To support optimal third-line antiretroviral therapy (ART) selection in Namibia, we investigated the prevalence of HIV drug resistance (HIVDR) at time of failure of second-line ART. A cross-sectional study was conducted between August 2016 and February 2017. HIV-infected people ≥15 years of age with confirmed virological failure while receiving ritonavir-boosted protease inhibitor (PI/r)-based second-line ART were identified at 15 high-volume ART clinics representing over >70% of the total population receiving second-line ART. HIVDR genotyping of dried blood spots obtained from these individuals was performed using standard population sequencing methods. The Stanford HIVDR algorithm was used to identify sequences with predicted resistance; genotypic susceptibility scores for potential third-line regimens were calculated. Two hundred thirty-eight individuals were enrolled; 57.6% were female. The median age and duration on PI/r-based ART at time of enrolment were 37 years and 3.46 years, respectively. 97.5% received lopinavir/ritonavir-based regimens. The prevalence of nucleoside reverse transcriptase inhibitor (NRTI), non-nucleoside reverse transcriptase inhibitor (NNRTI), and PI/r resistance was 50.6%, 63.1%, and 13.1%, respectively. No significant association was observed between HIVDR prevalence and age or sex. This study demonstrates high levels of NRTI and NNRTI resistance and moderate levels of PI resistance in people receiving PI/r-based second-line ART in Namibia. Findings underscore the need for objective and inexpensive measures of adherence to identify those in need of intensive adherence counselling, routine viral load monitoring to promptly detect virological failure, and HIVDR genotyping to optimize selection of third-line drugs in Namibia. Thus, comparably less information is available to guide selection of optimal regimens in people with virological failure while taking second-line ART.Global HIV drug resistance (HIVDR) surveillance efforts and the majority of studies performed in low- and middle-income countries have focused on estimating the prevalence of HIVDR prior to treatment initiation or after failure of first-line NNRTI-based therapies.ART in Namibia is delivered following a public health approach, which involves use of standardized first- and second-line regimens and simplified laboratory monitoring, including at least one viral load test per year. At the time of study enrolment, Namibia's national ART guidelines recommended first-line regimens consisting of two nucleoside reverse transcriptase inhibitors (NRTI), tenofovir (TDF)/emtricitibine (FTC) or lamividuine (3TC) administered with the NNRTI efavirenz (EFV). Recommended second-line regimens were three NRTI , or abacavir (ABC)) administered with a ritonavir-boosted protease inhibitor, either lopinavir/ritonavir (LPV/r) or atazanavir/ritonavir (ATV/r).At time of study initiation (2016), 140,241 adults out of an estimated 204,147 adults living with HIV were receiving ART in Namibia of which 3884 were taking PI/r-based second-line regimens . Globally and in Namibia, unanswered questions remain about the contribution of protease inhibitor (PI) drug resistance to second-line ART failure. In this study, we report the prevalence and patterns of HIVDR in people failing second-line ART in Namibia's public health ART program.22.1The 15 ART clinics with the largest number of people on PI/r-based ART in the country were selected. These 15 clinics captured 70% (2746 of 3884) of all people receiving PI/r-based ART; clinics were located in nine different geographical regions: Khomas, Ohangwena, Zambezi, Oshikoto, Oshana, Kavango East, Erongo, Omusati, and Otjozondjupa.Between August 2016 and February 2017, all HIV-infected people ≥15 years of age receiving second-line PI-based ART for at least six months and who had confirmed virological failure per Namibia national ART guidelines (two consecutive HIV RNA tests ≥1000 copies/mL separated by at least three months) were identified and asked to participate in the study during routine clinic visits. Written informed consent was obtained from all participants. Individuals with self-reported treatment interruption of 30 days or more at the time of enrolment were ineligible; no additional information was collected about this group.2.2 and were frozen at –80oC for storage. DBS from participants who had a third consecutive VL test ≥1000 copies/mL were brought to ambient temperature, re-packed into new gas impermeable bags with desiccant and humidity indicator cards and shipped to South Africa for HIVDR testing.At study sites, nurses drew 5 mL of whole blood via venepuncture for viral load (VL) testing (a third consecutive VL test). Whole blood specimens were transported in a cold box on the day of collection to the Namibian Institute of Pathology (NIP) for VL testing. On arrival at NIP, dried blood spot (DBS) specimens were prepared by pipetting 75 μL aliquots of whole blood on each of 5 circles of a Whatman 903 filter paper card . DBS were dried at room temperature for 24 hours and subsequently packed in individual gas impermeable bags per World Health Organization (WHO) guidanceVL testing was performed on plasma within 48 hours of specimen receipt using COBAS AmpliPrep/COBAS TaqMan HIV-1 Test . HIVDR testing was performed at the National Health Services Laboratory, University of Witwatersrand, Johannesburg, South Africa, a WHO-designated HIVDR genotyping laboratory. RECall was used to generate consensus sequences; quality assurance was performed following WHO recommendations. The Stanford HIV drug resistance algorithm (HIVdb), version 8.4, was used to identify HIVDR resistance profiles, mutations, and HIV subtypes; sequences classified as having low-, intermediate-, or high-level HIVDR were considered as resistant. The HIV-1 integrase region was not sequenced because at the time of this study integrase strand transfer inhibitors (InSTI) were not prescribed for treatment of HIV in Namibia's public health sector.HIV-1 protease and reverse transcriptase (RT) sequences were obtained using standard population sequencing adapted from previously described methods.,10 The algorithm was used becasue Namibia does not have an established algorithm for selection of third-line ART based on HIVDR genotype test results. Genotypic susceptibility scores (GSS) for individuals who would have been eligible for third-line ART  were calculated based on HIVdb penalty scores for each drug in the regimen that would have been prescribed following the algorithm. The ARV drugs used in the algorithm include darunavir/ritonavir (DRV/r), 3TC/FTC, ZDV or TDF , and an InSTI if the TDF or ZDV score is 30 or above or the DRV/r score is 15 or above. In addition, etravirine (ETR) is added if the TDF or ZDV score is 30 or above and the DRV/r score is 15 or above and the ETR score is below 30. One GSS point was given for each drug with a Stanford HIVdb penalty score <15; it was assumed that virus was fully susceptible to all approved InSTI.Potential third-line treatment options were considered based on an algorithm developed by South Africa's national third-line ART committee.2.3 Demographic and laboratory characteristics are summarized as prevalence values or median values and interquartile ranges (IQR). HIVDR prevalence is reported with 95% Wilson confidence intervals adjusting for site-level clustering using a robust standard error. Fisher's exact tests and Wilcoxon rank sum tests were used to conduct two-group comparisons with a significance level of 0.05.Analyses were performed using Stata .2.4The study received institutional review board (IRB) approval from the Ministry of Health and Social Services in Namibia and expedited review from the IRBs of Tufts University School of Medicine, the University of Witwatersrand, Johannesburg, South Africa. The study was reviewed in accordance with the Associate Director for Science at the Center for Global Health, United States Centers for Disease Control and Prevention CDC human research protection procedures and was determined to be research. CDC investigators did not interact with human subjects or have access to identifiable data or specimens for research purposes.3All 238 eligible individuals identified during the study period were enrolled; 96.6% (230/238) were receiving a regimen consisting of three NRTIs in combination with either ATV/r or LPV/r. Table HIVDR genotyping was successful for 160/238 (67.2%) participants. Overall 70.0% (112/160) of sequences had mutations conferring drug resistance to any drug; 50.6%, 63.1%, 13.1%, and 6.9% had any NRTI, any NNRTI, LPV/r or ATV/r, DRV/r resistance, respectively  had three or more major PI mutations Table . In addi,11 are presented in Table GSS scores for potential third-line regimens following guidance developed by South Africa's national third-line ART committee4This study of HIVDR in people failing PI/r-based second-line ART in Namibia has important findings. First, 30% of people failing PI/r-based ART had no predicted HIVDR and only 13% had PI resistance, suggesting that most virological failures were not due to drug resistant virus. This observation suggests that greatly intensified adherence support may be an optimal initial approach to patient management of viral non-suppression in people receiving PI/r ART in Namibia. Secondly, our findings support the importance of routine VL monitoring for early detection of suboptimal adherence, followed by HIVDR testing in individuals identified as failing therapy, an approach which is likely to minimize premature switch to more complex and costly third-line regimens.–14In this study, overall NRTI resistance levels were high and could lead to reduced efficacy of the NRTI backbone for half of the people evaluated for third-line PI/r-based regimens. However, the comparably low prevalence of PI resistance observed in this study is reassuring given that genotypic testing may not accurately predict regimen activity when providing PI/r-based ART after second-line PI failure: PI/r-based regimens have demonstrated efficacy in people with NRTI resistance, even when predicted NRTI resistance is complete, suggesting that NRTIs retain partial antiretroviral activity or that NRTI-resistant viruses have reduced fitness.Thirteen per cent of study participants failed with PI-resistant virus, emphasizing the need for HIVDR testing in people receiving PI/r-based second-line treatment to ensure the selection of highly potent third-line regimens  and to identify individuals with virological failure due to suboptimal adherence  who are likely to benefit from enhanced adherence counselling. Although 87% of patients in this cohort have no PI resistance, informal patient surveys in Namibia's national ART programme suggest that the majority of people failing PI/r-based ART report inconsistent adherence to the PI component of the regimen due to PI-specific toxicity or side effects; thus resulting in dual therapy . Because of expense and logistics, HIVDR genotyping remains a costly adherence test, presenting an additional challenge: the lack of robust and inexpensive objective measures adherence to ART and specifically the PI/r component, which may lead to delayed switch in the face of true HIVDR or premature switch in people with no HIVDR.–17 and in a recent retrospective non-representative convenience sample of 366 HIVDR genotypes obtained by Namibia's ART program during the period 2010 to 2015.The prevalence estimates of overall, drug class and drug-specific resistance observed in this study are broadly comparable to those observed in other countries In addition, in a recent meta-analysis, DRV/r monotherapy in ART-experienced people was shown to be effective. However, one-third (7 of 21) of people in this Namibian cohort would have been treated with regimens having suboptimal predicted efficacy because their viruses were fully susceptible to the InSTI only. Despite its low propensity to select for drug resistance mutations, there are an increasing number of reports of virological failure associated with InSTI resistance in ARV drug-experienced InSTI-naïve individuals receiving a dolutegravir-containing regimen,,21 underscoring the need for caution and close virological monitoring of this sub-population who may be at risk of virological non-suppression.The predicted activity of potential third-line regimens according to South Africa's national algorithm is somewhat encouraging, since most  patients were either susceptible to DRV/r or had at least two active drugs available. A recent publication assessing the efficacy of South Africa's third-line algorithm showed a high proportion of individuals on third-line had achieved HIV viral suppression  after a minimum of six months; therefore these results may not be generalizable to populations failing WHO-recommended second-line ART regimens consisting of only two NRTIs and PI/r. Nonetheless, our findings reflect the majority of those adults failing second-line ART in Namibia and provide important information that can be used programmatically for the management of people experiencing virological failure of PI/r-containing regimens in the country.Medication non-adherence appears to be an important cause of second-line ART failure in one third of people in this study. A limitation of this study is that pharmacy refill data, pill count, or plasma, hair, or urine drug levels were unavailable; therefore, objective measures of adherence could not be assessed. This study has additional limitations. Although study sites comprised over 70% of all people receiving PI/r in Namibia at the time of the study, sites may have differed from other regions in the country with respect to patient demographics or ARV drug treatment histories by virtue of their larger patient populations. Although amplification success rates were similar across all clinics, there is a possibility that specimens that failed to amplify may have more or less drug resistance than those that amplified; however, as described, there was no differences between people with and without genotypes available for analysis. In addition, nearly all study participants were receiving three NRTIs in addition to PI/r (as per Namibia guidelines5This study demonstrates high levels of NRTI resistance and moderate levels of PI resistance in people receiving PI/r-based second-line ART, supporting the need to optimizing treatment adherence to all three second-line ARV drugs, use of routine VL monitoring to promptly detect virological failure, and HIVDR genotyping to optimize selection of third-line drugs. In addition, programmatic concerns about selective non-adherence to PI/r argue for more tolerable highly potent and fixed-dose combination regimens.MRJ and SYH conceived the study. MRJ wrote the protocol, designed the study and wrote the first draft of the manuscript. NH, LB, SS, SA, ANS, TN, CLF-W, EGR, NM, CJH, and AMT supported implementation of the study and interpretation of data in light of Namibia's public health ART Programme. ND performed statistical analysis. KS, VK, SC perofmred HIVDR genotyping and quality assurance of sequences. MRJ and NP performed additional sequence quality assurance and performed the HIVDR interpretation and genotype susceptibility scoring. All authors contributed to the final draft of the manuscript and had access to the data.


Nonsignificant associations were observed between the other five flavonoid subclasses and 10-year incidence of hearing loss. Our findings do not support the hypothesis that the intake of dietary flavonoids protect against long-term risk of hearing loss. The association with isoflavone intake needs to be confirmed by other population-based studies.Dietary flavonoids are vasoactive phytochemicals with promising anti-inflammatory properties. We aimed to assess the associations between baseline intakes of six commonly consumed flavonoid subclasses and 10-year incidence of age-related hearing loss. At baseline, 1691 participants aged 50+ years had information on dietary intakes and hearing status. Hearing loss was defined as the pure-tone average of frequencies 0.5, 1.0, 2.0, and 4.0 kHz > 25 dB hearing level (HL). Dietary data were collected through a semi-quantitative food frequency questionnaire. The flavonoid content of foods was estimated using US databases. During the 10-year follow-up, 260 (31.6%) new cases of hearing loss (incident) were observed. After multivariable adjustment, participants in the fourth versus first quartile (reference group) of intake of dietary isoflavone had 36% lower risk of incident hearing loss after 10 years: odds ratios (OR) 0.64 ;  Age-related hearing loss is a decrease in hearing ability that occurs with age and is projected to be within the top 15 leading causes of the burden of disease by 2030 ,5. Age-rReactive oxygen metabolites and inflammatory processes may cause cochlear damage . TherefoHowever, to our best knowledge, no large prospective cohort study has assessed the temporal association between the intake of dietary flavonoids and age-related hearing loss. Therefore, we used a large, representative, population-based cohort to investigate longitudinal associations between six common flavonoid subclasses  and the 10-year incidence of hearing loss among adults aged 50+ years.The Blue Mountains Hearing Study (BMHS) is a survey of age-related hearing loss from 1997 to 2004 among participants of the Blue Mountains Eye Study (BMES) cohort as previously described . Briefly0.5-4KHz > 25 dB hearing level (HL) in the better of the two ears, i.e., bilateral hearing loss [We asked questions on family history of hearing loss and occupational noise exposure: ‘Have you ever worked in a noisy industry or noisy farm environment?’ Pure-tone audiometry was performed by audiologists in sound-treated booths, as previously described ,3,4,5. Wing loss .0.5-4KHz in the better ear of ≤25 dB HL at BMES-2 or BMES-3. Incident bilateral hearing loss was defined as a PTA0.5-4KHz >25 dB HL in the better ear at the 5- or 10-year follow-up examination among participants without hearing impairment.Risk of incident bilateral hearing loss during the 10 years (from BMES-2 to BMES-4) was considered among participants who had PTAA modified, 145-item, self-administered food frequency questionnaire (FFQ) was used to collect information on dietary intakes . The typFor each food, we assessed the intake of each individual flavonoid compound found in the food and determined the sum of measured flavonoids for each flavonoid class, as previously described . The flaSAS statistical software  version 9.4 was used for analyses. Associations between dietary flavonoid intakes (study factor) and 10-year incidence of age-related hearing loss (study outcome) were examined in discrete logistic regression models. Regression analysis first adjusted for age and sex and then for covariates that were found to be associated with incidence of age-related hearing loss in the BMHS cohort: Family history of hearing loss and exposure to noise at work . FindingThe Blue Mountains Eye and Hearing Studies were supported by the Australian National Health and Medical Research Council .Of the 2956 participants who had audiometric testing performed at BMES-2 , 1691 had complete information on dietary intakes. Of these 1691 baseline participants, 647 participants had bilateral hearing loss and were excluded from subsequent incidence analysis. Those who had hearing loss versus those who had normal hearing function at baseline were more likely to be male, older, and have a family history of hearing loss . Over thThe current study findings do not provide evidence that total flavonoid intake and the intakes of specific flavonoid subclasses influence the risk of age-related hearing loss. However, we observed a modest inverse association between the intakes of isoflavones and 10-year incidence of hearing loss, indicating a potential protective effect against the development of hearing loss.Although we demonstrated an association between a greater intake of dietary isoflavones and a lower risk of age-related hearing loss, we were unable to determine whether the observed association indicated a benefit of the isolated bioactive, per se, or whether it is a signal of the bioactives working together with the co-occurring phytochemicals within isoflavone-containing foods . We alsoNevertheless, we can posit a potential mechanism by which intake of isoflavones could protect against age-related hearing loss. For instance, isoflavones are considered to be ‘phytoestrogens’ with multiple potential cardioprotective benefits. They are structurally similar to estradiol and may reduce low-density lipoprotein cholesterol, induce nitric oxide production, and improve vascular reactivity ,32. ReceOverall, our results do not support a protective effect of most of the flavonoid classes against age-related hearing loss. The largely nonsignificant findings regarding total flavonoids and flavonoid subclasses could be due to the oral bioavailability of flavonoids being constrained by poor intrinsic transmembrane diffusion features . In addiThe strengths of our study include its representative sample of older adults with relatively high participation rates, minimizing selection bias. Furthermore, we used standardized, audiometric testing to measure hearing sensitivity and a validated food questionnaire to collect dietary information. Additionally, study participants were not aware of the study question and our dietary data were collected before detection of incident hearing loss, reducing indication bias arising from behavioral change after the hearing loss was diagnosed. However, this study is not without its limitations. First, flavonoid content of foods was solely estimated using US-based databases and, hence, this approach might have discounted any variation in the flavonoid content of Australian foods . We alsoIn summary, our study findings suggest that intakes of dietary flavonoid subclasses and compounds are unlikely to have a role in preserving healthy auditory function among older adults. However, we found that a higher intake of dietary isoflavones reduced the risk of incident hearing loss by over a third. Further large, prospective studies are now warranted to verify our findings and to establish whether aural benefits result from treatment with specific dietary flavonoids.


Tick-borne encephalitis (TBE) is a zoonotic disease that usually presents as a moderate febrile illness followed by severe encephalitis, and various neurological symptoms are observed depending on the distinct central nervous system (CNS) regions affected by the TBE virus (TBEV) infection. In Japan, TBE incidence is increasing and TBEV distributions are reported in wide areas, specifically in Hokkaido. However, an extensive epidemiological survey regarding TBEV has not been conducted yet. In this study, we conducted a retrospective study of the prevalence of antibodies against TBEV in patients with neurological disorders and healthy populations in a TBEV-endemic area in Hokkaido. Among 2000 patients, three patients with inflammatory diseases in the CNS had TBEV-specific IgM antibodies and neutralizing antibodies. The other four patients diagnosed clinically with other neurological diseases were positive for TBEV-specific IgG and neutralizing antibodies, indicating previous TBEV infection. In a total of 246 healthy residents in a TBEV-endemic region, one resident had TBEV-specific antibodies. These results demonstrated undiagnosed TBEV infections in Japan. Further surveys are required to reveal the actual epidemiological risk of TBE and to consider preventive measures, such as a vaccine program, for the control of TBE in Japan. Flavivirus. TBEV is transmitted by tick bites and is maintained in the zoonotic transmission cycle between ticks and wild vertebrate hosts. TBEV is prevalent over a wide area of the Eurasian continent, including Europe, Russia, and Far-Eastern Asia, including Japan [Tick-borne encephalitis (TBE) is a zoonotic disease that usually presents as a moderate febrile illness followed by severe encephalitis. TBE is caused by TBE virus (TBEV), a single-stranded, positive-sense RNA virus of the genus ng Japan ,2,3, andng Japan ; the Baing Japan ,6,7.After 7–10 days of incubation, TBE symptoms appear in a two-phase course but they vary in the subtypes. Approximately 30% of infected individuals remain asymptomatic. Flu-like symptoms are observed during the initial viremic phase of the illness, which include fever and headache ,9. AfterIxodes ovatus ticks in an area where the TBE patients were reported [In Japan, two patients infected with a virus within the TBE serocomplex had encephalitis during an epidemic of Japanese encephalitis (JE) in 1948 in the Tokyo area. The isolated virus, named Negishi virus, was retrospectively identified as a member of the louping ill virus through phylogenetic analyses performed decades later ,11. No sreported ,17,18,19reported ,21. In oreported ,23. ThesHere, we conducted a retrospective study of the prevalence of antibodies against TBEV in patients with neurological disorders who visited the Department of Neurology at Hokkaido University Hospital and the healthy population in a TBEV-endemic area in Hokkaido to investigate the epidemiological risk of TBEV endemic in this part of Japan.Blood and cerebrospinal fluid (CF) samples were collected from patients with neurological disorders, and blood samples from healthy volunteers in Yuni town between 2010 and 2018 were collected to identify previously undiagnosed TBE infections. Approval for this study was obtained from the Medical Ethics Committee of the Faculty of Veterinary Medicine, Hokkaido University Hospital . Written informed consent was obtained from all participants.2O2). Negative control antigens were prepared from the supernatant of untransfected HEK293T cells. The results were recorded as the P/N ratio , and a cutoff value of 1.26 was used.Anti-TBEV IgG antibodies were examined by SP-IgG-ELISA, previously developed in . Briefly2O2. The results were recorded as the P/N ratio, and a cutoff value of 1.30 was used.Anti-TBEV IgM antibodies were examined by SP-IgM-ELISA, previously developed in . BrieflyTBEV Oshima 5–10 strain  was incuSerum samples were first screened by IgG- and IgM-ELISA using SPs of TBEV. Sero-positive samples were examined by neutralization tests (NTs) against TBEV and JE virus (JEV) for further confirmation. The samples showing anti-TBEV neutralizing titer ≥ 20 and no anti-JEV neutralizing titer, or at least 4-times higher neutralizing titer against TBEV than JEV, were defined as TBEV infection. The serologic constellations were interpreted as described previously : IgM (+)CF samples suspected of recent TBEV infection were subjected to RT-PCR to detect TBEV genomic RNA. Total RNA was extracted using ISOGEN II  and reverse-transcribed using random primers and Superscript III reverse-transcriptase . TBEV-specific sequences were amplified using Platinum Taq polymerase . To amplify the envelope (E) protein gene of TBEV, we used the following primers specific for Far-Eastern TBEV: (forward) 5′-AGATTTTCTTGCACGTGCAT-3′ and (reverse) 5′-GCACACTGT1GTATGTAAGAC-3′.2. After 2–4 days, the cells were inspected for cytopathic effects (CPEs) and total RNA was extracted for RT-PCR.In order to isolate TBEV from the CF samples, they were inoculated into the BHK cells and incubated at 37 °C under 5% COTo investigate unconfirmed TBEV infection, we investigated anti-TBEV antibodies in 2000 patients with neurological disorders who visited the Department of Neurology in Hokkaido University Hospital from 2010 to 2018. The breakdown information of the patients is listed in As shown in 50 = 20). Three IgG-positive but IgM-negative samples had neutralizing antibodies against TBEV, but significant differences from neutralizing titers against JEV were not observed. One IgG-negative but IgM-positive sample had a low titer of neutralizing antibody against TBEV (NT50 = 10), but no significant difference was observed from that against JEV. One IgG-positive but IgM-negative sample and one IgG-negative but IgM positive sample were negative in the NT against both TBEV and JEV.Detailed information of the antibody-positive samples is shown in Detection of TBEV RNA and isolation of TBEV were performed using the CF samples with TBEV-specific IgM and neutralizing antibodies. However, TBEV RNA was not detected in the CF samples. BHK cells inoculated with the CF samples did not show any CPE and no TBEV RNA was detected in the cells.To investigate the natural infection rate in residents in a TBEV-endemic area, a serological survey was conducted in healthy volunteers in Yuni town in central Hokkaido, where TBEV antibodies were detected in wild animals. In total, 246 serum samples were collected and subjected to IgG-ELISA using SPs of TBEV, followed by NT against TBEV. One sample from an 80-year-old woman was positive for IgG and neutralizing antibody against TBEV.In this study, we conducted a large sero-epidemiological survey of TBEV infection in patients with neurological disorders and a healthy population in a TBEV-endemic area in Hokkaido, Japan. For the first screening of the subjects, we conducted IgG- and IgM-ELISA using SPs, which had higher specificity and sensitivity with minimum cross-reactivity with other flavivirus infections compared to the commercial ELISA using formalin-inactivated virion ,25. ELISThe positive samples by serological assays were speculated according to Dr. Dobler’s previous interpretation of serologic constellations  Table 3Table 3. Culex tritaeniorhynchus), and routine vaccination against JEV was not conducted until 2018. Therefore, many residents in Hokkaido do not possess JEV antibodies. Taking into consideration these regional situations, these subjects were considered to have previous TBEV infection.The other ten IgG- or IgM-positive samples were clinically diagnosed as other neurological diseases, not inflammatory diseases. Four of them had significantly higher titers of neutralizing antibodies against TBEV than JEV. In Japan, JEV is widely endemic and JE cases have been reported almost annually. However, no JE cases were reported in Hokkaido due to the limited distribution of mosquito vectors  were found to be positive . The posIxodes ovatus (0.05–0.33%) as compared to surveys in other endemic countries [I. ovatus because I. persulcatus is the primary vector of TBEV in Far-Eastern Russia and Asia.In Japan, no epidemiological survey has been conducted in the residents of TBEV-endemic regions. Our survey detected one person with the TBEV-specific antibody out of the 246 individuals included (0.4%). This seroprevalence rate was relatively lower than the usual ratio reported in studies in European countries (0–5%) ,33,34,35ountries ,19. ThesIn conclusion, our surveillance revealed undiagnosed TBE in patients with neurological disorders and previous TBEV infection in an endemic region in Hokkaido, Japan, for the first time. The incidence of TBE in Japan is increasing, and sero-epizootiological evidence for TBEV distribution has been reported not only in a wide area of Hokkaido but also in the main islands of Japan. However, awareness of TBE is significantly low, even in physicians, and only limited facilities can conduct laboratory diagnosis of TBEV infection in Japan. No vaccine against TBEV has been licensed in Japan. There is an urgent need to reveal the actual epidemiological risk of TBE and to consider preventive measures, such as a vaccine program, for the control of TBE in Japan.


We found no data in the literature on the embolization of the bronchial arteries in the context of hemoptysis associated with severe acute respiratory syndrome coronavirus 2. We therefore decided to share this experience.A 62-year-old patient with no significant medical history was admitted with acute respiratory distress. Chest computed tomography showed diffuse bilateral ground-glass opacities with limited consolidations. Diagnostic tests confirmed severe acute respiratory syndrome coronavirus 2 infection. The severity of respiratory failure required the implantation of veno-venous extracorporeal membrane oxygenation. The patient developed severe haemoptysis, which was successfully treated by bronchial artery embolisation.In the case of coronavirus-19 pneumonia, our experience suggests that the treatment of severe haemoptysis by bronchial artery embolisation is feasible and effective. The survival benefit should be assessed in the future. At the end of December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Hubei Province, China, leading to the global outbreak of coronavirus disease 2019 (COVID-19)  was performed on admission, revealing diffuse bilateral ground-glass opacities with limited consolidations  on a nasal swab confirmed COVID-19 diagnosis, and a bronchoalveolar lavage showed no bacterial infection. Following local guidelines, the patient was treated with hydroxychloroquine for 10 days. The patient developed renal failure that required dialysis. On day 2, refractory hypoxemia required the implantation of veno-venous extracorporeal membrane oxygenation (ECMO). Anticoagulant therapy with unfractionated heparin (UNFH) was immediately started after implantation as recommended by the Extracorporeal Life Support Organisation guidelines. According to local protocol, APTT was used to monitor and adjust UNFH therapy.On day 9, the patient developed life-threatening haemoptysis, and the chest x-ray showed increased condensation, particularly in the right lung. Several bronchoscopies were required to remove the recurring clots, particularly in the right main bronchus. Diffuse bleeding was seen on both sides. No heparin overdose was detected. Heparin treatment was stopped for 24 h. Viscoelastic coagulation tests (ROTEM®) were normal, while there was no effect of aerosolised tranexamic acid or intravenous desmopressin in the case of acquired Von Willebrand disease associated with ECMO therapy.Faced with recurrent haemoptysis, bronchial artery embolisation was considered. We found no data in the medical literature on COVID 19 and bronchial artery embolization. We contacted several colleagues in France and the United States, but none had encountered this situation. Faced with this massive hemoptysis and without embolization, the patient’s death seemed inevitable.Chest CT angiography (CTA) was performed prior to embolisation. It showed no pulmonary embolism or vascular abnormalities but the complete condensation of the right lung and three-quarter condensation of the left lung Fig. .Fig. 2CThrough a right arterial femoral access with a 4 French (F) introducer sheath, we performed selective bronchial arteriographies using a 4F Sidewinder 1 Catheter (Cordis) and 2.7F microcatheter , which showed the normal appearance and calibre of the right and left bronchial arteries Fig.  without After the embolisation procedure, no recurrence of haemoptysis occurred, even after resuming anticoagulant treatment with UNFH. The chest X-ray revealed a slight improvement in the ventilation of the right lung. Unfortunately, the patient died from non-haemorrhagic shock3 days post-embolisation.The full spectrum of COVID-19 disease is still emerging, but the most common signs and symptoms at onset are fever, dry cough, myalgia, anosmia and ageusia, fatigue, and dyspnoea (Guan et al. Pulmonary bleeding seems to be an atypical manifestation of SARS-CoV-2 infection, as COVID-19-associated haemoptysis has rarely been reported in the literature (Lapostolle et al. Very few major haemorrhagic complications have been described in hospitalised COVID-19 patients, with only a few cases of spontaneous abdominal internal bleeding and none at the bronchial level (Conti et al. A hallmark of severe COVID-19 is altered coagulation with a predominantly pro-thrombotic status and a high risk of thromboembolic events (Helms et al. The lungs are the target organ for SARS-CoV-2, and progressive respiratory failure is the primary cause of death in COVID-19 patients. Pathological examination of the lungs of deceased COVID-19 patients showed diffuse alveolar damage associated with both thrombotic and haemorrhagic lesions: severe endothelial injury, alveolar capillary microthrombi, and increased angiogenesis (Ackermann et al. In this context, we hypothesised that the life-threatening haemoptysis presented by our patient was consecutive to severe lung damage induced by SARS-CoV-2 combined with the anticoagulant effect of UNFH therapy, resulting in peripheral bronchial artery damage. In such cases, bronchial artery embolisation could be indicated.Faced with haemoptysis, chest CTA is recommended to specify the nature, extent, and topography of pulmonary lesions. CTA allows the evaluation of the bronchial artery anatomy and the exploration of pulmonary arteries, which are a less common cause of haemoptysis. A recent study showed that preprocedural CTA helps detect culprit ectopic bronchial arteries and non-bronchial systemic arteries originating from the subclavian and internal mammary arteries during bronchial artery embolisation (Li et al. At the time of writing, this is the first reported case of bronchial artery embolisation used for COVID-19-related haemoptysis. Our experience suggests that the embolisation of bronchial arteries is feasible and may help control bleeding in cases of COVID-19 pneumonia associated with life-threatening haemoptysis. However, the impact on survival remains uncertain. The risk incurred by medical and paramedical staff is an issue that must also be taken into account. We hope that other authors will report their similar experiences in order to provide answers to these questions.


We found that a heat-shock of 41°C for 6 h was sufficient to induce GCD and degeneration of a fraction of granule cells. Both of these factors, broadening of the granule cell layer (gcl) and increased neuronal cell death within the gcl, contributed to the development of a significantly reduced packaging density of granule cells. In contrast, Reelin expressing Cajal–Retzius (CR) cells in the molecular layer were heat-shock resistant. Thus, their number was not reduced, and we did not detect degenerating CR cells after heat-shock, implying that GCD was not caused by the loss of CR cells. Importantly, the heat-shock-induced deterioration of dg morphology was accompanied by a massive microgliosis, reflecting a robust heat-shock-induced immune response. In contrast, in the study that reported on GCD as a non-specific finding in pediatric patients, no microglia reaction was observed. Thus, our findings underpin the importance of microglia as a marker to distinguish pathological GCD from normal morphological variation.Granule cell dispersion (GCD) has been found in the dentate gyrus (dg) of patients with temporal lobe epilepsy (TLE) and a history of febrile seizures but was also recently observed in pediatric patients that did not suffer from epilepsy. This indicates that GCD might not always be disease related, but instead could reflect normal morphological variation. Thus, distribution of newborn granule cells within the hilar region is part of normal dg development at early stages but could be misinterpreted as pathological GCD. In turn, pathological GCD may be caused, for example, by genetic mutations, such as the reeler mutation. GCD in the reeler mutant goes along with an increased susceptibility to epileptiform activity. Pathological GCD in combination with epilepsy is caused by experimental administration of the glutamate receptor agonist kainic acid in rodents. In consequence, the interpretation of GCD and the role of febrile seizures remain controversial. Here, we asked whether febrile temperatures alone might be sufficient to trigger GCD and used hippocampal slice cultures as  Epilepsy encompasses a variety of neurological pathologies, in particular recurrent seizures of hyperexcitable nerve cells and the associated imbalance between excitation and inhibition . AlthougThe extracellular matrix protein Reelin controls several aspects of cortical development and function, in particular the positioning of radially migrating neurons and layer formation, including formation of the dentate gcl . In the An increasing number of different clinical and experimental studies point to a correlation between the pathophysiology of epilepsy and inflammation in the central nervous system, including the accumulation, proliferation, and activation of glial cells, in particular of microglia . Microglin vitro model to study development, function, and plasticity of hippocampal neurons and glial cells in health and disease in a tissue context that is similar to the in vivo situation  were used for hippocampal slice cultures. Animals were bred and maintained in accordance with the animal care guidelines of the institutional guidelines of the University of Bochum. All animals were housed at 22°C on a 12 h light/dark cycle with D-glucose). After 1 h of recovery, the slices were transferred to a separate membrane insert  and subjected to a different experimental condition . The cultures were incubated in vitro in a 37°C, 5% CO2 humidified incubator; 100 ml culture medium consisted of 50% MEM, 25% Hank’s balanced salt solution, and 25% heat-inactivated horse serum, supplemented with 1% glutamine (200 mM), 1.56% glucose (45% in aqua dest.), 0.58% NaHCO3 , 1% penicillin, and streptomycin (Thermo Fisher Scientific/Invitrogen). To simulate a fever situation in vitro, heat-shock of the cultures started at DIV 3. Thus, the incubation temperature for the hippocampal slice cultures was increased to 41°C for 6 h to achieve hyperthermia. Next, the slice cultures were again incubated at 37°C for 2 more days. Slices were subsequently fixed with 4% paraformaldehyde (PFA) in phosphate buffer (PBS) for 90 min. After that, the hippocampal sections were washed and transferred in PBS. The outcome was compared with the control slice cultures exposed all days at 37°C, 5% CO2.Rat hippocampal slice cultures were used for heat-shock studies. All solutions used for organotypic slice culture preparation were sterile, and all preparations were performed in a laminar air flow bench with horizontal counter flow . Five-days-old wistar rats (P5) were decapitated, and the hippocampus was dissected out and gently placed on the platform of a McIlwain tissue chopper. The hippocampus was cut perpendicular to the longitudinal axis into 400 μm thick slices, which were transferred into incubation solution at 4°C . After primary antibody incubation, sections were washed twice in 0.1% Tween-20 in PBS and once in PBS for 5 min, before secondary antibodies were applied for 3 h at room temperature: Alexa Fluor 594-coupled goat anti-rabbit IgGs  or Alexa Fluor 488-coupled goat anti-rabbit IgGs . Sections were washed again and treated with the primary antibodies diluted in 0.1 M PBS overnight at 4°C: anti-Reelin  or anti-Prox-1 . After washing, they were incubated with secondary antibodies conjugated with Alexa 488 or 594 fluorophores (1:400) for 2 h at room temperature. Nuclei were counterstained with the fluorescent dye 4′,6-diamidino-2-phenylindole (DAPI) . Finally, after three repetitive washing with PBS, free-floating sections were mounted on glass slides. All sections were embedded with fluorescent mounting medium , cover-slipped, and subsequently viewed and photographed using confocal fluorescence microscope (Nikon Spinning Disk). For image acquisition and quantification, a 20x air- and 60x water-magnification immersion objective was used. The sections were also processed by using the ImageJ 1.52k software. To control for specificity, sections were processed according to the protocol above with primary antibodies omitted.The collected slice cultures in PBS were subsequently processed free-floating and all incubations were performed darkened on a shaker. For immunostaining, tissue sections were permeabilized in 0.1% Triton-X-100 in PBS for 1 h at room temperature and then pre-incubated with blocking solution  overnight at 4°C. After washing 3x in 0.1 M PBS for 10 min, sections were incubated in primary antibodies diluted in 0.1 M PBS for two nights at 4°C. The following primary antibodies were used: rabbit polyclonal anti-Iba-1  or mouse monoclonal anti-ED-1  according to For quantitative analysis of cell distribution in hippocampal slice cultures, identical illumination for each section by a photographer who was blinded to the experimental condition was used. High-resolution images acquired with a 60x water immersion objective (Nikon 60xA/1.20) within subfields of the dg  was perfomed by counting cells that were immunopositive for CR-50 (CR-cells), Prox-1 (granule cells), Iba-1 or ED-1 (microglia), respectively. Cell numbers were normalized to the total number of DAPI stained cells per image subfield.Reelin positive interneurons were manually counted in optical sections from matched slices of five different animals per experimental condition.Propidium iodide (PI) staining was performed by applying 200 μl/ml of a 500 nM PI solution to living cell cultures 1 h before fixation with PFA. Slice cultures were then co-stained with Prox-1 as described earlier. Cell counts of PI positive cells in the gcl were achieved visually by an experimenter blinded to the experimental condition in one exemplary z-level of a slice.t-test was performed. In the case of non-normal distribution of the raw data, the Wilcoxon–Mann–Whitney test was used for statistics. Values were considered statistically significant at p < 0.05. Statistics reported in the text and figures are represented either by mean value and standard deviation or by boxplots giving the median (middle dash), the lower and upper quartile (box borders), and the minimum and maximum values (whiskers).Statistical analyses were performed with GraphPad Prism version 7.00  and in Microsoft Excel. For analysis, slices from the same animal incubated as control or treated as heat-shock were tested as pairs of groups. These pairs of groups were tested for normal distribution (D’Agostino test and Shapiro–Wilk test) and if so a paired Student’s To study the impact of a heat-shock on the morphology of the dg, we first analyzed the width of the gcl in matched hippocampal slice cultures after heat-shock and in untreated control cultures  by measuTo analyze a potentially deleterious effect of the heat-shock on survival of dentate granule cells, we determined the total cell number in confocal images of the gcl and mol in hippocampal slice cultures in control slices and after heat-shock  by countMicroglial cells can actively migrate to lesion sites with ameliorating effects supporting recovery after lesion . The ionHigh-magnification images of heat-shock-treated microglia stained against the Iba-1 protein demonstrate that the increased microglia staining resulted, at least in part, from mitotic cell divisions . NumerouAmmonshorn sclerosis and/or dispersion of dentate granule cell are frequently observed in epileptic hippocampal tissue, and febrile seizures early in life are suspected to be causally linked to these pathologies. Loss of Reelin, a protein known to control neuronal migration during development, has been discussed as a possible cause of pathological GCD. In turn, recent findings suggest that GCD may not always be disease related but could instead reflect normal morphological variation. For instance, the presence of newborn granule cells within the dentate hilar region during early dg development reflects the normal development of this region but could be misinterpreted as pathological GCD. Thus, the interpretation of GCD in a disease-related context remains controversial. To explore the potential impact of febrile temperature on granule cells and Reelin secreting CR cells, hippocampal slice cultures were subjected to a transient temperature increase (heat-shock). We found that a heat-shock caused both dentate GCD and degeneration of a fraction of granule cells. Surprisingly, the viability of CR cells was not affected. This precludes that heat-shock-induced GCD was related to depletion of the CR cell population. We also found that the heat-shock caused a massive increase in the number of microglial cells in the dg, indicating a robust immune response to the heat-shock-induced tissue damage.in vivo after administration of the epilepsy inducing drugs pilocarpine  development from neocortical development is the emergence of three consecutive germline matrices during dg development. In the tertiary proliferation zone located in the hilar region of the early postnatal dg, proliferating progenitors generate granule cells which migrate from the hilar region to their final positions in the compact gcl . Thus, tocarpine  or kainiocarpine .in vitro, and therefore we subjected hippocampal slice cultures from young postnatal rats to a heat-shock of 41°C for 6 h. Our experiments revealed that this transient heat-shock is sufficient to induce GCD in hippocampal slice cultures. What might be the underlying causes that induce GCD in our in vitro model? For instance, a reduction in the number of Reelin-secreting CR cells, implying reduced Reelin availability, had earlier been shown to be a potential trigger for GCD (We wondered whether a transient temperature increase might be sufficient to induce GCD  for GCD . Along t for GCD . Thus, r for GCD . However for GCD , might b for GCD . In our  for GCD , arguing for GCD . This inin vivo (An important question that is difficult to answer is whether GCD contributes to the development of seizures or rather has an anti-epileptic effect. Earlier studies suggested that individuals with GCD have an increased leakage of potassium ions from granule cells which reduces their excitability, pointing to an anti-epileptic effect . In turnin vivo . To analin vivo . Reeler in vivo . On the in vivo . In thisin vivo . Certainin vivo .in vitro model. The increased activation of microglia is one of the main components under pathological conditions, for instance, reflecting inflammatory changes in nerve tissue (Microglia cells, descendants of the monocyte-macrophage lineage, are involved in the detection of cerebral dysfunctions, infections or mechanical injuries and actively migrate to pathological lesion . Microgle tissue . Ammonshe tissue  and accoe tissue , and theIn our heat-shock model, we observed that the heat-shock-induced tissue damage was followed by a massive increase in the number of microglial cells in the dg, particularly in the gcl. Our findings suggest that subregion- and cell layer-specific differences exist in the microglial response to heat-shock see , 8, likein vitro exist. Here we provide evidence that microglia cells play a remarkable role in the heat-shock response in hippocampal slice cultures. The relationship between heat-shock, induced GCD and microglia activation is largely unexplored. Further experimental studies on the interplay between neuropathology and the microglia response may help to progress existing therapeutic options in the pathogenesis of febrile seizures and other neurological diseases.Another question that remains to be addressed is whether the transient temperature increase induces epileptic activity in the hippocampal slices. Studies on hyperthermia-induced seizures in immature rats were performed earlier , with elThe original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author/s.The animal study was reviewed and approved by Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen.JW and MM performed experimental work and statistical analysis of data, and contributed to data interpretation and manuscript writing. SR performed experimental work on Prox-1 in combination with PI staining. EF developed the concept of the study and was involved in data interpretation and manuscript writing. 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.


Health impact assessments of future environmental exposures are routinely conducted to quantify population burdens associated with the changing climate. It is well-recognized that simulations from climate models need to be bias-corrected against observations to estimate future exposures. Quantile mapping (QM) is a technique that has gained popularity in climate science because of its focus on bias-correcting the entire exposure distribution. Even though improved bias-correction at the extreme tails of exposure may be particularly important for estimating health burdens, the application of QM in health impact projection has been limited. In this paper we describe and apply five QM methods to estimate excess emergency department (ED) visits due to projected changes in warm-season minimum temperature in Atlanta, USA. We utilized temperature projections from an ensemble of regional climate models in the North American-Coordinated Regional Climate Downscaling Experiment (NA-CORDEX). Across QM methods, we estimated consistent increase in ED visits across climate model ensemble under RCP 8.5 during the period 2050 to 2099. We found that QM methods can significantly reduce between-model variation in health impact projections (50–70% decreases in between-model standard deviation). Particularly, the quantile delta mapping approach had the largest reduction and is recommended also because of its ability to preserve model-projected absolute temporal changes in quantiles. Global warming affects human health by increasing population exposures to hazardous environmental conditions, such as prolonged high temperature, elevated ambient air pollution, and extreme weather events  ,2. EvideHealth-impact projection is conducted by combining estimated health-exposure relationships with projected future exposures while considering various sources of uncertainties in the analysis . Future In order to assess temporal changes in meteorological trends, climate model simulations are performed for both historical (hindcast) and future (projection) periods. Generally, bias-correction for future simulation is accomplished in two steps. First, the bias between observations and simulations during the hindcast period is assessed. Then a correction algorithm is applied to future simulations by assuming the bias can be extrapolated to future periods. In a recent systematic literature review of heat-related mortality projections , among tQuantile mapping (QM) is a recent bias-correction approach that has gained popularity in climate science . It is mThe main objective of this paper is to investigate the use of QM methodologies in conducting health-impact projections. We aim to assess the robustness of health-impact estimates against different QM assumptions and assess the usefulness of QM in reducing uncertainties in a climate model ensemble. Our case study involves estimating future excess emergency department visits attributable to changes in daily warm-season minimum temperature in the Atlanta metropolitan area for the period 2050 to 2099. The focus on minimum temperature is motivated by historical trends of increasing humidity and minimum temperatures in the southeastern region of the United States ,17. MiniWe utilized climate model simulations from the North American-Coordinated Regional Climate Downscaling Experiment (NA-CORDEX) . The NA-RCM included the Canadian Regional Climate Model version 4 (CanRCM4), the Canadian Regional Climate Model version 5 by Université du Québec à Montréal (CRCM-UQAM), the High-Resolution Limited Area Model with ECHAM physics, version 5 (HIRHAM5), the Regional Climate Model version 4 (RegCM4), the Rossby Centre regional atmospheric model version 4 (RCA4), and the Weather Research and Forecasting model (WRF). GCM included the second-generation Canadian Earth System Model (CanESM2), the European community Earth-System Model (EC-EARTH), the Geophysical Fluid Dynamics Laboratory Earth System Models (GFDL-ESM2M), the Met Office Hadley Centre with the HadGEM2-ES Earth System model (HadGEM2-ES), and the coupled Max Planck Institute Earth System Model with mixed resolution (MPI-ESM-MR) and low resolution (MPI-ESM-LR). Specific RCM-GCM model combinations are given in Daily counts of emergency department (ED) visits for the 20-county Atlanta metropolitan area were obtained by aggregating patient-level records from individual hospitals and the Georgia Hospital Associations for the same historical period 1993 to 2004. We used the International Classification of Disease version 9 (ICD-9) codes to identify ED visits for all internal causes (ICD-9: 001–799) in the primary or secondary diagnosis fields. We restricted the analysis to warm months of May to September. The total number of ED visits was 6,994,110 with an average of 2286 per day.observation at time t during the historical period with cumulative distribution function (CDF) defined as z. For a quantile level τ, 0 ≤ τ ≤ 1 the quantile value  regression to estimate the quantile–quantile relation of the historical observed and modeled time series . For eacbove set . The aboa and b are obtained from fitting a linear least squares regression. The bias-corrected future projection at time t is then given by Linear QM that assumes a linear relationship between quantile functions of the observed and model time series during the historical period: +b×xm,pt .t is t via quantile mapping is given by QDM aims to preserve the model-projected absolute changes in quantiles following bias correction by QM . Hence, mσ and 0σ are the standard deviation of the modeled and observed datasets, respectively.To minimize the impact of seasonality, for each NA-CORDEX RCM/GCM combination, we applied the five QM methods by season  using the R packages qmap and MBC (for QDM) . To evaluate the model fit different quantile mapping methods on bias-correcting simulation data, we calculated the mean bias error (MBE), mean absolute error (MAE), root mean square error (RMSE), and normalized standard deviation (NSD) between the raw and bias-corrected model values during the historical period. Let We note that the above metrics are in-sample evaluation of model fit. Out-of-sample evaluations are challenging because climate model projections represent simulations under different emission assumptions.μ be the mean ED counts on day tWe used quasi-Poisson log–linear model to estimate the association between temperature and daily ED visit counts. Let t during the historical period , the attributable number (AN) associated with temperature exposure is given by:For each day t during the projection period , the AN is:t,HAN and t,PAN over the desired comparison time periods.For projected future ED visits, we used future temperature as counterfactual exposures. For each day t,HAN and t,PAN involved nonlinear functions of the estimated log relative risk. We obtained point-projection and projection uncertainty intervals via Monte Carlo simulations. Specifically, we first simulated 5000 realizations of the exposure-response function Calculations of In addition to evaluating health-impact projections by individual climate model, we further considered two ensemble methods for combing projections across models. In the first method, we calculated point and interval projections using the average health projections across the 10 models for each Monte Carlo realization. In the second method, we obtained point and interval projection by pooling all Monte Carlo realizations from individual models. The first method assumes the average projection as the ensemble estimate, while the second approach accounts for between-model variability.More importantly, applying quantile mappings reduced between-model variation. 0) at 7.07 °C. Finally, Quantile mapping (QM) has become widely used in bias-correcting climate-model simulations because of its ability to characterize distribution tails more flexibly. We describe, to the best of our knowledge, the first evaluation of different QM methods in projecting future health impacts associated with temperature. Using an ensemble of regional climate model simulations from the NA-CORDEX experiment, we found consistent increases in ED visits attributable to future changes in daily ambient minimum temperature under the RCP 8.5.From the recent review of heat-related mortality projections , delta cSeveral studies have compared results from applying different QM methods to bias-correct meteorology data. Murdock et al. (2015) suggested that traditional QM altered relative trends in precipitation extremes projected by GCMs, while quantile delta mapping (QDM) was able to preserve relative trends . Tong etThere are several additional variations of QM approaches that were not included in our analysis. First, smoothing splines (SSPLIN) is a nonparametric QM method in which a smoothing spline is used to fit the quantile–quantile plot of the observed and modeled data . HoweverBesides temperature, QM algorithms are also widely used in bias-correcting other simulated meteorology variables such as precipitation ,33,34 ancounterfactual scenario of future daily minimum temperature time series  occurring during the historical period 1993 to 2004. Moreover, changes in the at-risk population and baseline risks will likely have little influence on our comparison of different bias-correction methods because these parameters are not related to future exposure projections. Particularly, information regarding projected changes in population size and baseline ED rate can be incorporated in the health-impact calculations by replacing the parameter tn with its projected values.Finally, our health-impact projections of future ED visits have several limitations. First, we did not consider changes in population or changes in baseline ED visit rates. Population change is driven by migration, life expectancy and fertility, and these variables are interrelated to anthropogenic emission. Second, future baseline ED visit rates may change in response to the overall health of the population and health care access. Hence, our estimates should be interpreted as a In this case study of ED visits and daily minimum temperature in Atlanta, we estimated consistent increase in ED visits across climate models under RCP 8.5 during the period 2050 to 2099. We found that QM methods can significantly reduce between-model standard deviation by 50–70% in health projections. Particularly, the quantile delta mapping (QDM) approach is recommended because it gave the largest reduction in between-model variation, it resulted in good model fit during the hindcast historical period, and the approach aims to preserves model-projected absolute temporal changes in quantiles.


Anecdotal evidence suggests that Coronavirus disease 2019 (COVID-19), caused by the coronavirus SARS-CoV-2, exhibits differences in morbidity and mortality between sexes. Here, we present a meta-analysis of 3,111,714 reported global cases to demonstrate that, whilst there is no difference in the proportion of males and females with confirmed COVID-19, male patients have almost three times the odds of requiring intensive treatment unit (ITU) admission  and higher odds of death  compared to females. With few exceptions, the sex bias observed in COVID-19 is a worldwide phenomenon. An appreciation of how sex is influencing COVID-19 outcomes will have important implications for clinical management and mitigation strategies for this disease. Anecdotal reports suggest potential severity and outcome differences between sexes following infection by SARS-CoV-2. Here, the authors perform meta-analyses of more than 3 million cases collected from global public data to demonstrate that male patients with COVID-19 are 3 times more likely to require intensive care, and have ~40% higher death rate. Despite this, there are few analyses addressing whether this is a global rather than regional phenomenon. There are limited data that indicate whether the bias towards increased mortality in males is due to an increased proportion of infections in males, or a true representation of more severe disease. This gap in the literature highlights that sex remains an under-appreciated variable when interrogating outcomes in infectious diseases.A consistent feature of the ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)To address whether the reported sex-bias is validated by large-scale statistical analysis of global data, we have collected available case data from 90 reports including 46 different countries and 44 US states totalling 3,111,714 infected cases, and present a meta-analysis to investigate sex as a risk factor for SARS-CoV-2 infection, and COVID-19 morbidity and mortality. We demonstrate that while there is no difference in the proportion of males and females infected with SARS-CoV-2, males face higher odds of both intensive therapy unit (ITU) admission and death compared to females. The confirmation of this sex disparity with global data has important implications for the continuing public health response to this pandemic.st June 2020 . Most reports (n = 88) were government websites, one report was from the British Medical Journal Global Health Blog website, and a minority comprised published research articles (n = 3), from which data were captured from full text. Importantly, sex disaggregated data for the United States of America (USA) were only available at individual state levels. Data from individual states were therefore analysed as separate regions to account for differences in how the data may have been collected. Reports were excluded if they did not report the overall number of infections by sex , or had small case numbers (less than five). Of the remaining 102 reports, multiple early reports originated from China, which were carefully examined for duplication. Following the exclusion of nine reports due to possible duplication, and one report from Canada with a large percentage of cases with unknown sex, a total of 92 reports from 47 different countries were analysed further , demonstrating that males and females have similar numbers of infections    = 0.48,0.51; ons Fig. . Male seFunnel plots and sensitivity analyses indicated that the results for the estimated proportion of male cases and the estimated OR for ITU admission in men were unlikely to be influenced by reporting bias; however, the estimated OR for mortality in men may be an underestimate (Supplementary Information). Taken together these data highlight that, whilst there is no difference in the proportion of COVID-19 cases between sexes, men have a higher risk of ITU admission (OR 2.84) and death (OR 1.39).p = 1.86 × 10−10) and death . Despite this notable feature of the pandemic, sex is still not routinely reported in all available regional data.Here we present a large-scale global statistical analysis to show that whilst males and females are at equivalent risk of infection, male sex is associated with the development of severe disease as measured by ITU admission  for males103. During the same outbreak in Singapore, male sex was associated with an odds ratio of 3.10  for ITU admission or death104. The Saudi Arabian MERS outbreak in 2013 - 2014 exhibited a case fatality rate of 52% in men and 23% in women105. These data suggest that, whilst socio-economic factors may be influencing some aspects of the pandemic, fundamental differences in the immune response between males and females are likely to be a driving factor behind the significant sex-bias observed in the COVID-19 pandemic.Sex differences in the prevalence and outcomes of infectious diseases occur at all ages, with an overall higher burden of bacterial, viral, fungal and parasitic infections in human males111, more robust CD8+ T cell cytotoxic activity112, and increased B cell production of immunoglobulin compared to males113. Women report more severe local and systemic side effects, and produce higher antibody titres in response to the trivalent inactivated seasonal influenza vaccination (TIV)100, as well as to most other pathogen vaccines114. More specifically, females achieve equivalent protective antibody titres to males at half the dose of TIV115, with serum testosterone levels inversely correlating with TIV antibody titres116. Female B cells also produce more antigen-specific IgG in response to TIV117. These findings imply that females have an increased capacity to mount humoral immune responses compared to males, and together with the data from this meta-analysis, may have important implications for the development of vaccination strategies for COVID-19.Sex differences in both the innate and adaptive immune system have been previously reported and may account for the female advantage in COVID-19. Within the adaptive immune system, females have higher numbers of CD4+ T cells124, which is important for the early response in COVID-19125. Increased IFN production by females is associated with both sex hormone concentration and the number of X chromosomes present121. The X chromosome contains many immune-related genes126, as evidenced by the existence of many X-linked immunodeficiency disorders127. Furthermore X-encoded immune genes may be variably expressed on both alleles in immune cells in females, increasing immune response diversity129. Oestradiol offers an advantage against infectious disease by augmenting T cell responses132, increasing antibody production, somatic hyper-mutation and class switching133. Oestradiol also increases abundance of neutrophils134; and monocyte/macrophage cytokine production135.Females produce more type 1 interferon (IFN), a potent anti-viral cytokine, upon toll-like receptor 7 sensing of viral RNA than males136. This suggests a potential protective effect of oestradiol against the development of hyperinflammatory immune responses associated with mortality in COVID-19137. In contrast, the male sex hormone testosterone suppresses the immune system: hypo-androgenism is associated with increased inflammatory cytokines, antibody titres, CD4/CD8 ratios, and natural killer cells, and a decrease in regulatory T cells138. Interestingly, testosterone-deprivation therapy for prostate cancer has been associated with improved outcomes for COVID-19, suggesting that suppression of the immune response by testosterone, as well as the protective effect of oestrogen, may underlie the observed sex-bias139.An early report in pre-print suggested that the declining oestrogen levels in post-menopausal women may be associated with increased inflammatory cytokine production following infection with SARS-CoV-2140 and there is a marked association between morbidity/mortality and advanced age in COVID-19. For example, males show an age-related decline in B cells and a trend towards accelerated immune ageing142. This may further contribute to the sex bias seen in COVID-19.Age-related changes in the immune system are also different between sexes143 – is different between the sexes145. Oestradiol may influence ACE2 expression146, and the gene for ACE2 is located in the X chromosome147, which may render it susceptible to escaping X-inactivation in women.Other biological factors may influence the sex-bias observed in this study. Expression of angiotensin converting enzyme 2 (ACE2) receptors – which facilitate SARS-CoV-2 viral entry and human to human transmission148 or diabetes (9% of women vs 9.6% of men)149, the most common reported comorbidities in hospitalised COVID-19 patients150. Once more data become available, future studies can adjust for additional factors using techniques such as mediation analysis.Sex-based differences in co-morbidities that are associated with severe COVID-19 may also drive some of the differences observed in this study. However, due to the nature of these high level, publicly available summary data, metadata including age, ethnicity and comorbidities for individual cases are not available. The lack of adjustment for these factors limits our ability to accurately predict the role of sex in disease severity. Notably, there are no marked sex differences in the proportions of adults globally with hypertension (33% of women vs 36% of men)151, although smoking has not emerged as a clear risk factor for severe disease144. Men are less likely to wash their hands with soap after entering a restroom152, and in many cultures, men may be more likely to leave the house and enter crowded areas. Unequal access to healthcare and testing between sexes may skew towards a male bias in infection rates. The data, however, show no difference in the numbers of infected cases between sexes overall, so gender differences in hygiene behaviours and testing are unlikely to explain the sex disparity in disease severity. Regional gender differences in health-seeking behaviours and access to care may predispose men towards access to hospital and ITU admission156. However, the ubiquitous nature of the sex-bias in these data argues for a true biological difference in the response to SARS-CoV-2 between sexes.Gender-based socio-cultural and behavioural differences could contribute to the sex difference seen in COVID-19 disease severity. Men are more likely to smokeThese large-scale data demonstrate that although there is no sex difference in the proportion of people infected with SARS-CoV-2, males are at a significantly higher risk of severe disease and death than females. Previous reports describe fundamental differences between sexes in the immune response to infection, which include a more robust antiviral innate interferon response and increased adaptive immunity towards viral antigens in females. In people infected with SARS-CoV-2 these differences are likely to lead to more effective viral control in females, which may contribute to the relatively lower risk of developing severe disease. Although further studies are needed, these data have implications for the clinical management of COVID-19 and highlight the importance of considering sex as a variable in fundamental and clinical research.157, and an attempt was made to include as many regions reporting sex data as possible. Reports were translated using Google translate if they were not in English.An online search of government websites and published literature was performed by multiple researchers working remotely for regional data reports on COVID-19 cases that included sex as a variable from 01 January 2020 up until 01 June 2020 (Search terms: SARS-CoV-2/COVID-19/case/sex/country/data/death/ICU/ITU). In order to ensure unbiased representation from as many regions as possible, a cross check was done using the list of countries reporting data on ‘Worldometer’98 and 10.25375/uct.12952151.Reports were included if they contained sex as a variable in data describing case number, ITU admission, or mortality. Data were entered directly by individual researchers into an online structured data extraction table. For some sources, counts of male confirmed cases or male deaths were not provided, but percentages of male cases or male deaths were provided instead. To include these sources and avoid biases that might be introduced by their exclusion, we calculated counts of male confirmed cases and male deaths from the reported percentages with rounding to the nearest integer. We acknowledge that this approach assumes that the reported percentages are reflective of the true percentages. For some sources, data included confirmed cases and deaths of unknown sex. For these sources, the reported totals were used where the proportion of unknown sex was small  method was used to estimate an overall proportion. This yielded identical results, indicating the differing assumptions of these different methods were inconsequential for these data.Meta-analyses were also performed to estimate ORs with 95% CI associated with male sex for ITU admission and death, based on pooled average effect measures that were weighted according to the size and precision of each report. Random effects models were estimated and are reported rather than fixed effects models, since these do not assume uniformity across reports and account for variance between reports. The Mantel-Haenszel and DerSimonian-Laird methods were used to calculate the fixed effects and random effects estimates, respectively. Similar to the inverse variance weighting method, individual studies are weighted according to size and variance, and estimates were almost identical when the inverse variance weighting method was used. Both of these meta-analyses for the ITU admission and death outcomes were two-sided and tested the null hypothesis that the estimated OR was 1. Reports that did not contain the data required to calculate ORs were automatically excluded from meta-analyses.158. All R code and input data are available at https://github.com/claire-deakin/covid19-sex-bias and at 10.25375/uct.12952151.Meta-analyses were performed using R version 3.6.1 and the “meta” package version 4.11-0Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummaryDescription of Additional Supplementary FilesSupplementary Data 1


We sought to assess the relative effects of individual anaplastic lymphoma kinase (ALK) inhibitors for the treatment of non-small cell lung cancer (NSCLC).ALK- or ROS1-positive NSCLC who received any ALK inhibitor compared with placebo, another ALK inhibitor, or the same ALK inhibitor at a different dose. The primary outcome was treatment-related death. Secondary outcomes were overall survival (OS), progression-free survival (PFS), and serious adverse events. Data were pooled via meta-analysis and network meta-analysis, and risk of bias was assessed. PROSPERO: CRD42017077046.We searched MEDLINE, Embase, Cochrane CENTRAL, and grey literature  for randomized controlled trials (RCTs) that included participants with ALK-positive NSCLC were identified. Treatment-related deaths were rare, with 10 deaths attributed to crizotinib . All ALK inhibitors improved PSF relative to chemotherapy , while alectinib and brigatinib improved PFS over crizotinib and ceritinib . OS was improved with alectinib compared with chemotherapy (HR 0.57 [95% CrI 0.39–0.83]) and crizotinib (0.68 [0.48–0.96]). Use of crizotinib (odds ratio 2.08 [95% CrI 1.56–2.79]) and alectinib (1.60 [1.00–2.58]) but not ceritinib (1.25 [0.90–1.74), increased the risk of serious adverse events compared with chemotherapy. Results were generally consistent among treatment-experienced or naïve participants.Thirteen RCTs reporting outcomes of interest among participants with ALK-positive NSCLC. PFS may be improved by alectinib and brigatinib relative to other ALK inhibitors; however, the assessment of OS is likely confounded by treatment crossover and should be interpreted with caution.Treatment-related deaths were infrequent among  ALK) gene or ROS1 gene, respectively.[ALK gene renders the cancer sensitive to tyrosine kinase inhibitors, which bind to receptor tyrosine kinases and inhibit downstream signalling pathways.[Lung cancer ranks among the most common types of cancer in North America, with an estimated incidence of 222,500 in the US and 28,600 in Canada in 2017.[ectively. The prespathways.ALK-positive NSCLC patients in the US in 2011 and in Canada in 2012 based on initial results from single-arm phase I and II trials , which reported median progression-free survival of 8–10 months among participants with previous treatment experience. Subsequent randomized controlled trials (RCTs) comparing crizotinib with chemotherapy in treatment-experienced (PROFILE 1007) or naïve (PROFILE 1014) patients reported significant improvements in progression-free survival but no corresponding improvement in overall survival; however, a high proportion of patients who experienced disease progression on chemotherapy crossed over to crizotinib treatment, confounding the results. Acquired resistance to crizotinib is common, via secondary mutations in the ALK gene, which limits its efficacy to a median of about one year.[Four ALK inhibitors are approved for use in Canada and the US: crizotinib, ceritinib, and alectinib, and brigatinib. Crizotinib, the first-in-class tyrosine kinase inhibitor, was initially approved for use in ROS1 or ALK-positive NSCLC, and we used NMA methodology to provide an estimate of progression-free survival, overall survival, and SAEs associated with each individual ALK inhibitor.Previous systematic reviews of ALK inhibitors for the treatment of NSCLC have reported improved overall and progression-free survival with crizotinib and alectinib compared with chemotherapy., 7 Howeva priori (PROSPERO no.: CRD42017077046) and followed the Cochrane Handbook for Systematic reviews for Interventions[This review was registered rventions and the rventions . The search strategy was peer-reviewed by use of the PRESS checklist and utilTitles and abstracts of identified records, and the full-text of any potentially relevant record, were evaluated by two independent reviewers; disagreements were resolved by consensus. Decisions about study eligibility were based on information provided in the published records; study authors were not contacted to clarify eligibility.The following eligibility criteria were applied to each identified record to determine eligibility:ALK-positive and/or ROS1-positive NSLC.Treatment-naïve or experienced participants with phase III or IV ALK inhibitors .Placebo, chemotherapy, radiotherapy, another ALK inhibitor, or the same ALK inhibitor at a different dose.The primary outcome was treatment-related death. Secondary outcomes were overall survival, progression-free survival, and SAEs as reported by the study authors. Studies were not selected for inclusion based on reported outcomes.Randomized controlled trials.Data were extracted by one reviewer and verified for completeness and accuracy by a second reviewer, with disagreements resolved by discussion. We extracted study characteristics  and participant characteristics , as well as outcome data. We extracted event counts and denominators  for dichotomous outcomes , and we extracted hazard ratios (HRs) and 95% confidence intervals for time-to-event outcomes . Where PFS were assessed by both study investigators as well as by independent review committees, we extracted and analyzed data from the latter. We also extracted the percentage of participants who remained alive  or free of disease progression  after 12 months of treatment; these data are narratively described. We compared study and patient characteristics across studies to ensure that each record represented a unique publication of study data and to match up companion publications . Risk of bias (ROB) was assessed by two independent reviewers by use of the Cochrane Collaboration’s ROB tool for RCTs. SpecificI2 value, with I2 values above 75% considered to represent high heterogeneity; data were not pooled if the I2 value exceeded this threshold. We also considered clinical heterogeneity across RCTs by evaluating the similarity of included participants. Additionally, we assessed the model fit (fixed versus random effects) based on the deviance information criterion (DIC) and by comparing the residual deviance to the number of unconstrained data points for each analysis.[We first performed pair-wise meta-analysis to explore the class effect of treatment with any ALK inhibitor versus chemotherapy, followed by NMA to explore the effect of individual ALK inhibitors. Base-case analyses involving all participants were performed for all outcomes  as were subgroup analyses based on treatment experience. Complete case analyses were performed for dichotomous outcomes . For all other outcomes, analyses involved HRs reported by study authors, which accounted for participants censored from the study. Analyses were stratified by treatment experience (naive or experienced) for all meta-analyses and NMAs; no additional subgroup analyses were performed. Bayesian meta-analyses and NMA were performed by use of WinBUGS . Chemotherapy was selected as the reference group for the MA and NMA comparisons. We assessed heterogeneity by use of the analysis.2)) were assigned for basic parameters of the treatment effect in the model. Informative priors ) were applied for the between-study variance parameter in the random-effect binomial likelihood model for dichotomous outcomes to improve precision and reduce heterogeneity between studies.[In the Bayesian MA and NMAs, a normal likelihood with identity link model was applied for the time-to-event outcomes  using study-level summary measures (log HRs and their standard errors). A binomial likelihood model with logit link was used for the Bayesian MA and NMAs for the dichotomous outcomes  to estimate relative risk (RR) and risk difference (RD). Point estimates  and 95% credible intervals (CrIs) were estimated using Markov Chain Monte Carlo methods. For dichotomous outcomes, the RR was estimated based on the OR and the mean proportion of patients who experience the outcome in the reference group of the included studies. The conversion of OR to RR was based on the incidence of the event in the reference group. Vague priors  and allocation concealment 54%), although 38% and 46% of studies did not report details of randomization and allocation concealment, respectively  when assessed via pairwise meta-analysis. Similarly, there was no statistically significant difference between treatment naive  or experienced participants  (p for subgroups = 0.87). Relative risks are reported in Eleven treatment-related deaths were reported in 5 RCTs, 28, 29;I2 = 0%; 6 RCTs[I2 = 0%;) but not experienced  participants (p for subgroup differences p = 0.36). However, rate of crossover from chemotherapy to ALK inhibitor was high in some studies or was unreported but potentially high in others. Of note, the published overall survival data remain immature for several RCTs Next, we considered whether there was a differential effect of individual ALK inhibitors on overall survival via network meta-analysis. The evidence network for overall survival (based on HRs) included 2376 participants randomized to crizotinib, ceritinib, alectinib, brigatinib, or chemotherapy in 9 RCTs, 28, 29 I2 = 0%), with similar results among both treatment-experienced  and naive participants  (p for subgroups 0.99). The percentage of participants who were progression free at 12 months ranged from 18% to 68% among participants who received an ALK inhibitor, and between 6% and 39% among those who received chemotherapy.In total, 12 RCTs, 28, 29 The network meta-analysis for progression-free survival included 2583 participants randomized to crizotinib, ceritinib, alectinib, brigatinib, or chemotherapy in 10 RCTs, 28, 29 I2 = 62%) among all patients. The results were consistent among both treatment experienced  and naive participants  . Relative risks are reported in Nine RCTs, 28, 29 The network meta-analysis for SAEs included 2074 participants randomized to crizotinib, ceritinib, alectinib, or chemotherapy in 8 RCTs., 28, 29 Treatment-related deaths were infrequent among participants who received an ALK inhibitor, with no statistically significant difference in risk between crizotinib and chemotherapy; of note, no treatment-related deaths were reported for any other ALK inhibitor. In the NMA, we observed that all ALK inhibitors improved progression-free survival relative to chemotherapy, and that alectinib and brigatinib were associated with improved progression-free survival compared to crizotinib and ceritinib.In terms of overall survival, alectinib improved overall survival relative to chemotherapy and crizotinib, although the findings may have been influenced by crossover between treatment groups following disease progression. The net effect of such crossovers may be that the overall survival of patients receiving an ALK inhibitor is substantially longer than that of patients who received chemotherapy alone. This discrepancy is emphasized by differences in the duration of treatment exposure between patients randomized to chemotherapy and ALK inhibitors among RCTs that allowed cross-over. For example, in the ASCEND-5 trial, which peALK-positive NSCLC.There exists limited clinical trial data directly comparing the efficacy of individual ALK inhibitors. Three RCTs have directly compared crizotinib and alectinib in either treatment-naïve  or -expIn this review, use of crizotinib  was associated with an increased risk of SAEs. In the two head-to-head trials of crizotinib and chemotherapy  with crizotinib.[ROS1 rearrangements; additional research is needed to assess the benefits of other ALK inhibitors in this population.Clinical evidence for the treatment of NSCLC with izotinib.–48 Curreizotinib. and the izotinib. support We performed a comprehensive search of the published and grey literature for randomized and non-randomized studies, without language or date restrictions, and the protocol was registered a priori. We used NMA methodology to assess the relative efficacy and safety of individual ALK inhibitors. Previous reviews have used pair-wise meta-analysis to compare two treatments at a time or to pool the percentage of patients with an outcome from single-arm non-randomized studies., 50–52 WOur study has several limitations that merit consideration. First, we did not have access to individual participant data, and we are limited to describing the number and causes of deaths as reported in the published studies. Treatment-related deaths are uncommon with these agents, and the included RCTs may have been underpowered to detect differences in treatment-related deaths and other safety outcomes.Second, cause of death was not well reported, and the classification of a death as “treatment-related” was at the discretion of the investigators. As well, the duration of treatment varied between the RCTs, with participants exposed to ALK inhibitors for considerably longer than chemotherapy, likely owing to crossover to an ALK inhibitor after disease progression with chemotherapy. This has important implications for the assessment of several outcomes, including treatment-related deaths, overall survival, and SAEs, and the results should be interpreted with caution.Third, there are limitations associated with the outcomes progression-free and overall survival. Progression-free survival may be considered as a surrogate marker for overall survival, but it has not been validated as such. Overall Fourth, relatively few RCTs have assessed the efficacy of ALK inhibitors. As such, our analyses comparing the effects of individual ALK inhibitors involved few trials, and the findings may change with the publication of future trials. We were unable to assess publication bias because few studies contributed data to each outcome. Similarly, we were unable to fully assess the impact of patient characteristics on the outcomes, and future studies should consider other important variables such as age and sex, as well as the influence of cross-over between treatment groups.ALK-positive NSCLC. Among patients with ALK-positive NSCLC, progression-free survival was improved by crizotinib, ceritinib, alectinib, and brigatinib compared with chemotherapy, while alectinib and brigatinib were significantly better than crizotinib and ceritinib. Overall survival was improved only by alectinib; however, the findings are likely confounded by crossover between treatment groups and should be interpreted with caution. Few studies have enrolled participants with ROS1 mutations, and additional research is need in this area.Treatment-related deaths were infrequent among S1 FileAppendix 1: PRISMA NMA Checklist. Appendix 2: Search strategy. Appendix 3: Included studies. Appendix 4: Risk of bias. Appendix 5: Model diagnostics. Appendix 6: Treatment-related death. Appendix 7: Overall survival. Appendix 8: Progression-free survival. Appendix 9: Serious adverse events.(DOCX)Click here for additional data file.