Objectives: This study aimed to examine the prevalence of medication non-adherence among older adults with diabetes mellitus (DM) in Shandong province, China and to identify its influencing factors. Methods: A sample of 1002 older adults aged 60 or above with DM was analyzed. Medication adherence was measured using the Morisky–Green–Levine (MGL) Medication Adherence Scale. Descriptive statistical analysis, chi-square test, univariate and multivariate logistic regression analyses were employed. Results: The prevalence of self-reported medication non-adherence among older adults with DM was 19.9%. Female respondents (adjusted odds ratio (AOR) = 1.56, 95% CI: 1.09–2.24) and respondents who perceived medication adherence to be unimportant  were more likely to experience medication non-adherence. Respondents with 5 years of disease duration or longer were less likely  to experience medication non-adherence. Conclusions: This study showed that about one out of five older adults with DM in Shandong province, China, experienced medication non-adherence, and that gender, disease duration and perceived importance of medication adherence were associated with medication non-adherence in this population group. Provision of counseling and health education programs could be the future priority to raise patients’ awareness of the importance of medication adherence and improve patients’ self-management of DM. E. KoCharacterized by hyperglycemia, diabetes mellitus (DM) is a group of chronic, metabolic diseases in which the pancreas does not secrete enough insulin, a hormone regulating blood sugar, and/or the body develops resistance to insulin it produces ,3. As thAccording to the commonly used definition, medication adherence refers to ‘the extent to which patients take medications as prescribed by their healthcare providers’ . MedicatGiven the significant health and economic consequences caused by medication non-adherence, especially among older adults with DM, it is imperative to detect non-adherent patients and identify barriers to medication adherence, so as to prevent and control the prevalence and costs of this diagnosable and treatable epidemic from further expanding . Among vWith both the largest population with DM and the largest older population aged 60 years or above in the world, China, compared with many other countries, is at a higher risk of bearing heavy disease and economic burdens resulting from medication non-adherence in older patients with DM ,18. ThouThis study was based on the Shandong Province Elderly Family Health Service Survey conducted in 2017. Detailed sampling design has been described in previous publications . In geneAmong 7088 older adults who were selected and interviewed, 18 failed to complete the survey, resulting in a total sample of 7070 respondents. This study targeted a sample of 1002 older adults with DM. Respondents were identified as patients with DM if they were previously confirmed by physicians and were taking anti-diabetic medication/had taken anti-diabetic medication before. Respondents were perceived as having DM-related complication(s) if they received previous confirmation from physicians. Data with less than 10% of missing values were imputed with medians for continuous variables and modes for categorical or dichotomous variables. Before administering face-to-face interviews, informed consent for data collection and analysis was obtained from eligible participants who were willing and able to understand and answer interviewers’ questions.We used the MGL Medication Adherence Scale that was proved to have good concurrent and predictive validity and good internal consistency . Each ofIndependent variables were chosen from the survey questionnaire according to the five dimensions of adherence conceptual framework . Social p-value of less than 0.2 in the unadjusted analyses were selected as candidate independent variables for the adjusted model. We employed backward elimination on candidate independent variables with a p-value cutoff of 0.05 to identify independent variables significantly associated with medication non-adherence. Strength of associations between respondents’ characteristics and medication non-adherence was described using crude odds ratios (CORs) and adjusted odds ratios (AORs) with the corresponding 95% confidence intervals (CIs).All analyses were conducted using Stata 16.1 . Frequencies and percentages were calculated to describe categorical or dichotomous variables. We used chi-square test to determine the bivariate association between respondents’ characteristics and medication non-adherence. Unadjusted and adjusted logistic regression analyses were conducted to identify factors influencing medication non-adherence among older adults with DM. Independent variables with a The Research Ethics Committee of Shandong University granted the ethical approval for this survey (No. 20170110). Participation in the interview was voluntary with informed consent, and respondents’ data were collected anonymously.n = 707) had URRBMS, 67.4% (n = 675) had a disease duration of 5 years or above, 84.1% (n = 843) did not experience the incidence of complication, 84.4% only took OHA (n = 846), 90.6% (n = 908) perceived medication adherence to be important, and 75.4% (n = 756) had good mental health status. The overall prevalence of medication non-adherence was 19.9%, and the gender-specific prevalence was 15.4% in males and 21.8% in females.n = 463) were highly adherent, 33.9% (n = 340) had moderate adherence, and 19.9% (n = 199) had low adherence. In addition, the percentage of respondents reporting unintentional non-adherence was 44.5% (n = 446), the percentage of respondents reporting intentional non-adherence was 33.2% (n = 333), and the percentage of respondents reporting both unintentional and intentional non-adherence was 24.0% (n = 240).The unadjusted and adjusted associations between respondents’ characteristics and medication non-adherence are presented in Medication non-adherence is a serious challenge to the self-management of DM among adults with DM, especially among older adults, and its downstream effects will be multiplied if left unaddressed and will be manifested as increased incidence and prevalence of major complication and heavier disease and economic burdens of the disease. Therefore, the objectives of this study were to examine the prevalence of medication non-adherence among older adults with DM and to identify its influencing factors.The prevalence of self-reported medication non-adherence among older adults with DM in Shandong province was 19.9% in this study, which was close to the finding reported from Cambodia (17.2%)  but loweIn this current study, females were more likely to be non-adherent to their prescribed medications than males, which is consistent with some previous studies ,27. ConsOur study also demonstrated that respondents who perceived medication adherence as unimportant were more likely to experience medication non-adherence. This finding could be potentially attributed to the property of the MGL Medication Adherence Scale that it measures both intentional non-adherence (forgetfulness or carelessness) and un-intentional medication non-adherence (stop taking prescribed medication(s) when feeling better or worse) . In our This study has several strengths and limitations. The stratified multistage random sampling design of this study ensured that enrolled respondents represented different socioeconomic backgrounds. Additionally, the sample size of this study was larger compared with many previous studies and therefore is of better potential to identify factors associated with medication non-adherence. Furthermore, this study targeted older adults with DM, a population group that is vulnerable to the negative consequences of medication non-adherence, and provided evidence on the characteristics of older patients who were at higher risks of experiencing medication non-adherence.Several limitations of the present study should also be noted while interpreting the findings. First, this study was based on patients’ self-reported medication adherence and therefore was inevitably subject to recall bias and self-report bias. Second, the study did not distinguish between patients with type 1 diabetes mellitus (T1DM) and patients with type 2 diabetes mellitus (T2DM), nor did it distinguish between different types of DM complications. Third, the cross-sectional design of this study only allows for the establishment of association between respondents’ characteristics and medication adherence. Finally, this study only considered factors available in the survey questionnaire, and we acknowledge that there are some other confounding factors associated with medication non-adherence in addition to the factors included in this study. To better understand factors influencing medication non-adherence among older adults with DM and explore underlying mechanisms, future study need to pay more attention to the collection of DM-specific data, such as types of complications and types and doses of anti-diabetic medications.In conclusion, this study showed that about one out of five older adults with DM in Shandong province, China, experienced medication non-adherence, and that gender, disease duration and perceived importance of medication adherence were associated with medication non-adherence in this population group. Medication non-adherence among Chinese older adults with DM is a public health problem that demands attention and efforts from decision-makers and professionals in the healthcare sector. To counter this pressing challenge, future priorities could be to provide counseling and health education programs to raise patients’ awareness of the importance of medication adherence and improve patients’ self-management of DM.


The aim of this review is to provide an overview of the current scientific evidence concerning the role of stem cell transplantation in multiple myeloma. During the past decade, several new treatment options have become available, thereby questioning the role of stem cell transplantation for the management of multiple myeloma. This review focuses on these studies, demonstrating a benefit for autologous stem cell transplantation (auto-SCT). We also reviewed maintenance post auto-SCT and utility of allogeneic stem cell transplant.Autologous stem cell transplantation (auto-SCT) has been the standard of care in eligible newly diagnosed multiple myeloma (MM) patients. Outcomes of patients with MM have improved significantly due to the advent of several novel drugs. Upfront use of these drugs in induction therapy has significantly increased the rate and depth of responses that have translated into longer remission and survival. This has now raised a debate regarding the role and relevance of auto-SCT in the management of myeloma. However, clinical trials have confirmed the utility of auto-SCT even in the era of novel drugs. Tandem auto-SCT followed by maintenance has shown a progression-free survival (PFS) benefit in high-risk MM, and hence can be considered in young and fit patients with high-risk disease. Auto-SCT has the advantages of resetting the bone marrow microenvironment, short-lived toxicity compared to the long-term physical and financial toxicities of continued chemotherapy in the absence of SCT, very low transplant-related mortality (TRM) in high volume centers, and providing longer disease-free survival when followed by maintenance therapy. Allogeneic SCT is one potentially curative option for MM, albeit with an increased risk of death due to high TRM. Strategies to modulate the graft-versus-host disease (GVHD) while maintaining or improving the graft-versus-myeloma (GVM) effect could place allogeneic SCT back in the treatment armamentarium of MM. Multiple myeloma (MM) accounts for about 10% of all hematologic malignancies .About 35 years ago, alkylating agents and corticosteroids were the most effective conventional agents for the treatment of this disease but without any major improvement in the outcomes ,5. The fThe efficacy of high-dose chemotherapy is mostly related to damage to tumor-cell DNA. It has, however, been shown that agents such as thalidomide, bortezomib, and lenalidomide act not only on myeloma cells, but also on the tumor microenvironment . Hence, ASCT is offered as consolidation therapy to young patients and eligible elderly (older than 65 years) patients with newly diagnosed MM (NDMM). There is no age cut-off for ASCT eligibility, at least in the United States. In a study by Wildes et al. , after a2 was safe in both moderate and severe RI [2, however, have been seen to have lower associated mortality and be beneficial for renal recovery [2) are routinely used.Renal insufficiency (RI) is prevalent in 20–50% of the MM patients, and a portion of these patients are dialysis dependent . In the evere RI . Moderatrecovery ,25,26,27Immunomodulatory drugs and proteasome inhibitors are used along with steroids as backbone for induction therapy to reduce tumor burden and improve quality of life. This has led to improved patient outcomes, but there is ongoing research to improve upon this regimen. Current induction regimens consist of novel therapies, which include immune modulators (Imids) and proteasome inhibitors (PIs), such as bortezomib (V), lenalidomide (R), and dexamethasone (D) (VRD); V/thalidomide(T)/D (VTD); V/Cyclophosphamide (C) (VCD); and carfilzomib (K)/R/D (KRD). A direct comparison of thalidomide and lenalidomide as part of induction therapy was reported in the myeloma XI trial. Myeloma XI was a randomized trial that showed the superiority of lenalidomide over thalidomide in combination with cyclophosphamide in NDMM with 60% of patients achieving at least VGPR after induction with CRD. The results are comparable to other novel triple agent regimens . SeveralIt is widely known that ASCT is usually performed in patients who are responsive to chemotherapy, but there has been no consensus regarding the ideal pre-SCT response needed for ASCT that would improve survival. There have been conflicting reports regarding the effect of post-induction disease response on the benefit obtained from upfront ASCT. Studies have shown that the attainment of a deep response prior to ASCT was associated with a survival advantage , and fai2) followed by infusion of bone marrow graft in 1983 [T.J. McElwain and R.L. Powles hypothesized that using high doses of an effective agent such as melphalan or cyclophosphamide would lead to increased proportion of cell death and overcoming drug resistance of tumors. They reported the first successful treatment of nine patients with multiple myeloma, including one patient with plasma cell leukemia after conditioning with intravenous high dose melphalan 140 mg/m0 mg/m2 f in 1983 . Barlogi2) to bu-mel  showed significantly prolonged progression-free survival (PFS) with bu-mel but no difference in overall survival (OS). However, the incidence of mucositis, transaminitis, and febrile neutropenia was significantly higher with the addition of busulfan [2) conditioning was compared with a conventional therapy regimen consisting of vincristine, carmustine, melphalan, cyclophosphamide, and prednisone in the phase 3, intergroup SWOG 9321 study [2 remains the standard conditioning chemotherapy for ASCT in MM.Several drugs have been tested in combination with high dose melphalan as conditioning chemotherapy to improve the ASCT outcomes including busulfan, bendamustine, arsenic trioxide, ascorbic acid, bortezomib, and lenalidomide. None fared better than melphalan alone ,42,43,44busulfan . For relbusulfan ,47,48. Nbusulfan . A phasebusulfan . ASCT wi21 study . This st2) and total body irradiation with 8 Gy, followed by stem cell infusion, followed by maintenance with recombinant interferon alpha after hematologic reconstitution. Patients in the non-transplant arm received 18 cycles of chemotherapy with recombinant interferon-alpha given from cycle 9 until relapse. Patients in the ASCT arm had a higher probability of five-year event-free survival (EFS)  as well as five-year overall survival (OS) . These results were similar to those seen in the Southwest Oncology Group (SWOG) study done by Barlogie et al. comparing ASCT following “total therapy” with standard SWOG chemotherapy [High dose chemotherapy with autologous stem cell transplantation has been an integral part of management of young and fit elderly patients with NDMM for the last three decades. The IFM group conducted one of the earliest prospective trials in patients with NDMM, comparing the efficacy of high-dose chemotherapy followed by ASCT to conventional chemotherapy . After fotherapy . In 2003Several studies have been done in the last decade to investigate the role of ASCT in the era of novel drugs such as the proteasome inhibitors and immunomodulators. A study by the GIMEMA group randomized patients with NDMM to tandem transplant versus six cycles of MPR  following Rd  induction, with further randomization of each arm to maintenance with lenalidomide versus placebo. Both PFS and OS were superior in the ASCT group . In anotGiven the increase in the rate and depth of responses with novel drug combinations of both a PI and an IMiD that has become the standard of care for NDMM, the IFM/Dana-Farber Cancer Institute (DFCI) group conducted a prospective, randomized study to explore the role and timing of ASCT in MM. In this study, patients with NDMM up to 65 years of age received induction with three cycles of VRd followed by consolidation with either five cycles of VRd or ASCT followed by three additional cycles of VRd. Both the arms received lenalidomide maintenance until progression (in the DFCI US study) or for one year (in the IFM study). Patients in the chemotherapy only arm received ASCT at first relapse. While the DFCI study is still awaiting final outcomes, the IFM study showed that upfront ASCT in patients with NDMM resulted in significantly longer PFS compared to those without. The benefit was seen across all stages and cytogenetic risk groups . However2 (Mel 200) or Mel 200 followed 3–6 months later by another ASCT with Mel 120 mg/m2 and busulfan. Tandem ASCT resulted in significantly higher CR and near CR, EFS, and PFS with no difference in OS [p = 0.6; p = 0.8). However, in as treated analysis, six-year PFS for high-risk patients favored the tandem ASCT group ). Given the PFS benefit for tandem ASCT, it could be considered for young and fit patients with high-risk disease  [ASCT in MM is feasible and effective even when used for the treatment of relapsed disease ,71. In f< 0.001) . While R< 0.001) . Longer < 0.001) . This agp < 0.001), with no significant difference in three-year overall survival  [p < 0.001) with no difference in OS [Maintenance therapy has been one of the successful strategies to delay disease progression and prolong survival by deepening and sustaining the response achieved with ASCT. Prior to the era of novel drug therapy, interferon was used as post-SCT maintenance, but due to the better side effect profile and quality of life with immunomodulators and proteasome inhibitors, interferon is not used any more. Thalidomide was used as maintenance therapy with or without steroids with conflicting reports of survival benefit ,76,77. R = 0.14) . The IFMce in OS . HoweverGiven the conflicting reports on the OS benefit from maintenance therapy from these three trials, a meta-analysis of these studies was conducted by McCarthy et al., which confirmed the PFS benefit and demonstrated a significant OS benefit with lenalidomide maintenance after ASCT in patients with NDMM . LenalidBortezomib is often used as maintenance therapy in the United States, extrapolating the PFS benefit with its use in patients with high-risk cytogenetics in the HOVON 95/GMMG-HD4 study . PatientA recently published retrospective analysis comparing maintenance therapy with lenalidomide and bortezomib after upfront ASCT from two subsequent GMMG phase 3 trials showed a significant PFS benefit with lenalidomide maintenance with no difference in OS between the two groups .Ixazomib, an oral proteasome inhibitor, reduced the risk of progression or death by 28% compared to placebo in the post-ASCT maintenance setting in the TOURMALINE-MM3 study . Toxicitp = 0.0004) with no difference in OS [In the STaMINA trial, analysis at 38 months showed that patients who continued post-ASCT maintenance beyond three years had better PFS than those who discontinued it earlier . Median OS was 70.2  versus 71.8 months (tandem ASCT) (p = 0.856). The lack of overall survival benefit was due to the increased NRM from the toxicity of allo-SCT. Two-year NRM was higher with auto/allo . In patients with both del13q and del17p, median PFS and OS, respectively, were 37.5 and 61.5 months with auto/allo (n = 19) versus 6.1 and 23.4 months with tandem ASCT (n = 6) (p = 0.0002 and 0.032). Though the patient numbers were small, the study provided support for the use of allo-SCT in eligible patients with high-risk cytogenetics. A pooled analysis of the long-term survival of four randomized trials  comparing auto-auto vs. auto–allo after induction therapy based on availability of HLA-matched sibling donors in NDMM patients was recently published [n = 899) or auto-allo (matched-sibling) (n = 439). Median follow up of survivors was 118.5 months. Median OS was significantly better in the auto-allo group—78.0 months in auto-auto and 98.3 months in auto-allo . OS was 36.4% vs. 44.1% at 10 years (p = 0.01) for auto–auto and auto–allo, respectively. PFS was also better in auto-allo . However, the risk of NRM was higher in auto–allo , while risk of disease progression was higher in auto–auto . Median post relapse survival was 41.5 months in auto–auto and 62.3 months in auto–allo , supporting the role of durable graft versus myeloma (GVM) effect improving the efficacy of salvage therapies.The earliest large retrospective, comparative study of patients who received myeloablative allo-SCT compared to those who received ASCT was reported in 1996 by EBMT. OS was inferior in the allo-SCT group due to higher transplant-related mortality (TRM), in spite of lower relapse rate . Anotheritioning . No mainitioning ,103,104.itioning . Median ublished . PatientAllo-SCT in the relapsed setting may offer long-term PFS and OS, especially in young patients with high-risk disease and in those who relapsed within 18 months from initial ASCT ,108,109.Allo-SCT is traditionally performed with use of HLA-identical siblings or matched unrelated donors. Since such donors are not always available, allo-SCT from haploidentical related donors has been developed and increasingly used. Post-transplant cyclophosphamide (PT-Cy) has been used to selectively deplete allo-reactive T cells in haploidentical-SCT . High-doUnlike in the upfront setting, the role of allo-SCT in the treatment of RRMM patients has not been extensively studied. The following guidelines have been proposed by the International Myeloma Working Group together with the Blood and Marrow Transplant Clinical Trials Network, American Society of Blood and Marrow Transplantation, and the European Society of Blood and Marrow Transplantation committee. (1) Allogeneic HCT should be considered appropriate therapy for any eligible patient with early relapse (less than 24 months) after primary therapy that included an ASCT and/or high-risk features ; (2) allogeneic HCT should be performed in the context of a clinical trial if possible; (3) the role of post-allogeneic HCT maintenance therapy needs to be explored in the context of well-designed prospective trials; and (4) prospective randomized trials need to be performed to define the role of salvage allogeneic HCT in patients with MM relapsing after primary therapy .p = 0.01). It has been shown in several studies so far that MRD negativity is associated with longer PFS and OS, and if sustained, the first step towards functional cure for MM. The effect of MRD negativity on the outcomes is agnostic of the treatment regimen used or the method of determination. This provides a rationale to test the hypothesis that MRD testing could be used to determine the timing of ASCT in the upfront management of MM. Patients who fail to achieve MRD negativity after a fixed number of cycles of induction therapy can be taken for ASCT, while in those who achieve MRD negativity, transplant could be delayed with ongoing surveillance for resurgence of disease. Several ongoing clinical trials in the setting of post-SCT maintenance are incorporating MRD as a primary end-point to determine the optimal duration and intensity of therapy [ASCT in MM not only prolongs remission but also yields deeper responses, as was seen in the IFM/DFCI 2009 study. Patients in the transplant arm had higher MRD negativity rate compared to those in the non-transplant arm . Other methods to improve on PFS and OS, such as chimeric antigen T-cells (CAR-T) as consolidation post ASCT in high risk patients, CAR-T in patients who fail to achieve very-good partial response or better after at least six months of maintenance post ASCT, and dendritic cell vaccine post ASCT (BMT/CTN 1401), are in protocol stages, accruing and/or completed and awaiting results. Allo-SCT plays a critical role in high-risk patients, especially young patients who relapse within 18–24 months from initial ASCT. Methods to reduce GVHD and TRM are improving to allow allo-SCT to be utilized early in the disease course. However, despite the progress made so far, the problem of chemoresistant disease persists requiring the development of agents with novel mechanism of action for RRMM. Bispecific antibodies (BsAbs) are another immunotherapy option, whereby tumor cell death can be achieved by an enhanced interaction between immune cells (T cells) and tumor cells, with a potential role in the management of RRMM ,118. Sev


Defibrotide (DFB) effects on different endothelial cell pathways have been investigated focusing on a limited number of genes or molecules. This study explored the modulation of the gene expression profile of steady-state or lipopolysaccharide (LPS)-activated endothelial cells, following the DFB exposure. Starting from differentially regulated gene expression datasets, we utilized the Ingenuity Pathway Analysis (IPA) to infer novel information about the activity of this drug. We found that effects elicited by LPS deeply differ depending on cells were exposed to DFB and LPS at the same time, or if the DFB priming occurs before the LPS exposure. Only in the second case, we observed a significant down-regulation of various pathways activated by LPS. In IPA, the pathways most affected by DFB were leukocyte migration and activation, vasculogenesis, and inflammatory response. Furthermore, the activity of DFB seemed to be associated with the modulation of six key genes, including matrix-metalloproteinases 2 and 9, thrombin receptor, sphingosine-kinase1, alpha subunit of collagen XVIII, and endothelial-protein C receptor. Overall, our findings support a role for DFB in a wide range of diseases associated with an exaggerated inflammatory response of endothelial cells. It consosal DNA . Since tosal DNA , 4. Hepaosal DNA , 6. The in vitro and in vivo  are a rare cell population of circulating progenitors endowed with clonal potential, ability to give rise to mature endothelial cells, and promoting vascular formation  in vivo . The ECF in vivo , 9. The  in vivo . Therefo in vivo –15. Humae organs . Furthere organs . Wherease organs . Since bThis study aimed to understand which endothelial pathways are mainly regulated by DFB, to gather further information for its potential therapeutic use.in vitro effects in conditions mimicking in vivo administration for either preemptive or therapeutic DFB purposes. Before being exposed to LPS, endothelial cells were either or not preliminary incubated with DFB. DFB concentration used in the final experimental setting was preliminarily identified by evaluating the reduction of intercellular adhesion molecule-1 (ICAM-1) RNA expression after LPS exposure in the presence of increasing DFB concentrations . We selected the concentration of 100 ng/ml since it was the lowest DFB concentration at which ICAM-1 RNA expression was maximally inhibited. This concentration reproduces the DFB plasma level achieved in the clinical setting after intravenous administration at recommended doses for VOD/SOS treatment (The study was designed to explore the DFB-dependent reatment . The DFBa) control cultures: 6 h incubation in EBMTM-2; b) DFB-cultures: 6-h incubation in EBMTM-2 with DFB; c) LPS-cultures: 6-h incubation in EBMTM-2 with LPS; d) LPS + DFB-cultures: 6-h incubation in EBMTM-2 with LPS and DFB; e) preDFB-LPS + DFB-cultures: 2-h incubation in EBMTM-2 with DFB, followed by medium removal and then 6-h incubation in EBMTM-2 containing LPS and DFB. Cells were then washed in phosphate-buffered saline, directly lysed in the multiwell plates , and used for RNA extraction. A minimum of six overall experiments were carried out for each condition and in each set of experiments ECFCs derived from one single cord blood unit were utilized.ECFCs were obtained from cord blood units donated by the UNICATT Cord Blood Bank of Fondazione Policlinico Universitario A. Gemelli IRCCS  for transplant purposes. Units were collected from healthy full-term babies: units not suitable for transplantation for low volume or low cell content were used, according to the Institutional Ethics Committee approval . ECFCs were obtained according to the original method of Ingram et al . Briefly2 First Strand Kit (Qiagen). Real-time PCR was carried out by RT2 SYBR Green ROX qPCR Mastermix (Qiagen) using the Stratagene Mx3000P Real-Time PCR detection system . Gene expression profile was assessed by the Human Endothelial Cell Biology RT2 Profiler PCR Array , including primers for 84 tests and five housekeeping genes (−ΔΔCT method using the cycle threshold (CT) obtained at real-time PCR. Data analysis was performed using the web-based PCR array data analysis software, available at https://dataanalysis2.qiagen.com/pcr.Total RNA was extracted using the RNaesy MiniKit , according to manufacturer instructions. QIAxpert Spectrophotometer (Qiagen) was used to assess RNA integrity and concentration. Reverse transcription was performed with 0.5 µg of total RNA using the RTin silico analyses with Ingenuity Pathway Analysis  were performed. Data relative to genes with an average threshold cycle either not determined or greater than the defined cut-off (default 35), in investigated samples and controls were excluded from the analysis  and bio-functions’ analysis , 2) upstream regulators’ analysis, i.e. likely regulators that are connected to dataset genes through a set of direct and indirect relationships 3) interaction and causal network analysis, i.e. a generalized analysis of additional regulators that connect upstream regulators to genes significantly modulated in experimental datasets, including intermediate regulators   . Statistgulators . In ordeatterns) .All relevant data are contained within the article. The original contributions presented in the study are included in the supplementary material and further inquiries can be directed to the corresponding author.We carried out a preliminary exploratory heatmap analysis to evaluate gene expression profile clustering in different experimental conditions . The DFB pre-exposure significantly damped the activation of the above-mentioned pathways, likewise indicating a parallel inhibition of blood cell chemotaxis (γ), appeared to be common drivers of cell response to LPS and LPS + DFB, their role in DFB-pre-exposed cells was much less evident, with very low Z-score values  in various experimental settings are illustrated in For each experimental condition, we generated enriched interaction networks based on known relationships in the Qiagen Knowledge Base and ordered by decreasing IPA score. We identified six networks for LPS exposed cells, six networks for cells exposed to LPS with DFB, and eight networks for cells pre-exposed to DFB and then to LPS with DFB. The highest score network (score 11) in the first condition (LPS) was centered on five genes  . The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The purpose of this study is to report the clinical features and outcomes of Black/African American (AA) and Latino Hispanic patients with Coronavirus disease 2019 (COVID-19) hospitalized in an inter-city hospital in the state of New Jersey.This is a retrospective cohort study of AA and Latino Hispanic patients with COVID-19 admitted to a 665-bed quaternary care, teaching hospital located in Newark, New Jersey. The study included patients who had completed hospitalization between March 10, 2020, and April 10, 2020. We reviewed demographics, socioeconomic variables and incidence of in-hospital mortality and morbidity. Logistic regression was used to identify predictor of in-hospital death.Out of 416 patients, 251 (60%) had completed hospitalization as of April 10, 2020. The incidence of In-hospital mortality was 38.6% (n = 97). Most common symptoms at initial presentation were dyspnea 39% (n = 162) followed by cough 38%(n = 156) and fever 34% (n = 143). Patients were in the highest quartile for population’s density, number of housing units and disproportionately fell into the lowest median income quartile for the state of New Jersey. The incidence of septic shock, acute kidney injury (AKI) requiring hemodialysis and admission to an intensive care unit (ICU) was 24% (n = 59), 21% (n = 52), 33% (n = 82) respectively. Independent predictors of in-hospital mortality were older age, lower serum Hemoglobin < 10 mg/dl, elevated serum Ferritin and Creatinine phosphokinase levels > 1200 U/L and > 1000 U/L.Findings from an inter-city hospital’s experience with COVID-19 among underserved minority populations showed that, more than one of every three patients were at risk for in-hospital death or morbidity. Older age and elevated inflammatory markers at presentation were associated with in-hospital death. Since the first case of the novel coronavirus disease 2019 (COVID-19) was reported in Wuhan, China, more than 2.7 million cases have been confirmed globally . The COVAs the science surrounding this novel disease evolves, a greater attention must be paid to identifying individuals who suffer the most devastating effects. While recent studies have focused on describing the clinical disease course, outcomes and identifying modifiable risk factors, little is known about the effects of COVID-19 on minority populations in the United States.Newark is the second-most racially diverse city in New Jersey and houses more than 50% Black/African American and 33% Hispanic or Latino population. Here, we present the clinical characteristics and outcomes in Black/African American and Latino/Hispanic COVID-19 patients who were managed at a quaternary care hospital in Newark, New Jersey.Setting, Population and Data Collection:This study is a retrospective cohort study conducted at the Newark Beth Israel Medical Center (NBIMC), a 665-bed quaternary care, teaching hospital located in Newark, New Jersey. We reviewed data from a prospectively maintained COVID-19 database, which included all patients who had laboratory-confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. This was defined as a positive result on a reverse-transcriptase-polymerase reaction (RT-PCR) assay of a specimen collected on a nasopharyngeal swab.We included adults 18-years of age or older who were admitted between March 10, 2020 and April 10, 2020, with follow-up through April 20, 2020. Since most of the patient population served by NBIMC are Black/African Americans (AA) and Latino/Hispanics, non-AA or Latino/Hispanics were excluded from the study. Patients with full clinical data who had completed their hospitalization consisted the final study cohort. We compared outcomes between survivors vs. non-survivors.The NBIMC institutional review board approved this study and waived the need for an informed consent due to its retrospective nature. We extracted de-identified data, on epidemiological, demographic, clinical, laboratory, treatment, and outcomes from electronic Medical records by using a standardized data collection protocol.We extracted data on co-morbid conditions by interrogating physician documentation and confirming with their international classification of diseases (ICD-9/10) codes. Presenting symptoms were subjectively classified according to patients complains at initial presentation.An independent researcher (CS) who adjudicated any difference in interpretations between two senior author reviewers  crosschecked collected data.Other variables included population densities, housing units and median income representing the social determinants of health status among admitted patients. These variables were estimated by matching data from the United States Census Bureau , with 5-We defined sepsis and septic shock based on the 2016 Third International Consensus Definition for sepsis and septic shock . We inclWe summarized the data by using descriptive statistics, presenting continuous variables as median, interquartile range and categorical variables as proportions or percentages. We used the Mann- Whitney U test, Chi-square, or Fisher’s exact tests to compare differences between survivors and non-survivors.Univariate and multivariable logistic regression models were used to explore the risk factors associated with in-hospital mortality. We performed a stepwise regression model with backward selection using the minimum Akaike Information criterion to identify independent predictors of in-hospital mortality. To avoid overfitting, we tested for variables that were significantly associated with in-hospital mortality at a univariate level and only included them in the final model if they had a p value of < 0.05.Variables were not included in the univariate analysis if (a) their accuracy were not confirmed, such as self-reported presenting symptoms; (b) they showed collinearity or (c) their event rate was too low to calculate odds ratios. For laboratory values, we used sensitivity analysis to determine cut-offs based on their association with in-hospital death. Cut-offs were determined based on both clinical context and a sensitivity ≥80% in predicting in-hospital death. Statistical significance was considered when a two-sided alpha of less than 0.05 was reached. All analyses were performed using the JMP version 14.0.2 statistical software .Between March 10, 2020 and April 10, 2020, we identified 416 COVID-19 adult patients who had at least a follow up through April 20, 2020. We excluded 35 (8.4%) patients who were non-AA or Latino/Hispanics. Of the excluded patients, only 10 had completed hospitalization at the time of study analysis. As of April 20, there were 381 patients who had completed hospitalization. Among these, 130 were still hospitalized and 251 had either died or been discharged. In-hospital mortality was recorded in 97 (38.6%) patients.The most common symptoms at initial presentation were dyspnea 39% n=162) followed by cough 38% (n=156) and fever 34% (n=143). Other symptoms included myalgia, fatigue, nausea, and vomiting which occurred four to seven days prior to presentation.  infection was significantly higher among survivors than non-survivors (p = 0.008).We tracked major laboratory values at baseline and compared their levels among survivors and non-survivors. Figure The overall incidence of ARDS, Septic shock, acute kidney injury requiring hemodialysis and admission to an intensive care unit (ICU) for worsening clinical status was 33% (n = 82), 24% (n = 59), 21% (n = 52), 33% (n = 82) respectively. Of the 82 patients who required ICU admission, 70 (85%) had expired at the time of last censor  and only 11% (n = 6) of patients who required dialysis for AKI survived to discharge.After univariate analysis, the odds of in-hospital mortality were found to be higher in older patients, males, Black/African Americans, and those with hypertension, coronary artery disease and chronic kidney disease. Table  The presTo our knowledge, this is the first study to describe in detail clinical outcomes of minority patients hospitalized for COVID-19 in an inter-city hospital. We found an in-hospital death incidence of 38.6% among Black/African American and Latino/Hispanic patients. These patients resided in areas of New Jersey with reportedly higher population densities, housing units and lower income levels than the rest of the state. Minority patients were more likely to present later in the course of disease with worsening shortness of breath and cough while being older, with a high serum ferritin, and creatinine phosphokinase levels, were associated with in-hospital death.Earlier reports on outcomes after COVID-19 have highlighted the association between modifiable risk factors such as hypertension; diabetes and obesity with worse outcomes –12. ReceThe most common presenting symptom in our cohort was dyspnea followed by cough then fever. About 43% patients presented with dyspnea. Zhou et al., in their outline of the clinical course of major symptoms for COVID-19 infected patients, identified dyspnea as one of the common symptoms occurring on day 6 of the disease course .The mortality and morbidity associated with COVID-19 is reportedly high according to findings from previous studies , 13, 14.We identified three laboratory markers (namely➔hyperferritinemia, low serum hb and high level of CPK and older age, as independent predictors of in-hospital death. Similar to what was described by Ruan et al.,  we foundInterestingly, we found that, the presence of HIV infection was associated with survival in this patient population. This might be an important consideration in the understanding the pathophysiology of the disease process. First, despite an extremely high prevalence of HIV in the region where our center is located, only 5% of patients who required admission for COVID had HIV. Second, it was surprising to note that, all HIV patients survived until discharge except for one patient, aged 70-years who died in-hospital due to multi-organ failure. Most of the patients who survived were on anti-retroviral medications with normal CD4 counts and well controlled viral loads. In a Spanish experience with HIV and COVID-19, the authors treated five patients with both anti-SARS-COV-2 treatment on day of diagnosis followed by boosted-protease inhibitor anti-retroviral therapy. Concomitant antibacterial were given to three patients who had pneumonia, corticosteroids in other two and tocilizumab in one patient. The authors highlighted a discharge rate of 80% at the time of their writing . In factOur study has several limitations; first, this study is a retrospective study and is by all factors inherent to retrospective analysis. Second, the study does not include all laboratory variables previously described in the literature including interleukin levels and other variables needed to calculate risk assessment scores like SOFA scores at baseline. Third, although the patient population is predominantly Black/African American or Latino Hispanics, our results are limited by a single centers experience that may not be generalizable to the overall population. The exclusion of non-Latino/Hispanic patients from this study also limits the generalizability of the study findings. Our hospital population is mainly Black/AA or Hispanic patients, and there were only 10 non-Latino/Hispanic patients out of the who met the inclusion criteria. Fourth, due to the novel nature of COVID-19, there was no standard management protocol employed for all patients who were admitted. This would have likely contributed to the worse outcomes observed. Lastly, interpretation of our data should be done with caution due to the smaller sample size and lack of full knowledge on this disease.In conclusion, we present a single center’s experience with COVID-19 among underserved minority populations. These patients lived in densely populated areas with significantly lower median income and reported a higher in-hospital mortality rate than what is described in the literature. The incidence of in-hospital death was 38.6% and morbidity reported was like what has been described in the literature.


This meta-analysis evaluates the use of cumulative meta-analysis of rare events as a tool for safety monitoring of clinical trials using the example of coronary bioresorbable vascular scaffold–associated thrombosis. We retrospectively assessed how cumulative meta-analysis could serve as a safety monitoring tool to identify the time point when firm evidence for safety concerns of a rare outcome becomes available.The continued vigilance in safety monitoring in randomized clinical trials (RCTs) is critical as more data and experience are accumulated.PRISMA) reporting guideline.For this meta-analysis, we assessed the withdrawn polymeric everolimus-eluting coronary bioresorbable vascular scaffold (BVS) . The BVS received CE mark approval in January 2011 and US Food and Drug Administration approval in July 2016. In September 2017, the manufacturer voluntarily withdrew the device owing to safety concerns (increased risk of scaffold-related thrombosis) after it had been available for clinical use for more than 6 years in Europe and 1 year in the US. This study followed the Preferred Reporting Items for Systematic Reviews and Meta-analyses .We retrieved all available reports of RCTs comparing the BVS with metallic everolimus-eluting stents for percutaneous coronary interventions by searching PubMed, CENTRAL, and websites of major cardiology meetings occurring before May 31, 2019. Device-related (scaffold or stent) definite or probable thrombosis was the safety outcome of interest. We used Mantel-Haenszel (fixed-effects model) cumulative meta-analysis to summarize accumulated rare events over time and computed odds ratios (ORs) at each time point. All P = .06). The between-group difference became statistically significant on September 18, 2016 . Availability of longer follow-up and new trials resulted in an OR of 2.87  11 months before the Absorb BVS was withdrawn in September 2017. Between-group differences reached on March 18, 2017, had an OR of 3.15  with a lower limit of the 95% CI above 1.00 (P < .001), indicating that the experimental intervention was harmful.A total of 22 reports describing 8 RCTs including 8180 patients randomized to BVS (4553 patients) or everolimus-eluting stents (3627 patients) were included, with 96 and 20 device-related thromboses for each intervention, respectively. Patient recruitment took place over 6 years, with considerable overlap of recruitment periods . The cumove 1.00 . The fin4 Under these scenarios, false-positive rates for significance tests at the conventional P < .05 are typically too high, and naive interpretations of statistical significance should be avoided.6Timely recognition of safety signals is important to patients, physicians, regulators, and the medical community at large to avoid unnecessary, clinically important adverse events and to prevent waste of research efforts, especially in studies of the comparative effectiveness of medical devices. In the absence of large clinical trials, some adverse events may not be known a priori when a new device is used and additional mechanisms, such as regulatory oversight for unexpected events, may be needed; continuously updated cumulative meta-analyses may contribute to this purpose. Of note, although cumulative statistical testing can bias this approach, it is not of particular concern in the present analysis because it was performed retrospectively and was not associated with a stopping rule for the meta-analysis. However, in a prospectively designed cumulative meta-analysis, correction for multiple testing should be considered because the examination of multiple outcomes and repeated analysis of the data over time may exacerbate the risks associated with multiplicity and further adjustments may be warranted.


Plasmodium falciparum targets in 29,481 participants across three surveys. This included one community-based survey in which participants were enrolled during household visits and two sentinel group surveys in which participants were enrolled at schools and health facilities. First, we correlated continuous antibody responses with age (Spearman) to determine which showed strong age-related associations indicating accumulation over time with limited loss. AMA-1 and MSP-119 antibody levels showed the strongest correlation with age  in the community-based survey, which was most representative of the underlying age structure of the population, thus seropositivity to either of these antibodies was considered representative of cumulative exposure to malaria. Next, in the absence of a gold standard for recent exposure, we included antibody responses to the remaining targets to predict highly sensitive rapid diagnostic test (hsRDT) status using receiver operating characteristic curves. For this, only data from the survey with the highest hsRDT prevalence was used . The performance of the top two antigens in the training dataset —Etramp 5 ag 1 and GLURP-R0 —was confirmed in the test dataset . As no further improvement was seen by combining seropositivity to GLURP-R0 and Etramp 5 ag 1 (p = 0.266), seropositivity to Etramp 5 ag 1 alone was selected as representative of current or recent exposure to malaria. The validation of antibody responses associated with these exposure histories simplifies analyses and interpretation of antibody data and facilitates the application of results to evaluate programs.In our aim to eliminate malaria, more sensitive tools to detect residual transmission are quickly becoming essential. Antimalarial antibody responses persist in the blood after a malaria infection and provide a wider window to detect exposure to infection compared to parasite detection metrics. Here, we aimed to select antibody responses associated with recent and cumulative exposure to malaria using cross-sectional survey data from Haiti, an elimination setting. Using a multiplex bead assay, we generated data for antibody responses (immunoglobulin G) to 23  Globally, the number of countries in malaria pre-elimination and elimination phases is increasing . For malPlasmodium falciparum apical membrane antigen 1 (AMA-1) and the 19 kDa fragment of merozoite surface protein 1 (MSP-119)  . Age-speMSP-119) . Antibodon is 23 , while lon is 23  to 16  in Port-au-Prince weekly. Demographic and household characteristics were collected through a tablet-based questionnaire. Participants testing positive by cRDT were treated according to national guidelines.The island of Hispaniola, consisting of Haiti and the Dominican Republic, is the last remaining region in the Caribbean with malaria transmission. In 2016, 97% of the reported malaria cases on the island occurred in Haiti . In HaitP. falciparum antigens and peptides from asexual life-cycle stages were included in the panel  data were collected for all participants as previously described . Antibod19) were repeated  of the third point of the serial dilution for two out of three highly immunogenic antigens  were removed from further analyses  (10(MFI) participant data and the lower distribution was assumed to consist of seronegative individuals. Thresholds for seropositivity were defined as the mean of the lower distribution plus 5 SD  . In addi10MFIs) from participants were correlated with age; those that were most strongly correlated with age were considered to represent cumulative exposure. Analysis was conducted using the continuous antibody data instead of seropositivity to maximize the strength of any correlation. The HH-Artibonite survey was considered most informative in assessing correlations between antibodies and age, as it represents the full age-structure of the population sampled due to its community-based sampling strategy compared to sentinel group sampling in the EAG-surveys . Participants who were hsRDT positive were excluded to remove any antibody responses due to a current or recent infection. Spearman correlation coefficients (r) between all antibody responses and age (in years) were plotted using the R package corrplot  and a test dataset  using random selection. The numbers of hsRDT positive samples in the training dataset was 227 (7.1%) and in the test dataset was 120 (7.3%). The area-under-the-curve (AUC) of ROCs  was meapackage; ].10(MFI)'s, to most antigens showed weak positive associations with increasing age . The correlation coefficient between AMA-1 and MSP-119 was 0.67, p < 0.001  than the other two antigens  in the EAG-Grand'Anse survey. The validation of AMA-1 and MSP-119 as the most appropriate markers of cumulative exposure is consistent with the literature across multiple settings (19) was seen across all surveys .In the HH-Artibonite survey, antibody levels, log < 0.001 . Togethesettings , 40–42. Antibody levels to the remaining 14 targets were included to predict hsRDT status using ROCs and a training dataset consisting of two-thirds of the EAG-Grand'Anse participants. Etramp 5 ag 1 had the highest AUC for predicting hsRDT status (0.892), followed by GLURP-R0 (0.825); p = 0.118; children aged 1 to 15: p = 0.060; individuals aged 16 or older: p = 0.686) and the combined metric, representing seropositivity to either Etramp 5 ag 1 or GLURP-R0 . As no improvement was seen by adding GLURP-R0, Etramp 5 ag 1 alone was selected to represent recent exposure to falciparum malaria. Age-specific hsRDT and seroprevalence per survey is shown in The performance of antibodies to Etramp 5 ag 1 and GLURP-R0 as accurate markers for hsRDT status  was confirmed in the test dataset  as well 19 best reflected cumulative exposure over time, whilst those to Etramp 5 ag 1 showed the highest accuracy in predicting current or recent infection when compared to hsRDT status. For the latter, survey data was used from the area experiencing the highest malaria transmission (Grand'Anse).This study used multiplex serological data to select antigen-specific antibody responses associated with cumulative and current or recent malaria exposure in Haiti. IgG responses from almost 30,000 participants were analyzed from three surveys across two locations with differing levels of malaria transmission intensity. Results showed that IgG responses to antigens AMA-1 and MSP-119 were confirmed as the optimum representation of cumulative malaria exposure as they showed the strongest correlation with age. These antigens have been used extensively in sero-surveillance for P. falciparum transmission  recent exposure was higher in EAG-Grand'Anse compared to EAG-Artibonite which corresponds with the 2017 malaria incidence estimated at 18.1 and 0.6 per 1,000 inhabitants in Grand'Anse and Artibonite, respectively ; (2) recent exposure was higher in healthcare-seeking populations compared to (likely asymptomatic) children attending school in Grand'Anse; (3) recent exposure was similar in children visiting a health facility or attending school in Artibonite, indicating that in this low transmission setting, healthcare-seeking is likely not driven by malaria disease; (4) cumulative and recent exposure was lowest in the HH-Artibonite survey representing an asymptomatic, community-based population across all ages.19 as recent exposure markers (The gold standard for identification of antibodies with short (or longer) half-lives, is assessment of antibody acquisition and decay rates in longitudinal studies following naturally acquired malaria infections. However, such studies are costly, time and labor intensive, and thus rare. Moreover, no existing studies have used the same combination of the age range of the study population, antigen panel assessed and assay conditions , 25, 40. markers –45, 48 sP. vivax and P. knowlesi , we used age and hsRDT status to make this selection. Seropositivity to AMA-1 and/or MSP-119 was selected to represent cumulative malaria exposure, while seropositivity to Etramp 5 ag 1 was selected to represent recent exposure to malaria. The identification of these antimalarial antibodies will simplify analyses and interpretation where results are needed to inform program decisions in Haiti.Here we described the selection of antibodies associated with cumulative and recent malaria exposure in Haiti. We used antibody responses (IgG) to 23 The datasets generated for this study are available on request to the corresponding author.The EAG-Artibonite and EAG-Grand'Anse surveys were approved by the London School of Hygiene and Tropical Medicine (LSHTM) Research Ethics Committee (10393), Tulane Institutional Review Board (794709) and the National Bioethics Committee in Haiti (1516-30). The Center for Global Health Associate Director of Science reviewed and approved the protocols; Centers for Disease Control and Prevention (CDC) investigators were not considered to be engaged in human subjects research (protocol 2016-135a). The HH-Artibonite survey was approved by the CDC Institutional Review Board (6821), LSHTM Research Ethics Committee (10466) and the National Bioethics Committee in Haiti (1516-29 and 1617-31).For the EAG-Artibonite and EAG-Grand'Anse surveys, consent was collected from school directors following community meetings to inform parents and allow opt-out. At health facilities, adult participants provided consent directly, and consent for children (<18 years) was provided by a parent or guardian and children above 6 years gave assent to participate. Individuals aged 16 or 17 who were married, head of household, or a parent were considered ‘mature minors’ and consented directly. Thumbprint consent or assent (countersigned by a witness) was used for illiterate participants. For the HH-Artibonite survey, verbal consent for overall permission to conduct the survey was obtained from the head of household and/or primary caretaker and documented in the electronic data collection instrument. Individual-level verbal consent was sought from each present individual for the blood sample collection, and assent from persons aged 7–17 years.JL, MC, TPE, TD, RA, GS, CD, KH, and ER designed the surveys. TPE, TD, RA, VJ, GS, KH, and MC performed and supported field data collection. KT, JGB, SS and ER provided antigen constructs. JP, IR, TE, and GM performed laboratory data collection. LH, AE, JB, KT, CD, and ER supported laboratory data collection. LH, GS, KT, ER, and CD analyzed and interpreted the data. LH wrote the first draft of the manuscript with support from GS and CD. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Numerous studies conducted over the past 30 years have pointed to the presence of Epstein–Barr virus (EBV) in gastric cancer samples. This study was aimed to provide a meta-analytic review of the prevalence of EBV in gastric cancer patients, and to clarify the relationship between EBV infection and gastric cancer.A literature search was performed electronically using online databases for English language publications until July 1, 2019. The pooled EBV prevalence and 95% confidence intervals (CIs) were estimated using a random-effects model. To determine the association between EBV and gastric cancer, pooled odds ratio (OR) and its 95% CI were computed for case-control studies. Two separate analyses were performed on data from case-control studies with matched and non-match pairs designs to calculate the pooled estimates of ORs.2 = 83.2%). There were 20 studies with matched pairs design, including tumor and tumor-adjacent normal tissue pairs from 4116 gastric cancer patients. The pooled ORs were 18.56  for studies with matched pairs design and 3.31  for studies with non-matched pairs design. The proportion of EBV-associated gastric cancer among male cases was significantly higher than among female cases  (P < 0.0001). However, the pooled OR estimate for EBV-associated gastric cancer was significantly higher among females  than in males  (P = 0.06). EBV was more prevalent in the cardia (12.47%) and the body (11.68%) compared to the antrum (6.29%) (P = 0.0002).The pooled prevalence of EBV in 20,361 gastric cancer patients was 8.77% (95% CI: 7.73–9.92%; IEBV infection is associated with more than 18 times increase the risk of gastric cancer. Although the prevalence of EBV was higher in male patients than in female patients with gastric cancer, women are more likely than men to develop EBV-associated gastric cancer. Our findings showed that using tumor-adjacent normal tissues as the control group provides more robust and accurate results regarding the relationship between EBV infection and gastric cancer. According to GLOBOCAN statistics in 2018, gastric cancer is the fifth most frequently diagnosed cancer and the third leading cause of cancer-related mortality in the world accounted for 8.2% of all cancer deaths. Over 1,000,000 new cases of gastric cancer diagnosed in 2018 around the world, with an estimated 783,000 deaths [Herpesviridae family, is the first described human cancer virus and is responsible for approximately 1.8% of all human cancers, including Hodgkin lymphoma, Burkitt lymphoma, NK/T cell lymphoma, and nasopharyngeal carcinoma [Epstein-Barr virus (EBV), as a member of the arcinoma . Howeverarcinoma , and aftarcinoma . SubsequTo date, the mechanisms of EBV-associated gastric cancer are still not comprehensively clarified. Generally, virologic aspects, in conjunction with host genome abnormalities, co-potentiate the cancer progression. Regarding the virologic background, the EBV genome encodes oncoproteins, which target important cellular pathways. EBV-associated gastric cancer belongs to latency type I infection, in which only EBNA1, EBER, BamHI A rightward transcript (BART), and BART miRNAs are highly expressed, while the latent membrane protein 2A (LMP2A) can be detected in 40% of cases . Evidenc7 copies per cell) [The gold standard for the diagnosis of EBV infection in histopathologic samples is ISH, which detects EBV-encoded small RNA-1 (EBER1). EBER1 is highly expressed in latently EBV-infected cells up to 10er cell) . EBER1 ser cell) . PCR-baser cell .er cell) .There are several published meta-analyses addressing the prevalence of EBV among gastric cancer patients –16, howeThe present systematic review and meta-analysis was performed according to the recommendations of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement .A rigorous literature search was conducted using PubMed, Web of Science, Scopus, EMBASE, and Google scholar to identify all published articles reporting the prevalence of EBV in patients with gastric cancer. Databases were searched from inception to July 1, 2019. The bibliographies of all articles obtained were also reviewed for additional relevant publications. The list of keywords used for this systematic review and meta-analysis is provided in Additional file All records were imported to EndNote software version X8 , and duplicate entries were removed. The screening of the title and abstract of the remaining records was independently conducted by two researchers. The full-texts of the remaining records were then retrieved and reviewed, and any disagreements were resolved through discussion by a third investigator.Studies were considered eligible for inclusion in the present meta-analysis, if they met the following criteria: (1) Studies using cross-sectional and case-control designs reporting the prevalence of EBV infection in patients with different types of gastric carcinoma; (2) Studies using EBER-ISH technique to detect the presence of EBV transcripts or nucleic acids; (3) Studies using the formalin-fixed paraffin-embedded (FFPE) tissues and biopsies samples; (4) Studies published in peer-reviewed journals in the English language.Studies with following characteristics were excluded from the present meta-analysis: (1) Studies using serological techniques such as enzyme-linked immunosorbent assay (ELISA) to detect circulating antibodies to EBV infection; (2) Studies evaluating the presence of EBV in serum, plasma or peripheral blood mononuclear cell (PBMC) samples; (3) Studies assessing the presence of EBV in gastric carcinoma patients with underlying disorders; (4) Studies evaluating the presence of EBV by molecular methods such as PCR, nested-PCR and Real-Time PCR; (5) Studies addressing remnant gastric cancer, gastric lymphoma, and other types of gastric malignancies; (6) Studies using techniques other than EBER-ISH, (7) Studies published in languages other than English; (8) Reviews, letters to the editor, abstracts, and case reports.Two investigators independently extracted data from all eligible studies in a pre-designed data extraction form using Microsoft Excel 2013 . The two investigators cross-checked each other’s data extraction, and any disagreements were resolved by a third investigator. After retrieving the eligible articles, a modified checklist based on the guidelines of the strengthening the reporting of observational studies in epidemiology (STROBE) was used for assessing the risk of bias of the included studies , 20. TheThe present meta-analysis had two primary purposes; first, providing an updated estimate of the pooled prevalence of EBV among patients with gastric cancer, and secondly, investigating the association between EBV and the development of gastric cancer. A random-effect meta-analysis using the inverse variance method was applied to estimate the pooled prevalence of EBV (DerSimonian-Laird method) . The log2 statistics [P values less than 0.05 were considered statistically significant. Furthermore, for each case-control study with matched pairs design, we separately computed matched-pairs OR and its corresponding variance using the “escalc” function in the R “metafor” package [To evaluate the strength of the association between EBV infection and gastric cancer risk, the pooled odds ratios (ORs) with 95% CIs were generated from a random-effects model based on the DerSimonian-Laird method. For studies with a zero cell, a continuity correction of 0.5 was applied. We also conducted subgroup analyses to identify the possible sources of heterogeneity. The heterogeneity among the studies was assessed through Iatistics . To explatistics . All theatistics , 26, and package  , and only one paper  was failn = 15).Table 2 = 83.2%). The highest and lowest prevalence of EBV were found in gastric cancer patients from Poland and the United Kingdom, respectively . The proportion of EBV-positive gastric cancer among male cases was significantly higher than among female cases  (P < 0.0001) , whereas the pooled OR for studies with matched pairs design was 18.56 , indicating a solid significant positive relationship between EBV infection and gastric cancer .Among 30 case-control studies, 20 had matched pairs design, including tumor and tumor-adjacent normal tissue pairs from 4116 gastric cancer patients. The remaining ten non-matched case-control studies included 911 cases of gastric cancer and 436 controls. Using data obtained from studies with non-matched pairs design, the pooled OR of EBV infection was 3.31 . We chose strict inclusion and exclusion criteria to obtain pertinent studies and to increase the chance of finding a valid conclusion. The pooled prevalence and OR obtained in this meta-analysis were calculated from studies that detected EBV infection with the ISH method. All studies that investigated the presence of EBV by other methods, including different types of PCR assays, and even immunohistochemistry (IHC), did not consider in our analysis. The reason for this stems from the fact that the sensitivity and specificity of each detection method are different, and it is not reliable to draw a conclusion using the pooled data.Our meta-analysis showed that the pooled prevalence of EBV among gastric cancer patients from 26 countries is 8.77%  due to its high sensitivity and specificity to determine the precise intranuclear localization of the EBV-infected cells. The diagnosis of EBV-associated gastric cancer is confirmed by the presence of EBER within the tumor cells and its absence in the normal tissue adjacent to the tumor . Many st2 = 55.0%) and 18.56 , respectively. We performed two separate analyses for studies with match pairs and non-match pairs designs to demonstrate that the strength of association (ORs) between EBV infection and gastric cancer is the highest when tumor-adjacent normal tissues are used as a control group. This is due to the fact that confounding variables are eliminated from data analysis. Therefore, we can obtain more accurate and robust estimates of the association between EBV and gastric cancer. This finding of our study will be beneficial for researchers to design their future case-control studies appropriately. Using the tumor-adjacent normal tissues as the control group will provide more accurate results regarding the relationship between EBV infection and gastric cancer.One of the major strong points in this meta-analysis is that the pooled estimates of ORs were calculated from studies with matched pairs and non-matched pairs designs, separately, with different statistical methods. The detailed descriptions about the analysis of data for matched pairs and non-matched pairs studies are available in several previous studies . It has To date, several studies have attempted to discover the role of EBV infection in gastric cancer progression. EBV enters B lymphocytes in oropharyngeal lymphoid tissues. The virus then enters the gastric epithelial cells, either by the cell-to-cell contact between B lymphocytes and gastric epithelial cells or by direct entry into the gastric epithelia . It has P < 0.0001). However, the OR estimate for EBV-associated gastric cancer was significantly higher among females than in males (P = 0.06). According to these results, we concluded that women are more likely than men (1.5-fold) to develop EBV-associated gastric cancer. This novel finding can be explained by different genetic backgrounds, lifestyles, or hormonal conditions between the two genders.Another interesting finding of our meta-analysis is that the prevalence of EBV was 1.9-fold higher in male patients than in female patients with gastric cancer (P = 0.0002)  Table , althougP < 0.0001). It is well documented that there are several challenges when working with FFPE samples, such as the low amount of extracted nucleic acids, and fragmentation of genomes and transcripts during the processes of fixation and embedding in paraffin. Therefore, to prevent false-negative results, using biopsy samples is recommended.One prominent finding of the present meta-analysis is that EBV was detected more frequently in biopsy samples than in FFPE specimens from gastric cancer patients .According to Lauren’s histological classification, gastric carcinoma is classified into two distinct types, namely intestinal and diffuse types. There are many differences between intestinal and diffuse types based on their epidemiology, etiology, and pathology . HoweverSimilarly, our results did not indicate any significant difference in the prevalence of EBV-associated gastric cancer among different geographic regions, even between developed and developing countries. The same prevalence in developed and developing countries demonstrates that economic conditions are not related to EBV-associated gastric cancer risk.Helicobacter pylori infection and gastric cancer. Nevertheless, we did not consider data regarding the co-infection of EBV and Helicobacter pylori.There are some limitations in this study arose from the nature of the data sources used in the meta-analysis. Gastric cancer is a multifactorial disease affected by several risk factors. Age is considered as a risk factor for the development of EBV-associated gastric carcinoma. However, the majority of studies included in the current meta-analysis did not categorize EBV-infected and -uninfected gastric cancer patients based on the age group. Subsequently, we were not able to perform a subgroup analysis in this regard. Besides, there are some reports on the association between To sum up, our meta-analysis suggests that the pooled prevalence of EBV among patients with gastric cancer was 8.77%. To determine the association between EBV infection and gastric cancer, a matched-pairs analysis from case-control studies was performed, and the pooled OR was calculated 18.56. This finding indicates a robust positive association between EBV infection and gastric cancer risk. We recommend using biopsy instead of FFPE samples and the ISH technique instead of PCR methods to ensure the validity of results.Furthermore, the pooled prevalence of EBV was obtained from data from 26 countries in the world. Therefore, conducting studies in other geographical regions is strongly recommended to get more reliable estimates. Furthermore, we suggest that researchers use the tumor-adjacent normal tissues as the control group for their case-control studies to achieve more accurate results regarding the relationship between EBV infection and gastric cancer.Additional file 1.


In patients with left-sided tumours, treatment with chemotherapy plus cetuximab yielded longer OS than chemotherapy plus bevacizumab . mCRC patients with ETS and high DpR showed better OS than those lacking ETS and with low DpR . Moreover, ETS and/or high DpR achieved in patients with right-sided mCRC receiving chemotherapy plus cetuximab were associated with significantly better OS than in those lacking ETS and with low DpR . Taken together, our study demonstrates that primary tumour location is not only a well-known prognostic factor but also a relevant predictive factor in patients with mCRC receiving chemotherapy plus cetuximab. Additionally, both ETS and DpR could predict treatment outcomes and also potentially guide cetuximab treatment even in right-sided mCRCs.The primary tumour location is an important prognostic factor for previously untreated metastatic colorectal cancer (mCRC). However, the predictive efficacies of primary tumour location, early tumour shrinkage (ETS), and depth of response (DpR) on mCRC treatment has not been fully evaluated. This study aimed to investigate the predictive efficacies of these traits in mCRC patients treated with first-line 5-fluorouracil-based chemotherapy plus biologic agents, namely, cetuximab and bevacizumab. This was a retrospective analysis of the medical records of 110 patients with pathology-documented unresectable mCRC. Patients with left-sided mCRC receiving any first-line regimen showed better overall survival (OS) than those with right-sided mCRC [33.3 vs 16.3 months; hazard ratio (HR) 0.44; 95% confidence interval (CI) 0.27–0.74;  However, with advances in chemotherapeutic and biologic agents, diagnosis, and ablative techniques, the survival of patients with unresectable metastatic CRC (mCRC) has improved from 12 to over 30 months2. The standard first-line therapy for mCRC includes 5-fluorouracil plus folic acid combined with irinotecan (FOLFIRI) or oxaliplatin (FOLFOX)3. Further, in previous Phase-III randomised trials, compared to the FOLFIRI or FOLFOX regimen, administration of either an anti-epidermal growth factor receptor (EGFR) monoclonal antibody (cetuximab and panitumumab) or an anti-vascular endothelial growth factor (VEGF) antibody (bevacizumab) showed favourable OS, progression-free survival (PFS), and overall response rate (ORR) in the patients with CRC6. However, when the efficacies of combination therapies of cetuximab or bevacizumab with FOLFIRI or FOLFOX were compared, the outcomes were controversial. The FIRE-3 study randomised mCRC patients with KRAS exon 2 wild-type (wt) status into either FOLFIRI plus cetuximab or bevacizumab groups and the median OS was found to be better upon treatment with FOLFIRI plus cetuximab7. In contrast, Venook et al. in their CALGB/SWOG 80,405 study observed no significant difference in OS upon administration of cetuximab vs bevacizumab compared to the FOLFOX or FOLFIRI regimen for the treatment of patients with KRAS wt, advanced CRC or mCRC8. Therefore, a detailed analysis is required to comprehend the efficacy of either treatment modalities.Colorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide10. Therefore, it is essential to confirm the mutational status of RAS before administering anti-EGFR therapy to patients with mCRC. Furthermore, while the V600E BRAF mutation is a negative prognostic marker for mCRC, its utility in predicting response to anti-EGFR therapy is still uncertain11. The HER2 status may impact the response to anti-EGFR therapies since its activation substitutes EGFR dependence in a subset of mCRC patients, thus serving as a potentially negative predictor of the beneficial effects of anti-EGFR therapy12.Several studies have embarked upon identifying prognostic and predictive factors for targeted therapy using anti-EGFR antibodies in patients with mCRC. The anti-EGFR antibody did not show any clinical benefits for patients with any RAS mutation13, supported by post hoc analyses of large randomised controlled trials and meta-analyses. Holch et al.14 reported that in patients receiving bevacizumab and cetuximab, tumours originating in the right-side were associated with higher mortality than left-sided tumours. Additionally, several studies found the primary tumour location to be predictive of response to anti-EGFR therapy, although only in patients with a left-sided tumour harbouring a RAS wt status17. Furthermore, in the pooled analysis of six randomised trials, mCRC patients with left-sided tumours harbouring RAS wt status showed favourable ORR, OS, and PFS than those with right-sided tumours, and the analyses predicted better response upon treatment with chemotherapy plus anti-EGFR therapy than chemotherapy alone or chemotherapy plus anti-VEGF therapy18. Taken together, these findings suggest that the location of tumours on the left side can help predict the efficacy of anti-EGFR antibodies in mCRC patients with KRAS wt status.In contrast, the primary tumour location has been shown to predict prognosis in patients with previously untreated mCRC20. While ETS offers an early indication of sensitivity to treatment and is associated with improved PFS and OS irrespective of the treatment received, the DpR reveals the maximum tumour shrinkage achieved, which relates to the post-progression survival. However, their clinical relevance has not been firmly established, and there are limited data on these response assessments according to primary tumour location.Recent retrospective analyses of clinical trial data have included early tumour shrinkage (ETS) and depth of response (DpR) as measures for assessing tumour responseThe studies presented above are retrospective analyses of randomised clinical trials, majorly conducted in western nations, and their outcomes cannot be generalised to patients in actual clinics. Therefore, here, we investigated the prognostic and predictive efficacy of primary tumour location and the impact of ETS and/or DpR on therapeutic outcomes in a cohort of patients with mCRC treated with first-line chemotherapy plus bevacizumab or cetuximab in the real-world setting in a Japanese population.KRAS (RAS) status, tumour condition (advance or recurrence), comorbidity (with or without), and primary tumour status (resection or no resection), were comparable in the left-sided and right-sided tumours. However, significant differences were observed in the histological statuses, with 72 of the 76 left-sided tumours classified as adenocarcinoma (94.7%), whereas 10 of the 34 right-sided tumours were mucinous (29.4%). Further, the oxaliplatin-based regimen was the most widely used chemotherapy , although no significant differences were observed between irinotecan-based and oxaliplatin-based regimens for the treatment of left- and right-sided tumours. Additionally, bevacizumab-containing chemotherapy was preferred over cetuximab-containing regimen (60 of 110 (54.5%) vs 50 of 110 (45.5%) patients, respectively). However, no significant differences were noted for the use of biological agents between left- and right-sided tumours. Furthermore, 46 of 60 patients (76.7%) receiving bevacizumab had KRAS mutation.Based on the eligibility criteria, 110 patients with mCRC, who received first-line treatment were included in the study , treatment with cetuximab conferred better ORR in patients with left-sided tumours (77.8%) than in those with right-sided tumours , of which 4 showed complete response (3.6%) and 62 showed partial response (54.6%). Left-sided tumours showed significantly higher ORR than the right-sided tumours  months, respectively.p = 0.035)  , although the difference in PFS was not significant   and median OS  were not significant. , and the OS followed a similar trend . Further, we assessed the PFS and OS based on primary tumour location in patients treated with these agents , although the difference in PFS was not significant  , along with better median OS   Fig. C,D. Furt99) Fig. E, F.p = 0.0023)  vs 15 (44.1%), respectively). Further, patients with left-sided tumours treated with cetuximab showed high ETS than those treated with bevacizumab   in patients with right-sided tumours treated with bevacizumab . On the other hand, the median DpR in patients with right-sided mCRCs receiving cetuximab and bevacizumab was − 9.6% (range − 100 to 45) and − 33% (range − 58 to 3), respectively (p = 0.42)  and − 30.0% (range − 64 to 25), respectively (p < 0.001)   Fig. A. In theHere, we enrolled a cohort of consecutive patients with mCRC in real-world clinical setting, and assessed factors known to affect their prognosis and treatment outcomes, such as primary tumour location, biologic therapy (bevacizumab or cetuximab), and the more recently proposed indicators, EST and DpR.Our findings support the notion that primary tumour location is an important prognostic factor in mCRC and can help guide treatment decisions on the first-line regimen based on the findings that clinical benefits for cetuximab were observed in patients with left-sided mCRC. In addition, ETS (≥ 20% at week 8) and high DpR during treatment with first-line agents were associated with favourable outcomes in patients with mCRC, further supporting the use of these endpoints clinically. Furthermore, our study raises the possibility of a predictive relevance of ETS and DpR in guiding targeted therapy (cetuximab) in patients with right-sided mCRC.18. Further, we observed an association between the primary tumour location and treatment with cetuximab, where patients with left-sided tumours showed better survival outcomes when treated with cetuximab than with bevacizumab  is predominantly distributed in left-sided tumours and shows activated EGFR-dependent signalling defined by the over-expression of EGFR. Although right-sided tumours showed high proportions of the microsatellite unstable/immune CMS1 and the metabolic CMS3 subtypes24, these differences could be among the factors contributing to differential responses to treatment with cetuximab based on tumour location25. However, the main genetic drivers and crucial factors contributing to the vast differences remain largely unknown.Together, these observations suggest that mCRC is a heterogeneous disease with the primary tumours originating from different areas of the colon showing molecular differencesRAS is mutated (RAS mt) in about 55% of the mCRC cases, these patients need to be treated with bevacizumab26. However, the majority of recently published studies with randomised trials have focused on the RAS wt subpopulation alone; hence, data on treatment with bevacizumab for RAS mt mCRC involving the two primary tumour locations are limited28. Moreover, while several clinical studies have suggested that the tumour location does not predict benefit from bevacizumab treatment in patients with mCRC harbouring KRAS wt29 and undetermined RAS status30, other reports indicate better outcomes in left- than right-sided mCRC, with unselected RAS status, treated with bevacizumab30. Our analysis suggests that patients treated with bevacizumab had the RAS mt-mCRC predominantly, and left-sided mCRCs were associated with better PFS and OS than right-sided mCRCs  status of tumours was well balanced between the two cohorts. Finally, we may need to consider that treatment with bevacizumab may be less active in KRAS/RAS mutant mCRC patients, although such data are rare32.Third, a large proportion of the patients treated with bevacizumab were mutant for RAS wt left-sided tumours to provide better OS, while the right-sided tumours presented with poor prognosis with all treatment measures. However, mCRC patients with right-sided tumours harbouring RAS wt status, who received chemotherapy plus cetuximab and achieved ETS and/or high DpR, showed better clinical benefit than those without ETS and low DpR. These results suggest that in patients with RAS wt right-sided mCRCs, bevacizumab might not be the only treatment option, and cetuximab may be considered in a subtype of mCRC, wherein ETS and/or high DpR can be expected.In conclusion, our study supports the notion that the primary tumour location is a well-known prognostic factor and also a relevant predictive factor in patients with mCRCs receiving cetuximab. Further, our analysis indicates that chemotherapy plus cetuximab may serve as the preferred option for the treatment of patients with In this observational study, the medical records of 110 of 226 patients with pathology-documented metastatic Stage IV CRC, who underwent treatment between January 2011 and December 2016 at the Department of Gastroenterology, Hokkaido Cancer Center, Sapporo, Japan, were analysed retrospectively. The study was reviewed and approved by the Ethics Committee of Hokkaido Cancer Centre (No. 29-60), and the need for informed consent was waived. The patient records were anonymised and de-identified before analysis. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institution and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.KRAS wt or RAS wt status was considered in patients treated with cetuximab, while these statuses were neglected when treated with bevacizumab. The investigators evaluated the tumour response using computed tomography based on the guidelines of the response evaluation criteria in solid tumours . The ORR was defined as the sum of complete and partial response, the PFS was measured from the start of chemotherapy until the first documented progression or death, while the OS was measured from the date of commencing chemotherapy until death or the date of last evaluation. Primary tumours originating in the splenic flexure, descending colon, sigmoid colon or rectum were classified as left-sided mCRC, whereas, those originating in the cecum, ascending colon, hepatic flexure or transverse colon were classified as right-sided mCRC. ETS was defined as the relative change in the sum of the longest diameters of the RECIST target lesions from baseline till the 8th Week, and a 20% decrease was set as the cut-off value to discriminate early responders (ETS ( +)) from non-responders (ETS (−)) as described previously33.The inclusion criteria were as follows: (a) treatment with either FOLFOX or FOLFIRI plus cetuximab or bevacizumab; (b) patients above 20 years of age at the start of chemotherapy, with an Eastern Cooperative Oncology Group performance status of 0–1; and (c) adequate bone marrow reserves and renal/liver functions. Further, the 20. DpR was evaluated as a continuous variable . DpR had a negative value for tumour reduction, positive for tumour growth, and zero for no change. These median values were set as cut-off points. The last follow-up date was August 25, 2020.DpR was defined as the percentage change in the sum of the longest diameters of the RECIST target lesions at the nadir in the absence of new lesions or the progression of non-target lesions compared to the baselineThe Kaplan–Meier method was used to estimate PFS and OS, and these were compared with the log-rank test. A Cox proportional hazard model was used to calculate the hazard ratios (HR) and 95% confidence intervals (CI) for PFS and OS regarding the interaction between tumour origin (right or left), treatment (chemotherapy plus cetuximab or chemotherapy plus bevacizumab), ETS, and DpR as explanatory variables.p value < 0.05 was indicative of notable differences and considered statistically significant.Fisher’s exact test was used to analyse categorical variables of patient characteristics in 2 × 2 contingency tables. All statistical analyses were performed using Ekuseru–Toukei 2015 . Although these were post hoc analyses, a two-sided Supplementary Informations.Supplementary Legends.


Klebsiella pneumoniae is an emerging pathogen, which has become disseminated worldwide in humans but is rarely reported from other reservoirs. We report the first isolation of K. pneumoniae from an animal in Europe and also from a reptile, a captive tortoise, whose death it probably caused. Detection of this clone from an animal adds to evidence of niche expansion in non-human environments, where it may amplify, recycle and become of greater public health concern.The ST307 multidrug-resistant CTX-M-15-producing  Klebsiella pneumoniae ST307 has emerged globally in humans, being associated with sepsis, pneumonia and urinary tract infections caused by extended-spectrum β-lactamase (ESBL)- and carbapenemase-producing K. pneumoniae that can be multi- or extensively drug-resistant, and hence difficult to treat [K. pneumoniae ST307 is rare but the animal niche may be expanding. Previously, it has been identified from a dog in Brazil [Enterobacteriaceae [K. pneumoniae ST307 in a captive African spurred tortoise (Centrochelys sulcata) in a zoological park in the UK.The CTX-M-15-producing to treat . Amongstn Brazil  and from−1 injectable 2.5 % enrofloxacin every second day from 29 April and were tube-fed. They were kept in separate hospital tortoise boxes, enclosed indoors with separate heating and UVb lighting, medicated individually, given warm water baths daily and tube-fed if not eating.Four tortoises had been captive in a zoological park following seizure by customs upon entering the UK in 2013. They were kept in an enclosure that is separate from other animals in the zoo and there had been no previous apparent illness or treatment of any of the four tortoises during their time at the zoo. Keepers always wore disposable gloves for handling the tortoises and they were not handled by members of the public. Two of the tortoises had been under treatment for lethargy, inappetence and respiratory signs. Both were treated with 5 mg kgK. pneumoniae and a mixed fungal growth of Geotrichium candidum and Penicillium sp. were obtained.One of the sick tortoises died 28 May 2019 and was subject to a post-mortem examination at SRUC Veterinary Services Edinburgh within 24 h. Gross pathology diagnosis was pneumonia, and lung tissue was selected for culture on Columbia sheep blood agar, MacConkey agar without salt and Sabouraud dextrose agar with chloramphenicol . A confluent growth of K. pneumoniae isolated from lung tissue and whole-genome sequencing (WGS) was performed using Illumina NextSeq for detailed molecular characterization; the WGS results were submitted to ENA under PRJEB36913. Species classification by Kraken [K. pneumoniae and in silico multilocus sequence typing [K. pneumoniae ST307 isolates [https://github.com/katholt/Kleborate) was used to identify presence of known virulence factors associated with hypervirulence and invasive disease in human infections [DNA was extracted from the e typing  indicateisolates . Isolatefections . Only thfections  and the K. pneumoniae isolate, which was performed by disc diffusion according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) methodology and breakpoints, and confirmed by genotypes predicted from WGS using the APHA SeqFinder pipeline [blaCTX-M-15; for beta-lactamases it was blaTEM-1b, blaOXA-1; for gentamicin it was aac3-IIa; for ciprofloxacin/enrofloxacin it was qnrB1; for tetracycline it was tet(A); for sulphamethoxazole/trimethoprim they were sul2, dfrA14; and for chloramphenicol it was catB4. The AMR profile of the tortoise isolate was the same as that for a Norwegian human isolate (Norway-2014b_KP_NORM_BLD_2014_101814) that was present within the same sub-cluster, but different from those of the two human UK isolates also in this sub-cluster, which harboured variants of carbapenem resistance genes  [Klebsiella pneumoniae ST307, which may also harbour carbapenemase resistance, are to be avoided in future, whether in human or other environments.Although the group of tortoises in the premises in this study were not handled by the public, other zoos do encourage interactions with tortoises. A recent study of animals in petting zoos in Israel recovered ESBL-producing