Determining the role of fomites in the transmission of SARS-CoV-2 is essential in the hospital setting and will likely be important outside of medical facilities as governments around the world make plans to ease COVID-19 public health restrictions and attempt to safely reopen economies. Expanding COVID-19 testing to include environmental surfaces would ideally be performed with inexpensive swabs that could be transported safely without concern of being a source of new infections. However, CDC-approved clinical-grade sampling supplies and techniques using a synthetic swab are expensive, potentially expose laboratory workers to viable virus and prohibit analysis of the microbiome due to the presence of antibiotics in viral transport media (VTM). To this end, we performed a series of experiments comparing the diagnostic yield using five consumer-grade swabs  and one clinical grade swab for inhibition to RNA. For three of these swabs, we evaluated performance to detect SARS-CoV-2 in twenty intensive care unit (ICU) hospital rooms of patients including COVID-19+ patients. All swabs were placed in 95% ethanol and further evaluated in terms of RNase activity. SARS-CoV-2 was measured both directly from the swab and from the swab eluent.Compared to samples collected in VTM, 95% ethanol demonstrated significant inhibition properties against RNases. When extracting directly from the swab head as opposed to the eluent, RNA recovery was approximately 2-4x higher from all six swab types tested as compared to the clinical standard of testing the eluent from a CDC-approved synthetic (SYN) swab. The limit of detection (LoD) of SARSSARS-CoV-2 from floor samples collected using the consumer-grade plastic (CGp) or research-grade plastic The Microsetta Initiative (TMI) swabs was similar or better than the SYN swab, further suggesting that swab type does not impact RNA recovery as measured by the abundance of SARSSARS-CoV-2. The LoD for TMI was between 0-362.5 viral particles while SYN and CGp were both between 725-1450 particles. Lastly microbiome analyses (16S rRNA gene sequencing) of paired samples  collected using different swab types in triplicate indicated that microbial communities were not impacted by swab type, but instead driven by the patient and sample type.Compared to using a clinical-grade synthetic swab, detection of SARS-CoV-2 from environmental samples collected from ICU rooms of patients with COVID was similar using consumer grade swabs, stored in 95% ethanol. The yield was best from the swab head rather than the eluent and the low level of RNase activity and lack of antibiotics in these samples makes it possible to perform concomitant microbiome analyses. Since its appearance in early December of 2019, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has spread to 197 countries resulting in a total of 1,465,144 deaths and 62,844,837 confirmed cases as of December 1, 2020  was made at the following concentrations: 232000, 2320, 1160, 580, 290, 145, and 72.5 viral particles per μL. A total of 5 μL of each dilution, or water as a negative control, was pipetted onto each swab type in triplicate and then immediately placed into 95% EtOH. Swabs in EtOH were then stored overnight at −80°C until processing. Upon processing, an additional 24 ‘no swab’ controls were included whereby 5 μL of the dilutions were dispensed directly into the extraction plate lysis buffer. Samples were processed using the same nucleic extraction method as described for swab heads above, and eluted in 75 μL of elution buffer. For RT-qPCR, 5 μL of template was used for each marker N1 and Rp. To address potential issues of non-normality, total copies were compared across swab types at each individual dilution using Kruskal-Wallis tests with Benjamini-Hochberg FDR 0.05 post-hoc test.To estimate the limit of detection (LoD) and compare the viral yield across three swab types , a serial dilution of viral particles was spiked onto floor swabs. In brief, separate 25 cm x 25 cm areas of the floor from a low-traffic common room inside the Marine Biology Research building at UC San Diego, a building with no SARS-CoV-2 research activities, were swabbed with a total of 24 swabs per swab type. Swabs were processed in groups of six by swabbing a quarter of that 625-cmfacility . Swabs wca. 625-cm2 area) and the patient’s bedrail. All swabs were immediately placed in a collection tube containing 0.5-1.0 mL 95% EtOH, stored on dry ice, and processed for RNA or total nucleic acid extraction.All study patients were hospitalized with clinical concerns for COVID-19 and received standard diagnostic testing. Study samples were collected from subjects' nares or hospital surfaces using three dry swab types  under the UC San Diego Institutional Review Board protocol #150275 and #200613. Both nasal samples and hospital surfaces were collected using three dry swab types . Nasal samples were collected by inserting the swab into one nostril to the depth of approximately 2-3 cm and rotated for 5-10 seconds. Hospital surfaces sampled included the floor inside the patient’s room , and eluted into 70 μL buffer. For RT-qPCR, 5 μL sample was processed using the standard SARS-CoV-2 protocol provided by the CDC .A subset of 40 samples were processed for 16S rRNA gene sequencing using established EMP protocols . These iVisualizations and statistical comparisons performed using PRISM 8.0 and the limit of detection determination were consistent with CDC recommendations whereby samples with a Ct value greater than 40 were omitted (Cat# 2019-nCoVEUA-01) .Our experimental design sought to answer three primary questions: whether the efficacy of SARS-CoV-2 detection is influenced by the following three variables: 1) does the swab storage solution (95% EtOH vs 91% isopropanol) impact the sensitivity of detection; 2) which sample fraction, swab head or eluent, provides better detection fidelity; and 3) does the swab head material type matter? To do this we designed a series of experiments to compare RNA recovery as measured by RT-qPCR using multiple swab types and storage solutions. We additionally performed environmental sampling in a hospital environment with a subset of swab types for comparison.t-test p=0.03).To evaluate the feasibility of switching from VTM to a more readily-available, viral-inactivating sample collection solution, we compared the extraction efficiency of synthetic-tipped plastic-shafted nasopharyngeal (NP) SYN swab samples stored in VTM versus nasal samples collected using SYN swabs stored in 95% ethanol (EtOH) collected from two separate cohorts . When miTo more quantitatively determine the effects of alcohol-based preservation media, we extracted RNA from a pure, commercial sample of human RNA added to water, EtOH, or 91% isopropanol, and found no impact on extraction efficiency .2=0.56). TMI adsorbed the least  followed by plastic shafts  . CGp swabs had the highest recovery of RNA from the swab head, while TMI swabs had the highest overall recovery of RNA when combining both eluent and direct swab extractions  . All negative controls for floor swabs were indeed negative for SARS-CoV-2 using N1 and N2  . Specifiis test) . Rp yielca. 4.2 days, with a NP swab test occurring within 72 hours of the time of nasal sampling. Of the 12 nasal samples using the SYN swab preserved in EtOH from COVID-19(+) patients, nine were positive for the presence of SARS-CoV-2 or a false negative rate of 25% . The average time from diagnosis to sampling was e of 25%  comparede of 25% . These re of 25% . Bedraile of 25% . For SARe of 25% .The observed differences in detection among nares and environmental samples, taken in context of our previous experimental results demonstrating that the swab type does not inherently impact SARS-CoV-2 detection, suggests that variation in sample collection from the nares and other environmental samples has an important role in detection sensitivity. When swabbing an environmental surface or body site , there is inherent variation in the swabbing event which can be attributed both to stochastic differences in biomass  present and collected as well as the downstream processes such as nucleic acid extraction and RT-qPCR. To evaluate if certain sampling locations or swab types were more variable than others, we calculated the intra-assay coefficient of variance (CV) of the Rp gene Cq values. When comparing the variation across sample types , the CV was significantly higher in floor (Mann Whitney p=0.0056), patient nares eluent (P=0.0017), and patient nares swab  when compared to control (human RNA spike-in) samples. The median difference in nares swabs was greatest with a median difference in variance of 2.5 compared to controls . In this case, the CV of the positive control samples would demonstrate the variance in combined extraction and RTqPCR thus is indicative of the total variance in molecular sample processing. Next, we stratified for swab types. Swab types also demonstrated an effect with the inter-swab variation between values measured with CGp and SYN along with CPg and TMI swabs for controls and patient samples being significant, respectively  . Overall these findings demonstrate how variability in a given sampling (swabbing) event can influence SARs-CoV-2 detection.Staphylococcus, Corynebacterium, Pseudomonas, Streptococcus, and Enterobacteriaceae being the more dominant taxa. Nasal samples however were mostly enriched by either Staphylococcus or Corynebacterium, with patient 7 having a higher abundance of Lawsonella  spanning a total of three patients, two sample types  and the three swab types. After processing with Deblur, the total number of reads per sample were compared . Read cowsonella .When assessing whether it will be possible to adapt collection methodology to enable more affordable, more widely available, and more inter-assay compatible collection methods for SARS-CoV-2 monitoring, it is key to understand the feasibility of using both alternative swabs and sample storage solutions. Here we provide evidence that the variation observed in a given SARS-CoV-2 experiment is primarily driven by the time and method of sample collection rather than by the swab type, storage solution, and subsequent extraction and RT-qPCR. A critical caveat of our storage solution comparison was that different patient samples were used. An ideal experiment would be to collect two swabs from the same person and store each swab in two different buffers (VTM vs. 95% EtOH). However, the relative sizes of the nostril and swab heads would necessitate sequentially swabbing the same nostril or using a different swab in each nostril. The former approach would introduce the variable of reduced biomass from the first sampling, whilst the second would introduce the variable of nostril-nostril variation in biomass. Because our subsequent experiments showed equal biomass extraction efficiencies when compared to control extractions, we did not follow up on these additional experiments. In addition, we demonstrate that variability in the hospital sampling was primarily driven by the actual sample collection rather than swab type. When using alcohol-based storage solutions, we demonstrate that the nucleic acid or viral particles tend to become enriched on the swab head rather than the eluent and thus we recommend extracting directly from the swab head itself. We demonstrate that RNA can be successfully extracted from consumer-grade swabs stored in alcohol without compromising RNA integrity or yield. Of note, wooden-shafted swabs performed poorly only when extracting from the eluent, suggesting that RNA adsorption onto the shaft, rather than RT-qPCR inhibitors, may be the source of interference with current eluent-based testing methods for this swab type. As cotton-tipped swabs and alcohol-based storage solutions are compatible with standard microbiome- and metabolome analyses not feasible with VTM, these alternatives could enable more widespread assessment of the microbial context of SARS-CoV-2 RNA in human and environmental samples, including associated microbiome features.We also provide preliminary evidence that nasal samples collected using more widely available, consumer-grade, cotton-tipped swabs can be used to detect SARS-CoV-2 in the clinical setting. As cotton-tipped TMI swabs had only a marginally reduced performance compared to CDC-compliant SYN swabs for nasal samples compared to NP results, these swabs have potential as an attractive alternative for methods such as metabolomics that are complicated by the background from incompatible with synthetic-tipped swabs, as well as suggesting that the pool of available collection consumables could be expanded beyond medical-grade materials. Notably, this variation is less than that observed when comparing different methods for assessing the presence of SARS-CoV-2. Larger-scale testing will be needed to expand and confirm these findings, but our data suggests that these two swab types, in either 95% EtOH or isopropanol, would provide a valuable starting point.ca. 362.5 copies per extraction for TMI) and (750-1450 copies per extraction for CGp and SYN swabs) similar to that of other published studies (500 copies per extraction) [ca. 50% of COVID-19(+) participants’ bedrails and ca. 75% of floors, as well as the detection of SARS-CoV-2 on the floor of one non-COVID patient, suggests a potential reservoir for infections. Further studies should be completed to quantify the infectivity of these viral particles which could lead to policy implications such as increased cleaning measures. Indeed, the floor may be a potentially important reservoir for viral exposure, as shoe-covers are not currently recommended by the CDC or WHO. However, additional testing is needed to determine whether viable virus particles remain on these surfaces. Since detection largely does not differ across swab types, this suggests that differences seen in the quantitation of SARS-CoV-2 in the clinic in a given floor or nasal sample is due to variation in the swabbing event itself rather than a molecular processing problem. Because of this, we recommend standardization in medical devices used to collect both nasal and environmental samples specific to SARSs-CoV-2 to improve overall accuracy. Lastly, our efforts to quantify the total noise in a given sampling event and sample processing itself demonstrate how variation in the act of swabbing combined with sample processing may lead to variance and at times lower than expected specificity.When considering environmental sampling, our data suggest that TMI and CGp swabs may outperform or at least are similar to, CDC-compliant SYN swabs for collecting samples to detect SARS-CoV-2 from floor samples. We provide molecular evidence demonstrating the feasibility of detecting SARS-CoV-2 from floor samples with a limit of detection (raction) . Additioraction) -31, is wSecondary infections are an important and significant contributing factor to morbidity and mortality in COVID-19 patients ,33. WithIn summary, our results suggest detection of SARS-CoV-2 RNA in the environment could be performed using less expensive, consumer-grade materials and alcohol-based storage solutions. With the materials examined in this study, it is further conceivable that patients could collect samples from themselves, their environments at home, or their place of work, dramatically expanding the ability to deploy widespread methods for monitoring and predicting outbreak events. Additional confirmatory studies using consumer-grade swabs would greatly support COVID-19 screening worldwide, particularly in resource-limited communities.


How have second-generation diabetes drugs diffused into clinical practice among Medicare enrollees?In this cross-sectional study including data for 1 182 233 Medicare enrollees who first received diabetes drugs between 2007 and 2015, a time before second-generation diabetes drugs had demonstrated additional cardiovascular benefits and before they were recommended by clinical guidelines, there was substantial variation in their use across practices, with most of the early use concentrated among a few high-prescribing clinicians and practices.This finding suggests that there are potential shortfalls of certain traditional cost containment mechanisms and highlights opportunities to improve the value of early diabetes care. This cross-sectional study examines patient, prescriber, and practice characteristics associated with first- or second-generation diabetes drug initiation. Little is known about how new and expensive drugs diffuse into practice affects health care costs.To describe the variation in second-generation diabetes drug use among Medicare enrollees between 2007 and 2015.This population-based, cross-sectional study included data from 100% of Medicare Parts A, B, and D enrollees who first received diabetes drug therapy from January 1, 2007, to December 31, 2015. Patients with type 1 diabetes were excluded. Data were analyzed beginning in the spring of 2018, and revisions were completed in 2019.For each patient, the initial diabetes drug choice was determined; drugs were classified as first generation  or second generation .The primary outcome was the between-practice variation in use of second-generation diabetes drugs between 2007 and 2015. Practices with use rates of second-generation diabetes drugs more than 1 SD above the mean were considered high prescribing, while those with use rates more than 1 SD below the mean were considered low prescribing.Among 1 182 233 patients who initiated diabetes drug therapy at 42 977 practices between 2007 and 2015, 1 104 718 (93.4%) were prescribed a first-generation drug  and 77 515 (6.6%) were prescribed a second-generation drug . By December 2015, 22 457 practices (52.2%) had used DPP-4 inhibitors once, compared with 3593 practices (8.4%) that had used a GLP-1 receptor agonist once. Furthermore, 17 452 practices (40.6%) were using DPP-4 inhibitors in 10% of eligible patients, while 1286 practices (3.0%) were using GLP-1 receptor agonists in 10% of eligible patients, and SGLT-2 inhibitors, available after March 2013, were used at least once by 1716 practices (4.0%) and used in 10% of eligible patients by 872 practices (2.0%) by December 2015. According to Poisson random-effect regression models, beneficiaries in high-prescribing practices were more than 3-fold more likely to receive DPP-4 inhibitors , 24-fold more likely to receive GLP-1 receptor agonists  and 60-fold more likely to receive SGLT-2 inhibitors  compared with beneficiaries in low-prescribing practices.These findings suggest that there was substantial between-practice variation in the use of second-generation diabetes drugs between 2007 and 2015, with a concentration of use among a few prescribers and practices responsible for much of the early diffusion. An additional 1.5 million new cases were being diagnosed annually, the vast majority of which were new cases of type 2 diabetes.3 While each of these drugs offer certain clinical advantages, they are all considerably more expensive than older, first-generation drugs.6 In addition, insulin, a first-generation injectable drug, is a more potent hypoglycemic agent than the GLP-1 receptor agonists or amylin analogs (both injectable medications), and first-generation oral medications  are generally more potent hypoglycemic agents than DPP-4 inhibitors or SGLT-2 inhibitors.7Some of this increased spending may be driven by the multiple second-generation diabetes drugs approved by the US Food and Drug Administration (FDA) and released into the market since 2000. Since then, 4 new classes of diabetes drugs have been approved by the FDA for type 2 diabetes. In 2005, the FDA approved amylin analogs and glucagon-like peptide-1 (GLP-1) receptor agonists. The FDA approved dipeptidyl peptidase 4 (DPP-4) inhibitors in 2006 and approved sodium-glucose co-transporter-2 (SGLT-2) inhibitors in 2013.8 The cardiovascular benefits of GLP-1 receptor agonists were not proven until June 2016.9 Thus, the diffusion of second-generation diabetes drugs as initial therapy between 2007 and 2015 affords a unique opportunity to describe the diffusion and drivers of early drug adoption absent of clear evidence of clinical superiority. To that end, this study aims to use national claims data to quantify the variability in second-generation diabetes drug diffusion between 2007 and 2015 and describe the patient, prescriber, and practice characteristics associated with early diffusion.Importantly, between 2007 and 2015, the cardiovascular benefits of second-generation diabetes drugs were suspected but had not yet been proven. It was not until the Empagliflozin Reduces Mortality in Patients With Type 2 Diabetes at High Cardiovascular Risk (EMPA-REG OUTCOME) trial was published in November 2015 that SGLT-2 inhibitors first demonstrated additional cardiovascular benefits.STROBE) reporting guideline for observational studies.This project was reviewed and approved by the Harvard Medical School institutional review board. Informed consent was waived, as this was a deidentified, retrospective, claims-based study. This study is reported following the Strengthening the Reporting of Observational Studies in Epidemiology  number of the clinician who prescribed the first diabetes drug. That patient was then attributed to that NPI prescriber.13 A single practice or TIN can represent a solo practitioner, a small physician group, or a larger health care group. Large organizations will occasionally bill using multiple TINs. Face-to-face visits were identified using evaluation and management Current Procedural Terminology (CPT) codes 99201 to 99215, 99241 to 99245, G0402, G0438, or G0438, with clinicians who had specialty codes for family practice (08), internal medicine (11), geriatric medicine (38), general practice (01), or endocrinology (46). If patients had the same number of face-to-face visits to more than 1 practice, they were attributed to the practice with the greater sum of allowed spending. Beneficiaries who could not be attributed to a practice or who were attributed to a practice with fewer than 3 patients newly prescribed diabetes therapy in the same year were excluded to ensure adequate intrapractice variability , responsible for a plurality of their face-to-face office visits using a modified version of the Center for Medicare & Medicaid Services’ 2-step attribution rule.Diabetes drugs were divided into 2 categories based on their date of FDA approval: first-generation drugs were approved before 2000, and second-generation drugs were approved after 2000. All drugs were identified in Part D using National Drug Codes. First-generation diabetes drugs include metformin, sulfonylureas, thiazolidinediones, α-glucosidase inhibitors, meglitinides, and insulin. Second-generation diabetes drugs include amylin analogs , glucagon-like peptide-1 (GLP-1) receptor agonists , dipeptidyl peptidase 4 (DPP-4) inhibitors , and sodium-glucose cotransporter 2 (SGLT-2) inhibitors . Amylin analogues were not considered in our analysis owing to low use rates.Initiation was defined as the first diabetes drug for which a patient had 2 or more prescriptions, 30 or more days but 365 days or fewer apart. For patients with only 1 fill of multiple diabetes drugs, the first diabetes drug prescribed was considered the initiation drug. Patients who received a combination drug  or combination therapy  that included a first-generation drug and a second-generation drug, it was considered initiation with the second-generation drug.14 Patient geography was ascertained using the patient’s zip code and the Centers for Disease Control and Prevention’s census regions.15 Population density and urbanicity were obtained using the 2010 rural-urban commuting area codes.16 Using Chronic Conditions Warehouse flags, we identified patient comorbidities, including acute myocardial infarction, atrial fibrillation, chronic kidney disease, congestive heart failure, hyperlipidemia, hypertension, ischemic heart disease, stroke or transient ischemic attack, and cancer. The community hierarchical condition category score was computed for each beneficiary using Center for Medicare & Medicaid Services’ published algorithm.18We determined patient age, sex, race/ethnicity, dual-enrollment status, and disability status directly from the enrollment file. Zip code level estimates for socioeconomic variables, including median household income, percentage of zip code residents 25 years or older who graduated from high school, and percentage of households below the poverty line within the zip code were obtained using the patient’s zip code and 5-year estimates from the 2012 American Community Survey data.CPT evaluation and management codes) in each calendar year for each prescriber. Prescribers without any face-to-face office visits in the outpatient or carrier file  were considered a separate category because they comprised 6.7% of diabetes drug prescribers in 2007 and 10.8% in 2015. Based on a local random sample, these prescribers with no outpatient panel were a heterogeneous group of hospitalists, hospital-based advanced practice clinicians , and residents or fellows who bill for inpatient but not outpatient services. Finally, we determined the proportion of patients in a prescriber’s panel with diabetes  using Chronic Conditions Warehouse flags for diabetes.For each prescriber , we determined clinician type  based on the specialty code used most often for the clinician’s face-to-face visits. We also determined the prescriber’s outpatient panel size  using the number of unique face-to-face office visits , we determined the number of all Medicare beneficiaries attributed to the practice in each year, the number of clinicians affiliated with the practice, the ratio of affiliated clinicians to attributed beneficiaries, whether the practice was part of an academic medical center, and whether the practice was hospital-owned. Patients were attributed to the practice where they received most of their face-to-face primary care office visits.The primary outcome of this study was between-practice variation in the use of GLP-1 receptor agonists, DPP-4 inhibitors, and SGLT-2 inhibitors between 2007 and 2015. Between-practice variation was defined as the relative risk (RR) that a patient treated at a high-prescribing (defined as ≥1 SD above the mean) practice would receive a second-generation diabetes drug compared with a patient treated at a low-prescribing (≥1 SD below the mean) practice, after adjustment for predetermined patient, prescriber, and practice covariates. Secondary outcomes include the association of prespecified comorbidities with second-generation diabetes drug use rates and the association of clinician and practice characteristics with second-generation diabetes drug use rates.21) for each practice. This allowed us to create practice-level diffusion curves that show the percentage of practices that have used each second-generation diabetes drug at least once over time and the percentage of practices that are using each second-generation diabetes drug routinely over time. In our primary analysis, we determined the between-practice variation in use of second-generation diabetes drugs using Poisson random-effects regression models and calculated the RR that a patient treated at a high-prescribing practice would receive a second-generation diabetes drug compared with a patient treated at a low-prescribing practice, after adjustment for patient, prescriber, and practice characteristics that had been determined a priori based on clinical knowledge . Data were analyzed beginning in the spring of 2018, and revisions were completed in 2019.We analyzed data collected from January 1, 2007, to December 31, 2015. For continuous variables, we computed means with SDs and medians with interquartile ranges. For categorical variables, we calculated the number and percentage. We then determined the number and percentage of patients started on each diabetes drug in 2007, 2015, and pooled from 2007 to 2015. We determined time to first use of each second-generation diabetes drug for all practices and time to routine use of each second-generation diabetes drug  to receive a DPP-4 inhibitor compared with patients at low prescribing practices . Patient22 ischemic heart disease for GLP-1 receptor agonists,23 and heart failure for SGLT-2 inhibitors.8 We then estimated the RR of initiation with each second-generation drug in each disease subgroup (P < .001), and patients with concurrent ischemic heart disease had an RR of 1.13  to receive a GLP-1 receptor agonist (P < .001). Patients with congestive heart failure were not more likely to receive SGLT-2 inhibitors , although this may be limited by less than 2 years of data.To better understand the drivers of this variation, in secondary analyses, we considered predetermined patient, clinician, and practice characteristics. First, we created disease subgroups in which second-generation diabetes drugs had shown early evidence of advantage: chronic kidney disease for DPP-4 inhbitors,subgroup . PatientP < .001; DPP-4 inhibitors: RR, 1.44 ; P < .001; GLP-1 receptor agonists: RR, 2.35 ; P < .001) or when the prescribers’ panel had more than 50% of patients with diabetes . Prescribers without outpatient panels were more likely than endocrinologists to prescribe second-generation diabetes drugs . DPP-4 inhibitors diffused more slowly in practices that were large , part of an academic center , or hospital-owned . GLP-1 diffusion was not associated with any practice characteristics, but similar to DPP-4 inhibitors, SGLT-2 inhibitors also diffused more slowly in hospital-owned practices .Finally, we considered the associations of prescriber and practice characteristics with diffusion variability . We founBetween 2007 and 2015, 7% of Medicare patients newly treated for type 2 diabetes were prescribed comparatively expensive second-generation diabetes drugs. By 2015, the most commonly initiated second-generation diabetes drugs were DPP-4 inhibitors, but SGLT-2 inhibitors diffused rapidly after their FDA approval in 2013. Diffusion of GLP-1 was much slower. Overall, we found substantial between-practice variation in the use of these novel therapies, suggesting a concentration of use within a few high prescribers and practices responsible for much of the early diffusion.26 While claims data may miss some $4 generics fills when patients pay out of pocket, prior data suggest that among Medicare beneficiaries, the rate of use of $4 generic programs is comparatively low (16.3% of beneficiaries) and that even when the $4 generic program is used, a $4 charge for the fill can almost always still be observed in Part D data.27 Therefore, the fact that 7% of patients were prescribed markedly more expensive diabetes drugs, either as first or very early second-line therapy, that had not yet demonstrated any cardiovascular morbidity or mortality benefit beyond older first-generation drugs is an important observation.To illustrate the difference in cost, during this time, metformin, glipizide, and glyburide (first-generation diabetes drugs) were available as $4 generics while second-generation drugs cost between $300 and $500 per month.To explain the difference in diffusion trends among the second-generation diabetes drugs, we hypothesized that specific aspects of the drugs themselves affected early diffusion. For example, the higher rates of use of DPP-4 inhibitors and SGLT-2 inhibitors compared with GLP-1 receptor agonists could potentially be explained by the oral availability of DPP-4 inhibitors and SGLT-2 inhibitors. Normally, the longer drugs are available, the more they diffuse and the less their use becomes concentrated among a small number of clinicians with experience and comfort using them, as illustrated by the RR covariance of DPP-4 inhibitors compared with SGLT-2 inhibitors. In contrast however, the RR covariance of GLP-1 receptor agonists did not decrease over time, suggesting its use remained concentrated among a small group of prescribers and practices over the period of the study. This is likely because GLP-1 receptor agonists are injection drugs, and their use requires more expertise and the ancillary support capacity to teach injection techniques.29 Additionally, GLP-1 agonists frequently cause weight loss30 and so combined with preliminary data from the Liraglutide Effect and Action in Diabetes: Evaluation of Cardiovascular Outcome Results trial9 suggesting a possible decreased rate of cardiovascular death with liraglutide; specifically, GLP-1 agonists may have been particularly attractive for patients with type 2 diabetes and ischemic heart disease. Furthermore, SGLT-2 inhibitors increase glycosuria and thus enhance diuresis,31 and, combined with preliminary data from the EMPA-REG OUTCOME study,8 it is possible that the clinician anticipated an advantage for SGLT-2 inhibitors in patients with diabetes and congestive heart failure. However, when we examined early diffusion, we found that while there was a higher RR of use in these specific patient subgroups, most of the early use was concentrated among a few high-prescribing clinicians and practices and that these groups were the largest driver of early diffusion.Next, we hypothesized that part of the explanation for this higher than anticipated use of expensive second-generation diabetes drugs might have been early signals of benefit in patients with specific comorbidities. For example, as early as 2007, DPP-4 inhibitors were thought to be safer for patients with chronic kidney disease , a group of patients for whom metformin is often contraindicated and sulfonylureas have a higher risk of hypoglycemia.33 However, in contrast to prior studies, we also found that a novel group of clinicians—those without outpatient panels—were even more likely than endocrinologists to initiate second-generation diabetes drugs. To better understand who these prescribers were, we identified a random sample of 50 such prescribers from Massachusetts and found that this group was comprised of inpatient-based hospitalists, hospital-based advanced practice clinicians , and trainees . Prior data has suggested that inpatient clinicians may have more exposure to clinical trials32 or more experience with complex patients and that these factors may accelerate drug diffusion.33 Additionally, we hypothesize that initiating a novel therapy in the inpatient setting may be perceived as lower risk, because laboratory tests can be monitored closely. Moreover, many hospitals now have specialized inpatient services  and more focused experience to quickly develop a sense of comfort in using novel drugs.Consistent with prior work, we found that overall, novel second-generation drugs diffused more rapidly among specialists  and clinicians who cared for large numbers of patients with diabetes.Additionally, this observation raises questions on whether traditional tools that limit expensive drug use, such as prior authorizations, are as effective if the drug is initiated in the inpatient setting or at the time of discharge. In such a case, the burden to prior authorization paperwork may fall on the outpatient clinician. If the patient is doing well and the outpatient clinician is less familiar with the novel drug, the clinician may be more reluctant to change therapies, creating a stronger incentive for outpatient clinicians to scale the regulatory hurdles necessary to continue the drug. If true, this finding highlights the effect that hospital formularies may have on the diffusion of new therapies and suggests the need for alternative regulatory tools to monitor expensive novel therapies initiated in the hospital setting.Our study has several limitations. First, the results of this study must be interpreted within the context of the population studied. This study examined diffusion trends in older patients (age ≥66 years) with Medicare data. Therefore, the observed trends and patterns may not be applicable to younger populations or those with different insurance coverage. Second, like all claims-based studies, this study is limited by the lack of granular clinical data to further describe the characteristics of patients, clinicians, and practices initiating or being prescribed second-generation diabetes drugs. Third, this study is limited by the incomplete nature of claims data. For example, not all patients had a valid zip code or could be attributed to a valid practice with 3 or more eligible patients, which limited their inclusion in our study. The findings for these populations may be different, although our analysis of baseline characteristics would suggest that they are not (eTable 3 and eTable 4 in the The findings of this cross-sectional study suggest that there was significant variation among practices in their use of second-generation diabetes drugs as first-line therapy for diabetes between 2007 and 2015. Before there was any evidence of superior benefit, 7% of Medicare beneficiaries newly prescribed diabetes therapy received an expensive second-generation diabetes drug as first-line therapy. While early signals of benefit and patient comorbidities play a role, clinician and practice patterns played a more significant role. Much of the early use of these drugs was concentrated among a few clinicians and practices that drove most of the early diffusion. This variation in practice patterns highlights a potential shortfall of traditional cost containment mechanisms for prescription drugs and opportunities to improve the value of early diabetes care.


When preparing for the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the coronavirus infection disease (COVID-19) questions arose regarding various aspects concerning the anaesthetist. When reviewing the literature it became obvious that keeping up-to-date with all relevant publications is almost impossible. We searched for and summarised clinically relevant topics that could help making clinical decisions. This is a subjective analysis of literature concerning specific topics raised in our daily practice ; antiviral treatment) and we believe that these answers help colleagues in clinical decision-making. With ongoing treatment of severely ill COVID-19 patients other questions will come up. While respective guidelines on these topics will serve clinicians in clinical practice, regularly updating all guidelines concerning COVID-19 will be a necessary, although challenging task in the upcoming weeks and months. All recommendations during the current extremely rapid development of knowledge must be evaluated on a daily basis, as suggestions made today may be out-dated with the new evidence available tomorrow. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak emerged in Wuhan, China on 31 December 2019 and the resulting coronavirus infection disease (COVID-19) has been declared a pandemic by the World Health Organization on 11 March 2020. To date, four different common coronaviruses are known causing for millions of cases of respiratory infections all over the world. The SARS-CoV-2 is a single-stranded RNA virus and has, based on decoration with glycoprotein spikes, a crown-like membrane. The nucleocapsid includes proteins and the RNA genome, which encodes polyproteins, e.g., replicase, proteases and structural proteins. All these proteins can interfere with the human innate immune system. Angiotensin converting enzyme 2 (ACE2), a cellular receptor present in the lung, binds to viral proteins and serves as entry for the virus after host-viral membrane fusion. Subsequently, after endocytosis, the RNA genome is released into the host cell and translated and replicated within the cell. Detection of virus RNA by real-time reverse transcriptase polymerase chain reaction (RT-PCR)-based methods is a keystone in diagnosing current infection with SAS-CoV-2, while detection of immunoglobulins helps to test for previous infections and immunity. The latter can in the future hopefully be achieved by adequate vaccination, and different strategies are currently explored to find a respective vaccine, e.g., live attenuated vaccines, viral vector-based vaccines, recombinant protein-based vaccines, DNA or messenger RNA vaccines.Yearly, up to 2.6 million people die from respiratory infections, and the consequences of SARS-CoV-2 infection should be interpreted in the light of these numbers ,2. ProgrClinically severe infections  with a wide diversity of presenting symptoms, including altered mental status, myocardial, hepatic and kidney injury, and gastrointestinal symptoms like prolonged periods of nausea, vomiting and diarrhoea have been described. Most patients infected with SARS-CoV-2 have mild respiratory symptoms and a good prognosis after infection; these patients will not be presented to the anaesthetist or intensive care physician. However, some patients develop severe hypoxic respiratory failure early in the disease course, making non-invasive or invasive application of oxygen necessary. Next to hypoxemia, exhaustion from respiratory efforts may also be an indication for early endotracheal intubation.Any application of oxygen carries a significant risk of aerosolisation of virus material and, thereby, potentially disease transmission . PrelimiLungs of COVID-19 patients are characterised by the coexistence of severely affected lung areas with atelectasis that are not, or hardly, recruitable and adjacent to areas that are unaffected and remain remarkably compliant, which are at risk of over-distension. One important aim of invasive ventilation in general, including patients with COVID-19, is to prevent damage caused by the ventilator by protecting undamaged and otherwise fragile lung tissue. This overrules the aims of achieving normoxaemia and normocapnia.T) to 6 mL/kg ideal body weight (IBW) can reduce mortality by up to 25% in patients with acute respiratory distress syndrome (ARDS) [T, in particular for patients on assist ventilation . The use of a low VT of 6 mL/kg IBW, or even lower, is probably also beneficial in COVID-19 patients. Of note, IBW is not the same as actual body weight, and is calculated from the height of a patient; a simple calculation for a woman is “height (in cm)—110” and for a man “height (in cm)—105”. Shortly after intubation and start of invasive ventilation, a default VT of 350 mL in women and 400 in men turned out to be practical; this can be further adjusted after arrival in the ICU. When using a low VT, a higher respiratory rate, sometimes up to 35–40 per minute, has to be used to achieve an adequate minute volume. This high respiratory rate can generally be accepted, even when an increase in “dead space ventilation” results in hypercapnia, as long as the blood pH remains above 7.2 . Use of a low VT can also result in a slight deterioration in oxygenation, which can be compensated for with an increase in the inspired fraction of oxygen (FiO2). When the ventilator is converted from assist ventilation to pressure support ventilation, the VT will increase, sometimes well above 6 mL/kg IBW, which can be accepted if the driving pressure  remains low.Limiting tidal volumes (Ve (ARDS) . InternaDifferent morphologic phenotypes in ARDS exist: ‘focal, non-recruitable’ and ‘non-focal, recruitable’, needing different therapeutic approaches see . COVID-12O. Use of PEEP recruits collapsed lung areas and also keeps these areas partially open. However, aggressive lung recruitment manoeuvres with use of ‘preventive high PEEP’, with an intention to maximally recruit lung parenchyma, is harmful and associated with excess mortality in patients with ARDS [Patients with ARDS need more positive end-expiratory pressure (PEEP) than patients without ARDS, usually with an initial setting of 10 cm Hith ARDS . A major2 table, with a personalized approach based on lung morphology in which patients with ‘focal’ ARDS received low PEEP in the prone position, and patients with ‘non-focal’ ARDS received recruitment manoeuvres and high PEEP [2 table in combination with early prone positioning might be preferred. PEEP can lead to better oxygenation, which can be falsely interpreted as a ‘success’ of the intervention, because it signifies successful lung recruitment. However, it is important to remember that oxygenation is not the only goal, and that lungs of patients with COVID-19 are characterised by non- or hardly-recruitable, severely affected lung areas. Recruitment through increasing PEEP should only be pursued in case of severe hypoxaemia that does not respond to an increase in FiO2 well above 0.6. Too high PEEP (>10 to 12 H2O) will cause overdistension of unaffected lung areas, which can then result in an increase in driving pressure. For this reason the recommendation is to apply not more than 10–12 cm H2O PEEP, and only use higher PEEP levels when concomitant driving pressure is reduced. Last but not least, PEEP levels > 10 cm H2O can compromise cardiac performance, with negative effects on preload, resulting in reduced blood pressure and a possibly increased need for vasopressors and intravenous volume, which are both associated with increased morbidity in ARDS patients. For COVID-19 patients requiring large doses of vasopressors to maintain an adequate blood pressure, excessive PEEP should be reconsidered.One recent study that compared ventilation with PEEP according to a low PEEP and FiOigh PEEP , suggest2O, often as low as 5–7 cm H2O. The driving pressure, which is by approximation the difference between the maximum airway pressure (in pressure controlled ventilation) or the plateau pressure (in volume controlled ventilation), and the PEEP level, is associated with patient outcome [T or excessive PEEP. While there are currently no randomised studies, neither in patients with ARDS due to other diseases nor in COVID-19 patients, on interventions targeting driving pressure, we favour ventilation with a low driving pressure where possible. The easiest way to lower the driving pressure is to (further) limit VT. A reduction in PEEP may also have a beneficial effect on the driving pressure.COVID-19 patients show next to the affected lung areas, remarkably compliant unaffected lung areas, which can be ventilated with driving pressure well below 15 cm H outcome . DrivingProne positioning improves outcomes of patients with ARDS . It resuWe prefer to closely monitor the above, preferably with each nursing shift and maybe even more frequently. A practical list of what to monitor, and how to improve settings is provided in As for other respiratory viral infections, laboratory evidence of SARS-CoV-2 infection can be provided directly, by detection of the virus in clinical specimens, or indirectly, by detection of specific antibodies in blood. The benefits of both diagnostic strategies depend on the stage of the infection : viral sFor this reason, the current recommended strategy for laboratory diagnosis of COVID-19 during the acute stage of the infection is the detection of SARS-CoV-2 RNA in clinical specimens by real-time reverse transcriptase polymerase chain reaction (RT-PCR)-based methods. Based on viral genetic sequences from the initial isolated viruses during the early stages of the epidemic, highly sensitive and specific assays were immediately developed, targeted at genes encoding for the viral RNA-dependent RNA polymerase (RdRp) and envelope . This asThe sensitivity of PCR- or antigen-based virus detection assays is dependent on the quality of the specimen, hence adequate instruction and training in collection of specimens is essential. Nasopharyngeal or combined nasal- and oro-pharyngeal swabs are the preferred specimens for SARS-CoV-2 testing. Ideally, flocked swabs should be used as these increases the yield of cellular material, whilst swabs containing cotton or wood should be avoided as these may inhibit PCR reagents. After collection, swabs for PCR testing should be transferred to a transport medium that preserves viral RNA.Sensitivity of PCR-based diagnostics of upper respiratory tract specimens seems generally high during the acute stage of infection. Compared to mild infections, severe COVID-19 is associated with prolonged duration (longer than 10 days from illness onset) and higher levels of viral RNA . HoweverSerological assays that detect virus-specific antibodies, such as immunoglobulin (Ig) A (IgA), IgM or IgG, in blood or saliva are essential to diagnose recent or past infections; their diagnostic role during the acute stage of infection, early after symptom onset, is limited. Potential pitfalls of antibody detection assays include limited specificity, for example due to cross-reactivity with antibodies to other human coronaviruses, as well as limited sensitivity, which may pose a particular concern in diagnosing mild infections which may be associated with weaker antibody responses . AlthougStudies evaluating the diagnostic value of IgM and IgG detection in COVID-19 patients showed higher sensitivity of PCR during the first 5–7 days after illness onset and higher sensitivity of IgM detection thereafter ,28. HighIn summary, while PCR detection of viral RNA in respiratory specimens remains the mainstay of diagnostics in patients presenting with suspected COVID-19, there may be a role for serological tests in patients presenting late during the course of infection, for example with progressive or complicated disease, at a time when viral loads may have dropped, especially in the nasopharynx, while an antibody response is evolving . In addiThere is an ongoing discussion as to whether chest computer tomography (CT) can reliably prove SARS-CoV-2 infection and COVID-19, and whether every patient undergoing surgery needs to have a chest CT before surgery. Up to now, the role of CT scan for diagnosing COVID-19 is founded on a very limited number of patients and might be useful in patients with a clinically-suspected SARS-CoV-2 infection and a negative RT-PCR test . Differespecific method of diagnosis [In light of these data the CDC does not currently recommend chest CT to diagnose COVID-19. Viral testing remains the only iagnosis . Confirmiagnosis .Anosmia is increasingly recognised as a symptom in COVID-19 infection. It can accompany other mild respiratory symptoms, or can present as an isolated finding . In a EuThe SARS-CoV-2 has an extreme transmissibility, and even asymptomatic people can transmit the infection . High viA much higher prevalence of influenza than COVID-19 during the winter period made pneumonia as a result of other than SARS-CoV-2 infection likely during the beginning of the pandemic. This holds in particular true for children, infants and neonates: neonatal respiratory failure can result from a wide range of causes, and infection with other viruses are likely in this patient population .In the beginning of the pandemic it seemed that children were spared from COVID-19, but recent data show that children of all ages can be infected: a review of 45 publications revealed that 1–5% of the diagnosed COVID-19 cases were children, with more asymptomatic cases than in adults . Recent Although disease transmission seem to be primarily by aerosol or fomite, recent reports indicated a high percentage of SARS-CoV-2 positive rectal swabs outlasting the nasopharyngeal swab up to 24 days (range 6–24) ,56,57. TSpecial attention should be paid to the emotional burden in caring for paediatric patients. The health professional might be more willing to accept non-adherence to hygiene rules in order to act faster in an emergency situation with a child, or to make life more comfortable for the child. Similarly, parental fears are keeping children out of hospitals and emergency rooms and leads to delays in not-COVID related therapies/operations urgently needed ,59.Although generally in (suspected) COVID-19 patients as few persons as possible should be attending during induction of anaesthesia, attending parents can help to increase comfort and cooperation of the child. Furthermore, parents who choose to attend an emergency procedure of their child reported that they felt it was beneficial for themselves and for the child .The large proportion of asymptomatic children makes it difficult to identify paediatric patients, who might be a source for further transmission and spreading of the virus ,62. ChilPreliminary data from Italy suggest that among patients who are followed-up for cirrhosis, transplantation, autoimmune liver disease, chemotherapy for hepatoblastoma, none (children and adults) developed a clinical pulmonary disease, despite some patients tested positive for SARS-CoV-2 . AdditioThe SARS-CoV-2 gains entry to pulmonary cells after binding to membrane angiotensine converting enzyme (ACE) 2, which is a homologue of ACE1, the enzyme that converts angiotensin I to angiotensin II . The rolPublished data  suggestsIn China, the origin of the majority of available data today, researchers reported a prevalence of DM as high as 20% in COVID-19 patients ,74, whilEven more, several studies show an increased risk or poor outcome for patients with DM ,78,84. PNo verified antivirals specific to COVID-19 exist at present. The efficacy and safety of candidate drugs in the treatment of COVID-19 need to be confirmed in future preclinical and clinical trials. The transition from first respiratory symptoms to ARDS is probably based on uncontrolled cytokine release. Chloroquine has a promising inhibitory effect on cytokine release, however, its clinical use is limited and must be balanced against severe side effects: to be effective, chloroquine must be dosed in its toxic range. Hydroxychloroquine, which exhibits an antiviral effect highly similar to that of chloroquine, could attenuate the severe progression of COVID-19, inhibiting the cytokine storm by suppressing T cell activation. Hydroxychloroquine has a modulating effect on activated immune cells, downregulates the expression of Toll-like receptors and Toll-like receptor mediated signal transduction, and decreases the production of interleukin-6. However, clinical trials are necessary to confirm the hypothesis that hydroxychloroquine exerts beneficial effects in COVID-19 patients .Identification of candidate drugs and potential drug combinations targeting SARS-CoV-2 employing network proximity analyses of drug targets and human CoV (HCoV)-host interactions in the human interactome revealed 16 potential anti-HCoV repurposable drugs . Some ofRenal involvement in COVID-19 patients may be caused by direct infection, or may have a multitude of other etiologies, as occurs in critical illness in general. The human kidney might be a specific target for SARS-CoV-2 infection: post-mortem, SARS-CoV-2 antigens accumulated in kidney tubules, suggesting that SARS-CoV-2 infects the human kidney directly, inducing acute kidney injury (AKI) and contributing to viral spreading in the body . Early rclinicaltrials.gov) of remdesivir the presence of alanine aminotransferase or aspartate aminotransferase levels above five times the upper level of normal is a contraindication for inclusion.Hepatic involvement in COVID-19 may be part of multiple organ failure in severely affected patients. The liver might also be involved due to ACE2 expression in liver and bile duct cells , or by tThe role of NSAIDs and in particular ibuprofen is a good example for a “breaking news” having a higher “reproduction number” than the virus itself. It originates from reports in French media that four young patients with COVID-19 deteriorated after taking ibuprofen, leading to a recommendation to use paracetamol instead of ibuprofen by the minister of health in France . HoweverFurthermore, ibuprofen has respiratory and cardiovascular adverse effects which are relevant to patients at high-risk during a SARS-CoV-2 infection. The Centre for Evidence-Based Medicine of the University of Oxford generally questions the routine anti-pyretic administration of both paracetamol and NSAIDs in acute respiratory infections and COVID-19 . Their dBy giving answers to the above questions that were intensely discussed within our specialism, we hope to help colleagues in clinical decision-making. With ongoing treatment of severely ill COVID-19 patients other questions will come up, e.g., possible cardiac manifestations of the infection and influences of cardio-vascular co-morbidities on the course of the illness, whether plasma from convalescent patients could be used for therapy , or whet


Chimerism after orthotopic liver transplantation (OLT) has largely been investigated in intrahepatic cellular constituents. However, little is known about chimerism in the extrahepatic and large intrahepatic bile ducts. Our aim was to evaluate the presence and extent of chimerism after OLT in the peribiliary glands (PBG) and the luminal epithelium of the large donor bile ducts.in situ hybridization (FISH) for Y and X chromosomes was performed and the percentage of XY positive cells was determined among biliary epithelial cells. Immunohistochemistry was used to correlate chimerism with histological features.For this study, we examined six extrahepatic and large intrahepatic bile ducts from livers that were re-transplanted. In all cases there was a sex-mismatch between donor and recipient , which allowed to discriminate between donor- and recipient-derived cells. Specimens from female to female transplants were used as negative controls and male to male transplants as positive controls. Fluorescence Cholangiocellular chimerism in all studied specimens ranged from 14 to 52%. The degree of chimerism was not associated with biliary damage. Marked chimerism was present at 5 days post-OLT. Ki-67-positivity was detected in 1–8% of the epithelial cells at the time of liver re-transplantation, and this correlated inversely with the degree of chimerism.Recipient-derived cholangiocytes are present in the large bile ducts of the donor liver after OLT. The presence of chimerism in the large bile ducts suggests that recipient-derived cells may play a role in biliary regeneration following ischemia-induced injury during OLT. Post-transplant cholangiopathies continue to be a major challenge in clinical liver transplantation, in terms of morbidity and mortality. Incidence rates range between 1 and 30%, depending on variations in ischemia times and donor variables . After cex vivo model mimicking OLT, PBG cells started to proliferate 24 h after severe ischemic injury and formed new patches of biliary epithelium 72 h later (Peribiliary glands (PBG) are progenitor/stem cell niches arranged in a three-dimensional peribiliary network in the wall of large bile ducts . Besides h later . This su h later .While chimerism, characterized by the presence of recipient-derived cells among donor cells, has been shown to play a role in tissue remodeling and repair of the liver parenchyma after OLT , it is un = 3) and positive controls  were included. The reason for re-transplantation in the study group included distinct pathological liver and bile duct conditions  of six adult male patients that previously received a female full-sized liver graft were obtained during re-transplantation. Appropriate negative controls  overnight, for vWF with 0.2% pepsine pH 2.0 and for Ki-67 with Tris–EDTA pH 9.0 buffer in the microwave for 15 min. After PBS for 5 min, antibodies for CK19 , vWF , α-SMA , and Ki-67  were applied for an hour. Next, a 30 min-incubation was applied with peroxidase-labeled goat anti-rabbit antibody (for CK19 and vWF) or rabbit anti-mouse antibody (for α-SMA and Ki-67) in dilution 1:100. Rabbit anti-goat for CK19 and vWF and goat-anti rabbit for α-SMA and Ki-67 were used as third antibodies (1:100 dilution). The staining reaction was developed by 3,3’-diaminobenzidine (DAB) and counterstained with hematoxylin. Stained slides were scanned by a digital scanner and processed by ImageScope. The relative number of destroyed PBG was estimated by two (blinded) independent researchers and a semi-quantitative (SQ) score was applied . Likewise, a SQ score for the number of inflammatory cells around PBG in the section was evaluated by two independent researchers, but a different SQ score was appropriate for the observations . To quantify vWF, α-SMA, and Ki-67 expression, we used QuPath v0.2.0 to develop an appropriate classifier for each staining (Sections were prepared for hematoxylin and eosin (H&E) staining for histological injury assessment. All bile duct sections were examined in a blinded fashion by two experienced liver pathologists (MVDH and ASHG). Immunohistochemistry for cytokeratin 19 (CK19), von Willebrand factor (vWF), alpha-smooth muscle actin (α-SMA), Ki-67 and cluster of differentiation 45 (CD45) was performed for histological characterization of the sections. The immunostaining for CD45 was performed by using an automated immunostaining system  and by applying the Ultraview DAB detection kit of the same company. For all other immunostainings, tissue sections were deparaffinized through a graded alcohol series and rinsed in phosphate-buffered saline . Endogenous peroxidase activity was blocked by a 30-min incubation in Hstaining . For vWFBright495 (green)] and Y  probes  was applied to the air-dried sections. A cover slip was applied and sealed with Fixogum. Denaturation and hybridization were performed in a humidified chamber  at 80 degrees for 5 min and at 37 degrees for 16 h, respectively. Next, sections were covered in 2xSSC/0.1% Igepal at room temperature for 2 min followed by incubation with 0.4xSSC/0.3% Igepal at 72 degrees for 2 min and incubation with fresh 2xSSC/0.1% Igepal at room temperature for 2 min. Sections were dehydrated, airdried and counterstained with 4,6-diamidino-2-phenylindole (DAPI)/antifade 1 μg/ml.Tissue sections were deparaffinized in xylene for 2×15 min, dehydrated through a graded alcohol series, and then rinsed with distilled water for 4 min. We used a tissue digestion kit for all pretreatment and washing steps . Sections were incubated at room temperature for 30 min with 0.2 HCl and rinsed with distilled water. Then, to make DNA accessible for hybridization, tissue slides were treated with 8% sodium thiocyanate at 84 degrees for 40 min, rinsed with 2xSSC, and incubated at room temperature with a pepsin solution for 30 min that was 3 times renewed. Next, slides were rinsed with distilled water and 2xSSC followed by dehydration and air drying. 7–10 μl of a mixture of X [DXZ1, conjugated with Platinum-in situ hybridization (FISH) on the same section. The sections stained with CK19 and CD45 were first scanned with the Bioview duet-3 Software, put in xylene for 1 week, and, then, the sections were treated with FISH and scanned again with the same software. Localization of PBG and luminal epithelium was confirmed by the matching procedure in which the fluorescence section was matched with histology. A total of 25 fields of interest were selected using both the histology and the fluorescence scan. Next, 25 photomicrographs of each section were taken with green and red filter sets of Vysis . All photomicrographs were checked for accuracy. Photomicrographs in which no PBG and luminal epithelium were present or not well-recognizable were excluded. In addition, photomicrographs which showed areas without red and green signal were also excluded. In the end, two to nine photomicrographs of each biopsy in the study group were analyzed. Nuclei in which a red signal (Y chromosome) and green signal (X chromosome) were present were counted as recipient-derived cells. Nuclei that contained two green signals (X chromosomes) were counted as native donor cells. Nuclei that did not show clear signals were excluded from the counting.Fluorescence microscopy was performed using an Olympus BX63 microscope  equipped with a CCD video camera and Bioview duet-3 Software. We first scanned the cross-section at low power to allow for a wide field of view. To confirm that the counted cells were classified as epithelial cells – and not inflammatory cells - we performed immunohistochemistry for CK19 and CD45 prior to fluorescence Continuous variables were presented as mean ± standard deviation. GraphPad Prism 8  was used for presenting data in graphs. The Spearman correlation test and the Mann-Whitney U test to calculate significance for non-parametric data were performed using SPSS software version 23 for Windows .The initial indication for OLT, reason for re-transplantation, recipient age, ischemia times, time between primary OLT and re-transplantation, and donor age and type of the sex-mismatched transplantations are summarized in The diameter of the lumens of all examined donor bile ducts ranged from 1.7 to 7.8 mm, corresponding to the common bile duct or to the right or left hepatic bile duct; collectively called large ducts. Histologically, the bile duct biopsy of patient #1, re-transplanted for a post-transplant cholangiopathy, was marked by a largely intact epithelium and mild chronic inflammation in the bile duct wall with infiltration of lymphocytes and plasma cells. PBG showed hyperplasia of both deep and periluminal PBG, mild chronic inflammation with interstitial fibrosis between the acini, and dilated ducts. The biopsy taken from the large donor bile duct in patient #2, re-transplanted for recurrence of hepatitis C, was characterized by an absent luminal epithelium. The wall showed moderate chronic inflammation with fibrotic features. The periluminal PBG were relatively intact with desquamation of the epithelium and deep PBG were absent . PBG celFISH analysis of X- and Y-chromosomes was used to detect male recipient-derived cells in the female donor bile ducts. Fluorescence and histology images were matched  in orderR2 = −0.81; P = 0.037). Detected by the presence of XY chromosomes, recipient-derived cells were found in different ratios in all six large duct biopsies of patients that received a sex-mismatched liver graft . Y-positIn the present study, we demonstrated the presence of CK19 positive and CD45 negative recipient-derived cells in the donor large bile ducts. Epithelial chimerism ranged between 14 and 52% and was observed as soon as 5 days after OLT. Evidence of cell proliferation was observed in 1–8% of the biliary epithelial cells at time of liver re-transplantation, and this correlated inversely with the extent of chimerism. Clustered localization of recipient-derived cholangiocytes suggested a clonal expansion. While chimerism after OLT has largely been investigated in intrahepatic cellular constituents, this is the first study in which cholangiocellular chimerism is demonstrated in the large donor bile ducts.We could not find a relationship between the extent of chimerism and the time interval between OLT and sampling at re-transplantation. This is in accordance with an earlier study in which a time-dependent correlation was observed for hepatic chimerism, but not for chimerism of the intrahepatic small cholangiocytes. In that study, more hepatocellular chimerism was observed in liver biopsies taken at a longer time interval after OLT, compared to biopsies taken early after the procedure, whereas such pattern was not noted for the small intrahepatic cholangiocytes . In contThe extent of biliary injury did not seem to influence the engraftment of recipient-derived cells. Moreover, microvascular density, myofibroblast activation, and inflammation were not correlated with cholangiocellular chimerism. Most severe damage was observed in the bile ducts of patients #4 and #5 and the least severe biliary damage in patient #1. All these three patients suffered from post-transplant cholangiopathies, but cholangiocellular chimerism rates in these patients were moderate, compared to the others. As reflected by the radiographic presentation of post-transplant cholangiopathies, biliary lesions are often discontinuous along the biliary tree. The morphological abnormalities we observed in the present study may therefore have been influenced by “sampling error.” Moreover, damage or loss of epithelial cells may have influenced the extent of chimerism: Loss of recipient-derived epithelial cells in the donor bile duct later in the disease development due to ongoing injuries can, unfortunately, not be quantified, thus the accumulative number of recipient-derived cells remains elusive.Chimerism of intrahepatic small cholangiocytes in a study of Up to date, multiple studies have demonstrated recipient-derived cells in organ allografts . Two queRegarding the first question, various theories have been proposed to explain hepatic chimerism: fetal microchimerism, bone marrow-derived stem cells, cell fusion, and circulating epithelial cells. The first one refers to fetal cells that are known to persist in the maternal circulation after pregnancy which could explain non-self cells in the donor liver after OLT . Two preex vivo human bile duct slice model, PBG cells were shown to replicate, mature and migrate toward several denuded surfaces within 3 days  were not able to demonstrate a clear correlation between liver and bile duct injury and the extent of post-OLT hepatocellular or cholangiocellular chimerism . InteresIn addition, as applies equally for our study, detection of recipient-derived cells relies on molecular experiments  to label DNA fragments in archival frozen or paraffine-embedded specimens. These experiments are particularly challenging as proper preservation of DNA fragments requires optimal conditions during sample processing. Heterogeneity in sample processing makes quantification of recipient-derived cells therefore rather controversial and previously reported numbers may have been under- or overestimated .In conclusion, this study provides evidence for the presence of recipient-derived cells in the large donor bile ducts after OLT. Recipient-derived cells may contribute to regeneration of the damaged donor bile duct and distinctive origins of these cells could explain the divergent results obtained in liver chimerism studies up to now. This study provides an opportunity for a wide array of mechanistic studies, with the ultimate goal to identify methods that can be applied to stimulate biliary regeneration. These studies should focus on the source of recipient-derived cells in the large donor bile ducts and address the question what the drivers are behind biliary chimerism. To that end, lineage tracing studies could confirm the source of recipient-derived cells and expression profiles of high- and low-chimerism PBG could be compared for deeper insights regarding chimerism-stimulators.The datasets generated for this study are available on request to the corresponding authors.Ethical review and approval was not required for the study based on archived human biomaterials in accordance with national legislation and the code for usage of human remnant material .IJ participated in the research design, in performing laboratory experiments, interpreting the results, and writing the manuscript. MS contributed to the research design and set up of the experiments. MH participated in the histological analyses. AG participated in interpreting the results, histological analyses, and revising the manuscript. RP participated in the research design, interpreting the results, and revising the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The objectives of this study were to evaluate treatment in patients on current programmatic multidrug-resistant tuberculosis (MDR-TB) regimen and verify eligibility for the 9-month regimen and therapeutic drug monitoring (TDM).We performed a retrospective chart review of patients with MDR-TB receiving standardised regimen at the German Nepal TB Project Clinic, Nepal, between 2014 and 2016. Eligibility for the 9-month regimen and indications for TDM were evaluated.IQR) and culture conversion was 60 days (60–90 IQR). Observed side effects did not impact treatment outcomes. No difference in treatment success rates was observed between patients with predisposing risk factors and those without. Only 49% (36/74) of patients were eligible for the 9-month regimen and 23 patients for TDM according to American Thoracic Society guideline criteria.Out of 107 available patients’ medical records, 98 were included. In this centre, the MDR-TB treatment success rates were 69.0% in 2015, 86.6% in 2016 and 86.5% in 2017. The median time to sputum smear conversion was 60 days (60–90 Nepalese patients with MDR-TB on ambulatory care had good treatment outcome after programmatic treatment. Implementation of the new WHO oral MDR-TB treatment regimen may further improve treatment results. The 9-month regimen and TDM should be considered as part of programmatic care. The study evaluated treatment in patients on current programmatic multidrug-resistant tuberculosis (MDR-TB) regimen and verified eligibility for the 9-month regimen and therapeutic drug monitoring (TDM).The standardised MDR-TB regimen appears well tolerated in patients; none of the adverse events resulted in a change of regimen. Implementation of the new WHO oral MDR-TB treatment regimen may further improve treatment results. The 9-month regimen and TDM should be considered as part of programmatic care.The study provides complete treatment outcome data of patients with MDR-TB along with all significant clinical variables and drug susceptibility testing results (phenotypic and genotypic) make it relevant and provide a data-rich description of patients with MDR-TB treated in an outpatient setting.Mycobacterium tuberculosis that kills more people than HIV/AIDS every year. In 2018, an estimated 10 million people developed TB and 1.2 million died from it.Tuberculosis (TB) is the leading infectious disease caused by Later generation fluoroquinolones (levofloxacin and moxifloxacin) are key drugs in both standardised (20–24 months duration), short course 9-month and all-oral 20-month regimens in use for treating MDR-TB.1 10 13 15–17In the light of available evidence, it’s important to explore the link between various risk factors like diabetes mellitus, HIV, advanced age, low body weight and adverse events and treatment outcomes in patients under a standardised regimen for MDR-TB treatment in a local setting.A retrospective chart review was performed for all patients with MDR-TB receiving levofloxacin as part of their standard MDR-TB regimen enrolled for treatment at the Nepal Anti-Tuberculosis Association/German Nepal TB Project (NATA/GENETUP), Kathmandu, Nepal, between April 2014 and December 2016. Patients received 750–1000 mg levofloxacin one time a day. The other drugs included in the regimen were kanamycin at a median dose of 750–1000 mg, cycloserine and ethionamide both at one time a day dose of 500–750 mg and pyrazinamide at 1200–1600 mg one time a day dosing. Patients with missing records/incomplete information were excluded, and those who had pre-XDR TB (Extensively drug resistant) (with either fluoroquinolone resistance or resistance to second-line injectable agents) were also excluded. In the GENETUP clinic, all patients were tested for HIV and diabetes mellitus as a standard procedure. All patients with HIV were on antiretroviral therapy . Similarly, patients with diabetes mellitus were on hypoglycaemic agents.Primarily, patients were treated as outpatients from the start of treatment. Residential facilities  were provided to impoverished patients, those at the risk of treatment non-adherence and treatment failure. Due to limited room capacity, patients were qualified eligible for residential facilities on a case-by-case basis giving priority to those in dire need. This allowed healthcare providers at NATA/GENETUP to closely monitor and manage MDR-TB treatment in vulnerable subpopulations. For patients in ambulatory care, adherence was ensured by keeping contact information of two additional patient relatives along with their home address details. In some cases, community members were contacted if both patient and patient parties were unreachable. Since TB is considered a public health threat in Nepal, seldom, police authorities were involved to provide counselling to non-adherent patients.Medication safety was evaluated by recording adverse reactions in a standardised adverse effect monitoring form, every month until treatment completion. On daily visits to GENETUP clinic for DOTS  facilities, patients were given the opportunity to report serious side effects. Then, based on complaints and serum biochemistry results, offending agents were either discontinued for a period of time or replaced with other available drugs in the regimen.This being a retrospective study, patients were not involved.plus) molecular line probe assay was used for diagnosis of isoniazid (katG and inhA gene), and rifampicin (rpoB gene) resistance. For second-line drugs, molecular line probe assay  was used to identify resistance pattern to fluroroquinolones (gyrA gene), and aminoglycosides (rrs gene) and ethambutol (embB gene). Furthermore, the study evaluated impact of initial ethambutol resistance on MDR-TB treatment outcomes. Clinical variables included age, body weight at admission, body weight after 8 months of treatment, body weight below 35 kg, gender, HIV status, comorbidities, prior anti-TB therapy and presence of cavitary lesions. To evaluate the safety of the multidrug regimen, all recorded adverse events and lab test results at three different time periods  were retrieved from the medical records.Before initiation of treatment, two sputum samples were collected from patients for culture and second-line drug susceptibility testing (DST), and subsequently every month until 8 months followed by 10, 12, 16, 18, 20, 22 and 24 months for treatment monitoring by smear microscopy and culture on Löwenstein-Jensen media. Phenotypic first-line and second-line DST was carried out at the WHO critical concentrations.The MDR-TB treatment outcomes are defined based on WHO guidelines adopted by the NTP , Nepal.DST results on first-line and second-line drugs were obtained to evaluate potential eligibility of patients in a shorter 9-month MDR-TB regimen. Furthermore, an official clinical practice guideline from the American Thoracic Society was used to evaluate patients eligible for TDM. Criteria for TDM were patients with gastrointestinal problems that increase risk of malabsorption, concurrent HIV infection, impaired renal clearance, diabetes or patients not responding to MDR-TB therapy by the third month.16 21The association of variables with treatment outcomes (successful/unsuccessful) was studied using univariate logistic regression analysis . In the univariate analysis, variables with a p value of <0.25 were selected for entry in the final multivariate model. A p value of <0.05 was considered significant. Categorical data were expressed in frequencies and percentages whereas continuous variables were presented as median and IQR. Depending on the distribution of continuous variables, non-parametric tests were used for calculation of p values, where applicable.Out of 107 available patients’ medical records, a total of 98 MDR patients were included in this retrospective chart review see . BaselinIQR) and culture conversion (n=81) was also 60 days (60–90 IQR); two patients remained culture positive after intensive treatment characterised by the growth of NTM (Nontuberculous mycobacteria) later on (8th and 10th month), respectively. Treatment outcomes of the study population (n=98) were as follows: 73 (74.5%) patients were cured, 12 (12.2%) completed the treatment, 3 (3.1%) were lost to follow-up, 4 (4.1%) were transferred out, 5 (5.1%) died and 1 (1%) failed MDR-TB treatment. Among five deaths, three died during the first 2 months of TB treatment, one committed suicide at month 14 of treatment and one critically ill child died at an intensive care unit during the first month of TB treatment. In our study, 86.7% had successful outcome (n=98) based on standards proposed by Laserson et al.The median time to sputum smear conversion (n=75) was 60 days . Among 38 patients with MDR-TB who had initial resistance to ethambutol, all of them achieved favourable treatment outcomes (Laserson recommendations). Of 38 patients, 32 (84.2%) were deemed cure and 6 (15.8%) completed the treatment. However, ethambutol was not part of the longer 18–20 month MDR-TB regimen.Furthermore, we evaluated the proportion of patients with available DST results on first-line and second-line drugs for their potential eligibility in a shorter 9-month MDR-TB regimen. The results from both phenotypic and genotypic testing revealed that 52% of the patients with MDR-TB (n=74) had resistance to ethambutol. Based on results of this study, if ethambutol resistance were to be considered, only 48% of patients with MDR-TB would be eligible for shorter 9-month regimen. However, in programmatic settings like Nepal resistance to ethambutol is not taken into account because only fluoroquinolones and second-line injectables are considered important core drugs in a the shorter 9-month regimen. There is a documented evidence that the regimen fails only when the isolate is resistant to core drugs but not companion drugs .In our study, 23 patients categorised as high-risk group to poor MDR-TB treatment would have been eligible for TDM . Among tThe study evaluated treatment results of current programmatic regimen before implementation of all-oral 18–20 month regimen which includes new drugs like bedaquiline and delamanid, at the TB referral clinic of Nepal.et al, 70% of the outpatients with MDR-TB were reported to be cured on a regimen containing ofloxacin.We found a high treatment success rate of 86.7% for our MDR-TB cohort. In a previous study by Malla One of the striking observations from this study was that the majority of patients with MDR-TB had prior anti-TB therapy and 60% had failed a 6–8 month treatment regimen with first-line drugs and streptomycin. This is in contrast with the high success rate of drug-susceptible TB and not on par with the incidence of MDR-TB (15.4% of retreatment cases are MDR-TB based on drug resistance survey carried out in 2011/2012).et al argued that full susceptibility to ethambutol and isoniazid was not of paramount importance for patients to qualify for treatment with the shorter regimen.et al, successful outcomes were not affected by initial resistance to companion drugs .The side effects of MDR-TB drugs in patients remain a major concern but most were managed without change of the MDR-TB regimen. Regarding eligibility of patients for the shorter 9-month MDR-TB regimen, it is unclear if patients with resistant strains to ethambutol would qualify. Van Deun Since January 2018, the NTP of Nepal has endorsed the 9-month shorter regimen for the treatment of MDR-TB with weight band moxifloxacin dosing (600 mg or 800 mg) in all TB treatment centres, with the support of the Damien Foundation. At present, resistance to ethambutol and isoniazid (due to shortage of test kits) is not considered while determining eligibility. Patients are eligible if deemed susceptible to fluoroquinolones and aminoglycosides. Between July 2018 and July 2019, 72% (31/43) of the registered MDR-TB patients were eligible for a shorter 9-month treatment regimen in the GENETUP clinic. The remaining 28% received programmatic standardised MDR-TB regimen with injectable agents (kanamycin). Encouragingly, with the implementation of all-oral 18–20 month regimen, the trend is shifting with more patients being switched to all-oral regimen over shorter 9-month regimen containing injectable agents. The treatment outcome results from the shorter 9-month regimen in a high-incidence, high-fluoroquinolones-resistant setting like Nepal will be communicated to the TB community likely in 2020.max and AUC0-24 (Area under the concentration-time curve) of core drugs like moxifloxacin, linezolid and aminoglycosides, among others which has helped ensure adequate drug exposure and minimise associated toxicity. The treatment outcomes for MDR-TB in these centres are comparable to those reported globally for drug-susceptible TB (above 75%).In relation to drug exposure, 30% of the patients at the GENETUP clinic who were enrolled for a prospective pharmacokinetic study between 2016 and 2017 did not meet the established levofloxacin target for efficacy on one time a day 750–1000 mg dosing.This study has several limitations. First, being a retrospective study from a single centre, the generalisability is limited. However, due to the excellent programmatic care in this centre, it could serve as role model for other centres in the country and region. Second, lack of difference in treatment outcomes between predisposing risk factors and non-risk factors groups could be due to the low statistical power and high proportion of cure and completion rates compared with lost to follow-up, death and relapse, due to unavailability of patient information in the latter group. Pooling individual patient data from several treatment outcome studies as has been done by the Canadian team led by Menzies would likely improve the statistical power for future studies to detect a difference in response.In conclusion, the standardised MDR-TB regimen appears well tolerated in patients; none of the adverse events resulted in a change of regimen. Implementation of the new WHO oral MDR-TB treatment regimen may further improve treatment results. For patients resistant to fluoroquinolones or second-line injectables, all-oral regimen might be the best option and for those without resistance to fluoroquinolones or second-line injectables, the shorter regimen is indicated. The 9-month regimen and TDM should be considered as part of programmatic care.