To compare the diagnostic value of coronary CT angiography (CCTA) with use of 2 image postprocessing methods (CCTA_S) and (CCTA_OS) and original data (CCTA_O) for the assessment of heavily calcified plaques.Fifty patients  with suspected coronary artery disease who underwent CCTA and invasive coronary angiography (ICA) examinations were included in the study. Image data were postprocessed with “sharpen” and smooth reconstruction algorithms in comparison with the original data without undergoing any image postprocessing to determine the effects on suppressing blooming artifacts due to heavy calcification in the coronary arteries. Minimal lumen diameter and degree of stenosis were measured and compared between CCTA_S, CCTA_OS, and CCTA_O with ICA as the reference method. The area under the curve (AUC) by receiver-operating characteristic curve analysis (ROC) was also compared among these 3 CCTA techniques.P < 0.05), with no significance differences for detection of coronary stenosis in the left circumflex and right coronary arteries (P > 0.05).On a per-vessel assessment, the sensitivity, specificity, positive predictive value and negative predictive value, and 95% confidence interval (CI) were 100% , 33% , 41% , 100%  for CCTA_O, 94% , 66% , 57% , and 95%  for CCTA_S, 94% , 44% , 44% , and 97%  for CCTA_OS, respectively. The AUC by ROC curve analysis for CCTA_S showed significant improvement for detection of >50% coronary stenosis in left anterior descending coronary artery compared to that of CCTA_OS and CCTA_O methods (CCTA with “sharpen” reconstruction reduces blooming artifacts from heavy calcification, thus, leading to significant improvement of specificity and positive predictive value of CCTA in patients with heavily calcified plaques. However, specificity is still moderate and additional functional imaging may be needed. Studies in vitro and in vivo investigating the effects of different IR techniques on calcium score analysis showed that CCTA with use of IR significantly reduced calcium volume and calcium scores,17 leading to improved diagnostic value of CCTA in heavily calcified coronary arteries,18 while others reported no significant effects on measurements of calcified plaque volume using IR compared to FBP.19In recent years, the use of IR has been mainly focused on lowering radiation dose of CCTA in comparison with the conventional approach of filtered back projection (FBP).8 Due to limited information on the effects of image processing methods on the calcified coronary plaques,20 the purpose of this study is to apply the appropriate image postprocessing reconstruction derived from our preliminary findings to a group of patients with heavily calcified coronary plaques. We hypothesized that diagnostic value of CCTA, in particular, the specificity and positive predictive value (PPV) might be improved in the evaluation of heavily calcified plaques with use of the appropriate image processing method.We have recently reported our experience of using different image postprocessing algorithms in the assessment of CCTA images with high calcium scores, and showed the improved visualization of coronary artery lumen with use of image postprocessing “sharpen” algorithm.This retrospective study reviewed 100 consecutive patients who were referred for the assessment of suspected coronary artery disease (CAD) with CCTA on ≥64-slice CT scanners between March and August 2014. Eligible patients included adults with at least one coronary artery segment with calcified plaques identified on CCTA, with invasive coronary angiography (ICA) performed as the reference method for diagnosis of coronary artery stenosis. Patients were not eligible if they had any of the following conditions: known allergy for contrast media, prior history of coronary artery bypass surgery, prior coronary stenting, impaired renal function with serum creatinine level >1.5 mg/dL, or unable to control heart rate less than 65 beats per minute despite use of beta-blockers. Fifty patients were excluded due to the following reasons: coronary plaques other than calcified type  (n = 15), ICA was not available for comparison (n = 30), and coronary stenting (n = 5). The final cohort consisted of 50 patients with patient characteristics shown in Table CCTA image acquisition was obtained as part of clinical cardiac CT examinations; therefore, the need for institutional review board approval was waived by the local ethics committee for this study. No informed consent was obtained from the patients, given the retrospective nature of the study.21 Intravenous contrast medium  was administered during each scan with details described in our previous study.21 Images were reconstructed with a slice thickness of 0.6 to 0.75 mm and a reconstruction interval of 0.5 to 0.6 mm for the first- and second-generation dual-source CT, slice thickness of 0.5 mm and reconstruction interval of 0.25 mm for 640-slice CT, respectively.The patients were scanned with a first-generation dual-source CT , or a second-generation dual-source CT (Somatom Definition Flash); or 640-slice CT , with corresponding 20 patients (40%) for 64-slice CT, 15 patients (30%) for 128-slice CT, and 15 patients (30%) for 640-slice CT, respectively. The imaging protocol has been previously described in detail.21 Therefore, this study analysis is considered comparable between the different vendors.Although data were acquired with different CT scanners, our previous study did not show any significant difference in the diagnostic value between different multislice CT vendors.22 Details of the image processing algorithms used in this study have been described in our recent report. The ImageJ software provides default functions to perform the image processing conveniently. It also allows users to conduct nondefault image processing techniques with flexibility. For example, the default ImageJ “sharpen” function shows the most effective outcomes and it is actually a function to carry out the convolution filtering with the following kernel (convolution matrix/mask).Original 2D axial CT images in DICOM  and curved planar reformatted images were processed by a Windows 7 64-bit workstation with open source software ImageJ v 1.49b  using common spatial domain processing approaches.-1 -1 -1-1 12 -1-1 -1 -1The use of ImageJ “sharpen” function has been proved to effectively suppress artifacts due to heavy calcification, thus, this image postprocessing algorithm was selected for evaluation of CCTA images in this study. In addition, another commonly used approach, smoothed image subtracted from original data and the outcome integrated with the original image subsequently was also included in the image analysis. The default ImageJ function was used to smooth images in this approach. It is again based on the convolution filtering technique with the use of the following kernel.1 1 11 1 11 1 18 In summary, 3 different imaging datasets were analyzed in 50 patients, namely: CCTA original data (CCTA-O), CCTA with use of “sharpen” algorithm (CCTA-S), and CCTA original data integrated with the outcome of smoothed image subtracted from the original (CCTA-OS).This technique is more commonly known as the 3 × 3 mean filtering. The image subtraction process was handled by the default ImageJ “image calculator” function.21 Measurements were performed by 2 researchers  who were unaware of the results of ICA. Two reviewers assessed CCTA and ICA images separately with an interval of 2 weeks. Any discrepancy was resolved by a reading session in which both reviewers read images together to reach a consensus. Three consecutive measurements of the MLD at each coronary lesion were obtained, and the mean value was averaged to avoid intraobserver disagreement.Data were transferred to a separate workstation equipped with Analyze V 11.0  for image postprocessing and analysis. The minimal lumen diameter (MLD) of three main coronary arteries  was measured using the same approach as described in our previous study.The percentage of coronary lumen reduction (stenosis) was calculated using the following formula: 23 When there were multiple calcified plaques present in the coronary arteries, measurements were performed at the most severely calcified lesions as this study only focused on the heavily calcified plaques.RD refers to the reference normal diameter of coronary artery. The degree of coronary lumen stenosis was determined significant when the percentage of reduction was measured >50%.Invasive coronary angiography (ICA) was performed by femoral or radial approach. The MLD was measured in projections showing the most severe narrowing of three main coronary arteries  by a radiologist with more than 15 years of experience in cardiac imaging. Similarly, 3 consecutive measurements of the MLD within the same lesion were obtained, and the mean value was averaged.P-value of <0.05 was considered statistically significant.Statistical analyses were performed using SPSS 21.0 . All continuous variables were expressed as the mean ± standard deviation, while categorical variables were presented as frequencies or percentages. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for the detection of significant stenosis (>50%) on CCTA were calculated for individual three main coronary arteries with ICA as the gold standard. Receiver-operating characteristic (ROC) curve analysis was used to evaluate the diagnostic value of CCTA using different image postprocessing approaches in the measurement of MLD and detection of coronary stenosis compared to ICA. The areas under the ROC curves (AUCs) were compared among these three methods. A difference with A total of 102 calcified coronary arteries from 50 patients were detected and analyzed. Calcified plaques were found in 47 LAD, 28 LCx, and 27 RCA arterial branches, respectively. ICA showed coronary stenosis greater than 50% in 26 (52%) patients, with a total of 33 coronary arteries. More than half of calcified plaques  were found to involve the LAD. Twenty patients had 1-vessel disease, 5 patients had 2-vessel disease, and the remaining 1 patient had 3-vessel disease.P < 0.05).We randomly selected 20 cases (60 vessels) for interobserver variability and the comparison of MLD measurements between CCTA and ICA. The 2 readers blindly measured MLD and RD on these techniques individually, with results showing high correlation between these values performed by two observers  for CCTA_O, CCTA_S, and CCTA_OS in individual and all of the 3 coronary arteries are presented in Table P < 0.05)    Fig. A, with nFigure Findings of this study show that the use of image postprocessing method, the “sharpen” reconstruction algorithm significantly improves the specificity and PPV for CCTA detection of coronary stenosis in patients with heavily calcified plaques. These improvements are most likely due to the suppression of blooming artifacts resulting from the heavy calcification in the coronary arteries, thus, leading to better coronary lumen visualization and assessment of degree of stenosis.25 The specificity of CCTA was reported to be as low as 19% in patients with highly calcified plaques, according to these studies. Our results are consistent with these findings as the specificity of CCTA ranges from 18% to 44% in the CCTA images if no image postprocessing was implemented. The low specificity often leads to unnecessary downstream testing such as ICA or myocardial perfusion imaging, which increases additional cost and radiation dose to patients, thus restricting the clinical application of CCTA. It is therefore important to address this challenge in patients with heavily calcified plaques who undergo CCTA examination.Advances in CT technology have enhanced the diagnostic value of CCTA, making it a widely used less-invasive imaging modality in the diagnosis of CAD. However, studies based on single centre and multicentre experiences have demonstrated the negative impact of high calcium scores on the specificity and PPV of CCTA.28 A number of studies have applied this technique to patients with high calcium scores for assessment of diagnostic value of CCTA, but results are contradictory.29 Renker et al18 compared IR with FBP in 55 patients with Agatston scores of more than 400, and they found that the specificity and PPV were increased from 91.2% and 61.1% to 95.5% and 76.9% at per-segment level, 66.7% and 78.9% to 79.2% and 85.7% at per-patient level, corresponding to the CCTA with use of FBP and IR, respectively. Authors also reported that CCTA images using IR significantly lowered the calcification volume compared with those FBP images. This correlates with other studies indicating the significant reduction in calcium score and volume score with use of IR in CCTA compared to FBP images.17 However, other researchers showed no significant impact of IR on calcium volume or measurement of coronary lumen diameter.30 Thus, it is recommended that applying IR algorithms in CCTA images with high calcification be cautious due to these conflicting results.IR has been increasingly used in cardiac and noncardiac CT imaging because it improves image quality by reducing image noise, while in the meantime significantly lowering the radiation exposure to patients.20 compared subtracted with conventional CCTA images in 11 patients with 55 calcified coronary segments, with ICA as the reference method. At per-segment level, subtraction CCTA shows improvement in specificity which is 59%, compared to the 48.7% with conventional CCTA. The AUC by ROC analysis also shows significant improvement, increasing from 0.741 to 0.905, corresponding to conventional and subtraction CCTA, respectively. The present study was designed according to our previous report which tested a number of image postprocessing algorithms, with “sharpen” reconstruction algorithm confirmed to be most effective in reducing blooming artifacts.8 Results of this study further verify the usefulness of CCTA using “sharpen” algorithm in the assessment of calcified coronary arteries. Despite improved diagnostic value being achieved when comparing CCTA_S with CCTA_O or CCTA_OS, the specificity and PPV are still relatively low (<70%), which could generate more ICA or myocardial perfusion imaging. A study by Chow et al31 showed significant decrease of normal ICA examinations from 31.5% to 26.8% after CCTA was implemented into clinical practice. However, other studies reported no impact of introduction of CCTA on the number of ICA examinations.33 A recent study by Zorlak et al34 did not show any significant difference in downstream testing between CCTA and SPECT (single-photon emission computed tomography). Due to its limitation of being an anatomical test assessing coronary stenosis, combining CCTA and functional assessment tools has been shown to increase the specificity and positive predictive value, thereby improving the diagnostic workup of patients with significant coronary artery disease.36 This could be another research direction for assessment of heavily calcified plaques.Use of image postprocessing technique seems to be an alternative approach when dealing with highly calcified plaques in CCTA images. Despite limited data available in the literature, results of using image postprocessing algorithms in calcified coronary arteries are promising. Tanaka et al22 ICA remains the gold standard for the identification and detection of coronary stenosis, but it fails to provide information about coronary wall, plaque composition, or volume.37 Intravascular ultrasound (IVUS) and optical coherence tomography (OCT) are the invasive imaging modalities which allow for quantitative analysis of plaque characteristics, in particular, differentiation of plaque origin by demonstrating the plaque and coronary wall surface changes.39 Correlation of CCTA findings with IVUS or OCT will enable us to quantitatively assess these calcified plaque features, but this is not the focus of our present study.The limitations of CCTA overestimation of coronary lumen diameter and stenosis in the presence of severe calcification will not be completely overcome with image postprocessing methods. This has been confirmed in the present study, especially the very low values of AUC by MLD, indicating the low reliability of using MLD for evaluation of coronary lumen diameter due to calcified plaques. Although results in this study have shown significant improvements with use of “sharpen” reconstruction, the diagnostic value of specificity and PPV are still moderate (<70%) in the assessment of highly calcified plaques, indicating the high percentage of false positive results with CCTA. The origin of a calcified plaque may contribute to the moderate performance of CCTA. When a plaque is in close proximity to the coronary surface or intima, it may progress into luminal narrowing, which can be detected on ICA as lumen stenosis, while a plaque arising from the adventitia is likely to be viewed as significant stenosis on CCTA due to false positive results but it may be observed as normal or mild stenosis on ICA.41 Third, while this study clearly shows the feasibility of using “sharpen” reconstruction for reduction of blooming artifacts in calcified coronary plaques, this algorithm still needs to be optimized to further improve the diagnostic performance and clinical application of CCTA in clinical practice. Furthermore, other image processing algorithms including some advanced beam hardening algorithms were not applied to our images, thus, the potential benefit of these image processing algorithms cannot be commented. Finally, although our images were acquired with 64- or more slice CT scanners, the spatial resolution (0.5 mm) of the current standard CCTA is inferior to that of the high-definition CT (0.23 mm) which shows improved analysis of calcified plaques for high resolution CCTA.42 Further research of combining image processing algorithms with high resolution CT in the analysis of calcified plaques deserves to be investigated.Some limitations in this study should be acknowledged. First, the number of eligible patients is relatively low. The small sample size with a limited number of calcified plaques in all 3 coronary arteries limits the generalization of our results. Further studies in a large cohort are needed to verify our findings. Second, no calcium scoring was performed in this study group. Calcium scoring by noncontrast CT is usually performed to assess the coronary calcification which is regarded as a reliable predictor of cardiac events in patients with different age groups. However, recent studies showed no additional benefit to the addition of calcium scoring to contrast-enhanced CT as CCTA can be performed alone with acquisition of more diagnostic information.In conclusion, this study suggests that specificity and PPV of detecting coronary stenosis with CCTA in heavily calcified plaques can be significantly improved with use of “sharpen” reconstruction. However, specificity is still moderate and additional functional imaging may be needed. The enhanced diagnostic performance of CCTA may contribute to reducing the unnecessary invasive or follow-up procedures in patients with extensive calcification in the coronary arteries.


We examined network properties of genetic covariance between average cortical thickness (CT) and surface area (SA) within genetically-identified cortical parcellations that we previously derived from human cortical genetic maps using vertex-wise fuzzy clustering analysis with high spatial resolution. There were 24 hierarchical parcellations based on vertex-wise CT and 24 based on vertex-wise SA expansion/contraction; in both cases the 12 parcellations per hemisphere were largely symmetrical. We utilized three techniques—biometrical genetic modeling, cluster analysis, and graph theory—to examine genetic relationships and network properties within and between the 48 parcellation measures. Biometrical modeling indicated significant shared genetic covariance between size of several of the genetic parcellations. Cluster analysis suggested small distinct groupings of genetic covariance; networks highlighted several significant negative and positive genetic correlations between bilateral parcellations. Graph theoretical analysis suggested that small world, but not rich club, network properties may characterize the genetic relationships between these regional size measures. These findings suggest that cortical genetic parcellations exhibit short characteristic path lengths across a broad network of connections. This property may be protective against network failure. In contrast, previous research with structural data has observed strong rich club properties with tightly interconnected hub networks. Future studies of these genetic networks might provide powerful phenotypes for genetic studies of normal and pathological brain development, aging, and function. Discovery of organizational principles within the structure of human cortex will advance understanding of normal and abnormal behavior. Gene expression findings  aged 51–60 participated in the primary VETSA project, with a mean age = 55.4 years (SD = 2.5). Participants were predominantly Caucasian (89.7%), with an average education of 13.8 years (SD = 2.1). In comparison to U.S. census data, participants in the VETSA are similar in health and lifestyle characteristics to American men in their age range , a longitudinal study of cognitive and brain aging with baseline in midlife  or Boston University for a day-long protocol of medical, physical, psychosocial, and neurocognitive assessments. Informed consent was obtained from all participants prior to data collection, the data collection was approved by the relevant institutional review boards, and all research conformed to relevant regulatory standards. Standard MRI exclusion criteria were imposed in order to ensure participant safety .Neuroimaging was performed at either UCSD or the Massachusetts General Hospital (MGH) in Boston. All images were acquired on Siemens 1.5 Tesla scanners. Sagittal T1-weighted MPRAGE sequences were employed with a TI = 1000 ms, TE = 3.31 ms, TR = 2730 ms, a flip angle = 7°, slice thickness = 1.33 mm, and voxel size of 1.3 × 1.0 × 1.3 mm. Raw DICOM MRI scans from both sites were transferred to MGH for initial post-processing and quality control, and then to UCSD for further image analysis.SA expansion and CT mapping were conducted using methods based on the publicly available FreeSurfer software package  and all possible bivariate analyses of the entire set of parcellations (n = 1128). Cluster and graph theory analyses were employed to investigate the network properties of the genetic covariance matrices.Structural equation modeling was used to examine genetic contributions to individual differences in global-adjusted CT and SA across the 48 parcellations  using a standard model for bivariate twin data. Details of biometric modeling procedures are described below. Maximum likelihood-based genetic analysis of CT and SA of all 48 parcellations in one multivariate model was not practical due to sample size. Instead, we conducted a combination of univariate  influences, common or shared environmental (C) influences, and unique environmental (E) influences  asymptotically follows a χa2) and environmental (e2) origin. In this analysis, we left out a shared environmental variance component (c2) from the models due to a lack of any significant shared environmental variance across all univariate analyses of the regional measures. A simplified diagram of this bivariate Cholesky model is presented in Figure To analyze the pattern of genetic relationships between the regional measures of SA and CT, we adopted a bivariate AE approach. The bivariate Cholesky decomposition of each pair of regional size measures partitions the covariance due to genetic  were defined by genetic correlations significant at α < 0.05. No correction for multiple testing was applied because these analyses were exploratory. We identified significant edges by comparing the fit of a bivariate Cholesky decomposition with a submodel in which the path allowing for genetic covariance was removed.i edges, two nodes were identified by sampling from a pool of 48 nodes (with replacement) with uniform probability. Analysis and visualization of graphs was performed using Watts–Strogatz functions in the igraph 0.6.5  and the clustering coefficient (C)  = Φk/Φunc(k) provides a weighted ratio in which values >1 indicate the presence of rich club properties.We also examined the extent to which networks evidenced rich club properties. Rich club properties were operationalized using the Variance decomposition demonstrated substantial heritability of SA within a majority of genetically-derived SA parcellations  and for mean CT within some genetically-derived CT parcellations . Tables c2) was excluded from the models due to a lack of any significant shared environmental variance across all univariate analyses of the regional measures. Genetic correlations derived from AE models between SA of the parcellations ranged from −0.56 to 1.0. Genetic correlations between parcellation-based mean CT ranged from −0.71 to 1.0. As can be seen on the diagonal of the heatmaps, bilaterally homologous regions were more likely to be positively genetically correlated.Color mapping of the genetic correlation matrix is shown in Figure Pictured in Figure p < 0.05 as a threshold for edge placement, we used only the lower half of the matrix and identified 222 unique edges connecting the 48 parcellation-based measures of CT and SA. We used binarized edges for the network analyses resulting in undirected, non-weighted graphs. The mean degree of the network (4.63) was greater than the natural log of the number of nodes (3.87), thus small world properties were estimable  is 3.17, only the CT network mean degree met this threshold, and only small world values for CT were estimable.It is possible that the small world properties could be an artifact of sparse connective properties across two different types of size measures, rather than within genetic CT- and SA-values. Therefore, we also separated parcellation-based CT- and SA-values and examined Φunck = 0.75, Φk = 1.23, E = 72, N = 13), while within-CT did indicate possible rich club properties, such that the weighted ratio ρunck was > 1.0 . When parcellations are graphed such that edge length corresponds to strength of correlation, increased local genetic connections appear to surround right frontal SA and left medial CT parcellations  genetic covariances between structures in SA are less than in CT; (2) the genetic covariance between CT and SA for each structure is low; (3) the genetic covariance between SA and CT overall is low and basically negative; (4) the genetic correlations between homologous structures were the highest for SA and CT, and all positive.This study used biometric modeling and graph theoretical approaches to examine emergent organizational principles from genetic associations among regional measures of brain size (both CT and SA), where the boundaries of the parcellations were determined Our finding of generally higher heritability for SA compared to CT measures is consistent with our previous findings within regions whose boundaries were determined based on sulcal/gyral anatomy (Kremen et al., A relatively dense local genetic network was observed between CT of orbitofrontal, superior temporal, precuneus, and dorsolateral prefrontal parcellations. Preliminary evidence of tight interconnected CT nodes, and sparse nodes of lower degree, indicate the possibility of a genetic CT network with rich club properties.Graph theory has been applied to the brain in a wide variety of biological contexts, such as mapping of human brain circuitry and the human connectome (for a review, see Bullmore and Sporns, Notably, we found in the present study that SA was significantly negatively genetically associated with CT across a number of the connected parcellations. One possible explanation for negative genetic associations could be a competition or a “balancing out” between regions, e.g., for space within the physical confines of the skull, whose maximum size may be evolutionarily limited by the size of the birth canal. Another possibility is that cortical stretching might lead to a complex configuration of regional relationships between SA and CT. Globally, SA and CT have been observed to be genetically distinct (Panizzon et al., Research has identified unique rich club networks of the cortical connectome across species, and within human samples such networks have succeeded in differentiating healthy individuals from individuals with psychiatric disorders (van den Heuvel and Sporns, One caveat to these findings relates to the relatively small number of nodes examined. We chose 12 nodes in each hemisphere because that was the preferred number of clusters for both SA and CT in the original genetic parcellation study (Chen et al., MT, WK, and CEF provided resources and coordination of data collection and processing. LM, LE, CF, and MP oversaw MRI processing and analyses. CC performed the genetic parcellation cluster analyses. AD and MN planned and undertook the statistical analyses, and MP and BV contributed to the biometrical analyses of the parcellation data. AD and CS conducted network analyses and AD drafted the first version of the manuscript. CS, MP, MN, LE, CF, CEF, CC, and WK contributed to subsequent drafts. All authors have reviewed and approved the final submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Aedes aegypti. One of the world’s largest urban agglomerations infested by Ae. aegypti is the Brazilian megalopolis of Sao Paulo, where >26,900 cases of dengue were reported until June 2015. Unfortunately, the dynamics of the genetic variability of Ae. aegypti in the Sao Paulo area have not been well studied. To reduce this knowledge gap, we assessed the morphogenetic variability of a population of Ae. aegypti from a densely urbanised neighbourhood of Sao Paulo. We tested if allelic patterns could vary over a short term and if wing shape could be a predictor of the genetic variation. Over a period of 14 months, we examined the variation of genetic (microsatellites loci) and morphological (wing geometry) markers in Ae. aegypti. Polymorphisms were detected, as revealed by the variability of 20 microsatellite loci  and 18 wing landmarks (quantitative estimator Qst = 0.4732). These levels of polymorphism are higher than typically expected to an exotic species. Allelic frequencies of the loci changed over time and temporal variation in the wing shape was even more pronounced, permitting high reclassification levels of chronological samples. In spite of the fact that both markers underwent temporal variation, no correlation was detected between their dynamics. We concluded that microevolution was detected despite the short observational period, but the intensities of change of the markers were discrepant. Wing shape failed from predicting allelic temporal variation. Possibly, natural selection (Qst>Fst) or variance of expressivity of wing phenotype are involved in this discrepancy. Other possibly influential factors on microevolution of Ae. aegypti are worth searching. Additionally, the implications of the rapid evolution and high polymorphism of this mosquito vector on the efficacy of control methods have yet to be investigated.Scientific research into the epidemiology of dengue frequently focuses on the microevolution and dispersion of the mosquito  Aedes (Stegomyia) aegypti Linnaeus 1762 is a widely distributed mosquito vector of dengue, the most important arboviral disease in humans. The prevention and control of the dengue virus currently depend on controlling its mosquito vector. Different methods have been proposed for dengue vector control, but many of these methods are limited by the microevolution of mosquitoes.In this context, the demography, dispersion, and evolution of these insects within urban areas have been frequently investigated. Additionally, these biological issues are of importance to professionals engaged in vector control and surveillance initiatives but are far from being satisfactorily understood. Dispersal is preferably estimated by population genetics indicators, such as gene flow, migration and genetic variability, given the difficulty and low reproducibility of mark-release-recapture methods for small mosquitoes –3.Ae. aegypti throughout the world [Aedes [Genotypic markers such as Single Nucleotide Polymorphisms (SNPs) and microsatellite loci have been largely used to investigate the microevolution of he world –8, but the world , 10. Wind [Aedes .Aedes spp. [Ae. aegypti changes over short evolutionary time periods; 2) genetic microevolution can be assessed based on wing phenotype.Despite the usefulness of microsatellite loci and wing shape, these markers have been seldom combined in a single study of biological patterns of des spp. , and as des spp.  and Vidades spp. , we formAe. aegypti over 14 months using microsatellites and wing geometry as biological markers. The chosen population came from "Subprefeitura Butanta", a small, homogeneous and densely urbanised neighbourhood of Sao Paulo City (Brazil). Globally, this city is one of the largest urban agglomerations infested by Ae. aegypti, with approximately 11,000,000 people distributed over an area of 1,523 sq. km. There were 31,101 cases of dengue reported in this City in 2014, and more than 26,900 cases were reported from January to June 2015 [To test these hypotheses, we evaluated the temporal morphogenetic variations of a single population of une 2015 .Ae. aegypti were collected in “Subprefeitura Butanta”, a relatively small, geopolitically delimited neighbourhood comprising approximately 3% of the Sao Paulo municipality, which is homogeneously and densely urbanised . Specimens were obtained from six traps placed throughout the neighbourhood , winter 2011 (WIN), spring 2011 (SPR), summer 2011/12 (SUM) and autumn 2012 (AUT12) . For the loci described by Slotman et al. [Genomic DNA was extracted with the DNeasy Blood and Tissue Kit  or standard DNA isolation   estimated as described by Weir and Cockerham [st and D were computed using Genetix V4.05 [To assess the genetic stability over time, several analyses were done. Differentiation among chronological samples was examined by Wright’s F-statistics .The right wings of all 150 females were detached, mounted on slides-coverslip and photographed according to Vidal et al. . On eachWing size was estimated by calculating the “centroid size” vector . To asseWe estimated the allometric influence of wing size on wing shape through multiple regression analysis of the Procrustes coordinates vs. centroid size. We tested the statistical significance of allometry by means of nonparametric permutation tests with 10,000 iterations using MorphoJ V1.05 software. To geometrically describe wings independently from size variation, allometric effect was removed using multivariate regression.Ae. aegypti were compared regarding wing size and shape in order to evaluate the morphological variation during the observational interim (14 months). Univariate parametric statistic tests were used to compare centroid sizes: ANOVA with Tukey post-hoc test implemented using InStat [Chronological samples of g InStat .Discriminant analysis was used to explore the degree of wing shape dissimilarity among individuals in a morphospace of canonical variables . To better visualise the shape disparity among the samples, thin-plate splines were constructed based on the regression of CV1 scores on shape coordinates (TpsRegr V1.31 ). Discrist) and metric disparity among samples were estimated and statistically compared by nonparametric permutation tests  using COV software [Quantitative estimator of intra/inter group differentiation  in the morphospace of PCs is proportional to the morphological variability of that population set. In the present case, the amount of dispersion was estimated using the procedure developed by one of us (L.S.) in  and was st scores and morphological Qst scores. Based on the FCA plot, we calculated an index of “genetic diversity” using a similar method to that used to estimate the “morphological diversity” . These genetic and morphological diversities were then compared using linear correlation analysis (α = 0.05).In order to confront molecular and morphological data, a Pearson´s correlation analysis was done (α = 0.05) between phenetic and genetic distances . We also compared between FAll 149 individuals of the five chronological samples were successfully genotyped using 20 microsatellite loci. All loci were polymorphic and comprised in total 115 alleles , which were distributed as follows: 2 (AED19), 4 (C2A8), 7 (T3A7), 7 (A10), 13 (B07), 8 (B19), 5 (AC1), 3 (AC2), 2 (AC4), 6 (AC7), 5 (AG1), 18 (AG2), 5 (AG3), 4 (AG4), 5 (AG5), 7 (AG7), 3 (CT2), 5 (A1), 2 (B2) and 4 (B3). The following unique alleles were detected in AUT11: allele 229 ; allele 179 ; 168 ; 133 ; 121 and 133 ; 149 ; 176 , WIN: 177 ; 207 ; 131 ; 154  and SUM: 151   comparisons, and most loci (13 out of 20) were in HWE.Only some departures from equilibria were significant. The pair of loci AG1-AG3 was in linkage in all chronological samples. The B07, B19, AG1, AG2 and AG4 loci occasionally presented heterozygote deficits . The T3Ast), genetic distance (D), factorial correspondence analysis (FCA) and clustering (Bayesian model) based on allele frequencies indicated that genetic structure changed over time. Some private alleles were found but all of them appeared in low frequencies. Considering that high levels of null alleles and linkage of loci could bias our interpretation, we performed the analysis with and without C2A8, T3A7 and AG1 loci but the results were quite similar (not shown). The AG1 locus was discarded from the presented analysis  because it appeared in linkage not only with AG3 locus but also with other 6 loci.The analyses of genetic fixation . Pairwise D values calculated over all loci between samples ranged from 0.027 to 0.063 and revealed slight genetic differentiation. The UPGMA dendrogram based on the genetic distance matrix , as shown in st and morphological Qst, were lower in the sub-analysis .In order to evaluate the morpho-genetic syntopic variation (in a single trap location), we also did a sub-analysis with only the samples from trap T6. In this case, the most likely k was equal to 3, which is in accordance to the three seasons sampled in trap T6 . Thus, aThe mean shape temporal variation can be visualised in the thin-plate-spline series shown in st) was 0.4732.The phenogram of chronological samples based on Mahalanobis distances  corroborStatistical analysis  of the consecutive chronological comparisons revealed that wing size increased from AUT11 to WIN, remained stable during SPR, decreased to the lowest level in SUM and reached its highest point in AUT12 , which is the most syntopic sampling, is a clear demonstration of the how microevolution may occur during a short period (three consecutive seasons).The population genetics parameters  indicated that our genetic markers are representative and our geographically-restricted collecting has successfully sampled within a single deme. Variability at the microsatellite loci was high, and the rate of change was not homogeneous. Allelic frequency profiles  indicateAe. aegypti. Even in the colder season (WIN), the genetic variability was high, what is unexpected to an exotic species (Ae. aegypti is not native from the Neotropics), as introduced populations usually undergo genetic drift and founder effect. This may reflect a rich genetic patrimony and may imply adaptation of this species to new environments, which is a limiting factor to control efforts.It is not possible to do a complete comparison between our genetic results and the data available in the literature because, as far as we know, there are not other studies which comprise all the 20 loci set employed here. We can say that, in general, the population was highly polymorphic, because the observed allelic diversity and richness are seldom found in a single population of Ae. aegypti populations from other locations across the world [The loci used here exhibited, on average, similar polymorphism levels as those reported for he world , 39–42. he world  and popuhe world . At the he world , and popAe. aegypti over time has been occasionally studied. Ordinarily, allelic frequencies of microsatellites do not change greatly in a few years. For example, another population of Ae. aegypti from Sao Paulo State (Brazil) exhibited dimorphic SNPs that remained stable for 6 years [Ae. aegypti exhibited allelic variation during the transition of dry/wet seasons, possibly driven by a bottleneck effect [Aedes japonicus, which arguably occurred as a result of the merging of two genetically distinct lineages [Allelic variation in  6 years . Genetic 6 years  and betwk effect  and Olank effect  detectedlineages .Ae. aegypti from the Brazilian neighbourhood “Subprefeitura Butanta” changed over the study interval. This is the first investigative study of the temporal variation of the wing characters of Ae. aegypti.The wing shape/size patterns of Aedes albopictus [Regarding wing shape, the significant metric disparity among all samples and the high scores of cross-validated reclassifications showed that wing shape evolved and diverged during the 14 months interval, a remarkably short time period. The levels of temporal divergence were so high as to be compatible with partial population substitution, which, however, was not supported by the microsatellite results. The most similar case in the literature is a study of the congeneric bopictus , the winst indicator (0.4732), and the level of polymorphism remained high in each chronological sample during the study interval, as indicated by the morphological diversity estimates  is a nationally important centre of trade and people travelling. Even when a single trap was analysed (trap T6), both morphological and genetic variations occurred, indicating that microevolution can be detected over just a few syntopic generations.Apparently, the velocity of the microevolution of Biological and instrumental limitations prevent wings and microsatellites from being completely mutually congruent. The analysis and interpretation of microsatellites are not equivalent to that of wing morphometrics because of the distinct nature of these markers. Microsatellites are diploid, presumably neutral and independent multiallelic loci , whereas2 = 0.0845; r = -0.2908; p = 0.4151). The observed Qst value was several times higher than the Fst value, indicating that morphological variability was lower intra-sample than inter-sample. This pattern is typically interpreted as if natural selection is acting on wing shape [In fact, results of the two types of markers were discrepant and wings patterns changed faster. The oscillation of morphological diversity was uncorrelated to the genetic diversity. This analysis was not shown, but it is noticeable by comparison between Figs ng shape .Ae. aegypti from Sao Paulo City (including older samples from Subprefeitura Butanta) and observed Qst>Fst; however, wings morphology appeared to be more stable than microsatellite allelic patterns. To understand this partial contradiction between that study [Similarly, a previous paper of ours  analysedat study  and the at study  was atemIt would be reasonable to think that wing shape is under selective pressure because wings are important to flight and mating behaviour . HoweverAn alternative explanation for the observed rapid variation of the wing characters is that quantitative polygenic traits (as supposedly occurs to insect wings ) may exhAlthough the wing does not permit us to predict fine parameters of population genetics, it may be a semi-quantitative indicator of variation and variability. The fact that wing shape is sensitive to short temporal units ascribes discriminant power to these markers. However, the absence of correlation with neutral genetic markers indicate that wing shape may not be suitable to assess microevolution. This conclusion is different from that about the usefulness of wing geometry to macroevolutionary approaches. For instance, wing shape has been increasingly proven to be adequate to diagnose species and cryptic taxa , 51–55.Ae. aegypti individuals is not broad because Subprefeitura Butanta has numerous breeding sites. This assumption is in accordance with previous observations that dispersal is restricted when breeding sites are more available [In this specific ecological context, we assume that the dispersal of vailable , 56.Microevolution has relevant implications in epidemiology. Rapid allelic frequency changes may involve non-neutral characters and occasionally affect the susceptibility of mosquitoes to insecticides and chemical repellents. The successful insertion of transgenic or benign mosquito lineages may also be influenced by microevolution.Ae. aegypti should be further investigated and taken into consideration when redesigning vector control and surveillance protocols.The development of these processes is multi-causal, and a central factor is the ecological context. A comprehensive understanding of the ecological context requires careful studies that consider biotic/abiotic local peculiarities. We propose that not only the static biological patterns but also the evolutionary dynamics of S1 FigSignificant distinctions: AUT11 X AUT12 p<0.01, WIN X SUM p<0.01, SPR X SUM p<0.01 and SUM X AUT12 p<0.001.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Tableo, observed heterozygosity; He, expected heterozygosity; Fis, inbreeding coefficient. In bold, significant p-values (α<0.05) following Bonferroni correction (corrected α<0.0005) rejecting Hardy—Weinberg equilibrium.—not applicable.N, sample size (number of mosquitoes); AR, allelic richness; H(XLSX)Click here for additional data file.S3 TableIn bold, significant p-values (p>0.01).—not applicable.(XLSX)Click here for additional data file.


A Mediterranean dietary pattern is widely recommended for the prevention of chronic disease. We sought to define the most likely effects of the Mediterranean diet on vascular disease and mortality.We searched MEDLINE, EMBASE and the Cochrane Central Register without language restriction for randomized controlled trials comparing Mediterranean to control diets. Data on study design, patient characteristics, interventions, follow-up duration, outcomes and adverse events were sought. Individual study relative risks (RR) were pooled to create summary estimates.Six studies with a total of 10950 participants were included. Effects on major vascular events (n = 477), death (n = 693) and vascular deaths (n = 315) were reported for 3, 5 and 4 studies respectively. For one large study (n = 1000) there were serious concerns about the integrity of the data. When data for all studies were combined there was evidence of protection against major vascular events , coronary events , stroke  and heart failure  but not for all-cause mortality  or cardiovascular mortality . After the study of concern was excluded the benefit for vascular events  and stroke  persisted but apparently positive findings for coronary events  and heart failure  disappeared.The Mediterranean diet may protect against vascular disease. However, both the quantity and quality of the available evidence is limited and highly variable. Results must be interpreted with caution. Cardiovascular disease (CVD) is a leading cause of mortality worldwide accounting for one quarter of the estimated 52 million deaths in 2010.Populations in the Mediterranean region have been reported to experience lower morbidity and mortality rates from CVD compared with northern European populations, which hAlthough there are randomized controlled trials (RCTs) assessing the effect of the Mediterranean diet on the primary or secondary prevention of CVD,14 the lwww.prisma-statement.org)  . Relevanent.org) . The seaThe literature search, data extraction and quality assessment were conducted independently by two authors using a standardised approach (TL and AW). All completed RCTs assessing the effects of a Mediterranean diet compared to any control diet involving adults, with follow up period longer than three months, that reported one or more of the relevant outcomes were eligible for inclusion. Although there is considerable variability, a diet with non-restricted fat intake and with at least two of the following seven components was defined as a Mediterranean style diet for the purposes of this review; 1) use of olive oil as the main cooking oil, 2) moderate red wine consumption, 3) high consumption of nuts and/or legumes, 4) high consumption of grains and cereals, 5) high consumption of fruits and vegetables, 6) high consumption of fish with low consumption of meat and meat products and 7) low to moderate consumption of milk and dairy products.Published reports were obtained for each trial and standard information about each trial was extracted into a spreadsheet. The data sought included summary metrics for each trial about baseline patient characteristics , details of the dietary intervention, follow-up duration, outcome events and adverse events. Study quality was judged according to the Cochrane risk of bias assessment tool. Any disWe sought data on total cardiovascular events , coronary events , cerebrovascular events , heart failure, total mortality, incidence of type 2 diabetes mellitus, end-stage kidney disease, adverse effects  and quality of life.Individual study relative risks (RRs) and 95% confidence intervals (CIs) were calculated from event numbers extracted from each trial. In calculating RRs, the total number of patients randomized in each group was used as the denominator. If no events were reported in either treatment or control group of any trial, 0.5 was used as the numerator. For the three arm PREDIMED study, the oliThe literature search yielded 4637 articles, of which 51 were reviewed in full text . Of thesThe trials had a sample size that ranged from 48 to 7447 participants . Two stuIn regard to study quality , four of·63, 95% CI: 0·53–0·75, p< 0·001) compared to control diet. Exclusion of the study with serious concerns about its integrity  did not result in a change of overall direction of the effect   .·00, 95% CI: 0·86–1·15, p = 0·97) or cardiovascular mortality  compared to control  (·65 (95% CI 0·48–0·88) for cerebrovascular events. Only two studies[·30 (95% CI: 0·17–0·56). Exclusion of the data from the trial by Singh et al resulted in null findings for all of the cause specific outcomes except cerebrovascular events which continued to remain significant    for coroo studies,19 invol = 0·01) .·005) but no effect on any other domain of that score or the Medical Outcome Study (MOS -12) short form general health survey[One study that enrolled 279 participants reported data on quality of life. There wth survey  are three societies that all recommend the Mediterranean diet to reduce cardiovascular risk. All are careful to base this advice on the observed favorable although modest effects of the Mediterranean diet on intermediate cardiovascular risk factors. Clearly, recommendations based upon definitive clinical outcomes would be far preferable, and are supported by the findings of this review, however the strength of conclusions should remain circumspect because of the weakness of the evidence base. It seems unlikely that clinicians and patients fully appreciate the limitations of the data and the extent of the uncertainty. This is an area that warrants another well-conducted, adequately powered trial able to precisely and reliably define the overall balance of benefits and risks associated with the Mediterranean diet.The American Heart Association/American College of Cardiology, the Eurhttp://content.onlinejacc.org/article.aspx?articleid=2198774This work has been presented at the American College of Cardiology Annual Scientific Sessions in 2015 in abstract form. The abstract is available from the following link: S1 Fig(TIFF)Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 File(RTF)Click here for additional data file.S4 File(DOC)Click here for additional data file.


Using Illumina sequencing, we characterized and compared the bacterial community from two Lobophora  species. The two species presented distinctive bacterial communities. Both species shared approximately half of their OTUs, mainly the most abundant bacteria. Species-specific OTUs belong to Planctomycetes, Proteobacteria, and Bacteroidetes. In total, 16 culturable bacterial strain were isolated and identified from the Lobophora surface, consisting of 10 genera , some of which are not known as, but are related to pathogens involved in coral diseases, and others are naturally associated to corals. When patches of marine agar with 24 h cultures of each of these bacteria were placed in direct contact with the branches of the scleractinian coral Acropora muricata, they caused severe bleaching after 24 h exposure. Results suggest that regardless of taxonomic affinities, increase in density of these bacteria can be adverse to corals. Nevertheless, the microbial community associated to macroalgal surface may not represent a threat to corals, because the specific bacterial screening and control exerted by the alga preventing specific bacterial proliferation.While reef degradation is occurring worldwide, it is not uncommon to see phase shifts from coral to macroalgal dominated reefs. Numerous studies have addressed the mechanisms by which macroalgae may outcompete corals and a few recent studies highlighted the putative role of bacteria at the interface between macroalgae and corals. Some studies suggest that macroalgae may act as vectors and/or foster proliferation of microorganisms pathogenic for corals. Using a combination of high throughput sequencing, bacterial culturing, and  Competition between benthic macroalgae and corals, two ecosystem engineers of tropical reefs, is a key process shaping the structure of reef communities. Contrary to healthy reefs where macroalgae and corals maintain a stable coexistence, in disturbed reef ecosystems, macroalgae often gain dominance over scleractinian corals. While declines in coral cover are generally associated with increases in the abundance of fleshy Hughes, , and cruLobophora  plays a particularly important ecological role in algal–coral–grazing interactions and competition  represents one of the most important algal groups, and the genus Lobophora-associated bacteria, characterizes the bacterial community associated to the macroalgae, and assesses the adversity of bacterial isolates of this macroalgal-holobiont on the Scleractinian coral Acropora muricata Linnaeus  and Lobophora rosacea in Ricaudy at 2 m depth  under a biological safety cabinet.This study was performed in Noumea lagoon, New Caledonia, where two species of L. rosaceae and L. monticola) using the Quick-gDNA kit (Zymo Research™) applying the manufacturer protocol.DNA was extracted from five freeze-dried replicates (a replicate = a unique specimen) of each species  on a MiSeq following the manufacturer's guidelines.The total 16S rDNA region was amplified using the universal primers 27F and 1494R Lane,  with theAll the diversity analyses were performed using the program QIIME 1.8.0: Quantitative Insights Into Microbial Ecology  and the Simpson indices of diversity (1 – D). The non-parametric ACE and the Chao 1 richness index were calculated with the software estimates  was performed in R  analysis was performed in R using the “vegan” package.In order to obtain direct descriptors of the diversity of bacterial assemblages, we calculated two widely used diversity indices, the Shannon  plate. Then the swab cotton was introduced into a sterile Falcon tube with 10 mL of Marine Broth  and shaken vigorously for 1 min and incubated 48 h at 30°C, under shaking conditions (120 rpm). The second method consists of inoculating a MA petri dish by directly placing an algal thallus on it. After firmly pressing it against the agar, the thallus was left 5 min on it. The process was renewed with the other side of the thallus on the same petri dish and the plates were incubated at 30°C during 48 h. In the third method, a thallus was placed into a 15 mL sterile tube with 10 mL of sterile saline water and shaken for 1 min. Three MA plates were inoculated by spreading, respectively, 100 μL of the 1/10, 1/100 dilutions and pure suspension. Plates were then incubated 48 h at 30°C.After the 48 h incubation, bacteria presenting visually diverse colony morphologies were selected for isolation. A small portion of a bacterial colony was gently scooped with a disposable sterile inoculating loop and streaked into a MA plate, which was then incubated 48 h at 30°C. This process was renewed two more times to assure pure isolates. Several colonies of a given bacteria were then placed into 10 mL of MB within a 50 mL sterile tube and incubated 24 h at 30°C. Hundred microliters of the resulting culture was spread onto a MA plate and incubated 24 h at 30°C. Finally, colonies picked from the marine agar plates and introduced in 800 μL of MB or 800 μL of bacteria from the liquid culture were put together with 800 μL sterile 25% glycerol solution into a cryotube and stored at −80°C.Bacterial DNA was extracted with the DNeasy® Blood and Tissue Kit , following the manufacturer's instructions. The 16S rRNA gene (1500 pb) was used for bacterial identification. Amplification of the 16S rRNA gene by PCR, using the universal 16S rRNA primers Table , was fol2 were cut from the petri dish under the biosafety cabinet and put into sterile re-sealable zipper storage bags and stored into a cooler until application. The agar strips were then applied on the coral branches and fixed to it with sterile labeled tulle bands  test. Statistical analyses were performed using R.Pulse Amplitude Modulated (PAM) fluorometry was used to assess the effects of bacteria on coral health (effective quantum yield). PAM fluorometry measurements were carried out with a Diving-PAM  directly after removal of the strips. PAM fluorometry measures the photosynthetic efficiency of photosystem II within the endosymbiotic Lobophora specimens studied, 3247 and 3313 OTUs were identified from L. rosacea and L. monticola, respectively. On average each sample had 1386 ± 162 OTUs (Table H′), and 0.977 ± 0.010 and 0.989 ± 0.003 (1 – D), for L. monticola and L. rosacea, respectively. The Shannon  and Simpson  indices were not statistically significant different between the two species. The non-parametric estimators predicted an average number per sample of 2777 ± 606 and 2839 ± 386 OTUs (ACE), and of 2805 ± 646 and 2881 ± 604 (Chao1) OTUs for L. monticola and L. rosacea, respectively. Chao1  and ACE  both richness estimators were not statistically significant different between the two species. Comparison of the core OTUs shared by at least four replicas per species between the two Lobophora species is presented in Figure 467520195.3–4675204.3.In a dataset of more than 644,377 sequences obtained from the 10 Us Table . ShannonR = 0.488−0.94; p = 0.006−0.012) between the two Lobophora species. The most influential taxa, determined with the SIMPER analysis, are given in Table Lobophora species is species-specific. The four analyses resulted in the classification of the ten samples into two distinct clusters corresponding to the two Lobophora species. The first component that contributed significantly to explaining the relationships among the samples , by clearly disjointing the two species, accounted for 40–54% of the variance depending on the taxonomic level, with a stronger value at the phylum level  were not detected and four of these families were abundantly characterized in the Illumina 16S rRNA amplicon sequencing .Bacteria grew quite well after 48 h of incubation, as visually assessed. We managed to culture 20 strains, 10 for each species of ns Table . The twoA. muricata in direct contact with each of the macroalgae-associated culturable bacterial patches, showed severe visual bleaching ,  and the other to evenness (Simpson) were not significantly different between the two species. In contrast, multivariate analyses conducted on Illumina sequencing results clearly showed the species-specificity of the bacterial assemblages associated to individuals of L. rosacea and L. monticola. The two species mainly differ in the most abundant bacterial taxa among those shared by both species. Results indicate that the two Lobophora species have comparable richness of bacterial assemblages but contrasting composition. The two Lobophora species grow in habitats with contrasting environmental conditions. Considering the habitat specificity of these two species, it is questionable whether the bacterial assemblage species-specificity could be linked to the different niches that the two species inhabit. Transplant or common garden experiments will allow concluding whether/to what extent Lobophora species bacterial community composition is controlled by ecological factors or the evolutionary history of the host.This is the first bacterial community characterization of species from the genus Lobophora species. They also shared four bacterial families , three genera , and two OTUs (Microbulbifer sp. and Ruegeria sp.1). Barott et al. , and established a microbial fingerprint for each group. When comparing their results with ours it is clear that our Lobophora fingerprints (relative abundance percentage) do not resemble any of the algal groups studied by Barott et al. , but did not present Firmicutes. Furthermore, Lobophora presented one additional major phylum (Verrucomicrobia) not detected in the other algal groups.Similar phyla were found with relatively similar percentages between the two t et al.  performeAcropora spp., and the Acroporid white syndrome affecting A. hyacinthus. While none of the isolated bacterial OTUs have yet been reported in the literature as coral pathogens the majority of the isolated genera   and Alteromonadales (10 and 9%), and three rare orders . Similarly, Barott et al. (Dictyota species harbored the highest percentages (39.6 and 40.8%) of potential pathogens associated with coral diseases, which supports the idea that members of the Dictyotales hold a reservoir of potential coral pathogens.The present bioassay results evoke symptoms of “white” diseases, such as the white plague syndrome affecting massive and encrusting corals, the white band disease affecting r) Table  were doct et al.  showed tIt is only recently that the microbial component has been considered in the interactions between algae and corals, and we have only begun to scratch the surface of understanding the complex interactions between coral, algae, and microbes Table . Recent Lobophora hosts surface and core bacterial orders present in diseased and bleached corals, and epibacteria experimentally capable of bleaching corals, and that (2) all tested bacteria can equally induce strong and quick coral bleaching. The bleaching potential from the tested bacteria is apparently not restricted to bacteria from the genus Lobophora considering the panel of bacteria isolated which belong to a variety of taxa but also the natural presence of some of these taxa in healthy corals. In other words, regardless of their taxonomic affinity, dense mature bacterial films can intrinsically bleach corals. But while these results seem to concur with recent findings on the role of macroalgae as conveyors and fosterers of coral pathogens, this does not imply necessarily that macroalgae represent a threat to corals. The mere presence of bacteria is not a threat, as the consequences depend on conditions allowing them to proliferate. In fact, corals themselves also naturally host pathogenic bacteria that can be detrimental to the health of the host under certain conditions. Nonetheless, only specific bacteria have actually demonstrated the capacity to efficiently proliferate and bleach corals. Smith et al. (Our findings suggest that (1) h et al.  previoush et al. , bacterih et al.  supportsh et al. , and theLobophora natural products have experimentally demonstrated a broad-spectrum of antibacterial activities  and abiotic  factors is regulating microbial composition and abundance on both the coral and the alga Ritchie, . ComparaFuture studies should be directed at exploring and clearly identifying factors that lead to changes in the microbial community composition.in situ bioassays; AE, ES, and TA performed the Illumina sequencing analyses; AE, ES, TA, and CV analyzed the Illumina sequencing data; CV and FH analyzed the bioassays data; and CV, AE, TA, LG, ES, FH, CP, and OD co-wrote the paper. All authors critically revisited this work for important intellectual content and approved the final version to be published. All authors agree to be accountable for all aspects of the work.CV, AE, OD, and CP designed research; CV, LG, FH, and JG performed the bacterial culture and the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Heparin is routinely administered in postoperative abdominal surgery aiming to prevent adhesions formation; however, there is no consensus indicating its effectiveness. This study evaluated the effect of heparin on peritoneal reactivity after abdominal surgery, through the association between peritoneal fluid features and ultrasonographic and laparoscopic examination. Ten adult horses were used: control group (CG) and treated group (TG). Both groups underwent laparotomy and small colon enterotomy. TG received subcutaneous heparin at 150 IU/kg every 12 hours for 5 days. The animals underwent ultrasonography and peritoneal fluid examination prior to enterotomy (M0) 12 hours (M1), 1 day (M2), 2 days (M3), 4 days (M4), 6 days (M5), 10 days (M6), and 14 days after enterotomy (M7) with laparoscopic examination being performed on the fifth postoperative day. Peritoneal inflammatory response was observed in both groups. The peritoneal fluid of TG animals showed higher echogenicity during heparin therapy. No inflammatory difference was observed between groups through peritoneal fluid features, except for the higher D-dimer concentration in CG. On laparoscopy, slightly diffuse peritoneal reactivity for both groups was observed, being higher for TG. Laparoscopy and ultrasonography association allowed detailed access to the abdominal cavity. Ultrasonography assessed the diffuse peritoneal inflammation, and laparoscopy allowed the detailed analysis of the segments. No gross beneficial reactions resulting from the use of heparin on peritoneal reactivity were observed; however, it was observed by D-dimer evaluation that the TG had less fibrin deposition, which is directly related to a lower rate of abdominal adhesions formation. Enterotomies are often employed for enteroliths, foreign bodies, and impacted material removal , 2. SurgAfter surgical trauma, peritoneal inflammation occurs concurrently with the coagulation process focusing on tissue reparative order. Thrombin is the major link between inflammation and coagulation system, since the enzyme is responsible for cleavage of circulating soluble fibrinogen to insoluble fibrin clot . The coaIf a persistent postsurgical inflammatory stimulus is present, an imbalance between the formation and lyses of fibrin may occur, giving rise to fibrinous adhesions which subsequently form the basis for fibrous abdominal adhesions development –10.In this sense, the use of anticoagulants in order to prevent fibrin clots formation possibly inhibits adhesions formation , 11. HepDue to the large size of the horse abdomen, there is no method able to evaluate fully the abdominal cavity, being necessary the combination of different methods to better evaluation. It is described that the association of ultrasonography and laparoscopy allows greater accuracy in horses digestive system diseases evaluation , 15.Abdominal ultrasonography is a noninvasive and widely available modality that can easily integrate the examination of the abdominal cavity , 16. LapThe physical chemical and cytological evaluation of peritoneal fluid  is seen Besides this, quantification of D-dimers can also be used as a tool for peritoneal response evaluation; D-dimer is a fragment that is released exclusively during fibrin lysis via the action of plasmin. In the serum, plasmin levels are correlated with the destruction of fibrin after hyperfibrinogenic and hypercoagulable states and serve as a marker of coagulation and fibrinolytic activity –23.Few studies have been conducted using peritoneal fluid, and most of these have focused on human patients. However, measurement of the D-dimer concentration in the peritoneal fluid of equines with severe gastrointestinal disorders demonstrated marked hyperfibrinolysis related to increased fibrin formation and degradation . A D-dimThis study aimed to evaluate heparin effect on peritoneal reactivity after laparotomy and experimental small colon enterotomy, associating peritoneal fluid features, laparoscopy, and ultrasonography examinations, since heparin is routinely used in the postoperative of abdominal surgery as a prophylactic strategy for adhesion formation inhibition, but without the existence of consistent results of its effectiveness .Ten adult horses underwent a laparotomy and experimental small colon enterotomy in the standing position. Animals were divided into two groups of five animals: control group (CG) and treated group (TG). The TG immediately after surgery and every 12 hours for 5 days received heparin at a dose of 150 IU/kg subcutaneously. The CG did not receive treatment.2 (6.6 mg/kg intravenously), and flunixin meglumine (1.1 mg/kg intravenously). The procedure was performed in the standing position and under continuous infusion sedation with detomidine (5 μg/kg bolus followed by continuous infusion of 20 μg/kg/h) associated with butorphanol (20 μg/kg followed by continuous infusion of 13 μg/kg/h). After antisepsis, local anesthesia was performed on line incision and adjacent deep tissues with 2% lidocaine associated with vasoconstrictor.Prior to surgery, the right abdominal region was shaved and the horses of both groups received a combination of potassium penicillin (30.000 IU/kg intravenously), gentamicin7 was applied. After suturing, the serosal layer was subjected to abrasion with dry gauze in 50 repeated moves for each adjacent portion of the band, which results in a hemorrhagic local serosa. The segment was repositioned and abdominal wall synthesis was performed in Sultan pattern with number 2 polyglactin 910; skin suture was performed in Wolff pattern with 0-monofilamentar nylon.A ten cm incision was performed on paralumbar fossa skin; subcutaneous tissue was divulsioned and muscle sectioned; the peritoneum was punctured and abdominal cavity accessed. Small colon identification was proceeded by direct palpation, and the further aboral segment was externalized; after delimitation and emptying of the enterotomy site segment, two coprostasis forceps were applied in order to prevent fecal flow. Subsequently two Allis clamps were applied for segment positioning and enterotomy realization in the band region; a simple separate suture pattern with 3–0 CatgutAfter surgery, benzathine penicillin was administered (30.000 IU/kg intramuscularly every 72 hours for 10 days), and gentamicin (6.6 mg/kg intravenously every 24 hours for 05 days), flunixin meglumine (1.1 mg/kg intramuscularly every 24 hours for 5 days), and antitetanus serum (10.000 UI subcutaneously in a single application) were administered for both groups.The animals underwent ultrasonography and peritoneal fluid evaluation prior to enterotomy (M0); 12 hours (M1); 1 day (M2); 2 days (M3); 4 days (M4); 6 days (M5); 10 days (M6), and 14 days after enterotomy (M7).The ultrasonography evaluation of small colon exam was performed with a convex transducer (5 MHz and 25–35% gain). To review the peritoneum and free peritoneal fluid a linear transducer (13 MHz and a gain of 80–90%) was used.Ultrasonography examination was performed by transabdominal via and aimed to evaluate the small colon and peritoneal fluid. The small colon was visualized in the left dorsal quadrant, using as reference the left kidney, since the small colon is usually located caudal to it and dorsal to pelvic flexure. The small colon was evaluated for its topographical location, architecture, and wall thickening.The parietal peritoneum was evaluated in the ventral portion of the abdomen. The presence of irregularities, fibrin deposition, and peritoneal thickening from possible peritoneal inflammation were evaluated. In the same region, the quantity, echogenicity, and presence of debris on free peritoneal fluid were assessed.For ultrasonographic findings standardization a score scheme to quantify peritoneal fluid and evaluate its echogenicity was created.Because the dynamic feature of ultrasonography examination, it was observed that during intestinal peristalsis the distance between the peritoneum and the viscera adjacent to the ventral region variated, showing not to be a good evaluation tool to quantify peritoneal fluid. The association of videos findings, notes, and images obtained during ultrasonography examination was used for quantification.The amount of free peritoneal fluid was standardized in a graduation system as follows: −1 (reduced), 0 , 1 (slightly increased), 2 (moderately increased), and 3 (strongly increased), and normality was standardized as the more frequently image observed in animals prior to surgery .Echogenicity was graded in a system of scores, as follows: 0 , 1 (slightly increased), 2 (moderately increased), and 3 , and normality was established as the appearance of liquid prior to surgical procedure .The peritoneal fluid samples were collected at the most ventral region of the abdomen using a 40 × 12 mm hypodermic needle and tubes containing 3.8% sodium citrate at a 9 : 1 (liquid : anticoagulant) ratio (v/v) for D-dimer evaluation, EDTA tubes for cytology count, and tubes with no anticoagulant for physicochemical tests. The samples for the D-dimer were centrifuged for 15 minutes immediately after collection, using a 1000 G rotation. D-dimer analysis was performed using the latex agglutination test, according to the manufacturer's instructions.Fifteen days after the initial surgery, a new surgical approach was performed. The animals received 700 g of magnesium suphate 36 hours before the procedure and were subjected to 24 hours fasting, in order to small colon emtying and easier handling of the segments. Prior to surgery, the left abdomen was shaved. Rectal palpation was proceeded in order to assess the anatomic topography at the site of cannulae insertion, avoiding iatrogenic punctures. The procedure was performed in standing position with identical sedation protocol to that used in laparotomy.2, Light Source Xenon 180 W, illumination cable, microcamera with processor, rigid endoscope  cannula EndoTIP TM (10 mm), laparoscopic and conventional surgical instruments, TV monitor, image recording system comprising a notebook, and a capture plate.Laparoscopic equipment was composed of electronic insufflator COThe surgical access area underwent local anesthesia with 2% lidocaine associated with vasoconstrictor. Antisepsis was performed; the skin incision was approximately 2 cm; the access was created through the introduction of video-assisted cannula (EndoTIP TM) with rigid endoscope inside. The pneumoperitoneum was induced. The abdominal pressure was maintained during the procedure between 12 and 15 mmHg, varying according to each animal supported volume. A systematic evaluation of abdominal cavity was established to perform a complete scan of structures amenable to evaluation through the left observation.After abdominal cavity evaluation, two new anesthetic blocks points of approximately 1.5 cm where performed for cannulas insertion (11 mm); atraumatic intestinal graspers (Babcock) were introduced. A detailed evaluation of the small colon and enterotomy region was performed with Babcock forceps, supporting mesocolon dorsally; the optical was advanced to the right side of the abdomen in order to observe the laparotomy wound region. The images were stored for later evaluation.2; the cannulas were removed and finally withdrew the EndoTIP by performing rotational movements that promoted muscle layers approximation; the skin synthesis was performed in simple separated standard with nylon 0.The graspers were removed and the safety valves of the cannulas were opened allowing the release of the COIn order to facilitate the images evaluation scores of diffuse peritoneal reactivity were created, being 0 (no diffuse reactivity), 1 (mild diffuse reactivity), and 2 (moderately diffuse reactivity) .P < 0.05.The Wilcoxon test  was usedThe applied ultrasonographic methodology allowed the evaluation of small colon segments in approximately 85% of patients in different times, and possibly among these, only in a small proportion, the segment subjected to enterotomy was found, being rarely observed segments with wall thickening and architecture regularity loss.Ventral abdomen ultrasonograph examination allowed the assessment of peritoneal fluid quantity, echogenicity, homogeneity, flocculation, and fibrin presence.No difference between groups was observed for the amount of free peritoneal fluid; however an individual variation among animals was observed. Based on employed graduation scale it was found that animals maintained their individual volume regardless of the surgical procedure. After the 10th day after surgery there was a slight increase in the amount of free fluid in both groups, which is represented by the increase of one score in scale.Relying on the proposed graduation scale, peritoneal echogenicity increased after 12 hours of surgery for both groups. Increased echogenicity persisted until the 14th day, however, with progressive reduction after the 6th day postoperatively. Although there was no significant difference between groups, the TG showed persistence of increased echogenicity in relation to the CG .Fibrin clots and irregularities in the parietal peritoneum were present between the 4th and 6th day in both groups.P < 0.0470), with 48% of red samples in CG and 72% for TG. In subsequent analysis, peritoneal fluid acquired gradually decreasing turbidity and coloration tends to be normal.After 12 hours of surgery, all peritoneal fluid samples presented red color; for TG the red color was maintained throughout the treatment period. Significant difference was obtained between groups  when all times were combined  and at M3 (P < 0.0358) and M4 (P < 0.0122)  .P = 0.0745) between groups when all time-points were combined or not   with detd or not .No changes in visceral topography were observed on laparoscopic exam. The quantity and quality of peritoneal fluid evaluated macroscopically were compatible with ultrasonographic findings and did not differ between groups.During small colon manipulation with atraumatic forceps, it was observed that adjacent regions to enterotomy had become covered with a thin layer of fibrinous exudate and presented reactive and friable to manipulation, rapidly becoming bleeding in both groups, but with greater intensity in TG.Although there was no significant difference in diffuse peritoneal reactivity between groups, there was evidently greater reactivity in TG. CG showed 60% of score 0, 20% of score 1, and 20% of score 2 and TG showed 60% of score 1 and 40% of score 2.Invariably, the region of the enterotomy to both groups was covered by fibrin. On the 15th day after enterotomy the suture performed with catgut was preserved. No difference between groups was observed for fibrin deposition over enterotomy region .Moore and Hinchcliff  describeAlthough transrectal ultrasonography is the most appropriate method for evaluating small colon , transabIncreased echogenicity observed 12 hours after surgery in both groups was assigned to cellular migration in response to abdominal inflammatory process and intracavitary surgical bleeding. The difference observed between groups for echogenicity possibly occurred due to heparin treatment, which resulted in maintenance of the displacement of RBCs for peritoneal fluid, since the high erythrocyte count, macroscopic aspect, and a gradual decreased in echogenicity for TG after the suspension of treatment.Despite the fact that the difference in echogenicity did not show statistical significance between groups, it was observed that the TG showed higher echogenicity. Probably a study with a large number of animals should demonstrate statistical difference.Postoperative peritonitis, fibrin presence, and peritoneal irregularities observed during this study corroborate with literature information that reports increased echogenicity due to high cellularity, loss of homogeneity, fibrin deposition, and presence of cellular debris , 31.Although TG had presented higher peritoneal fluid echogenicity, it cannot be inferred by ultrasonographic examination that these animals had higher peritoneal reactivity, since the high liquid cellularity corresponded mainly to erythrocytes, with no difference between groups for the presence of fibrin, cellular debris, and thickening of the peritoneum and intestinal walls.Ultrasonography allowed peritoneal cavity inflammation and bleeding due to heparin treatment identification but did not allow detailed tracing of small colon. Transabdominal ultrasound use may have influenced the sensitivity to detect small colon changes.Although there was no significant difference in the D-dimer concentration between groups, higher values tended to be observed for animals in the CG, which is consistent with the notion that abdominal surgery results in a state of hyperfibrogenesis and hyperfibrinolysis . BecauseD-dimer values are directly related to poor prognosis and high mortality; the indication for the use of heparin in hypercoagulable states and after abdominal surgery in equines was strengthened by the results of our study.Although the animals in the present study did not show abdominal adhesions, our results highlight the potential use of heparin for prophylaxis in cases of abdominal adhesions. However, further clinical studies should evaluate the use of heparin in equines suffering from colic syndrome and those subjected to laparotomy.Laparoscopy was performed through the left abdominal approach, a procedure which according to Galuppo et al.  allows sLaparoscopic examination allowed a detailed abdominal cavity and enterotomy area analysis, demonstrating no significant difference on fibrin deposition over enterotomy region; however without statistical significance, more diffuse peritoneal reactivity to visceral manipulation on TG was observed.The association of peritoneal fluid evaluation, laparoscopy, and ultrasonography allowed detailed access to the abdominal cavity. Ultrasonography and peritoneal fluid evaluation allowed the assessment of diffuse peritoneal inflammation, while laparoscopy allowed the evaluation of peritoneal inflammation and a detailed analysis of the segment subjected to enterotomy.No gross beneficial reactions resulting from the use of heparin on peritoneal reactivity were observed; however, it was observed by D-dimer evaluation that the TG had less fibrin deposition, which is directly related to a lower rate of abdominal adhesions formation.


Low uptake of routinely recommended adult immunizations is a public health concern. Using data from the peer-reviewed literature, government disease-surveillance programs, and the US Census, we developed a customizable model to estimate human and economic burden caused by four major adult vaccine-preventable diseases (VPD) in 2013 in the United States, and for each US state individually. To estimate the number of cases for each adult VPD for a given population, we multiplied age-specific incidence rates obtained from the literature by age-specific 2013 Census population data. We then multiplied the estimated number of cases for a given population by age-specific, estimated medical and indirect  costs per case. Adult VPDs examined were: (1) influenza, (2) pneumococcal disease (both invasive disease and pneumonia), (3) herpes zoster (shingles), and (4) pertussis (whooping cough). Sensitivity analyses simulated the impact of various epidemiological scenarios on the total estimated economic burden. Estimated US annual cost for the four adult VPDs was $26.5 billion (B) among adults aged 50 years and older, $15.3B (58 %) of which was attributable to those 65 and older. Among adults 50 and older, influenza, pneumococcal disease, herpes zoster, and pertussis made up $16.0B (60 %), $5.1B (19 %), $5.0B (19 %), and $0.4B (2 %) of the cost, respectively. Among those 65 and older, they made up $8.3B (54 %), $3.8B (25 %), $3.0B (20 %), and 0.2B (1 %) of the cost, respectively. Most (80–85 %) pneumococcal costs stemmed from nonbacteremic pneumococcal pneumonia (NPP). Cost attributable to adult VPD in the United States is substantial. Broadening adult immunization efforts beyond influenza only may help reduce the economic burden of adult VPD, and a pneumococcal vaccination effort, primarily focused on reducing NPP, may constitute a logical starting place. Sensitivity analyses revealed that a pandemic influenza season or change in size of the US elderly population could increase these costs dramatically. Health care in the United States is facing serious challenges: access and affordability issues, discordant reimbursement systems, unnavigable health care delivery, complex reform, and underutilization of prevention, to name only a few. Although it is intuitive to scrutinize direct delivery systems and individual health outcomes to look for solutions, the use of population health analysis generally provides the best perspective on where the greatest impact with health care reform can be achieved. In a health care environment of limited resources and a rapidly aging population, it is critical to begin exploring ways to obtain data about public health interventions to ensure appropriate stewardship of scarce health care dollars. According to the Centers for Disease Control and Prevention (CDC), low uptake of routinely recommended adult immunizations remains one area of public health practice that has been insufficiently addressed at local, state, and national levels CDC, . We deteInfluenza (flu) is a contagious respiratory illness caused by influenza viruses. The best way to prevent influenza is by getting vaccinated each year per recommendation by CDC Advisory Committee on Immunization Practices . Pneumonia with empyema and/or bacteremia, febrile bacteremia, and meningitis are the most common manifestations of invasive pneumococcal disease (IPD). Nonbacteremic pneumococcal pneumonia (NPP), middle-ear infections, sinusitis, and bronchitis are non-invasive and less severe manifestations of pneumococcal infection, but are considerably more common than IPD. As of September 2014, ACIP now recommends that adults 65 years and older receive both 13-valent pneumococcal conjugate vaccine (PCV13) and 23-valent pneumococcal polysaccharide vaccine (PPSV23). Adults 65 and older who have never received a pneumococcal vaccination should receive PCV13 first, followed by PPSV23 6–12 months later. Adults who are already vaccinated with PPSV23 should receive PCV13 at least a year later  is caused by the varicella zoster virus, which also causes chickenpox. For reasons that are not fully understood but that are likely due, in part, to immunosenescence with age, the virus can reactivate years after chickenpox, manifesting as shingles. The most common complication of herpes zoster is post-herpetic neuralgia (PHN) which causes severe and debilitating pain for weeks, months, or (in rare cases) years. PHN occurs in as many as a third of untreated adults aged 60 and older. Herpes zoster may also lead to serious complications involving the eye and, very rarely, pneumonia, hearing problems, blindness, brain inflammation , or death. ACIP recommends routine vaccination of all persons 60 and older with one dose of zoster vaccine  dose of tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis (Tdap) vaccine. In addition, certain adults 65 and older who anticipate having close contact with an infant  should receive a single dose of Tdap to protect against pertussis and reduce the likelihood of transmission  at least one US Food and Drug Administration (FDA)-approved vaccine available for the prevention of vaccine-type disease, and (2) broad, age-based ACIP-recommendations for use in adults. Simply stated, for the four adult VPDs chosen, vaccination represents a safe and effective medical strategy for disease prevention that remains underutilized, and more information is needed to understand the true burden of these diseases—with the ultimate goal of raising awareness about the need for improvements in prevention efforts.We derived burden of disease estimates for four major adult VPDs: (1) influenza, (2) pneumococcal disease , (3) herpes zoster (shingles), and (4) pertussis (whooping cough). We obtained estimates of adult VPD incidence and associated costs from the literature  for each of the four diseases  the estimated number of cases for a given population per year, (2) the estimated medical costs of a single case , and (3) estimated indirect costs associated with a single case. The combination of these three estimates provided the backbone for the economic model that estimated the annual burden of adult VPD from a societal perspective. The basic formula for estimating total cost based on these three measures was: the estiFor each of the four major adult VPDs, we presented results for two age groups: individuals aged 50 and older and those 65 and older. We chose these age groups for two main reasons. First, these age groups closely align with FDA indications and ACIP recommendations for vaccines that protect against the four major adult VPDs evaluated in this study. Secondly, these are the most commonly used age groups when discussing adult immunizations as they reflect the point during the life course where immunosenescence has typically begun. Thus, at these two age groups, incidence rates of infectious disease begin to increase markedly and vaccination becomes particularly important. We used sensitivity analyses to simulate the impact of various epidemiological scenarios on the total estimated economic burden, and constructed 95 % confidence intervals around point estimates using a Monte Carlo simulation model. Customized estimates, with varying inputs to population, incidence, and cost parameters can be obtained for nearly any population. Interested parties should contact the manuscript’s corresponding author.Combining the estimated number of cases for each of the four major adult VPDs in the United States with the respective estimated medical and indirect cost per case yielded estimated total annual costs of: $16.0 billion (B)  for influenza, $5.1B  for pneumococcal disease, $5.0B  for herpes zoster, and $397.7 million (M)  for pertussis. Thus, for these four adult VPDs, the combined estimated cost burden in 2013 for US adults aged 50 and older was $26.5B    was attributable to adults 65 and older  is considerable, and that in 2013, the economic impact attributable to adult VPD for US adults aged 50 and older was $26.5B ($15.3B for those 65 and older). This economic burden would be even greater if mortality costs  and leisure time costs  were included in the analyses. Moreover, results of the model showed that although influenza (often the focus of most adult immunization programs) accounted for the majority of cases of adult VPD (81 % of adults 50 and older and 77 % of adults 65 and older), from a cost perspective, pneumococcal disease and herpes zoster both represent a significant economic burden in addition to influenza. Specifically, among adults 65 and older, although pneumococcal disease represented only 8 % of total cases, it contributed to one-fourth of the total costs, with the large majority (85 %) of pneumococcal burden due to NPP. Likewise, herpes zoster made up 11 % of total cases, but 20 % of total adult VPD costs among those 65 and older. Thus, broadening adult immunization efforts beyond influenza only may help reduce the economic burden of disease.Based on this model and consistent with previous research  has been a paradigm of success for immunizing adults Nichol, , 1998. HThis epidemiological model is not without limitations. The model relied on the human capital methodology of calculating cost, which does not tend to capture the monetary or nonmonetary value of pain, suffering, and premature death due to disease. In 2013, it was estimated that approximately 54,000 individuals aged 50 and older died from influenza and pneumonia. The large majority of which  occurred among adults 65 and older (Murphy et al., In spite of these limitations, this model gives a broad overview of the societal cost attributable to four major adult VPDs for the United States as a whole and for each state. Although modeling of this type is not an exact science, the model uses peer-reviewed inputs for age-adjusted, disease-specific incidence and cost, and provides an educated ‘best guess,’ with sensitivity analyses, as to what the burden of disease due to adult VPD was in the United States in 2013.Childhood immunization programs in the United States have nearly eliminated diseases that once claimed the lives of countless children. Adult immunization programs, however, have been slow to evolve. Many reasons have been identified for the nation’s poor adult immunization rates. These include: variable insurance coverage among patients, a lack of incentives for health care providers to stock and deliver adult immunizations, a lack of a ‘medical home’ for adult patients, the absence of reminder-recall and vaccination history tracking systems, insufficient development of standing order programs for vaccination, and lack of assessment of practice-level vaccination rates with feedback to staff members in medical practices (Lau et al., In order for adult immunization efforts to increase, the value of preventing these diseases, in both human and economic terms, must be recognized. Results from this analysis provide an estimate of the cost attributable to four major adult VPDs, and highlights the importance of addressing adult vaccination uptake. Sensitivity analyses suggested that as the US population ages over the next decade and beyond (Ortman et al.,