Access to hepatitis C virus (HCV) testing and treatment is limited in Myanmar. We assessed an integrated HIV and viral hepatitis testing and HCV treatment strategy. Sofosbuvir/velpatasvir (SOF/VEL) ± weight‐based ribavirin for 12 weeks was provided at three treatment sites in Myanmar and sustained virologic response (SVR) assessed at 12 weeks after treatment. Participants co‐infected with HBV were treated concurrently with tenofovir. Cost estimates in 2018 USD were made at Yangon and Mandalay using standard micro‐costing methods.803 participants initiated SOF/VEL; 4.8% were lost to follow‐up. SVR was achieved in 680/803 (84.6%) by intention‐to‐treat analysis. SVR amongst people who inject drugs (PWID) was 79.7% (381/497), but 92.5% among PWID on opioid substitution therapy (OST) (74/80), and 97.4% among non‐PWID (298/306). Utilizing data from 492 participants, of whom 93% achieved SVR, the estimated average cost of treatment per patient initiated was $1030 (of which 54% were medication costs), with a production cost per successful outcome (SVR) of $1109 and real‐world estimate of $1250. High SVR rates were achieved for non‐PWID and PWID on OST. However, the estimated average cost of the intervention  of $1250/patient is unaffordable for a national elimination strategy. Reductions in the cost of antivirals and linkage to social and behavioural health services including substance use disorder treatment to increase retention and adherence to treatment are critical to HCV elimination in this population. Availability and access to HCV testing and treatment, fear of prosecution for drug use, and high stigma for key populations, results in most presenting for HCV and HIV treatment are already well advanced in their illness. EQUIP launched a single arm demonstration project to evaluate an integrated, simplified protocol for testing and treating HCV and HIV among key populations in Myanmar. We describe the treatment outcomes and estimated costs for simplified HCV testing and DAA treatment with SOF/VEL with or without RBV for patients with and without HIV co‐infection.22.1Enrolment occured between December 2017 and November 2018 at three clinical facilities in Myanmar: the Than Sitt Charity Clinics in Yangon and Mandalay, both operated by the Myanmar Liver Foundation (MLF), and the Asian Harm Reduction Network (AHRN) Clinic in Waimaw, Kachin. Patients were referred by peers, community workers, civil society groups, the General Practitioners Society, the National AIDS Program (NAP), the Myanmar Anti‐Narcotic Association (MANA), Medicins Sans Frontier Holland (MSFH) and Médecins du Monde (MdM). The target populations were PLHIV, PWID, MSM and FSW, although initial enrolment included populations outside of these, most of whom could not afford private treatment.2.2Eligible participants were HCV viremic, HCV treatment naïve or experienced (prior pegylated interferon [PegIFN] and RBV only), and 18 years or older, with HCV genotype 1, 2, 3, 4, 5 or 6, with or without HIV‐1 co‐infection. Patients with compensated cirrhosis (Child‐Pugh Class A) and HBV infection were eligible; those with decompensated cirrhosis (Child‐Pugh Class B or C) or prior treatment with HCV DAAs were not eligible. Patients who were ineligible for HCV treatment were referred to other treatment centres. All participants provided written informed consent.2.3The intervention combined HCV and HIV testing, simplified HCV treatment, and HIV treatment initiation for those with HIV co‐infection not yet on antiretroviral therapy (ART). HCV treatment was with fixed‐dose combination sofosbuvir 400 mg/velpatasvir 100 mg (SOF/VEL) ± weight‐based ribavirin for 12 weeks. Ribavirin was initially included in the regimen for genotype 3 participants who were cirrhotic or who had previously failed an interferon‐based treatment regimen. In April 2018, 5 months into enrolment, based on more recent findings on treatment response rates with SOF/VEL without ribavirin for genotype 3 infection, including amongst PWID and in resource‐limited settings in Asia,Participants with hepatitis B surface antigen positivity were concurrently treated with tenofovir disoproxil fumarate (TDF). Provision of HIV and hepatitis B treatment was derived from national or USAID supported HIV treatment programmes. The first‐line HIV treatment regimen in Myanmar—TDF/lamivudine (3TC)/efavirenz—posed a challenge as efavirenz could not be co‐administered with velpatasvir due to drug‐drug interactions. As such, the NAP supported the substitution of dolutegravir or another alternative (such as lopinavir/ritonavir) for efavirenz. Participants were followed for 24 weeks (through 12 weeks after treatment completion), including assessments of HCV treatment outcomes and safety. Intervention steps are illustrated in Figure 2.42.4.1Medical history and clinical assessment were undertaken at baseline and repeated at week 24; additional clinical assessments were performed at clinicians’ discretion. Concomitant medications were recorded. Liver disease stage was defined based on either ultrasound , AST‐to‐platelet ratio index (APRI) score ≥2.0, or Fibroscan ≥12.5 kPa. For cirrhotic patients, Child‐Pugh score was calculated; compensated cirrhosis was defined as Child‐Pugh Score ≤6. SOF/VEL and TDF were dispensed at weeks 0, 4 and 8. Ribavirin was dispensed at weeks 0, 2, 4 and 8. A pharmacist provided drug information at each visit. Counselling was provided at baseline and weeks 4, 8, 12 and 24 by a social worker, or peer counsellor on study procedures and treatment adherence, HCV transmission and re‐infection risks and harm reduction, with referral to harm reduction services as appropriate for substance use comorbidities and HIV and HCV risk factors.2.4.2+ T‐cell count for HIV‐infected participants. Laboratory results were also obtained from the medical record if reported within the specified window . HCV RNA testing was undertaken for all participants with positive HCV antibody results, as described below. HCV genotyping, subtyping and resistance testing for all HCV viremic patients were performed at the Department of Medical Research, Ministry of Health (Yangon) using the Advanced Biological Laboratories (ABL) UltraGene‐HCV assay on Thermofisher QuanStudio 3 qPCR system and resistance was performed using the ABL DeepChek assay on the Thermofisher SeqStudio sequencer. All participants who initiated HCV treatment had the following laboratory tests at week 24: HCV RNA, haemoglobin, platelets, AST, ALT, bilirubin, albumin, creatinine, blood urea nitrogen, prothrombin time/INR and HIV RNA (if HIV‐infected). Participants receiving ribavirin additionally had haemoglobin monitoring at weeks 2, 4 and 8. Pregnancy testing was required for all females of childbearing potential at screening and at weeks 4, 8, 12 and 24 for those assigned to ribavirin. Participants with eGFR < 60 at screening and also receiving tenofovir had creatinine monitoring at weeks 4 and 12. Participants with isolated hepatitis B core Ab positivity (HBcAb+/HBsAg‐/HBsAb‐) had hepatic liver tests  at weeks 4, 8 and 12 to assess for potential HBV reactivation.The screening laboratory evaluations included serum HCV antibody if not already available in the medical record, haemoglobin, platelets, AST, ALT, total bilirubin, albumin, creatinine, blood urea nitrogen, pregnancy testing, prothrombin time/International Normalized Ratio (INR), point‐of‐care (POC) HBV serologies with WHO qualified SD Bioline , POC rapid HIV testing  and CD42.4.3® HCV Viral Load  and/or Roche (COBAS®AmpliPrep/COBAS ®TaqMan ®HCV quantitative) assay. Xpert® and Roche HCV viral load was performed at Ni‐Ni Laboratory in Yangon for the Yangon and Mandalay sites. Xpert® HCV viral load was performed on site at AHRN Waimaw clinic, Kachin. Roche HCV viral load estimation served as the standard of care VL assay prior to validation of Xpert® HCV VL. The limits of quantification were 10 IU/mL for Xpert, and 15 IU/mL for Xpert® and Roche assays respectively.HCV viral load was measured using near POC Cepheid Xpert2.5HCV genotyping and subtyping were performed at the Department of Medical Research, Myanmar Ministry of Health (Yangon) using Advanced Biological Laboratories (ABL) UltraGene‐HCV assay (lower limit of detection of 20 IU/mL) and DeepChek‐HCV genotyping assay (ABL SA) on Thermofisher QuanStudio 3 qPCR and ThermoFisher SeqStudio sequencer systems. The DeepChek‐HCV genotyping assay differentiates all six HCV genotypes and can discriminate HCV 1b/2k chimeras and subtypes 1a and 1b. After RNA extraction from plasma and RT‐PCR of the HCV genome according to the manufacturer's instructions, amplicons were obtained for 5’UTR, NS5B and NS5A regions. DNA products were sequenced with specific primers by capillary electrophoresis according to the manufacturer's instructions. Sanger sequencing, the BigDye X Terminator  were used.The sequences obtained were read and aligned with the DeepChek‐HCV. A consensus sequence was generated for each sample and compared with the HCV genomic Basic Local Alignment Search Tool (BLAST) bank for determination of the HCV genotype and subtype. A threshold for similarity of minimum 85% was used to consider the genotype or subtype.2.6The two primary outcomes of the analysis were sustained virologic response (SVR) at 12 weeks after the end of treatment and cost per patient with SVR. Other outcomes included safety (adverse events) during the treatment period. For the cost analysis, we assigned each patient one of four outcomes at 24 weeks after treatment initiation: (a) treatment success (SVR 12 weeks after therapy completion); (b) treatment failure (HCV viraemia greater than the lower limit of detection 12 weeks after therapy completion); (c) loss to follow‐up ; or (d) death (died within the 24‐week study period).2.72.7.1P‐level of less than .2 in the bivariate analysis was used to select variables for multivariable analysis. A final model was determined using stepwise backward elimination method and only P‐level of less than .05 was considered for the final model. Multi collinearity test, specification error testing and Hosmer‐Lemeshow test (HL test) for logistic regression were undertaken on the final model. Data were analysed using STATA version 15 SE .Baseline sociodemographic, clinical and laboratory characteristics of the screened participants were calculated using proportions or means (standard deviations) or medians (interquartile ranges) for continuous data. The proportion with SVR was calculated for each clinical site and overall, by intention‐to‐treat (ITT). Differences between those successfully treated SVR achieved compared to those who failed treatment were assessed by t test or chi‐square test as appropriate, and by multivariable logistic regression. A Cost estimates were made at two sites, Yangon and Mandalay, using data from the first 492 patients from these sites. Costs were estimated from the provider perspective from initial screening date until assessment of outcome using standard economic methods described previously.® testing were donated and are also excluded. Costs attributable to the research study but unlikely to be incurred in routine implementation  were included only in Scenario 2.Resources incurring costs included events, laboratory assessments, HCV and HBV medications, clinic staff, indirect costs, education and outreach during the 24‐week follow‐up period. Events included physical examinations (physician visit), counselling visits and Fibroscans (supplies only). Laboratory assessments included HCV RNA, HCV genotyping, HIV and HBV testing, pregnancy testing, PT/INR, and blood counts and chemistries. Indirect costs included support staff/personnel, building costs, equipment and office supplies. HIV medications were utilized but were paid for separately by the government HIV programme and are excluded from the cost analysis. Equipment and technicians’ time for FibroscanFor the cost analysis, we considered four scenarios: (a) observed costs excluding research‐related expenses ; (b) observed costs including research expenses; (c) observed costs excluding research related expenses and excluding routine HCV genotype testing; and (d) observed costs excluding research‐related expenses and increasing the treatment failure rate to 14% to simulate potential poorer treatment adherence and/or effectiveness in a real‐world setting.We also evaluated the average resource utilization per patient by cost category and HCV treatment outcome and calculated 95% confidence intervals by outcome category. We calculated the production cost of a successful outcome by dividing the sum of all costs by the number of successful outcomes. We applied the average exchange rate for 2018 of 1.00 United States dollar (USD) = 1547.71 Myanmar Kyat (MMK).2.8The study was reviewed by Myanmar Ministry of Health and Sports Institutional Technical and Ethical Review Board, University of Public Health (ITERB‐2017/Research/18), the University of the Witwatersrand Human Research Ethics Committee (M17078) and the UCLA Medical Institutional Review Board (#18‐00003). The Boston University Institutional Review Board approved analysis of a de‐identified analytic dataset (H‐37820). All patients provided written informed consent. The study is registered at ClinicalTrials.gov (NCT03579576).33.1We screened 1007 HCV antibody positive patients and enrolled those eligible at the three study sites between 18th December 2017 and 11th November. We excluded 193 of those screened for the reasons listed in Figure Six‐hundred and eighty participants had HCV genotyping results available at the time of this analysis. Of these, the most common genotypes were 3 (48.8%) and 6 (41.4%). The subtypes were mostly 3b (36%) followed by 6n (19%) and many new genotype 6 subtypes were found in Myanmar.3.2Treatment outcomes by site are reported in Table After treatment initiation, 39 participants were counted as lost to follow‐up (LTFU), 1 died, and 23 were known to have completed treatment but missed the week 24 SVR assessment. A total of 764 participants were assessed for SVR at 24 weeks. By intention‐to‐treat analysis (ITT), 123 failed (HCV viraemia above the limit of quantification at week 24), for an overall treatment success rate of 680/803 (85%). SVR rates differed significantly by site, risk group, and HIV co‐infection status, with the greatest number of treatment failures and losses to follow‐up in Kachin. Treatment failure was greater among PWID than other groups. Out of 803 who initiated treatment, 61.9% (497) were PWID and of the PWID assessed at 24 weeks, 76.7%. achieved SVR. Amongst PWID who initiated HCV treatment, 80 were on opioid substitution therapy (OST) at treatment initiation; those on OST were more likely to achieve SVR by ITT: 92.5% vs 73.6% among PWID not on OST.3.3P = .674; hat, P = .47), no evidence of multicollinearity (mean variance inflation factor ‐VIF = 1.5) and an overall good fit .In Table 3.4Resource utilization for the first 492 participants enrolled at the Yangon and Mandalay study sites and locally collected unit costs is presented in Table 4In this demonstration project, treatment of HCV among key populations in three regions in Myanmar, including 61.9% PWID, 43.7% HIV co‐infected, and 2.5% HBV co‐infected, utilizing an approach with limited laboratory testing and frequent counselling led to a moderately high SVR rate of 85% amongst all who initiated treatment. Across all sites, virologic failure was demonstrated in 10%, and loss to follow‐up in 5% of participants. Treatment success varied widely by site, with Yangon achieving 96% SVR, Mandalay 83%, and Kachin only 72%. PWID status was associated with treatment failure, and rates of both virologic failure and LTFU were higher in Mandalay and Kachin sites where the majority treated were PWID. While adherence to HCV treatment was not measured, barriers to adherence to treatment amongst PWID may have contributed to low SVR rates. Notably, the SVR rate amongst PWID on OST was significantly higher than for PWID not on OST, suggesting that PWID status alone may not adversely impact treatment success if PWID are linked to substance use and harm reduction treatment. Indeed, other studies have demonstrated high rates of success with HCV treatment co‐located with substance use treatment services and amongst PWID receiving OST.https://knoema.com/atlas/Myanmar/Health‐expenditure‐per‐capita). Nearly half the cost estimated per patient is attributed to the cost of SOF/VEL alone. Reduced drug costs by drug companies in addition to donations by manufacturers and/or foreign donor investments will likely be needed for scale‐up of treatment.The cost per patient treated estimated in our real‐world scenario, $1030, likely understates the true cost of program implementation, as it excludes costs for scaling up and maintaining the treatment program, such as procurement, training, management and oversight. It also excludes the costs of screening and confirmatory testing for those who do not further engage in care. For budgeting purposes, under the assumption of no genotype testing and decreased real‐world effectiveness but with approximately 20% added for program management, screening and confirmatory testing, an average cost of $1250/patient is likely a reasonable estimate for this intervention in 2019. Although much less than seen in high‐income countries, a cost of $1250/patient will likely be challenging to pay for a country with per capita health expenditures estimated at $62/year (The production cost for a successful outcome reported here is modelled utilizing confirmatory HCV RNA pricing negotiated for the project through Ni‐Ni laboratories. Other confirmatory HCV tests, including those that can be performed at or near the point of care, may allow further reductions in the laboratory cost components, though reliance on foreign imports for parts and servicing may offset cost savings. Other ways to reduce costs include omitting Fibroscan testing, which is not required for an elimination program.This analysis has several limitations. Given the LTFU and inability to assess SVR in some participants who completed treatment, the true SVR rate is likely slightly underestimated, although the overall LTFU was still somewhat small. While the four cost scenarios presented aimed to provide a reasonable estimate of the costs across a range of strategies in Myanmar, the cost results were based on Yangon and Mandalay treatment outcomes and costs only, excluding those from Kachin. The poorer treatment outcomes and likely higher cost of resource inputs in Kachin, resulting from its more remote location, were not taken into account. Inclusion of rural like Kachin would likely benefit from cost benefit analysis for upscale of simplified HCV treatment protocols.Several major barriers to universal treatment and elimination of HCV in Myanmar were identified, including proximity to clinics, the cost of DAAs, the cost and feasibility of HCV RNA testing, and significant virologic failure among PWID not on OST. Identification of HCV viremic patients may be improved, with a point of care assay to detect HCV viraemia or antigenemia. Increased access to substance use treatment and harm reduction services is further necessary to improve SVR rates among PWID.It is critical to appreciate that markers of marginalization such as enforcement‐oriented drug policies, poor patient‐provider relationships, institutionalized stigma within health care systems and social exclusion of PWID are key drivers of challenged treatment compliance and adherence, which impact health seeking behaviour and the ability to access and complete HCV treatment.A multidisciplinary approach to HCV treatment where treatment and counselling services are offered in a ‘one stop shop’ has been shown to improve treatment uptake and adherence to therapyKWC has received a research grant to the institution from Merck Sharp & Dohme.


Three patients diagnosed with severe COVID‐19 pneumonia received treatment with hydroxychloroquine combined with lopinavir, ritonavir, and favipiravir. Two patients diagnosed early, received tocilizumab when the pneumonia became severe and survived. The thrid patient was diagnosed late and received tocilizumab when the disease progressed to acute respiratory distress syndrome, and died. Tocilizumab could be used in patients with severe COVID‐19 pneumonia who did not show response to a combination of antiviral therapy. However, the use of a combination antiviral therapy should be further confirmed in clinical trials. In cases of severe COVID‐19 pneumonia, IL‐6 levels should be monitored.A previous study demonstrated a mortality rate of 8.1% among patients with severe COVID‐19 pneumonia, and only 0.1% of them had nonsevere COVID‐19.In addition, a systematic review indicated that an anti‐IL‐6 receptor antibody may benefit patients with severe COVID‐19 pneumonia, although the effectiveness of anti‐IL‐6 receptor antibody, such as tocilizumab for COVID‐19 remains uncertain.Herein, we report three patients with confirmed SARS‐CoV‐2 infection who developed severe pneumonia that was refractory to treatment combination of hydroxychloroquine, lopinavir plus ritonavir, and favipiravir. Tocilizumab was then administered, and the disease severity was monitored using IL‐6 and hs‐CRP levels.22.12, who had chronic medical illness of hypertension, hyperlipidemia, and coronary artery disease presented to the emergency department with symptoms of fever with chills, productive cough with white phlegm, sneezing, sore throat, and myalgia. The patient received hydroxychloroquine, lopinavir plus ritonavir, and favipiravir for 10 days combined with azithromycin for 5 days. His recent medications included manidipine , atenolol , rosuvastatin , clopidogrel , and aspirin . He was a healthcare worker who had been in contact with a hospitalized patient with confirmed SARS‐CoV‐2 infection on day ‐17 of hospitalization and was quarantined thereafter. On day ‐3 of hospitalization, both samples of nasopharyngeal swab and oropharyngeal swab showed undetectable levels of SARS‐CoV‐2 RNA using real‐time reverse transcription‐polymerase chain reaction (RT‐PCR), and the patient showed no symptoms.A 58‐year‐old Thai man with a body weight of 70 kg, height of 175 cm, body mass index (BMI) of 22.8 kg/m2 97% in room air (Table 3). The patient was admitted to the negative pressure isolation ward and received treatment of hydroxychloroquine , darunavir plus ritonavir  as antiviral treatment, starting on day‐ 1 of hospitalization. Azithromycin  was administered as an anti‐microbial and anti‐inflammatory agent  and azithromycin , but the symptoms did not improve. On the day of hospitalization, body temperature 37.9 ºC, heart rate 90 beats/min, blood pressure 136/85 mmHg, respiratory rate 20 breaths/min, and SpOir Table . RT‐PCR 2 92% in room air; Table Chest radiograph on day‐ 2 of hospitalization showed progression of interstitial infiltration in the right middle and left lower lung zones Figure . On day‐2 88% in room air). He was then transferred to the intensive care unit (ICU) with negative pressure isolation. Chest radiograph showed new alveolar infiltration in the right upper lung zone and increased alveolar infiltration in the right middle, left upper, and left lower lung zones , increased hs‐CRP (164.7 mg/L), increased levels of IL‐6 (46.6 pg/mL), and increased levels of LDH (409 U/L). Tocilizumab (8 mg/kg/dose intravenous drip for 90 minutes with repeated dose in the next 12 hours) was administered due to progression of hypoxemia and increased lung infiltration. The patient also received supplement oxygen of 5 L/min via a nasal cannula in the prone position to keep SpO2 >94%.On day‐ 8 of hospitalization Table , the pats Figure . Laborat2 (91% in room air). Laboratory findings showed low ALC (351 cells/mm3), decreased hs‐CRP levels (155.8 mg/L), and mildly elevated alanine aminotransferase (ALT) levels (41 U/L). Chest radiograph showed slightly decreased infiltration in both upper lung zones, but infiltration in both lower lung zones was unchanged. On day‐ 10 of hospitalization (Table 3) and markedly decreased levels of hs‐CRP (63.1 mg/L).On day‐ 9 of hospitalization Table , the IL‐3), decreased hs‐CRP levels (31.6 mg/L), and mildly elevated ALT levels (56 U/L). Chest radiograph showed markedly decreased infiltration on day‐ 18 of hospitalization and was normal on day‐ 27 of hospitalization  and sore throat. Physical examination revealed enlargement of both tonsils with exudate. She was diagnosed with acute exudative tonsillitis and received treatment with amoxicillin plus clavulanate . She was a healthcare worker and had had contact with a hospitalized patient with confirmed SARS‐CoV‐2 infection on day ‐18 of hospitalization and was thereafter quarantined. On day ‐4 of hospitalization, both samples of nasopharyngeal swab and throat swab showed undetectable levels of SARS‐CoV‐2 RNA, and the patient had no symptoms. Both samples of nasopharyngeal and oropharyngeal swabs were retested for SARS‐CoV‐2 RNA on day ‐1 of hospitalization. The results showed detectable SARS‐CoV‐2 RNA levels in the region of ORF1ab, N gene, NS‐1, and NS‐2 in both samples  and lopinavir plus ritonavir  combined with azithromycin  for anti‐inflammation. Amoxicillin plus clavulanate  was given for continued treatment of acute exudative tonsillitis. Favipiravir  was added as an antiviral treatment on day‐ 2 of hospitalization, after awaiting drug delivery from the hospital of Ministry of Public Health in Thailand.3).On day‐ 5 of hospitalization Table , her fev2 (87%) in room air and increased shortness of breath with a respiratory rate of 40 breaths/min. The patient received supplement oxygen via a face mask in the prone position in order to keep SpO2 >94%. Tocilizumab (8 mg/kg/dose intravenous drip for 90 min with repeated dose for the next 12 hours) was then administered to attenuate lung inflammation, as hypoxemia developed. Laboratory findings showed low ALC (1158 cells/mm3) and increased levels of hs‐CRP (145.4 mg/L), IL‐6 (49.8 pg/mL), and LDH (380 U/L) . Laboratory findings showed low ALC (1013 cells/mm3), decreased levels of hs‐CRP (120.8 mg/L) and LDH (369 U/L), and increased levels of IL‐6 (865.0 pg/mL) (Table 3) and markedly decreased hs‐CRP levels (5.4 mg/L) , and SpOOn day‐ 10 of hospitalization, a maculopapular rash with itching was observed on both arms and thighs. The patient received antihistamine medications, including chlorpheniramine , diphenhydramine , and cetirizine . Generalized maculopapular rash was then found to have spread throughout the face, neck, and trunk on day‐ 12 of hospitalization Figure . The rasOn day‐ 11 of hospitalization, ALT levels (115 U/L) were elevated and rose to 303 U/L on day‐ 16 of hospitalization Table , but dec2 increased (96% in room air) on day‐ 13 of hospitalization  score of 8 (E3M1V4), blood pressure of 187/84 mm Hg, and motor power grade II on the right extremities. The patient received normal saline infusion, and blood pressure was controlled in the ICU. The GCS score increased to 12 (E4M2V6), and motor power increased to grade III on the right extremities within 24 hours after treatment  with neutrophilia (9767 cells/mm3), and urinalysis revealed 30‐50 white blood cells/high power field. Doripenem  was then given due to suspected hospital‐acquired pneumonia and urinary tract infection. Two samples of hemoculture were negative, but sputum culture showed Klebsiella pneumoniae, and urine culture showed Escherichia coli >105 CFU/mL. Both organisms were sensitive to ceftriaxone, and the antibiotic was then tapered to ceftriaxone . Between days‐ 5 and 9 of hospitalization, the patient had no fever, and chest radiograph showed decreased infiltration in both lower lung zones , but motor power was still grade III on the right extremities. The patient had no cough and no shortness of breath, but lungs showed crepitation in both lower lobes. Chest radiograph showed increased alveolar infiltration in both the lower lung zones Figure . Laborats Figure .2 (94%), and lung crepitation at the right upper lobe (Table 3).On day‐ 10 of hospitalization, the patient had no fever, but she had fatigue, watery diarrhea, increased respiratory rate (22 breaths/min), decreased SpObe Table . Chest rOn day‐ 13 of hospitalization, the patient had dry cough, fatigue, and watery diarrhea (Table 3) and mildly elevated ALT levels (51 U/L). The patient was transferred to the negative pressure isolation ward and received antiviral treatment with hydroxychloroquine , darunavir plus ritonavir , and favipiravir . Azithromycin  was given for anti‐inflammation.On day‐ 15 of hospitalization, the patient still had dry cough, fatigue, and watery diarrhea, but shortness of breath with decreased SpO2 (88% in room air) developed Table . She had2 >94%. On day‐ 18 of hospitalization, the patient had shortness of breath with decreased SpO2 (90% in room air) and received endotracheal tube intubation with ventilator support due to hypoxic respiratory failure. Chest radiograph showed increased alveolar infiltration and pleural effusion in both lungs. On day‐ 19 of hospitalization, she had no fever but still had dry cough and worsening hypoxemia, with a low SpO2/FiO2 ratio (98), determining the occurrence of severe ARDS. Laboratory findings showed a low ALC (619 cells/mm3) and increased serum creatinine levels (>0.3 mg/dL) from baseline, determining the diagnosis of acute kidney injury (AKI) , cough, fatigue, watery diarrhea, and shortness of breath Table . She wasOn day‐ 26 of hospitalization Table , the patOn day‐ 27 of hospitalization Table , the patOn day‐ 30 of hospitalization, the patient still had shock Table . Laborat32 <94% in room air. All patients had chronic medical illness, shown to be more common among patients with severe pneumonia (38.7%) compared to those without (21.0%).2 ≤100 mm Hg with positive end‐expiratory pressure ≥5 cm H2O, septic shock, sick sinus syndrome, and AKI. A previous study showed that older patients with confirmed SARS‐CoV‐2 infection and aged ≥65 years are at risk of developing ARDS (hazard ratio 3.26) and of progressing from ARDS to death (hazard ratio 6.17).We reported three cases of patients with confirmed SARS‐CoV‐2 infection with severe pneumonia according to the definition of WHO,Regarding exposure history, two of the patients (cases 1 and 2) were healthcare workers and had a history of exposure to a patient with COVID‐19, with a time to illness development after exposure of 15 days in case 1 and 17 days in case 2. The time elapsed from exposure to symptoms was unknown in case 3. A previous study showed a median incubation period of 4 days among patients with COVID‐19 pneumonia, which was shorter than that among our patients.The duration from the onset of symptoms to the development of pneumonia was only a few days: 3 days in case 1 and 2 days in case 2. Severe pneumonia, defined according to WHO,3). A previous study reported that lymphopenia was present in 83.2% of patients with confirmed SARS‐CoV‐2 infection, and particularly in those with severe pneumonia (96.1%), those admitted to ICU, used mechanical ventilation, or died (92.6%).Laboratory findings in our patients showed normal white blood count with lymphopenia  developed severe ARDS and one dose of tocilizumab (8 mg/kg/dose for 90 min) was administered with dexamethasone (12 mg daily). After receiving treatments with tocilizumab and the corticosteroid, his hs‐CRP levels were still high, and IL‐6 levels increased. The patient died due to cobacterial infection within 2 weeks after ICU admission. A previous study showed common complications among patients with severe COVID‐19 pneumonia, including ARDS (15.6%), septic shock (6.4%), and AKI (2.9%),No clinical trial has been carried out on the effectiveness of a combination antiviral therapy. However, several studies have been conducted on pharmacological treatments with potential clinical benefit and adjunctive pharmacological treatments.A systematic review showed that tocilizumab may be used as a potential adjunctive pharmacological treatment for patients with COVID‐19, particularly for those with high IL‐6 levels.4Early diagnosis and timely treatment of severe COVID‐19 pneumonia may be important for the survival of patients to prevent the development of a cytokine storm, which is a critical mediator of respiratory failure, shock, and organ dysfunction. Tocilizumab could be used in patients with severe COVID‐19 pneumonia who did not respond to a combination of antiviral therapy. However, the use of a combination antiviral therapy should be further confirmed in clinical trials or national regulations with close monitoring.5Informed consent was obtained from the patients.None declared.MS, VT, DS, and VD: performed literature review. MS, YK, SK, TR, WL, SH, SJ, and SL: conceived study and patients’ care. MS, VT, SC, DS, and VD: analyzed data. MS, YK, VT, SK, SC, TR, WL, DS, SH, SJ, SL, and VD: contributed to the writing of the manuscript.


E2F2 and E2F3, and this may result in LUSC carcinogenesis.Here, we show that miRNA‐126‐3p is significantly underexpressed in 685 cases of lung squamous cell carcinoma (LUSC) as compared to 298 nontumor lung tissues, by combining various methods including in‐house RT‐qPCR, miRNA‐sequencing, and miRNA microarrays. The downregulation of miRNA‐126‐3p may regulate cell cycle signaling by targeting  SOX2, E2F2, and E2F3 were selected as hub genes from the PPI network for further analysis. In summary, our results suggest that the low expression of miRNA‐126‐3p may play a role in promoting the development of LUSC and miRNA‐126‐3p may be a biomarker for LUSC early diagnosis and prognosis.Lung squamous cell carcinoma (LUSC) is the main pathological type of pulmonary malignant tumors; at present, less than 10% of patients with advanced metastatic LUSC live for more than 5 years. We previously reported that low expression of miRNA‐126‐3p is associated with the occurrence and progression of lung adenocarcinoma (LUAD). Here, we examined expression of miRNA‐126‐3p in 23 samples from patients with LUSCs and 23 normal control specimens by quantitative real‐time PCR (RT‐qPCR). Associations between miRNA‐126‐3p expression and clinical features were studied from materials derived from Gene Expression Omnibus (GEO) chips and The Cancer Genome Atlas (TCGA) database. Twelve online platforms were used to identify candidate target genes of miRNA‐126‐3p. Further analyses of the Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and protein–protein interaction (PPI) network were performed on the target genes. GEO microarray analysis, TCGA data mining, RT‐qPCR, and integration analysis consistently reported low expression of miRNA‐126‐3p in LUSC. A total of 42 genes were identified as potential target genes of miRNA‐126‐3p from online platforms, GEO microarrays, and the TCGA database. GO and KEGG analyses demonstrated that the target genes are involved in several biological processes that promote the progression of LUSC.  AUCarea under the curveBPbiological processCCcellular componentCIconfidence intervalDAVIDDatabase for Annotation, Visualization and Integrated DiscoveryDEGsdifferentially expressed genesFFPEformalin‐fixed, paraffin‐embeddedGEOGene Expression OmnibusGOGene OntologyKEGGKyoto Encyclopedia of Genes and GenomesLRlikelihood ratioLUADlung adenocarcinomaLUSClung squamous cell carcinomaMFmolecular functionNSCLCnon‐small‐cell lung carcinomaORodds ratioOSoverall survivalPPIprotein–protein interactionQquartileROCreceiver operating characteristicRT‐qPCRquantitative real‐time PCRSCLCsmall cell lung cancerSDstandard deviationSEstandard errorSMDstandard mean differenceSROCsummary receiver operating characteristicSTRINGSearch Tool for the Retrieval of Interacting GenesTCGAThe Cancer Genome AtlasTFstranscription factorsLung cancer is a neoplastic disease of the lower respiratory tract; it includes a variety of histological subtypes and biologically distinct genotypes that have a high mortality rate . The annet al. , and GO–MF . Transcription regulation plays a pivotal role in orchestrating normal development and disease progression [SOX2, E2F2, and E2F3 act as TFs and can affect the biological process of LUSC through mutual regulation with miRNA126‐3p. In the KEGG pathway analysis, we noted that small cell lung cancer (SCLC), the cell cycle, pathways in cancer, prostate cancer, and microRNAs in cancer were all closely related to human cancer. Wang et al. [et al. [E2F2 and E2F3, were all involved in these cancer‐related pathways. Hence, we hypothesized that miRNA‐126‐3p regulated the biological function of LUSC by targeting E2F2 and E2F3.To explore further the molecular mechanism of the miRNA‐126‐3p function in LUSC, we executed a functional analysis of bioinformatics on the predicted 42 target genes. Three functional analyses of GO showed that gression . It had gression . Severalgression . Therefog et al.  reported [et al.  pointed  [et al. , 35, 36.SOX2, E2F2, and E2F3. Because we used overexpressed genes in TCGA and GEO microarrays when we were predicting target genes, the expressions of these genes in tumor tissues were upregulated. Based on the miRNA‐126‐3p downregulated in tumor tissues, these overlapping and most frequently occurring genes can be used as target genes of miRNA‐126‐3p. The TF SOX2 acted as a key regulator in biological processes, such as transcriptional regulation, cell proliferation, and oncogenesis [SOX2 has been confirmed in various cancers. de Vicente et al. [SOX2 expression was an important predictive biomarker of oral cancer risk in early stage patients, while Zhan et al. [SOX2 can inhibit esophageal squamous cell carcinoma development. Furthermore, an epigenomics analysis revealed that SOX2 can drive tumor heterogeneity in LUSC [SOX2 axis might be an underlying therapeutic direction in NSCLC. Although SOX2 has been reported in NSCLC, there are few in‐depth studies on the mechanism of action between SOX2 and MiRNA‐126‐3p in LUSC.We selected three genes with the highest frequency as the hub genes from the PPI network: ogenesis , and thee et al.  revealedn et al.  identifi in LUSC . Chang [ in LUSC  found thE2F2 and E2F3 are two members of E2F family, and E2F1‐3, a subfamily of the E2F family, serves as a transcriptional activator [E2F2 and E2F3 may act as vital regulators in several cancers. Lin et al. [E2F2. Moreover, it is well documented that E2F2 can serve as an attractive therapeutic target and biomarker for ovarian cancer [E2F2 worked as an activator in the tumor invasion of NSCLC, and it was implicated in the proliferation and poor prognosis of NSCLC cells [E2F3 has been reported in the literature on hepatocellular carcinoma [E2F2‐3 and miRNA‐126‐3p in LUSC have not yet been reported. We speculate that miRNA‐126‐3p may target E2F2 and E2F3 to restrain the cell proliferation and cancer progression of LUSC. We consider that miRNA‐126‐3p regulates the elevated expressions of core genes, SOX2, E2F2, and E2F3, and it may help elucidate the carcinostatic functions of miRNA‐126‐3p in LUSC. This must also be confirmed by further experimental function studies.The E2F family plays an important role in cell cycle regulation, tissue homeostasis, and apoptosis , 43. TFsn et al.  confirmen cancer . E2F2 woLC cells . The spearcinoma , bladderarcinoma , ovarianarcinoma , LUAD [4arcinoma , and gasarcinoma . The mecThis is the first in‐depth study on and discussion of the mechanism of miRNA‐126‐3p in LUSC. By combining RT‐qPCR, bioinformatics, and an integrated analysis, we confirmed that the downregulation of miRNA‐126‐3p in LUSC was involved in several biological processes that promote the occurrence, progression, and poor prognosis of LUSC. The key role of miRNA‐126‐3p in LUSC has provided new ideas and directions for the diagnosis of and molecular therapy for LUSC in the future.in vitro studies to validate the effect of miRNA‐126‐3p knockdown or overexpression in LUSC. In future studies, it will be necessary to add well‐designed in vitro experiments to validate the results and hypotheses of this study. Third, to investigate the early diagnostic value of miRNA‐126‐3p for LUSC, we attempted to screen the high throughput data from the GEO, ArrayExpress, SRA, and Oncomine datasets. Unfortunately, no data on the miRNA‐126‐3p expression in LUSC patients were available from blood, serum, or plasma samples. Five microarrays  were obtained from blood in NSCLC patients, which could reflect the potential diagnostic value for the whole NSCLC population. Indeed, the expression of miRNA‐126‐3p in NSCLC patients was significantly lower than in healthy controls (data not shown). However, the clinical implication of miRNA‐126‐3p in the early diagnosis of LUSC patients must be confirmed in future.The study has several shortcomings. First, it is a single‐center study, so the number of clinical samples used for the RT‐qPCR analysis is small, and more clinical tissue samples are needed for more accurate and persuasive results. Second, it lacks In this study, a comprehensive analysis of RT‐qPCR, TCGA, and GEO datasets illustrated that miRNA‐126‐3p is underexpressed in LUSC tissues. A low expression of miRNA‐126‐3p suppressed the development of LUSC, whereas a high expression of miRNA‐126‐3p was associated with a better LUSC prognosis. MiRNA‐126‐3p may act as a tumor inhibitor in LUSC, and it suppresses the proliferation of LUSC cells. In summary, miRNA‐126‐3p may be a promising biomarker for LUSC diagnosis and prognosis.The authors declare no conflict of interest.All authors have contributed to this study for submission. S‐WC performed the data extraction and statistical analysis and drafted the paper. S‐WC and H‐PL conceived and designed the study and edited the manuscript. GC, JY, H‐FZ, and W‐JM contributed to the design of the study, supervised all experiments, and corrected the paper. W‐YH, X‐MW, S‐PH, LG, JLi, Z‐WF, PC, and G‐QZ collected, extracted, and analyzed the data. X‐JL, Z‐GH, Z‐YL, JLu, D‐PY, and T‐QG critically revised the manuscript. All authors have contributed to, read, and approved the final manuscript for submission.Fig. S1. Stability of 13 potential references in lung squamous cell carcinoma (LUSC) tissues and cells evaluated by geNorm and NormFinder. A total of 12 cases of LUSC and their corresponding adjacent non‐tumor lung tissues was used to test the references . Five different siRNAs were transfected into CALU1 cells as well .Table S1. Stability values of potential internal references based on geNorm and NormFinder.Click here for additional data file.


Focal segmental glomerulosclerosis (FSGS) is a serious condition leading to kidney failure. We aimed to investigate the clinical characteristics of FSGS and its differences compared with minimal change disease (MCD) using cross-sectional data from the Japan Renal Biopsy Registry. In Analysis 1, primary FSGS (n = 996) were stratified by age into three groups: pediatric (< 18 years), adult (18–64 years), and elderly (≥ 65 years), and clinical characteristics were compared. Clinical diagnosis of nephrotic syndrome (NS) was given to 73.5% (97/132) of the pediatric, 41.2% (256/622) of the adult, and 65.7% (159/242) of the elderly group. In Analysis 2, primary FSGS (n = 306) and MCD (n = 1303) whose clinical diagnosis was nephrotic syndrome (NS) and laboratory data were consistent with NS, were enrolled. Logistic regression analysis was conducted to elucidate the variables which can distinguish FSGS from MCD. On multivariable analysis, higher systolic blood pressure, higher serum albumin, lower eGFR, and presence of hematuria associated with FSGS. In Japanese nationwide registry, primary FSGS patients aged 18–64 years showed lower rate of NS than those in other ages. Among primary nephrotic cases, FSGS showed distinct clinical features from MCD. FSGS originally referred to steroid-resistant nephrotic syndrome in pediatric patients with segmental obliteration of glomerular capillaries, with or without hyalinosis on light microscopy2. The entity of FSGS has been expanded and FSGS is currently regarded as a group of kidney diseases sharing common glomerular lesions3. There are two pathophysiological types of FSGS: primary and secondary ones. In primary FSGS, it is postulated that circulating permeability factors cause podocyte injury, inducing NS4. Whereas, secondary cases have underlying etiologies such as hypertension, obesity, viruses, drugs, genetic mutations, or adaptive conditions5, and they do not always manifest NS. Therefore, the clinical presentations of FSGS are diverse. Cases with NS  show a different clinical course compared with minimal change disease (MCD) i.e. poor therapeutic response, worse renal prognosis6, and rapid recurrence of proteinuria after kidney transplantation7. However, it is difficult to clearly distinguish nephrotic FSGS from MCD because of their similar clinical presentations and the sampling limitations of pathological specimens9. To date, few studies have described the clinical features of FSGS, as well as the distinction between nephrotic FSGS and MCD using a large sample size.Focal segmental glomerulosclerosis (FSGS) presents with proteinuria, often accompanied by nephrotic syndrome (NS), and may lead to end-stage renal disease10. Cross-sectional studies using this database have described the clinical features of several kidney diseases in Japan19.The Japan Renal Biopsy Registry (J-RBR) is a nationwide, web-based, registry of renal biopsies established by the Japanese Society of Nephrology in 2007The present study aimed to clarify the clinical characteristics of FSGS patients in Japan by age stratification of them and comparison with MCD patients through an analysis of data from the J-RBR compiled between July 2007 and June 2016.Among 30,949 patients who registered to J-RBR between July 2007 and June 2016, 1,410 cases were extracted for the evaluation of FSGS. Additionally, 3656 patients were also extracted for the evaluation of MCD . Fewer patients who were consistent with NS in laboratory data at biopsy were found in the pediatric group and adult group than in the elderly group .For Analysis 1, 996 primary FSGS cases were divided into three age groups: pediatric group (n = 132), adult group (n = 622), and elderly group (n = 242). The detailed age-stratified distribution of the prevalence and proportion of patients with FSGS and MCD are shown in Supplementary Fig. 2). FSGS patients also had higher serum albumin (1.9 vs. 1.7 g/dL), lower daily urinary protein levels (6.28 vs. 7.00 g/day) and higher prevalence of hematuria (52.9 vs. 29.8%) at biopsy.Of 1410 patients with “focal segmental glomerulosclerosis” and 3656 with “minor glomerular abnormalities” on histological diagnosis (histopathology), 306 with nephrotic FSGS and 1303 with MCD were included in Analysis 2 , and presence of hematuria . In Model 2, higher systolic blood pressure , higher serum albumin , and presence of hematuria .As summarized in Table The present study used the data from a Japanese nationwide kidney biopsy registry. This is a unique study focusing on the clinical manifestations of FSGS with a large sample size. Furthermore, this study is the first one to describe the clinical features of primary FSGS from two different viewpoints: age stratification and comparison between nephrotic FSGS and MCD.20. Therefore, approximately 800 patients per year were inferred to be diagnosed with FSGS by biopsy in our country. The prevalence of FSGS varied among races and countries21. An international survey revealed that FSGS accounted for 19.1% of primary glomerular disease in North America, 14.9% in Europe, 6.9% in Asia, and 15.8% in Latin America22. A part of this epidemiological difference could be explained by the presence of genetic variants in apolipoprotein L1 (APOL1) among people with sub-Saharan ancestry23. Additionally, several studies reported that the incidence of FSGS was increasing, especially in the US26. The present study shows that incidence of FSGS in Japan was lower than that in reports from other regions21 and it had not changed for the past decade.In the J-RBR database, FSGS occupied 3.4–4.5% in total number of kidney biopsy. The Research Group on Progressive Renal Disease from the Ministry of Health, Labor and Welfare of Japan reported that the annual number of native kidney biopsy in Japan was estimated 18,000 to 21,00027. However, distinguishing secondary FSGS is challenging and the incidence of secondary FSGS remains unclear. D’Agati reported that 10–20% of FSGS patients are secondary ones3. In J-RBR, secondary cases constituted 16.2% of the total FSGS patients, and our results showed that the etiologies in these patients were different among the age groups; 10.8%, 17.4% and 16.0% among the pediatric, adult and elderly group, respectively. As the etiology, hypertension and obesity were the most common in total or among the adult and elderly group. Unilateral kidney or renal dysplasia, low birth weight and genetic disorders were leading causes of FSGS among the pediatric group . However, the urinary protein level was the lowest in this group. This discrepancy between clinical diagnosis and laboratory data could be explained by the effect of immunosuppressive treatment. Because of the quite high occupancy of MCD in NS of pediatric age, children with NS typically receive empiric treatment centering glucocorticoids and the indication of kidney biopsy are limited to the cases with refractory clinical course i.e., steroid-resistant or frequent relapse. Therefore, it is possible that most pediatric FSGS patients had already received immunosuppressive treatment by the time of biopsy. However, we cannot distinguish whether the data was obtained before or after initiation of immunosuppressive treatment, in the J-RBR data. The adult group showed the lowest prevalence of NS (41.2%) and the highest prevalence of chronic nephritic syndrome (54.3%). Whereas, the elderly group showed a higher prevalence of NS (65.7%) than the adult group. This suggested the differences in indications for renal biopsy among age groups. In Japan, a nationwide health examination program for all community residents, including urinalysis screening, has been conducted for over 40 years28. This regular screening enables early detection of urine abnormalities and early referral to a nephrologist. The suggested indication for renal biopsy in adult patients in Japan is ≥ 0.5 g/day of proteinuria or any amount of proteinuria with hematuria29. Many patients in the adult group may have undergone biopsy based on these suggested indications. Therefore, it is likely that the non-nephrotic cases with a substantial number of secondary FSGS were included to this age group. In contrast, it was also possible that the threshold of indications for renal biopsy in elderly patients might be higher i.e., clinicians attempted to perform biopsy for NS patients but not for the non-nephrotic patients in this age group. The clinical features of overall FSGS patients including secondary FSGS in the J-RBR and their age-group comparison were shown in Supplementary Table Analysis 1 described the clinical characteristics of primary FSGS in three age groups. We showed that the proportion of NS was different in each age group. Previous studies reported that nephrotic-range proteinuria is more frequently seen in children than in adults in FSGS30 and secondary cases with identifiable etiologies had been excluded. Even though classic articles suggested that hypertension, renal insufficiency and hematuria at the onset of disease are helpful to distinguish FSGS from MCD1, none have ever been statistically evaluated for their clinical significance in FSGS with a large sample size. We found that higher blood pressure, higher serum albumin, and incidence of hematuria at biopsy were associated with nephrotic FSGS in two multivariable models. FSGS was associated with older age and lower eGFR in univariate analysis. However, association between higher age and FSGS was not observed in multivariable Model 2, which suggested that there was confounding between age and eGFR. As described above, pediatric patients could represent biased population with the necessity of single or repeat biopsy for steroid-resistant or frequent relapse NS. We conducted sub-analysis only including the patients aged ≥ 18 years  and obtained similar results as original analysis  were consistent with NS31. Another limitation of this study is the lack of information regarding pathological subgroups of FSGS: variants of the Columbia classification27. Previous studies reported that morphologic variants of FSGS demonstrate distinct features in their clinical presentation and prognosis37. A longitudinal investigation is currently underway based on J-RBR database including the additional information regarding Columbia classification.This study has several limitations. First, the J-RBR system did not collect detailed information regarding the etiology of FSGS. All the information was provided by the local coinvestigators. The J-RBR also lacked the information of the findings in electron microscopy and detailed genetic testing that could help discriminating primary and secondary FSGS. It is possible that primary FSGS may include some secondary cases and, therefore, the percentage of secondary FSGS may be underestimated in our study. Second, the J-RBR did not collect the information about total number of glomeruli. Therefore, it was possible that we included the patients without adequate number of glomeruli to distinguish FSGS from MCD in the present study. Third, this study was based on cross-sectional data. Although eGFR was lower in FSGS than in MCD, it was difficult to determine whether the impaired kidney function indicated chronic kidney disease or acute kidney injury. Fourth, as mentioned above, the J-RBR data do not include information on whether the data were obtained before or after the initiation of immunosuppressive treatment. Modification of the J-RBR system has been conducted to achieve a more accurate description of pathological diagnoses, including etiological informationIn conclusion, nation-wide registry system revealed the characteristics and clinical features of FSGS in Japan. The results show that the incidence of FSGS in Japan is lower than that in other countries. When primary FSGS patients were divided into three age groups, the incidence of NS was lowest in the adult group (18–64 years), suggesting that the indications for renal biopsy might have been stricter among pediatric and elderly patients. When the analysis was focused on primary NS at the time of renal biopsy, FSGS showed distinct clinical features, such as younger age, higher blood pressure, higher serum albumin, lower eGFR, lower daily urinary protein level and presence of hematuria compared with MCD.10. As of June 2016, 143 nephrology centers participated in this registry, which included 30,949 patients. The J-RBR enters patient clinical information at biopsy. The J-RBR diagnosis consists of three components: (i) a clinical diagnosis, (ii) a histological diagnosis by pathogenesis, and (iii) a histological diagnosis by histopathology.This cross-sectional study used the data from the J-RBR. The details of the J-RBR system were described in a previous publicationAmong the patients who registered to J-RBR between July 2007 and June 2016, cases whose histological diagnosis (histopathology) was “focal segmental glomerulosclerosis” were extracted for the evaluation of FSGS. Additionally, patients whose histological diagnosis (histopathology) was “minor glomerular abnormalities” were also extracted for the evaluation of MCD  of “focal segmental glomerulosclerosis” without multiple registration (n = 1409), the patients whose histological diagnosis (pathogenesis) was “primary glomerular disease” were defined as primary FSGS unless they had information of etiologies. The patients with identified information of their etiologies  were defined as secondary FSGS. For example, when hypertension was considered to be a cause of FSGS, the case was classified into secondary FSGS. When hypertension was considered as just a concomitant condition, the case was classified into primary FSGS. FSGS lesions in other diseases  and renal graft were excluded. The details of selection for FSGS patients were described in Supplementary Fig. The annual incidence of FSGS and proportion of primary and secondary FSGS and were described. Then, two main analyses were conducted as follows.The cases with primary FSGS were divided into three age groups: pediatric group < 18 years), adult group (18–64 years), and elderly group (≥ 65 years)  and urinary protein ≥ 3.5 g/day or ≥ 3.5 g/gCr and serum albumin ≤ 3.0 g/dL for adult and elderly patients (age ≥ 18). The body surface area (m2) of pediatric patients was calculated by the DuBois formula: height (cm)0.725 × body weight (kg)0.425 × 0.007184.(i) primary cases, (ii) clinical diagnosis of NS, and (iii) laboratory data consistent with NS, which was defined as urinary protein ≥ 40 mg/h/mFrom the patients with histological diagnosis (histopathology) of “minor glomerular abnormalities”, following patients were included: (i) primary MCD, (ii) clinical diagnosis of NS, and (iii) laboratory data consistent with NS. Patients with multiple registrations, secondary MCD, other diseases with no obvious light microscopic glomerular findings , and renal graft were excluded. The details of selection for MCD patients were described in Supplementary Fig. Clinical parameters were compared between nephrotic FSGS and MCD patients.Clinical data at biopsy such as patient characteristics , comorbidities (concomitant hypertension and diabetes), urinary findings , and blood test findings  were extracted from the J-RBR database.38: eGFR [mL/min/1.73 m2] = 110.2 × reference-sCr/sCr (mg/dL) + 2.93, reference-sCr =  − 1.259 × Height (m)5 + 7.815 × Height (m)4 − 18.57 × Height (m)3 + 21.9 × Height (m)2 − 11.71 × Height (m) + 2.628 , reference sCr =  − 4.536 × Height (m)5 + 27.16 × Height (m)4 − 63.47 × Height (m)3 + 72.43 × Height (m)2 − 40.06 × Height (m) + 8.778  and adults (age ≥ 18)39: eGFR [mL/min/1.73 m2] = 194 × sCr (mg/dL)−1.094 × Age−0.287 , eGFR [mL/min/1.73 m2] = 194 × sCr (mg/dL)−1.094 × Age−0.287 × 0.739 . Additional diagnostic information, including details of etiology were also extracted.eGFR was calculated using equations based on serum creatinine (sCr) level for Japanese children (age < 18)Clinical characteristics were expressed as median/interquartile range and frequency number/percentage. We used the Kruskal–Wallis test to compare continuous variables and the chi-squared test to compare the proportions of categorical variables among the age groups.We used the Mann–Whitney U test to compare continuous variables and the chi-squared test to compare the proportions of categorical variables between nephrotic FSGS and MCD. Logistic regression analysis was used to evaluate the association between each variable and diagnosis of FSGS compared to MCD. Variables with P < 0.05 in univariate analysis and variables with clinical importance  were included in the multivariable model, while we excluded variables with strong correlations (Pearson’s correlation coefficients > 0.40) or clinical relevance.Statistical analyses were conducted using STATA IC version 14.0 . The statistical significance level was set at P < 0.05.Written informed consent was obtained from all participants and/or their legal guardians. All procedures performed in the present study were in accordance with the standards of the ethics committee of the Japanese Society of Nephrology  and the ethics committee of Nagoya University , and with the Helsinki Declaration of 1975 and its later amendments. J-RBR is registered in the UMIN Clinical Trial Registry (UMIN000000618).Supplementary Information.


Colorectal cancer, common in both men and women, occurs when tumors form in the linings of the colon. Common treatments of colorectal cancer include surgery, chemotherapy, and radiation therapy; however, many colorectal cancer treatments often damage healthy tissues and cells, inducing severe side effects. Conventional chemotherapeutic agents such as doxorubicin (Dox) can be potentially used for the treatment of colorectal cancer; however, they suffer from limited targeting and lack of selectivity. Here, we report that doxorubicin complexed to hyaluronic acid (HA) (HA‐Dox) exhibits an unusual behavior of high accumulation in the intestines for at least 24 hr when injected intravenously. Intravenous administrations of HA‐Dox effectively preserved the mucosal epithelial intestinal integrity in a chemical induced colon cancer model in mice. Moreover, treatment with HA‐Dox decreased the expression of intestinal apoptotic and inflammatory markers. The results suggest that HA‐Dox could effectively inhibit the development of colorectal cancer in a safe manner, which potentially be used a promising therapeutic option. In 2019, over 100,000 new cases of colon cancer have been reported; it is expected to cause about 50,000 deaths. However, early diagnosis, often by screenings, resulting in the removal of colorectal pulps before they develop into cancers, and conventional therapies have resulted significant decrease in mortality.Encapsulation of drugs in liposomes offers a potential means to reduce toxicity. To address this, efforts have been made to design liposomal formulations for the treatment of colorectal cancer. MM‐398 and IHL‐305, a liposomal formulation of Irinotecan, were designed to augment tumor efficacy while minimizing toxicity. Subcutaneous injection of MM‐398 showed improved efficacy while reducing toxicity in nude mice with colorectal cancer, however patients receiving one dose of MM‐398 experienced neutropenia, diarrhea, as well as vomiting and abdomen pain.Adriamycin, commonly known as doxorubicin (Dox), has been used, in combinations with other drugs, to treat many different types of cancers, such as breast, lung, ovarian, and bladder cancers, as well as neuroblastoma, leukemia, Hodgkin's and non‐Hodgkin's lymphoma. Doxorubicin encapsulated in a liposome (Doxil), administrated intravenously, has been used to treat breast cancer, ovarian cancer, Kaposi's sarcoma, and other solid tumors.Hyaluronic acid (HA), a naturally found polymer found in the body, has been shown to be useful in cancer treatment as a therapeutic. HA is able to bind to receptors on the cell membrane of tumor cells, which slows metastasis and cell migrationA combination of Dox and HA can be used as treatment. Recently, a Dox‐loaded hyaluronic acid ceramide nanoassembly releasing microspheres, administered intra‐arterially, was used to treat tumors.22.1The drug doxorubicin (Dox), was chemically linked to 50 KDa hyaluronic acid (HA) polymer via nucleophilic acyl substitution reactions. Briefly, the polymer was dissolved in a mixture of deionized water and DMSO at a ratio of 1:1 volume. The catalyst 4‐Dimethylaminopyridine (DMAP) and the activator 1‐ethyl‐3‐3‐dimethylaminopropyl) carbodiimide hydrochloride (EDC) were added to the solvent mixture at a molar ratio of 1:1 relative to the HA monomers. Following 30 min of activation, Dox was added into the reaction mixture at molar ratios 0.4:1 relative to the HA monomer and left stirring for 24 hr. Following reaction, the product was purified by size exclusion chromatography via Sephadex G‐25 PD‐10 desalting columns  followed by overnight dialysis ) against DI water. The samples were then lyophilized, stored at 4°C and reconstituted in PBS before use. The amount of Dox incorporated was measured using its respective fluorescence spectra at Ex/Em 470/590.2.2The size and morphology of HA‐Dox were examined by transmission electron microscopy. Briefly, 2.0 μl of HA‐Dox suspensions were allowed to air‐dry on Formvar carbon‐coated cupper grids. Transmission electron microscopy (TEM) was performed on a JEOL JEM‐1400 TEM instrument, operating at a voltage of 100 kV . Particle zeta potential was measured by dynamic light scattering (DLS) on Malvern Zetasizer . The mean particle size of HA‐Dox was estimated with a NanoSight NS3000 .2.3Lyophilized HA‐Dox was dissolved in PBS at 1 mg/mL DOX concentration (pH 7.4) and incubated for 5 days at 37°C in Slide‐A‐Lyzer MINI dialysis devices . The devices were inserted in microcentrifuge tubes holding 1 ml of PBS. The 100 μl of Dox‐release medium in the microcentrifuge tubes was collected at the indicated time points and its concentration was measured via fluorescence using the TECAN plate reader. The amount of Dox released at each time point was divided with the initial amount loaded to obtain the cumulative release. All measurements were carried out in triplicate, and the results were indicated as the mean ± SD.2.4All animal studies were carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals as adopted by National Institute of Health, approved by Harvard University IACUC. Mice were housed in cages with free access to water and food, located in a well‐ventilated temperature‐controlled room between 18 and 23°C with relative humidity ranging from 40 to 60% under a 12‐hour light/dark period.2.5g, 15 min, 4°C; supernatant was removed and this wash step was repeated three times.Healthy female balb/c mice (50–56 days) were purchased from the Charles River Laboratory . Whole blood, collected in EDTA coated tubes (BD Microtainer) or serum collecting tubes (BD Microtainer), was spun at 1,000 g for 10 min at 4°C. Plasma and well as the buffy layer containing white blood cells and platelets, was removed; serum was stored at 4°C for 1 hr. Isolated erythrocytes (RBCs) were washed by adding ice cold 1x Dulbecco's‐phosphate‐buffered‐saline (DPBS) pH 7.4 up to 12 ml total volume and pipetting gently up and down to mix RBC extensively. RBC suspensions were centrifuged at 6002.6HA‐Dox was adsorbed onto murine RBC at RT in 55% serum. Washed naïve RBC and washed RBC: HA‐Dox suspensions (1% Hematocrit) were dispensed onto a 96 U‐shaped plate and visually accessed after 24 hr at room temperature after RBC suspension had fully sedimented. Carboxylated polystyrene beads was used as a positive control.2.7, HA‐AlexaFluro647, as well as HA‐Dox‐AlexaFluro647 were intravenously injected into healthy female Balb/c mice weighing between ~18 and 20 g. The biodistribution studies of Dox and HA‐Dox were performed after 0.08, 0.5, 6, and 24 hr post‐injection. At these time intervals, the blood was collected from inferior vena cava (IVC) and organs were isolated. Organs were weighed, homogenized in 10% water (w/v) and centrifuged 20,000g for 20 min. Homogenates (supernatant) were collected; drug was then extracted with 6x methanol, incubated for 30 min with gentle vortex, and centrifuged 20,000g for 20 min. Supernatant was read on a SpectraMax I3 plate reader was interpreted as a percentage of injected dose/tissue. The biodistribution studies of HA‐AlexaFluro647, as well as HA‐Dox‐AlexaFluro647 were performed after 0.08 hr and 0.5 hr post‐injection. Intestines were harvested and far red fluorescence signals were imaged using Perkin Elmer IVIS small animal imaging system.Dox, HA‐Dox2.82 for 15 min. After incubation, an aliquot was aspirated into a BD LSRFortessa cell analyzer (BD Biosciences) for analysis, gated at 10,000 events. Results were expressed as percentage of Annexin V positive RBCs. Data were analyzed using FCS Express Version 6 (DeNovo Software). Homogenates (100 μl) from intestinal organs , diluted in PBS (900 μl) were gently vortexed, and directly aspirated into a BD LSRFortessa cell analyzer (BD Biosciences) for analysis, gated at 10,000 events.HA‐Dox was adsorbed onto murine RBC at RT in 55% serum. Washed naïve RBC and washed RBC:HA‐Dox suspensions (5 μl) at 10% Hematocrit were added to 995 μl of PBS, gently vortexed, and ran on a BD LSRFortessa cell analyzer , gated at 10,000 events. Data were analyzed using FCS Express Version 6 . Washed RBC and HA‐Dox suspensions at 10% Hemocrit were incubated at room temperature with annexin V‐647 in buffer containing 2 mM CaCl2.9All experiments involving naive and colon cancer mice were carried out in strict accordance with Guide for the Care and Use of Laboratory Animals, adopted by National Institutes of Health, approved by Harvard University IACUC. Briefly, 50–56‐day‐old female balb/c mice were maintained in a temperature and humidity‐controlled facility under a 12 hr light/dark cycle. Water and standard diet were given ad libitum. After 1 week of acclamation, animals were subcutaneously injected once with azoxymethane (AOM) (20 mg/kg body weight). After 7 days, 2.0% dextran sulfate sodium (DSS) (Sigma‐Aldrich) was given in drinking water over 7 days followed by regular drinking water for 1 week. Saline, 105 μg Dox and 105 μg HA‐Dox was injected i.v. three times post DSS feeding period. After 1 week, mice were euthanized and intestines were harvested.2.10Immediately after mice were euthanized, intestines were harvested in and placed in 10% formalin overnight and then processed for embedding in paraffin. Paraffin‐embedded sections (5 μm) were treated with xylene; rehydrated in decreased ethanol series, and stained with hematoxylin and eosin (H and E) or immunohistochemistry and examined under a light microscope. To access the severity of AOM‐DSS‐induced colon carcinogenesis, the intensity of apoptotic and inflammatory markers was determined using IHC analysis. Colon and other intestinal tissues were immune stained with Caspase‐3 , Bax , iNOS (Abcam), and COX‐2 (Abcam) as previously described and examined under a light microscope. Colon and other intestinal tissues were also stained with 1:200 dilution of anti‐KI67 (Abcam) and later with 1:200 dilution of anti‐rabbit secondary antibody (Abcam). The sections were then counterstained with hematoxylin and finally dehydrated and covered with coverslips.33.1HA‐Dox was synthesized by conjugation of doxorubicin to HA through nucleophilic acyl substitution reaction chemistry. The morphology of HA‐Dox was examined by transmission electron microscopy (TEM). HA and Dox are spherical particles possessing a core–shell morphology Figure . TypicalDynamic light scattering (DLS) measurements also showed that these HA‐Dox possessed a zeta potential of −4.8 ± 1.06 mV Figure , which c3.2In vitro release rates of HA‐Dox indicated a slow and steady release of the drug from HA. Approximately, 20.6 ± 0.8 wt% Dox was released at the end of 120 hr . The in vitro release profile is similar to the pattern obtained previously attributing the successful incorporation of Dox and its slow release via amide hydrolysis.3.3Pharmacokinetics of HA‐Dox was evaluated in healthy Balb/c mice (105 μg of either free Dox or HA‐Dox). HA‐Dox exhibited longer circulation compared to free Dox. Dox and HA‐Dox exhibited classical elimination behavior, however, a significant difference in the elimination half‐life was found. While only 20% ID of free Dox was detected in blood 0.08 hr after injection, 40% ID of HA‐Dox could be detected in the blood at the time point Figure . The difThe mechanisms of enhanced persistence of HA‐Dox in blood were studied. Hyaluronic acid by itself has a blood half‐life of 3–5 min in the blood. Hence, simple association of dox with HA is not expected to render long circulation. We assessed whether association of dox‐HA with red blood cells (RBCs) may be responsible for extended circulation. Prior literature has shown that RBC‐association enhances the circulation of polymeric nanoparticles.Adsorption of HA‐Dox onto RBCs did not induce agglutination Figure , inset, 3.4Biodistribution of Dox and HA‐Dox in mice was assessed. Significant accumulation of HA‐Dox was found in intestine (~45% ID), while only ~7% ID was found in the liver 0.5 hr after administration Figure . MinimalWithin the intestines, most HA‐Dox as well as free Dox was found to accumulate in the duodenum and jejunum after 0.08 hr. Unlike free Dox, a significant accumulation of HA‐Dox was also observed in the ileum, colon, and cecum Figure . CompareTo our knowledge, there have been no reports of free Dox or any Dox complexes accumulating in the intestines via intravenous injection. To confirm HA‐Dox accumulates in the intestines, duodenum and jejunum, ileum and colon, and cecum homogenate were obtained from mice 24 hr after IV administration and analyzed using a BD LSRFortessa cell analyzer. For each intestinal homogenate, a mean fluorescence intensity (MFI) shift in the Dox channel (488 nm‐582/42) compared to the noninjected mice was observed . FurtherTo assess the role of HA in targeting of HA‐Dox in the intestine, localization of AlexaFluro647‐labeled HA was measured. Signal intensities were observed in the stomach 0.08 hr after administration. Some signal was observed in all parts of the intestine , suggesting that HA accumulates in the intestines . Althoug3.5The therapeutic potential of HA‐Dox was tested using AOM‐DDS‐induced colon cancer. Exposure to AOM‐DDS induced colon cancer‐like symptoms including significant shortening of the colon and intestines. Treatment of mice with saline alone did not revert the length reduction Figure . In contTo investigate the effect of HA‐Dox in chemical‐induced colon cancer, we first performed histological analysis of AOM/DSS mice intestinal tissues. Microscopic evaluation of H&E stained small intestine and colon sections of AOM/DSS‐treated mice displayed increased intestinal injuries compared to healthy mice Figure . Saline 3.6To evaluate the effect of HA‐Dox on the proliferation and differentiation ability of intestinal crypt cells, Ki67 were assessed by immunohistochemistry staining Figure . The exp3.7The effect of HA‐Dox on the inflammation in AOM/DSS mice was also assessed in terms of (cyclooxygenase‐2) COX‐2 and inducible nitric oxide synthase (iNOS) expression levels by immunohistochemistry . Althoug3.8There have been studies that have shown that Dox induces apoptosis by activation of caspase 3 in cultured cardiomyocytes in vitro and rat cardiac ventricles in vivo. It is unclear whether administration of Dox can cause the increased activation of caspase‐3 in the intestines with the concomitant apoptosis and shedding of villus in healthy or sick mice. To specifically address the type of cell death responsible for intestinal epithelial cell (IEC) shedding and loss from the villus, we performed IHC for active caspase‐3 as well as Bax2 in healthy and AOM/DSS mice . Interes4Doxorubicin has been used to treat cancers such as leukemia, lymphoma, as well as breast cancer, lung cancer, and thyroid cancer. However, there are no reports of the use of doxorubicin to intestinal and colon cancers. To our knowledge, this is the first report that intravenous injections of doxorubicin variants can target the intestines. There are many studies have shown that drugs taken orally are absorbed systemically from the small intestine, making oral drug delivery to be the traditional choice for intestinal targeting. However, oral administration of doxorubicin remains a challenge for several reasons. Doxorubicin undergoes acid hydrolysis in the stomach and any remaining doxorubicin reaching the intestine is discharged by P‐glycoprotein, found in the epithelial cells lining the small intestine and colon. Thus, doxorubicin exhibits low oral bioavailability and traditional drug delivery pathway to the intestines is hindered. It is only available on the market as intravenous formulations, often laden with cardiotoxicity and a drastically reduced ability to specifically target the intestines and colon. However, our variant of doxorubicin conjugated to hyaluronic acid is biocompatible and does not exhibit the issues present in the current intravenous injection therapy.The mechanism that enables HA‐Dox to target and remain in the intestine after being intravenously injected is unclear; however, the presence of CD44 and RHAMM in the intestine may offer insight into the phenomenon. HA‐Dox, a 150 nm nanoparticle‐like complex, was observed 0.08 hr (5 min) after administration in the blood, however, over time, HA‐Dox was cleared from the bloodstream. HA‐Dox is able to absorb onto RBC in the presence of serum; however, it detaches quickly. Unlike most nanoparticles that are able to adsorb onto RBC,In addition, there have been studies focusing on the inter‐ and intra‐cellular interactions between hepatic cells and nanoparticles as well as the elimination of nanoparticles through the hepatobiliary system.We thus analyzed whether intravenous injection of HA‐Dox in AOM/DSS mice could be used to prevent colorectal cancer progression. The accumulation and therapeutic effects of HA‐Dox in the intestinal tissues are probably enhanced by both an increase in blood flow and in vascular permeability; both being well documented in inflammatory bowel disease (IBD). Dox slows or stops the growth of cancer by blocking topoisomerase 2. There have been studies that have shown HA a promising anti‐cancer agent. There have been studies that have shown that HA can act as inhibitor of tumor vascularization, tumor metastasis, cancer formation, and cancer growth. Mechanism of its action include either to promote the creation of a capsule from the connective tissue, resulting in the encompassing of the tumor, proteolytical activation of CD44, or binding to the cell membrane of the tumor cells.There is evidence that a link between inflammation and colorectal cancer exists.There are many reports that show that chemotherapy causes extensive damage to the DNA present in the intestinal cell wall. Researchers have been looking into reducing the damage done to the intestinal cell walls from chemotherapy, as it would render the treatment more bearable and allow for a higher rate of implementation of chemotherapy.In conclusion, our study revealed that HA‐Dox may be an effective therapeutic agent to prevent or reduce the risk of colorectal cancer development, particularly for people who already suffer from IBD.Supplemental Figure 1 Accumulation of HA‐Dox in Perfused Intestines:. The amount of HA‐Dox represented as percentage of injected dose (%ID) for free Dox and HA‐Dox following I.V. injection in mice at 24 h after perfusion compared to no perfusion remaining in different parts of murine intestine. Each data point represent means ± SEM (n = 3).*, p < 0.05; non paired, two‐tailed t‐test.Supplemental Figure 2: Accumulation of HA‐Dox in Intestines: Representative flow cytometry histograms portraying the presence of HA‐Dox following I.V. injection in mice at 24 h in (A) Duodenum and Jejunum (B) Ileum and Colon and (C) Cecum homogenates. Dox spiked in was used as a positive control.Supplemental Figure 3: Accumulation of HA‐Dox in Intestines: Representative confocal images portraying the presence of HA‐Dox represented by dots following I.V. injection in mice at (A) 0.08 h and (B) 6 h in Duodenum and Jejunum Ileum and Colon and Cecum. Images were taken with a 40x objectiveSupplemental Figure 4: Biodistribution of HA‐647 in Intestines: Representative ex vivo fluorescence image obtained with IVIS of GI organs (A) 0.08 h and (B) 0.5 h after I.V. administration of HA conjugated with alexafluor 647. Organs: (1) Stomach; (2) Duodenum; (3) Jejunum; (4) Ileum; (5) Cecum; and (6) Colon. A scale of the radiance efficiency is presented to the right of excised mouse organ image.Supplemental Figure 5: Biodistribution of HA‐Dox‐647 in Intestine: Representative ex vivo fluorescence image obtained with IVIS of GI organs (A) 0.08 h and (B) 0.5 h after I.V. administration of HA‐Dox conjugated with Alexafluor 647. Organs (1) Stomach; (2) Duodenum; (3) Jejunum; (4) Ileum; (5) Cecum; (6) Colon. A scale of the radiance efficiency is presented to the right of excised mouse organ image. Representative flow cytometric analysis of Dox presence in (C) Duodenum and Jejunum (D) Ileum and Colon (E) Cecum homogenates 0.08 h and 0.5 h after I.V. administrationSupplemental Figure 6: Inflammation analysis of Murine Intestinal Tissues Induced with Chemical Induced Colon Cancer: Immunohistochemistry of Intestinal Tissues treated with azoxymethane and dextran sulfate treated with saline, Dox, and HA‐Dox, determined by (A) Cox‐2 and (B) iNOS staining. Representative images were taken at 400x. Red scale bar: 50 umSupplemental Figure 7: Apoptotic analysis of Murine Intestinal Tissues Induced with Chemical Induced Colon Cancer: Immunohistochemistry of Intestinal Tissues treated with azoxymethane and dextran sulfate treated with saline, Dox, and HA‐Dox, determined by (A) Caspase‐3 and (B) Bax staining. Representative images were taken at 400x. Red scale bar: 50 umClick here for additional data file.


Prescription opioids are commonly used for postoperative pain relief in older adults, but have the potential for misuse. Both opioid side effects and uncontrolled pain have detrimental impacts. Frailty syndrome (reduced reserve in response to stressors), pain, and chronic opioid consumption are all complex phenomena that impair function, nutrition, psychologic well-being, and increase mortality, but links among these conditions in the acute postoperative setting have not been described. This study seeks to understand the relationship between frailty and patterns of postoperative opioid consumption in older adults.Patients ≥ 65 years undergoing elective surgery with a planned hospital stay of at least one postoperative day were recruited for this cohort study at pre-anesthesia clinic visits. Preoperatively, frailty was assessed by Edmonton Frailty and Clinical Frailty Scales, pain was assessed by Visual Analog and Pain Catastrophizing Scales, and opioid consumption was recorded. On the day of surgery and subsequent hospitalization days, average pain ratings and total opioid consumption were recorded daily. Seven days after hospital discharge, patients were interviewed using uniform questionnaires to measure opioid prescription use and pain rating.p = 0.01). Doses of opioids prescribed at hospital discharge and the prescribed morphine milligram equivalents (MME) at discharge did not differ between groups. Seven days after discharge, the cumulative MME used were similar between cohorts. However, patients with frailty used a larger fraction of opioids prescribed to them  vs. 25.0 % , p = 0.007) and were more likely  to use 50 % and greater of opioids prescribed to them. Patients with frailty had higher pain scores before surgery and seven days after discharge compared to patients without frailty.One hundred seventeen patients , 64 % male), were evaluated preoperatively and 90 completed one-week post discharge follow-up. Preoperatively, patients with frailty were more likely than patients without frailty to use opioids (46.2 % vs. 20.9 %, Patterns of postoperative opioid use after discharge were different between patients with and without frailty. Patients with frailty tended to use almost all the opioids prescribed while patients without frailty tended to use almost none of the opioids prescribed. Prescription drug overdose deaths in the United States, where opioids are among the agents implicated, have increased between 1999 and 2010 in parallel with a 4 fold increase in morphine milligram equivalents (MME) per capita of opioid prescribing . ImproviAging is accompanied by cumulative declines across multiple physiologic systems, which may result in frailty. Frailty is defined by decreased reserve and resistance to stressors and increased vulnerability to adverse outcomes . FrailtyA prospective cohort study of older surgical patients was conducted over six months during 2018 and 2019 in a single academic institution that serves as both a Level 1 Trauma Center and a safety net hospital . This study was approved by the institutional review board at the University of Washington (IRB# STUDY00002781) and follows the STROBE guidelines. Patients 65 years and older undergoing elective surgery and anticipated to be admitted to the hospital for an overnight stay were recruited during their visit to the pre-anesthesia clinic. After providing written informed consent, patients were evaluated in the pre-anesthesia clinic before surgery, followed daily during their hospitalization after surgery, and interviewed seven days after their discharge from the hospital. Demographic data was extracted from the electronic medical record.Preoperative opioid use was evaluated by patients' self-report. Electronic medical records and nurses’ medication administration records were used to track daily opioid administration for each day of hospital stay and opioid prescriptions at discharge Fig. . Opioid Pain catastrophizing was assessed using the Pain Catastrophizing Scale (PCS) during preoperative evaluation by a trained researcher Fig. . The PCSFrailty was assessed preoperatively by a trained researcher utilizing the Clinical Frailty Scale (CFS) Fig. . The CFSP value of < 0.05 was considered statistically significant for all analyses.Statistical analyses were performed using SPSS version 25 . We tested all variables for normality and found them to be not normally distributed. Continuous variables are presented as median and interquartile range (IQR). To test the differences in continuous variables between two groups, with frailty and without frailty, the Mann–Whitney U test was performed. To compare differences between two groups, with frailty and without frailty, for categorical variables (e.g. with frailty/without frailty and using 50 % and greater of the opioids prescribed) we performed the Pearson’s Chi-Square test. The Spearman correlation was performed to test correlation between different frailty scores. A multivariate logistic regression analysis was performed to compare between the groups with frailty and without frailty in using 50 % and greater of the opioids prescribed with adjustment for age and sex. Odds ratio (OR) and 95 % confidence interval (CI) are presented. p≤0.001). The median age of the population with and without frailty was not different , but the population with frailty was more likely to be female . Preoperatively, a greater percentage of patients with frailty used opioids . Of opioids used preoperatively, oxycodone (41.5 %), tramadol (17.1 %), hydrocodone (12.2 %) were used most commonly, and codeine, fentanyl, hydromorphone, methadone, and morphine each made up < 10 % of prescriptions (Table p = 0.01) as well as pain catastrophizing scores  vs. 4 , p = 0.001). Surgical procedures were classified as spine (n = 42), vascular (n = 18), neurosurgery (n = 20), orthopedic (n = 18) (foot and ankle (n = 13), femur (n = 4), and humerus (n = 1)), and other  (n = 19)  , hospital day 1  vs. 22.5 , p = 0.507), hospital day 2  vs. 22.5 , p = 0.644), and hospital day 3  vs. 27.8 , p = 0.494). In the hospital, oxycodone (33.6 %), fentanyl (23.2 %), hydromorphone (18.1 %), and morphine (15.1 %) were ordered most commonly, with methadone and tramadol each being ordered < 10 % of the time. All patients with frailty (100 %) and most patients without frailty (82.9 %) were discharged from the hospital with an opioid prescription. Discharge prescriptions were most commonly oxycodone (79.3 %) and hydromorphone (16.1 %), with hydrocodone, methadone, morphine, and tramadol each prescribed < 10 % of the time (Table p = 0.906). Seven days after discharge, the total consumed MME did not significantly differ in patients with and without frailty  vs. 33.8 , p = 0.129). However, patients with frailty used a larger fraction of the total opioids prescribed to them  vs. 25.0 % , p = 0.007).There was great variability in quantity of opioids used in both patients with and without frailty. Although median preoperative opioid consumption was zero in both groups, since less than half of both study populations consumed any opioids preoperatively Table , patient07) Fig. . During p = 0.018). After adjusting for sex and age, the comparison of frailty with 50 % and higher consumption remained significant . Furthermore, the frail population used greater than 80 % of their prescription more often than the non-frail . The small sample size did not allow for further adjustments (as indicated by the wide CI). In contrast, the patients without frailty were more likely to use less than 20 % of their prescription , an average over prescription of 164.8 MME which is the equivalent of twenty-one 5 mg oxycodone pills.The primary outcome of our study was the use of less than 50 % of opioid prescriptions by patients seven days after hospital discharge Fig. . Ninety p = 0.01), however; during their hospitalization pain scores were similar. Seven days after discharge from the hospital, pain scores were different again: patients with frailty reported higher pain scores than patients without frailty  vs. 3 , p = 0.01)  than patients without frailty , 42. MorOpioid doses were variable, and patients with and without frailty did not differ in the amount of opioid medications used in the hospital, or the amount provided at discharge. The novel finding in this study is that frailty is also associated with a unique pattern of opioid use after hospital discharge. First, opioid use in our cohort does not follow a bell-shaped normal distribution. In fact, most patients use very little or almost all of the opioids prescribed. Second, the pattern of discharge opioid use was very different between patients with frailty , and patients without frailty . The findings of this study could potentially be explained by greater familiarity with opioid use in the population with frailty (who are more commonly prescribed opioids before surgery), lower tolerance for postoperative pain or reduced coping in patients with frailty (as suggested by higher pain catastrophizing scores before surgery), or more caregiver encouragement for use of pain medication in patients with frailty. Greater opioid usage in patients with frailty may put them at increased risk for side effects of their prescription and subsequent functional difficulties . ConversPatient use of opioids is often reflective of the amount prescribed . Opioid This study has several limitations: the study population is a small cohort of elective surgical patients at a single institution that is an academic, urban, safety net hospital. Furthermore, opioid use postoperatively in the United States may be higher than in other countries , which lA simple, rapid frailty assessment may be useful in predicting patterns of opioid use after elective surgery in older adults.