Can the outcomes associated with an antimicrobial stewardship program be sustained in the long term?In this cohort study, after implementation of prospective review and feedback, the use trends increased for piperacillin-tazobactam and carbapenems and declined for other broad-spectrum antibiotics. After the addition of a computerized decision support system, the use trends decreased for piperacillin-tazobactam and carbapenems and for other broad-spectrum antibiotics.These findings suggest that concurrent use of prospective review and feedback and a computerized decision support system may help to sustain the outcomes associated with antimicrobial stewardship programs. This cohort study examines associations of sequential implementation of antimicrobial stewardship initiatives with trends in antibiotic use and incidence of multidrug resistant organisms. There is a lack of studies comparing the intended and unintended consequences of prospective review and feedback (PRF) with computerized decision support systems (CDSS), especially in the longer term in antimicrobial stewardship.To examine the outcomes associated with the sequential implementation of PRF and CDSS and changes to these interventions with long-term use of antibiotics for and incidence of multidrug resistant organisms (MDROs) and other unintended outcomes.This cohort study used an interrupted time series with segmented regression analysis of data from January 2007 to December 2018. Data were extracted from the electronic medical records of patients admitted at a large university teaching hospital with high rates of antibiotic resistance in Singapore. Data were analyzed from June 2019 to June 2020.PRF of piperacillin-tazobactam and carbapenems , with the addition of hospital-wide CDSS , and lifting of CDSS for half of the hospital wards for 6 months .Monthly antimicrobial use was measured in defined daily doses (DDDs) per 1000 patient-days. The monthly incidence of MDROs was calculated as number of clinical isolates detected per 1000 inpatient-days over a 6-month period. Unintended outcomes examined included in-hospital mortality and age-adjusted length of stay (LOS).Clostridioides difficile decreased .The number of inpatients increased from 56 263 in 2007 to 63 572 in 2018. During the same period, the mean monthly patient days increased from 33 929 in 2007 to 45 603 in 2018, and the proportion of patients older than 65 years increased from 45.5% in 2007 to 56.6% in 2018. After intervention 1, there were 0.33  more DDDs per 1000 patient-days per month of piperacillin-tazobactam and carbapenems and −11.05  fewer DDDs per 1000 patient-days per month for other broad-spectrum antibiotics. After intervention 2, there were −0.22  fewer DDDs per 1000 patient-days per month of piperacillin-tazobactam and carbapenems and −2.10  fewer DDDs per 1000 patient-days per month for other broad-spectrum antibiotics. After intervention 3, use of piperacillin-tazobactam and carbapenem increased by 0.28  DDDs per 1000 patient-days per month. After intervention 2, incidence of In this cohort study, concurrent PRF and CDSS were associated with limiting the use of piperacillin-tazobactam and carbapenems while reducing use of other antibiotics. In 2017, the World Health Organization (WHO) categorized antibiotics into Access, Watch, and Reserve categories. Seven classes of antimicrobials, fluoroquinolones, third-generation cephalosporins, glycopeptides, antipseudomonal penicillins with β-lactamase inhibitors, and carbapenems, were on the Watch list.7 However, there is a lack of studies comparing PRF with CDSS. In addition, there are calls for adopting robust study designs using interrupted time series (ITS) analysis to evaluate intended and unintended consequences.8The effectiveness of antimicrobial stewardship activities, such as prospective review and feedback (PRF) and computerized decision support systems (CDSS), in hospitals has been demonstrated.Enterobacterales were not susceptible to third-generation cephalosporins and 7.0% were not susceptible to carbapenems.9 We aimed to examine the associations of the sequential implementation and changes to these interventions with the long-term use of antibiotics with high use, incidence of multidrug-resistant organisms (MDROs), and other unintended outcomes.Two main interventions, PRF and CDSS, were implemented sequentially at a university teaching hospital in Singapore, where up to 37.7% of all STROBE) reporting guideline for cohort studies.This cohort study was approved by the Domain Specific Review Board of the National Healthcare Group, Singapore, which waived the requirement for informed consent because the study was based on aggregated nonidentifiable data. This study was reported following the Strengthening the Reporting of Observational Studies in Epidemiology . PRF was continued for all wards throughout the 6 months. A total of 19 acute care wards  and 12 subacute care wards were excluded. However, as the 32 acute care wards in the main hospital building contributed two-thirds of total patient days, it was considered a significant intervention. A crossover design was adopted to adjust for bias and contamination between clusters owing to Hawthorne and spillover effects because of physicians’ rotation and patient transfers.10 In both study groups, more than 80% of patients had their physicians accepting CDSS and PRF recommendations. However, patients in the voluntary CDSS wards received significantly fewer CDSS recommendations (132 recommendations for 641 prescriptions [21%]) compared with the compulsory CDSS arm (612 recommendations for 616 prescriptions [99%]).10We conducted an interrupted time series with segmented regression analysis of antibiotic use, MDRO incidence, and other unintended outcomes at Tan Tock Seng Hospital (TTSH), Singapore, a university teaching hospital, over 11 years, from January 2007 to December 2018. All operational wards were included. In 2007, antimicrobial stewardship interventions included antibiotic restrictions for vancomycin, linezolid, carbapenems, and polymyxin B. In 2009, a multidisciplinary antimicrobial stewardship team, including infectious disease physicians and antimicrobial stewardship–trained pharmacists, was established. The hospital implemented empirical antibiotic guidelines in January 2009, and daily real-time PRF of piperacillin-tazobactam and carbapenems was conducted on weekdays starting in April 2009 (intervention 1). Antibiotic restrictions were thereafter limited to only linezolid and polymyxin B. During the PRF, the antimicrobial stewardship team conducted audits and provided recommendations concurrently to the prescribers, who could accept or reject them. A CDSS that covered 52 infective syndromes and followed hospital empirical antibiotic guidelines was introduced in April 2011 (intervention 2). Details of the CDSS have been described elsewhere.Klebsiella pneumoniae and Escherichia coli, Clostridioides difficile, carbapenem-resistant Pseudomonas aeruginosa and carbapenem-resistant Acinetobacter baumannii. The monthly incidence was calculated as number of clinical isolates per 1000 inpatient-days. Only the first of repeated isolates  from the same patient was counted during a 6-month period. Unintended outcomes were examined in terms of in-hospital mortality and age-adjusted length of stay (LOS). These outcome data were obtained from the hospital quality management information system. In-hospital mortality was defined as number of deaths per 100 inpatient discharges (including death discharges). Age-adjusted mean LOS was calculated by dividing total patient-days in a month with number of inpatient discharges in the same month, adjusted by age, using year 2007 as the reference year. Since there was a substantial expansion of subacute care wards in the hospital over the years, we reexamined the time-series again after excluding subacute care wards.Data on antibiotic use were extracted from the pharmacy dispensing system, and data on incidence of MDROs in TTSH were collected from the hospital electronic medical record system. We included piperacillin-tazobactam and carbapenems targeted by PRF and CDSS. In addition, antibiotics that were considered broad-spectrum and had high use were included, defined as levofloxacin, ciprofloxacin, ceftriaxone, ceftazidime, cefepime, vancomycin, and intravenous amoxicillin-clavulanate. Monthly antimicrobial use was measured in defined daily doses (DDDs) per 1000 patient-days to adjust for fluctuations in patient load. For MDROs, we included the incidence of third-generation cephalosporin–resistant (3GCR) 12 We applied ordinary least squares regression with Newey-West SEs to account for an error structure that was assumed to be heteroskedastic and autocorrelated at lag 2 for piperacillin-tazobactam and carbapenems and at lag 3 for other broad-spectrum antibiotics. The autocorrelation of each model was determined using Cumby-Huizinga general test and visual inspection of autocorrelation and partial autocorrelation plots. No apparent seasonality was detected using periodogram, cycle plot, and visual inspection of autocorrelation and partial autocorrelation plots. Nonstationarity was not detected in all the time series using Dickey-Fuller test.Sample size calculation was not conducted, as data from all patients at the hospital were included. A segmented regression analysis of interrupted time series was used to examine changes in the level and trend. Separate intercepts and slopes were estimated in each preintervention and postintervention segment of the period.14 The MDRO incidence time series was also filtered with the same model. The cross-correlation function was computed between the filtered time series for each antibiotic use and MDRO incidence at lags of up to 12 months. For cross-correlations that were statistically significant, we noted the lag in the cross-correlation function corresponding to the maximum correlation coefficient (r), to determine the lead time of antibiotic use relative to 3GCR K pneumoniae and E coli, C difficile, carbapenem-resistant P aeruginosa, and carbapenem-resistant A baumannii.15We examined the lead-lag association between antimicrobial resistance and antibiotic use. To identify meaningful associations between 2 time series, each time series was prewhitened to remove autocorrelation and temporal patterns. An autoregressive integrated moving average CC model was fitted to an antibiotic use time series, which was filtered to obtain white noise residuals.P values were 2-sided, and statistical significance was set at P < .05. Segmented regression analyses were performed using Stata Standard Edition version 16 (StataCorp) and correlation analyses were performed using R version 3.5.0 . Data were analyzed from June 2019 to June 2020.All P < .001). The mean (range) monthly number of operational acute care wards was 42 (40-44) wards in 2007 and 53 (53-56) wards in 2018, and operational subacute wards increased from 1 in 2007 to 10 in 2018. The mean (range) monthly patient-days also increased from 33 929 (29 723-35 028) patient-days per month in 2007 to 45 603 (43 101-48 740) patient-days per month in 2018.The annual number of inpatients increased from 56 263 in 2007 to 63 572 in 2018. During the same period, the proportion of inpatients aged older than 65 years increased significantly, from 45.5% in 2007 to 56.6% in 2018  DDDs per 1000 patient-days, and use increased at a mean monthly trend of 1.17  more DDDs per 1000 patient-days. When empirical antibiotic guidelines and PRF were implemented in April 2009 (intervention 1), use of piperacillin-tazobactam and carbapenems increased at a slower monthly trend of 0.33  more DDDs per 1000 patient-days. When compulsory CDSS for piperacillin-tazobactam and carbapenem prescription and voluntary use of CDSS for other antibiotics were introduced in April 2011 (intervention 2), use decreased at a trend of −0.22  fewer DDDs per 1000 patient-days per month for piperacillin-tazobactam and carbapenems. However, there was a significant increase of 0.28  more DDDs per 1000 patient-days per month when compulsory use of CDSS for piperacillin-tazobactam and carbapenems was lifted in March 2017 for half of the acute wards (intervention 3). All 3 interventions were associated with significant change in use level in the month of implementation and trend change A.In January 2007, the level of other broad-spectrum antibiotic use was 1424.51  DDDs per 1000 patient-days, and the monthly use trend remained stable at −0.05  DDDs per 1000 patient days. After intervention 1, there was a significant increase of 103.46  more DDDs per 1000 patient-days in use level, followed by a significant decrease of −11.05  fewer DDDs per 1000 patient-days per month in monthly trend compared with the preintervention trend. After intervention 2, there was a significant increase in monthly trend of 9.00  more DDDs 1000 patient-days per month compared with the postintervention 1 trend, resulting in a lower rate of use at −2.10  fewer DDDs per 1000 patient-days per month. After intervention 3, use level increased by 109.2  DDDs per 1000 patient-days in the month of implementation and the monthly trend of other broad-spectrum antibiotics remained stable. B AntibioK pneumoniae and E coli after any of the interventions except for an increase in level in the month of implementation of intervention 2.  fewer cases per 1000 patient-days per month. The incidence density increased at a trend of 0.02  more cases per 1000 patient-days per month after intervention 1, and it decreased at a trend of −0.0026  fewer cases per 1000 patient-days per month after intervention 2 , with a lead time of 3 months . Use of intravenous amoxicillin-clavulanate was also correlated with C difficile incidence density .In January 2007, the incidence density of ention 2 . There wP aeruginosa was 0.42 cases per 1000 patient-days. In the month of intervention 1, carbapenem-resistant P aeruginosa recorded a level reduction of 0.11  fewer cases per 1000 patient-days. After intervention 3, there was a significant increase of 0.006  more cases per 1000 patient-days per month in the monthly trend compared with the postintervention 2 trend  and carbapenems   fewer cases per 1000 patient-days per month after intervention 2 and 0.007  fewer cases per 1000 patient-days per month after intervention 3  fewer deaths per 100 discharges per month A. Age-adC difficile and carbapenem-resistant P aeruginosa after intervention 2. In-hospital mortality remained stable throughout the study, but there was an increase in age-adjusted LOS after interventions 1 and 2.This cohort study found that implementation of PRF and CDSS was associated with limiting the use of piperacillin-tazobactam and carbapenems while reducing use of other broad-spectrum antibiotics in a hospital with high–antibiotic resistance rates. The continued use of CDSS was needed to sustain the decrease in use of piperacillin-tazobactam and carbapenem. There was a reduction in the incidence density of 16 One of the concerns for implementation of antimicrobial stewardship programs is the undesirable consequence of the “squeezing the balloon” phenomenon,17 which is an increase in use of some antibiotics owing to restriction of the use of other antibiotics. Although we were not able to report total antibiotic use, we attempted to address this limitation by adding intravenous amoxicillin-clavulanate acid and other broad-spectrum antibiotics from the WHO Watch list.1The reduction in piperacillin-tazobactam, carbapenems, and other broad-spectrum antibiotic use observed in our study was consistent with other successful antimicrobial stewardship program implementations.18 The continued availability of the voluntary use of CDSS may have helped prescribers learn and adopt hospital empirical antibiotic guidelines, which were focused on antibiotic choice and duration, sustaining the trend of other broad-spectrum antibiotics.After compulsory CDSS use for piperacillin-tazobactam and carbapenems (intervention 3) was removed for half of the acute care wards, the monthly use of piperacillin-tazobactam and carbapenems increased while that of broad-spectrum antibiotics remained stable. Without compulsory CDSS, there could have been fewer opportunities for prescribers to consider alternate antibiotics and potentially an increase in use of piperacillin-tazobactam and carbapenems.K pneumoniae and E coli remained stable throughout the study while that of C difficile decreased when compulsory CDSS was added. The decrease in C difficile also correlated with the decrease in other broad-spectrum antibiotic use. Carbapenem-resistant P aeruginosa incidence decreased after interventions 1 and 2 and increased after intervention 3, and changes correlated significantly with the use of carbapenems and piperacillin-tazobactam. Although the overall incidence of carbapenem-resistant A baumannii decreased throughout the years, it did not correlate with antibiotic use, which suggests that the decreasing trend could have been the result of infection control measures.19 Antimicrobial stewardship has been demonstrated to reduce the incidence of multidrug resistant and extended-spectrum β-lactamase (ESBL)–producing gram-negative bacteria in addition to incidence of C difficile infections.23 The presence of community acquired ESBL-producing gram negative bacteria, with reports ranging from 12% to 26% of all ESBL-producing gram-negative bacteremia being community acquired, may explain the stable trend of 3GCR K pneumoniae and E.coli in our study.25 A study by Bond et al26 demonstrated a similar decrease in trend of health care–associated C difficile infection after implementation of CDSS with concurrent guideline dissemination.The incidence density of 3GCR 27 In our study, there was an increase in age-adjusted LOS, which could be attributed to increases in proportions of patients older than 65 years and number of subacute care wards. Our previous study on the safety of PRF did not find a significant increase in LOS of patients who had carbapenem therapy deescalation.28 In addition, to address the challenges of an aging patient population, TTSH introduced its transitional care services in July 2016 to support timely transition from a hospital to the community, which may lead to reduction in age-adjusted LOS after intervention 3.29 A Cochrane review of antimicrobial stewardship interventions found consistent reduction in LOS in the included studies.30 No changes in in-hospital mortality rate were observed after antimicrobial stewardship interventions, which suggest that that our antimicrobial stewardship interventions were not associated with adverse outcomes in patients.We evaluated our antimicrobial stewardship activities according to a structured framework of expected, desirable outcomes ; expected, undesirable outcomes (intervention trade-offs); unexpected, undesirable outcomes (unpleasant surprises); and unexpected, desirable outcomes (pleasant surprises).31 as these were regarded as targets of stewardship programs and monitoring. We were unable to determine if the MDROs were community-acquired or health care–associated and did not account for the possible impact of infection control measures. There was no control group for comparative analysis, and confounding effects from other hospitalwide activities were not accounted for. The antibiotic use, resistance, and other outcomes may not have a direct association with PRF and CDSS, as prescribers may learn from interactions with our interventions and modified their overall antibiotic prescribing. We attempted to partially mitigate the possibility of chance finding by examining antibiotic use by individual antibiotics to check whether the trend was consistent across antibiotic groups.This study has several limitations. We did not include overall antibiotic use in the hospital and only focused on commonly used broad-spectrum antibiotics from the Watch list, based on WHO classification,C difficile and carbapenem-resistant P aeruginosa incidence without increasing mortality in a large university teaching hospital with high rates of antibiotic use and resistance over a 11-year period.This cohort study found that implementation of PRF and CDSS was associated with limiting the use of piperacillin-tazobactam and carbapenems while reducing that of other antibiotics. The continued use of CDSS may be needed to sustain the reduction in piperacillin-tazobactam and carbapenems. Our antibiotic stewardship activities were associated with a reduction in


Biliary tract cancer (BTC) is a group of rare and aggressive malignancies with a dismal prognosis. There is currently a significant lack in effective treatment options for BTC, with gemcitabine-cisplatin remaining the first-line standard of care treatment for over a decade. A wave of investigational therapies, including new chemotherapy combinations, immunotherapy, and biomarker-driven targeted therapy have demonstrated promising results in BTC, and there is hope for many of these therapies to be incorporated into the Canadian treatment landscape in the near future. This review discusses the emerging therapies under investigation for BTC and provides a perspective on how they may fit into Canadian practice, with a focus on the barriers to treatment access. Biliary tract cancer (BTC) is a group of aggressive malignancies arising from the gallbladder , intrahepatic or extrahepatic bile ducts , or ampulla of Vater (ampullary cancer) . There iCurrently, there is a lack of effective therapeutic options for patients with advanced BTC. For patients with resectable tumours, curative intent therapy with surgery followed by adjuvant chemotherapy is the standard of care, resulting in a median overall survival (OS) of over 4 years . RadiothFollowing progression on gemcitabine-cisplatin, many patients are not well enough to receive second-line therapy and there is a lack of effective treatment options for those fit to receive subsequent therapy . While tTherapeutic development in BTC has flourished in the last few years, and after many unsuccessful clinical trials, there are finally novel treatments that can provide hope for patients with advanced, unresectable BTC. Several new regimens are expected to enter the treatment landscape in Canada within the next 5–10 years; however, significant barriers to accessing these therapies exist. Most therapies under investigation fit under the categories of new chemotherapy combinations, immunotherapy, and biomarker-driven targeted therapy . This reA number of chemotherapy combinations have been evaluated in clinical trials for the first-line treatment of advanced or metastatic BTC. None of these trials demonstrated survival outcomes beyond those achieved with gemcitabine-cisplatin in the ABC-02 trial; however, a recent phase II study evaluating albumin-bound paclitaxel added to gemcitabine-cisplatin in advanced BTC reported a median OS of 19.2 months, which compares favorably to the OS reported in the ABC-02 trial . In addiOther triplet chemotherapy regimens have demonstrated less encouraging results when compared to gemcitabine-cisplatin. The PRODIGE38 study did not show an improved 6-month PFS rate for modified FOLFIRINOX compared to gemcitabine-cisplatin, which was the primary endpoint of the phase II portion of the study (44.6% vs. 47.3%) . The medp = 0.031). This regimen has since become the most common second-line treatment option for patients with advanced BTC in Canada. Most recently, the phase II NIFTY trial, enrolling advanced BTC patients who progressed on first-line gemcitabine-cisplatin, reported a significantly longer median OS with liposomal irinotecan and 5-FU versus 5-FU alone  [Few randomized controlled trials have demonstrated efficacy of chemotherapy combinations in the second-line setting. The phase III ABC-06 study evaluating modified FOLFOX in patients with advanced BTC following first-line gemcitabine-cisplatin demonstrated a 15% improvement in 6- and 12-month survival rates compared to active symptom control alone . The med= 0.035) .Therapies targeting immune checkpoint pathways, including the programmed death-1 (PD-1)/programmed death-ligand 1(PD-L1) axis, have demonstrated activity in other cancers and have shown success in prolonging PFS and OS in both biomarker-selected and unselected populations . The ratSingle-agent immunotherapy with the anti-PD-1 agents pembrolizumab and nivolumab or the anti-PD-L1 agent durvalumab have demonstrated modest activity in phase II clinical trials of chemorefractory BTC, with overall response rates (ORRs) between 5 to 22% in unselected patients and 41% in patients with microsatellite instability/mismatch repair deficiency ,24,25.  Dual cheThere is evidence to suggest that chemotherapy may act synergistically with immunotherapy through several mechanisms of immunomodulation, providing a rationale to explore combination therapies in BTC . Both nip = 0.021) [p = 0.001) and increased ORR (27% vs. 19%) [The TOPAZ-1 study was subsequently conducted, which was a phase III, double-blind placebo-controlled trial randomizing patients with unresectable or metastatic BTC to receive gemcitabine-cisplatin (for up to eight cycles) plus durvalumab or gemcitabine-cisplatin (for up to eight cycles) plus placebo as first-line treatment . The stu= 0.021) . Additiovs. 19%) . Rates ovs. 19%) . Anothervs. 19%) . Immune checkpoint inhibitors in combination with other novel agents and locoregional therapy are also being studied in BTC. Thus far, results have been reported for a phase II study evaluating the anti-VEGF agent lenvatinib in combination with pembrolizumab in advanced previously treated BTC. In this trial, pembrolizumab-lenvatinib achieved an ORR of 25%, median PFS of 4.9 months, and median OS of 11.0 months . Several targeted agents have shown encouraging efficacy in patients with advanced BTC who harbor specific genomic alterations, although together, this represents a small fraction of patients with BTC and many of these patients may not be identified due to the limited access to biomarker testing for BTC in Canada. Recently, two agents targeting the Fibroblast Growth Factor Receptor (FGFR) family genes—pemigatinib and infigratinib—have been approved by Health Canada for previously treated, unresectable, or metastatic CCA with FGFR2 gene rearrangements. FGFR2 gene fusions occur in 10−16% of CCA cases, and almost exclusively occur in the intrahepatic subtype. The approval of pemigatinib and infigratinib were based on phase II studies where a median PFS of approximately 7 months was reached in patients with chemo-refractory cholangiocarcinoma with FGFR2 gene fusions ,36. Tab A similaAlthough rarely occurring in CCA (<1% of cases), tumors with gene fusions involving the neurotrophic tyrosine receptor kinase (NTRK) family proteins have been reported to respond to TRK inhibitors in a small number of patients evaluated in basket studies  ,48. EntrActivating mutations in isocitrate dehydrogenase 1/2 (IDH1/2) occur in 10–20% of patients with CCA. The phase III ClarIDHy study randomized patients with previously treated advanced CCA harboring IDH1/2 mutations to treatment with the IDH1 inhibitor ivosidinib or placebo ,49. IvosMutations in the BRAF gene have been reported in 3–7% of BTC cases, and are also enriched in those with iCCA. In a basket study exploring the activity of the BRAF and MEK inhibitors dabrafenib and trametinib in rare tumours harboring the BRAFV600E mutation, the cohort of 43 patients with BTC achieved an ORR of 51% and median OS of 14 months [HER2 amplification or overexpression occurs in 10–16% of gallbladder carcinomas and 5–11% of eCCA . Given tIncreasingly sophisticated genomic analyses in BTC are expected to reveal insights on additional biomarkers of treatment response and new therapeutic targets. For example, a multi-omics analysis in iCCA tumor samples identified an IDH mutant-enriched subtype that correlated with hypermethylation and decreased expression of ARID1A, suggesting that inhibition of chromatin modifiers such as EZH2 may be effective in this subtype . Other sWith several recent BTC trials reporting encouraging results, the question on many Canadian oncologists’ minds is which therapies will be made available to our patients and funded by the Canadian provinces. Following Health Canada approval, the decision to provide Canadians with access to drugs through public reimbursement programs is made at the provincial level. Funding decisions are largely aided by recommendations from Health Technology Assessment (HTA) bodies, including the Canadian Agency for Drugs and Technologies in Health (CADTH) and Quebec’s Institut National d’Excellence en Santé et en Services Sociaux (INESSS). These HTA bodies consider cost-effectiveness, patient-based values, and adoption feasibility in addition to clinical benefit, when appraising drugs for reimbursement recommendations . As BTC is a rare, aggressive disease with extremely poor prognosis and limited treatment advances, access to new therapies can be hindered if HTA assessments  are measured against the same standards as all oncology drugs. For example, although phase III randomized controlled trials are the gold standard for determining clinical benefit, this trial design may be difficult to achieve in BTC, particularly in the second-line setting, as trial enrollment is challenged by a small overall population of eligible patients. Evaluation of targeted agents that require the selection of even smaller genetically defined subpopulations adds to enrollment difficulty. This challenge is illustrated in the negative recommendation issued by CADTH for the reimbursement of pemigatinib in patients with previously treated CCA with FGFR2 fusions . This reIn contrast to the negative recommendation for pemigatinib, the TRK inhibitor larotrectinib has been issued a conditional positive recommendation for funding by CADTH, for any patient with an advanced solid tumor harboring NTRK fusions who have no other effective treatment options . This foAnother challenge with assessing new therapies in BTC is deciding what constitutes a clinically meaningful benefit. Both the American Society of Clinical Oncology (ASCO) and the European Society for Medical Oncology (ESMO) have published consensus documents proposing how a clinically meaningful benefit may be measured across different tumor types and scenarios. In ASCO’s publication, working groups for colon, pancreatic, lung, and breast cancer all selected median OS as the primary end point of interest. In general, they deemed an HR between 0.6 to 0.8 and a median OS improvement between 2.5 to 6 months over standard therapy as a clinically meaningful outcome . HoweverResponse rate, duration of response, and PFS, although not validated as surrogate markers for OS, are also clinically meaningful in BTC. A large, durable response can downstage patients, as was reported in 20% of patients in the phase II trial of albumin-bound paclitaxel plus gemcitabine-cisplatin . This maQuality of life is also of great importance to patients with BTC and must be considered in the value assessment of a therapy . The MagThe prospect of patients with BTC gaining access to new treatments is exciting. However, the only regimen currently being reviewed for Health Canada approval is durvalumab in combination with gemcitabine-cisplatin for first-line treatment of BTC. This is based on results from the phase III TOPAZ-1 trial, which is the first phase III randomized, double-blind, placebo-controlled trial in over a decade to demonstrate a statistically significant improvement in OS, without increasing toxicity or reducing quality of life, compared to gemcitabine-cisplatin alone ,33. HoweAs demonstrated in other clinical trials, median OS alone does not appropriately capture the extended right-sided tail commonly observed in the survival curves for chemoimmunotherapy regimens, which represents a portion of patients with long-term survival ,69,70. IAnother potential limitation of the TOPAZ-1 trial comes from the dosing schedule of gemcitabine-cisplatin. In TOPAZ-1, gemcitabine-cisplatin was stopped after eight cycles of therapy in both arms, similar to the dosing regimen used in the ABC-02 trial. However, anecdotally, many Canadian oncologists will give gemcitabine-cisplatin until progression or dose-limiting toxicity (such as neuropathy related to cisplatin), or treatment is continued with gemcitabine monotherapy after cycle 8. This may complicate the implementation of durvalumab for the first-line treatment of BTC specifically in Canada. The practice of giving gemcitabine-cisplatin until progression or toxicity is largely based on studies from other tumor types such as breast cancer, which demonstrate improved survival with continued palliative chemotherapy . One retThe current landscape of systemic treatments for advanced, unresectable BTC is extremely limited in Canada, consisting mainly of chemotherapy options including standard of care first-line treatment with gemcitabine-cisplatin. Although biomarker-driven agents targeted against NTRK  and FGFR  are approved by Health Canada for second-line therapy, there are several barriers to accessing these agents for patients with advanced BTC. These include lack of provincial funding and access to timely biomarker testing. In addition, the pool of patients who harbor FGFR2 or NTRK fusions and who are fit to receive second-line therapy and beyond represent less than 5% of the BTC population. Therefore, there is still a high unmet need for more effective therapies for all patients with BTC, particularly in the first-line setting.The wave of chemo-immunotherapy and biomarker-driven treatments showing activity in clinical trials for advanced BTC is reminiscent of the therapeutic evolution in non-small cell lung cancer that began 10 years ago, which provided much needed new treatment options for patients with a very poor prognosis. However, in contrast to the rarity of BTC, the high frequency of lung cancer in Canada and throughout the world allowed for large, randomized phase III trials to be done which demonstrated a clear clinical benefit. Given these large trials are challenging to conduct in BTC, different considerations for value assessment beyond median OS are needed. Progression-free survival, response rate, duration of response, and quality of life are particularly important outcomes in BTC. Landmark OS analyses that can capture whether a portion of patients can achieve long-term survival from an experimental treatment are also valuable. To better understand how new therapies might provide value to patients, clinical trials and real-world studies should aim to capture outcomes such as biliary stenting, hospitalizations, and down-staging, and explore whether these correlate with patient quality of life. Together, this may help to shape the definition of a clinically meaningful benefit for patients with BTC and improve access to therapies that meet the needs of patients.


Guidelines on COVID-19 management are developed as we learn from this pandemic. However, most research has been done on hospitalised patients and the impact of the disease on non-hospitalised and their role in transmission are not yet well understood. The COVID HOME study conducts research among COVID-19 patients and their family members who were not hospitalised during acute disease, to guide patient care and inform public health guidelines for infection prevention and control in the community and household.An ongoing prospective longitudinal observational study of COVID-19 outpatients was established in March 2020 at the beginning of the COVID-19 pandemic in the Netherlands. Laboratory confirmed SARS-CoV-2 infected individuals of all ages that did not merit hospitalisation, and their household (HH) members, were enrolled after written informed consent. Enrolled participants were visited at home within 48 hours after initial diagnosis, and then weekly on days 7, 14 and 21 to obtain clinical data, a blood sample for biochemical parameters/cytokines and serological determination; and a nasopharyngeal/throat swab plus urine, stool and sperm or vaginal secretion (if consenting) to test for SARS-CoV-2 by RT-PCR  and for viral culturing. Weekly nasopharyngeal/throat swabs and stool samples, plus a blood sample on days 0 and 21 were also taken from HH members to determine whether and when they became infected. All participants were invited to continue follow-up at 3-, 6-, 12- and 18-months post-infection to assess long-term sequelae and immunological status. Knowledge on COVID-19, the disease caused by coronavirus SARS-CoV-2, is evolving at an enormous pace since the virus emerged in Wuhan, China at the end of 2019  and rapihttps://www.umcg.nl/-/covid-home) was set up in March 2020 by researchers from the Department of Medical Microbiology and Infection Prevention of the University Medical Center Groningen (UMCG), the Netherlands in collaboration with the Municipal Health Services (GGD in Dutch) of the north of the Netherlands. The project focusses on the impact of COVID-19 on non-hospitalised individuals, their acute and long-term clinical presentation, infectious period and transmission routes. We aimed to answer research questions that would inform public health guidelines on biosafety and transmission-control measures, as well as understand COVID-19 disease and its management in home-isolated patients.To answer these questions, the COVID HOME study (Guidelines for quarantine and isolation measures vary by country and between regions within countries. Understanding the relevance of long-term (infectious) viral shedding in infected individuals  is key tCharacterisation of the clinical phenotypes of patients with ‘mild’ disease is still lacking. The WHO clinical progression scale  classifiHere we report the design of a prospective observational cohort study of home-isolated SARS-CoV-2 infected individuals and their HH members. The main strength of this study is the early, systematic, prospective follow-up with an intense sampling/data collection of non-hospitalised COVID-19 patients during the acute disease evolution with a medium and long-term follow-up. The comprehensiveness, diverse data collection and analysis of the study described here will allow us and other researchers to add knowledge to the already ongoing fast research on COVID-19.The project received funding from the Netherlands Organisation for Health Research and Development (ZonMw) , grant 1The objectives of the study have been divided into two work packages (WP) and their respective sub-objectives.11.1Determining the viral load kinetics and duration of viral shedding of SARS-CoV-2 RNA in bodily specimens through real-time reverse transcription polymerase chain reaction 1.2Evaluating the viability of the virus in different specimens and at various time points of patient follow-up, by viral culturing1.3Determining the relation of viral load kinetics and the appearance and kinetics of anti-SARS-CoV-2 IgM, and IgG antibodies over time1.4Determining the genetic diversity of SARS-CoV-2 through whole-genome sequencing (WGS)1.5Determining the chain of transmission within the HH and into the working environment, especially for healthcare settings, through viral WGSIn WP1 we aim to measure the duration and routes of viral shedding, genetic diversity, and development of immunity of non-hospitalised individuals with COVID-19 to improve guidelines for biosafety and patient isolation by:22.1Describing the clinical spectrum of COVID-19 in non-hospitalised individuals and determine potential risk factors for mild versus more severe clinical evolution2.2Assessing the development of long-term sequelae and post-viral fatigue syndrome (PVFS) in non-hospitalised COVID-19 individuals2.3Correlating laboratory parameters and cytokine changes with clinical evolution2.4Determining the relation of viral loads & anti-SARS-CoV-2 antibody kinetics with clinical evolutionWithin WP2 we expect to aid in establish guidelines for the management of  COVID-19 patients at home, including early detection of predicting factors for severity and long-term consequences by:WP1 will inform public health guidelines on biosafety, duration of home isolation and de-escalation measures as well as improve our understanding of transmission chains in the community . In 1.1 we aim to understand whether viral detection in different body specimens in non-hospitalised individuals differs from what is being found in hospitalised patients, thus understanding the contribution of outpatients to the overall transmission of COVID-19. Here, we tested for SARS-CoV-2 RNA on systematically and simultaneously collected clinical specimens from different body sites  for at least 4 weeks, giving detailed data to compare to the clinical evolution and other parameters such as immune response (1.3) and viral genotypes (1.4). In previous studies SARS-CoV-2 RNA was found in all of these specimens with conflicting results for vaginal fluid [1.2, the RNA-positive specimens were cultured to ascertain if infectious virus could be found in the samples mentioned in 1.1, at different time points during the clinical evolution. The presence and duration of infectious virus in different specimens influences infection prevention measures. Recent studies showed shedding of infectious virus in respiratory samples from critical hospitalised patients to last up to 20 days (median=8 days) since symptom onset [The data gathered in al fluid , 15; wital fluid , 25, 26.om onset , while os median= days sinom onset .In 1.3 anti-SARS-CoV-2 antibody dynamics of our cohort of patients were measured and are being aligned with the viral shedding and virus culture results, to evaluate antibody kinetics, seroconversion rates and duration and the relation of serological response with viral clearance and virus viability. To assess long-term immunity, IgG data will be obtained up to 18 months post-infection in consenting patients  and cluster analysis using viral WGS. The transmission of SARS-CoV-2 from/to the community by HCW and their family members is being addressed here.lacement , 33. WGSlacement . TransmiWP2 we strive to describe the spectrum of acute clinical presentation and the interrelation with paraclinical parameters in COVID-19 infected individuals that do not merit or get admitted to hospital. As we have enrolled HH members of COVID-19 positive individuals  factors for mild versus more severe clinical evolution will include age, gender, BMI and specific blood parameters that have been related with severe disease progression such as C-reactive protein, hepatic transaminases, lactate dehydrogenase (LDH), leptin, interleukin (IL)-6, ferritin & other [In uals see , we are  & other .2.1 is used in 2.2 to identify clinical and laboratory parameters , and clinical phenotypes, associated with the development of long-term sequelae. Immunopathology appears to play a very important role in SARS-CoV-2 pathogenesis. Critical disease has been found to be associated with a ‘cytokine storm’, the overproduction of inflammatory cytokines, ultimately resulting in acute respiratory distress syndrome (ARDS) [2.3 the dynamics of these cytokines will be determined during the acute disease and the long-term clinical evolution. Finally, in 2.4 we test the relationship of viral load dynamics and anti-SARS-CoV-2 antibody kinetics (collected in WP1 sections 1.1-1.3) with the clinical evolution. Viral loads of asymptomatic individuals have been reported to be similar to symptomatic patients [Clinical evidence on the subacute and long-term persistence of symptoms after SARS-CoV-2 infection or Long COVID, was recognised during the first few months of the COVID-19 pandemic , 38. Thee (ARDS) . In hospe (ARDS) . Cytokine (ARDS) . In 2.3 patients . HoweverThe study is carried out amongst residents of the Northern Provinces of the Netherlands  comprising around 1.7 million inhabitants. Laboratory confirmed SARS-CoV-2 infected individuals of all ages (index cases) that do not merit hospitalisation, and their HH members, were invited to join the study and enrolled after signing a written informed consent. Consenting parents/guardians of infected children (≤16 years) signed the informed consent form. The complete HH, i.e. all persons (contacts) living in the same home, was to join the study at enrolment. If this requirement was not fulfilled, the HH was excluded from the study.An ongoing prospective longitudinal observational study of SARS-CoV-2 infected individuals that did not require hospitalisation and their HH members was initiated on March 2020, at the start of the pandemic in the Netherlands.Enrolled individuals comprised the identified laboratory COVID-19 index positive patients plus all their HH members. The average number of HH inhabitants in the Netherlands and the northern region is 2.14 , thus foThe index participant was the first laboratory-confirmed SARS-CoV-2 infected individual of any age identified in a HH and invited to join the study together with the complete HH. The requirements for inclusion were:Onset of symptoms <5 days before inclusionPositive diagnostic RT-PCR for SARS-CoV-2 performed within 48h of inclusionIndex person should supply all required samples in the protocol for the duration of the studyAll HH members should join the studySigning of informed consent formhttps://www.umcg.nl/-/covid-home) for further information. Patients were also given time to ask questions and/or to think before deciding to join.Potential index cases were identified at the virology facility of the Department of Medical Microbiology and Infection Prevention  and at the laboratories reporting to the GGD of the Northern Provinces. Suspected SARS-CoV-2 infected individuals were diagnosed by real-time RT-PCR performed on material from nasopharyngeal/throat (NPT) swabs. Laboratory SARS-CoV-2 positive individuals were contacted by phone by an infection prevention expert from either the UMCG MMBI department or the GGD who explained the study objectives and procedures and invited the positive individual and the HH members into the study if the inclusion criteria were met. Eligible individuals were also directed to the project’s website  was planned within the next 24 hours to enrol participants into the study after signing a written informed consent corresponding to the participant’s age . Participants were asked to supply their individual e-mail address in the informed consent and to authorise it’s use to receive electronic questionnaires and information from the COVID HOME researchers via the project’s email. Participants were also encouraged to use the project’s email to ask questions or to inform of any event. A schedule was drawn so that study participants were visited weekly for at least 21 days by project staff to collect data and samples according to the different protocols. Those not tested or who were negative for SARS-CoV-2 RT-PCR, were enrolled into the protocol for HH members (section d).A systematic weekly follow-up at the patient’s home was set up to acquire data and samples to determine kinetics, length, viability and routes of viral shedding (objectives 1.1 & 1.2), serological dynamics (objective 1.3), cytokine kinetics (objective 2.3) and evolution of haematological & biochemical blood parameters and their relationship with the patient’s clinical progression .Enrolled patients were visited at home soon after initial diagnosis (Day 0), and then weekly on days 7, 14 and 21 after confirmed infection  to obtaiResearch staff visiting infected individuals at home followed safety measure protocols designed according to UMCG and national guidelines. Standard operating procedures (SOP) including flow-charts were designed for: i) safety procedures during home visits and at work, ii) handing/sending and retrieving CRFs from participants, iii) preparation of sampling materials and transport requirements, iv) sampling strategy , and v) The following CRF/eCRFs were/are applied to infected individuals :A self-completing clinical questionnaire (CRF-02) was given to patients at enrolment to record socio-demographic data, comorbidities, treatment and daily clinical history (symptoms and signs) during at least 21 days, and further if symptoms were still present. Participants still negative by RT-PCR but presenting symptoms were encouraged to fill this form as well.An individual HH questionnaire (CRF-03) was applied at enrolment to record socioeconomic characteristics, preventive measures, behaviour and intradomiciliary isolation measures taken.A phone interview (CRF-04) was carried out at 21 days, and at 3 and 6 months to assess smell and taste alterations in detail in consenting individuals.The fatigue screening questionnaire (eCRF-05) to establish presence or absence of fatigue/tiredness, length of time of fatigue/tiredness, and any loss of activityAdditional fatigue questionnaire (eCRF-06) to identify presence and severity of symptoms associated with development of PVFSHealth-related Quality of Life (RAND-36) questionnaire (eCRF-07) and tothe Hospital Depression and Anxiety Scale (HADS) questionnaire (eCRF-08)To evaluate the development of PVFS as part of the assessment of Long COVID, participants older than 16 years were asked to answer on Day 14 and then at 3, 6, 12 and 18 months post-infection to:At 3, 6, 12 and 18 months post-infection, both adults and children receive eCRF-10 to assess Long COVID related symptoms, new comorbidities and re-infection.™ (Research Electronic Data Capture) secure web-based software platform [CRF-02 and CRF-03 were given to participants to fill in paper while the rest were eCRFs and were answered online using the REDCapplatform . AutomatHH members who had not been tested or were negative for SARS-CoV-2 RT-PCR at enrolment were followed with weekly testing of NPT swabs and stool samples for 3 weeks to determine if and when each individual became infected. At the beginning of the study, it became apparent that some individuals, especially children, stayed RT-PCR negative for NPT swabs, but became positive for SARS-CoV-2 infection when stool samples were tested by RT-PCR. Therefore, both NPT swabs and stool samples were collected weekly from (still) negative individuals. This information is being used to understand transmission chains, timing of infection between close contacts (family/HH members) and family cluster dynamics (objective 1.5). Moreover, to ascertain if RT-PCR negative individuals had been previously infected or seroconvert during follow-up, blood samples were drawn at enrolment and on Day 21 for IgM and IgG serology testing.All HH members were asked to fill CRF-03 at enrolment and CRF-02 if presenting symptoms regardless of RT-PCR results. Once an individual was confirmed by real-time RT-PCR of any of the samples (NPT swabs and/or stool), the protocol for COVID-19 infected individuals (section c.) was followed.At 3, 6, 12 and 18 months post-infection, SARS-CoV-2 positive participants are invited to answer PVFS and eCRF-10 questionnaires sent digitally to determine post-COVID long-term sequelae (section d.) and provide blood samples for serologic determination.Likewise, individuals that remained RT-PCR negative for SARS-CoV-2 infection have a blood sample taken at 3, 6, 12 and 18 months after enrolment for serological testing and a slightly modified version of eCRF-10 (eCRF-10a) is applied to attempt to differentiate long-term symptoms associated to COVID-19 from other causes, such as lockdown measures.Smell and taste alterations are common in the acute phase during COVID-19 infection . AlteratConsenting study participants were called three weeks after COVID-19 diagnosis to fill in a questionnaire (CRF-04) regarding smell and taste alterations by phone. The questionnaire was based on a previously used questionnaire which was designed to study taste and smell disturbances in patients with gastrointestinal stromal tumors treated with tyrosine-kinase inhibitors . The queProtocols and SOPs for sample processing were designed and updated according to the evolving knowledge on SARS-CoV-2 diagnosis and clinical evolution.et al. [Viral load kinetics and duration of viral shedding of SARS-CoV-2 RNA in bodily specimens of all origins  were determined using an in-house protocol (in-house qPCR) . Nucleicet al.  with minThe presence of infectious virus in different SARS-CoV-2 positive samples  was ascertained through virus isolation in the BSLII facility of the UMCG. Samples cleared were spun down and filtered through 0.2 μm filter and characterised by a confirmatory RT-PCR positive test for COVID-19 and negative for a panel of other respiratory viruses. The presence of infectious SARS-CoV-2 virus was monitored observing for cytopathic effect (CPE) according to protocol for 7 days, and at day 2 and 7 using an in-house flow cytometry-based assay detecting viral antigen. Samples were considered positive for the presence of infectious SARS-CoV-2 virus if the flow cytometry-based assay showed more than two standard deviations from the mock-infected cells. Samples scoring positive in CPE but negative in flow cytometry-based assay were considered negative.Serological measurements (objective 1.3) are performed on the Abbott Architect platform, using a chemiluminescent magnetic microparticle immunoassay performed at the virology facility of the UMCG. Samples obtained during the acute disease were tested with the Semi-quantitative Architect IgM (AdviseDx SARS-CoV-2 IgMSP assay), and Semi-quantitative Architect IgG (Abbott Alinity i SARS-CoV-2 anti-nucleocapsid protein IgG assay), while long-term samples at 1, 3, 6, 12 & 18 months are tested with the Quantitative Architect IgG (AdviseDx SARS-CoV-2 IgG II) . These rTo fulfil objective 1.4, nucleic acid extraction, purification and concentration from positive samples was performed after which, cDNA was prepared using a sequence-independent single-primer amplification (SISPA) approach, based on the round A/B methodology. Amplified cDNA was purified using a 1:0.8 ratio of AMPure XP beads , and quantified using a Qubit high-sensitivity double-stranded DNA (dsDNA) kit (Thermo Fisher), both according to the manufacturers’ instructions. Multiplex sequencing libraries was prepared using 250 ng of cDNA from up to 5 samples as input to the SQK-LSK109 and barcoded individually using the EXP-NBD104 Native barcodes (Oxford Nanopore Technologies). Libraries were sequenced on FLO-MIN106 flow cells on the MinION Mk1b device (Oxford Nanopore Technologies), with sequencing proceeding for up to 24 h.Consensus sequences will be aligned with relevant sequences from SARS-CoV-2 lineages using MAFFT v7.313 . MaximumAs part of objectives 2.1-2.4, blood samples were processed at the routine UMCG laboratory to determine full blood count, haematocrit, C-reactive protein, creatinine, glycaemia, albumin, hepatic transaminases , bilirubin, creatine kinase (CK), CK-MB isoenzyme, LDH, Prothrombin time (PT-INR), troponin, ferritin, procalcitonin, D-dimer, leptin and IL-6.A panel of cytokines and chemokines  is being measured using Luminex-based multiplex technology. Serum samples and associated clinical and Luminex/ELISA data will be combined and shared between different UMCG-COVID-19 research teams working on complementary studies addressing different COVID-19-related research questions. These markers will be used to assess prediction of disease severity and of the development of long-term symptoms.Due to the exploratory and mostly descriptive nature of the analysis, we did not conduct an exact sample size calculation. However, we focused on the prevalence of long-term sequelae as initial observations indicated that close to half of the infected individuals continue to suffer from symptoms after 4 weeks from infection , 54. TheClinical presentation during the acute disease will be firstly presented in a descriptive analysis where the systematic prospective data collection will be used to better characterise mild clinical cases. We will attempt to define clinical phenotypes as endpoints and factors related to each phenotype. Principal component analysis-based cluster analysis will be used to group relevant variables of interest such as age, gender, comorbidities, signs, symptoms, haematological and biochemical parameters into phenotypes. Multinomial logistic regression models will be fitted to estimate associations of phenotypes with exposure variables such as viral loads, genetic sequence of SARS-CoV-2, anti-SARS-CoV-2 antibody kinetics, laboratory parameters and cytokine/chemokine levels. Clinical phenotypes will be categorised from milder to more severe disease presentation and potential predicting factors (measured at presentation) will be identified using generalized linear mixed models.Generalised mixed models will be also used to assess the associations between the duration and severity of signs/symptoms of COVID-19 disease and viral loads, anti-SARS-CoV-2 antibody kinetics and change in laboratory parameters and cytokines, after adjusting for age and gender. The likelihood ratio test will assess statistical significance of the estimates at the 5% level. Univariate and multivariate analyses of potential risk factors for disease severity (mild versus more severe) will be performed using logistic regression comparing crude and adjusted odds ratios. Variables with a P-value ≤ 0.2 after adjusting by age and gender will be fitted into multivariate logistic regression models and adjusted for further confounders. Effect modification will be analysed and resulting models compared by likelihood ratio test.A generalised linear regression model (Poisson distribution and log-link function) will be fitted to analyse the relationship between the duration of viral/RNA shedding and the exposure variables . Significance will be determined at the 5% level.Phylogenetic analysis will be performed to determine viral diversity and potential chains of transmission within HHs and into the community as explained under Rationale above.The presence of long-term sequelae at 3,6,12 & 18 months will be defined as reporting any symptoms and/or not feeling fully recovered at each of these time-points, as well as assessing the health-related quality of life using the SF-36 score at the different time points. These three definitions will be used as endpoints. A descriptive analysis will be performed to compare symptom presentation between time points and symptoms characteristics, such as number of symptoms, type of symptoms, symptom presentation , and severity. Univariate and multivariate analyses will be carried out to assess risk factors for Long COVID from relevant variables of the acute disease such as comorbidities, number and duration of symptoms, type of symptoms and socio-demographic variables of interest. Analysis strategy will be as described above.This study has been approved by the Medical Ethical Review Committee of the UMCG (METc 2020/158) and follows international standards for the ethical conduct of research involving human subjects. All procedures employed in the clinical and related laboratory studies comply with national and European legislation in respect of research involving human subjects.Informed consents including participant information sheets (available in Dutch and English) were obtained before the collection of participants’ data and samples. At enrolment, participants invited to join the study received a written and oral explanation of what the research entails. Adult informed consents were applied to individuals older than 16 years, while a parent/guardian informed consent was signed by the child’s parent/guardian and together with their child if aged between 12-16 years. Individuals may withdraw from the study or may not consent to parts of the study, without any consequences.A detailed data management plan (DMP) has been prepared by the project data steward and approved by the principal investigator describing how data will be handled both during research and after the project is completed. The UMCG has a comprehensive ISO27001 certificate and is compliant covering all of its strategic activities: Healthcare, Research and Education. The UMCG Research Code was drawn based on this certification. The COVID HOME DMP complies with the UMCG quality policy, UMCG and University of Groningen (RUG) Research Code and national guidelines on ethical research.All information obtained during the course of this study, including hospital records, personal data and research data will be kept strictly confidential according to the General Data Protection Regulation (GDPR) of the Netherlands. Data protection and privacy is coordinated by the data protection officer which supervises that research conducted within the UMCG is performed in accordance with the GDPR. Study data were/are collected and managed using REDCap electronic data capture tools hosted at UMCG. Electronic web-based eCRFs have been developed using REDCap (Research Electronic Data Capture) which is a secure, web-based software platform designed to support data capture for research studies . ParticiAll data  are stored in password protected databases and archived in institutional research drives secured under encryption by our UMCG Information and Communication Technology department that can only be accessed by authorized project personnel. Data storage is backed-up automatically every day on the servers of the UMCG ensuring data safety. Samples are stored and managed locally according to UMCG SOPs. Patient information sheets and informed consents are kept in a locked cabinet. The data will be stored at the UMCG for 15 years. Data that may be reported in scientific journals will not include any information that identifies the participants in this study. We follow a FAIR  data policy. After an embargo period of approximately 12 months (subject to change) from the project culmination and publication of main results, the processed, pseudonymised data will be made available for re-use. Requests for re-use of data will be evaluated by the principal investigator, project manager and data steward who will check whether the research question falls within the scope of the informed consent. If data will be shared it will not include directly identifiable information and can only be (re-)used within the scope of the informed consent. Third party use of data is governed in part by intellectual property rules and agreements, which will become available after publication of the main results.This study aims to close a knowledge gap on the impact and consequences of COVID-19 on non-hospitalised patients, in contrast to most studies that naturally, have concentrated on hospitalised and critically ill patients. Enrolment of participants was initiated soon after the first cases were detected in the Netherlands , howeverOur COVID HOME study addresses key questions of relevance to guide patient care and inform public health guidelines for infection prevention and control in the community and HH situation in a single study. The enrolment of patients right after diagnosis will allow the early identification of parameters potentially indicative for progression to severe disease. This early identification cannot be achieved in hospitalised individuals who are usually admitted a week or more after disease onset. The parameters that will be collected in this study, such as viral load, antibody response, cytokine changes and clinical and laboratory parameters of patient evolution will consent the identification of independent factors associated with disease evolution, insight in other transmission routes, and immune response dynamics in non-hospitalised patients. Moreover, as HH members of COVID-19 positive individuals were also enrolled, we have been able to determine if other family members become infected , how long does it take to get infected and which are the transmission dynamics within the HH. It will be important to determine transmission chains between the HH and workplace of adults, especially for healthcare settings to inform biosafety guidelines. A better understanding of the clinical evolution of non-hospitalised patients and the factors influencing disease progress  can be rapidly turned into “home standard of care” guidelines for patients in home isolation and their HH members, as well as shed light on potential new therapies and/or strategies preventing progression to severe COVID-19 disease. Determining the length of infectiousness of COVID-19 patients will either confirm current guidelines or prompt their change in relation to quarantine and isolation measures, contributing to limit the spread of COVID-19 in the Netherlands and elsewhere. Hence the importance of the collaboration with public health institutions  and policy makers.In the Netherlands, the first indications of long-term consequences of SARS-CoV-2 infections (what is now known as Long COVID or Post-COVID syndrome) appeared though a survey carried out by the Dutch Lung Foundation in June 2020 . 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The International Maternal Pediatric Adolescent AIDS Clinical Trials Network (IMPAACT) P1104s study evaluated neuropsychological outcomes over 96 weeks in children living with HIV (CLHIV) aged 5–11 years at 6 Sub-Saharan African sites to explore associations between HIV-illness related biomarkers and neuropsychological outcomes.Children living with HIV had participated in IMPAACT P1060, which compared efficacy of nevirapine versus lopinavir/ritonavir in children initiating ART at <3 years of age. At age 5–11, neuropsychological evaluations of KABC cognitive ability, TOVA attention-impulsivity and BOT-2 motor domains were assessed and repeated after 48 and 96 weeks. Clinical, antiretroviral therapy (ART) and laboratory  parameters were used to predict neuropsychological outcomes using linear mixed-effects multivariable regression models, controlling for child and caregiver characteristics.246 CLHIV  began ART at a median age 14.9 months . Nadir CD4 percentage was 14.7% ; the median peak viral load (VL) was 750 000 copies/ml  and 63% had ≥WHO stage 3 clinical disease; 164 (67%) were on lopinavir/ritonavir, 71 (29%) were on nevirapine and 7 (3%) were on efavirenz. Other antiretrovirals were similar. Nevirapine at P1104s study start or later was associated with poorer neuropsychological scores across all domains except Global Executive Composite, even when controlling for nadir CD4 percent and time-varying HIV VL. Other predictors of poorer scores in KABC domains included low birth weight, WHO stage 4 disease and serious illness history and elevated VL was associated with worse BOT-2 scores.Children receiving nevirapine had poorer neuropsychological scores than those on lopinavir/ritonavir. Antiretroviral choice might adversely impact neuropsychological performance. In addition, low birth weight and markers of severe HIV disease: advanced WHO clinical HIV disease, history of serious illness and an elevated VL, were associated with lower neuropsychological scores. Across 23 endemic countries, 1.5 million children still live with HIV . Almost Early ART initiation has a substantial positive impact on neurocognitive function , 7. NumeThis analysis included children from two related clinical studies : The P10In April 2009, P1060 cohort 1 closed due to superiority of LPV/r over NVP. Cohort 2 was unblinded and closed in October 2010, again following superiority of LPV/r over NVP. The findings from P1060 have previously been reported and detailed findings are not reported here , 20. TheP1104s was conducted between October 2013 and December 2016 in South Africa, Zimbabwe, Malawi and Uganda. This study compared neuropsychological outcomes in children aged 5–≤11 years from the P1060 study compared to age-matched HIV-exposed uninfected children (HEU) and children not exposed to HIV, recruited from similar neighborhoods as the CLHIV enrolled in P1060. These results have previously been reported , 10. NinNeuropsychological evaluations were conducted at 0, 48, and 96 weeks. These evaluations included the Kaufman Assessment Battery for Children, 2nd edition  assessing cognitive ability, The Test of Variables of Attention  for measuring attention-impulsivity, the Bruininks–Oseretsky Test, 2nd edition (BOT-2) testing motor proficiency. The Behavior Rating Inventory for Executive Function (BRIEF) was used to evaluate behavioral and cognitive problems through interviewing parents or caregivers, reported as the Global Executive Composite (GEC) score.The KABC-II is a comprehensive performance-based assessment of cognitive ability for children 3 to 18 years of age and was administered and scored using the Luria model of neuropsychological ability . Van Wyha priori in relationship to various biomarkers and clinical predictors of pediatric HIV infection progression and severity, the measure of choice is the MPI , Simultaneous Processing , Learning , and Planning/Reasoning (for children 7 years of age and older). In the scoring and interpretation of KABC-II performance, these four domains  are combined into a composite score of overall cognitive ability, called the Mental Processing Index (MPI). That was our primary outcome in the present study, in that it is intended to be the one measure that represents overall cognitive ability performance on the KABC-II assessment battery from a neuropsychological perspective. A second composite performance measure is also available with the KABC-II call the Non-verbal Index (NVI), which is comprised of various subtests across the four domains that do not depend on spoken language or an understanding of English to complete. In addition to the MPI score, we have also sometimes used NVI an an important outcome in our P1104S and other study assessments , 26, whe the MPI .In terms of a comprehensive assessment of neuropsychological outcomes for pediatric HIV, two important domains that are not directly assessed by the KABC-II are deficits in attention and enhanced impulsivity (attention deficit and hyperactivity disorder of ADHD), and a separate measure of overall motor proficiency. Because of the importance of these domains in assessment clinical predictors for HIV disease progress and severity in children , 32, we In the present analyses we included the Tests of Variables of Attention  to asseThe short version of the BOT-2 provides only one standardized measure of overall motor proficiency which includes 14 items; with one or two items pertaining to each of the following motor domains: fine motor precision, fine motor integration, manual dexterity, bilateral coordination, balance, running speed and agility, upper-limb coordination, and strength. The TOVA and BOT-2 findings for our P1104s study groups  were included along with the KABC-II in the validation analyses by Chernoff et al.  and the For all of these African-based studies in CLHIV cited above, the one non-performance-based neuropsychological measure also included in our pediatric HIV research in subSaharan Africa was the Behavior Rating Inventory of Executive Function (BRIEF) for school-age children (age 6 years or older) completed by the mother or principal caregiver of the child on study . This 86Clinical , antiretroviral (ARV)  and laboratory data  were collected prospectively from P1060 and this data was included for the current analysis. Clinical and laboratory events were graded according to DAIDS Table for Grading the Severity of Adult and Pediatric AEs, V1.0, 12/2004 . We comb10 viral load or the lowest CD4% ever reported at or prior to study entry . For these analyses, we focused on three outcomes, KABC MPI, BOT-2 and TOVA D-Prime. An additional analysis that incorporated a study week by regimen interaction effect explored whether the association between ARV regimen at P1104S study entry and each neuropsychological outcome changed across the study period.Linear mixed models with restricted maximum likelihood estimation (REML) and robust fixed effect error estimates were used to explore whether neuropsychological test scores were associated across time with growth, clinical history, HIV disease severity and treatment markers (VL and CD4+ percentage and count) as well as ARV regimen, and to estimate these associations. To account for participant-level variability, an intercept-only random effect term was included. Each regression analysis included a separate potential predictor. In our analysis, GEC scores were transformed so that a higher score indicated better function. To explore whether nadir CD4% or time-varying viral load may explain some of the effects of the target variables , models were re-fit to include either a covariate corresponding to current logChild and caregiver characteristics, individual research sites and study week were controlled for. Slope estimates and adjusted means with 95% confidence intervals were presented. Tests of statistical significance were two-sided, and 5% error rates were used. In results tables, significant results are highlighted with shading based on the Wald test results. All models were fit using the “PROC MIXED” procedure with the “empirical” option in SAS/STAT software v9.4.Baseline characteristics of all children enrolled into P1060 and then into P1104s were similar . Of 246 At P1104s entry, children had been on ART for a median of 4.6 years and had higher weight-for-age and height-for-age z-scores than at entry to P1060. ART regimens at P1104s entry were as follows; 164 (67%) were receiving LPV/r, 71 (29%) were on NVP and 7 (3%) children were on EFV (NNRTI)-based ART. More children were receiving LPV/r-based regimens at P1104s baseline than on P1060 entry.During the P1104s study, clinical, immunological, and virological characteristics remained stable across the week 48 and 96 time points. There were few regimen changes in the year preceding the study and during the P1104s study .Clinical risk factors  for lowe10 VL were associated with higher KABC-MPI scores, [1.01 ] but few children had high VL during the study  and D-Prime scores but similarly so for both study groups, with no statistically significant time by group interactions . ParticiThis study provides data from a well-characterized, randomized pediatric CLHIV cohort initiating ART between 6 months and 3 years and includes clinical, HIV disease-related and ART-associated risk factors for neuropsychological test scores. Unexpectedly, NNRTI-based ART was a consistent risk factor for lower neuropsychological scores than PI-based ART across all domains except executive functioning when tested between 5 and 11 years of age. This finding was apparent at baseline, P1104s start and remained consistent at the 48-week and 96-week time-points in children who had initiated either LPV/r or NVP in P1060 and those who switched from NNRTI-based to PI-based ART prior to enrollment in P1104s. In children who switched from NVP, few switches occurred the year prior to P1104s start and almost no children switched during P1104s, therefore most were stable on a LPV/r regimen at P1104s start.As only a small number of children received efavirenz in this study, comparisons for EFV are not possible. However these findings support a class effect for NNRTI-based ART. Nalwanga et al., from Uganda, compared neurocognitive scores in children receiving PI- versus non-PI (nevirapine or Efavirenz-based) ART. They found higher BOT-2 scores in those receiving PI-based ART, indicating improved motor function. Although there were no differences in the KABC or TOVA scores, this study was limited by a small sample size, less than a third of P1104s .Children randomized to the NVP arm in early childhood, and then maintained on that arm until P1104s study entry at school age, do most poorly across all the global performance-based neuropsychology measures  compared to children who remain on a PI-based regimen. Although there were very few, the next most poorly performing group at P1104s study entry are those children switched to NVP from their initial PI treatment regimen in P1060.Most of the PI vs. NVP-based between-group differences in KABC neuropsychological scores averaged around 5 standard points across the three P1104s assessment points, with no overlap in the corresponding 95% CIs. Based on our prior published validation work with the KABC assessment in P1104s , this inThese same conclusions are evident for the KABC performance domains of Simultaneous Processing and Planning especially across the three assessment points as the children get older in P1104s. Our past work with CLHIV in sub-Saharan Africa have related these differences to VL , and mea3   . HoweverAside from possible neurotoxic or CSF VL suppression when comparing ART treatment regiments in early childhood, poorer clinical outcomes in early childhood associated with the NVP treatment arm in P1060 , very liNevirapine has been replaced by LPV/r and dolutegravir in most countries, as recommended by international and country-specific guidelines. NVP is still recommended in premature infants and neonates less than 2 weeks post-gestational age, with raltegravir as an alternative option. Infants are switched to LPV/r weeks to months later depending on in-country guidance. In some countries children may still receive NVP as part of a fixed dose combination therapy where either LPV/r or dolutegravir are unavailable. Additionally, NVP is widely used for postnatal prophylaxis in HIV-exposed infants for at least 6 weeks –50. AlthThe GEC was not different between groups, despite differences shown in other tests which measure some elements of Executive Functioning . It is likely that the BRIEF was a not sensitive enough measure as it is reliant on parent/caregiver report, compared to the other tests which were from direct observation and measurement of abilities.10 copies. The CHER study, where children were randomized to initiate on ART with first early diagnosis between 6 and 12 weeks or later based on clinical and immunological guidance at the time, early ART prevented neurocognitive delay, although locomotor scores were lower in CLHIV than controls in early years. All children were on LPV/r  , 53 and  years , on LPV/r .Undoubtably, the key issue in neuropsychological function preservation in children is early HIV diagnosis and treatment, ideally through birth diagnosis or within the first few weeks of life. Not only does this limit HIV-encephalopathy and neurocognitive impairment, but also all the comorbidities associated with untreated HIV infection , 7, 54. Prematurity and LBW were also associated with lower neurocognitive scores. Prematurity, in the absence of HIV, is associated with lower cognitive scores in children entering school, directly proportionate to the gestational age and maturity at birth . In turnSurprisingly, thrombocytopenia, pulmonary disease, malnutrition, low height-for-age z-score were statistically protective for some of the neuropsychological scores. These factors usually result in worse outcomes even once initiated on ART  and are Our study is not without statistical limitations. In this analysis, we screened many potentially predictive variables and did not adjust for multiple testing. In addition, the P1104S study was not powered for this secondary study objective. As a result, we could have observed spurious significant results. However, we considered this an exploratory analysis and looked for consistency in the findings. Except for our analysis of entry ARV regimens, we did not explore how associations between screening variables and neuropsychological outcomes may have changed over time since our aim was to understand broadly the overall associations between screening variables and neuropsychological outcomes.HIV has shifted from being a severe, frequently fatal disease to a manageable chronic disease. More CLHIV are entering older childhood, adolescence, and adulthood. As neurocognitive scores in CLHIV tend to be lower than HEU and HU children, early recognition and supportive therapy remain important but are often limited in countries with the highest burden of HIV disease , 29. ImpUse of NVP at P1104s study start or during follow-up was associated with poorer neuropsychological scores than LPV/r, even when controlling for nadir CD4+ percent and time-varying HIV VL. Other predictors of poorer scores in KABC domains included LBW, WHO stage 4 disease and serious illness history but not elevated VL on P1060 or P1104s. Our findings have important implications on neurocognitive function among CLHIV as newer ARVs become available. In addition, low birth weight and markers of severe HIV disease: advanced WHO clinical HIV disease, history of serious illness and an elevated VL, were associated with lower neuropsychological scores (4841).The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by relevant Human Research Ethics Committees (HRECs) in each country where the study was done. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.MC conducted the statistical analysis for this manuscript. LF, MFC, and MB developed the first draft and led the development of subsequent drafts of this manuscript. All authors assisted with conceptualizing this manuscript approved the manuscript and contributed significantly to the work.


This study aimed to analyze the effect of aquatic plyometric training (APT) on jump performance in volleyball players. Twelve female athletes (16.6 ± 0.9 years) were assessed through the following jump tests: spike height (SH), squat jump (SJ), countermovement jump (CMJ) and CMJ with an arm swing (CMJA). Jump height in each test and the eccentric utilization ratio (EUR) were the outcome measures. APT consisted of sets of drop jumps for 6 weeks (2 sessions/week) at a water depth of 0.75 m. Tests were performed at the beginning of a five-week pre-season period, before and after APT, and four weeks later for the follow-up. Repeated measures ANOVAs were used to analyze data and Hedges’ g to estimate effect size (ES). Performance of all jumps did not change from baseline to Pre-APT. Performance improved in SH , the SJ  and the CMJA  after APT when compared to Pre-APT. No changes were observed after the follow-up period. In conclusion, including six weeks of APT in the training routine of youth volleyball players improved performance of a sport-specific task (SH), the SJ and CMJA, with gains preserved after a four-week follow-up. In volleyball, vertical jumps occur in several game situations, such as the serve, set, attack and block. High level jump performance during these movements may provide decisive advantages when attempting to score. Due to the frequent repetition of those game situations, athletes perform many jumps during a match . Thus, iPlyometrics is a method used to develop jumping ability in volleyball . PreviouAquatic plyometric training (APT) has demonstrated a positive effect on athletic performance  in non-athletes  and athlimprovement observed in some variables is equal  or even The physical properties of water make movements slower and attenuate the reaction forces during jumps . Thus, iTo the best of our knowledge, there is no evidence regarding the effect of APT on performance of volleyball-specific tasks, such as the spike. Improving spike height is particularly important, since it may differentiate between the players´ technical level . AttackiSample size was calculated based on data from a pilot study analyzing the effect of APT on spike height, using the G*Power software version 3.1.9.2 . Considering a mean difference of 8 cm between pre- and post-APT, a standard deviation of 10 cm and an alpha of 0.05, twelve participants were required to reach a power of 80%.Twelve female volleyball players ) participated in the study. All the players took part in national competitions and belonged to the team that won the Brazilian Under-18 Scholar Championship in 2019, with a technical-tactical training routine of 5 weekly sessions of 2.5-hour each. This study was approved by the local ethics committee and all individuals  provided written informed consent before participating.Participants were instructed to avoid strenuous exercise in the 48 hours preceding a test session and to arrive in a rested and fully hydrated state. All tests were performed at the same time of day to minimize the effects of diurnal biological variation on the results. Participants were verbally and visually instructed on test execution 1 day before the baseline assessment.Assessments were conducted in the team’s training gymnasium. Participants performed 5 min of light running to warm up and were instructed to execute joint mobilization exercises. After a familiarization period consisting of submaximal jumps, jump tests were carried out in the following order: (1) spike height (SH); (2) countermovement jump (CMJ); (3) squat jump (SJ); and (4) countermovement jump with an arm swing (CMJA). Three valid attempts were collected for all the tests and a minimum of 1 min rest was granted between each repetition.height(cm) = Sports, São Paulo, Brazil). The eccentric utilization ratio (EUR) was calculated as the ratio between CMJ and SJ height. A portable device designed and constructed for this study, similar to the Vertec Jump Tester, was used for spike height (SH). The device consists of 80 metal swivel vanes (arranged in 0.5-cm increments) attached to a metal pole. It was positioned near the volleyball net (0.4 m) in the left front position (position 4). Testing facility conditions  wereCMJ, SJ and CMJA height was calculated using the flight time  for within-trial SH reliability was ≥ 0.96 for all the assessments.consistent throughout the tests. Participants performed a full spike movement  , aimed aThe best execution of each participant in each test and each assessment session was considered for further statistical analysis.Participants were submitted to an initial assessment (Baseline - week 1). After a 5-week period a new assessment (Pre-APT - week 6) was performed. During this period, termed a Pre-season, athletes continued with their regular training routines. Next, a 6-week APT protocol was included in the team’s training routine, and another assessment was carried out after the intervention (Post-APT - week 13). A final assessment was performed four weeks after the end of APT (Follow-up - week 17) .A total of 12 training sessions were conducted over a 6-week period, with a minimum of 24 hours between sessions. Training was carried out in a swimming pool with water temperature of 27 ± 1ºC and 0.75 m of immersion . A previous study showed that vertical ground reaction force during the drop jump at this immersion level was significantly lower than on land .Each plyometric training session lasted an average of 30 minutes. The training protocol followed the proposal of During the Pre-season, the team training routine and competition consisted of 23 technical-tactical sessions, and no competitions occurred. During the intervention (APT), 26 technical-tactical training sessions were held, and the team participated in three competitions (36 sets). Between the Post-APT and Follow-up assessments, the team underwent 18 technical-tactical sessions and took part in three competitions (25 sets).Regular training routine consisted of 5 technical-tactical sessions per week. Training was organized based on the game complexes, with two days for complex 1 , two days for complex 2  and one day for a combination of both complexes and their transitions. In addition, resistance training was held three times a week (three sets of 12 to 15 repetitions with 65-70% of 1RM), without power exercises during the study period (from baseline to the follow-up).Means and standard deviations were calculated for each variable and data normality was tested using the Shapiro-Wilk’s test. Four one-factor repeated measures analyses of variance (ANOVA) were used to analyze the effect of time on SH, SJ, CMJ, EUR and CMJA. The Bonferroni’s post-hoc test was used for multiple comparisons. Given the objectives and hypotheses of this study, comparisons were made only for the chronological sequence of the assessments, that is, the pre-season (baseline vs. pre-APT), intervention (pre-APT vs. post-APT), and follow-up (post-APT vs. follow-up). Hedges’ g was used for the estimation of the effect size (ES) and values were interpreted as follows : <0.19 =p > 0.05). Performance improved in SH , SJ  and CMJA  after APT when compared to Pre-APT. No changes compared to Post-APT were observed during the follow-up period (p > 0.05).The results of ANOVA and post-hoc comparisons are presented in The aim of this study was to analyze the effect of a 6-week APT program on jump performance of youth volleyball players. Our main findings demonstrate that adding APT to the training routine of athletes improved SH, SJ and CMJA performance. Four weeks after the end of APT, the performance gains were maintained.After the pre-season which included the first five weeks of the annual training program, jump performance did not change. During this time, training sessions were aimed at improving technical skills and overall physical conditioning . Although this approach has proven to be important in improving game performance with regard to serving, reception, setting and attacking , it seemAfter APT, SH improved substantially (average difference of 12 cm). Spike height is very important for athletic performance and professional practice . A higheThe enhanced performance in non-specific vertical jumps (i.e. SJ and CMJA) observed after APT corroborates previous findings reporting the positive effect of both land-  and wateAn increase in jump performance is related to enhanced muscle power . In the In drop jumps, such as those used in the intervention, longer contact times are associated to greater force and higher jump height . In wateThe intervention period did not change the EUR, corroborating the results of a previous study which analyzed the effect of land-based plyometrics in a group of college-aged males . The durThe gains in SH, SJ and CMJA observed after APT were maintained four weeks after the end of the intervention. To the best of our knowledge, no other study investigating the effects of APT on athletic performance included a follow-up period. Our results demonstrate the potential of this type of intervention for volleyball players, given that performance was maintained for around one month after the last APT session, within the competitive period.In athletes, most studies involving plyometrics address only the immediate effects after intervention, and information on the maintenance (or not) of changes after a certain period is scarce . This inDespite the novelty of the data presented here, some limitations should be noted. All the APT sessions were held after technical-tactical training, despite its being recommended before . This waIn conclusion, a 6-week aquatic plyometric training protocol with 0.75 m immersion improved squat jump, countermovement jump with an arm swing and spike height performance in female volleyball players. Four weeks after the intervention, these gains were maintained. Our results suggest that APT can be considered an option for physical training of volleyball players in a setting where the mechanical load on landing is reduced. This would enable coaches to increase the load of other types of stimuli, such as those included in technical-tactical training. The proposed protocol, consisting of 30-min sessions twice a week for a 6-week period, which is feasible to be implemented at different times during the season, promoted important improvements in the athletes’ jump performance.