N = 37), suicide completers (N = 22) with no evidence of heroin use and from control subjects who did not abuse illicit drugs and died of non-suicide causes (N = 28). We identified 1298 differentially methylated CpG sites (DMSs) between heroin users and controls, and 454 DMSs between suicide completers and controls (p < 0.001). DMSs and corresponding genes (DMGs) in heroin users showed significant differences in the preferential context of hyper and hypo DM. HyperDMSs were enriched in gene bodies and exons but depleted in promoters, whereas hypoDMSs were enriched in promoters and enhancers. In addition, hyperDMGs showed preference for genes expressed specifically by glutamatergic as opposed to GABAergic neurons and enrichment for axonogenesis- and synaptic-related gene ontology categories, whereas hypoDMGs were enriched for transcription factor activity- and gene expression regulation-related terms. Finally, we found that the DNAm-based “epigenetic age” of neurons from heroin users was younger than that in controls. Suicide-related results were more difficult to interpret. Collectively, these findings suggest that the observed DNAm differences could represent functionally significant marks of heroin-associated plasticity in the OFC.We employed Illumina 450 K Infinium microarrays to profile DNA methylation (DNAm) in neuronal nuclei separated by fluorescence-activated sorting from the postmortem orbitofrontal cortex (OFC) of heroin users who died from heroin overdose ( Opioid overdose is now the second leading cause of accidental death among adults in the U.S. ,2. PrescAs with other drugs of abuse, the susceptibility to opioid addiction is known to be influenced roughly equally by genetic and environmental factors ,7,8, sugDNAm is a stable mostly repressive epigenetic modification that is extremely important for both the establishment of cell-type-specific phenotypes in the nervous system  and the m) prefrontal cortex (PFC) in drug self-administration and the long-lasting propensity to relapse [Several features of DNAm were considered in the design of the present study. First, recent studies indicate that the composition and dynamics of DNAm are not only distinct in the brain compared to other tissues ,24, but  relapse ,29,30. M relapse ,32,33 an relapse ,35,36 st relapse ,38.In addition, a number of studies have reported DNAm alterations in the human brain that are associated with addiction . TheRecently, a highly accurate multi-tissue epigenetic biomarker of tissue age  based on DNAm levels has been introduced . This apIn the present report, we compared DNAm in neuronal populations of the mOFC among individuals who abused heroin and died of heroin overdose, suicide completers without any evidence of heroin use, and a control group consisting of specimens from psychiatrically normal individuals who died of non-suicide causes and did not abuse illicit drugs. All brain specimens came from the same brain collection. Suicide completers were included as a comparison group with a different pathophysiology. The study revealed significant methylation disturbances in heroin abusers that may represent a functionally relevant epigenetic signature of heroin addiction in the human brain. Moreover, while the epigenetic age of neurons from the suicide and control individuals did not differ from their biological age, the epigenetic age of neurons from heroin users was younger. N = 88) consists of a Hungarian/Swedish population of heroin abusers who died of heroin overdose (N = 37), suicide completers without any evidence of heroin use (N = 22), and control subjects who did not abuse illicit drugs and died of non-suicide causes   was 3.75 years (ranging from 0.5 to 10 years). The control and suicide groups were negative for blood opiates and had no history of any drug addiction. All suicide subjects died from hanging. The control subjects died from myocardial infarction, pulmonary embolism, electric shock or viral infection. Subjects from all 3 groups showed negative toxicology for other common drugs of abuse and common therapeutic agents. EtOH was detected in the blood and/or urine of 6 heroin, 5 suicide, and 3 control subjects and EtOH levels were not significantly different among groups. Nicotine toxicology was not conducted, but tobacco use is frequent in the general population from which all the subjects were collected. Human postmortem brain specimens were obtained from our Brain Collection at the Icahn School of Medicine at Mount Sinai that has been extensively used for many molecular and epigenetic studies ,67,68,69 N = 29) . One of Autopsy brain specimens were cut coronally in 1 cm slabs, frozen, and kept at −80 °C. From these specimens we harvested the ventral extent of the PFC commonly referred to as the orbital frontal cortex . Specifically, we dissected the regions containing the medial orbital (MOrG) and inferior orbital (IOrG) gyri just anterior to the transversely running orbital sulcus. The region was dissected in a single block bounded by the olfactory sulcus medially and the inferior orbital sulcus laterally as described previously .2, 3 mM MgCl2, 10 mM Tris-HCl), filtered through a cell strainer, and centrifuged for 5 min at 300 g. The pellet was resuspended in Blocking Buffer  and incubated for 45 min with Alexa488-conjugated anti-NeuN antibodies  (1:1000 dilution). Next, a second centrifugation step  through a layer of 1.1 M sucrose was done, and the resulting pellet was resuspended in PBS. The DNA dye 7-AAD  was added to a final concentration of 2 μg/mL, and the sample was subjected to the FANS procedure using Vantage with DiVa (excitation wavelength 488 nm). Finally, the sorted nuclear fractions were precipitated by centrifugation at 4000 rpm for 20 min at 4 °C and stored frozen at −80 °C until DNA isolation. The latter was performed using proteinase K treatment and two rounds of phenol extraction followed by ethanol precipitation. Cell structure is not preserved in frozen autopsy brain specimens. However, the nuclei of different cell types remain intact. Antibodies against the RNA-binding protein NeuN, which is expressed exclusively in the neuronal nuclei, have been used to separate neuronal from glial nuclei using FANS  and NeuN(−) nuclear fractions, with the width of separation reaching up to an order of magnitude of the NeuN signal intensity . In addiFor each specimen, DNA was extracted from the neuronal fraction and subjected to sodium bisulfite treatment to generate methylation-specific base changes before hybridization. Batch effect was minimized by performing the bisulfite treatment simultaneously for all 88 specimens, and by randomizing the placement of heroin, suicide and control samples across the arrays.DNA samples were bisulfite converted using EZ DNA methylation kit . Specifically, 500 ng of high quality genomic DNA  was denatured by incubation with NaOH-containing Zymo M-Dilution buffer for 15 min at 37 °C. Next, the denatured DNA was incubated with bisulfite-containing CT-conversion reagent for 16 h at 50 °C in a thermocycler. Every 60 min the reaction was heated to 95 °C for 30 s. All 88 samples were processed on the same plate. The Infinium methylation assay was carried out as described . In shorM and U intensities greater than 10.5. All but one sample passed a test for concordance between estimated and reported sex. This sample was removed from the subsequent analyses  and the KnownGene data set from the University of California, Santa Cruiz) (UCSC) (http://genome.ucsc.edu).For differential methylation analysis, we employed limma R/Bioconductor package to perform linear regression and moderated t-statistics  adjusting for age, sex and tissue pH and the first four “negative control” principal components, which typically capture batch and slide effects as described previously . DiffereR software scripts and in a web-based calculator. The measure of epigenetic age acceleration was defined as a raw residual resulting from regressing DNAm age on chronological age. By definition, epigenetic age acceleration does not correlat with chronological age (r = 0).DNAm age  was calculated from the neuronal samples profiled with the Illumina Infinium 450 K platform as described in . Brieflywww.webgestalt.org, [p-values < 0.01 (Benjamini-Hochberg multiple test adjustment) were considered statistically significant. In addition, we applied the cutoff of N ≥ 10 genes for the minimal number of genes associated with a specific GO term. For the GO analysis, we included the genes which had both hyperDMSs and hypoDMSs into both hyperDM gene (G) and hypoDMG lists. Gene ontology (GO) analysis was performed with the online software tool WebGestalt . Enrichhttps://github.com/vsbuffalo/scythe, https://github.com/najoshi/sickle)), further trimmed by 3 bp from the 5′ end of read 1 as suggested in the library kit protocol, and then mapped to human hg19 genome with STAR software [We employed our recently developed FANS-based protocol  to isolasoftware . EdgeR Rsoftware  was usedr = 0.85, p = 1.2 × 10−25; N = 28) and suicide subjects (N = 22), the heroin subjects (N=37) showed a younger DNAm vs. chronological age  captures aspects of the biological age of the brain tissue . We estip-value < 0.001). This resulted in 454 DM sites (DMSs) between suicide and control subjects (DMsui sites) and 1311 sites that were DM between heroin users and controls (DMher sites)  CpGs sites between control and suicide specimens using FDR < 0.1, whereas there were 3 sites that were DM between heroin users and controls. We then used a more liberal threshold of significance  , and we r sites) . N = 10 oldest control individuals to balance the groups by age, analyzing N = 18 controls and N = 37 heroin individuals. In this new cohort there were no significant difference in age between the heroin and control subjects (p = 0.09). We detected no noticeable effect of age on the regression estimates for the heroin vs. control comparison  which did not have the years-of-use information, and using this filtered cohort, compared the results of the differential methylation analyses employing two different linear models: (1) heroin/non-heroin status, and the covariates from the initial analysis, and (2) Log10 (Years of Heroin Use + 1) and the covariates from the initial analysis. We found no significant differences between the results from these two models  based on the position of the DMSs . There were a total of 1159 unique DMGs associated with DMher sites (DMher genes) and 407 genes associated with DMsui sites (DM genes) ; 87 geneWe first asked if heroin- or suicide-associated hyper- or hypoDMSs were enriched (or depleted) within genic features , putative regulatory elements  or CpG island-related features . We also tested if such enrichments could be specific to the direction of the DNAm changes (hyper- vs. hypoDM).We detected clear differences in the patterns of enrichment or depletion between hyper- and hypoDMSs within many of the assessed features in the neurons from heroin users . We perBoth hyper- and hypoDMher sites were depleted from the genomic areas outside of CpG islands or shores (the latter is defined as 2000 bp-width-areas at ≤2000 bp from the nearest CpG island). In addition, hyperDMSs were slightly enriched in CpG islands, whereas hypoDMSs were enriched within shores.When the distribution of the suicide-associated DMSs within different genic features was analyzed, only few significant results were detected. HyperDMsui sites were depleted within promoters as well as within proximal H3K27ac peaks, and were moderately enriched in exons, introns and gene bodies . No enriwww.webgestalt.org [To obtain insight into the possible biological implications of the detected DNAm changes in the OFC of heroin users and suicide completers, we performed a functional annotation and enrichment analysis of the DMGs using the online software tool WebGestalt . This tN = 660) and hypoDMher genes (N = 531) were analyzed, we obtained essentially non-overlapping sets of enriched GO terms (NGF).Notably, when hyperDMher genes  or somatostatin (SST) [Our analysis of DM between heroin users or suicide subjects and controls was performed in FANS-separated neuronal nuclei. This experimental design could not distinguish between the contributions of specific neuronal subpopulations that exist in human PFC  to the observed differences in DNAm. To investigate neuronal subtype-specificity of the hyper- and hypoDM genes, we compared the DMG sets with genes that were differentially expressed (DE) between Glu projection neurons and MGE-derived GABA interneurons in the PFC. MGE-derived GABA interneurons comprise ~60–70% of all cortical GABA neurons, and express parvalbumin (in (SST) . This poin (SST) ,85,86,87in (SST)  and thatN = 1339 and 817 genes, respectively), Next, we overlapped the DEG and DM gene sets and assessed the enrichment using the hypergeometric distribution test. A significant difference in patterns of enrichment was observed between hyper- and hypoDM genes , DM genes . For botDM genes . In contDM genes .p-value (<0.001) we detected 454 CpG sites that were DM between suicide and control subjects, and 1298 CpG DM sites between heroin users and controls. We then assessed genomic features, neuron-subtype specificity as well as functional categories and pathways that were associated with these DNAm differences.In this study we employed the Illumina 450 K Infinium DNA methylation array to probe the differences in DNAm in the OFC of heroin abusers who died of heroin overdose, suicide completers, and control subjects who died of non-suicide causes. There was no evidence of any illicit drug use for the latter two groups. We analyzed differential DNAm (1) between suicide completers and controls and (2) between heroin users and controls. Applying a conservative nominal Whereas the number of DM sites and genes identified in the suicide vs. control comparison was low and the results were difficult to interpret, we found noticeable differences for heroin-associated DM sites and genes. In particular: (1) hyperDMher sites preferentially occurred in exons and more generally in gene body regions. In contrast, no such enrichments were detected for hypoDMher sites; (2) hypoDMher sites were significantly enriched in promoters and in the TSS-proximal H3K27ac ChIP-seq peaks, whereas hyperDMher sites were dramatically depleted in these regions; (3) hypoDMher sites were strongly enriched in TSS-distal H3K27ac ChIP-seq peaks (predicted enhancers), whereas hyperDMher sites were not significantly overrepresented in these regulatory regions; (4) at the gene level, hyperDMher genes were strongly enriched in Glu-specific, but not in GABA-specific genes; (5) significant differences in gene ontology (GO) category enrichments were found between hyper- and hypoDMher gene sets: hyperDMher genes were enriched in GO categories for genes implicated in axonal and synaptic localization, as well as in synaptic transmission. In contrast, hypoDMher genes were not enriched in these categories. Instead, hypoDMher sites were overrepresented among genes with DNA binding and transcription factor activity. Thus, the hyper- and hypomethylation in the specimens from heroin users are enriched in different neuronal subtypes and different genomic contexts. These observations imply that different mechanisms of epigenetic regulation might be recruited in different neuronal subtypes or specifically targeted to particular genic features  in the context of opioid exposure. Collectively, these findings suggest that the observed DNAm differences could represent functionally significant marks of heroin-associated maladaptive plasticity in the OFC.Notably, heroin-associated enrichment of the hyperDMGs in Glu-specific genes as well as in synaptic and axonal genes is in agreement with the notion that drug-associated alterations in the ventral PFC lead to reduced glutamatergic output from this region . Our finSLC17A7 , OPRL1 . The latter encodes one of the 3 Ten-Eleven Translocation enzymes that oxidize methylcytosine to produce the hydromethylcytosine DNA mark, and thus could alter the inhibitory character of DNAm and/or contribute to DNA demethylation. Of note, TET1, another member of the TET family, has been recently implicated in the mechanism of cocaine action in the NAc [ARC (cg08387463). ARC is an immediate early gene (IEG), which can be upregulated in an activity-dependent manner [ARC, at ~1300 bp distance from the TSS. Increased DNAm of ARC (which contrasts with increased ARC expression in the NAc as found in other studies) might be a specific signature of heroin addiction in the OFC.Whereas our approach was mostly focused on the analysis of sets of DMSs or DMGs, inspecting these data allowed us to identify a number of individual genes that could potentially have major implications for heroin addiction or suicide. Among the heroin-associated DMGs are  the NAc . Anothert manner . Here wePRRT1 and SORBS2. For both of these genes, more than one hyperDMS was detected in suicide completers vs. controls, —the largest number of sites for any suicide-associated DMG in our study, and 3 hyperDMSs for SORBS2, cg09555153, cg04392082, cg22328746). PRRT1 encodes a little-studied auxiliary subunit of the AMPA receptor, and was shown to colocalize with extrasynaptic GluA1 puncta in primary neuronal cultures [SORBS2 gene encodes ArgBP2 protein and is alternatively spliced in the brain to encode a neuron-specific nArgBP2 isoform. In mice, nArgBP2 is highly expressed in many brain regions, including cortex, and localizes at dendritic spines. Knockout of Sorbs2 leads to reduced dendritic complexity, as well as to a reduced acoustic startle response, and defective long-term object recognition memory and contextual fear memory [Among the suicide-associated DNAm genes, of particular interest are r memory . It was r memory .Another intriguing finding of our study is the discovery that the OFC neurons of heroin users show a younger epigenetic age than the control subjects. This finding will require confirmation by other studies and is not straightforward to interpret. However, significant cellular and molecular changes were reported with repeated exposure to drugs of abuse, and the “neural rejuvenation” hypothesis has been recently proposed to conceptualize some of these changes, with one of the main defining features being the formation of immature, silent synapses . Silent To the best of our knowledge, the present study constitutes the first genome-wide DNAm profiling of human postmortem brain samples  of heroin abusers vs. control subjects. It also contributes to a growing list of studies exploring DNAm in the suicide brain ,97,98. IWe investigated the genome-wide DNAme differences in the OFC of individuals who abused heroin and died of heroin overdose and of individuals who did not use heroin and died by suicide vs. control subjects who did not use illicit drugs and died of non-suicide causes. We identified numerous CpG sites that were differentially methylated between heroin users and control individuals as well as between suicide victims and controls. We found that the hyper- and hypomethylated CpG sites and associated genes in the heroin users were enriched in different neuronal subtypes and different genomic contexts. These observations suggest that different modes of epigenetic regulation might be recruited in different neuronal subtypes or specifically targeted to particular genic features and/or regulatory elements  in the context of heroin exposure. We also found that the DNAm-based “epigenetic age” of neurons from heroin users was younger than that in controls. Suicide-related differences in DNAm were subtle and difficult to interpret. Collectively, these findings implicate heroin-induced epigenetic remodeling in the previously reported functional abnormalities of the ventral mPFC in heroin abusers.


The correct name is: Richard Guest. The correct citation is: Blanco-Gonzalo R, Lunerti C, Sanchez-Reillo R, Guest R (2018) Biometrics: Accessibility challenge or opportunity? PLoS ONE 13(3): e0194111.


Clinicians are encouraged to use guidelines to assist in providing evidence-based secondary prevention to patients with coronary heart disease. However, the expanding number of publications providing guidance about exercise training may confuse cardiac rehabilitation clinicians. We therefore sought to explore the number, scope, publication characteristics, methodological quality, and clinical usefulness of published exercise-based cardiac rehabilitation guidance.We included publications recommending physical activity, exercise or cardiac rehabilitation for patients with coronary heart disease. These included systematically developed clinical practice guidelines, as well as other publications intended to support clinician decision making, such as position papers or consensus statements. Publications were obtained via electronic searches of preventive cardiology societies, guideline databases and PubMed, to November 2016. Publication characteristics were extracted, and two independent assessors evaluated quality using the 23-item Appraisal of Guidelines Research and Evaluation II (AGREE) tool.n = 35; 65%) and were freely accessible . Thirty (56%) publications contained only broad recommendations for physical activity and cardiac rehabilitation referral, while 24 (44%) contained the necessary detailed exercise training recommendations. Many were labelled as “guidelines”, however publications with other titles (e.g. scientific statements) were common . This latter group of publications contained a significantly greater proportion of detailed exercise training recommendations than clinical guidelines (p = 0.017). Wide variation in quality also existed, with ‘applicability’ the worst scoring AGREE II domain for clinical guidelines (mean score 53%) and ‘rigour of development’ rated lowest for other guidance types (mean score 33%).Fifty-four international publications from 1994 to 2016 were identified. Most were found on preventive cardiology association websites  contains supplementary material, which is available to authorized users. The aim of guidelines is to assist in the achievement of consistent, effective, and appropriate health care for clinical conditions, thereby bridging the gap between research evidence and practice.Clinical practice guidelines have become a key tool in delivering evidence-based, high quality health care. Ideally, they provide a systematic synthesis of the expansive base of research evidence, and support clinicians with recommendations based on “an assessment of the benefits and harms of alternative care options” (p4). TheThe number of guidelines providing recommendations for the use of exercise training and cardiac rehabilitation in the secondary prevention of coronary heart disease has rapidly expanded. These publications are produced by a variety of bodies, including both professional societies and government organisations. A search of the National Guideline Clearinghouse identifies 78 publications related to cardiac rehabilitation , and mosRecommendations intended to assist in the delivery of evidence-based practice may additionally be found in other types of guidance documents such as scientific statements and position papers. These types of publications, often produced by professional associations, are useful to clinicians as they are typically designed to provide more detailed information about topics (such as exercise training) which are considered too specific to address in broader disease focused clinical guidelines . Until rDespite the proliferation and widespread availability of both clinical practice guidelines and other guidance documents in cardiac rehabilitation, there is limited research documenting the total number, characteristics, quality, and scope of these publications. A previous examination  includedAnalysis of a cross-sectional sample of publications.To be included, publications were required to provide recommendations intended to optimise the use of cardiac rehabilitation, physical activity, or exercise for people with established coronary heart disease . We excluded publications concerned solely with the primary prevention of coronary heart disease. To capture the full spectrum of clinical guidance available to clinicians, we included systematically developed clinical practice guidelines , as wellInitially we searched for appropriate references within the web-based resources and publications of cardiology societies and cardiac rehabilitation associations in Australia, New Zealand, the United Kingdom, the United States, Canada, and Europe  were collected using a standardised Excel spreadsheet. We additionally used the title, abstract, preamble, and introduction to classify publications according to three different scopes of information provided: (a) the general diagnosis and management of cardiovascular conditions or cardiovascular risk; (b) guidance about cardiac rehabilitation interventions; or (c) guidance about exercise or physical activity for cardiovascular disease. Publications were also classified based on the type of recommendations provided, either: (a) broad recommendations about the need for cardiac rehabilitation referral and/or general physical activity advice; or (b) detailed and specific recommendations for exercise training or protocols during cardiac rehabilitation. Finally, we classified publications according to guidance document type , based on their self-reported title.The scientific and methodological quality of each included publication was independently evaluated by two researchers using the Appraisal of Guidelines Research and Evaluation II (AGREE II) instrument . Both reThe online My AGREE PLUS platform  and assoDescriptive statistics  were used to analyse the characteristics and scope of included publications. Chi-squared tests were used to examine the hypotheses that there were differences in the main scope and clinical usefulness (detailed vs broad exercise recommendations) of publications based on guidance document classification . A Bonferroni adjusted alpha level of 0.025 was used due to multiple comparisons. All analyses were performed using SPSS Statistics 23.0 (SPSS Inc. Chicago IL).Figure The included publications were dated between 1994 and 2016, with almost two-thirds (61%) published within the previous 5 years, and most as updates to previous guidance (69%) Table  and all Just over half 52%) of the included publications focussed on the general diagnosis and management of a range of cardiovascular conditions such as acute coronary syndromes, percutaneous coronary intervention, or coronary artery bypass grafting  advocated referral to formal cardiac rehabilitation programs for most patients, but did not offer further guidance about the format these programs should take (Table Detailed recommendations for the exercise training or protocols to be used during cardiac rehabilitation were provided in 24 publications. Nineteen (79%) of these were found within the resources and publication sections of national cardiac rehabilitation associations or cardiology societies. Seven of these documents could only be found via these sources, and were not indexed in PubMed or listed in guideline databases.The majority of guidance documents (56%) described themselves as clinical guidelines. Exercise and physical activity recommendations were also found however in other publications with titles such as scientific statements (13%) and position papers (13%) Table .p = 0.0004). Cardiovascular disease management and diagnosis were more likely to be the main scope of publications classified as clinical guidelines , while other types of guidance publication had a greater focus on topics such as exercise, physical activity, and cardiac rehabilitation . Additionally, the proportion of publications which contained detailed exercise training recommendations  varied significantly by the type of guidance document, with clinical guidelines less likely to contain more detailed recommendations compared to other guidance types (p = 0.017). Just 30% of the publications classified as clinical guidelines contained these detailed recommendations compared to almost two-thirds of other guidance types .A comparison of the clinical usefulness and main scope of these different types of document is presented in Table Standardised domain scores from the AGREE II assessment are displayed for each included guidance publication in Additional file Domain scores from the AGREE II assessment of clinical practice guidelines are displayed in Table Table This study systematically explored the scope, characteristics, and methodological quality of a range of publications which provide guidance for exercise-based cardiac rehabilitation and physical activity in coronary heart disease. We identified 54 guidance publications which were produced by numerous national and international associations, many overlapping in clinical focus. Not all of these were published in journals, nor titled as clinical guidelines, nor indexed in PubMed, but most were freely available and recently produced. Methodological quality was generally modest, and varied within and between clinical guidelines and other types of guidance. The domains of ‘applicability’, ‘stakeholder involvement', and ‘rigour of development’ were particularly poor across most publications.While the methodological quality of guidelines in many areas of healthcare has been assessed, there is limited research exploring the publication characteristics of these documents. Studies which have appraised guideline quality in other clinical areas have observed similar findings to those in our sample, in that a large proportion of guidance is produced in the United States , 19, is A recent study  which apOur finding that the domain of ‘applicability’ scored poorly in exercise and cardiac rehabilitation guidance is not unique to this area. ‘Applicability’ has been identified as the lowest rated domain in guidelines for cancer-related fatigue , biologiIt is important to also recognised that AGREE scores have not been shown to consistently relate to the uptake of a guideline in practice . While tThe modest AGREE domain scores observed for both clinical guidelines and other guidance publications in this sample may not solely be due to inadequacies in the development processes. Incomplete reporting in many of the publications also contributed to modest scores, as items with missing information could not be rated, whether or not the process actually occurred during development. While this raises the need for better attention to reporting in the future, this poor reporting is not unique to guidelines, but rather is an ongoing problem in many areas of research, including trial methodology , 27 and A range of potential barriers to guideline implementation in practice have been previously identified and issues with the guideline publications themselves are frequently cited , 31. AcrWhile clinical practice guidelines and other types of guidance aim to provide recommendations to optimise patient care, the observed differences in methodological quality between these groups of publications is not unexpected, given their varying aims and development processes. Unlike guidelines, other forms of guidance do not necessarily use a systematic review and grading of the relevant evidence. Instead they are more likely to provide the collective opinion of an expert multidisciplinary panel concerning evidence-based approaches to advancing patient care in particular areas , 32, 33.Furthermore, clinical guidelines are generally designed to provide recommendations where there is a strong evidence base, while guidance such as consensus statements may be developed to inform clinical practice where the evidence is less clear or still evolving . For exaFinally, the notion that clinical guidelines act as a concise and evidence-based “what to do” rather than “how to do” appears to be consistent with the findings of this study. While these publications do provide broad evidence-based recommendations for exercise-based cardiac rehabilitation, clinicians requiring specific guidance about the “how to” of translating exercise training interventions into practice, would most likely need to source this information from other types of guidance document. The role of publications such as scientific statements in providing the “how to” of evidence translation has previously been acknowledged , 4. GiveGiven these findings, clinicians should be cautious when searching for and implementing recommendations for exercise-based cardiac rehabilitation in practice. Potential users need to understand the inherent differences between these publication types in order to make judgements about the potential risk of bias, and the necessary trade-off often required between methodological quality and clinical usefulness. The summary of guidance documents provided in this article may assist clinicians in selecting the most trustworthy and relevant guidance publication for their practice, or at least provide an aid in assessing methodological quality. Where it exists, a clinical guideline should be considered above other types of guidance, however this may often not be possible as currently clinicians have access to very few recently developed, rigorous guidelines which also provide the specific and detailed recommendations necessary to implement exercise and cardiac rehabilitation. Relevant journals and professional associations are therefore encouraged to use the AGREE II tool and IOM criteria when developing or updating clinical guidelines. Other types of publications, such as scientific statements, should at least provide clear reporting of the methodology employed and links to the evidence to promote increased transparency. Additionally, the relationships between publications from the same organisation need to be made clearer, as we often had difficulty determining which had overlapping content or had been replaced by newer versions.A strength of our study is the use of a comprehensive search strategy with broad selection criteria. This enabled us to identify publications which are representative of all English-language guidance currently available to clinicians working in the field of exercise-based cardiac rehabilitation. We also used a validated tool (AGREE II) to systematically assess and compare the quality and transparency of these publications. By using a standardised checklist for all guidance publications, we were able to assess whether biases in development had been adequately addressed. While the AGREE tool is not specifically designed for assessing guidance documents such as scientific statements, and it could therefore be argued that these non-guideline publications would naturally have lower scores, no other suitable appraisal tool currently exists . As thesFinally, in analysis we chose to classify publications as clinical guidelines based on their title rather than the established IOM criteria. Consequently, two publications  were classified as clinical guidelines in our study, even though they failed to explicitly report a systematic search or assessment of the evidence used to formulated recommendations. However, we felt clinicians who may not be aware of the intricacies of the guideline definition may make the same classification, given that they are titled guidelines and are currently two of the most widely used cardiac rehabilitation resources, particularly in North America.We have shown that while an expansive volume of exercise-based cardiac rehabilitation guidance exists, much of it has limitations (in either rigour or usefulness) which may hinder integration of evidence into cardiac rehabilitation practice. While publications are often easily located via professional associations or guideline developers, clinicians need to be aware that these may vary substantially in terms of quality, guidance type, and usefulness of recommendations. There is room for improving the quality of all these publications, and in particular for providing transparent, rigorously developed, evidence-based clinical guidelines with a specific focus on implementable exercise prescriptions for people with coronary heart disease.Additional file 1:a Cardiac Rehabilitation and Cardiology associations searched. b Guideline databases searched. (DOCX 15 kb)Sources searched for publications. Additional file 2:Table. The 6 AGREE II domains and corresponding 23 items. (DOCX 12 kb)Additional file 3:Table. Details of all included publications. (DOCX 68 kb)Additional file 4:Table. AGREE II domain scores (%) for all included publications. (DOCX 27 kb)


Opioid dependence is a major health concern across the world and does also occur in adolescents. While opioid substitution treatment (OST) has been thoroughly evaluated in adult populations, very few studies have examined its use in adolescents. There are concerns that OST is underutilised in adolescents with heroin dependence. We sought to measure changes in drug use among adolescents receiving OST and also to examine treatment attrition during the first 12 months of this treatment.We included all heroin dependent patients aged under 18.5 years commencing OST at one outpatient multidisciplinary adolescent addiction treatment service in Dublin, Ireland. Psycho-social needs were also addressed during treatment. Drug use was monitored by twice weekly urine drugs screens (UDS). Change in the proportion of UDS negative for heroin was examined using the Wilcoxon signed rank test. Attrition was explored via a Cox Regression multivariate analysis.p < 0.001) and from month three to month 12 (p = 0.01). Use of other drugs did not change significantly. People using cocaine during month 12 were more likely to be also using heroin (p = 0.02). Unplanned exit occurred in 25% patients by 120 days. The independent predictors of attrition were having children, single parent family of origin, not being in an intimate relationship with another heroin user and evidence of cocaine use just before treatment entry.OST was commenced by 120 patients . Among the 39 patients who persisted with OST until month 12, heroin abstinence was 21%  at month three and it was 46% (95% CI = 30–63%) at month 12. Heroin use declined significantly from baseline to month three  contains supplementary material, which is available to authorized users. There is global ongoing concern regarding opioid dependence. While this is primarily focused on non-prescribed use of pharmaceutical opioids in USA currently, heroin has been the main concern elsewhere, including Ireland , 2. The Opioid substitution treatment (OST) is now the main first line treatment intervention for heroin dependence among adults . The maiThere are no clinical trials examining outcome of OST beyond three months in people aged under 18 years old . There aThe DARP study of adolescents in USA in the 1970s did report a progressive decline in heroin use over the first year of MT but it did not provide any specific information on the actual rates of heroin use during treatment . More reAdolescents entering addiction treatment typically present with polysubstance use and this is also the case for those with primary dependence upon heroin –13. An aDrop out from OST has been examined by a small number of studies in this age range. Studies of MT indicate that 25% of patients drop out within approximately four months , 8, 15. Given this paucity of outcome studies of OST in adolescent heroin dependence, we sought to address a number of questions which are of importance to clinicians and patients in order to make informed decisions. What proportion of adolescents ceases heroin use while on OST? Does abstinence increase incrementally or simply stabilise beyond three months on OST? What is the rate of drop out? Are there any baseline or treatment characteristics which are associated with either abstinence or drop out?This study was based at the Young Persons’ Programme (YPP) within the National Drug Treatment Centre (NDTC) in Dublin, Ireland. The approach to assessment and treatment of heroin dependent adolescents at this service is summarised below but has been described in detail elsewhere , 21. AltAssessment typically involved three clinic visits to meet different members of the multi-disciplinary team, a supervised urine drug screen (UDS) being obtained at each visit. In addition to assessing for heroin dependence using ICD-10 criteria, co-existing psychological, developmental, social and physical needs were identified.The main pillars of treatment involved OST (methadone or buprenorphine), counselling and in some cases, family therapy. Induction onto a stable dose of methadone typically occurred over a period of about two weeks while buprenorphine induction took 2–3 days . If patiPatients provided two supervised UDS per week for on-site drug toxicology testing during treatment. These samples were screened for opiates, EDDP (a methadone metabolite), cocaine, benzodiazepines and alcohol. They were intermittently screened for amphetamines and cannabis. The provision of ‘take-away’ doses of medication was utilised as a contingent reinforcer of drug abstinence, as evidenced by urine toxicology.Patients were also provided with treatment of co-morbid medical or psychiatric problems . They weThe overarching goal of the service was to reduce the harm which young people were experiencing related to their drug use. It held an aspiration, but not an insistence, that patients progress towards complete abstinence via gradual OST dose reduction and ultimate cessation of this medication. This ultimately occurs in about one in every four cases . Where dThe inclusion criteria were:- heroin dependence, aged under 18.5 years and commenced OST. The cohort under study included patients who commenced treatment between May 2000 and June 2016. Patients with a primary diagnosis of dependence on prescription opioids were excluded, as such patients have been rarely encountered in Ireland.The study was approved by the Research Ethics Committee of the NDTC. Baseline descriptive characteristics were obtained from the patients’ initial structured assessment, this being adapted from the Maudsley Addiction Profile . We recoThose who were referred to and commenced on another OST program were categorised as “transfers for ongoing treatment”. This typically involved transfer to an adult OST program, this option being available to patients after their 18th birthday. However, most opted to persist with treatment on the YPP beyond this age . PatientTo examine changes in drug use, five periods during the first year of treatment contact were examined for each patient. The results of all urine drug screens (UDS) provided by each patient during each period was collated. The periods in question were (a) pre treatment assessment phase which was typically 7 to 10 days in duration and involved provision of 2–4 UDS, (b) induction phase which comprised the remaining days in that first month of patient contact after commencing OST, (c) third entire month of treatment, (d) sixth entire month of treatment and (e) twelfth entire month of treatment. During phases (b) to (e), patients were typically providing two UDS per week. For each patient, a dichotomous outcome variable to indicate abstinence/use was created for each of the phases they were being treated (a) to (e). They were deemed to be using heroin if any one UDS sample tested positive for opiates during that phase. If all UDS were opiate negative, they were deemed heroin abstinent. Similarly, a single positive for cocaine in a month resulted in that month being deemed one of cocaine use.In order to conduct an exploratory analysis, we used Pearson chi square test to examine association between categorical covariates and dichotomous outcome variables of interest, except in instances where a predicted cell value was less than five, where Fisher’s Exact test was utilised. Odds ratios and their 95% confidence intervals were calculated to indicate the direction and magnitude of associations. Continuous variables were converted into categorical variables, by choosing the median value as the point of split, apart from age where we used 18th birthday as the cut-off.When reporting the proportion of patients who were heroin abstinent, we also calculated the 95% confidence intervals of those rates using the exact confidence limits for binomial proportions. McNemar’s paired proportions test with continuity correction was employed to determine whether OST was associated with changes in the binary category of drug use. We sought to confirm that heroin abstinence in month three was greater than baseline, and that abstinence at months six and twelve were greater than month three. We also examined changes in the proportion of samples which tested negative for heroin using the related samples Wilcoxon signed rank test. For each period, we calculated this proportion by dividing the number of negative tests by the total number of UDS obtained. All of these analyses were confined to the subgroup of patients who persisted with OST for a full year.To examine drop out and factors associated with same, the Kaplan-Meier test was conducted. The event of interest was unplanned exit from treatment via drop out or imprisonment. The time to treatment exit, in days, was recorded for each patient who left their index treatment episode during the first year, whether by unplanned exit or via planned exit (following completion of dose taper or via transfer to another OST service). For patients who persisted with OST, the number of days entered was 365 days. The Log Rank was used to test for the equality of the survival distributions within each covariate. To facilitate interpretation of these differences, the estimated number of days to drop out by 25% is reported. In order to explore independent predictors of unplanned exit, we then conducted a multivariate analysis using Cox Regression. Covariates were selected for entry into the final equation using the forward and backward selection technique. All analyses were conducted using IBM SPSS Statistics, version 21.There were 120 eligible patients who commenced OST. One patient with primary methadone dependence was excluded and there were no presentations with primary prescription opioid dependence. The patient characteristics are provided in Table Table Table p = 0.02). Abstinence was not significantly associated with higher medication dose (p = 0.88). All of the patients who were using cocaine during month 12 were also using heroin (p = 0.02). Early reductions in heroin use, as evidenced by provision of at least one heroin negative sample during induction tended to be associated with reduced likelihood of heroin abstinence at month 12 (p = 0.07).There were very few pre-treatment characteristics significantly associated with heroin abstinence at month 12. Full results of this analysis are presented in Additional file p = 0.02). No other baseline characteristic was significantly associated with abstinence in that analysis with imputed results.We repeated the analysis, with imputed positive heroin results for the 43 patients who had an unplanned discharge. This indicated that heroin abstinence was significantly associated with being in an intimate/sexual relationship with another heroin user . To further assist understanding of the analysis and the pattern of drop out, an example is provided in Fig. Information on attrition during treatment is reported in Table The Cox regression analysis  had switched to methadone at that point. Only one person (2%) had switched from methadone to buprenorphine by month three. Seven people who commenced buprenorphine remained in treatment for 12 months, by which time six has switched to methadone. Only two of the 32 commenced on methadone had switched to buprenorphine by month 12.Heroin use is associated with a vast array of risks , 5, 25. The rate of heroin abstinence in month twelve constituted a significant improvement compared to the third month of treatment, at which point one in five demonstrated abstinence. This is consistent with one other study in adolescents and also with research on adult patients showing that longer treatment with OST builds incremental improvements in outcome , 15, 26.The confirmation that adolescents on OST can achieve substantial reductions in heroin use, with many doing so very early in treatment, should act as source of optimism for clinicians. It suggests that it is realistic for staff and treatment services to have a high expectation of reduced heroin use early in treatment and to be confident that many patients will achieve periods of sustained abstinence during treatment.We found no indication of a relationship between medication dose and heroin abstinence. There was no upper ceiling dose used in this service and our finding may indicate that dose was appropriately titrated against clinical need. There is research to indicate increased heroin use at lower OST doses in both adolescents and adults , 27. In The second main focus of this study was treatment retention. We found that one in four patients had an unplanned treatment exit by 120 days. Retention rates were similar in DARP in the 1970s and in the more recent study by Kellogg et al. , 15. ThoUnfortunately, half of those who were heroin abstinent demonstrated some ongoing use of benzodiazepines. Overall, the rates of abstinence from drugs other than heroin did not increase significantly from pre-treatment levels. Studies of OST in adults suggest that they can achieve reductions in use of other drugs , 26. HowAt this service, efforts were made to address cocaine use. Where use occurred it was discussed in counselling sessions and also during key working interventions. Our failure to successfully reduce cocaine use appears important for this patient group. Although cocaine use was not extensive among these heroin dependent adolescents, it seems to act as a negative prognostic factor for those who use it. Cocaine use at baseline was a significant predictor of treatment drop out. Cocaine use during OST was correlated with ongoing heroin use. A correlation between persistent heroin use and cocaine use has also been demonstrated by others in both adolescents and adults , 27, 28.Other potential prognostic factors were identified in the exploratory analysis, many of which suggested the importance of the family and social support system around the adolescent. Some characteristics of family systems may provide support to an adolescent who is facing a severe drug use disorder while other family types may add to the strain on an adolescent who is already coping poorly , 33. OurWe found that adolescents from families with two parents had better retention. Crome et al. also found better outcome with increased family support . InvolveA final systemic factor which was correlated with outcome was being a young parent. While only a small number of these adolescent patients had a child themselves, those who were faced with this additional strain on their personal resources were less likely to persist with treatment. Drop out occurred in spite of the efforts by the service to support these young parents via provision of crèche facilities on site. Others have highlighted the particular challenges faced by single parents with addiction issues, and some services have developed targeted programs of support such as the Parents under Pressure intervention .Those who had a previous psychiatric admission appeared less likely to be abstinent. Crome et al. also reported poorer outcome in those with a psychiatric history . Howeverp value via a Bonferoni correction, this raises the possibility that some significant findings may also constitute type 1 statistical errors.The strengths of this study include the sample size which is quite large relative to other studies of OST in this age range. However, the sample size is still relatively small for statistical purposes, with limited power. In view of the large number of statistical tests conducted in this study, and the decision not to alter the The treatment intervention was delivered at a single site making it easier for clinicians elsewhere to determine how applicable our findings may be to their own treatment setting. The monitoring of drug use by twice weekly supervised UDS constitutes a very thorough level of evaluation of drug use and we can be reasonably confident that the patients whom we determined to be abstinent are truly abstinent. A limitation can arise from assessing change via very regular urine screens. For example, a patient could change from injecting heroin four times a day at treatment entry to smoking heroin just twice a week. While this constitutes a very positive achievement from a harm reduction perspective, all UDS would be likely to remain opiate positive and our results would indicate no change in such a scenario.This study adds to the currently limited evidence base for OST in adolescents who are heroin dependent. The positive outcomes appear broadly similar to those achieved in adults, among whom there is widespread acceptance of this treatment. One of the lingering doubts about OST in adolescents since the DARP study relates to the limited success in achieving reductions in use of other drugs . In spitOverall, the efforts currently being made to ensure greater provision of OST to adolescents by groups such as the American Academy of Pediatrics appear appropriate and timely . Also, wAdditional file 1:Table S1. Heroin abstinence during month 12 among 39 heroin dependent adolescents on opioid agonist treatment. (DOCX 19 kb)


C) based on the experimental data. The comparison results demonstrate that the proposed damage evolution model can accurately describe the experimental results.This paper presents an experimental study on circular stirrup-confined concrete specimens under uniaxial and monotonic load. The effects of stirrup volume ratio, stirrup yield strength and concrete strength on damage evolution of stirrup-confined concrete were investigated. The experimental results showed that the strength and ductility of concrete are improved by appropriate arrangement of the stirrup confinement. Firstly, the concrete damage evolution can be relatively restrained with the increase of the stirrup volume ratio. Secondly, higher stirrup yield strength usually causes larger confining pressures and slower concrete damage evolution. In contrast, higher concrete strength leads to higher brittleness, which accelerates the concrete damage evolution. A plastic strain expression is obtained through curve fitting, and a damage evolution equation for circular stirrup-confined concrete is proposed by introducing a confinement factor ( The cracking process of concrete is different from those of other brittle materials. Research indicated that when maximum principal stress reaches a certain level, a large number of microcracks may exist in concrete, especially at the interface between coarse aggregates and mortar ,29. ConcC). The proposed damage evolution equation can represent the experimental results reasonably.This paper aims to investigate the damage evolution in circular stirrup-confined concrete specimens through experimental study. The tangent module degradation was defined as a damage indicator, and it was subsequently used to obtain damage evolution curves. Furthermore, the effects of stirrup volume ratio, stirrup yield strength and concrete strength on damage evolution of the stirrup-confined concrete are discussed. To describe the effects of various confinement parameters on concrete damage evolution, a damage evolution equation is proposed by introducing a confinement factor  was used in each test. Because of the discrepancy in strain between specimens, two loading conditions with different strain increments were selected in the tests. For unconfined concrete, the magnitudes of strain increment (Δε) in two loading conditions were 5.0 × 10The monotonic stress-strain curves of all specimens are shown in When the load reached a certain level, complete unloading always led to plastic deformation. The strain of unloading points increased with the loading/unloading process progressed.The curves showed obvious stiffness degradation during unloading process. With loading cycles increased, the unloading elastic modulus decreased continuously.The stirrup-confined concrete bore more loading/unloading cycles with higher ultimate strain compared with the unconfined concrete when failure occurred.In the cyclic stress-strain curves, three characteristic behaviors can be observed: In this study, two typical cyclic loading curves were selected for discussion. Curves of the remaining 14 specimens showed similar features, shown in c0) for three groups of unconfined concrete specimens was evaluated as 1.94 × 10−3, while those of the stirrup-confined concrete (εcc) were in the range of 2.56 × 10−3–3.76 × 10−3. The ultimate compressive strain was defined as the strain when stress softened to 50% peak compressive stress. The average ultimate compressive strain (εcu) of three groups of unconfined concrete was 3.33 × 10−3, while those of the stirrup-confined concrete (εccu) were in the range of 5.59 × 10−3–8.76× 10−3. The peak compressive stress ratios (fcc/cf0) and the corresponding peak compressive strain ratios (εcc/εc0) between stirrup-confined and unconfined concrete specimens are all greater than 1 for all specimens. The values of ccf/cf0 were in range of 1.07–1.19. Compared with the concrete strength improvement, the peak strain showed a more remarkable enhancement; εcc/εc0 were in the range of 1.43–2.12. In addition, the ultimate compressive strain ratios (εccu/εcu) were in the range of 1.70–2.79. It clearly indicated the stirrup confinement improved concrete ductility.The test results for all specimens are listed in cc/fcf0) and corresponding peak compressive strain ratio (εcc/εc0). In the table, for Kent and Park model [cc/εc0 may be caused by using the same expression to estimate enhancements in both strength and strain, which is relatively approximate. The model proposed by Saatcioglu and Razvi [et al. [rk model , the undnd Razvi  appears  [et al. , the tesThe cyclic loading tests in this study showed that after a certain load limit was reached, the complete unloading of each loading cycle generated irreversible residual deformation, known as plastic deformation ,52. The p-ε relationship experimentally obtained, two functions were obtained for unconfined and stirrup-confined concrete, respectively. For simplicity, a unified function was proposed for both stirrup-confined and unconfined concrete specimens.By applying a quadratic polynomial expression to the ε−3–5.0 × 10−3, and quadratic polynomial fitting is close to an average curve in that range. With parameters c = 6.283 and d = 0.831, the proposed function of stirrup-confined concrete shows good correlation with the experimental data with R2 = 0.997, as shown in R2 = 0.988 is observed, as shown in For comparison, a linear function is fitted, and the results are plotted together with the test data shown in D was defined to represent the evolution of concrete damage by Lemaitre [D can be calculated by means of Equation (4), where E0 is initial elastic modulus and uE is unloading secant stiffness of the line connecting present unloading point to next loading point in the loading cycles.The cyclic loading tests clarify that the concrete stiffness degrades because of the development of microcracks with the increase of plastic strain. In order to describe this phenomenon, a damage indicator Lemaitre . AccordiThe effects of the stirrup volume ratio, stirrup yield strength and concrete strength on concrete damage evolution were investigated by comparing the test results, which are illustrated in −3 and the curves increased sharply, suggesting rapid damage evolution. With stirrups allocated, damage emerged at strain in the range of 2.0 × 10−3–4.0 × 10−3, indicating that the damage evolution was delayed significantly. As compared with unconfined concrete, the damage evolution curve was gradual and longer. It is also observed in d2σ/dε2 = 0 at the interval point). Based on the strain at interval point εin, the plastic phase could be divided into two stages: plastic stage 1 and plastic stage 2. The damage evolution also presented distinct features in the different states:D was very small .In the elastic stage  is modified from cD by multiplying a confinement factor (C) as follows cD is the damage indicator of unconfined concrete defined by Li and Li [cY is damage energy release rate , which ca, cb, cl and ck are parameters of unconfined concrete (Li and Li [In Equations (6) and (7), i and Li ) that neC) is given by inC is confinement factor corresponding to εin × inC = 0.5 is suggested through the calibration with test data. The parameters m, n, and mA in Equation (8), which are obtained by the regression analysis of experimental results, are given by: Considering the different features of damage evolution before and after the interval point mentioned above, the expression of confinement factor  was introduced to the proposed damage evolution equation, to describe the effects of various confinement parameters on concrete damage evolution. The established damage evolution model can well represent the whole damage evolution process in circular stirrup-confined concrete. Because of less confinement parameters involved, the model can be conveniently applied to evaluate the plastic damage behavior of circular stirrup-confined concrete with reasonable accuracy.Based on experimental results, a plastic strain expression was proposed, and a confinement factor (Evidently, more test results are needed to fully validate the proposed model. In addition, the size effect of specimen on damage evolution model was not considered in the present study. As such, the proposed damage evolution model for circular stirrup-confined concrete can be refined in future research works.A study of damage evolution of circular stirrup-confined concrete specimens under monotonic and cyclic compression loadings was presented. The effects of stirrup volume ratio, stirrup yield strength and concrete strength on stress-strain curves and damage evolution of stirrup-confined concrete were investigated. Based on experimental results, expressions for plastic deformation and damage evolution were proposed. The following conclusions can be drawn from this study:


P = 0.00001) and overall survival . In conclusion, this meta-analysis demonstrates that c-Met overexpression is a significant prognostic marker for poor survival in patients who underwent surgical resection for PAC.The overexpression of c-Met protein has been detected in pancreatic adenocarcinoma (PAC). However, its prognostic impact remains unclear. We performed this meta-analysis to evaluate the prognostic value of c-Met overexpression in PAC. A systematic computerized search of the electronic databases such as PubMed, Embase, and Google Scholar was carried out. From 5 studies, 423 patients who underwent surgical resection for PAC were included in the meta-analysis. Compared with patients with PAC showing low c-Met expression, patients with c-Met-high tumor had significantly worse disease-free survival (hazard ratio = 1.94 ,  Despite the recent advances in diagnostic and therapeutic modalities, pancreatic adenocarcninoma (PAC) is still among lethal malignancies with 5-year survival rates of less than 10% . SurgicaWith more understanding of molecular mechanisms of carcinogenesis, novel molecular agents targeting epidermal growth factor receptor, vascular epithelial growth factor receptor, or c-Met has been proposed for the treatment of PAC , 8. HoweMET is a proto-oncogene that encodes tyrosine kinase receptor for hepatocyte growth factor (HGF) [MET gene) and initiates auto-phosphorylation of an intracellular kinase on the beta-subunit of the receptor. This interaction allows the binding and activation of multiple signaling molecules such as Src, PI3K, Gab1, SOS, or MEK1/2 [or (HGF) . HGF, alr MEK1/2 , 10. Thir MEK1/2 , 12. Ther MEK1/2 , lung car MEK1/2 , gastricr MEK1/2 , colorecr MEK1/2 , cervix r MEK1/2 , or hepar MEK1/2 . Severalr MEK1/2 –17.The overexpression of c-Met protein has also been detected in PAC –25. HoweFigure Table low or c-Methigh). IHC criteria were briefly summarized in the Table c-Met expression was assessed by immunohistochemistry (IHC). There was a marked heterogeneity between the thresholds used to dichotomize c-Met status (c-MetP = 0.00001) .From four studies , 23–25, ) Figure . The fixP = 0.006) .From four studies –25, 382 ) Figure . The ranVisual inspection of the funnel plots for DFS and OS showed symmetry, indicating there were no publication biases Figure .In this meta-analysis, we evaluated the prognostic impact of c-Met overexpression in patients with resected PAC. The results show that high c-Met expression is associated with significantly poor DFS or OS. To our knowledge, this is the first meta-analysis suggesting that c-Met overexpression represent an adverse prognostic marker in patients with PAC.PAC shows unfavorable prognosis with the most aggressive tumor biology. The traditional post-operative prognostic factors such as tumor size, lymph node involvement, or status of resection margin are insufficient to predict patients with a high risk of recurrence or metastasis. Therefore, the identification of reliable predictive markers and potential therapeutic targets is essential to guide individual treatment strategies and improve prognosis in patients with PAC. c-Met has been proven to play a critical role in the pathogenesis and progression of many tumor types –12.et al., patients with PAC showing diffuse staining for c-Met showed better OS than those with tumors showing no or focal staining (P = 0.026 by log-rank test). However, this study had a very small sample size (27 patients for OS comparison) and classified patients by the c-Met positivity or negativity, not by the c-Met expression status (low or high). In the current meta-analysis, we only included studies comparing survivals (DFS or OS) according to the c-Met expression status. Patients with c-Met-high PAC showed significantly shorter DFS  and OS , compared with those with c-Met-low tumor. Our results indicate that high c-Met expression is a significant prognostic marker for poor survival in patients with resected PAC.The enhanced expression of c-Met has also been observed in PAC –26. BecaMultiple studies also demonstrated that high expression of c-Met was associated with poor survival in various cancers –18. Thusin situ hybridization, or real-time quantitative PCR are currently used for assessing c-Met status [However, the major challenge for clinical development of c-Met inhibitors is that there are no standardized methods and criteria for c-Met overexpression. A variety of methods such as IHC, Western blot, fluorescence t status . In thisOur study has inherent limitations that should be noted. First, the meta-analysis included a small number of studies with a limited sample size. Second, the included studies were all retrospectively performed. Third, of the five studies, four were conducted in Asia. Finally, as we already mentioned, IHC criteria to stratify c-Met status were various among studies.In conclusion, our meta-analysis demonstrates that c-Met overexpression is a significant prognostic marker for poor survival in patients who underwent surgical resection for PAC. However, larger studies using standardized methods are still needed to verify the prognostic role of c-Met expression in PAC.This study was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines . We perfEligible studies should meet the following inclusion criteria: (i) patients had a diagnosis of PAC; (ii) DFS and/or OS were analyzed by c-Met expression status; (iii) HRs with 95% CIs for DFS or OS were reported or could be calculated from the data provided; (iv) papers were written in English.Data extraction was carried out independently by two investigators (BJK and HSK). If these two authors did not agree, other investigators (JHK and HJJ) were consulted to resolve the dispute.The following data were extracted from all eligible studies: first author's name, year of publication, country, number of patients, tumor stage, treatment, methodology of IHC, the criteria used to dichotomize c-Met expression as ‘high’ or ‘low’, and HR with 95% CIs for DFS or OS.I2 inconsistency test: P < 0.1 and I2 > 50% indicated the presence of significant heterogeneity. The fixed-effects model (Mantel-Haenszel method) was selected to calculate the pooled HR when substantial heterogeneity was not observed. When significant heterogeneity was detected across studies, we adopted the random-effects model (DerSimonian-Laird method).Statistical values used in this meta-analysis were obtained directly from the original articles. When papers had no HR and 95% CI, the Engauge Digitizer version 9.1 was used to estimate the needed data from Kaplan-Meier curves. The effect size of DFS and OS was combined through HR and its 95% CI. Heterogeneity among studies was estimated using the chi-square-based Cochran's Q statistic and P-values were two-sided and P < 0.05 was considered statistically significant. Publication bias was assessed graphically by the funnel plot method [The RevMan version 5.2 was used to combine the data. The plots show a summary estimate of the results from all the studies combined. The size of the squares represents the estimate from each study and reflects the statistical ‘weight’ of the study (relative contribution to the summary estimate). Results are presented as forest plots with diamonds representing estimate of the pooled effect and the width of diamond representing its precision. The line of no effect is number one for binary outcomes, which depicts statistical significance if not crossed by the diamond . All rept method .


Activation of the MET receptor tyrosine kinase by its ligand, hepatocyte growth factor (HGF), has been implicated in a variety of cellular processes, including cell proliferation, survival, migration, motility and invasion, all of which may be enhanced in human cancers. Aberrantly activated MET/HGF signaling correlates with tumorigenesis and metastasis, and is regarded as a robust target for the development of novel anti-cancer treatments. Various clinical trials were conducted to evaluate the safety and efficacy of selective HGF/MET inhibitors in cancer patients. There is currently no optimal or standardized method for accurate and reliable assessment of MET levels, or other biomarkers that are predictive of the patient response to MET-targeted therapeutics. In this review, we discuss the importance of accurate HGF/MET signal detection as a predictive biomarker to guide patient selection for clinical trials of MET-targeted therapies in human cancers. Receptor tyrosine kinases (RTKs) are involved in many vital processes including mammalian development, cell function and tissue homeostasis. The RTK MET and its ligand hepatocyte growth factor (HGF) activate multiple signaling pathways mediating embryogenesis, tissue regeneration and wound repair under normal physiological conditions . HoweverThe MET proto-oncogene, located on chromosome 7 (7q21-31), is widely expressed in the epithelial cells of organs such as the liver, lung, gastrointestinal tract, and kidney during embryogenesis and adulthood . HGF binGab-1 binding to MET through GRB2 primarily leads to activation of the phosphoinositide 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK)/ERK pathways. In the MAPK cascade, MET activation stimulates SHC and GRB2 recruitment to Gab-1, leading to MAPK activation , along wInteractive crosstalk between MET and other cell membrane proteins has been heavily investigated due to the development of drug resistance in targeted therapies. For example, the adhesion protein CD44 variant (CD44v6) is thought to promote downstream activation of the Ras pathway by complex formation between GRB2 and ezrin, radixin and moesin . MET canvia MET gene amplification  dCTP-labeled MET-H probe  detected MET amplification in 10% of chemotherapy-naïve primary gastric carcinomas compared with the surrounding normal mucosa  = 0.70) and PFS  [vs. 5.7 months [4.2-10.4]). Conversely, MET-negative patients had slightly reduced OS when treated with rilotumumab plus ECX compared with ECX alone (11.1 months [6.9-13.2] vs. 11.5 months [5.5-20.5]). A similar trend was observed for PFS [In a phase II trial (NTC00719550), rilotumumab in combination with the cytotoxic agents epirubicin, cisplatin or capecitabine (ECX) was evaluated in 121 patients with advanced or metastatic gastric or esophageal junction (G/EGJ) cancer. Rilotumumab plus ECX had an anti-tumor efficacy compared to ECX alone, with modestly improved OS  . The MET for PFS .vs. 11.5; HR = 1.37) and PFS . OS, PFS and ORR were statistically worse in the rilotumumab arm. Significantly more patients in the placebo arm achieved 12-month OS  and ORR  compared to the rilotumumab arm. No benefit from treatment with rilotumumab was observed, including patients with higher MET expression, and a higher incidence of fetal adverse events occurred in the rilotumumab arm [A phase III study to assess the efficacy and safety of rilotumumab in advanced gastric cancer did not confirm the phase II findings and was terminated due to futility results and casualties with an increased number of deaths as compared to chemotherapy . Rilotumumab arm . We suggumab arm .p = 0.69) or OS  was noted in patients receiving onartuzumab plus erlotinib vs. placebo plus erlotinib. However, in MET-positive (≥50% of tumor cells with a staining intensity of 2+ or 3+) NSCLC patients , onartuzumab plus erlotinib was associated with improved PFS  and OS . In contrast, MET-negative patients demonstrated worse OS after treatment with onartuzumab compared with patients treated with erlotinib alone. Despite improved PFS and OS in the MET-positive population, the ORR in this subset was not different from that of the placebo plus erlotinib arm [Onartuzumab  is a single-armed humanized modified 5D5 anti-MET antibody that binds to the MET sema domain and effectively prevents HGF from binding to MET . Preclininib arm , possiblinib arm .Because MET is involved in the regulation of tumor cell survival and metastasis, a better understanding of individual patient sensitivities to MET inhibitors can help guide clinical trial design. Therefore, the key to success for MET-targeted therapies in human cancers may lie in MET-driven patient selection, which is determined by many factors.via IHC [First, an integrated platform that incorporates accurate, validated methods and reagent kits, such as antibodies and substrates for the characterization of MET alterations, will help to improve MET-driven population selection. For example, a phase III trial of onartuzumab showed a lack of efficacy in patients with advanced NSCLC, who were selected for the trial based on MET overexpression detected via IHC . HoweverNext, MET-positive patient selection must also take into consideration of tumor stage and type, along with sample storage conditions, and must incorporate stringent clinical inclusion criteria. A single biomarker or a combination of biomarkers may serve as prognostic factors to fully inform patient selection. For example, in the phase III onartuzumab clinical trial, defining IHC MET positivity as 50% of tumor cells with moderate staining intensity might have resulted in the inclusion of patients with false-positive MET-overexpressing tumors.MET/CEP7 ratio of two or more or a mean of ≥5 MET copies per cell were defined as MET FISH positivity, which may result in the inclusion of patients with no MET-amplified tumors [MET gene amplification analysis by FISH for improved accuracy and reduced false positive detection rates.In a phase II trial, however, IHC analysis correlated better with significant improvements in OS and PFS compared with FISH analysis. In this study, a d tumors . These cd tumors . AdditioAdditionally, MET-driven or MET-positive cancer should be determined by the dominant activation of MET and related oncogenic pathways. MET signaling rarely occurs independently. Other oncogenic pathways, such as EGFR and TGF-β, appear to be involved in tumor progression independent of MET signaling . HoweverThus far, no MET inhibitors or antibodies have been approved for clinical use. Evidence is mounting that fighting MET-driven cancers may depend on many elements, such as the specific drug target, disease stage or type, optimal methods for tumor MET status assessment, and, perhaps most importantly, the identification of robust biomarkers as predictors of patient benefit from MET-targeting therapeutics.


IgG4-related disease is a rare and novel disease entity that tends to involve multiple organs. The pulmonary manifestation of this disease is highly variable and may mimic lung cancer, pneumonia, interstitial lung disease (ILD), sarcoidosis, and so forth. Small airway disease is rarely reported in IgG4-related lung disease (IgG4-RLD). In the current study, we describe a rare case of IgG4-RLD with patterns of ILD and bronchiolitis.A 43-year-old man had chronic cough and dyspnea on exertion for 4 years. Initial chest radiography showed diffuse interstitial infiltration. Follow-up chest computed tomography 4 years later revealed bilateral diffuse centrilobular nodules with tree-in-bud pattern, bronchial wall thickening, and mediastinal lymph nodes. Bilateral diffuse multifocal ground-glass opacities and mosaic attenuation were also observed. Pulmonary function test revealed mixed restrictive and obstructive ventilatory impairment.Video-assisted thoracoscopic surgery (VATS) lung biopsy showed interstitial fibrosis with lymphoplasmacytic infiltration rich in IgG4-positive plasma cells. Serum IgG4 level also showed remarkable elevation. Therefore, IgG4-RLD is confirmed.VATS wedge resection of right upper lobe and mediastinal lymph node.The patient responded well to steroid and immunosuppression therapy, and was regular followed-up in outpatient clinic.IgG4-RLD should be considered not only in ILD, but also in small airway disease. Serum IgG4 level may be a useful tool for screening. Although the lung is occasionally involved in IgG4-RD, the clinical and image presentations are highly variable, with a disease spectrum involving mediastinal lymphadenopathy, interstitial pneumonia, pleural effusion, and uncommonly, airway disease. Small airway disease is a rare presentation in this disease entity. In this report, we present a biopsy-proven case of IgG4-related lung disease (IgG4-RLD) with interstitial lung disease and bronchiolitis.IgG4-related disease (IgG4-RD) is a novel recognized fibro-inflammatory disorder and usually manifests as a mass-like lesion or generalized swelling in various organs, with characteristic pathological findings showing IgG4-rich plasma cell infiltration, chronic inflammation, and fibrosis. Nearly any organ can be involved, and many cases have elevated serum IgG4 concentrations.2Written informed consent was obtained from the patient for publication of the article.A 43-year-old man presented with chronic exertional dyspnea and cough for 4 years. The initial chest radiograph Fig. A showed Initial differential diagnosis includes diffuse panbronchiolitis, sarcoidosis and hypersensitivity pneumonitis. Connective tissue related ILD was excluded because of no associated symptoms such as arthralgia or skin rash, and there was no remarkable elevation of autoimmune marker. Pulmonary infection including tuberculosis was also unlikely due to no infection sign, negative sputum culture, and acid-fast stain. The patient received video-assisted thoracoscopic wedge resection of the right upper lobe and lymph node dissection. Both pathological findings showed prominent interstitial lymphoplasmacytic infiltrates, mild eosinophil infiltration, and fibrosis involving the alveolar septa. On immunohistochemistry, the number of IgG4-positive plasma cells was >50/high-power field (HPF), and the IgG4/IgG-positive plasma cell ratio was >40% Fig. . The serWe started anti-inflammatory treatment with prednisolone 20 mg/day and azathioprine 50 mg/day, and his respiratory symptoms and chest image improved after 3 months of treatment. The eosinophilia and anemia also improved. His serum IgG4 level gradually decreased but was still higher than the upper normal limit. The follow-up pulmonary function test revealed the following results: FEV1, 2.55 L (73.1% predicted); FEV1/FVC, 75%; TLC, 7.05 L (111.2% predicted); RV, 3.42 L (178.1% predicted); RV/TLC, 48.5% (160.2% predicted). Currently, he is in stable condition and regularly followed up in the outpatient clinic.3 IgG4 elevation in association with autoimmune pancreatitis was first recognized in 2001 and, later on, was also described in various idiopathic diseases which was previously thought to be confined to a single organ.,2 In the early 2010 s, the understanding of IgG4-RD had become more comprehensive. Now, it is considered as a systemic disease and usually presents with multiple-organ involvement and, typically, with mass formation or diffuse enlargement of the affected organs. Autoimmune pancreatitis, sialadenitis, dacryoadenitis, and retroperitoneal fibrosis are the most common and typical presentation of IgG4-RD. Multiple-organ involvement is common, and nearly all organs can be involved by this unique disease, although the prevalence of each organ involvement is still unclear.,2 The pathogenesis of IgG4-RD remained unclear. Whether the excess of IgG4 antibodies results in tissue destruction and fibrosis, or is merely a disease marker in response to the inflammation needs to be elucidated. Many patients are associated with allergic diseases such as asthma and atopy. Eosinophilia and elevated serum IgE level were also observed in 40% of patients. Therefore, IgG4-RD is suggested to be possibly associated with autoimmunity.The IgG4 antibody is a subclass of IgG and accounts for <5% of the total IgG in normal conditions. IgG4 was traditionally thought to be an anti-inflammatory immunoglobulin because of its inability to activate classical complement pathways, aggregate antigens, and form immune complexes. Increase of IgG4 positive plasma cells in tissue is helpful for diagnosis, especially when correlating with the morphological features on pathological examination. Elevated serum IgG4 concentration is also a useful screening tool but is not reliable enough to make a diagnosis. In a meta-analysis conducted by Hao et al, the pooling accuracy of serum IgG4 levels of >135–144 mg/dL yielded a sensitivity of 87% and specificity of 83% for diagnosing IgG4-RD. Serum IgG4 level may also increase in other diseases such as bronchiectasis, asthma, idiopathic pulmonary fibrosis, emphysema, hypersensitivity pneumonitis, primary sclerosing cholangitis, inflammatory bowel disease, Hashimoto's thyroiditis, and in approximately 5% of the normal population.,6 As IgG4-RD may have some histological variations between different organs, Japanese experts proposed comprehensive diagnostic criteria for this disease, which are practical for general physicians and nonspecialists. Patients who fulfill the following 3 criteria are diagnosed as having IgG4-RD: organ involvement or damage consistent with IgG4-RD; serum IgG4 level of >135 mg/dL; and histopathology showing marked lymphoplasmacytic infiltration, fibrosis, IgG4/IgG-positive cells of >40%, and >10 IgG4-positive cells per high power field. Our case fulfilled all 3 comprehensive diagnostic criteria for IgG4-RD and involved only the lung; therefore, the diagnosis of IgG4-RLD was made.The definite diagnosis of IgG4-RD must rely on histopathological analysis. The key morphological features are dense lymphoplasmacytic infiltrates, storiform fibrosis, obliterative phlebitis, and mild-to-moderate eosinophil infiltrates. However, the typical storiform fibrosis may not be apparent in the lung.The percentage of pulmonary involvement in IgG4-RD is uncertain. According to previous literatures, lung involvement was occasionally found in IgG4-RD patients. Pulmonary involvement of IgG4-RD is highly variable and can mimic lung cancer, usual interstitial pneumonia, nonspecific interstitial pneumonia, organizing pneumonia, and sarcoidosis. Four patterns were observed in IgG4-RLD, which include mediastinal, parenchymal, pleural, and airway involvement. Mediastinal and hilar lymphadenopathy are the most common pulmonary patterns. Parenchymal involvement includes nodules, masses, ground-glass opacities, consolidation, reticular opacities, bronchovascular thickening, and even honeycombing. Pleural effusion or nodules as predominant presentations were also reported but were uncommon. Airway involvement in IgG4-RLD is rare, including tracheobronchial stenosis and traction bronchiectasis.–12 Our case is the first IgG4-RLD with bronchiolitis in Taiwan, and the mosaic attenuation hasn’t been reported previously. The bronchial thickening and centrilobular nodules on chest CT are also clear evidences of large and small airway narrowing. The pulmonary function test of our patient showed mixed obstructive and restrictive pattern, which was compatible to ILD and small airway disease. However, similar to 2 previously reported cases,–12 our case also had normal DLCO value, and we suggest this is because of more extent of small airway involvement than interstitium.The bronchiolitis pattern of IgG4-RLD is unique. To our knowledge, only 6 cases of IgG4-RD with bronchiolitis were reported in literature, and all of them were Japanese patients Table .[8–12] O,6 However, some patients may not achieve complete resolution and relapse may occur. For steroid-refractory patients, immunosuppressants such as azathioprine, mycophenolate mofetil, and cyclosporine can be considered. Anti CD20 antibody (Rituximab) is also a reasonable treatment choice for refractory cases. The long-term survival in IgG4-RLD remains unclear because of the limited clinical follow-up data.,6Steroid therapy effectively relieved our patient's dyspnea and pulmonary lesions. The initial mixed restrictive and obstructive pattern in the pulmonary function test also resolved after steroid therapy. The mainstay treatment for IgG4-RLD is similar to that for IgG4-RD, which is corticosteroid therapy, and most patients have favorable response in 2 weeks. The usual dose for oral prednisone is between 0.6 and 1 mg/(kg·day) for 2 to 4 weeks, and then the dose is gradually tapered every 2 to 4 weeks to maintain a dose of 2.5 to 5 mg/day and then discontinued within 3 years. This disease should be considered not only in ILD, but also in small airway disease. Elevation of serum IgG4 level and associated multiorgan involvement are useful clinical clues, but definitive diagnosis requires surgical biopsy.This is the first case of IgG4-RLD with bronchiolitis in Taiwan. In conclusion, chest physicians should keep IgG4-RLD in mind because this novel disease is frequently underdiagnosed but potentially treatable.


A descriptive, retrospective and cross sectional study was conducted to assess the prescribing practices and antibiotic use patterns in the Accident and Emergency department of the Bahawal Victoria Hospital, Bahawalpur, Pakistan. A sample of 4320 prescriptions (systematic random sampling) was drawn out of a total of 1,080,000 prescriptions written during the period 1st January–31st December 2014. The standard World Health Organization/International Network for Rational Use of Drugs prescribing indicators were used to determine the prescribing practices of physicians. Published ideal standards for each of the indicators were used to identify irrational drug use. We also utilized an additional indicator to report the percentage share of antibiotics prescribed. The average number of drugs prescribed per encounter was 2.3 (SD = 1.3) . Drugs prescribed by generic name occurred 83.1% of the time . Antibiotics and injections were prescribed 52.4%  and 98.0%  of the time respectively. Drugs prescribed from the Essential Drugs List equated to 81.5% . Out of 52.4% (n = 2262) prescriptions with antibiotics prescribed, 77.7% (n = 1758) had one antibiotic, 22.1% (n = 499) included two antibiotics, and 0.2% (n = 5) had three antibiotics. Cephalosporins were the most commonly prescribed class of antibiotics (81.5%) followed by penicillins (6.4%) and fluoroquinolones (6.2%). Among the individual antibiotics, ceftriaxone contributed the highest percentage share at 71.8% followed by cefotaxime (5.6%) and metronidazole (4.7%). The most frequently prescribed antibiotic combination was ciprofloxacin with metronidazole (52.1%). Irrational prescribing practices were common. Continuous education and training of physicians is required to ensure rational prescribing at Bahawal Victoria Hospital in the future. The importance of assessment and evaluation of quality in healthcare is gaining global recognition and medicines play a pivotal role in the healthcare delivery system. The appropriate use of medicines is a crucial element in the quality of health outcomes and in the appropriate medical care provided to patients  in collaboration with the International Network of Rational Use of Drugs (INRUD) developed a set of indicators to measure the performance of healthcare facilities related to the utilization of drugs ’ , the economic burden of AMR amongst outpatients is estimated to be between $400 million and $18.6 billion. The costs associated with inpatients are thought to be much higher  department of the Bahawal Victoria Hospital (BVH), Bahawalpur, Pakistan. We also report the antibiotic use patterns in this same context. The results of this study could assist the hospital administrators to develop and implement appropriate interventions to improve rational prescribing of medicines in the A & E department of the BVH. The study findings may also be applicable to other hospitals that are suspected to have similar drug use practices.This study was carried out in the A & E department of the BVH, Bahawalpur, Pakistan. The BVH is a 1600 bed tertiary care hospital with established surgical and medical facilities. A total of 350 physicians, 10 pharmacists, 400 nurses and 3000 paramedical staff serve an average of 90,000 patients per month. The A & E department was established in 2005 with a capacity of 100 beds. The staff includes 25 doctors, one pharmacist and 54 nurses who provide care for between 2000 and 2500 patients per day.A retrospective, cross-sectional study design was employed to evaluate the prescribing practices for all medicines, as well as antibiotic utilization patterns. The optimal values for the prescribing indicators were adopted from previous studies Desalegn . The preInclusion All prescriptions written for the patients attending the A & E department of the BVH from 1st January 2014–31st December 2014 irrespective of age, gender and diagnosis were included in the study.The sample size for this study was 4320 prescriptions collected retrospectively from 1,080,000 written during the 1-year period of study. The prescriptions written each month were separated and then 360 prescriptions from each month were selected using a systematic random sampling technique.The data relating to the prescribing indicators was noted for each prescription and then recorded into a standard WHO prescribing indicator form  was used for the analysis of data. Descriptive statistics such as frequencies, percentages, averages and standard deviations (SD) were used to present data.The average number of drugs per encounter was 2.3 SD = 1.3). The percentage of drugs prescribed by generic name was 83.1% (SD = 1.1). The percentage of encounters with an antibiotic prescribed was 52.4% (SD = 0.5) and 98% (SD = 0.3) of drugs were given by the injectable route. Over three quarters (81.5%) of drugs were prescribed from the EDL or some other formulary (SD = 1.1)  and of these over three-quarters  had one antibiotic, 22.1% (n = 499) included two antibiotics and 0.2% (n = 5) had three antibiotics. Cephalosporins (81.5%), penicillins (6.4%) and fluoroquinolones (6.2%) were the most frequently prescribed antibiotic classes (Table Ceftriaxone was the most commonly prescribed (71.8%) antibiotic. Thereafter, cefotaxime (5.6%), metronidazole (4.7%) and amoxicillin (4.7%) were most common Table .Table 3MThe most frequently prescribed antibiotic combinations were ciprofloxacin with metronidazole (52.1%) and ceftriaxone with metronidazole (38.8%) Table .Table 4MIrrational prescribing practices exist all over the world and eventually they lead to unwanted effects in patients    Table . This woIn our study, 98% of prescriptions included at least one injectable product  Table  and KuwaWith regard to drugs prescribed from the EDL, our findings were comparable to other studies conducted in the Lao People’s Republic (86.2%)  prescriptions containing antibiotics, 77.7% (n = 1758) contained one antibiotic, 22.1% (n = 499) included two antibiotics and 0.2% (n = 5) had three antibiotics. These findings can be compared with a study conducted in Jordan , penicillins (6.4%) and fluoroquinolones 6.2%) (Table % Table . In a siThe increasingly growing threat of AMR and the lack of a significant pipeline of new antibiotics in development has drawn attention toward multi-drug therapies (Golan et al. There are certain other risks and adverse effects associated with the use of antibiotics and antimicrobial combinations. The major associated risks include development of super-infections, augmented toxicity and greater cost. The well described adverse effects include hypersensitivity reactions, diarrhea, nephrotoxicity and coagulopathy (Rybak and McGrath The current study demonstrates irrational prescribing practices in the A & E department of a tertiary level hospital in Pakistan. The findings in terms of prescribing indicators are divergent with the expected norms. Cephalosporins contributed the highest percentage share amongst all the antibiotic classes, while ceftriaxone was the most commonly prescribed antibiotic. The extent of antimicrobial prescribing in this setting raises concerns of AMR and highlights an ignorance of doctors towards their prescribing behavior.This study contributes to the international literature by addressing medicines use in the context of the A & E department; a point of first contact with the health system in many developing countries. Further, this study adds to the body of knowledge surrounding antibiotic use in this A & E setting and compares use in Pakistan with other developing countries. The study adds further evidence that policy-makers, hospital administrators, doctors, nurses and pharmacists should all be wholly concerned about the developing world’s ongoing contribution to AMR. There are few antibiotic medicines in the drug development pipeline and therefore few therapeutic solutions when current agents stop being effective. The strategy must immediately be focused on curbing the current rise in AMR.The study findings support a recommendation that continuous training and educational programs for medical staff concerning rational use of injections and antibiotics should be implemented and monitored so that the required changes in prescribing become sustainable. A feedback monitoring system on doctors’ antibiotic prescriptions would be expected to significantly improve their prescribing behavior. Knowledge and compliance with updated clinical guidelines is also recommended to enhance the degree to which generic prescribing occurs.Further studies are encouraged to find the reasons for irrational use of medicines and to demonstrate the factors driving the irrational prescribing of antimicrobials by the physicians at BHV. Appropriate interventions regarding rational prescribing of antimicrobials should be designed and implemented in the hospital and an evaluation is required to determine whether there has been any improvement in the prescribing practices of physicians.There are limitations with pharmaco-epidemiological approaches that compare studies across countries where the hospital context might be different. However, the authors are left with few options when the literature is scarce and limited within the context of A and E antibiotic prescribing in developing countries. In addition to international comparisons the findings from this study should not be generalized to the whole of Pakistan; but they do give a robust indication of what is happening in one hospital A and E department and may precipitate other hospitals in Pakistan to begin an audit which will give a sense of whether the high use of IV ceftriaxone is a national phenomenon. Based on the fact that a uniform healthcare policy is implemented throughout the country, and medical graduates from various institutions are working in Bahawalpur city the practices may be similar to A & E departments of other tertiary care hospitals in Pakistan. This needs to be further instigated and if it is the case then a national policy will need to be developed to address this issue. In this study, we did not explore the reasons for irrational drug use, which could be considered in future studies.


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