In brain-injured patients intracranial pressure (ICP) is monitored invasively by a ventricular or intraparenchymal transducer. The procedure requires specific expertise and exposes the patient to complications such as malposition, hemorrhage or infection. As inner-ear fluid compartments are connected to the cerebrospinal fluid space, ICP changes elicit subtle changes in the physiology of the inner ear. Notably, we previously demonstrated that the phase of cochlear microphonic potential (CM) generated by sound stimuli rotates with ICP. The aim of our study was to validate the monitoring of CM as a noninvasive method to follow ICP.Non-invasive measure of CM-phase was compared to ICP recorded invasively in a prospective series of patients with acute brain injury managed in a neuro-intensive care unit. The study focused on patients with varying ICP and normal middle-ear function.In the 24 patients with less than 4 days of endotracheal ventilation and whose ICP fluctuated (50-hour data), we demonstrated close correlation between CM-phase rotation and ICP (average 1.26 degrees/mmHg). As a binary classifier, CM phase changes of 7–10 degrees signaled 7.5-mmHg ICP increases with a sensitivity of 83% and 19% fallout.Reference methods to measure ICP require the surgical placement of a pressure transducer. Noninvasive CM-based monitoring of ICP might be beneficial to early management of brain-injured patients with initially preserved consciousness and to the diagnosis of neurological conditions, whenever invasive monitoring cannot be performed.NCT01685476, registered on 30 August 2012.ClinicalTrials.gov The online version of this article (doi:10.1186/s13054-017-1616-2) contains supplementary material, which is available to authorized users. In brain-injured patients, intracranial pressure (ICP) is monitored to manage cerebral perfusion pressure and prevent secondary lesions , 2. The Cochlear sensory-cell responses to sound stimuli may address this challenge. Their detection is routinely applied in screening of neonatal hearing, by paramedics after minimal training. The sensitivity of cochlear sensory-cell responses to ICP changes results from a balance between cerebrospinal and intralabyrinthine fluid pressures –8, such The objective of the research was to confront noninvasive ICP monitoring using CM with invasive ICP monitoring in patients with acute brain injury managed in a neuro-intensive care unit (NICU). During the one-year enrolment period, 53 consecutive patients were enrolled in this prospective, observational study. The inclusion criterion was the possibility of organizing a monitoring session without disturbing the NICU schedule, and prior to this session, of obtaining informed consent from an authorized person. In view of the collected raw data, all patients measured more than 4 days after NICU referral were excluded from further analysis. The main reason was the lack of sufficient ICP fluctuations in these patients, as the intended search for correlation between CM and ICP is valid only if the independent variable, ICP, exhibits enough variability.-controlled infusions [The patients had been referred to the NICU for head trauma, including brain hemorrhage, tumors, stroke or meningitis. They were intubated, mechanically ventilated and sedated with sufentanil plus midazolam or remifentanil using targetnfusions .-trode™, active) inserted in the external acoustic meatus and skin electrodes pasted on the forehead (passive and ground). The CM-eliciting stimulus is a tone sent by a calibrated earphone . The only action undertaken by the NICU nurses was positioning of the electrodes. The fastest rate of CM sampling was one point every 10–20 s in the NICU environment. Here, for monitoring sessions scheduled to exceed 60 minutes, one CM data point was collected every 5 minutes. For monitoring sessions of 30–60 minutes, the sampling rate increased to 1/minute and for even shorter sessions, to 3/minute. In parallel, ICP was continuously monitored through an intraparenchymal probe . Data collection was stopped when nursing procedures required the monitoring equipment to be disconnected. Accidental earplug-electrode dislocation, which occurred once, led to invalid data, which was identified and discarded offline.The CM technique detects a phase rotation due to the change in stiffness of the ossicular chain induced by ICP. If middle-ear mechanics are modified by a confounding factor, e.g., a poorly ventilated or fluid-filled middle-ear cavity, the CM phase may undergo an additional rotation and the sensitivity of the CM-phase-to-ICP relationship may change. After video-otoscopic examination of the tympanic membrane, the tip of a tympanometer  was inserted. By sweeping the air pressure in the sealed ear canal, tympanometry determines whether the tympanic membrane optimally transmits sound pressure to the cochlea at atmospheric pressure. Insufficient middle-ear ventilation or middle-ear effusion is thus readily identified. As these two conditions are known to be induced by long-lasting intubation and the resulting auditory-tube dysfunction , 13, a sThe protocol was approved by the local Ethics Committee and National ANSM Agency, and registered in the Clinical trials register under the number NTC01685476 . Informed consent was initially given by an authorized person, pending the patient's own consent, recovery permitting.R), the determination coefficient (R2) and the 95% confidence interval for the correlation coefficient as outcomes. The slope of the linear regression line relating CM-phase change to ICP change was computed for each ear. Its correlation with other factors, namely age and severity indexes, was studied.Pearson's correlation between ICP and CM phase changes were analyzed for each monitoring session, with the correlation coefficient (Receiver-operating characteristics (ROC) were plotted, representing sensitivity against 1 - specificity of the CM-based binary classifier, with each point corresponding to a gradually increasing discrimination threshold, defined as the CM-phase-change boundary between pass and refer diagnoses. Several ROC analyses were performed, using two different gold standards for ICP increase (against which CM phase increases were tested), 5 mm and 7.5 mmHg; and two different gold standards for ICP decrease (against which CM phase increases were tested), 5 mm and 7.5 mmHg. The optimal CM-phase-change limit was determined either according to the Youden index, or from the best trade-off between the risk of false negatives that dismiss genuinely threatening ICP increases, and of false positives that might induce an over-reaction to harmless ICP changes.Although all 53 patients who were tested had valid monitoring signals from the intraparenchymal ICP transducer and the CM electrodes (at least in one ear), only the data from the 24 patients who had not been intubated for more than 4 days when tested were further analyzed. These patients were aged between 19 and 76 years . Seventeen patients had been referred with head trauma, three with brain hemorrhage and four with a tumor, stroke or meningitis. At referral, which occurred on average 3.9 h (SD 4.4) after symptom onset, the mean Glasgow score was 5.3 (SD 3.4), mean ICP was 18.9 mmHg (SD 11.4), with ICP over 20 mmHg in 11 patients. Severity was 54.8 (SD 14.3) on the Simplified Acute Physiology Score (SAPS-II score) and 8.2 (SD 2.2) on the Sequential Organ Failure Assessment score (SOFA score).When their CM was monitored, all of these 24 patients had normal middle-ear function as assessed by tympanometry. Conversely, none of the excluded patients (n = 29) who had been endotracheally ventilated for more than 4 days when tested, had a normal tympanometry . In the subgroup of patients who received everyday tympanometry, it was possible to check that middle-ear negative pressure, then effusion, developed from the fifth day post intubation . Individual slopes of the CM-phase-to-ICP linear regression displayed some variability  possibly unduly influenced by two outliers. The slope of the CM-phase-to-ICP changes in degrees/mmHg displayed no significant correlation with age . Severity indexes did not explain the variability of outcomes .Age was weakly correlated with the quality of CM monitoring in relation to ICP , namely the ability to warn of ICP changes of +5 and -5 mmHg. For the +5 mmHg criterion , and specificity remaining good at 81%. In the mirror situation of a 5 mmHg decrease in ICP  over periods of 3–4 h, in patients with no middle-ear impairment.The performance of the resulting classification compared favorably to those of other methods that targeted similar ICP changes , with seA conservative use of ROC analysis thus chooses a more stringent CM-phase limit than that of Youden's index. False negatives decreased to 17% when an ICP increase of 7.5 mmHg was targeted. Conversely, when ICP variation remained less than 5 mmHg, the rate of false alarms that a drifting CM phase would trigger was less than 20%. The tracking of ICP decreases performed even slightly better. An important target of the present method, i.e., patients with a steady ICP increase, 5 mmHg then 7.5 and more after the accident, would thus be spotted early and efficiently enough to be directed to an appropriate emergency ward with little risk of undue alarm.Abnormal transmission of sound through the middle ear must be ruled out as it may influence the CM phase in an unpredictable manner. Tympanometry, which can be performed by paramedics after minimal training (and might be implemented in CM measuring devices), is recommended in the category of patients with head trauma with fractures, to rule out bilateral middle-ear involvement. In patients intubated for fewer than 5 days, middle-ear status remains normal, ensuring large CM-to-ICP correlations. In patients who stay longer in NICU, middle-ear effusion develops in relation to intubation and unconsciousness . In trauThe CM monitoring procedure conducted with an extratympanic electrode is harmless, complying with security requirements on exposure to loud sounds, which tolerate an 8-h, 80-dB (in Europe) or 85-dB (in the USA) daily exposure. With the chosen sampling rate, suitable for detecting slow changes in ICP, patients were exposed to sound for about 20 s every 5 minutes and showed no reaction to sound presentation.CM measurements have been routinely used for decades in audiology . The preMost non-invasive methods require initial invasive calibration  except tInvasive ICP measurements are not exempt from technical problems. The baseline drift of ICP microtransducers reaches up to 7.3 mmHg so that the standard deviation of the error may vary from 3.4 to 8.5 mmHg . ExternaThe patients in this study were selected because the gold standard of invasive ICP monitoring was required to assess the CM technique. They do not constitute the realistic target population for noninvasive ICP monitoring. The present preliminary results suggest a potential interest for the CM technique to be used whenever invasive monitoring of ICP is unavailable or contraindicated, keeping in mind the two limitations of CM, i.e., the need for calibration to access absolute ICP and the risk of false negatives due to the imperfect sensitivity.With a strategy based upon the detection of an increase in ICP of 7.5 mmHg, the same order of magnitude as the standard deviation of the error of the best noninvasive absolute methods published so far, CM monitoring would spot significant ICP trends in patients starting from a clinically satisfactory status, suggesting a likely normal initial ICP. At the scene of an accident, particularly in a remote area far from acute-care hospitals, a noninvasive method of detecting an increase in ICP could be crucial in patients initially categorized as having “mild brain injury” before the onset of consciousness disorders. Mild traumatic brain injuries are usually managed in a general hospital if early computed tomography is normal. A significant change in CM could signal the formation of an intracranial hematoma and, together with the clinical methods currently in use, help assess whether transfer to a neurosurgical center is appropriate.Management of cerebrovascular accidents provides similar indications. In patients with ambiguous clinical assessment, our method could contribute to early detection of brain edema when ICP starts increasing. Patients under the influence of alcohol or treated with platelet anti-aggregants or anticoagulants require special attention because they are susceptible to worsening and difficult to monitor clinically. In these cases, non-invasive monitoring of ICP could help decide whether transfer to an NICU is required. Other indications include the pediatric population whose lack of compliance might make medical surveillance difficult, and the follow up of patients referred to emergency wards for severe headache on suspicion of intracranial hyperpressure, when invasive methods cannot be performed. In addition to primarily being used for screening in departments without the possibility of invasive monitoring, CM may serve as an outpatient tool in patients with long-lasting ICP problems, after initial calibration by an invasive ICP measurement.In conclusion, the portability and cost-effectiveness of the CM method, usable by paramedics in emergency vehicles or in patients’ homes, makes it promising for use in patients who cannot currently benefit from ICP monitoring.


One possible solution to try and overcome GCR, is to identify molecules that prevent or revert GCR by hyper-stimulating the biological activity of the GR. To this purpose, we screened for compounds that potentiate the dexamethasone (Dex)-induced transcriptional activity of GR. To monitor GR transcriptional activity, the screen was performed using the lung epithelial cell line A549 in which a glucocorticoid responsive element (GRE) coupled to a luciferase reporter gene construct was stably integrated. Histone deacetylase inhibitors (HDACi) such as Vorinostat and Belinostat are two broad-spectrum HDACi that strongly increased the Dex-induced luciferase expression in our screening system. In sharp contrast herewith, results from a genome-wide transcriptome analysis of Dex-induced transcripts using RNAseq, revealed that Belinostat impairs the ability of GR to transactivate target genes. The stimulatory effect of Belinostat in the luciferase screen further depends on the nature of the reporter construct. In conclusion, a profound discrepancy was observed between HDACi effects on two different synthetic promoter-luciferase reporter systems. The favorable effect of HDACi on gene expression should be evaluated with care, when considering them as potential therapeutic agents. GEO accession number GSE96649.Glucocorticoid resistance (GCR),  Glucocorticoids (GC) are a most effective therapy for the treatment of many inflammatory disorders, such as asthma, rheumatoid arthritis and inflammatory bowel disease –3. GlucoThe therapeutic success of GCs is limited due to two main drawbacks. High doses and/or prolonged administration of synthetic GCs often result in side-effects, including glaucoma, type 2 diabetes, growth retardation and skin thinning , 10. Morin vivo as well as in vitro using cell lines, induces a status of GCR  Vector) plasmid was purchased from Promega. SAHA  was obtained from Sigma Aldrich and Belinostat , CI994, Class I HDACi  and Abexinostat, pan HDACi, mostly targeting HDAC1 (PCI-24781) were purchased from Selleckchem. For all compounds, a stock dilution of 10-2M was prepared in dimethylsulfoxide (DMSO) and stored in -20°C. Dexamethasone  was purchased from Sigma Aldrich and dissolved in water. Recombinant human TNF was produced in E. coli and purified in our department.The GRE-luc plasmid was described previously . Briefly-6M Dex for five hours. Cells were then harvested and luciferase activity, expressed in arbitrary light units, was quantified with the Glomax instrument, measuring the D-luciferin  conversion. For transiently transfected cells, luciferase activity was corrected for the protein concentration in the sample by normalization to constitutive β-Gal levels. β-Gal levels were quantified with a chemiluminescent reporter assay, using CPRG substrate (Sigma Aldrich).X-tremeGENE HP DNA transfection reagent from Roche was used to transfect the A549 cells in 24-wells plate according to manufacturer’s instructions. Six hours after transfection medium was changed to Optimem Medium  and cells were exposed to HDACi. After the indicated duration, cells were stimulated with 105’-ATACAAAGGATATCAGGTGG- 3’ and the reverse primer: 5’- TTGCGTCGAGTTTTCCGG-3’. Results are given as relative expression values normalized to the geometric mean of the housekeeping genes 36B4 and Cyclophilin. The stability of these housekeeping genes in the appropriate experimental model and condition was verified using the geNorm algorithm [Total RNA was isolated from A549 cells using TRIzol  and the InviTrap Spin Universal RNA Mini Kit  according to the manufacturer’s instructions. RNA concentration as measured with Nanodrop (Thermo Scientific), and 1μg of RNA was used to prepare cDNA by reverse transcription with the iScript advanced cDNA synthesis kit (Bio-Rad). qPCR was performed with SYBR Green Master Mix (Bioline) using the Roch LightCycler 480 system (Applied Biosystems). Luciferase mRNA was detected by RT-qPCR using the forward primer: lgorithm .6 cells/well) in DMEM. Cells were exposed to Belinostat (1μM) equal dilution of DMSO, 16 hours before Dex  or PBS stimulation. Total RNA was isolated using TRIzol (Gibco Life Technologies) and the InviTrap Spin Universal RNA Mini Kit  according to the manufacturer’s instructions. RNA concentration was measured and quality was checked with the Agilent RNA 6000 Pico Kit (Agilent Technologies Belgium), and 5μg of RNA was sent to the Nucleomics core facility of VIB (http://www.nucleomics.be/) for sequencing on an Illumina Genome Analyzer. Library preparation was performed according to the Illumina truseq RNA stranded library protocol. The library was subjected to single end sequencing. Raw sequencing data underwent a quality control analysis with FastQC [2 fold change |LFC| of 1 for comparisons to the control condition. To detect subtler changes between Dex and Belinostat (single and co-treatment) the LFC cut-off was lowered to 0.5 for the direct comparisons between these conditions (FDR was kept at 1%). RNAseq data are deposited under accession number GSE96649.A549 cells were seeded in 6-well plates . One compound, SAHA, known as a broad-spectrum HDAC inhibitor [et all. who demonstrated GCR in vivo [The success rate of glucocorticoid therapy as a treatment for inflammatory disorders is limited by side-effects and GCR , 65. Scrnhibitor  strongly in vivo . Interes-6M Dex on the transcriptome of A549 cells was significant, and numerous GRE genes were strongly induced, e.g. FKBP5 (21-fold induction) and SGK (4.89-fold induction). In total, 938 genes were strongly and significantly affected by Dex (To next study the impact of Belinostat on endogenous GR-induced (GRE-driven) genes, we performed a transcriptome profiling using RNA sequencing in A549 cells. The impact of a 5h incubation of 10d by Dex , i.e. wid by Dex  , but no stimulatory effects of Belinostat  were observed in the absence or presence of Dex. On the contrary, Belinostat reduces the luciferase expression , would still stand in other experimental system.We and others have shown before that under TNF-induced GCR conditions, the transcriptional activity of GR is heavily compromised . One way-6M) was increased about four- to ten-fold  of the glucocorticoid signaling pathway. The maximal luciferase fold induction obtained with Dex alone (10ten-fold .AGATCACTCTGTGCA). We also checked the genes that did show a basal effect of Belinostat alone with a Dex effect for GRE elements and surrounding sequence. However, we could not find GRE elements any more than could be expected by random chance in these groups. Hence, the RNAseq experiment did not correlate in a direct manner to the reporter-based assay. We subsequently investigated several mechanisms that could explain the contradictory effects of Belinostat including the chromatin status, as transformed GRE-luc, integrated in the A549 genome is accepted to be wrapped around histones. These histones contain an N-terminal histone tail which can undergo posttranslational modifications including lysine ubiquitination, sumoylation, phosphorylation but also acetylation [When studying the impact of Belinostat on a genome-wide scale, only a minority of Dex-induced genes was further enhanced in the presence of Belinostat, or more repressed in case of Dex repressed genes. Belinostat alone had a huge effect on the mRNA levels of thousands of genes. When taking the impact of Belinostat into consideration on Dex-regulated genes without basal Belinostat effect, only 5 genes were found to be further regulated in the same direction by Belinostat . Among ttylation . The lastylation . HDACi atylation , 22. Theet al. demonstrated that the histone acetyltransferase p300 is recruited to the TAT-GRE promoter in a Dex responsive way, indicating a role of p300 in GR transcriptional activity [et al identified HDAC1 as coactivator for GR-induced transcription of the MMTV promoter [Finally, by testing Belinostat effects in another, and differentially regulated validated GC reporter system, i.e. the MMTV-luciferase reporter, no co-stimulatory effect of Belinostat was observed, but on the contrary, a repressive effect was seen. Although additional experiments will be needed to further pinpoint the exact mechanisms of action of Belinostat on the GRE-luc and MMTV-luc reporter construct, it is of interest that signalling pathways involving different GR responsive elements can be modulated differentially by Belinostat. We have considered a possible mechanism by which Belinostat have an altered effect on GRE-luc and MMTV-luc expression. It has been proposed that the bound DNA sequence acts as an allosteric ligand that alters GR conformation and hence interactions with other proteins such as cofactors which ultimately affect the transcriptional output . Consequactivity . On the promoter . More sppromoter . These lOur work has contributed to identify opposite GRE-specific regulatory effects by the HDACi Belinostat, with a stimulatory effect on the classic palindromic GRE derived from the TAT promoter and an inhibitory effect on the specific GRE from the MMTV promoter. To extract generalizing conclusions based on reporter gene assays, our take-home message would be to not solely rely on one type of promoter-luciferase reporter, but invest in different ones and also invest in non-reporter dependent validation assays.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Most patients with advanced non-small cell lung cancer (NSCLC) have a poor prognosis and receive limited benefit from conventional treatments, especially in later lines of therapy. In recent years, several novel therapies have been approved for second- and third-line treatment of advanced NSCLC. In light of these approvals, it is valuable to understand the uptake of these new treatments in routine clinical practice and their impact on patient care. A systematic literature search was conducted in multiple scientific databases to identify observational cohort studies published between January 2010 and March 2017 that described second- or third-line treatment patterns and clinical outcomes in patients with advanced NSCLC. A qualitative data synthesis was performed because a meta-analysis was not possible due to the heterogeneity of the study populations. A total of 12 different study cohorts in 15 articles were identified. In these cohorts, single-agent chemotherapy was the most commonly administered treatment in both the second- and third-line settings. In the 5 studies that described survival from the time of second-line treatment initiation, median overall survival ranged from 4.6 months  to 12.8 months . There was limited information on the use of biomarker-directed therapy in these patient populations. This systematic literature review offers insights into the adoption of novel therapies into routine clinical practice for second- and third-line treatment of patients with advanced NSCLC. This information provides a valuable real-world context for the impact of recently approved treatments for advanced NSCLC. Globally, lung cancer is the second most common newly diagnosed cancer and the leading cause of cancer death. In 2012, there were an estimated 1.8 million new lung cancer cases and almost 1.6 million deaths –5.Lung cancer imposes a substantial economic and societal burden . In EuroSystemic therapy can provide a meaningful clinical benefit for patients with advanced NSCLC, and several new therapies have been approved since 2010 .–25.13–25In 10 studies that provided detailed information on prescribed treatment regimens, single-agent chemotherapy was the most commonly used second-line treatment regimen except in one study from Japan. In the Japanese study, single-agent targeted therapy was the most commonly prescribed second-line treatment . DocetaxPemetrexed was one of the most commonly prescribed second-line treatments in the studies that included patients treated after its initial approval in 2004 , 25, 27.Second-line treatment with EGFR-TKIs increased in the European studies after initial approval of erlotinib in 2005 , 24, 25.Platinum-based doublet chemotherapy was administered in all 7 studies that described the use of second-line combination chemotherapy regimens , 24–26. Nine of the 12 studies reported detailed information on patients who received third-line therapy , 21–25. All 12 studies reported details about the distribution of first-line treatment regimens. The most frequently administered first-line treatment across all countries was platinum-doublet chemotherapy . Three EIn Japan, fourth-line chemotherapy was reported in 17.7% of the patients who received first-line chemotherapy. The top 3 anti-cancer treatment regimens in the fourth-line setting were single-agent S-1 (22%), docetaxel (21%), and gefitinib (21%) . In BrazEleven of 12 studies included in this review presented overall survival (OS) data  13, 15––15–27. FIn 5 studies that described median OS from time of second-line treatment initiation in routine clinical practice, median OS ranged from 4.6 months  to 12.8 months  , 25, 27.Two studies reported biomarker testing and treatments prescribed based on biomarker status, one in Italy (LIFE) and one in the US that included only patients with adenocarcinoma  15, 16,,16, 27. EGFR mutations received erlotinib as second-line treatment compared with 17% of the 89 EGFR wild-type patients. The use of erlotinib was significantly more common in patients with EGFR mutations compared with EGFR-wild type patients (P < .001) [Both studies provided evidence of biomarker-driven therapy choices within their cohorts. The LIFE study reported that 26 of 37 (70%) patients who received EGFR-TKIs as first-line therapy were known to have EGFR-activating mutations prior to first-line treatment , 16. In  < .001) .The objective of this study was to describe real-world treatment patterns and survival outcomes for patients with advanced NSCLC who received second-line or later treatments, through a review of recently published observational studies. To our knowledge, there are no systematic reviews that summarize this information. By qualitatively reviewing this information from multiple countries, we provide a broad overview of how patients are managed in this setting around the world.Overall, the retrieved studies showed how newly approved therapies are rapidly integrated into clinical practice, and how clinical practice evolves in response to therapeutic advances. In addition, most studies demonstrated a high level of clinician adherence to international treatment guidelines, such as those from European Society of Medical Oncology and the US National Comprehensive Cancer Network , 29. AmoSurvival after initiation of second-line treatment was reported in most studies, and was generally consistent with outcomes reported from pemetrexed, docetaxel, and erlotinib clinical trials –33. The In most studies that reported the proportion of patients with advanced NSCLC treated across lines of therapy, between one-third to one-half of those who received first-line treatment also received second-line therapy , 23–26. ALK mutations provides further evidence that biomarker-directed therapy is an important treatment option for these patients [There was limited information reported on biomarker-guided therapy decisions in the studies included in this review, likely due to the time periods covered by the studies. In the studies covering more recent time periods, the rapid uptake seen in the use of targeted therapies and biomarker testing is encouraging. It reveals that biomarker-driven treatment decisions are being integrated into routine clinical practice. This information provides a positive outlook for the uptake of newer targeted therapies in the second- and third-line settings. The rapid uptake of anaplastic lymphoma kinase (ALK) inhibitors such as crizotinib and ceritinib in patients with advanced NSCLC harboring patients .EGFR mutation testing in Europe after erlotinib approval. This suggests a trend among clinicians of using new therapies when clinically appropriate, despite limitations in access to biomarker testing. These trends imply positive uptake of new immunotherapies like programmed death-ligand 1/programmed death 1 (PD-L1/PD-1) checkpoint inhibitors despite evolving biomarker testing guidelines.Biomarker testing may impact uptake of newly approved targeted therapies. More information on how and when biomarker testing is performed in routine clinical practice is needed, since it is unclear in the included studies whether biomarker testing was more commonly performed prior to first-line versus second-line treatment. Across the European studies, erlotinib use increased despite approval of A few limitations should be considered when interpreting the information provided in this review. A meta-analysis could not be conducted due to the heterogeneity of the study designs and data presentation in the included studies. There were substantial differences in patient selection procedures, data collection methods, and outcome measure definitions. Thus, a qualitative data synthesis was the most appropriate way to summarize these studies . MoreoveReal-world studies of second- and third-line treatment patterns in advanced NSCLC provide insights into how evidence from clinical trials impacts clinical practice. The studies included in this qualitative systematic review demonstrate the limited clinical benefit of the second- and third-line treatments for advanced NSCLC that were available at the time these studies were conducted. Within recent years, several novel therapies have been approved for use in these settings, and it will be important to determine the benefit that these and other new treatments may provide to patients with advanced NSCLC.S1 TableThis table contains the search string that was used to perform the systematic review.(DOCX)Click here for additional data file.S2 TableThis table contains the assessment of bias for each study.(DOCX)Click here for additional data file.S3 TableThis table contains the summary of third-line treatments by country.(DOCX)Click here for additional data file.S1 FileThis document contains a completed PRISMA checklist.(DOC)Click here for additional data file.


Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii, and may be intrinsic or mediated by transferable carbapenemase-encoding genes. This type of resistance genes are already widespread in certain parts of the world, particularly Europe, Asia and South America, while the situation in other places such as sub-Saharan Africa is not well documented. In this paper, we provide an in-depth review of carbapenem resistance providing up-to-date information on the subject.Carbapenem resistance is a major and an on-going public health problem globally. It occurs mainly among Gram-negative pathogens such as  Over seventy years the antimicrobial era has been marked by successive discoveries of a wide range of antibiotics and the subsequent emergence of antibiotic resistance. Bacterial resistance continues to increase, and drug researchers and manufacturing industries are not producing new drugs to replace the existing antimicrobials against which resistance has developed. The effects on current infection rates cannot be simply estimated ,2. The eIn recent times, development of antimicrobial resistance is rapidly changing, and the impending public health challenges these may cause in many health sectors need worldwide coordinated interventions. Many deaths have occurred as a result of this in Europe, and the European Centre for Disease Prevention and Control (ECDC) had estimated that 25,000 people may die each year from infections related to antimicrobial resistance ,4. GlobaCarbapenems possess broad spectrum antibacterial activity and have a unique structure that is defined by a carbapenem coupled to a β-lactam ring which confers protection against most β lactamases such as metallo-β-lactamase (MBL) as well as extended spectrum β-lactamases . ConsequCarbapenems are bactericidal β-lactam antimicrobials with proven efficacy in severe infections caused by extended spectrum β-lactamase (ESBL) producing bacteria . There aCarbapenems are potent members of the β-lactam family of antimicrobials that are structurally related to the penicillins. Mode of action of carbapenems is initiated first by penetrating the bacterial cell wall and binding to enzymes known as penicillin-binding proteins (PBPs) ,13. The Current understanding describes transpeptidase inhibition as the main enzyme target of carbapenems during bacterial cell wall synthesis. Through transpeptidation, a co-valent bond is formed by PBPs comprising of carboxypeptidase and transpeptidase enzymes which in effect prevent their peptide cross-linking activities during peptidoglycan biosynthesis. The lethal effects are thought to result in cell death by autolytic action within the bacterial cell . AccordiGenerally, carbapenems are preferred over other types of antimicrobials in treating invasive or life-threatening infections because of their concentration-independent killing effect on the infecting bacteria ,20. TheyAcinetobacter baumannii and Pseudomonas aeruginosa [Pseudomonas aeruginosa is relatively limited when compared with meropenem or imipenem. Studies also confirmed that Acinetobacter baumannii has increased susceptibility to imipenem or doripenem compared with that of meropenem [Comparatively, among the carbapenems, doripenem is highly stable against hydrolysis of most β-lactamases ,22, but ruginosa ,24. Evidruginosa . Ertapeneropenem ,26. SevePseudomonas species and other non-fermentative Gram-negative bacteria. Currently, different kinds of carbapenems are used in clinical practice as antipseudomonal agents; doripenem, imipenem and meropenem. The agents; ertapenem, imipenem and meropenem are poorly absorbed orally and require parenteral administration to be effective. Recently approved doripenem is of value among the current available carbapenems for treating serious infections [Carbapenemases as a mechanism of resistance have been reviewed in the context of the effective use of carbapenems in combination therapy to enhance successes in patient outcomes . Many enfections ,29.Pseudomonas aeruginosa, as a specific example.Carbapenems have trivial hepatic metabolism effects leading to hepatotoxicity with jaundice, although this is an uncommon medical condition for these agents ,30. MostInfection with Carbapenem-Resistant Enterobacteriaceae (CRE) is emerging as an important challenge in health-care settings and a growing concern worldwide ,32. CarbDevelopment of resistance to carbapenems may be due to intrinsic or acquired resistance mechanisms or both. Large numbers of bacteria, both commensals and pathogens, naturally tend to be resistant to certain classes of antimicrobial agents. This insensitivity is termed intrinsic resistance, the occurrence of which limits and complicates drug selections for treatment. This can increase the risk for development of acquired resistance. For instance, Gram-negative organisms reduce the uptake of β-lactam drugs, by selectively altering their cell membrane porin channels. Reduction of outer membrane permeability in this manner prevents the β-lactams from reaching their targets.Stenotrophomonas maltophilia, Bacillus cereus and Aeromonas species [Stenotrophomonas maltophilia is the only organism commonly associated with frequent hospital acquired infections. Others in the same category, often considered commensals, typically carry chromosomal metallo-β-lactamase enzymes, which are not transferrable, from the groups which had a serine-based hydrolytic mechanism of action [Pseudomonas aeruginosa and subsequently Acinetobacter species in clinical specimens. In recent times, MBL genetic materials are mobile in the Enterobacteriaceae family [Forsberg et al.  have sho species . These bf action ,38. In te family ,39.Bacteria have acquired multiple mechanisms of resistance including enzymatic inactivation, target site mutation and efflux pumps. Of these, the development and emergence of inactivating enzymes were established early following the discovery and clinical introduction of the β-lactam class of antibiotics. Over time, the β-lactam-hydrolysing enzymes extended their spectra of activity beginning with penicillinases, followed by cephalosporinases, then to ESBLs and most recently, to the MBLs and other carbapenemases. The MBLs have hugely impacted the utility of carbapenems (often considered as last resort drugs) which are used for the management of multi-resistant Gram-negative bacilli .Klebsiella, Enterobacter species and Escherichia coli as well as other genera. Among the carbapenems, ertapenem is the worst affected; isolates may continue to be susceptible to other carbapenems. In most cases, reduced susceptibility to ertapenem depends largely on the degree of AmpC/ESBL presence and exact changes of the porins. The overexpression of efflux pumps and loss of OprD porin are the most common mechanism of carbapenem resistance in Pseudomonas aeruginosa, notably to imipenem. Other β-lactams may be affected by this mechanism. Pseudomonas aeruginosa efflux-pump overexpression occurs more regularly when meropenem is used when compared with imipenem. Both commensals and pathogenic bacteria have different mechanisms of using their efflux pumps to remove amphipathic or lipophilic substances in and out of the cells. These mechanisms have been recognised in other organisms such as Enterobacter aerogenes and Klebsiella species against imipenem agent [Many of the acquired carbapenemases found in Enterobacteriaceae are plasmid-mediated and there are several ways by which they can be spread amongst bacterial isolates. In addition, there are other important mechanisms conferring carbapenem-resistance that have been observed in recent times. First, the carbapenems that are resistant to hydrolysis can be destroyed in the presence of plasmid AmpCs in combination with ESBL enzymes, making Gram-negative bacteria insusceptible to carbapenem agents . A signiem agent .Generally, Gram-negative bacteria are more resistant to a large number of antimicrobials and other chemotherapeutic agents than Gram-positive bacteria due to cell wall differences, external decreased membrane permeability, efflux pumps and the presence of various broad-spectrum-β-lactamases . The resistance may be attributed to the presence of broad-specificity drug-efflux pumps ,44. The Escherichia coli as an example; the orthologous inner membrane pump and outer membrane porin constituents AcrB and To1C have been resolved to 3.5 and 2.1 Å resolution respectively, while the structure of periplasmic component MexA from Pseudomonas aeruginosa has been solved to 3.5 Å resolution. The structural basis of the inner membrane pump, AcrB, has been determined in the presence of numerous hydrophobic small molecular compounds, which indicates a varied binding mode for each ligand, at least in this efflux pump element [One of the structures of drug-efflux pump systems extensively reviewed by Poole  was MexA element . The thr element  this has element .Acinetobacter isolates. Weak detection levels have been found for Oxacillin-hydrolysing (OXA) carbapenemases, more than their absence [Thirdly, non-carbapenemase specific mechanisms such as mixture of porin loss in addition with specific efflux pump systems have been reported in  absence ,50. Carb absence . AccordiKlebsiella pneumoniae, have been encountered most commonly in ventilator-associated pneumonia, bacteremia, urinary tract and surgical site infections. Generally, Klebsiella pneumoniae carbapenemase (KPC)-producing carbapenemases in the β-lactamase molecular class A are rare, however, the variants are rapidly spreading, with KPC-2 and KPC-3 frequently reported in the UK [There are a number of factors that predispose persons to infections by CRE and other multi-drug resistant (MDR) Gram-negatives including ESBL producers. Exposure to these resistant organisms can cause serious infections in patients with the following reported risk factors: immune-suppression, advanced age, admission to intensive care unit (ICU), mechanical ventilation, previous exposure to antimicrobials, organ or stem-cell transplantation and prolonged hospital stay ,53. Withn the UK . The genn the UK ,58. In cn the UK ,60.Klebsiella pneumoniae bloodstream infections at a hospital in Ohio, USA while Ben-David et al. [There are reports which suggest that long-term care facilities play a crucial role in the spread of CRE. Neuner et al.  reportedd et al.  reportedd et al. . Growingd et al. . A prospd et al.  concluded et al. .Enterobacteriaceae belonging to 3 classes of β-lactamases: the Ambler classes A, B and D β-lactamases. These classes are of greatest clinical importance among nosocomial pathogens. Class A, C and D β-lactamases all share a serine residue in the active site. The clinical role of a fourth class (Ambler class C) is unknown but possesses some extended activity towards carbapenems [A large variety of carbapenemases have been identified in bapenems ,66.Serratia marcescens enzyme), IMI-1 (Imipenem-hydrolysing β-lactamase), SFC-1 (Serratia fonticola carbapenemase-1), with the others being plasmid encoded—KPC (KPC-2 to KPC-13), IMI (IMI-1 to IMI-3), derivatives (GES-1 to GES-20) of GES (Guiana extended spectrum), but all actively hydrolyse carbapenems and are partially inhibited by clavulanic acid [blaKPC existing globally [blaKPC genes [Serratia marcescens (1), Escherichia coli (1), Klebsiella oxytoca (1) and Klebsiella pneumoniae (4) from a hospital and used multilocus sequence typing (MLST) to identify these organisms. This study revealed ST512 as a single-locus variant from the original known ST258 Klebsiella pneumoniae isolates widespread in Greece. The indication was that there was interspecies and intra-species were plasmid-mediated transfer belonging to the repFIIA replicon type. In another incidence, an elderly patient who visited Greece in 2012 contracted community acquired KPC-2-positive Klebsiella pneumoniae. This was isolated from the patient’s urine confirming it is widespread in Greece [A number of this class of enzymes have been identified; some are chromosomally encoded—NmcA , SME , Imipenem-resistant Pseudomonas (IMP)-type carbapenemases, VIM , GIM (German imipenemase) and SIM (Seoul imipenemase). The genes encoding these enzymes are often located within a variety of integron structures and incorporated in to the gene cassettes [These enzymes are mainly in the class of β-lactamases having the ability to hydrolyse carbapenems but are susceptible to inhibition by ethylenediaminetetraacetic acid (EDTA), a chelator of Znassettes ,74.Klebsiella pneumoniae and Escherichia coli isolates but have also been found in association with Acinetobacter baumannii and Pseudomonas aeruginosa organisms [By mid-2010, the NDM-1 gene may have been acquired by bacteria from the community and introduced to other countries including Europe and the United States through tourists travelling around the globe, while the same strains have been found in environmental samples in India ,76. At lrganisms ,77,78.Acinetobacter and Pseudomonas species as well as those in the Enterobacteriaceae family. The IMP genes have also been found in Brazil and Canada after first reports from Europe in 1997. These gene types have spread slowly to other countries in the Far East and subsequently to the United States and Australia [Currently, up to 18 varieties of IMP-type carbapenemases have been identified. This type was first recognized in the 1990s in Japan. The majority of these enzymes were investigated in ustralia ,79.Pseudomonas aeruginosa and Pseudomonas putida. Another growing family of carbapenemases first isolated in Verona, Italy, in 1997, and exhibited predominantly integron-associated metallo-β-lactamases. The family consists of 14 members. The VIM and IMP families have some similarities in terms of which plasmids they are carried on and that they are integron-associated. The VIM family has amino acid sequence diversity of up to 10%, 15% for IMP and 70% between the two families. Both hydrolyse all β-lactams except monobactams, and are susceptible to all β-lactam inhibitors [VIM genes are rarely encountered in Enterobacteriaceae but occur most commonly in hibitors .Acinetobacter baumannii, Pseudomonas aeruginosa and rarely in isolates of Enterobacteriaceae family in most countries including the United Kingdom and in the United States [Acinetobacter baumannii isolated in 1985 from a patient in Edinburgh, Scotland. Activity of OXA carbapenemases is increased by upstream elements which control gene expression [Klebsiella pneumoniae in Turkey, the Middle-East, North Africa and Europe [Acinetobacter species while the spread of OXA-23 type which occurs globally, is more encountered in USA and Europe. The OXA-58 group had been described significantly over the globe in several outbreaks [These enzymes are serine-β-lactamases poorly inhibited by EDTA or clavulanic acid. These carbapenemases are of the OXA enzyme type, and have a weak activity against carbapenems. The enzymes are found primarily in non-fermenter organisms such, d States ,82. The d States , and thepression . The majd Europe . Non-nosutbreaks . OXA-48–utbreaks ,37.The presence of a carbapenemase can be detected by a number of methods in clinical laboratories. These include automated systems or disc diffusion, MICs, selective agar, modified Hodge test, synergy tests , spectrometrics, whole genome sequencing and molecular methods. The majority of the genes controlling carbapenemase production are transferrable by plasmids. The presence of carbapenemases signifies some relevance to clinicians; however, care has to be taken in the management of patients to carbapenem therapy which may change depending on the mechanism of resistance existing at that time. Currently, detection of the enzymes is difficult because of the different mechanisms involved and unreliable techniques practised in some clinical laboratories . On the The baseline test that first predicts carbapenemase enzyme production is disc diffusion or the use of automated systems. Prior to this susceptibility testing, a bacterial species identification is required which takes about two days for common bacterial pathogens, and although it is highly accurate, there are inherent problems in differentiation between acquired and intrinsic resistance ,89.Acinetobacter species, Pseudomonas species and Enterobacteriaceae have been documented [Klebsiella pneumoniae study isolates as being susceptible to imipenem or meropenem.For disc diffusion, impregnated discs containing a standard amount of an antibiotic agent are placed on agar plate seeded with a bacterium to be tested. As the organism grows during overnight incubation, the antibiotic agent diffuses into the agar medium. Susceptibility of the test organism is proportional to the zone of inhibition produced by the antibiotic used. With the automated systems, instruments are used to analyse antibiotic susceptibility testing with standardised inoculum for the test strain and diluted in a specialised broth with a drop of antimicrobial susceptibility testing indicator added. The final inoculum turbidity is adjusted to 0.5 McFarland Standard. The inoculum is poured into a panel, closed in a secured place and the inoculated panel is then placed into, for example, instrument for VITEK or VITEK 2 automated system. The panel is then read automatically by the instrument, the data generated is analysed with preliminary algorithms and compared to the controlled results as appropriate. Either one or both methods have shown variability in their ability relating to the underlying mechanism of carbapenem resistance. Several reports of carbapenemase detection using phenotypic based methods in cumented ,91. Refecumented , than E-cumented  identifiAcinetobacter species [The determination of ertapenem MICs by automated testing tends to identify cases which are ertapenem-resistant, which in effect is recommended as a sensitive laboratory test of KPC production irrespective of the method employed . Carbape species . Perez a species  establis species  and CLSI species  revised Acinetobacter . Culture media are made selective by adding chromogenic substrates and agents that inhibit growth of other Gram-positive, Gram-negative and yeast isolates. The CHROMagar™ culture media were originally formulated for the screening of patients in the ICU and later redesigned so that Acinetobacter species appear as bright salmon red colonies. A new formulation, with the addition of ‘Klebsiella pneumoniae carbapenemase supplement’ was able to pick out carbapenem-resistant Acinetobacter baumannii. Recent modifications to CHROMagar Acinetobacter have improved selective growth for organisms that are resistant to carbapenems [The use of chromogenic agar preparation has emerged for identification of MDR organisms from surveillance cultures such as CHROMagar KPC and CHROMagar™ bapenems ,98. Accobapenems , CHROMagbapenems .Acinetobacter baumannii isolates and found negative results for all tested NDM-producing isolates and only weak positive results for VIM-, IMP- and OXA-type producers [The modified Hodge test has been used extensively and is a phenotypic technique for detecting carbapenemase activity routinely used in clinical pathology laboratories. This test was recommended by the CLSI in 2009 , howeverroducers . There aroducers ,102.Acinetobacter species and 100% for Pseudomonas species [Double-disc synergy testing has several versions; carbapenem with -clavulanate, -cloxacillin -EDTA or 2-mercaptoproionic acid, and one which utilises a double sided E-test , imipenem versus imipenem with EDTA, and are used as a screening test for MBL producers. This method is efficient for detection of MBL carbapenemases with high resistance, but may be deficient for detecting MBL producers with low resistance to imipenem according to Walsh et al. . Other r species ,104. A r species  indicate species . Accordi species , resista species .Pseudomonas species harbouring carbapenemases [Pseudomonas aeruginosa and Enterobacteriaceae isolates with only 80% specificity [Acinetobacter species with high-level of sensitivity and specificity [The newly developed Carba Nordmann-Poirel test, derived from the name “Carbapenemase Nordmann-Poirel”, is in use to detect carbapenemase producers in Enterobacteriaceae. The technique requires no expertise, is reproducible, inexpensive and may easily be adapted as an additional important test in any clinical laboratory. The test may help healthcare facilities to curb and implement rapid containment measures to limit the spread of carbapenemase producers in hospitals . Nordmanenemases . In contenemases  observedcificity . Anothercificity ,109. A rcificity . Comparecificity .Klebsiella pneumoniae in a long-term facility for children and young adults [The recent changes in the breakpoints for carbapenems  have furg adults .During the period of transition, there may be inconsistencies between laboratories regarding identification of ESBLs and CREs. Many different laboratory interpretations of MIC levels may have led to confusion among clinicians about when it is suitable to use an extended spectrum cephalosporin drug, a carbapenem, or neither of them. These determinants are also important for infection prevention and control practices. Pharmacists need to be responsive to these issues so that they can offer the best advice and other method in recommending appropriate antimicrobial choice. Pereira et al.  offered Molecular techniques have become an efficient tool for carbapenemase detection. Polymerase chain reaction performed on colonies may give results within 4 to 6 h with excellent sensitivity and specificity and this may reduce the chance of spreading organisms in hospitals. Wang et al.  reported102 DNA microarray, Check direct CPE assay and Hyplex-Super-Bug ID kit are on the market for diagnostic testing purposes. Excellent detection ability and reproducibility with these assays have been reported [In relation to clinical laboratory practice, innovations of molecular techniques have been evaluated and are currently in use for the detection of carbapenemase producers. Check-MDR CTreported ,119,120.reported .Klebsiella isolates from blood cultures (BCs) and to obtain proteomic profiles enable to discriminate between carbapenemase-producing and non-carbapenemase-producing strains. MALDI-TOF MS is available in most research laboratories for confirmation of a host of bacterial resistance enzymes, with sensitivity and specificity of 98.9 and 97.1% respectively [A study by Sparbier et al.  describeectively . Howeverectively . Cost anectively .Several molecular techniques have been developed for characterisation of bacterial pathogens including CRE during period of outbreaks and for use in routine clinical laboratories. Notable among these techniques are multilocus sequence typing, multilocus enzyme electrophoresis and DNA fingerprinting methods such as pulsed field gel electrophoresis, amplified fragment length polymorphism and multiple locus variable-number tandem repeat analysis . Whole GAeromonas hydrophila isolate in the 1980s. Sequentially, followed in London (1982) by Seoul imipenemase (SME-1) from Serratia marcescens, imipenemases (IMI-1) in California (1984) and NMC-A in France (1990), both from Enterobacter cloacae [Across the globe, first reports of carbapenemases occurred in the 1980s. Its subsequent spread raised a number of concerns, and these have coincided with years in which no new antimicrobial agents against Gram-negatives have been developed. Japan reported the first carbapenemase from an  cloacae .Klebsiella pneumoniae carbapenemases predominate worldwide [Klebsiella pneumoniae ST258 type strain, and throughout the USA in particular, MLST, a specific molecular tool was used to find out the global epidemiology linkages and help understanding the dissemination of KPC genes. There are fresh fears and challenges on-going and under discussion by researchers as to how to curb the spread of carbapenem resistance across the globe for epidemiological reasons. Detection has been difficult due to differences in their genetic makeup within and across countries. Researchers have observed divergent resistant patterns from CREs in Europe, the United States and South America. The unusual resistance factors have been monitored through a surveillance programme, named the Intensive Care Antimicrobial Resistance Epidemiology (ICARE) by the CDC from 2000 onwards. Earlier CRE infections, particularly KPC, were confined to hospitals in New York. Between 2000 and 2010, the infection rate had increased from 1% to 12% in 42 States respectively [Klebsiella pneumoniae isolates to be categorised while the majority have been found in Pseudomonas aeruginosa [Pseudomonas putida and others in the Enterobacteriaceae family [Carbapenem resistance involves one or more of several diverse mechanisms of actions in multi-resistant Gram-negative bacteria. The most frequently encountered β-lactamase enzymes are the acquired carbapenemases in the Ambler class A group of which orldwide ,130,131.orldwide ,132. Folectively  with a 5ectively . Thirty-ectively . The occruginosa , Pseudome family .Klebsiella pneumoniae isolates, contrasting with 12% and 20% for carbapenem-susceptible Klebsiella pneumoniae respectively [Studies have shown that the spread of KPC-positive organisms in some parts of the USA was not related to travel. Most infections seemed to occur in hospitals, with the majority of the outbreaks contracted in long-term care facilities (LTCFs) for care receivers with severe medical conditions who needed longer stays for their clinical management ,138,139.ectively . The cliectively ,135. Theectively .Enterobacter species from a blood sample and the first case of a known Klebsiella pneumoniae producing KPC-positive isolate, all from Scotland [Klebsiella pneumoniae ST258 the most frequent strain to be captured by MLST (10 out of 12). The source was traced to Greece, Cyprus or Israel, with patients having travelled to those countries in the time prior to admission. Between 2010 and the first six months of 2012, the epidemiological pattern rapidly changed in terms of numbers identified, 231 KPC-positive strains in 2010, 368 in 2011 and 293 for the first six months in 2012 were referred for further confirmation at the national reference laboratory [Currently, 39 countries in the European Union including France and the United Kingdom have come together to fight the burden of CREs in their respective countries. The study is being coordinated by the European Centre for Disease Prevention and Control in Stockholm to keep epidemiological data of patients who test positive for such bacteria in their health-care institutions ,143. BetScotland ,144. Theboratory .Klebsiella pneumoniae in 2005 from a patient who had had 3 months admission in a New York City  hospital. The strain type was isolated from blood and urine cultures. From 2009 to 2012, there have been several CREs investigated. In a recent study, five cases out of 20 were confirmed and linked their sources to countries such as Kuwait, China, Italy and Israel. Community acquired strains were found to be rare and most of the documented cases in France were from persons colonisation with the strains [Rapid spread of CREs across Europe was observed in late 2005 when an identified KPC isolate in Israel was later found to be genetically related to a type strain in New York. France acquired its first KPC-2-positive  strains ,145.Escherichia coli were harbouring NDM, exhibiting more resistant traits than KPC-positive Klebsiella isolates [Klebsiella bacteria including those with NDM-1 genes into hospitals. For instance, following a Colorado episode, the CDC used whole-genome sequencing, a technology utilised for the first time to resolve outbreaks in the hospital. Researchers were in doubt as to exactly how NDM was escalating among the population. Studies later revealed that bacteria carrying the NDM enzyme found its way outside the boundary of hospital settings into community water and sewage environments with some epidemiological link to parts of Southern Asia [Carbapenemases became a public health concern in 2007 when a total of 1275 CRE infections were identified across their health care centres. KPC-positive organisms found its way in to other countries including Italy, Colombia and the United Kingdom ,146,147.isolates . There wern Asia ,149.Klebsiella pneumoniae ST258 [Acinetobacter baumannii and Pseudomonas aeruginosa) in other countries [Citrobacter freundii. The earlier isolates, none were attributed to travels outside the country [Escherichia coli isolate was recovered from river water although there were no reports of medical cases in connection of KPC-positive isolates in recent times from their hospitals [In Italy, the enzyme type described in Israel ST258 isolates located in transposon Tn4401 was detected in KPC-positive isolate (KPC-3 gene type) . Over thae ST258 . The sitountries ,153. Spaountries . A later country . Howeverospitals .Klebsiella pneumoniae isolates were confirmed in Sweden and France in 2007 [Klebsiella pneumoniae was reported afterwards in a tertiary hospital in Heraklion [Poland has a history of high prevalence of CREs carrying KPC genes. A KPC-positive case was confirmed in Warsaw. Later, a number of cases were confirmed by the national reference laboratory in five Warsaw hospitals towards the end of 2008, with more identified from 2009 to 2012 (not published). Most common sources of infections were either through stool or urine and ST258 type of strains were high among the isolates countrywide ,158. In  in 2007 ,160. An eraklion , and twoeraklion ,163.Escherichia coli carrying KPC-2-positive gene contributed to 40% infections outside ICUs [Klebsiella pneumoniae from Greece have been reported in other countries most of them following international travel [Currently, KPC-positive organisms are frequently encountered in Greece and are not only found in the ICU, patients on surgical and medical wards have been victims of such infections. Outbreaks in long-term care facilities have been described, ide ICUs . Howeverl travel . There hl travel ,167.Israel is one such country with a successful story of controlling CRE infections after strain ST258 was suspected to have been imported from the USA in 2005 and eventually spread over all Israeli health-care facilities . Most coKlebsiella pneumoniae, Proteus mirabilis and Escherichia coli [blaKPC gene in which 8 Klebsiella pneumoniae isolates were with 7 KPC-2 and 1 KPC-3, and 1 Escherichia coli isolate with KPC-2. Interestingly, one of Klebsiella pneumoniae isolates was co-harbouring KPC-2 with OXA-1, NDM-1, TEM-1, CTX-M-15 and SHV-12 β-lactamases, and RmtB known to be transferring aminoglycoside resistance [Escherichia coli suspected to have been imported from India by a patient who had been colonised [The most hit areas of the Asian continent by the CRE isolates were India, Hong Kong, Taiwan, South Korea, and central, north-west, eastern and south-west parts of China. In India in particular, KPC-positive isolates were observed during a phase-3 clinical trial of tigecycline drug between 2002 and 2006 from three organisms; hia coli . By the sistance . The SENsistance . Earlierolonised .Klebsiella species. Zhejiang province recorded KPC-positive Klebsiella pneumoniae in China in the year 2004 from a mid-seventy-year-old ICU patient [Klebsiella pneumoniae that showed susceptibility to carbapenem agents were later found to be carrying KPC-2 genes, closely related to ST258 type [Citrobacter freundii and Escherichia coli respectively and infections were mainly from urine and sputum specimens. In many instances, the KPC-3 gene is more common in Enterobacter cloacae and Klebsiella pneumoniae isolates found abroad [Enterobacter cloacae and Citrobacter freundii in hospital sewage, raising awareness of possible pollution of water bodies and reservoirs [The most common carbapenemase gene in China is KPC-2 type found in a number of  patient , and the258 type . The Shad abroad . China hservoirs  while Geservoirs  studied Klebsiella pneumoniae isolates from two patients in 2005 [Pseudomonas aeruginosa [Klebsiella pneumoniae outbreak occurred in which 20 died out of 32 patients. Fourteen of the death tolls were due to infections, with one patient travelling from Israel, a country with several outbreaks of KPC-3 genes [Pseudomonas aeruginosa ST235, ST308, ST1006 and ST1060, while in Klebsiella pneumoniae, ST258 and ST51233 were identified [Klebsiella pneumoniae identified in 2006.Endemicity of KPC genes had been identified in these regions of Latin America, specifically, across Colombia among the Enterobacteriaceae family. Reported cases of KPC-positive isolates of KPC-2 type were found in  in 2005 ,179, andruginosa . After t-3 genes . A numbeentified ,182. Subentified . ArgentiPseudomonas aeruginosa isolates were carrying KPC genes, and most had genetic relatedness to ST654, different from the ST258 Klebsiella pneumoniae type which suddenly spread up to mid-2009 in Argentina [Klebsiella pneumoniae isolates in an intensive care unit with no trace of overseas travels [Klebsiella pneumoniae isolates [Pseudomonas harbouring KPC-2 genes from hospital sittings. Venezuela and Chile, in particular, had no evidence of endemicity, however, several outbreaks of KPC-positive carriers from the Enterobacteriaceae group were latterly recorded [By the end of 2010 following SENTRY studies carried out for 2 years, substantial increases in KPC-positive isolates in Argentina and Brazil were recorded . In anotrgentina ,186. A K travels . Subsequisolates . Brazil recorded .blaNDM-1 in Acinetobacter baumannii, blaVIM-1 in Pseudomonas species and blaOXA-48 in Klebsiella pneumoniae. In Ghana, multi-drug resistance of Gram-negative bacteria has reached alarming rates [Many countries have not instituted active surveillance studies specifically monitoring the KPC threat since its inception around the globe, the majority of which are in the African continent. Countries in Africa, including Morocco, Kenya and South Africa have reported NDM-1 as the most dominant carbapenemase gene ,190. Howng rates ,195,196.ng rates ,135. RecAcinetobacter baumannii in most hospitals [Antimicrobial treatment of CRE infections has been challenged by the emergence of more complex resistance phenotypes as well as economic and regulatory pressures. Agents such as polymyxins and tigecycline have recently seen resurgence in their clinical usage (particularly colistin) in the management of multidrug-resistant Gram-negative infections, particularly CRE including carbapenem-resistant ospitals .Acinetobacter baumannii isolates when combined with another antimicrobial agents such as tigecycline. In a study conducted by Lee et al. [Klebsiella pneumoniae evaluated that three out of twelve managed with polymyxin monotherapy experienced polymyxin resistance in their treatment. None of the four cases which were treated with polymyxin in combined therapy with tigecycline detected resistance to either antimicrobial agent [The polymyxins are active agents and attain sufficient serum levels in the treatment of serious bloodstream and CRE infections. The agents produce additive or synergistic effects on humans against multidrug-resistant organisms including e et al. , 16 patial agent ,199. Outal agent . In an oal agent .Escherichia coli. The drug is licensed for most complicated intra-abdominal, skin and soft tissue infections. Interestingly, a study reports success in various infections caused by carbapenemase producers [Tigecycline, a glycylcycline, is active in vitro against most carbapenem-resistant roducers . A laterroducers  reportedroducers . Howeverroducers ,203.Combination therapy with tigecycline proved successful in many instances with positive clinical outcomes in the majority of infections involving KPC bacteria . During Klebsiella pneumoniae infections are considered ineffective, and therefore not recommended for clinical management of patients [Aminoglycosides, notably gentamicin, amikacin and tobramycin have different in vitro activities. Treatments of CRE infections may depend on the susceptible organism as studies have shown gentamicin activity against gentamicin-susceptible strains in urinary tract infections have positive outcomes. Of the aminoglycosides, amikacin appears to be the more active against CREs when compared with gentamicin or tobramycin . While ipatients ,205.Klebsiella pneumoniae isolates [Pseudomonas aeruginosa as a urinary pathogen. Many patients that developed treatment failure were immunosuppressed or had urethral stents due to the use of fosfomycin as monotherapy in kidney transplant cases.Fosfomycin, a bactericidal antibiotic that inhibits bacterial cell wall biogenesis has seen its use renewed globally in response to the recent threat of antimicrobial resistance including carbapenem-resistant isolates ,207. TheKlebsiella pneumoniae isolates [Combination therapies have shown remarkable outcomes in dealing with MDR and CRE infections. The most commonly used combinations are colistin, polymyxin B or tigecycline combined with a carbapenem. In a retrospective study by Qureshi et al.  on patieisolates . Lee andisolates  also stuisolates . In futuisolates ,203,210.The threat must be answered by strict adherence to infection control strategies and policy guidelines designed for individual countries ,105. PerDevelopment of novel antimicrobials remains the prime objective for CRE control, a look at critical interventions aimed at preventing the transmission and infections with these organisms are of great significance ,41. In wAcinetobacter and Pseudomonas species, less encountered in hospitals are the main organisms spreading into the community. These organisms have the potential to spread resistance to other bacterial isolates into the community. Despite efforts to control carbapenem resistance, a definite solution to the problem is still far from achievement. Carbapenemase-encoding genes are already widespread in certain parts of the world particularly, Europe, Asia and South America while the situation in other places such as sub-Saharan Africa is not well documented. There is the need for active surveillance of carbapenemase-encoding genes as major step to controlling the menance.The growing threat to public health includes the rapid spread of CRE into the community. Multidrug-resistant Gram-negative bacteria such as


The expression level of circ-ITCH was significantly lower in HCC tissues, compared with that in adjacent tissues . Cox regression analysis indicated that high expression of circ-ITCH was associated with favorable survival of HCC . These results indicate that circ-ITCH may have an inhibitory effect on HCC, and could serve as susceptibility and prognostic biomarkers for HCC patients.Hepatocellular carcinoma (HCC) ranks the sixth most common cancer and the third cause of cancer-related mortality worldwide. Recent studies identified that circ-ITCH Suppresses mutiple cancers proliferation via inhibiting the Wnt/beta-Catenin pathway. In current study, conducted a genetic association study together with epidemiological follow-up study to delineate the role of circ-ITCH in the development and progression of HCC. we found rs10485505 (adjusted OR =1.18; 95% CI=1.06-1.31; P value =3.1×10 Hepatocellular carcinoma (HCC) ranks the sixth most common cancer and the third cause of cancer-related mortality worldwide , 2. In ACircular RNAs (circRNAs) are an enigmatic class of RNA with unknown function . With thRecently, Li et al  found thA totally of 1,600 HCC cases and 1,800 cancer-free controls were recruited for the genetic association study. Table -3) and rs4911154  were significantly associated with HCC risk. For rs10485505, compared with the genotype CC, genotype CT  and TT  were significantly associated with increased HCC risk; while for rs4911154, compared with the genotype GG, genotype GA  and AA  were significantly associated with increased HCC risk.Figure To explore the influence of expression of circ-ITCH on occurrence of HCC, a TaqMan-based qRT-PCR assay was used for the divergent primer set to determine the levels of ITCH in 288 HCC samples and matched adjacent tissues. As shown in Figure Furthermore, to determine whether circ-ITCH could serve as prognostic marker for HCC, we analyzed the association between expression of circ-ITCH and prognosis of HCC in the 288 HCC cases with follow-up data. As shown in Figure In this study, we have used a candidate gene approach to examine the associations between genetic variations of circ-ITCH and susceptibility of HCC. We also evaluated the possibility of the expression of circ-ITCH as an susceptibility and prognostic biomarker for HCC. To the best of our knowledge, This should be the first study which aims to investigate the role of circ-ITCH in the development and progression of HCC. Our findings demonstrated that rs10485505 and rs4911154 were significantly associated with increased risk of HCC, and the expression of circ-ITCH could serve as susceptibility and prognostic biomarkers for HCC patients.CircRNAs are a novel type of RNAs, which are generated primarily through a type of alternative RNA splicing called “back-splicing,” a downstream splice donor being joined to an upstream splice acceptor through splice skipping or direct splice . They coCirc-ITCH was located on chromosome 20q11.22, and spans exons 6–13 of gene ITCH, which encodes a member of the Nedd4 family of HECT domain E3 ubiquitin ligases . MutatioOur study has several strengths. First, the large sample size provides enough statistical power to detect such moderate associations. Second, we have included the genetic-homogenous participants, which were all Han Chinese population. Although our study revealed some interesting findings, there still exist some limitations. One limitation was that the findings were not validated in an independent cohort. Secondly, further mechanism of circ-ITCH and its SNP affecting susceptibility and prognosis of HCC was not explored here . HoweverIn summary, our study showed that the expression level of circ-ITCH in HCC tissues was significantly lower than the level in adjacent tissues, and high expression of circ-ITCH was associated with favorable survival of HCC. We also found that rs10485505 and rs4911154 in circ-ITCH were significantly associated with increased risk of HCC. This study indicates that circ-ITCH not only has prognostic significance, but also susceptibility biomarker for HCC. Meanwhile, genetic variation of circ-ITCH can also be used as the biomarkers for prediction and screening of HCC. These findings should be verified by larger, well-designed epidemiologic and functional studies.Totally included in current study were 1,600 HCC cases and 1,800 cancer-free controls. All cases were pathologically confirmed as HCC, and all subjects in this study were homogenous Han Chinese. The control subjects were randomly enrolled at the same period when they visit the hospital for physical examinations, matched by age, gender, HBV infection status, and education level. Demographic data and environmental exposure history of each participants were collected by face to face interviews. Blood (10ml) was collected for each patient. Plasma was separated immediately and stored at -80°C before use, while tissue samples were snap-frozen in liquid nitrogen immediately after dissection and then stored at −80°C before RNA extraction. The study was approved by the institutional review board of the hospital, and a written informed consent was collected from all individual participants of the study.Total RNA was extracted from 288 randomly selected HCC tissue samples and matched adjacent tissues using TRIzol reagent  according to the manufacturer's protocol. The quality of the RNA samples were measured using Agilent 2100 bioanalyzer, and an Integrity Number value above 6.0 indicated acceptable RNA integrity for RT-PCR assays. The relative gene expression of circ-ITCH  was determined using the ABI Prism 7900 sequence detection system , and GAPDH was used as an internal standard control. All reactions were performed in triplicate.https://snpinfo.niehs.nih.gov/) was used for the tagSNP selection of circ-ITCH. As shown in Figure SNPinfo Web Server (Student's t test and Pearson's chi-squared (χ2) test were used to assess the differences in demographic factors between HCC cases and controls. Hardy-Weinberg equilibrium (HWE) of the controls was done by a goodness-of-fit χ2 test. The ORs and 95% CIs were calculated by an unconditional logistic regression model for the associations between genotype distribution and HCC susceptibility. Mann–Whitney U-test was used to analyze the expression difference of circ-ITCH between HCC tissues and adjacent non-cancerous tissues. Patients were divided into low or high expression groups of circ-ITCH according to the median value. Kaplan–Meier and Cox regression analyses were utilized to assess the association between the circ-ITCH and overall survival of HCC patients. Statistical analyses were performed by using SPSS software package , and all reported p values were two-sided with a significance level of 0.05.


Acquired resistance is a bottleneck that restricts the efficacy of epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) for lung cancer. Ginsenoside Rg3 is an antiangiogenic agent which can down-regulate the expressions of vascular endothelial growth factor (VEGF) and EGFR. Combination of EGFR-TKI and ginsenoside Rg3 may be a promising strategy to delay acquired resistance. This retrospective study explored the efficacy and safety of this combined regimen in patients with EGFR mutation and advanced non-small cell lung cancer (NSCLC).th 2016, the median follow-up period reached 22.9 months. The median PFS was significantly longer in group A than in group B . In addition, ORR was significantly higher in group A than in group B . The median OS in group A showed no extended tendency compared with that in group B . No significant difference in side effects was found between the two groups.By the deadline of March 31A total of 124 patients with advanced NSCLC and EGFR active mutation were collected and analyzed. All of them were treated with first-line EGFR-TKI and divided into two groups. In group A (n=52), patients were administered EGFR-TKI plus ginsenoside Rg3 at standard doses. In group B (n=72), patients received EGFR-TKI alone. Progression-free survival (PFS), overall survival (OS), objective response rate (ORR) and side effects were analyzed.Ginsenoside Rg3 improves median PFS and ORR of first-line EGFR-TKI treatment in EGFR-mutant advanced NSCLC patients, thus providing a new regimen to delay acquired resistance of EGFR-TKI. Lung cancer is the malignancy that causes the highest morbidity and mortality worldwide. The 5-year survival rate for lung cancer is less than 20%, despite progress in chemotherapy, radiotherapy, targeted molecular therapy and immunotherapy . Non-smaTumor growth and progression depends on angiogenesis. Currently, antiangiogenic agents have become a potential anticancer strategy . PreviouA total of 720 patients with EGFR mutation attended the three affiliated hospitals of Third Military Medical University  from January 2012 to January 2015. Among them, 252 patients met the inclusion criteria. However, 20 patients had no complete information, 30 patients took ginsenoside Rg3 for no more than 8 weeks or uncontinuously, 19 patients adopted other therapies without disease progression, 26 patients were treated with EGFR-TKI for less than 2 months, and 33 patients took EGFR-TKI discontinuously. The remaining 124 patients were collected into the study, aged from 27 to 84 yrs (median age was 58 yrs)  continuously during the course of EGFR-TKI. No significant difference existed in gender, age, smoking status, ECOG PS, treatment intervention, distant metastases, clinical stage, histological type, EGFR active mutation sites or treatment line, except for EGFR-TKI products between the two groups. There were 14 cases of erlotinib, 36 cases of gefitinib and 2 cases of icotinib in group A, while 47 cases of erlotinib, 24 cases of gefitinib and 1 cases of icotinib in group B . In groups A and B, median PFS were 12.4 months  and 9.9 months  respectively. The median PFS was significantly prolonged in group A compared with group B  had occurred in group A and 42 events (58.3%) in group B. Median OS of 124 patients was 24 months . The median OS was 25.4 months in group A  and 21.4 months in group B . Different from median PFS, ginsenoside Rg3 addition showed no obvious tendency to extend median OS  in group A and 39 patients (54.2 %) in group B showed grade ≥ 2 of adverse events and there was no significant difference between the two groups , which needs to be further explored in the future prospective study.Previous studies have reported that EGFR-TKI has a tendency to be more effective in patients with EGFR exon 19del than in those with exon 21 L858R mutation , 44. In As ginsenoside Rg3 and bevacizumab have the commonality that they are both antiangiogenic agents against VEGF. The analogy between the two agents is to better introduce the feasibility of EGFR-TKI combined with antiangiogenesis. However, bevacizumab had been shown to be associated with some special side effects, mainly proteinuria and haemorrhagic events in the studies of JO25567  and OLCSWe have also tried our best to control and reduce the probable biases during the current study. All advanced NSCLC patients harboring EGFR active mutation underwent the screening and selection during the research period to minimize the confounding effects of retrospective cohort study. On this basis, our results revealed that the median PFS in patients receiving EGFR-TKI alone was consistent with prior studies . MoreoveInevitably, there are still several limitations in our study. First, retrospective study is not as powerful as prospective trial. It is difficult to avoid nonrandomized design or distributional difference in EGFR-TKI product selection, which might decrease the credibility of results. Second, a limited sample size is collected. We only included the patients between January 2012 and January 2015 in three hospitals, since that few patients took both EGFR-TKI and ginsenoside Rg3 before 2012 and the observation after 2015 was not long enough to do statistic analyses. Third, there was rare data being collected to further explore the possible relationships between the efficacy of ginsenoside Rg3 and special types of EGFR mutation, such as exon 20 mutation, T790M mutation and so on. Fourth, this study didn't involve the detection of EGFR resistance mutation and molecular changes of cancer tissues. T790M mutation was not a usual examination in hospitals in China during the period of this observation. And, it is hard to obtain cancer tissues to detect relevant signaling pathway by immunohistochemistry (IHC) during the process of EGFR-TKI treatment. Considering the above limitations, we plan to conduct a cohort of patients with prospective randomized design in the near future.In conclusion, our study provides the first evidence that the addition of ginsenoside Rg3 to EGFR-TKI confers a reduced susceptibility to drug resistance in treating NSCLC patients with EGFR active mutation, and that ginsenoside Rg3 can be a considerable agent to improve patient median PFS and delay acquired resistance to EGFR-TKI. No increased toxicity seems to be found after the addition of ginsenoside Rg3. It also confirms the combination of EGFR-TKI and antiangiogenesis as a new regimen. However, by the limitation of sample size in this study, the definite efficacy and safety of combining ginsenoside Rg3 and EGFR-TKI still warrant further prospective investigation.th edition of the International Association for the Study of Lung Cancer (IASLC) [Patients who were histologically or cytologically diagnosed with unresectable stage III or IV advanced NSCLC and attended the three affiliated hospitals of Third Military Medical University  between January 2012 and January 2015 were eligible and the medical records of them were retrospectively collected and analyzed. Tumor, node, and metastasis (TNM) classification of lung cancer was based on the 7 (IASLC) . InclusiThe patients were divided into two groups. In group A, patients were treated with EGFR-TKI plus ginsenoside Rg3 for continuous 8 weeks or longer . And in group B, patients received EGFR-TKI alone. EGFR-TKI products  were administered at conventional doses . Subsequent chemotherapy drugs after EGFR-TKI failure contained Paclitaxel, Gemcitabine, Docetaxel, Pemetrexed, Cisplatin, Nedaplatin, Carboplatin, etc. And local treatment after local recurrence consisted of palliative surgery, external-beam radiotherapy, interstitial irradiation and thermal therapy.th 2016, the medical records were reviewed to obtain demographic, clinical and pathological information. After 8 weeks’ TKI treatment, tumor response was assessed according to Response Evaluation Criteria in Solid Tumors (RECIST 1.1), including complete response (CR), partial remission (PR), progressive disease (PD) and stable disease (SD). The values of ORR, disease control rate (DCR), median PFS and OS were also analyzed. PFS was defined as the time from the initiating EGFR-TKI treatment to disease progression or death. Likely, OS was defined as the time from the first dose of EGFR-TKI to the death of disease. All patients were followed up at an interval of 1 to 2 months during and after EGFR-TKI treatment, which was also an often follow-up visit [By the last follow-up on March 31up visit . RoutineThe ORR, DCR and side effects were compared using the standard Chi-square test or Fisher exact test. The median PFS and OS were estimated using the Kaplan-Meier survival analysis. Differences between groups were compared using the stratified log-rank test. Hazard ratio (HR) and 95% CI were also calculated by Cox proportional hazard regression models. All statistical analyses were performed using SPSS 18.0 software package . Statistical significance was set at P < 0.05.


Autoimmune hepatitis (AIH) is a chronic inflammation of liver with unclear etiology. It is frequently associated other autoimmune diseases, and its association with celiac disease (CD) is well established. In this article, we describe the case of a 50-year-old male with long-standing AIH taking azathioprine for 10 years, evaluated for flares in transaminases. Despite adding high-dose corticosteroids, his transaminases and bilirubin remained high. Serology for CD was ordered, which revealed elevated tissue transglutaminase antibody IgG and endomysial IgA, which was further confirmed by endoscopic biopsy. Strict gluten-free diet was advised and now for over 2 years he is in remission with azathioprine and budesonide. This emphasizes the role of gluten-free diet in reversing liver dysfunction in patients with AIH, and clinicians should consider screening for CD in patients with AIH with persistent elevation of liver enzymes despite immunosuppressant treatment. Extensive hepatic workup along with workup for CD showed celiac-related AIH, and immunosuppression along with strict gluten-free diet (GFD) helped achieve remission.Celiac disease (CD), also called gluten-sensitive enteropathy, is an autoimmune disease triggered by the ingestion of the gliadin fraction of gluten and/or other cereal prolamins in genetically predisposed patients.A 50-year-old male with a history of hypertension and long-standing AIH taking azathioprine for 10 years was evaluated for flares in transaminases, aspartate transaminase 123 U/L, alanine transaminase 115 U/L, bilirubin 2.6 mg/dL, international normalized ratio 1.3, and thrombocytopenia. There was no relevant family, alcohol, or drug history, and he had never received a blood transfusion. He denies any use of over-the-counter drugs or herbal supplements. Serum markers for hepatitis A virus, hepatitis B virus, and hepatitis C virus were negative. Serum antinuclear antibodies were found to be positive but anti-smooth muscle antibodies and anti-mitochondrial antibodies were negative, as were antibodies against cytomegalovirus or Epstein-Barr virus. He had normal iron, thyroid-stimulating hormone, α-1 antitrypsin, and ceruloplasmin. Abdominal ultrasound showed heterogeneous and nodular liver. Liver biopsy showed grade 3 portal fibrosis concerning for cirrhosis, which could also be the likely cause of his thrombocytopenia. Budesonide was added to his drug regimen. Despite high-dose steroids, his transaminases remained high and bilirubin continued to rise. He did not show any signs and symptoms of decompensated liver disease. Serology for CD was done and revealed elevated tissue transglutaminase antibodies  and positive endomysial immunoglobulin A antibody. Endoscopic biopsy of duodenum showed flattening of villi with intraepithelial lymphocytes, which confirmed the diagnosis of CD  and 2. S5 Conversely, the prevalence of CD in AIH is significantly higher than that found in the general population, with 2 studies reporting the prevalence as 6.4% and 4%.7 AIH and CD share a common immunological basis. Approximately 95% of CD patients express HLA-DQ2, and the remaining patients have HLA-DQ8.8HLA-DR3 expressed in AIH has a strong linkage with HLA-DQ2 and may account for the association between these diseases.9 This linkage probably explains damage to hepatocytes as well as the intestinal mucosa by the immunologic mechanism generated by tTG acting both as a self-antigen or generating neo-antigen.10Celiac disease is associated with many autoimmune diseases like type 1 diabetes mellitus, autoimmune thyroiditis, psoriasis, AIH, inflammatory bowel disease, vitiligo, and so on.10 Commonly used serological tests used for detecting CD are anti-gliadin antibodies, anti-endomysial antibodies(EmA), and anti-tTG antibodies. Anti-gliadin antibody testing is no longer used routinely because of its low specificity and sensitivity. The IgA anti-tTG is an optimal screening test with its 96% sensitivity, and IgA-EmA is diagnostic (specificity nearly 100%). Intestinal biopsy findings remain the gold standard and are used to confirm positive antibody test results. In children aged less than 5 years, EmA and tTG antibody testing are less reliable and require additional study.11Studies demonstrate that early serological screening testing for CD is strongly recommended in all patients with AIH.1214 The role of GFD particularly in patients with AIH is still not fully explained. Kaukinen et al10 demonstrated in their study that 3 out of 4 patients with severe liver disease and concomitant CD were found to be no longer meeting the criteria for liver transplantation after maintained on a GFD, with reversal of hepatic dysfunction noticed in all 4 patients. A possible explanation of the benefit from GFD is that it heals the gastrointestinal mucosa and decreases any further triggers of autoimmunity.15 In another study, GFD along with steroids resulted in higher remission rates in pediatric patients with AIH coexisting with CD, compared with AIH without CD.16GFD is an effective treatment for most patients with CD and extraintestinal manifestations.17 from their study delineate that a portion of adults that developed a complete mucosal recovery could have a lower risk of all-cause mortality than patients with persistent mucosal damage. However, they did not find complete mucosal recovery in a significant number of adults with CD, despite considerable improvement in clinical response after treatment with a GFD. This could be explained either due to poor adherence to GFD or gluten from unidentifiable sources, or even a longer duration of study is required in adults for the effect to be measured. On the other hand, children were found to have achieved mucosal recovery as much as 95% after 2 years of treatment with a GFD.17Rubio-Tapia5 as well as mortality,18 but further studies are required to shed light on its implication in achieving remission in AIH.In summary, growing evidence suggests considerable benefits of GFD in decreasing the risk of subsequent autoimmune disease, malignancy, osteoporosis,Clinician should consider screening of CD in patients with AIH with persistent elevation of liver enzymes despite immunosuppressant and corticosteroid treatment. Early application of GFD can prevent further liver damage and can help achieve remission in these patients.