Borderline tumors of the ovary (BOT) are a distinct entity of ovarian tumors, characterized by lack of stromal invasion. Recent studies postulated that the presence of invasive implants, incomplete staging, fertility sparing surgery and residual tumor after surgery are major prognostic factors for BOT. There are no biomarkers that can predict BOT or the presence of invasive implants.The aim of our study was to assess the value of CA125 and HE4 alone, or within ROMA score for detecting BOT, and for predicting the presence of invasive implants.Retrospective, monocentric study on 167 women diagnosed with BOT or benign ovarian masses. Serum HE4, CA125 levels and ROMA were assessed preoperatively. Due to low number of BOT with invasive implants, we performed an unmatched analysis (consecutive patients) and a matched analysis (according to age and histology) to compare BOT with invasive implants, BOT without invasive implants and benign disease.There were no significant differences in the HE4 and CA125 expressions in the three groups of patients . The ROC analysis showed that CA125 alone is superior to ROMA and HE4 in discriminating patients with BOT with invasive implants from patients with benign diseases and BOT without invasive implants. A newly established score, ROMABOT, did not perform better than ROMA. The analysis of the matched groups revealed similar results as the analysis of all samples.Both HE4 and CA125 are not reliable biomarkers for the diagnosis of BOT or for predicting the presence of invasive implants. Borderline tumors of the ovary (BOT) are an independent entity of ovarian tumors, being characterized by the presence of cellular proliferation and nuclear atypia without any destructive stromal invasion ,2. HowevBOT affect younger women with one third of the patients being under 40 years of age at time of initial diagnosis . In contTo date, the cancer antigen 125 (CA125) is the most commonly used tumor marker in the evaluation and clinical management of an ovarian mass, but since it has a low specificity, especially in premenopausal women ,14, the Until now, there are no clinical or biomarkers to predict the presence of invasive implants in BOT. In this study we analyzed the value of individual CA125, HE4 and the combination of both markers in the ROMA score for detecting BOT, and for predicting the presence of invasive implants.In the current study samples from a total of 167 patients with either benign gynecological diseases or BOT with or BOT without invasive peritoneal implants were collected prospectively, within the Tumor Bank Ovarian Cancer project (TOC).All patients received surgical treatment in our comprehensive center for ovarian cancer treatment at the Department of Gynecology, Virchow Campus Clinic, Charité Medical University of Berlin.Written informed consent was obtained before the collection of serum samples. Ethical approval for this study was provided by the ethics committee at the Charité Medical University of Berlin (EK207/2003).Serum samples were obtained before surgery. After centrifugation and aliquotation into cryovials, samples were frozen at −80°C until further usage.HE4 concentrations in serum were measured using the HE4 EIA assay . Each sample was analyzed in duplicate. The appropriate controls were within the ranges provided by the manufacturer.CA125 was determined in serum during the routine analysis by using Roche Kits.To stratify pre- and postmenopausal patients into either low or high-risk groups for the presence of a malignant pelvic mass, we used the following equations in order to calculate the ROMA score  ,20:Therefore the ROMA Index is mainly determined by HE4 in premenopausal status and by both biomarkers in postmenopausal patients.The clinical data were collected and entered in a SPSS database. All analyses were performed with PASW 21.0 .Furthermore, we identified a cohort of 14 BOT patients with invasive implants that were matched by histology and correspondent age in order to minimize bias with another 14 patients presenting with non-invasive implants. In addition, every BOT patient with invasive implants was matched with a total of 4 age-correspondent patients with benign ovarian masses.Continuous variables were compared with the Kruskal-Wallis test; p-values for pairwise comparisons were presented without adjustment for multiple testing. Receiver operator characteristics (ROC) curve analysis was performed to evaluate the predictive accuracy of HE4 and CA125 expression for discriminating patients with borderline or benign tumors from patients with BOT and invasive peritoneal implants.Adjusted hazard ratios (HR) and 95% CI for prognostic factors were calculated. A two-tailed p-value < 0.05 was considered as statistically significant.We enrolled 167 patients with BOT and benign diseases. A total of 104 patients were diagnosed with benign masses, including ovarian fibrothecoma and uterine leiomyomata. Forty-eight patients presented with BOT without invasive implants, and 15 patients with invasive implants. The menopausal status of all patients was assessed stratifying them into either being premenopausal (N = 104) or postmenopausal (N = 61), respectively. Two patients were excluded since the menopausal status was unclear. Patient characteristics are presented in Table Median HE4 expression in serum of premenopausal patients was 49 pM (range: 20-1410 pM) in benign diseases, 37 pM (range: 35–94.6 pM) and 45.1 pM (range: 29-2135 pM) in BOT patients with and without invasive implants, respectively.Preoperative median CA125 serum concentrations in premenopausal patients were 21 U/ml (range: 7-251 U/ml) in benign diseases, 32 U/ml (range: 19-232 U/ml) and 24.5 U/ml (range: 3-725 U/ml) in BOT with and without invasive implants, respectively.Among premenopausal patients there was no significant differences in the HE4 (p = 0.984) and CA125 (p = 0.141) expressions in the three groups of patients.The AUC for HE4 and CA125 were 0.515 (95% CI. 0.212-0.818) and 0.710 (95% CI 0.512-0.908), respectively.Furthermore, we evaluated the role of ROMA in predicting BOT and presence of invasive implants. The AUC for ROMA algorithm was 0.521 (95% CI 0.223-0.820).The ROC analysis showed that in premenopausal patients, CA125 alone is superior to HE4 and ROMA in discriminating patients with BOT with invasive implants from patients with benign diseases and BOT without invasive implants , with 50 pM (range: 27-241 pM) in benign diseases, 76.55 pM (range: 40-461 pM) and 68 pM (range: 41-334 pM) in BOT patients with and without invasive implants, respectively. HE4 concentrations in benign diseases differed significantly from those concentrations found in BOT with (p = 0.007) and without invasive implants (p = 0.025), respectively. In contrast, there was no significant difference (p = 0.418) in the HE4 concentrations between the two BOT subgroups.In postmenopausal patients CA125 values were significantly different in the three groups of patients (p = 0.003). Median CA125 expression in serum was 18 U/ml (range: 5-334 U/ml) in benign diseases, 76.5 U/ml (range: 12–259 U/ml) and 31.5 U/ml (range: 9-134 U/ml) in BOT patients with and without invasive implants, respectively. CA125 levels in BOT patients with invasive implants were higher compared to BOT without invasive implants, but no statistical significance has been reached (p = 0.159).The ROC analysis revealed that CA125 alone is slightly superior to HE4 and similar to ROMA in discriminating postmenopausal patients with BOT with invasive implants from patients with benign diseases and BOT without invasive implants. AUC for CA125, HE4 and ROMA were 0.778 , 0.732  and 0.782 , respectively  with 48 years (range: 19–86 years) in benign diseases, 55 years (range: 25–87 years) and 44 years (range: 16–83 years) in BOT with and without invasive implants, respectively. Biomarkers usually can be expressed differently according to age. Therefore, and due to the low number of patients with invasive implants, we decided to match the BOT patients with invasive implants  according to age and histology. Every patient was matched with one BOT patient without invasive implants and with 4 other patients with benign diseases. All analyses presented above were repeated within this setting.Likewise the analysis of patients of all three groups, the matching process revealed significant results for HE4 concentrations for BOT with (p = 0.009) and without (p = 0.020) invasive implants compared to benign diseases. No significant difference (p = 0.822) between BOT with and without invasive implants was found. The CA125 concentration for BOT without invasive implants compared to benign diseases showed no significant difference (p = 0.087). CA125 concentration of BOT with invasive implants differed significantly (p = 0.001) compared to benign diseases. Similar to the median HE4 concentrations, there was no significant difference (p = 0.201) in CA125 concentrations between BOT with and without invasive implants.The ROC curve analyses showed that CA125 is superior to HE4 and ROMA in discriminating patients with BOT with invasive implants from patients with benign diseases and BOT without invasive implants, even within this setting. AUC for CA125, HE4 and ROMA were 0.788 , 0.660  and 0.744 , respectively.In postmenopausal patients, CA125 performed slightly better than ROMA and both performed better than HE4 alone in discriminating borderline tumors from benign conditions, as well as to predict the presence of invasive implants. AUC for CA125, HE4 and ROMA were 0.762 , 0.723  and 0.728 , respectively.The aim of our study was to assess whether preoperatively measured serum concentrations of HE4 and CA125 alone or within ROMA score can detect the presence of borderline tumors of the ovary (BOT) or predict the presence of invasive implants. HE4 was detected using EIA(e) technology. Previous study showed no significant differences between performance of the HE4 kits and methods being used .Current study showed that CA125 is superior in both pre- and postmenopausal patients to ROMA and both of them are superior to HE4 alone in discriminating borderline tumors from benign diseases. The introduction of ROMABOT as a new score for the prediction of invasive implants didn’t increase the performance of the test. Both CA125 and HE4 couldn’t predict the presence of invasive implants. To the best of our knowledge, this study is the first one investigating the role of HE4 and CA125 in predicting BOT with invasive and non-invasive implants.The limitation of this study is the low number of BOT specimens, especially the BOT with invasive implants. The presence of invasive implants is a rare condition of the BOT. In the multicentric study of Du Bois et al. , invasivThe lack of early cancer biomarkers is currently a major obstacle for blood-based early detection, within ovarian cancer patients . The quaCurrents studies suggested that some of the BOT lesions might be possible precancerous condition that might evolve to low grade serous ovarian cancer -26. HE4 Shih et al. analyzed the impact of histological and clinical features in the process of relapse in 266 patients with BOT. In this study 143 (53.8%) presented with elevated serum CA125 values (>35 IU/L). Patients with normal baseline CA125 levels had a significant longer 3-year PFS compared to patients with elevated CA125 levels at time of diagnosis . The samMost authors conclude that CA125 concentrations in BOT patients are often negative or at least not significantly increased ,31. Du BIn the recent publication of Kaijser et al., the authors analysed the value of serum HE4 or ROMA score as second-stage tests for tumors thought to be difficult to characterize on the basis of ultrasound findings . From 36In our study HE4 and CA125 were significantly differently expressed in benign diseases vs. BOT and BOT with invasive implants only in postmenopausal patients. Nevertheless no biomarker could differentiate between the two subgroups of BOT.Large population studies showed that both CA125 and HE4 vary with the age at diagnosis. Urban et al., showed that HE4 increases slowly with the age and particularly in women aged > =55 years, where increases in about 27.1% per decade were reported . TherefoOur study revealed that HE4 and ROMA are not better in predicting the presence of invasive implants in BOT than CA125 alone. The lack of sensitivity and specificity of HE4 and CA125 biomarkers in predicting the presence of BOT, underline once more that these tumors are a specific entity of ovarian tumors, which pathogenicity is not yet understood.Due to small number of patients presenting with invasive implants, the long duration of follow-up needed for accurate evaluation and the lack of literature multicentric, international studies having BOT as main focus are needed. Serial HE4 and CA125 serum values similar to UKCTOCS trial  togetherThe authors declare no competing interests.EIB participated in study design, HE4 analysis, patients’ inclusion and article writing. TvanG carried out CA125 analysis and participated to the article reviewing. RR carried out the statistics and reviewed the article. MN documented the clinical data and drafted the manuscript. RC participated at article reviewing. KG participated at documentation of clinical data and article editing. Prof. DT participated at article reviewing and design. Prof. IV participated at article reviewing. Prof. JS was involved in study design and article reviewing. All authors read and approved the final manuscript.


The aim of study was to assess the relationship between zinc-α2-glycoprotein (ZAG) and androgen excess with insulin resistance in polycystic ovary syndrome (PCOS) women. 99 PCOS women and 100 healthy controls were recruited. Euglycemic-hyperinsulinemic clamp (EHC) was preformed to assess their insulin sensitivity. Circulating ZAG was determined with an ELISA kit. In healthy subjects, circulating ZAG levels exhibited a characteristic diurnal rhythm in humans, with a major nocturnal rise occurring between midnight and early morning. Circulating ZAG and M-value were much lower in PCOS women than in the controls. In all population, overweight/obese subjects had significantly lower circulating ZAG levels than lean individuals. Multiple linear regression analysis revealed that only M-value and the area under the curve for glucose were independently related factors to circulating ZAG in PCOS women. Multivariate logistic regression analysis showed that circulating ZAG was significantly associated with PCOS even after controlling for anthropometric variables, blood pressure, lipid profile and hormone levels. The PCOS women with high ZAG had fewer MetS, IGT and polycystic ovaries as compared with the low ZAG PCOS women. Taken together, circulating ZAG levels are reduced in women with PCOS and ZAG may be a cytokine associated with insulin resistance in PCOS women. Polycystic ovary syndrome (PCOS) is characterized by elevated circulating androgen levels, chronic anovulation, and polycystic ovaries2346810et al. reported that silencing ZAG resulted in reduced adiponectin (ADI), insulin receptor substrate-1(IRS-1) and glucose transporters-4 (GLUT4) gene expression in primary human adipocytes indicating that ZAG plays an important role in modulating whole-body and adipose tissue insulin sensitivityZinc-alpha-2-glycoprotein (ZAG) is a 41-kDa glycoprotein assigned to the Major Histocompatibility Complex (MHC) class I family of proteinsglucose), the area under the curve for insulin (AUCinsulin), HOMA-IR, prolactin (PRL), luteinizing hormone (LH), total testosterone (TEST), and free androgen index (FAI) were higher, whereas M-value and sex hormone binding globulin (SHBG) were lower in the PCOS women than in the controls , and their blood samples were collected from 0800 on day 1 to 0800 on day 2 to determine whether circulating ZAG is regulated by circadian-related factors. The result showed that a significant increase in circulating ZAG was observed at midnight in both male and female subjects. ZAG started to rise at 20:00 hr, peaked at 24:00 hr, and then decreased to nadir at 8:00 hr . In addition, fasting ADI levels were also significantly lower in women with PCOS than in the controls  had significantly lower circulating ZAG levels than lean individuals (BMI < 25 kg/m2) . In addition, overweight/obese PCOS women also had significantly lower circulating ADI levels than that of lean PCOS women  and YZAG-Normal = 18.5 + 4.02 X M-value −0.71 X AUCglucose + 0.02 X DHEA-S . Multivariate logistic regression analysis showed that circulating ZAG levels were significantly associated with PCOS even after controlling for anthropometric variables, blood pressure, lipid profile and hormone levels . Importantly, the PCOS subgroup with high ZAG had fewer MetS, IGT and polycystic ovaries as compared with the low ZAG subgroup  in the 92 healthy women with normal weight. High M-vlue was defined by M-value ≥6.28subgroup . PCOS wosubgroup .To investigate the predictive value of ZAG for risk stratification of dysmetabolism in PCOS, MetS, IGT and IR , we analyzed the receiver operator characteristic (ROC) curves of circulating ZAG. The ROC curve analyses revealed that the cutoff value for circulating ZAG to predict PCOS was 42.6 mg/L  that women with PCOS or overweight/obese are more IR than control subjects and have different circulating ZAG levels. In PCOS or overweight/obese women, circulating ZAG levels were significantly decreased. Furthermore, in PCOS subjects, ZAG levels correlated with other IR markers, such as HOMA-IR, M-value, and obesity related parameters, such as BMI, WHR and FAT%. These changes were similar to that of ADI, a known insulin sensitizer, in an insulin-resistant state, thus further suggesting ZAG is associated with IR and obesity.The EHC is considered as the ‘gold standard’ measure of insulin sensitivity. EHC is also the method with the fewest drawbacks and is closest to the real measure of insulin sensitivityIn subgroup analysis, we further found that PCOS women with high ZAG had lower HOMA-IR and higher M-value. These women had also fewer MetS and IGT as compared with PCOS women with low ZAG. Therefore, these data further suggest that ZAG may be a useful marker of IR, MetS and glucose dysmetabolism.In vitro, insulin may enhance LH-dependent ovarian androgen production, especially in theca cells from PCOS women22In vivo, sustained hyperinsulinemia amplifies both GnRH- agonist induced ovarian steroidogenesis and ACTH-stimulated adrenal steroidogenesis2728Some data support a bidirectional relationship between hyperandrogenism and IR. To determine the predictive value of ZAG for IR, PCOS, IGT and MetS, we analyzed the ROC curves of circulating ZAG and these metabolic phenotypes. The ROC curve analyses indicate that the ZAG might be a useful marker for the diagnosis of PCOS and for the prediction of MetS, IGT, and IR in PCOS women. We also determined the optimal cutoff value of the ZAG for identifying individuals with IR, IGT, MetS or PCOS in the Chinese population. However, the low sensitivity and specificity for MetS and IGT in ROC analysis probably indicate the heterogeneity of MetS and IGT in women with PCOS. In fact, PCOS is characterized by heterogeneity in phenotypic manifestations mainly related to reproductive and hormonal aberrations and the presence of metabolic disturbances, including IGT and MetS30In addition, we analyzed the association of circulating ZAG with the other components involved in PCOS diagnosis, such as hyperandrogenemia, anovulation and polycystic ovarian morphology by ROC curves. We found that ZAG was not a predictor for these components. Therefore, we believe that the association of ZAG with PCOS is due to the high prevalence of IR in these women. Therefore, the main strengths of this study are 1) it’s prospective design with inclusion of newly diagnosed PCOS women prevents pharmacotherapy and other confounding variables such as age and sex; 2) the association between ZAG and IR is investigated by EHC, a gold standard for evaluating insulin sensitivity; 3) importantly, the predictive values of ZAG for IR, PCOS, IGT and MetS are evaluated.Our study has also some limitations. First, our cross-sectional design limits any firm conclusion about the possible causative role of ZAG in IR and PCOS. This would require longitudinal intervention studies and warrants future investigation. Secondly, our sample constituted entirely of Chinese women. Therefore extrapolation of these results to other ethnic groups should be undertaken with caution. In addition, the study was also limited by a relatively small sample, although the number of subjects included would provide more than 90% power to demonstrate associations at the conventional α < 0.05 level. Nonetheless, this study is sufficient to demonstrate novel associations of circulating ZAG with hormonal, metabolic parameters, and IR in PCOS women.Overall, our findings demonstrate that decreased ZAG levels are a feature of IR and MetS, and hyperandrogenism may contribute to IR in PCOS women. We also found that the main predictor of circulating ZAG concentrations was M-value, whereas the main predictors of FAI levels were Mets, LH/FSH ratio, and HOMA-IR. However, further longitudinal and interventional studies are needed to clarify the clinical and pathophysiological significance of a decreased ZAG level in women with PCOS.2; 2) diabetes was confirmed or FBG ≥6.1 mmol/L and/or 2h- postprandial glucose (2h- BG) ≥7.8 mmol/L (IGT); 3) high blood pressure was confirmed or SBP/DBP ≥140/90 mmHg ; 4) triglyceride ≥1.7 mmol/L and/or high-density lipoprotein cholesterol (HDL-C) <0.9 mmol/L in male or HDL-C <1.0 mmol/L in female2, known cardiovascular disease, thyroid disease, neoplasms, smoking, diabetes, and renal impairment (serum creatinine 120 μmol/L). Oral contraceptives or other drugs involving in carbohydrate metabolism, if administered, were discontinued for at least 3 months before the study. All subjects gave their written informed consent before entering the study, which was conducted in accordance with the Declaration of Helsinki and approved by the ethical committee of the Second Affiliated Hospital of Chongqing Medical University.The PCOS group comprised 99 women who were referred to the Department of Endocrinology and Gynecology of the Second Affiliated Hospital of Chongqing Medical University from January 2013 to January 2014 due to menstrual irregularities. The enrolled women with PCOS were in good health and not suffering from chronic or acute diseases. The diagnosis of PCOS was based on the 2003 Rotterdam consensus (The Rotterdam ESHRE/ASRM-sponsored PCOS consensus workshop group) with at least two of the following featuresAt 0800 h on the study days, after an 8–10 h overnight fast, an OGTT was performed on all subjects. These subjects ingested 75 g glucose, and venous blood was drawn at 0, 30, 60, and 120 min for the measurement of glucose and insulin.EHC was performed on 99 women with PCOS and 100 healthy women as previously describedIn another separate sub-study, the healthy subjects (5 women and 5 men) aged 18–35 were admitted to the metabolic ward at 0600 and were provided with a standardized meal. Blood samples were drawn from an indwelling venous catheter in the forearm at 0800, 1000, 1200, 1600, 2000, 2400, and 0400 over a period of 24 h to investigate the daily ZAG levels. Blood was immediately centrifuged, and the plasma was separated and stored at −80 °C for measurement of ZAG.2 were measured with well-established electrochemi-luminescence immunoassay using COBAS E immunoassay analyzers (Roche Diagnostics GmbH). Total testosterone levels were measured by coated tube RIA . DHEA-S and SHBG were performed using an automated analyzer . FAI was calculated as FAI = (testosterone/SHBG) ×100.Blood samples were collected in the early-follicular phase  in the control group. Blood samples were collected after a spontaneous bleeding episode or upon first examination in PCOS women. Plasma glucose and HbA1c were measured by the glucose oxidase method and anion exchange high-performance liquid chromatography, respectively. Insulin was measured by radioimmunoassay using human insulin as standard . Free fatty acid (FFA) was measured with a commercial kit . TC, HDL-C, LDL-C, and triglyceride were analyzed enzymatically using an autoanalyzer . Serum hormonal concentrations including LH, FSH, testosterone and Prog, PRL and Evs. the ZAG concentrations of the calibrators, and concentrations of plasma samples were determined by using this calibration curve. The limit of detection was 1.95 ng/mL, and intra-assay and inter-assay variations were <10% and <12%, respectively. The assay has high sensitivity and excellent specificity for detection of human ZAG with no significant cross-reactivity or interference. Human ZAG ELISA was performed in duplicate. If duplicates had 10% CV, the sample was repeated. Circulating ADI level was also measured by ELISA as we previously describedCirculating ZAG levels were determined with an ELISA kit obtained from Ray Biotech  following the manufacturer’s protocol with slight modification. Briefly, 100 μl human plasma was collected from subjects who had fasted overnight using EDTA-containing tubes, and samples were applied to the test wells along with ZAG standards of concentration 7.8–500 μg/ml. Then, 100 μl of specific biotin- conjugated anti-human ZAG was added to each well and incubated at 37 °C for 1 h. Each well was then washed three times with PBS containing 0.05% Tween-20 (pH 7.2–7.4). Colorimetric reaction was performed for 20 min with the use of horseradish peroxidase– conjugated streptavidin  as substrate. Optical densities were measured at 450 nm. A calibration curve was constructed by plotting the absorbance values at 450 nm glucose and AUCInsulin during the OGTT were calculated geometrically using the trapezoidal rule.BMI was calculated as weight divided by height squared. The percentage of body fat (FAT %) was measured by bioelectrical impedance . The HOMA-IR = FIns (mU/L) × FBG (mmol)/22.5. HOMA-β = 20 × FIns (mU/L)/FBG (mmol) −3.5t test was used in comparisons between two groups. Pearson correlation analysis was used to evaluate the relationship of ZAG with IR and other covariates. Multiple linear regression analysis using a stepwise method  for the introduction of independent variables was used to identify the main determinants of ZAG levels and FAI among the variables showing a statistically significant correlation in univariate analysis. Receiver operating characteristics (ROC) curves of ZAG levels were constructed to determine the optimal cutoff point for the prediction of PCOS, MetS, FAI and IR . P values < 0.05 (two-tailed) were considered statistically significant.The stratified Mantel Haenszel row mean score test was used to assess whether there was a change in the prevalence rate of PCOS and ordered categories of ZAG level was row mean scores differ on average across all subjects. Trend tests of PCOS incidences across the three categories of ZAG levels were conducted by Cochran-Armitage tend tests. Row mean score test and Cochran-Armitage trend tests were conducted using SAS software version 9.30 , while the other statistical analyses were conducted using SPSS software version 19.0 . Results are expressed as mean ± SD or median (interquartile range) unless stated otherwise. Variables with a non-normal distribution were transformed by logarithm or square-root before analysis. Independent sample How to cite this article: Lai, Y. et al. Circulating Zinc-α2-Glycoprotein levels and Insulin Resistance in Polycystic Ovary Syndrome. Sci. Rep.6, 25934; doi: 10.1038/srep25934 (2016).


Healthcare workers (HCWs) in low-incidence settings are often serially tested for latent TB infection (LTBI) with the QuantiFERON-TB Gold In-Tube (QFT) assay, which exhibits frequent conversions and reversions. The clinical impact of such variability on serial testing remains unknown. We used a microsimulation Markov model that accounts for major sources of variability to project diagnostic outcomes in a simulated North American HCW cohort. Serial testing using a single QFT with the recommended conversion cutoff (IFN-g > 0.35 IU/mL) resulted in 24.6%  of the entire population testing false-positive over ten years. Raising the cutoff to >1.0 IU/mL or confirming initial positive results with a (presumed independent) second test reduced this false-positive percentage to 2.3% (95%UR: 2.0–2.6%) or 4.1% (95%UR: 3.7–4.5%), but also reduced the proportion of true incident infections detected within the first year of infection from 76.5% (95%UR: 66.3–84.6%) to 54.8% (95%UR: 44.6–64.5%) or 61.5% (95%UR: 51.6–70.9%), respectively. Serial QFT testing of HCWs in North America may result in tremendous over-diagnosis and over-treatment of LTBI, with nearly thirty false-positives for every true infection diagnosed. Using higher cutoffs for conversion or confirmatory tests  can mitigate these effects, but will also diagnose fewer true infections. As a result, many hospitals in the United States have begun to use IGRAs for serial testing of HCWs; this practice has revealed unexpectedly frequent conversions and reversions among IGRA results89101112Health care workers (HCWs) are at risk for exposure to tuberculosis (TB) and are routinely tested for latent TB infection (LTBI) as part of most infection control programs in North American hospitals12156IGRA results are known to have multiple sources of variability (unrelated to TB infection), which may contribute to the observed variation in test resultsWe used a microsimulation Markov model to project diagnostic outcomes in a simulated HCW cohort under different serial screening strategies. The simulated cohort comprised 10,000 hypothetical HCWs at a representative North American institution undergoing serial LTBI screening using QFT every year over a period of ten years. The model incorporates the major sources of variability in serial QFT results as well as different serial screening strategies, including different cutoffs for a QFT conversion and confirmatory testing with different degrees of correlation (modeled as Pearson correlation coefficients) between initial and confirmatory results .To accomplish the model objectives, the model first assigns each individual HCW to one of three “QFT classes” based on the IFN-gamma response /mL [“class 1”], 0.20–0.99 IU/mL [“class 2”], or ≥1.00 IU/mL [“class 3”]), reflecting the distribution of initial QFT results from a large, multi-center study of HCWs at low risk of TB infection who were tested serially under semi-controlled conditionsIn subsequent years, in addition to the within-subject variability as described above, an annual risk of true TB infection is applied. In the baseline scenario, we assume a high-risk HCW population in a North American healthcare institution in which 0.1% of the cohort acquires LTBI each year16After assigning an “underlying” QFT value to each simulated HCW in each year (to account for within-subject variability and the possibility of true infection)810202223ast) in each year, additional variability in QFT results is added according to The model can thus be represented mathematically as follows. Given an “underlying” QFT value  as described above, k represents the additional sources of variability for which sufficient data were available in the systematic review , and xskt is a value randomly drawn from a beta distribution fit to the measured variability in QFT results among people whose initial QFT value was in class s, and whose repeat tests were known to have been exposed to each item k, using data from the systematic reviewwhere After incorporating these sources of variability and true LTBI infection, measured QFT results are truncated between 0 and 10 IU/mL (corresponding to manufacturer’s guidelines), and a test result (positive or negative) is assigned at each year, according to different cutoffs for a positive result and different testing strategies . Based on the test result and true LTBI status  at each year, individuals in the population cycle between four Markov states: true negative, false negative, true positive, and false positive . All elith and 97.5th percentile of results across all simulations.We evaluated outcomes using the manufacturer’s recommended conversion cutoff for a positive test result (≥0.35 IU/mL), as well as higher cutoffs at regular intervals . In addition, we modeled the correlation between initial and confirmatory tests in separate analyses as a Pearson correlation coefficient of 0.0 (presumed independent tests), 0.5, and 0.9 (best reflection of available data)In the base case scenario , 0.9% (95%UR: 0.7–1.1%) of the population experienced a true incident TB infection within ten years. Under a testing strategy in which a QFT conversion was defined as an initial value of <0.35 IU/mL followed by a subsequent test ≥0.35 IU/mL–and with no confirmatory testing–24.6% (95%UR: 23.8–25.5%) of the population was projected to test false positive by the end of ten years, versus 0.85% (95% UR: 0.68–1.04%) true positives: a ratio of 29 false positives for every true positive TB infection diagnosed. Under this strategy, 23.5% (95%UR: 15.4 −33.7%) of patients with true incident infections were missed on the first test after infection. Including variability due to TST boosting nearly doubled the number of false positives, while removing variability due to incubation delays and blood volume  did not materially change results (data not shown).We then evaluated different strategies for reducing the false positive: true positive ratio, by instituting a higher cutoff for conversion and/or requiring confirmation of positive tests, under different assumptions about the correlation between the initial and confirmatory test . IncreasThe value of a confirmatory test depended strongly on the modeled correlation between initial and confirmatory test results. If the hypothetical correlation between initial and confirmatory test was high, outcomes were very similar whether or not confirmatory testing was performed.  can substantially reduce the number of false-positive results, but at the consequence of diagnosing fewer people within the first year after true infection. Furthermore, in settings with a low annual risk of TB infection , the positive predictive value of serial QFT testing, even with high cutoffs and independent confirmatory testing, can be 60% or lower. For example, if preventive therapy costs $430 per treatment courseConfirmatory testing has been discussed before30As with any modeling study, we are limited by the accuracy and appropriateness of the model and parameter assumptions employed. While we performed sensitivity analyses around the sources of variability, distributions employed, cutoffs for the underlying QFT categories and other parameters, we cannot exclude the possibility of having not modeled or not modeled accurately a particular unknown source of variability. In the assignment of initial QFT values and the distribution of within-subject variability, our analysis relied heavily on data from the largest study of IGRAs in North American HCWs [8]; however, this study collated QFT measurements from laboratories employing potentially diverse methodologies. Such pooling of data from different settings may increase uncertainty in the data underlying our results. In choosing categories and cutoffs we employed conservative estimates likely to be favorable to QFT; to the extent that we have underestimated the probability of QFT conversions under each source of variability, our results may underestimate the number of false-positive results that could be expected under each serial QFT testing strategy. Furthermore, as the focus of this modeling work was on serial QFT testing, we did not consider serial TST or T-SPOT.TB algorithms; further work comparing serial QFT to serial TST or T-SPOT.TB testing–incorporating year-to-year variability in each test–could add further value.This model demonstrates the potential pitfalls of blindly using cutoffs designed for single tests in the setting of serial testing, particularly in settings of low TB incidence . Simulation can help us understand these consequences in the absence of large prospective cohort studies. In realistic settings of low risk of TB infection, even with high cutoffs and confirmation, the post-test probability of a positive QFT test on serial testing may still only approach 60%, while annual testing with a single QFT using the recommended diagnostic cutoff may cause over 25% of individuals to have a false-positive test over a span of ten years. The value of serial testing of HCWs should therefore be discouraged in the North American setting, where TB incidence is now at an all-time low.How to cite this article: Moses, M. W. et al. Serial testing for latent tuberculosis using QuantiFERON-TB Gold In-Tube: A Markov model. Sci. Rep.6, 30781; doi: 10.1038/srep30781 (2016).


Chromobacterium violaceum 12472 and in S. aureus strain with agr:blaZ fusion over a broad range of subinhibitory concentrations, indicating that the extracts contain high concentration of molecules that can interfere with the QS mechanisms mediated by AHL as well as AIP.Quorum sensing (QS) mechanisms have been demonstrated to have significance in expression of pathogenicity in infectious bacteria. In Gram negative bacteria the autoinducer molecules that mediate QS are acyl homoserine lactones (AHL) and in Gram positive bacteria they are peptides called autoinducing peptides (AIP). A screening of tannin-rich medicinal plants was attempted to identify extracts that could interrupt the QS mechanisms in both Gram positive and Gram negative bacteria over a wide range of concentrations and therefore potentially be potent agents that could act as broad spectrum QS inhibitors. Six out of the twelve Indian medicinal plant extracts that were analyzed exhibited anti-QS activity in N-acylated homoserine lactones (AHLs) whereas in Gram positives they are small peptides called autoinducing peptides (AIPs) [ luxI encodes an autoinducer synthase that catalyzes the formation of the signal molecule, AHL, that on reaching the quorum activates luxR that codes for the receptor of AHL. This complex then binds to the QS regulated promoters and leads to transcription of all genes controlled by QS system [ agr locus in Staphylococcus aureus that codes for its pathogenicity determinants. The agr locus consists of two divergent operons that are activated by promoters P2 and P3, respectively. P2 acts as a promoter for genes that synthesize the autoinducer. P3 codes for RNAIII, which is the regulatory effector of the agr regulon and initiates the transcription of genes that encode a variety of exoproteins responsible for pathogenicity [Unlike the early views on unicellular bacteria that suggested solitary behavior and little interactive capacity within a population, recent developments in ecological sciences suggest that communities, rather than individuals, play a major role in the maintenance of ecological stability , 2. Expls (AIPs) . QS in mS system , 4, 7, 8genicity , 9–11. Vibrio cholerae, Escherichia coli, Bacillus cereus [ Pseudomonas aeruginosa, Staphylococcus aureus [ Acinetobacter baumannii [Because QS controls the virulons in many infectious bacteria likes cereus , 12, Pses aureus , and Aciaumannii , blockinaumannii . Rasmussaumannii  have sug Penicillium spp. and Chlamydomonas reinhardtii [In the recent years, screening for anti-QS substances in eukaryotes like plants, algae, and fungi is gaining significance. As these eukaryotes do not possess active immune systems, they rely on physical and chemical defense mechanisms. It is therefore hypothesized that secondary metabolites produced by them can aid in preventing colonization by pathogens. Such natural quorum sensing interfering compounds are reported in exudates of several vegetables and spices and are also found to be secreted bynhardtii , 14–17.agr activity have also been made [agr activity of tannin-rich plant extracts with the aim of determining spectrum of activity and of suggesting their potencies as anti-QS substances.Considering the enormous number of plant varieties and the chemical diversity that plants inherently possess, screening plants for medicinally significant compounds seems rational. Various medicinal plants have been screened for their anti-QS potential –22, and een made –34. In t Syzygium cumini and Embelia ribes were acquired from the local market. Fruits of Phyllanthus emblica, Terminalia bellirica, and Terminalia chebula; pericarp of Punica granatum; flowers and seed kernel of Mangifera indica; and the barks of Acacia arabica, Terminalia arjuna, Thespesia populnea, and Casuarina equisetifolia were collected from Mumbai and the respective plant samples were authenticated at Agharkar Institute, Pune, and The Blatter Herbarium, St. Xavier's College, Mumbai. The plant parts were washed, dried, and powdered. Methanolic extracts were prepared by soaking the powders (1 gm) in methanol (10 mL) overnight. These extracts were used for phytochemical screening. For the quantitative biological assays the dried methanolic extracts were dissolved in 7.8% DMSO to known concentrations and stored at −25°C [Powders of seeds ofat −25°C .Extracts were qualitatively analyzed for presence of phytochemicals, namely, saponins, alkaloids, flavonoids, di- and triterpenes, phenols, and tannins using methods suggested by Roopashree et al. (2008) . The aciChromobacterium violaceum ATCC 12472, wild type strain that produces a purple pigment in response to QS (C6 AHL) molecules that it produces;Chromobacterium violaceum ATCC 31532, nonpigmenting strain that overproduces C6-AHL, the autoinducer;Chromobacterium violaceum 026, a mini Tn5 mutant of Chromobacterium violaceum 31532, which is unable to produce the autoinducer C6-AHL but responds to an exogenous supply of C6-AHL by producing the purple pigment [ pigment .All three strains were maintained on Luria agar.Three biomonitor strains were used for anti-AHL activity: Staphylococcus aureus agrP3::blaZ RN6390 pRN8826 that contains agrP3-blaZ fusion plasmid and produces β-lactamase spontaneously due to agr expression was used for detection of anti-QS activity that targeted AIP mediated QS. This strain was maintained on Luria agar with 10 μg/mL chloramphenicol.Biomonitor strainμL in 96-well microtitre plates. Wells were inoculated with 5 μL of a bacterial suspension (108 CFU/mL) of the C. violaceum 12472/S. aureus agrP3::blaZ RN6390. All experiments were performed in triplicate and plates were incubated at 37°C for 24 h. After incubation 50 μL of 0.2 mg/mL alcoholic solution of INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium) (Hi media) was added and plates were incubated at 37°C for 30 min. Reduction of INT to its formazan product was a clear indication of growth/no growth. Change in color to pink was noted as growth. Concentrations lower than the cidal concentration were then used as SICs.MICs of the extracts were determined essentially to decide subinhibitory concentrations (SICs) that were used for quantitative anti-QS activity in Gram positive and Gram negative biosensor strains. MIC was determined using the microdilution method given by Valgas et al. (2007) . 8 mg/mL C. violaceum ATCC 12472. Concentrations of the extracts that were selected were below the MIC and equaled 0.5MIC (N/2), 0.25MIC (N/4), 0.125MIC (N/8), 0.0625MIC (N/16), and 0.03125MIC (N/32) of each extract and were prepared in Luria broth. To 1 mL of each SIC 50 μL of overnight culture (107 cfu/mL) of C. violaceum ATCC 12472 was added and tubes were incubated for 24 hours at room temperature. Medium without the extract served as a positive control, medium with 7.8% DMSO served as vehicle control, and medium with subinhibitory concentrations of extract but no culture served as blanks. After incubation, amount of growth in each tube was accessed by measuring absorbance at 655 nm. The pigment produced was extracted by adding equal quantity of acetone to the harvested biomass [For quantitative studies of the anti-QS activity, a modified method of Blosser and Gray (2000)  was used biomass  and abso N-acyl homoserine lactone response (luxR effect) or due to its effect on synthesis of AHL (luxI effect), method of Mihalik et al. (2008) [μL of methanolic plant extracts were placed on Luria agar plates. C. violaceum strains, CV026 and CV31532, were streaked in circles 8–10 mm apart such that in one plate strain CV026 formed the inner circle and strain CV31532 the outer circle, while in the other plate it was vice versa. On incubation at RT for 24 hours pigmentation in the CV026 strain was used to determine the luxR/luxI effect. A lowered signal in the form of pigmentation of CV026 when it forms the outer circle suggested that LuxI was the target while a lowered signal in the form of pigmentation of CV026 when it forms the inner circle suggested that LuxR was the target.To determine whether inhibition of QS was due to the effect on. (2008)  was used agr activity was done using the S. aureus agrP3::blaZ reporter assay, originally described by Ji et al. (1995) [β-lactamase activity using the chromogenic cephalosporin, nitrocefin, as a substrate. S. aureus agrP3-blaZ strain was grown in CYGP broth overnight at 37°C. The overnight broth culture was further diluted 1/100 with fresh medium and incubated at 37°C to attain logarithmic phase . In a microtitre plate, 5 μL of tannin-rich plant extract prepared in CYGP broth was added to 45 μL of log phase culture such that it reached a subinhibitory concentration equivalent to 0.5MIC (N/2), 0.25MIC (N/4), 0.125MIC (N/8), 0.0625MIC (N/16), and 0.03125MIC (N/32). In the positive control well 5 μL of supernatant of the overnight broth culture was added as a source of AIP to 45 μL of the log phase culture, whereas 5 μL of 7.8% DMSO was added to 45 μL of the log phase culture as vehicle control. The plate was incubated at 37°C on Selec microplate shaker for 55 minutes. After incubation 50 μL of CYGP broth containing 5 mM sodium azide was added, followed by 50 μL of 132 μg/mL nitrocefin (Toku-E) prepared in 0.1 M sodium phosphate buffer, pH 5.8. β-lactamase activity was measured in Biorad imark microplate reader using kinetic mode at ε490–ε650 nm. One unit of β-lactamase activity was defined as an increase in 0.001 OD units per min. All assays were performed in triplicate and average percent enzyme activity was calculated with respect to the vehicle control. % enzyme activity values were reported with SD that was calculated using the Microsoft EXCEL software. Minimum concentration of extract in mg/mL that showed 50% inhibition in activity was determined and designated as the minimum quorum sensing inhibitory concentration (MQSIC) [Quantitative measurement of. (1995) . This as125MIC N/. In the 3. The protein precipitation test too was strongly positive for all extracts. This confirmed that all the shortlisted plants were rich in tannins. Tannins are primarily of two types: condensed and hydrolysable. Qualitative analysis for condensed tannins showed that the extracts of Acacia arabica, Terminalia arjuna, Casuarina equisetifolia, Thespesia populnea, Mangifera indica (seed kernel), and Embelia ribes were rich in condensed tannins. In contrast, extracts of Phyllanthus emblica, Terminalia bellirica, Terminalia chebula, Punica granatum and Mangifera indica (flower), and Syzygium cumini were rich in hydrolysable tannins as they showed high tannin content but gave a negative acid butanol test [All the methanolic extracts when screened for their phytochemical constituents showed strong reactions with FeClnol test –49. C. violaceum 12472 on exposure to subinhibitory concentrations of extracts showed an increase with decreasing concentration of extract. All the twelve extracts showed anti-QS activity at subinhibitory concentrations  in their MIC and MQSIC. In contrast, extracts of E. ribes, A. arabica and M. indica (seed), T. arjuna, C. equisetifolia, and T. populnea had a very low range of concentration at which they could prevent QS, as the difference between their MIC and MQSIC was only 4-fold, while extracts of P. emblica, T. bellirica, and M. indica showed N/8 as their MQSIC value.Amount of pigment produced bytrations . All theextracts , therefoluxI effect) or a transcription response (luxR effect) the luxI/luxR assay was done. Amongst the hydrolysable tannin-rich extracts studied T. bellirica, T. chebula, S. cumini, and M. indica (seed) showed a putative luxI effect, indicating that they would affect production of the C6 HSL by CV31532, whereas P. emblica showed a putative luxR effect indicating that it would interfere with the binding of the autoinducer to receptors on CV026. It is interesting to note that P. granatum and M. indica (flower) showed both luxI and luxR effect  too showed low activity at this concentration. A comparison of the MQSIC with the MIC of the extracts  was 16 times lower than its MIC and in T. populnea and A. arabica it was found to be 8 times lesser, while in C. equisetifolia it was 4 times lesser than its MIC.Z strain . The mosextracts  was used Punica granatum and an ethyl acetate fraction of S. cumini leaves has also been reported [ P. emblica fruit extract can suppress quorum sensing genes in S. mutans. These reports draw attention to the fact that tannin-rich extracts of medicinal plants influence quorum sensing mechanisms.In a qualitative study, Shukla and Bhathena (2014)  have repreported , 51. Hasreported  have repagr activity of tannin-rich plants. One of the striking inferences of this study is that extracts rich in condensed tannins are unable to prevent AHL as well as agr mediated bacterial communication over a wide range of subinhibitory concentrations. On the other hand, extracts rich in hydrolysable tannins, namely, P. emblica, T. bellirica, T. chebula, P. granatum, S. cumini, and M. indica (flower), exhibit a broad spectrum anti-QS activity, that is, affecting activity of acyl homoserine lactones as well as autoinducers over a wide range of subinhibitory concentrations. Okuda and Ito (2011) [ T. chebula, P. granatum, and S. cumini can be categorized as potent quorum sensing inhibitors as they demonstrate this activity at concentrations that are several times lower than their respective MIC values. Anti-AHL activity of pericarp of Punica granatum using a qualitative method was reported by Zahin et al. (2010) [ P. granatum is effective over a wide range of concentrations much lesser than its MIC and also that it affects AHL activity by influencing both production and its binding. The anti-QS property of P. granatum gets augmented, as it also shows significant anti-agr activity. Since all the extracts show distinct protein binding ability, these extracts may be disrupting QS either by inactivating enzymes responsible for the synthesis of the autoinducers or by binding to protein receptors of QS signals as demonstrated in the luxI/luxR assay.The current study is a quantitative evaluation of anti-AHL and anti-o (2011)  have att. (2010) . The cur Phyllanthus emblica, Terminalia bellirica, Terminalia chebula, Punica granatum, Syzygium cumini, and Mangifera indica (flower), are effective QSIs that can interrupt QS in both Gram positive and Gram negative bacteria over a wide range of subinhibitory concentrations, suggesting the presence of a high concentration of anti-QS molecules. Thus plant extracts rich in hydrolysable tannins can be used as broad spectrum antipathogenic drugs at concentrations that will not impose selection pressure. As this study has been done on crude extracts, purification and identification of the active principle along with verification of its anti-QS activity using in vivo studies are essential. However, such a pharmacognosy approach to the so-called alternative systems of medicine can be of immense help in the search for novel phytocompounds with medical applications.Although the discovery of antibiotics has been a boon to human civilization, the current traditional antimicrobials need to be reviewed for their efficacy because of the increasing occurrence of multidrug resistant strains. One novel therapeutic approach to overcome the problem of resistance is the use of antipathogenic drugs that target key regulatory bacterial systems responsible for the expression of virulence factors. Since quorum sensing leads to expression of virulence genes in several known pathogens, agents that can interrupt bacterial communication can be used as antipathogenic drugs. The study on tannin-rich crude extracts from Indian medicinal plants used in Ayurveda showed that extracts of hydrolysable tannin-rich plants,


Aims. We investigated the incidence, risk factors, and prognostic impact of silent atrial fibrillation (AF) after coronary artery bypass graft (CABG) surgery. Methods. This observational study prospectively included 100 patients referred for CABG surgery. Holter ECG monitoring was used to record every arrhythmic event for 7 days. AF was defined as at least one episode >30 s. Episodes recorded on Holter ECG monitoring but not clinically identified were classified as silent AF. Results. Among 34 patients who developed new-onset AF, 13 had silent AF. Compared with patients with maintained sinus rhythm (SR), silent AF patients had a significantly higher logistic EuroSCORE (2.9 (1.5–5.2) versus 2.3 (1.4–3.7), p = 0.017) and were more likely to have previous sleep apnea  and left atrial diameter >45 mm . At one-year follow-up, 30% of silent AF patients had developed symptomatic AF versus 7% in the SR group (p = 0.03) and 11% in the clinical AF group (p = 0.21). Conclusion. After CABG surgery, silent AF is common and may be associated with a higher incidence of recurrences at one-year follow-up than clinical AF. Improved screening for silent AF may help to reduce thromboembolic events in this high-risk population. Atrial fibrillation is the most common cardiac arrhythmia, and its prevalence is growing with the ageing of the population , 2. PostSilent AF is defined as nonsymptomatic AF, not diagnosed by clinicians during hospitalization . AlthougThis observational study was designed to characterize the incidence of post CABG silent AF, to determine its associated factors, and to quantify its impact on AF recurrence at one year.The independent ethics committee of Dijon University Hospital approved the study protocol  and written informed consent was obtained from the patients. The study was designed as a prospective observational study. All investigations were conducted in accordance with the principles outlined in the Declaration of Helsinki.All of the patients undergoing CABG surgery at Dijon University Hospital from September 2011 to March 2013 were screened for participation in this prospective, observational study. The exclusion criteria were age <18 years or >80 years, previous atrial fibrillation/flutter, previous treatment with amiodarone, and previous cardiac surgery and emergency surgery. This observational study was designed to recruit the first 50 patients planned for on-pump (ONP) surgery and the first 50 patients planned for off-pump (OFP) surgery among patients meeting the inclusion criteria. This recruiting strategy has already been used in the particular setting of CABG [Exhaustive clinical data were collected at admission and the following variables were recorded: age, sex, cardiovascular risk factors, cardiovascular and pulmonary diseases, previous regular medication, and preoperative echocardiographic parameters. Medications administered during the hospital stay (but not the doses) were recorded. Previous renal failure was defined as preoperative estimated glomerular filtration rate (eGFR) <60 mL/min using the MDRD formula. After discharge, each patient's daily medical files and treatment sheets were analyzed by a physician unaware of the Holter ECG monitoring results.All the patients underwent preoperative transthoracic echocardiography. The left ventricular ejection fraction (LVEF) was calculated using the Simpson method and was dichotomized at 45%. Left atrial (LA) anteroposterior diameter was dichotomized at 45 mm following classical cut-off values .Routine cardiac medications were continued until the morning of the surgery, except for clopidogrel, which was stopped at least 5 days earlier. Before the induction of anesthesia, a complete hemodynamic monitoring system was set up in the operating room. Off-pump surgery was performed with the use of Cor vasc  to stabilize the heart. Patients were heparinized with 150 IU/kg intravenously to achieve an activated clotting time >300 s. The anastomoses were performed using temporary occlusion with LeGoo gel   started immediately after the surgery. The Holter ECG device was programmed to record every arrhythmic event for 7 days and was removed after 7 days of recording or at death (whichever occurred first). AF was diagnosed according to the European Society of Cardiology guidelines; that is, any arrhythmia that presents the ECG characteristics of AF and lasting at least 30 s on a rhythm strip should be considered AF . Silent Overall AF duration episodes were collected, and the total time in AF per day (AF burden) was calculated.2SVAS2C score [In cases of clinical new-onset AF, patients received amiodarone and anticoagulants according to the CHAD2C score . As sileWe recorded clinical outcomes over one-year of follow-up, namely, length of stay in the ICU and in the hospital and death in the ICU, at discharge , and at one year. The presence of new-onset AF and the occurrence of stroke between discharge and the follow-up time points were recorded. Follow-up information at one year was obtained by phone call to the patient, the patient's family, or the general practitioner. One-year follow-up was obtained for all patients.p value <0.05 was considered significant. All analyses were performed using SPSS 20.0 0 .Continuous variables are presented as means ± standard deviations (SD) when normally distributed or medians and ranges otherwise, and categorical variables as numbers (percentages). Patients were classified into the clinical AF, silent AF, or sinus rhythm (SR) group according to the onset of atrial fibrillation on the Holter tracings and to the medical records. An exploratory identification of the characteristics of both groups was performed using the one-way ANOVA for continuous variables with Tukey post hoc analysis as appropriate and Fisher's exact test for categorical variables. All the tests were two-sided, and a p < 0.001).One hundred patients were prospectively included in the study . Among, p < 0.05), were more likely to have an impaired LVEF <45% (6 (29%) versus 2 (3%), p < 0.05), and were less often smokers (2 (10%) versus 25 (38%), p < 0.05).When compared with maintained sinus rhythm patients (SR), clinical AF patients were significantly older , were more likely to have been diagnosed with sleep apnea syndrome (4 (31%) versus 5 (8%), p < 0.05), and were more likely to have LA enlargement (diameter > 45 mm) (4 (36%) versus 3 (5%), p < 0.05).When compared with SR patients, silent AF patients had a significantly higher logistic EuroSCORE (2.9 (1.5–5.2) versus 2.3 (1.4–3.7), However, there was no significant difference between the clinical AF group and the silent AF group regarding baseline characteristics. Moreover, the preoperative CHAD2DS2-VASc score did not differ significantly between the three groups.p < 0.05) and a longer aortic cross-clamp duration . CPB and aortic cross-clamp durations were longer in silent AF patients than in clinical AF patients (119 (103–145) versus 95 (75–113) minutes, p < 0.05; 96 ± 21 versus 71 ± 19 minutes, p < 0.05, resp.).p < 0.05, 14 (67%) versus 13 (20%), p < 0.05, and 8 (40%) versus 0, p < 0.05, resp.). Furthermore, clinical AF patients were more likely than SR patients to suffer from infectious complications (3 (14%) versus 1 (2%), p < 0.05).Concerning postoperative characteristics, inotropic catecholamines, amiodarone, and vitamin K oral anticoagulants (VKA) were more often given to clinical AF patients than to SR patients (6 (29%) versus 5 (8%), p < 0.05). Both AF groups had a similar mean heart rate . The onset of the first AF episode was significantly later in the silent AF group than in the clinical AF group, within the 4 first days of monitoring (7 (54%) versus 21 (100%), p = 0.001). Not surprisingly, the AF burden during the first seven days of recording was shorter in the silent than in the clinical AF group (35 (3–120) versus 869 min (161–2366), p = 0.001).p = 0.023). These patients were also more often on oral anticoagulants . Two ischemic strokes occurred within the first year of follow-up, one in the SR group and one in the silent AF group. However, the small number of events did not allow a statistical comparison between the three groups regarding stroke or death at one year.At one-year follow-up, symptomatic  AF was more frequently diagnosed in patients from the silent AF group than those from the SR group .The risk profile in patients who develop silent AF after CABG is different from that in SR or clinical AF patients, suggesting that silent AF may be triggered by other mechanisms.Despite the small number of events, silent AF patients were more likely to develop symptomatic AF at one year than were SR or clinical AF patients. These results highlight the importance of screening for silent AF, which may eventually become a clinically relevant arrhythmia.Silent AF was defined as nonsymptomatic AF and AF not diagnosed by clinicians during the hospitalization. Its incidence is underestimated as it can only be identified by several days of continuous cardiac monitoring using a Holter ECG or with implantable loop recorders. In fact, silent AF is mostly underdiagnosed because of its short duration and its lack of associated symptoms. Patients with silent AF may therefore not receive appropriate anticoagulation therapy, and the disease may be unmasked by a cerebrovascular event as its first manifestation [Our observational study was designed to characterize underdiagnosed silent AF in a post-CABG setting. We also aimed to identify risk factors for silent AF and its prognostic impact at one-year follow-up. The main results of our work are as follows:estation . Previouestation . Recentlestation . In the estation . Howeverestation .When the Holter recordings of silent AF and clinical AF patients were compared, several significant differences were noted: the burden of AF in the silent AF group was lower than that in the clinical AF group during the Holter ECG monitoring, and in the silent AF group the onset of AF was more likely to occur after the first 4 days following the cardiac surgery. These results are closely linked to the definition of silent AF. It is obvious that the longer the AF episode lasts, the greater the chance it will be identified by heart rate monitoring, systematic ECG, or a clinical examination. Moreover, most patients were continuously monitored in the ICU during the first three days. We may then hypothesize that screening for AF was more intensive during the ICU stay than in the surgery ward. However, this hypothesis cannot explain the whole pattern of silent AF. Indeed, 6/13 of silent AF patients (46%) had the first onset of AF during their ICU stay. In fact, unlike Holter ECG recordings, tracings from ECG monitors are not usually reanalyzed afterwards. Besides, monitor alarms may not have been set up appropriately for the detection of AF as opposed to life-threatening arrhythmias like ventricular fibrillation (VF).It has recently been demonstrated that early (within the first 5 days after cardiac surgery) and late (6 to 30 days after cardiac surgery) POAF have different predictors . NotablyFinally, silent AF patients were more likely than SR patients and clinical AF patients to develop symptomatic AF at one year. We may hypothesize that clinical AF patients were more often on antiarrhythmic drugs, which may in turn have protected patients from AF recurrences. However, silent AF could also be interpreted as incidental AF diagnosed during the hospital stay thanks to the Holter monitoring, whereas clinical AF would more likely be related to the acute cardiac injury triggered by the surgery. Bidar et al. demonstrated that 30-day monitoring using a transtelephonic loop recorder, with an autotrigger algorithm for AF, improved its screening: 25% of total POAF recorded occurred between day 6 and day 30 . This obOur study has several limitations. First of all, given the relatively small number of patients , our results should be confirmed in larger population studies. Another limitation must be highlighted: even though the patients undergoing on-pump and off-pump surgery were comparable, the study was not designed as a randomized controlled trial to compare the incidence of POAF in these two groups. Moreover, the assessment of LA dimensions could have been improved by the measurement of LA surface area and volume. As the follow-up data were recorded at one year, we did not collect the date of each event and we were unable to conduct survival analyses on our data.Silent AF after CABG surgery is common (1/3 of recorded AF) and mostly underdiagnosed even with the use of ECG monitors in the ICU. We have shown in this study that silent AF has to be considered for appropriate medications , as it is associated with a higher incidence of recurrences at one year than is the case for clinical AF. Although this is a small observational study, we think that it is relevant to improving AF screening during hospital stays by using Holter monitoring in order to prevent thromboembolic events in this high-risk population.


Studies looking at air temperature (Ta) and birth outcomes are rare.We investigated the association between birth outcomes and daily Ta during various prenatal exposure periods in Massachusetts (USA) using both traditional Ta stations and modeled addresses.2.5), random intercept for census tract, and mother’s health.We evaluated birth outcomes and average daily Ta during various prenatal exposure periods in Massachusetts (USA) using both traditional Ta stations and modeled address Ta. We used linear and logistic mixed models and accelerated failure time models to estimate associations between Ta and the following outcomes among live births > 22 weeks: term birth weight (≥ 37 weeks), low birth weight , gestational age, and preterm delivery . Models were adjusted for individual-level socioeconomic status, traffic density, particulate matter ≤ 2.5 μm  in association with an interquartile range increase (8.4°C) in Ta during the last trimester. Ta over the entire pregnancy was positively associated with PT  and LBW .Ta during pregnancy was associated with lower birth weight and shorter gestational age in our study population.http://dx.doi.org/10.1289/ehp.1308075Kloog I, Melly SJ, Coull BA, Nordio F, Schwartz JD. 2015. Using satellite-based spatiotemporal resolved air temperature exposure to study the association between ambient air temperature and birth outcomes in Massachusetts. Environ Health Perspect 123:1053–1058;  The increase in temperatures over the last century and continued increases in emissions of greenhouse gases have focused attention on the effects of increasing heat . Relativin utero growth restriction  also contribute to perinatal morbidity and mortality , early-term delivery (delivery at 37–38 weeks gestation), and ortality . Evidencortality . The patortality . Social ortality . Environortality . Furtherhttp://modis.gsfc.nasa.gov/data/) surface temperature (Ts) over a large area with varying geographical characteristics (covering the entire Northeast and Mid-Atlantic areas of the United States) at a 1 × 1 km spatial resolution. We incorporated land use and meteorological variables to predict daily 24-hr mean Ta for grid cells even when satellite Ts measures were not available. A similar model has previously been developed for PM2.5 on the same resolution  among term births, preterm birth (< 37 weeks), and gestational age.http://www.mass.gov/eohhs/gov/departments/dph/programs/admin/dmoa/vitals/). The term birth weight and LBW  analyses included 453,658 births ≥ 37 weeks gestational age, and the gestational age and preterm birth (< 37 weeks) analyses included 473,977 births. The study and the use of birth data was approved by the Massachusetts Department of Public Health and the human subjects committee of the Harvard T.H. Chan School of Public Health. Informed consent was not required because we used anonymous administrative data.In the analysis we included the entire state of Massachusetts . The stuhttp://www7.ncdc.noaa.gov/CDO/) monitoring stations, and residence-specific cumulative traffic density. We describe each metric in more detail below.For exposure data we used three different indicators: predicted 1 × 1 km Ta from our model, ground Ta from the nearest National Climatic Data Center . Mean out-of-sample R2 values for days with and without Ts data were 0.947 and 0.940, respectively, indicating excellent model performance. Mean out-of-sample temporal and spatial R2 values also were high  (on model . In thesctively) .To estimate Ta exposure, we linked each mother’s residence at the time of delivery to its corresponding grid cell . Daily T2.5.PM Particulate matter ≤ 2.5 μm (PM2.5) was estimated on a 1 × 1 km grid from the same MODIS satellite, using daily measures of aerosol optical depth using a similar methodology  as the temperature model. Further details have been published previously  2002 Road inventory. These data are based on automatic vehicle counts on major highways, periodic counts on other major roads, and estimated counts for all other roads . The percent of each census tract that was open space was then calculated and assigned to birth addresses belonging to that tract.Percent of open space. The percent of open space data was obtained from the office of geographic information Commonwealth of Massachusetts, information technology division MassGIS . The subSocioeconomic indicators. Socioeconomic data at the individual level were obtained from the Massachusetts birth registry. Information included the mother’s race/ethnicity , mother’s years of education, and the Kotelchuck adequacy of prenatal care utilization index (APNCU). The APNCU is based on the number and the time of start of mother’s prenatal visits  and was Median income. Data were obtained from the U.S. Census Bureau 1999 median household income  for everIndividual-level covariates. Maternal age, parity, gestational age , number of cigarettes smoked per day during and before pregnancy, chronic conditions of mother or conditions of pregnancy , previous occurrence of a preterm birth, whether the mother ever had a previous infant weighing ≥ 4,000 g, and sex of infant were all obtained through the Massachusetts Birth Registry  through the index child’s birth certificate.To identify factors affecting birth weight, we used linear mixed regression models to estimate associations between both monitor and modeled Ta during different time windows and term birth weight, and logistic mixed regression to estimate associations with preterm birth (< 37 weeks) and LBW  . SeasonaSpecifically, we fit the following models:ij =BW (α j+ u) + β1iTa + β2i +PM γXi + eij(ju) ~ N and [1]Logit (ij/LBWij = 1|XPT) = (α j+ u) + β1iTa + β2i +PM γXi + eij(ju) ~ N, [2]ijBW, ij,PT and ijLBW represent birth weight, preterm, and LBW, respectively, for the ith subject in census tract j; α and ju are the fixed and random (tract-specific) intercepts, respectively; γXi denote the set of variables included in the model, which include predicted ambient air temperature, predicted ambient PM2.5, cumulative traffic density, percent of open spaces, age of mother, median income, gestational age, chronic conditions of mother or conditions of pregnancy , parity, previous infant weighing ≥ 4,000 g and sex of infant, sine and cosine , APNCU , mother’s race , mother’s education , and previous preterm occurrences. ije is the error term and finally, σ2u is the variance of the tract random effects, and jje ~ N.where We estimated associations between Ta during different time windows and gestational age using an accelerated failure time model (AFT).T) is given bySuch models are a form of survival analysis that model the survival time directly instead of the hazard. Gestational age is used as a continuous outcome in the AFT model. The log-linear form of the AFT model with respect to time . A two-sided p-value < 0.05 was considered statistically significant.where μ is the intercept, σ is a scale parameter, and εWe also ran analyses stratified on subject residence < 30 km or ≥ 30 km of a Ta monitor . Statistical analyses were performed in SAS  and R . Cases wDescriptive statistics are presented in Term birth weights were negatively associated with predicted Ta in almost all exposure time windows . In geneThe OR for low term birth weight with a 2.7°C increase in model-based Ta over the entire pregnancy was 1.04 , compared with 1.07  for monitor-based Ta . The OR A 2.7°C increase in Ta over the entire pregnancy was associated with a 0.26% decrease in gestational age , and an 8.4°C increase in Ta over the last trimester before birth was associated with a 0.15% decrease in gestational age  . For monp-value = 0.26).The association between an IQR increase in predicted Ta during the entire pregnancy and birth weight was stronger among births to mothers with residences in urban areas  compared with births to mothers residing in rural areas , though the differences were not statistically significant , regardless of the distance between a participant’s residence and the closest Ta monitor.We found a consistent negative association between Ta and birth weight for infants who were born full term after adjusting for other potential risk factors, such as previous and current mother’s health conditions, socioeconomic factors, and physical environment risk factors such as traffic density in surrounding grid cells. The association with Ta over the entire pregnancy was stronger in more urban areas (< 30 km from a monitor) than in more rural areas (≥ 30 km from a monitor), though the difference was not statistically significant. In contrast to the associations found with our modeled predicted Ta, associations between birth weight and Ta measured at the nearest ground monitor stations were close to the null, suggesting that predicted Ta classified exposure more accurately than monitor-based estimates. Interestingly, for the AFT analysis we found that an increase in Ta over both periods were associated with a decrease in gestational age; yet in the monitored Ta analysis, these associations were significantly associated with an increase of gestational age. These findings need to be further explored in future studies.A key advantage of the presented study is the exposure assignment. Because our model allows us to predict temporally and spatially resolved Ta, we can assign daily Ta exposure to the entire study population, avoiding potential selection bias that would yield a nonrepresentative sample. It also captures the urban heat island effect, as shown in The literature on the potential impact of Ta on birth weight and its determinants is still very limited. Increased Ta may affect birth weight through direct or indirect means. The causes of preterm birth and LBW are largely unknown, but are likely to be a complex mix of genetic, behavioral, socioeconomic, and environmental factors . Heat stRace, ethnicity, education, and other socioeconomic status factors are often clustered spatially and can act as potential confounders since they do not vary by time but do vary by space. We use a random-effects model with a random intercept for FIPS  code while controlling for seasonality to reduce bias as well.There are several limitations in the present study. First, the spatial resolution of the exposures was 1 × 1 km. As satellite remote sensing evolves and progresses, higher spatial resolution data should become available in the coming years, which will further reduce exposure error. Such increased resolution should enable us to more precisely estimate daily intraurban exposures and how these vary across spatial locations. Other limitations include the lack of some health and personal-level data such as maternal weight, body mass index, differences across different locations in physical activity, and pollen exposure. We also lacked data on indoor temperature exposure and information on air conditioning use in households. Finally, another limitation is the lack of information on road noise as in some recent pregnancy outcome studies .In summary, our findings suggest that higher Ta during pregnancy may be a risk factor for lower birth weight.