had an increased risk of cough with mucus among the non-exposed subjects.Glutathione S-transferases showed some effect on symptoms of the lower airways, in particular on cough with mucus. However, the effect was not observed among the exposed workers, but rather among the non-exposed subjects. One possibility is that these symptoms are caused by different mechanisms among exposed and non-exposed individuals, i.e. GSTs are not involved in TDI-related airway obstruction. Piirilä and co-workers , found asubjects ,46. PiirGSTM1 and GSTP1 for cough with mucus among non-exposed workers. Nevertheless, there may still be significant gene-gene interactions present for the symptoms analysed, however, for detection of such effects a larger study population had been required.Gene-gene interactions are of relevance to study, considering the complex metabolism of isocyanates as well as responses of the immune system. However, since the results differed for most polymorphisms for exposed and non-exposed, we had to analyse the gene-gene interactions in each group. The gene-gene combination analysis did not indicate any evidence for departure from an additive effect. The exception was the results from the combination of The low prevalence of certain risk genotypes are reflected in the large confidence interval for ORs obtained. Thus, the data should at this point be cautiously interpreted and new studies are needed for replication of the results. Another aspect to consider is the issue of multiple interferences. We examined fifteen polymorphic loci, and five symptoms for both exposed and non-exposed workers which might result in a large number of false positives. Nevertheless, no correction for multiple testing has been performed. Adjustments such as the Bonferroni correction are not applicable as they cannot take linkage between polymorphisms into account, which occurs among some genes investigated in our study. We do not stress individual significant findings, but rather believe that consistent association patterns between genotypes and symptoms indicate true findings.By studying TDI-exposed as well as non-exposed individuals, specific gene-TDI interactions were indicated for symptoms of the eyes and lower airways. The finding that certain genotypes modified the effect of symptoms arising in different tissues suggests that these symptoms are associated with each other and have common mechanisms.We did not expect to find individuals diagnosed with asthma among the exposed workers, since asthmatics should not be employed in isocyanate workplaces in Sweden today, and if workers acquire asthmatic symptoms after employment, they should be transferred to non-exposed jobs. Surprisingly, increased risks of severe symptoms of the airways, e.g., wheeze and/or chest tightness, which are associated with asthma (46), were recorded in the present study. Thus, bearing in mind that our study is a cross-sectional one, with potential for a healthy-worker-selection, our findings may be an underestimation of the true risk. Furthermore, gene-exposure interactions appear to be present in early signs of health effects.The authors declare that they have no competing interests.KB carried out part of the molecular genetic analyses, performed part of the statistical analyses and drafted the manuscript. HT carried out the exposure assessments and participated in the design of the study. AA performed part of the statistical analyses. MW and AR carried out part of the molecular analyses and the design of the study. ML conceived of the study, participated in its design and coordination, and performed the health investigations. All authors read and approved the final manuscript.