Furthermore, hypothesis test using a computational experiment reinforced our conclusion. Taken together, we suggest that bioinformatics analyses with peripheral blood RNA-seq could unveil the cause of insidious late islet graft loss.Clinical islet transplantation has recently been a promising treatment option for intractable type 1 diabetes patients. Although early graft loss has been well studied and controlled, the mechanisms of late graft loss largely remains obscure. Since long-term islet graft survival had not been achieved in islet xenotransplantation, it has been impossible to explore the mechanism of late islet graft loss. Fortunately, recent advances where consistent long-term survival (≥6 months) of adult porcine islet grafts was achieved in five independent, diabetic nonhuman primates (NHPs) enabled us to investigate on the late graft loss. Regardless of the conventional immune monitoring methods applied in the post-transplant period, the initiation of late graft loss could rarely be detected before the overt graft loss observed via uncontrolled blood glucose level. Thus, we retrospectively analyzed the gene expression profiles in 2 rhesus monkey recipients using peripheral blood RNA-sequencing (RNA-seq) data to find out the potential cause(s) of late graft loss. Bioinformatic analyses showed that highly relevant immunological pathways were activated in the animal which experienced late graft failure. Further connectivity analyses revealed that the activation of T cell signaling pathways was the most prominent, suggesting that T cell-mediated graft rejection could be the cause of the late-phase islet loss. Indeed, the porcine islets in the biopsied monkey liver samples were heavily infiltrated with CD3 However, over half of the patients transplanted with human islets returned to the insulin-dependent, diabetic state within 5 years4. The causes for this late islet graft loss are still controversial. Previous reports encompass a higher rate of islet apoptosis due to endoplasmic reticulum (ER) stress6, hypoxia8 in end-portal venules within the liver, and recurrent autoimmunity9. In addition, there were evidences of metabolic deterioration due to lipid accumulated around the islets (lipotoxicity)11 and toxicity of immunosuppressive drugs13, which could all result in graft loss. Last but not least, insufficient immunosuppression could also be an important cause of chronic islet loss especially due to antibody-mediated rejection processes14. However, none of the above could clearly and single-handedly explain the exact causes of chronic islet graft loss.The Edmonton protocol was introduced in 200015. This unique opportunity allowed us to examine how the porcine islets are lost in the late phase of islet xenotransplantation. Here, we selected two monkeys with the same immunosuppressive regimen to analyze the cause of late graft loss in islet xenotransplantation: one (R051) had stable graft function for the entire follow-up periods and the other (R080) lost graft function around 160 days post-transplantation (DPT). Peripheral blood RNA-seq and subsequent bioinformatics analyses using Time-series RNA-seq analysis package (TRAP)16 hinted on the possibility of immune rejection in R080. Further in silico analyses focused on the interactions of graft loss period-related activated pathways (GLPAPs) proposed that lymphocytes- or platelet-mediated rejection might have been the cause of late graft loss.Recently, we reported consistent long-term (≥6 months) porcine islet graft survivals in five independent monkeys15). However, vital signs and routine laboratory examinations including complete blood cell count (CBC), liver function test (LFT), C-reactive protein (CRP), kidney function test (blood urine nitrogen/creatinine), electrolyte panel , lipase and amylase had shown no abnormal findings in both of the monkeys (data not shown). Also, monitoring of peripheral blood lymphocyte subsets by flow cytometry and ELISPOT  exhibited relatively early hyperglycemia around DPT160, suggesting a graft failure. Intravenous glucose tolerance test (IVGTT) had shown that the porcine islet graft loss in R080 was in progress between DPT120 and 180  select up-regulated pathways with p-value under 0.05 from the results yielded by TRAP comparing t3 and t2 in R080, ii) select up-regulated pathways with p-value under 0.05 from the results yielded by comparing R080 and R051 at t3 and then take the pathways belonging to the intersection of those sets  profile was established. Then, a protein-protein interaction (PPI) network was mapped with the seed genes and the global effects were measured using network propagation. After the genes were ranked under each hypothesis, Pearson’s correlation analysis was performed between the DEG profile ranks and the ranks calculated by the network propagation. This was to test which of the five hypotheses represented by the seed genes produced the gene expression profile similar to the DEG profile we measured. In other words, we tried to see how much the participation of genes in the actual biological process that had induced the graft loss coincided with the perturbation in the expression of genes in the given condition. Furthermore, we performed random simulations for 1000 times and calculated empirical p-values to test the significance of the coefficient as shown in Methods. The result is shown in Table To reconfirm our findings, we sought to computationally test each hypothesis which could explain the islet graft loss. We selected five hypotheses that are known to cause the late graft loss31. Although the engraftment of islets has been successful short-term, it relatively lacked long-term durability, resulting in late-phase graft failure in some islet transplant recipients2. Likewise, we and others have found that the porcine islet grafts were also lost in the transplant recipient monkeys within 6~30 months after transplantation in pig-to-NHP islet xenotransplantation32. Luckily, we were able to experience consistent, prolonged graft survival in rhesus monkeys15, and thus this retrospective analysis was performed in hopes to unearth the cause of late graft loss in pig-to-NHP islet xenotransplantation. To our knowledge, there have not been studies that were focused on the cause of late graft loss nor the mechanism of immune rejection in islet transplantation models of higher mammals.Pancreatic islet transplantation is currently one of the best treatment options for end-stage type 1 diabetes patientsBecause we were not able to find any definitive explanation of islet loss with routine laboratory tests including biochemical and immunological assays in peripheral blood from R080, we used RNA-seq technology to quantify the amount of transcript in the samples obtained from whole blood taken at various time points after transplantation. Then, we carried out pathway analysis and pathway interaction network analysis based on TRAP and PINTnet, respectively. By performing pathway analyses, we found 59 activated pathways and named those GLPAPs Table . Further29. There also have been reports indicative of platelet’s contribution in the chronic rejection of transplanted organs33. T cells have been mentioned very little regarding the chronic rejection of transplanted organs, but our results might have discovered that T cells may partake significantly in the chronic rejection of transplanted islets, at least in pig-to-NHP settings.Computationally, the above findings were reinforced by hypothesis testing. We can measure the influence of some nodes of interests to other nodes on a network using the network propagation. Likewise, we can measure the influence of genes on a biological network or a gene regulatory network. If we map genes on a curated biological network, select some genes as seed genes and run network propagation, we can measure the influence of the seed genes to other genes and rank the genes by the influence they received. Top 100 genes, for example, are the most influenced 100 genes. In our study, network propagation and subsequent correlation analysis revealed that chronic rejection-related genes had been the most related to the late graft loss Table . It is nIt would be of dire importance to show that the RNA-seq bioinformatics data truly represented the immunobiology of the recipient which experienced late graft loss. We retrieved the archives of liver biopsy materials of R080, and performed IHC that could prove the pertinence of the three GLPAPs  to the rejection. Accordingly, we could observe the infiltration of T cells to the graft site as well as antibody and fibrinogen accumulation Fig. . Since t34, and it was reported by Xie et al. that microbiota alteration could result in acute rejection of rat liver allografts35. Because intestinal blood is drained to the liver via portal vein, we hypothesized that intestinal infection might have activated strong innate immunity and this in turn triggered adaptive immune response to the graft through heterologous immunity. In line with this notion, the immunologically hostile effects of infection on the transplanted allografts were reported36 and infection even might break down established tolerance to the graft in murine heart transplantation models37.It is intriguing that only R080 experience immune system activation compared with R051 despite the usage of the same immunosuppressive regimen. To find out putative reason(s), we carefully reviewed pre-clinical symptoms, signs and laboratory data. Interestingly, we noticed that R080 had experienced two times of severe giardiasis-induced diarrhea around DPT90 and DPT120. This finding indicated that intestinal infections preceded the sampling for the RNA-seq about one month. There is increasing evidence that microbiota products could activate the innate immune system of the liverThere are some limitations in our work. First, we only had one monkey which experienced relative early graft loss within the chronic phase after transplantation. Thus, our study cannot give a definite conclusion, but rather an intriguing insight to our field. We are planning to scale up our study using more animals to validate our bioinformatic analysis method. Second, although we suggested that three pathways might have been involved in immune rejection in the chronic phase, we only found the presence of the effectors in each pathway and could not present their actual involvement. Third, because rhesus pathways in KEGG were relatively insufficient, we were not able to analyze our data in high-resolution maps. For example, even though we were interested in CD40L or IL-6 signaling pathways in our setting, we were not able to analyze them because KEGG did not support those pathways. Lastly, we were not able to pinpoint single candidate molecule or a set of molecules which could be critically responsible for graft rejection. Our next works will focus on these questions.Macaca mulatta), 3–4 years of age were used in our study. All animal experiment procedures were performed in compliance with the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health . The experiments were approved by Seoul National University (SNU) Institutional Animal Care and Use Committee (IACUC no. 15-0297-S1A0). Islet donor pigs, the Seoul National University (SNU) miniature pigs were bred as in designated pathogen-free (DPF) grade38.Rhesus monkeys  was administered subcutaneously 2~3 hrs before islet infusion with dose of 5 mg/kg. 106 to 107ex-vivo expanded regulatory T cells were adoptively transferred after ATG depletion.Induction immunosuppression included a regimen with anti-human CD154 monoclonal antibody, sirolimus , and anti-thymocyte globulin . Sirolimus was administered daily to achieve stable trough levels between 3 and 8 ng/ml. CVF  was administered on day -1 of the transplant to prevent complement activation. TNF-α neutralizing monoclonal antibody, adalimumab . The resulting spots were counted on a computer-assisted ELISpot Reader System .ELISPOT analysis was performed by the method previously describedFlow cytometry analyses of peripheral blood leukocytes were performed using the following monoclonal antibodies: CD3-FITC , CD4-APC-Cy7 , CD8-PE-Cy7 , FoxP3-PE , HLA-DR-PerCP-eFluor710 , CD20-PE , CD14-Alexa488 , CD16-APC . Absolute counts of leukocytes were measured with 123count eBeadsTM (Thermo Fisher Scientific). FACSCanto II flow cytometer  and FACSDiva software (BD Biosciences) were used for analyses.TM-Blood kit (Thermo Fisher Scientific) as recommended by the manufacturer. 500 μl of peripheral blood from the recipient monkeys was used for each sample. Eluted total RNA was stored in −80 °C. Next, RNA sequencing was performed. Purified total RNA was sequenced by Theragen Etex (Korea).Total RNA from peripheral blood of rhesus monkey was extracted using the Ambion Ribopure15. Briefly, liver biopsy samples from the recipient monkeys were fixed in 10% neutral buffered formalin, and embedded in paraffin. Paraffin-embedded tissues were 4-μm sectioned using a microtome. Each de-paraffinized and hydrated section was incubated for 30 min with primary antibody cocktails for insulin , CD3 , CD4 (Santa cruz Biotechnologies), CD8, CD20, and CD68 , and then washed four times in TBST. After staining procedure, all of the stained slides were dried at 60 °C, and mounted with aqueous mounting medium (Thermo Fisher Scientific). For immunofluorescence staining, anti-fibrinogen antibody and anti-insulin antibody (both from Abcam) were used as primary antibodies and, Alexa 488-conjugated anti-rabbit IgG and Alexa 647-conjugated anti-mouse IgG were used as secondary antibodies, respectively. FITC-conjugated anti-monkey IgG  was used to detect antibody involvement at the graft site. The stained sample was observed by Carl Zeiss Axio Imager A1 microscope and images were taken with a micrograph with AxioVision software .Biopsy and immunohistochemical staining was conducted as described previously16 and pathway interaction network20 were performed as described previously. Network propagation was performed as follows: The genes related to the five hypotheses were collected as seed genes by the literature search and domain knowledge, and each hypothesis was represented by a set of genes. Next, DEG profile was established by measuring the expression change of each gene with calculating the log2 fold change between R080 and R051 at time point 3 and ranking the genes. At that time, we removed the genes of which the expression value was smaller than 1 in either R080 or R051 to prevent extremely high or low fold change yielded by the comparison between small numbers. Then, we mapped the seed genes on a PPI network. The number of nodes and edges in the network are 6,780 and 117,963 respectively. The number of seed genes are ten, nine, eight, ten, and nine for ER stress, islet exhaustion, lipotoxicity, chronic graft rejection, and toxicity of immunosuppressant, respectively. After that, the global effect of the seed genes was measured using network propagation and the genes in the PPI network were ranked for each hypothesis. Then, Pearson’s correlation coefficients were calculated between the ranking in the DEG profile and the ranking by the network propagation for each hypothesis. One thousand random simulations were run and empirical p-values were calculated to test the significance of the coefficient, as shown in the equation below:i indicates each hypothesis and it ranges from 1 to 5. pi indicates the empirical p-value of i-th hypothesis. N is the number of random simulation and it is 1,000. j indicates the j-th random simulation. cij is the coefficient of j-th random simulation of i-th hypothesis. i-th hypothesis.TRAPSupplementary Information


Coronary artery ectasia (CAE) can present as an acute coronary syndrome (ACS) with a high clot burden in ectatic coronary arteries. Thrombectomy with intracoronary thrombolysis often does not ensure immediate blood flow. Also, there have not been clear guidelines regarding long-term management in such cases.A 40-year-old male presented with anginal chest discomfort and a working diagnosis of non-ST elevation myocardial infarction (NSTEMI) was made. The initial angiography showed thrombotic occlusion of several large and ectatic coronary arteries with visibly swirling blood flow. The culprit lesions were treated with balloon angioplasty and multiple rounds of thrombectomy yielding red thrombi. Interestingly, the post-intervention antegrade flow decreased in both vessels  score: 0), possibly because of the distal migration of the clots. Peri-procedure, the patient received two boluses of eptifibatide, 180 mcg/kg each, followed by a continuous infusion of 2 mcg/kg/minute for 18 hours. Afterward, the patient was started on ticagrelor and continued on daily aspirin, high-intensity statin, beta blocker, and Coumadin® with heparin bridge. During the one year follow-up period, the Coumadin was switched to rivaroxaban, ticagrelor was stopped after six months, and the patient was continued on guideline-directed medical therapy (GDMT) for coronary artery disease (CAD) with favorable outcomes.The presented case gives us an insight into not only the intra-procedural but also the post-procedural management of ACS in the setting of CAE, and that is thrombectomy alone followed by longer duration oral anticoagulation in addition to GDMT for CAD. However, it will be interesting to see future studies aimed toward defining the duration as well as the choice of anticoagulation, i.e., dual antiplatelet therapy (DAPT) alone or in combination with warfarin/novel oral anticoagulants (NOACs). Coronary artery ectasia (CAE) often presents in the form of an acute coronary syndrome (ACS) due to slow flow leading to thrombus formation in ectatic coronary arteries, and getting rid of this high thrombus burden during the percutaneous coronary intervention (PCI) can be a challenging task. For this purpose, thrombectomy with intracoronary thrombolysis has been utilized for restoring the blood flow. However, there have been cases showing migration of the clots into distal coronary vessels during thrombectomy attempts, making it difficult to establish immediate flow in all segments. Such cases were managed with extended duration oral anticoagulation, and thrombus clearance was demonstrated with serial follow-up angiographies. We hereby describe a case of diffuse CAE presented as ACS. The high clot burden was successfully dealt with using thrombectomy and a glycoprotein IIb/IIIa inhibitor followed by extended duration oral anticoagulation, thus avoiding intracoronary thrombolysis and negating the need for follow-up angiographies.A 40-year-old male with a past medical history of hypertension and a family history of premature myocardial infarctions (MIs) in a number of first-degree relatives came to the emergency department (ED) with chest pain of two hours’ duration. The patient described it as sudden onset retrosternal pressure which was constant, non-progressive, 10/10, non-radiating, and without any aggravating or alleviating factors. Symptoms started at rest and were associated with mild shortness of breath, left arm heaviness, vomiting, and a syncopal episode. The patient reported that his mother experienced myocardial infarction at 38 years of age and two of his maternal uncles and three first cousins died of myocardial infarction in their 40s. Enroute to the ED, the patient received aspirin (162 mg) and sublingual nitroglycerin with minimal improvement. Vital signs were remarkable for a heart rate of 55 beats/minute and normal blood pressure, respiratory rate, and oxygen saturation. Physical examination revealed normal heart sounds and clear lungs. The initial electrocardiogram (ECG) showed sinus bradycardia with a first-degree atrioventricular (AV) block but without any ST-T wave changes. The initial troponin-T was negative and a total creatine kinase (CK) was 248. The patient received Plavix (600 mg), atorvastatin (80 mg), morphine for pain, and nitroglycerin and heparin infusions for presumed unstable angina. Beta-blocker was not given due to bradycardia. A subsequent ECG four hours later showed prominent Q-waves in the inferior leads and the troponin-T and CK rose to 0.2 and 624, respectively. Interventional Cardiology was consulted and the patient was taken to the catheterization lab for further management of the non-ST elevation myocardial infarction (NSTEMI). The coronary vessels on initial angiography were large and ectatic with visibly swirling blood flow Figures -2. ThereThe patient received eptifibatide (180 mcg/kg double bolus) immediately before the initiation of PCI, followed by a continuous infusion of 2 mcg/kg/minute. The infusion was continued for 18 hours after which the patient was started on ticagrelor, 90 mg orally twice a day (maintenance dose), and continued on daily aspirin, high-intensity statin, a beta blocker, and Coumadin bridged with heparin. Echocardiography done on the following day showed basal lateral and basal-mid inferolateral wall akinesis and an estimated ejection fraction of 55.0%. Owing to a personal and family history of premature MI, the patient underwent extensive rheumatologic workup which included complement levels (C3 and C4), anti-myeloperoxidase antibody, anti-proteinase-3 antibody, anti-dsDNA-antibody, and anti-Smith antibody, but all results were unremarkable. Interestingly, the patient had an elevated antinuclear antibody (ANA) and a low positive Scl-70 antibody titer, but a final diagnosis of scleroderma or any other connective tissue disorder was not entertained given the absence of suggestive clinical signs and symptoms. The hypercoagulability workup was kept limited to JAK2 kinase mutation analysis, Factor-V Leiden, and prothrombin gene mutational analysis as the patient had received anticoagulants, as well as antithrombotics, in the acute setting. The patient also underwent MRA (magnetic resonance angiography) of the whole body which failed to show any aneurysmal dilation of vasculature elsewhere. The hospital course remained uneventful and the patient was discharged on aspirin, ticagrelor, and Coumadin after achieving therapeutic INR . During the one year follow-up period, the Coumadin was switched to rivaroxaban, ticagrelor was stopped after six months, and the patient was continued on guideline-directed medical therapy (GDMT) for coronary artery disease (CAD) with favorable outcomes. The patient has been playing full-court basketball games without any further complaints or hospitalization.CAE is defined as the local or diffuse dilation of coronary arteries ≥ 1.5 times the diameter of the adjacent normal segment of the same vessel or the diameter of the patient’s largest coronary artery . CAE is The case we present had a high thrombus burden during PCI with a very low post-thrombectomy TIMI flow due to possible embolization of the thrombi into distal coronary segments. However, based upon the utility of longer duration oral anticoagulation (as described in the cited studies) in clearing the thrombi, as well as in reducing the occurrence of MACE, we opted to start our patient on extended duration oral anticoagulation rather than using intracoronary thrombolysis and multiple follow-up angiograms. The one-year follow-up results in our case were favorable. Thus, it can be suggested to remove the maximum clot burden with mechanical thrombectomy alone and provide secondary prophylaxis with oral anticoagulation in addition to GDMT for CAD. Since there are no current guidelines regarding the best management of this disease, the future studies may address the duration as well as the choice of anticoagulation/antithrombin therapy.


There is a need to develop sustainable emergency health care systems in low-resource settings, but data that analyses emergency health care needs in these settings are scarce. We aimed at assessing presenting complaints (PCs) and post-discharge mortality in a large emergency department population in Nepal.Characteristics of adult patients who entered the emergency department (ED) in a hospital in Nepal were prospectively recorded in the local emergency registry from September 2013 until December 2016. To assess post-ED mortality, patient households were followed-up by telephone interviews at 90 days.In 21892 included adults, the major PC categories were injuries (29%), abdominal complaints (23%), and infections (16%). Median age was 40 years and sex distribution was balanced. Among 3793 patients followed at 90 days, 8% had died. For respiratory and cardiovascular PCs, 90-day mortality were 25% and 23%. The highest mortality was in individuals with known chronic lung disease, in this group 32% had died by 90 days of ED discharge, regardless of PC. In women, illiteracy compared to literacy (adjusted odds ratio (aOR) = 7.0, 95% confidence interval (CI) = 2.1-23.6) and being both exposed to tobacco-smoking and traditional cooking stove compared to no smoke  were associated with mortality. The mortality was much higher among family-initiated discharged patients  compared to doctor-initiated discharged (3%).Our report suggests that nearly one in ten patients seeking emergency health care died within 90 days. This finding is alarming and novel. Post-discharge studies need to be replicated and appropriate follow-up programs in low-resource settings where primary health care is underdeveloped are urgently needed. The Disease Control Priorities project has estimated that almost half of the deaths and over a third of the disabilities in low- and middle-income countries (LMICs) could be addressed through effective emergency care . Top priIn Nepal, emergency care systems are underdeveloped ,9. PatieTo add knowledge in this area, we aimed to describe 1) characteristics of adult ED patients across presenting complaints (PCs) in a hospital in Nepal; 2) mortality until 90 days after presentation and assess factors that are associated with mortality in this population. We took advantage of increasing access to mobile phones in Nepal and follow-up information was assessed by telephone interviews.A prospective observational study was conducted in the ED of a non-government university hospital with 375 beds . The hosDemographic and clinical information was prospectively registered in systematic emergency forms by ED nurses, paramedics and doctors, and was extracted into an electronic database by a research nurse  or urban . Ethnicity was categorized into four groups recognized by Nepali authorities; Brahmin and Chhetri, Janajati, Dalit, and others. Brahmin and Chhetri are generally considered as a group having a higher socioeconomic status and Dalit typically have a lower socioeconomic status .Time of presentation at ED was categorized into; daytime (08-16 weekdays) and after working hours (16-08) or holidays. ED disposition was categorized into; hospitalized, non-hospitalized or dead in the ED. Hospitalizations were further categorized: admitted to general wards, directly transferred to ICU (Intensive Care Unit) or OT (Operating Theatre), or referred to other hospitals from ED. Non-hospitalized patients were categorized into; doctor-initiated discharge or family-initiated discharge (FID).The presenting complaints from the emergency forms were translated into “International Classification of Primary Care-2 (ICPC-2)” codes , and claAt ED disposition all patients were asked for consent to be telephone-interviewed at 90 days after initial presentation to the ED, and trained research nurses called and interviewed the patient or a family member  and unadjusted odds ratios (ORs) with 95% confidence intervals (CIs) are presented. Data analyses were performed using STATA 13.1 .The study was approved by the institutional ethical review committee of Kathmandu University School of Medical Sciences in Nepal (58/13) and the Regional Committee for Medical and Health Research Ethics in South East Norway (2014/1246). As this study is based on routinely collected pseudo anonymized patient information in the hospital, informed consent from the patients was not obtained in individuals, as approved by the local ethical committee. Verbal consent was taken for information on telephone numbers, and at the beginning of the telephone interviews.During the study period, 33435 patients were enrolled. In total 21892 patients were included in the analysis  of the patients were hospitalized . Falls from heights were the most common cause of injury (36%) followed by traffic injuries (28%)  were men  (Table).Self-harm was the main PC for 381 patients (panel A and panel B).Of the 21892 included patients, a total of 12540 household phone numbers were recorded in the patient registry. Of these 12540 patients, 4331 households  participated in the structured telephone interview at 90 days . No such association was found in men (interaction P < 0.001). In women, the association between exposure to smoke and 90-day mortality was strong: aORs for mortality in women exposed to traditional cooking stoves, tobacco smoking, and tobacco plus traditional cooking stove compared to no smoking exposure were 1.2 (95% CI = 0.7-1.9), 1.8 (95% CI = 1.0-3.2) and 2.8 (95% CI = 1.6-4.9) respectively.We assessed evidence of interactions by sex for associations between demographic factors and 90-day mortality. Thus, results for associations between demographic factors and 90-day mortality are presented separately for men and women. In women, literacy was strongly associated with 90-day mortality  compared to literate group  than reported in other studies from Cambodia 60%)  and Paki0%  and PThe rate of FID was high (10%). A study from India found this proportion to be (4%)  and in tRoad traffic injuries in Sub-Saharan Africa and Southeast Asia have increased by 10%-50%, and are projected to be the sixth-leading cause of deaths and third-highest cause of DALYs in this region . In NepaAlthough a relatively small proportion of ED patients, self-harm and suicide are increasingly recognized as a health problem in LICs, particularly in women. The maternal mortality and morbidity report from Nepal in 2008/09 had reported that suicide was the single leading cause of death among women of reproductive age (16%) compared to maternal related issues (12%) . SuicideMortality by 90 days after emergency health care in the current study was more than 20-fold the ED mortality. The ED mortality in the current study was lower (0.3%) than previously reported from Pakistan (1.3%), and a systematic review in LMICs reported a median ED mortality of 1.8% (IQR 0.2%-5.1%) ,44.Very little evidence exists on mortality after emergency care in LMIC’s. To the best of our knowledge, only one study has previously attempted to assess mortality after emergency care. The study from a tertiary level Vietnamese hospital assessed 30-day mortality in two much smaller ED populations and reported mortality of 9.8% and 7.8%, respectively . HoweverPatients with respiratory and cardiovascular complaints had particularly high mortality. Thus, nearly one in four patients with these complaints died within 90-day. This is much higher than reports from HICs. The 60-day mortality among patients with respiratory complaints in a Spanish study was 6.3% . SafwenbMany factors may contribute to the high post-discharge mortality that we observed. We suspect that patients in the present study presented to health care at a late stage of chronic diseases, since health services are unaffordable for many of these patients ,47. TypiInterestingly, we found illiteracy independently associated with increased mortality at 90 days in women but not in men. Secondary analysis of 2011 Nepal Demographic and Health Survey reports that illiterate Nepalese women are less aware of health risks, and that could result in less health seeking behavior . FurtherThis is a single-center study, thus generalizations should be done with caution. However, the cohort comprised a large population from both rural and urban regions and the distribution of patient characteristics show that the patient population is highly representative for the region in respect to age, gender, geography and ethnicity. Also, the long data collection period is a strength; the study includes data from a three year period, covering possible seasonal variations. However, it is a limitation that the study had a low follow-up rate for the 90 days telephone interviews. For the interviews, the results should interpreted with caution, although we show that those lost to follow-up have similar characteristics and PCs. We cannot rule out the possibility of selection bias in the interviewed patients, leading to possible underestimation of mortality and a higher follow-up in healthier and more resourceful families. Patients who did not provide their telephone number were not interviewed and these patients might not have had a phone due to economic conditions, and could also be frailer than the ones available for interviews. Also, it is possible that those who were called and did not answer were more likely to have died.Classification of presenting complaints was performed using a hierarchy approach which may result in an underestimation of frequency of complaints lower in the hierarchy .The study revealed that nearly one in ten died within 90-day of emergency care. However, follow-up rates were low and findings suggest a need for replication of post-discharge mortality studies with adequate systems that limit loss to follow-up. These studies should be performed as multi-center studies and include information on local health care systems that may affect post-discharge mortality. It will be important to study factors that affect post-discharge mortality, such as availability of follow-up systems, transportation and affordable medication. We argue that post-discharge mortality is a particularly important indicator on quality of care in low-resource settings, where primary care health systems are limited, and transportation and economic issues may halter adequate follow-up and treatment for complications or chronic diseases.


Interventional ablation has been demonstrated to represent an effective therapy in patients with atrial fibrillation (AF), leading to restoration and maintenance of sinus rhythm in the majority of cases. However, recurrence of AF is encountered in 35% to 40% of cases, and the causes for this frequent complication have not been elucidated so far.Here we present the study protocol of the FIBRO-RISK trial, a prospective, single-center, cohort study which aims to investigate the impact of inflammatory-mediated myocardial fibrosis on the risk of recurrence after successful catheter ablation of atrial fibrillation. The level of systemic inflammation in the pre-ablation and immediate post-ablation period will be assessed on the basis of serum levels of inflammatory biomarkers , while the level of cardiac fibrosis will be determined based on cardiac magnetic resonance imaging associated with complex post-processing techniques for mapping myocardial fibrosis at the level of left atrium and left ventricle. At the same time, the amount of epicardial fat will serve as an indirect marker of localized inflammation and will be determined at different levels in the heart , while ventricular function will be assessed on the basis of serum levels of NT-proBNP prior to the procedure. All these parameters will be investigated in patients with successful ablation of AF, who will be divided into 2 groups: group 1 – patients who develop AF recurrence at 1-year, and group 2 – patients with no recurrence of AF at 1-year. In all patients, the following biomarkers will be determined: serum levels of inflammatory biomarkers and NT-proBNP at 24 hours and 1-year post procedure, the amount of myocardial fibrosis at the level of left atrium and left ventricle at baseline +/− 7 days, and the amount of epicardial fat surrounding left atrium, right atrium and the entire heart at baseline +/− 7 days.The primary endpoint of the study will be represented by the rate of AF recurrence at 1-year post ablation, documented by either ECG or Holter monitoring. The secondary endpoints of the study will consist in:1.the rate of re-hospitalization,2.the rate of survival without relapse, and3.the rate of major adverse cardiovascular events .In conclusion, FIBRO-RISK will be the first CMR-based study that will investigate the impact of inflammation-mediated myocardial fibrosis and ventricular remodeling on the risk of recurrence after successful ablation of AF, aiming to validate inflammatory biomarkers and myocardial fibrosis as predictors for AF recurrence. Several risk factors for the occurrence and recurrence of AF have been described in the literature, including left atrial (LA) enlargement, left ventricular (LV) dysfunction, the amount of epicardial fat surrounding cardiac structures, or myocardial fibrosis. However, the relationship between inflammatory-mediated myocardial fibrosis and AF recurrence after radiofrequency catheter ablation has not been elucidated so far.Atrial fibrillation (AF) has an increasing prevalence, being currently the most frequent type of supraventricular arrhythmia and representing a significant burden for the healthcare system. In most of the cases, myocardial tissue located at the level of the pulmonary veins represents the origin of AF. The pathophysiology of AF is still incompletely understood, several studies suggesting that profibrotic and inflammatory processes can play a crucial role in the development of AF.Here we present the study protocol of the FIBRO-RISK trial, a prospective, single-center, cohort study which aims to investigate the impact of inflammatory-mediated myocardial fibrosis on the risk of recurrence after successful catheter ablation of AF.1.2primary objective of the study is to investigate the correlation between the degree of LA and LV fibrosis quantified by cardiac magnetic resonance (CMR), inflammatory biomarkers, and the risk of AF recurrence post catheter ablation.The secondary objective of the study is to investigate the correlation between structural remodeling of the left and right atrium, epicardial fat tissue volume and serum inflammatory biomarkers in patients with AF undergoing successful catheter ablation and their impact on long term outcomes.The 22.1This is a clinical prospective, non-randomized, cohort, single-center study to investigate the relationship between inflammatory status, inflammation-mediated atrial and ventricular fibrosis and remodeling, and the risk of AF recurrence. The duration of the study is 2 years which include the initial screening and the follow-up period for the recurrence of AF.2.2Declaration of Helsinki of 1975, and all patients will sign an informed consent and will be checked for the inclusion and exclusion criteria prior to enrollment in the study.The study protocol was approved by the Ethics Committee for Scientific Research of the University of Medicine and Pharmacy of Tirgu Mures  and the Ethics Committee for Scientific Research of the Cardio Med Medical Center . All study procedures comply with the 2.3The study will include 100 subjects with AF suitable for catheter ablation. Imaging biomarkers and laboratory analyses such as: high sensitive C Reactive Protein (hsCRP), matrix metalloproteases (MMPs), interleukin-6 (IL6) and N-Terminal Pro–B-Type Natriuretic Peptide (NT pro-BNP) will be determined in the first 24 hours after the procedure. The anatomy of pulmonary veins, atrial fibrosis, atrial volumes and the amount of epicardial fat will be evaluated and quantified with late gadolinium-enhancement cardiac magnetic resonance (LGE-CMR). All these parameters will be investigated in patients with successful ablation of AF, who will be divided into 2 groups: group 1 – patients who develop AF recurrence at 1 year, and group 2 – patients with no recurrence of AF at 1 year. In all patients, the following biomarkers will be determined: serum levels of inflammatory biomarkers and NT-proBNP at 24 hours and 1-year post procedure, the amount of myocardial fibrosis at the level of left atrium and left ventricle at baseline +/− 7 days, and the amount of epicardial fat surrounding left atrium, right atrium and the entire heart at baseline +/− 7 days.The study will be conducted over a period of 2 years, in which patients will be examined at baseline, and will be followed-up for recurrence of AF, as shown by the FIBRO-RISK diagram with the flowchart that will be used in the study Fig. .Inclusion criteria:Patients with non-valvular paroxysmal or persistent AF who undergo successful catheter ablation, either by cryoablation or by radiofrequency advanced 3D mapping system;Ability to provide informed consent;Patients aged at least 18 years;Exclusion criteria:Patients with valvular AF;Patients with acute coronary syndrome in the last 30 days;Patients in whom AF is presumed to be caused by hyperthyreosis;Patients with long-standing persistent or permanent AF;Unwillingness or incapacity to provide informed consent;Allergy to gadolinium contrast media;Absolute or relative contraindications to magnetic resonance imaging;Pregnancy or lactation;Women with childbearing potential in absence of any contraceptive treatment;Renal insufficiency (creatinine greater than 1.5 mg/dL) or renal failure requiring dialysis;Active malignancy or malignancy within the last 5 year prior to enrollment;Conditions associated with an estimated life expectancy of under 2 years;2.4The study will be conducted in the Center of Advanced Research in Multimodality Cardiac Imaging of the Cardio Med Medical Center, and is funded by the Romanian Ministry of European Funds, the Romanian Government and the European Union, as part of the research grant number 103545/2016 – CardioIMAGE (contract 43/05.09.2016), which was selected for funding following an international peer-review procedure within the national competition of research grants.2.5One hundred patients with successful ablation of AF who meet the selection criteria will be divided into 2 groups, namely subjects who develop AF recurrence at 1-year post ablation (group 1) and subjects with no recurrence of AF at 1-year post ablation (group 2).2.6The following information will be collected from all patients: baseline demographics, history and physical examination findings, cardiovascular risk factors. Conventional 12-lead EGC was performed in each patient at baseline, 1, 3, 6, 12 months follow-up visit.2.6.1Complete blood count and biochemistry will be evaluated at the central laboratory of the County Clinical Emergency Hospital of Tirgu Mures as per routine clinical practice, at baseline.Inflammatory status will be assessed at baseline, respectively at 1-year post procedure. hsCRP levels will be assayed by immunoturbidimetric assay . Matrix metalloprotease 9 and interleukin-6 levels will be assessed via enzyme immunoassay (ELISA) method . Determination of all inflammatory biomarkers will be performed at the Advanced Medical and Pharmaceutical Research Center of the University of Medicine and Pharmacy Targu-Mures. NT-proBNP serum levels will be determined prior to the procedure, at 24 hours, and repeated after 1 year, at the central laboratory of the Emergency Clinical County Hospital of Tirgu-Mures using an electrochemiluminescent immunoassay .2.6.2All patients will undergo transthoracic echocardiography with simultaneous ECG recording at baseline and 1-year post ablation procedure. Echocardiographic assessment will be performed with a Vivid E9 echocardiographic equipment , at rest, in the lateral decubitus position. Standard M-mode, 2-dimensional and Doppler images will be acquired as per American Society of Echocardiography standards. Left ventricular volumes and ejection fraction will be quantified and calculated according to Simpson rule, using images acquired in the apical 4-chamber plane. All measurements will be averaged for at least 3 heart cycles.2.6.3All CMR examinations will be performed with an 1.5 T Siemens Magnetom Aera magnetic resonance equipment . LV function will be assessed using cine sequences in long-axis (2-chamber and 4-chamber) and short axis. Short-axis volumetry will be used for determination of LV mass, LV ejection fraction, LV end-diastolic volume (LVEDV) and end-systolic volume (LVESV). Ventricular and atrial fibrosis will be assessed using late postgadolinium myocardial enhancement sequences (LGE) at 10 minutes after intravenous injection of gadolinium extracellular contrast agents. Acquired LGE images will be post-processed using the Medis QMass 8.1 software  with definition of the epicardial and endocardial contours and manual setting of the hyper-enhancement threshold for the recorded sequence of slices. Delayed signal intensity (DSI) analysis performed in 10 to 12 consecutive short-axis LGE images. The full width at half maximum will be used for image segmentation, using an automated hyper-enhancement threshold and fibrotic areas will be considered those with slow washout kinetic.Quantification of epicardial fat volume will be performed by tracing and manually adjusting epicardial contours, as part of the post-processing protocols.2.6.4Seldinger technique will be used for venous access, with 1 sheath positioned on the left and 2 sheaths on the right femoral veins. A decapolar diagnostic catheter will be placed in the coronary sinus, after being advanced via left femoral access site and secured with a 6F sheath.Transesophageal echocardiography will be used to guide interatrial septal puncture. A bolus of 5000 to 7500 U of heparin followed by 1000 to 2000 U/h will be administrated and the following heparin dosage will be adjusted using activated clotting time, determined every 15 to 30 minutes.TM NavXTM system, Model EE3000 , improved by fusing images acquired with CMR images. After inserting the mapping catheter in the ostium of the pulmonary vein (PV), radiofrequency catheter ablation will be performed to obtain pulmonary vein isolation. Ablation will consist in applying a 30 W energy and a 10 to 30 g pressure with temperature adjusted at 50°C, by an irrigated (5–20 ml/min) contact force-sensing catheter positioned on the left atrial wall. A PV will be considered electrically isolated by eliminating all electrical potentials, or by demonstrating successful PV potential dissociation.In case of radiofrequency ablation, 1 non-steerable sheath will be used for placing the duodecapolar mapping catheter (Lasso) and the other steerable sheath for placing the 3.5 mm tip externally irrigated contact force-sensing ablation catheter. A continuous heparinized saline wash of the long sheaths will be provided during the entire procedure. The 3-D electro-anatomical map will be acquired with the EnSiteIn case of cryoablation procedure, a cryoablation balloon  will be advanced with transesophageal control via transseptal route and will be placed at the ostium of each pulmonary vein, delivering minus 40 to 70° cooling energy, during 180 to 240 seconds application. The ablation will be considered efficient when complete electrical block between atrial tissue and pulmonary vein will be achieved.2.7The clinical trial will be conducted from January 2019 to December 2020.2.8primary outcome of the study is represented by the rate of AF recurrence at 1-year post ablation, documented by either ECG or Holter monitoring.The Secondary outcome refers to rate of re-hospitalization, rate of survival without relapse and rate of major adverse cardiovascular events 2.9Baseline (day 0)oObtain and document consent from participant on study consent form.oVerify inclusion/exclusion criteria.oObtain demographic information, medical history, medication history, alcohol and tobacco use history.oRecord results of physical examinations and 12-lead ECG.oCollect blood specimens .oImaging procedures: transthoracic 2-D echocardiography, late gadolinium-enhancement CMRoCatheter ablation (radiofrequency or cryoablation)Follow-up visits oRecord results of physical examinations, 12-lead ECG and medical history.oImaging procedures: transthoracic 2-D echocardiographyFinal study visit (month 12)oRecord results of physical examinations, medical history,12-lead ECG and determination of serum levels of inflammatory biomarkers and NT-proBNPoImaging procedures: transthoracic 2-D echocardiographyoEnd-point evaluation.2.10Medical history, clinical examination, laboratory tests ;12-lead ECG2D transthoracic echocardiography with measurement of: cardiac diameters, volumes, valvular function and regurgitation, pressure gradients, left ventricular systolic and diastolic function and ejection fraction.Late Gadolinium-Enhancement CMR with the evaluation of: left and right atrial volume, volume of epicardial adipose tissue, degree of atrial and ventricular fibrosisPulmonary vein isolation radiofrequency ablation with advanced 3D mapping system or cryoablation.2.11All the information will be collected in a database that consists of patient's background, medical history, medication, imaging features provided by cardiac ultrasound, CMR and imaging post-processing.2.12The study will include 100 subjects with AF undergoing successful ablation of pulmonary veins via interventional route. After analysis of follow-up data, patients will be distributed in two groups according to the presence or absence of recurrent episodes of AF during the follow-up.Sample size calculation was performed using the StatMate 2.0 software. For sample calculation, the proportion of event-free population was estimated at 60%. According to this calculation, a sample size of 50 patients in each subgroup has a 90% power to detect an increase in MACE-free rates proportion of 0.29, with a significance level  of 0.05 (2-tailed). Therefore, the total sample size was established at 100 patients.2.13t test. Non-normally distributed variables will be analyzed using the Mann-Whitney test. Categorical variables will be expressed in numbers and percentages and will be compared using the Fisher exact test.Statistical analysis will be performed using Graph Pad InStat 3.10 software at the level of significance 5%. All data will be checked for normality. Continuous variables with normal distribution will be presented as mean ± standard deviation and will be compared using 3This study presets the protocol of a prospective, single-center, cohort study which aims to investigate the impact of inflammatory-mediated myocardial fibrosis on the risk of recurrence after successful catheter ablation of AF.,4 In the latest years, a large number of studies tried to identify predictors for a successful outcome on long term after AF ablation, however reliable clinical markers to predict sinus rhythm maintenance or AF recurrence have not been identified so far. The main contribution of the present study will be to validate new clinical features, such as inflammatory biomarkers, epicardial fat, ventricular remodeling or myocardial fibrosis, as predictors of AF recurrence in patients who are scheduled for catheter ablation. In this trial we hypothesize that several imaging markers associated with cardiac structural alteration, provided by CMR , associated with serum inflammatory biomarkers  could form a powerful prediction panel to predict the risk of AF recurrence in the post ablation period, reflecting the impact of inflammatory-mediated myocardial fibrosis on the risk of arrhythmia.Although AF ablation procedures are constantly improving, recurrences continue to be problematic as a significant proportion of patients present with AF relapse despite an optimum management in the post-ablation period. It is well known that a higher amount of atrial fibrotic tissue can favor atrial arrhythmogenesis.,6 At the same time, the amount of contrast enhancement in the LA wall, considered to represent the volume of fibrotic tissue of the total LA wall, has been recently described by prospective trials to be independently associated with the likelihood of recurrent arrhythmia after ablation.,6,7 Previous studies showed that pre-existing fibrosis on LGE-CMR was the strongest independent predictor of ablation procedure failure and arrhythmia recurrence. McGann et al proved that subjects with >30% atrial fibrosis on LGE images at baseline had a 71% chance of AF recurrence at 1 year.The relationship between inflammation, myocardial injury and atrial fibrillation has been extensively studied in the recent years. Several recent studies managed to assess the feasibility of LGE-MRI in detecting both pre-existing and post-ablation atrial and ventricular fibrosis.,11–14 Their results raised interest in the use of CMR as a periprocedural risk assessment tool. Despite these promising results, the assessment of atrial and ventricular fibrosis has not yet been widely adopted in clinical practice.–18 We should not forget that the capacity of LGE-CMR to detect fibrosis in the atria is still controversial due to the lack of reproducibility. Thus, this trial aims to validate myocardial fibrosis, targeting both atrial and ventricular fibrosis quantified by CMR, as predictor for AF recurrence.Beside baseline evaluation, several studies suggest that quantification of post ablation LA scarring may play a role in determination of the AF recurrence risk.,19–22 Therefore, the role of inflammation in AF recurrence has been recently proposed as a new and challenging hypothesis to explain the high rate of recurrence after AF conversion.,21,23–26 As the exact pathways from inflammation to AF are still unclear, it is presumed that inflammatory cytokines may bind to atrial myocytes inducing tissue damage. In line with this hypothesis, the FIBRO-RISK trial aims to investigate the correlation between inflammatory biomarkers and cardiac fibrosis, evaluated via LGE-CMR, in order to prove the impact of inflammatory-related myocardial fibrosis on the risk of recurrence after successful catheter ablation of AF.Previous observational studies showed that elevation of several inflammatory serum biomarkers, such as hsCRP or IL-6, are associated with occurrence and recurrence of AF, since patients with AF present significantly higher levels of inflammatory biomarkers as compared with subjects maintaining sinus rhythm.,21,23–26 A very recent study demonstrated that in patients admitted in an intensive care facility, the presence of ventricular remodeling leading to ventricular dysfunction was associated with a higher incidence of AF and conduction abnormalities. NT-proBNP is a biomarker reflecting ventricular dysfunction, which has been shown to predict AF recurrence unrelated to ablation procedure.,29 However, it still remains unclear if these biomarkers are useful predictors of AF recurrence after ablation. In the FIBRO-RISK study, we aim to validate, for the first time in the literature, the role of ventricular remodeling, serum biomarkers associated with ventricular dysfunction, inflammation and myocardial fibrosis, assessed by LGE-CMR, as independent predictors of AF recurrence after successful catheter ablation, in order to prove that inflammation can play a significant role in the complex interrelation between myocardial dysfunction, remodeling and AF recurrence.Besides inflammation, neurohormonal activation, classically associated with ventricular dysfunction, has been shown to play a significant role in the recurrence of AF following pulmonary vein isolation.4FIBRO-RISK will be the first CMR-based study that will investigate the impact of inflammation-mediated myocardial fibrosis on the risk of recurrence after successful catheter ablation of AF, aiming to validate inflammatory biomarkers and myocardial fibrosis as predictors for AF recurrence.Submission to ethical committee was performed by Korodi Szilamer. The radiologist responsible for CMR imaging interpretation is Mihaela Ratiu. Korodi Szilamer, Roxana Hodas and Theodora Benedek will perform data statistical analysis. All the authors approved the final manuscript.Conceptualization: Szilamer Korodi, Rodica Toganel, Roxana Hodas, Mihaela Ratiu, Andras Mester, Istvan Kovacs, Imre Benedek.Data curation: Theodora Benedek, Kovacs Istvan.Formal analysis: Andras Mester, Theodora Benedek.Investigation: Mihaela Ratiu, Theodora Benedek, Istvan Kovacs.Methodology: Szilamer Korodi, Rodica Toganel, Imre Benedek, Theodora Benedek.Resources: Imre Benedek.Supervision: Imre Benedek, Theodora Benedek.Validation: Mihaela Ratiu.Visualization: Mihaela Ratiu, Andras Mester.Writing – original draft: Szilamer Korodi, Rodica Toganel, Roxana Hodas, Mihaela Ratiu, Andras Mester, Istvan Kovacs, Imre Benedek.Rodica Toganel orcid: 0000-0001-6510-9646.


China’s national tuberculosis programme does not have cohort wise information regarding attrition and delays in the multidrug resistant tuberculosis (MDR-TB) diagnosis and treatment pathway.Under the Global Fund programmatic management of drug-resistant TB (2006–13), we assessed the attrition and delay in the pathway and the factors associated.Cohort study involving secondary programme data. All patients identified as presumptive MDR-TB (defined as i) previously treated TB patients which included recurrent TB, return after loss to follow up, treatment after failure and ii) new TB patients that were non-converters at three months of treatment or in close contact with a known MDR-TB patient) during October 2006 to June 2013 were eligible for phenotypic drug susceptibility testing (DST). Pre-diagnosis attrition (presumptive MDR-TB not undergoing culture and DST) and pre-treatment attrition (confirmed MDR-TB patients not initiated on treatment) was calculated. Diagnosis delay was the time interval from DST eligibility to DST result, treatment initiation delay was fom DST result to treatment initiation and total delay was from DST eligbility to treatment initiation. Factors associated with attrition and delay were identified using log binomial regression and linear regression, respectively.Of 78 564 presumptive MDR-TB patients, 2 470 (3.1%) underwent pre-diagnosis attrition. Of 9 283 MDR-TB patients, 3 361 (36.2%) underwent pre-treatment attrition. Median(IQR) diagnosis delay was 84  days; treatment initation delay was 23 days and total delay was 117 days. Long diagnosis delay was an independent predictor of pre-treatment attrition in a dose response relationship. While pre-treatment attrition was less likely among presumptive criterion ‘previously treated’ and with increasing time period, it was more likey among elderly and in east and west region. While the diagnosis delay increased with time period, treatment initiation delay and total delay reduced with time period. Short diagnosis delay was associated with west region, smear negative patients and presumptive criterion ‘treatment after lost to follow up’. Short treatment initiation delay was associatied with east and west regions while long treatment initiation delay was associated with elderly and presumptive criterion ‘recurrent TB’. Total delay predictors were similar to treatment initiation delay. In addition, short total delay was associated with presumptive criterion ‘treatment after failure’.The diagnosis and treatment delay were long and the pre-treatment attrition was considerable high. Long diagnosis delay is likely to predict pre-treatment attrition. Multidrug-resistant TB (MDR-TB), defined as resistance to at least isoniazid and rifampicin, along with rifampicin-resistant tuberculosis (RR-TB), is a major public health concern in many countries and threatens global attempts to end TB . Timely Majority of MDR-TB patients are lost in the diagnosis and treatment pathway. Globally in 2017, there were an estimated 558 000 MDR/RR-TB patients, of whom only 160 684 (29%) were diagnosed and 139 114 (25%) were initiated on treatment [Globally, the treatment success rates for MDR-TB are between 55–56% ,17. DiagChina contributes to 13% of the global MDR/RR-TB patients. Seven percent of new TB patients and 24% of previously treated patients have MDR-TB. In 2017, there were an estimated 73 000 MDR/RR-TB patients of whom only 13 069(18%) were diagnosed and 5943(8%) were initiated on treatment . Median With the support of the Global Fund, China initiated a programme for MDR-TB ) between October 2006 and June 2014 in a phased manner . In thisThis was a cohort study involving record review of programme data. All presumptive pulmonary MDR-TB patients belonging to the GFPMDT sites and eligible for DST between October 2006 and June 2013 were included. By June 2013, the GFPMDT covered 67 prefectures across 24 provinces in China .Presumptive MDR-TB were defined as i) previously treated TB patients which included recurrent TB, return after loss to follow up, treatment after failure and ii) new TB patients that were non-converters at three months of treatment or in close contact with a known MDR-TB patient.China, the world’s most populous country, is a unitary sovereign state in East Asia with a population of over 1.4 billion . It has The launching criteria for PMDT sites included good ‘directly observed treatment–short course’ foundation, sound local government support and willingness to pilot PMDT. Hence, most of the project sites were located in east and middle region where the economic situation was better than the west region.Patients presented at county-level health institutions (basic management units (BMUs)), where sputum testing was conducted and drug-resistance risk assessment was initiated. Sputum specimen from presumptive MDR-TB patients were transported to prefecture or provincial-level laboratory (culture DST facility) for culture. For specimen with a positive culture result, the proportion method was used to determine the susceptibility of isolates against rifampicin and isoniazid. There was one laboratory register at country / BMU level for routine sputum microscopy. There was a culture DST register at prefecture level laboratory or provincial-level reference laboratory. The culture DST facility shared the DST results with the BMU. All the demographic and clinical data including laboratory test results were recorded in the presumptive MDR-TB register at the BMU.Patients diagnosed with MDR-TB were referred to the designated hospital at prefecture-level. Prior to initiating treatment, patients underwent a thorough medical examination. They received a standardized treatment regimen for 24 months that consisted of 6–8 months of intensive phase  and 18 months of continuation phase  [During January to April 2014, data were collected from the presumptive MDR-TB register at BMU and MDR-TB register at prefecture level designated hospitals. Data on baseline characteristics like province, prefecture, region, age, gender, GFPMDT phase, sputum smear status at TB diagnosis, presumptive MDR-TB criteria, dates of eligibility for DSTand DST result were collected. Among confirmed MDR-TB, date of DST results, treatment initiation (yes/no) and date of treatment initation were extracted. The procedure of data collection and management included the following steps: 1) the local staff from prefectural level filled the questionnaire which was designed at national level; 2) the provincial-level staff conducted data quality assessment and reconciled discrepancies 3) the national-level staff conducted final data review and assessment to eliminate missing data and errors.Data were single-entered in an MS Excel database in December 2014. The dataset was analyzed using STATA .The following three time intervals were calculated: between eligibility for DST and DST results (diagnosis delay), between diagnosis and treatment initiation (treatment initiation delay), and between eligibility for DST and treatment initiation . Delays were summarized using median (inter-quartile range-IQR). Pre-diagnosis attrition and pre-treatment attrition were summarized using frequency and proportion.Predictive modelling using log binomial regression was performed to identify risk factors for attrition. While using the diagnosis delay variable as one of the potential factors associated with pre-treatment attrition, the diagnosis delay was categorized based on quartiles. Linear regression was used to determine the factors associated with delays . In all the multivariable analyses, variables with unadjusted p<0.2 were included.The associations in the log binominal models were summarized using relative risks (unadjusted and adjusted–RR and aRR) and 0.95 confidence interval (CI). The associations in linear regression models were summarized using Beta (β) coefficients and 0.95 CI. The β coefficient indicated the adjusted mean difference of delay (in days) between the sub-category of interest and the reference sub-category .As we were dealing with very large numbers of presumptive MDR-TB patients, we assessed the programmatic significance of the β coefficients before assessing the statistical significance. Hence, for the risk factor analysis of diagnosis delay, we considered a β coefficient of at least seven days as programmatically significant association.The study was approved by the Ethics Committee of Chinese Center for Disease Control and Prevention. (number 201807 dated 9 April 2018) and Ethics Advisory Group of the International Union Against Tuberculosis and Lung Disease (The Union), Paris, France (EAG number 23/18 dated 17 April 2018). The study was proposed by National center for TB prevention and control, which was approved by the research department of Chinese Center for Disease Control and Prevention. As this study involved analysis of secondary data, waiver of informed consent was sought and approved by the ethics committees.A total of 78 564 presumptive MDR-TB patients were included. Their mean (standard deviation) age was 48.1(18.2) years, 58 392 (74.3%) were males and 43 717 (55.6%) were from east region. Most  patients were sputum smear positive and 51 635 (65.7%) were ‘new’ TB patients eligible for DST .Of 78 564, 77 372 (98.5%) underwent culture. Of those who tested culture positive, 99.2% (64 852/65 353) underwent DST. Therefore, among 78 564, a total of 2470 (3.1%) underwent pre-diagnosis attrition .A total of 9283 were diagnosed as MDR-TB and of them 3361 (36.2%) underwent pre-treatment attrition .Median(IQR) diagnosis delay was 84  days; treatment initation delay was 23 days and total delay was 117 days .As the pre-diagnosis attrition was very low and not programmatically significant, we are not presenting the risk factor analysis. Pre-treatment attrition was very high. Pre-treatment attrition was significantly i) lower in round 5-phase 2 and round 7 when compared to round 5-phase 1 of GFPMDT phase, ii) higher among elderly when compared to patients in 15–44 year age group, iii) higher in east and west regions when compared to middle region, iv) higher among patients with recurrent TB, treatment after failure of category I/II regimen and previously treated–others, when comared to new TB patients, and v) high among those with long diagnosis delays (dose response relationship seen) .The diagnosis delay increased in all the phases when compared to round 5 phase 1. It was significantly i) lower among patients with negative sptum smear microscopy status when compared to those with positive smear, and ii) lower among previously treated patients who were lost to follow-up when compared to new TB patients .The treatment initiation delay was significantly i) lower in all phases when compared to round 5 phase 1, ii) lower in east and west region when compared to middle region, iii) higher in elderly when compared to patients in 15–44 year age group, and iv) higher among recurrent TB patients when compared to new TB patients .The total delay was significantly i) lower in round 5 phase 2 and single stream framework when compared to round 5 phase 1, ii) lower in east and west region when compared to middle region, iii) higher in elderly and 45–64 year age group when compared to patients in 15–44 year age group, and iv) higher among recurrent TB patients and treatment after failure in category I/II patients when compared to new TB patients .This is the first study from China to offer many seminal observations on the MDR-TB diagnosis and treatment pathway—a holistic look at flow of a cohort of presumptive MDR-TB patients which includes pretreatment delay calculation from eligibility for DST and the effect of diagnosis delay on pre-treatment attrition. To the best of our knowledge, ours is the first study to fill the ‘evidence gap’ regarding the association between long diagnosis delay and pre-treatment attrition in the MDR-TB care pathway.We acknowledge some limitations in this study. First, it is likely that not all the presumptive MDR-TB were identified, therefore the selection bias might have led to an underestimation of attrition, especially pre-diagnosis attrition. Second, the treatment outcomes for a significant cohort of MDR-TB patients was not tracked and collected. Hence, we were not able to study the association between delays and MDR-TB outcomes, evidence for which is limited globally . Third, Limitations notwithstanding, there were some key findings. First, the long total delay was majorly contributed by long diagnosis delay (due to the use of phenotypic DST which happens over two rounds: one round for culture of Mycobacterium tuberculosis 6–8 weeks) and another for DST). The total delay was longer than globally reported average figures with phenotypic DST during 2000–15 (117 vs 108 days), probably due to eligbility of DST (not sputum specimen receipt at DST facility) being the starting point for delay calculation [ weeks anSecond, the pre-treatment attrition was very high when compared to the global figures of 24% during 2000–15 (reduced to 13% in 2017) . Almost Third, long diagnosis delay contributed to pre-treatment attrition possibly due to patients being very sick at diagnosis to move to a prefecture level facility.Finally, within the project, around two-thirds of the presumptive MDR-TB patients were new TB patients. In India, majority of the patients were previously treated patients and non-identification of presumptive MDR-TB contributed largely to pre-diagnosis attrition –7,15,16.There are important implications for China. First, though diagnosis delay was increasing over time period, we expect that by the introduction of rapid molecular DST, we should be able to reduce this delay which will in turn reduce pre-treatment attrition, the total delay and potentially result in better outcomes. China has recently updated the national TB guidelines (end of 2018). Along with the world health organization guidelines , this stSecond, to reduce the pre-treatment attrition and treatment initiation delays, we recommend decentralized MDR-TB treatment. This may be tried at least for those patients who are not ill at diagnosis . A commuThird, low contribution of previously treated patients in the presumptive MDR-TB cohort indicates that there is a critical need to assess this situation nationally in China. The national estimates of presumptive MDR-TB patients based on the presumptive criteria and the number of TB patients that actually undergo culture and phenotypic DST and/or rapid molecular testing  may be compared.Finally, a qualitative systematic enquiry is recommended to understand why certain risk groups are more prone to experience attrition and longer delays .In this MDR-TB care pathway from the Global Fund PMDT project in China (2006–13), we holistically documented the attrition, delays and their associated factors. Not only does this study emphasize the importance of early treatment initiation but also brings the focus on early diagnosis by testing patients as soon as they are eligible which can reduce pre-treatment attrition as well as potentially improve treatment outcomes. As China prepares to expand the coverage of rapid molecular DST at the level of counties, similar studies are recommended in future to monitor the reduction of delay and attrition along the MDR-TB care pathway, and the effect of pre-treatment delays (starting from DST eligibility) on treatment outcomes. This is vital if we are to end TB in China and globally by 2035 .S1 Annex(TIF)Click here for additional data file.S2 Annex(XLSX)Click here for additional data file.


With total and out-of-pocket spending for oral oncolytics rising, there is increased interest in choosing oncology treatments based on their clinical value relative to cost.To determine if out-of-pocket spending varied for higher versus lower benefit oral oncology drugs reimbursed by commercial insurers.This study was a retrospective analysis of commercial insurer prescription drug claims filed between 2007 and 2014 for 13 oral oncolytics approved before 2009. We calculated mean monthly out-of-pocket payment for each fill by patient. We then categorized oral oncolytics by their overall and progression-free survival benefits for each FDA-approved indication, using evidence from published studies. We assessed the relationship of survival benefit with mean monthly out-of-pocket payment, adjusting for demographic and plan characteristics.Our population included 44,113 patients aged 18-65 years (mean 52.5 [SD 9.4]) with a cancer diagnosis who filled 731,354 prescriptions. The most commonly represented oncolytics were imatinib (37.4% of fills), lenalidomide (17.7% of fills), and dasatinib (10.0% of fills). Approximately 32.3% of fills were for drug-indication pairs with an overall survival benefit of 4+ years. In adjusted analyses, there was no clear pattern to suggest that out-of-pocket payments differed with drug indication-specific survival benefits. Drugs for indications providing > 0 to 1 year of overall survival benefit were significantly more likely to have a lower out-of-pocket payment versus those prescribed off-label, but there were no significant differences in out-of-pocket payments between drugs and associated indications in any other survival category versus drugs used off-label.Out-of-pocket payments for oral oncolytics were not clearly related to indication-specific value in commercially insured patients. This finding suggests that despite increased attention to value- and indication-based drug pricing, cost sharing for oral oncolytics does not currently reflect these goals. What is already known about this subjectSpending on cancer drugs, particularly oral oncolytics, continues to rise in the United States, with 1 study showing an average monthly out-of-pocket spending increase from $88 in 2001 to $198 in 2011.There is interest in choosing treatments based on their value, and institution of lower-cost sharing levels for interventions with greater efficacy has been shown to increase adherence and improve clinical outcomes.What this study addsOut-of-pocket payments for oral oncolytics from 2007-2014 were not clearly related to indication-specific value.Despite increasing interest in value-based pricing, out-of-pocket payment design is not congruent with the goal of aligning costs with health outcomes.1 and spending on targeted oral oncolytics has increased particularly rapidly, with mean monthly expenditures in the year of product launch increasing from $1,869 in 2000 to $11,325 in 2014.2 Rising drug prices and expanded use of cost sharing have translated to higher out-of-pocket spending for targeted oral oncolytics.3 For example, 1 study found that average out-of-pocket monthly expenditures rose from $88 in 2001 to $198 in 2011.4Cancer drug costs continue to rise in the United States,5—with definitions of outcomes in oncology integrating clinical benefits, toxicities, and patient preferences.7 Evidence of the relationship between drug prices and outcomes in oncology is mixed but largely suggests that overall price is not correlated with benefit.10 With regards to prices that patients see, there is a lack of evidence that insurers have employed cost sharing to preferentially encourage use of higher- versus lower-efficacy oncology drugs. Institution of lower cost-sharing levels for interventions with greater evidence of efficacy is known as value-based insurance design and has previously been shown to increase adherence to medications and improve clinical outcomes.11 Given the known inverse relationship between out-of-pocket spending and adherence to or continuation of oncology drugs,13 use of value-based cost sharing could potentially drive patient adherence to high-efficacy oral oncolytics and thus enhance outcomes.Concurrently, there is increasing interest in choosing treatments based on their value—or health outcomes relative to their cost6Within this context, we sought to describe out-of-pocket spending by patients with commercial insurance who had a diagnosis of cancer during 2007-2014 and were treated with oral cancer drugs approved before 2009. We specifically assessed if patients using drugs with higher versus lower benefit had lower out-of-pocket spending, with benefit defined in terms of overall or progression-free survival as per the American Society of Clinical Oncology’s (ASCO) 2016 Value Framework.14 We restricted our analysis to the 13 drugs approved before 2009 to ensure availability of 5 years of data for analysis, thus, allowing for adjustment for changes in spending over time. The MarketScan database represents > 50 million employees and their dependents enrolled in commercial health insurance plans sponsored by approximately 100 large and medium-sized U.S.-based employers. About 56% of the non-elderly U.S. population was covered by employer-sponsored coverage in 2014, making the MarketScan database largely representative of our population of interest.15 We identified 731,354 prescription drug claims filled by 44,113 patients with a cancer diagnosis in the year of the fill. We excluded 5,063 claims with patient or plan payment < $0 or days supplied < 0.We used the MarketScan Commercial Claims and Encounters Database for 2007-2014 to examine drug use and expenditures among cancer patients who had filled any orally administered, targeted nonhormonal anticancer medications approved before 2009.16We summed monthly copayments, coinsurance, and deductibles for each fill. We adjusted fills for > 1-month supply to a single month supply to generate mean monthly total out-of-pocket payments. We did not consider employee contributions to commercial health insurance coverage via monthly paycheck deductions because we sought to estimate the cost to patients of an oral oncolytic at the time of prescription fill. Patient cost sharing at the point of service is a formulary tool that may influence a patient’s use of a prescription drug.We categorized drugs by overall survival benefit  and progression-free survival benefit  for each indication approved by the U.S. Food and Drug Administration (FDA) for the particular oncolytic (as described in the product label for that oncolytic). We examined evidence from randomized controlled clinical trials that compared the drug against standard of care or placebo for each particular FDA-approved indication. We reported overall or progression-free survival benefit as a relative survival benefit . If multiple similar trials were available for a drug and indication, we selected the trial presenting the largest estimate of survival differences between patients receiving treatment versus control. If median overall survival or progression-free survival were not reached in published trials , we used estimates from cost-effectiveness analyses and, where appropriate, focused on the survival measure for which a significant effect had been shown in randomized controlled trials.Drugs and associated indications were partitioned into the following 8 categories of increasing evidence for benefit, based on the relative distribution of survival estimates for all fills: (1) off-label use ; (2) no survival benefit for an on-label indication; (3) > 0-5 months progression-free survival benefit; (4) > 5-10 months progression-free survival benefit; (5) 10+ months progression-free survival benefit; (6) > 0-12 months overall survival benefit; (7) > 12-48 months overall survival benefit; and (8) 48+ months overall survival benefit. If a drug was associated with statistically significant overall survival and progression-free survival benefits, it was only put into an overall survival category, given the clinical superiority of overall survival benefits over progression-free survival benefits. The distribution of each drug and its associated assignment into these categories, as well as the study from which information about benefit was derived, is described in 17 use of the drug was associated with a progression-free survival benefit of 4.6 months, while use of placebo was associated with a progression-free survival benefit of 1.6 months. We thus calculated the relative progression-free survival benefit as 3 months. This placed pazopanib for an indication of soft-tissue sarcoma in category number 3 for > 0-5 months of progression-free survival benefit.As an example of this process, pazopanib is approved for use in soft tissue sarcoma. In a 2012 study by van der Graaf et al.,18 we used estimates of these metrics from economic modeling studies based on randomized controlled trial data.19 Given that randomized controlled trials had demonstrated an overall survival benefit of imatinib versus interferon alpha, we defined the relative survival benefit of imatinib as the difference between the 19.1 remaining life-years associated with first-line imatinib versus the 9.1 life-years associated with first-line interferon alpha.19In the case of imatinib, since median overall and progression-free survival was not reached even in trials with longer-term follow-up,20 We also measured total monthly drug reimbursement  to account for differences in drug prices. Variables are categorized in For each patient in each year, we documented age, health plan type, and census region, as reported in the MarketScan database. We also calculated previous year comorbidity based on the Klabunde modification of the Charlson comorbidity score, using diagnoses reported in MarketScan for each patient.21 predicting changes in out-of-pocket payments by drug survival benefit and adjusting for the previously described covariates. The first part of the model consisted of a logit function that assessed the association of drug survival benefit with having any out-of-pocket spending (vs. none). The second part used a generalized linear model with a gamma distribution and a log-link function to assess the association of drug survival benefit with level of non-zero out-of-pocket spending. Estimates from these 2 models were combined using a margins function.21After describing out-of-pocket expenditures by drug benefit and patient characteristics, we estimated a 2-part model,In sensitivity analyses, we conducted stratified analyses, first, considering overall survival as a continuous variable among patients treated with drugs with evidence for an overall survival benefit and, second, considering progression-free survival as a continuous variable among patients treated with drugs, with evidence for only progression-free survival benefit. Finally, in a sensitivity analysis, we conducted a version of the original 2-part model excluding the top 5% of monthly out-of-pocket spending claims. All dollars were inflation adjusted to 2014 U.S. dollars based on the medical component of the Consumer Price Index.9 Accordingly, in a secondary analysis, we reassigned the survival benefit for each drug and all its associated indications based on the benefits associated with the drug’s first approved indication. Finally, we repeated analyses within subgroups of drugs or indications, including among tyrosine kinase inhibitors only, among drugs for treating renal cell carcinoma, and for a single drug  when considering all of its indications.Because pricing decisions are often made at launch when only 1 indication for a drug is known, it is possible that cost sharing may be more closely related to the first indication’s benefit rather than that of subsequent indications.P values < 0.05 were considered statistically significant. Data were analyzed using SAS version 9.4 . This study was deemed not human subjects research by the Harvard University Institutional Review Board.Two-sided The 44,113 patients who filled 731,354 prescriptions for oral anticancer drugs between 2007 and 2014 had a mean age of 52.5 (standard deviation [SD] 9.4) years, and approximately half of the patients were male . The mosP < 0.001). In addition, fills for drugs with higher total costs (plan plus patient payment) were associated with higher out-of-pocket spending .Results of sensitivity analyses including only (a) patients filling drugs with evidence of an overall survival benefit or (b) patients filling drugs with a progression-free survival benefit and examining overall or progression-free survival as continuous variables similarly revealed no significant relationship between out-of-pocket payment and survival benefit . Our findings also held within subgroups of drugs or indications such as among only tyrosine kinase inhibitors, only drugs for renal cell carcinoma, and only for lenalidomide when considering all of its indications .Finally, analyses examining survival benefit based on the benefit reported for the drug’s first approved indication also showed no association of out-of-pocket payments with benefit .7 we found that out-of-pocket payments for oral oncolytics from 2007-2014 were not clearly related to indication-specific survival benefit in a commercial population. Although drugs with > 0-1 year of overall survival benefit were associated with significantly lower out-of-pocket spending, there was no difference in out-of-pocket payments for drugs in any other survival category versus drugs used off-label. In addition, while previous analyses have examined drug pricing in relation to first indication survival benefit,9 our analysis did not show any relationship between the survival benefit of the first approved indication and out-of-pocket payment.Despite consistently rising out-of-pocket spending for oral oncolytics and increasing interest in value-based pricing,22 and there is evidence that beneficiaries with higher-out-of-pocket costs are more likely to discontinue cancer therapy.13 Notably, substantial financial toxicity of cancer therapy has been associated with worse outcomes, including early mortality.23 Value-based insurance designs have been shown to improve adherence in nononcology clinical areas,26 and ideally, cost sharing could be aligned to encourage greater use of and adherence to high-value cancer treatments. However, we saw no association between out-of-pocket payments and drug survival benefit. This finding suggests that despite some payers and pharmacy benefit managers announcing or considering value-based drug pricing,7,27 the complex U.S. drug reimbursement and distribution system still does not lend itself to cost sharing that reflects value.7,28Cancer therapy is associated with significant financial toxicity, including bankruptcy,12 In addition, several payers have publicly cited interest in value-based pricing for oncology drugs.27,29 However, the fact that a relationship does not exist between value as defined by survival and out-of-pocket costs has not been previously demonstrated in the oncology realm. Possibly reflecting the increasing prevalence of oral chemotherapy parity laws,31 we observed lower out-of-pocket payments for fills in more recent calendar years. As expected, we observed higher out-of-pocket payments for more expensive drugs.Previous work has demonstrated that out-of-pocket spending increased substantially between 2001 and 2011 for oral oncolytics and that Medicare enrollees are more likely to discontinue more expensive cancer drugs.Our study has limitations that need to be considered. Given the composition of the MarketScan database, we studied only commercial health insurance claims for individuals with employer-sponsored insurance; out-of-pocket payments and their relation to drug benefit may differ for other payers, including individuals enrolled in Medicare Part D. Although the MarketScan database provides some information about employee health plan types, it does not provide more granular data regarding copay, coinsurance, or drug coverage arrangements for individual plans, which limited our ability to adjust for more granular differences in coverage. The database also does not provide information regarding employee contributions to health insurance coverage, such as paycheck deductions. We were also limited to evaluating filled prescriptions, which by nature of patient-pharmacy interactions and the composition of the MarketScan database means those for which claims were paid in full. It is possible that patients prescribed lower value treatments were less likely to fill them if faced with high out-of-pocket costs. Prescriptions not filled were excluded from our analysis, thus, potentially excluding a segment of our population of interest facing significant costs.6 However, there are certainly other aspects that contribute to a therapy’s clinical value, including safety, tolerability, and ease of use. Further analyses should examine the relationship between these additional measures and out-of-pocket costs.Our analysis included only oral oncolytics approved through 2009, so findings could differ for more recently approved drugs, particularly given the recent focus on the value of care delivered in the context of health care reform. In addition, we described value in terms of survival benefit. This metric has been designated as a cornerstone of defining value in ASCO’s 2016 Value Framework.Finally, since information about cancer stage is not available in the MarketScan database, it was assumed that any drug used for a particular FDA-approved indication was used for the correct stage of disease based on oncologist expertise. While this was largely true, there may have been exceptions not captured in our analysis.Our study demonstrated that for commercially insured patients, out-of-pocket payments were not associated with indication-specific survival for oral oncolytics approved before 2009.This finding highlights opportunities for commercial health insurers to address the complexities of implementing value-based cost sharing for oncolytics in a health care landscape that will increasingly emphasize benefit reaped for dollars spent.


OBJECTIVES/SPECIFIC AIMS: Pembrolizumab is an anti-PD-1 immune checkpoint antibody that has demonstrated promising anti-tumor activity in patients with solid tumor malignancies, including patients with brain metastases from malignant melanoma and non-small cell lung cancer. Leptomeningeal disease (LMD) is a rare form of malignant spread to the central nervous system (CNS), that occurs in 2%–10% of patients with solid tumors, most commonly in breast cancer and non-small cell lung cancer. We propose an open-label phase II study of pembrolizumab in patients with LMD from advanced solid tumors (NCT03091478). This study aims to determine if pembrolizumab therapy can lead to a radiologic, cytologic or clinical response in the CNS, in patients with LMD. METHODS/STUDY POPULATION: Patients with pathologically confirmed advanced solid tumors, and either radiologic or cytologic evidence of LMD, will be identified at a single institution. Radiologic LMD will be defined as a >4 mm area of measurable LMD on gadolinium-enhanced MRI brain/total-spine; and cytologic LMD will be defined as the presence of malignant cells on CSF cytology. Patients will be excluded if they have: active autoimmune conditions that require immunosuppression, received radiation therapy to the only area of measurable LMD within 3 months of study enrollment, have an ECOG performance status <1. Once enrolled, patients will receive pembrolizumab 200 mg intravenously every 3-weeks, until disease progression or unacceptable toxicity. Patients will have CSF sample sampling, blood draws, radiologic imaging of the body (CT), brain/total-spine (gadolinium-enhanced MRI) pre-treatment, after 2 and after 4 cycles of therapy, for response assessment and correlative studies. The primary endpoint of the study is CNS response assessed at 12 weeks/after 4 cycles of pembrolizumab, defined either as radiologic response (reduction in size of LMD on gadolinium-enhanced MRI) and/or cytologic response (conversion of positive to negative CSF cytology on 2 consecutive samples) and/or clinical response. Secondary endpoints will include progression-free survival, overall survival, and safety. To explore the mechanisms by which pembrolizumab may affect LMD, we will assess dynamic changes in genomic and immunologic markers in the CSF and serum pre and post pembrolizumab using next-generation sequencing and multi-color flow cytometry, respectively. RESULTS/ANTICIPATED RESULTS: We will aim to accrue a total of 20 patients, allowing for a 10% drop-out rate, the final sample size will include 18 patients who have received at least 1 dose of pembrolizumab. CNS-response at 12 weeks will be assessed radiologically +/− cytologically, and the proportion of patients with CNS response and associated 95% confidence interval with be reported. CNS-progression-free survival and overall survival will be assessed using the Kaplan-Meier method. Cause of death will be recorded. Safety will be assessed as detailed above, and monitored as per an institutional Data Safety and Monitoring Plan. Exploratory endpoints will include genomic testing of tumor cells and cell-free DNA in CSF and serum, and immunologic studies of immune cells in CSF and serum at pre-defined timepoints. These data will be presented descriptively. We conservatively estimate that we will accrue 1 patient per month at our institution. Study duration will be approximately 24 months, allowing 18 months for accrual and 6 months for follow-up and data analysis. DISCUSSION/SIGNIFICANCE OF IMPACT: There are no currently FDA-approved therapies for patients with LMD from solid tumors. Anti-PD-1 immunotherapy is a promising class of agents, with known efficacy in patients with CNS metastatic disease, across tumor types. This study seeks to identify whether pembrolizumab may lead to CNS responses in patients with LMD. Additionally, genomic and immunologic analyses in CSF and blood pre and post-pembrolizumab may identify mechanisms by which immunotherapy affects the CNS in patients with LMD.