PurposeTo investigate the potential to perform linear accelerator output quality assurance (QA) with the ScandiDos Delta4 Discover (Discover) onboard transmission detector.Materials and methodsUsing the ScandiDos Delta4 software (version 8), a conversion factor from raw signal to output was obtained via cross-calibration with an accredited dosimetry calibration laboratory (ADCL) calibrated ionization chamber for each photon energy, including flattening-filter-free (FFF) energies. With the calibration factor for 6 MV (6x) photon energy, output measurements were taken with both the Delta4 Discover and ion chamber and compared for output as a function of gantry angle and dose-rate dependence. Monitor unit (MU) linearity for 6x was measured and compared with ion chamber measurements. Additionally, the Discover was used to take output measurements, for 6x, approximately every hour throughout the course of a treatment day, and compared with ion chamber output measurements at the beginning and end of the treatment day.ResultsOutput measurements for each photon energy were comparable with a maximum difference of -0.57% for flattened beams (6x) and 0.21% for FFF beams (10FFF). Output measurements using the Discover matched ion chamber output measurements at every dose rate within 2%, and within 1% for output as a function of gantry angle. MU linearity test agreed with ion chamber measurements with a maximum difference of 0.41%. Output measurements using the Discover showed a daily drift in output throughout the course of a treatment day of around 2% and correlated very well with ion chamber outputs measured at the beginning and end of the treatment day (within 0.2%).ConclusionsThe ScandiDos Delta4 Discover onboard transmission detector is able to accurately measure linear accelerator output comparable to ion chamber measurements. One of the medical physicist’s primary responsibilities is to ensure the safety and quality of linear accelerator performance -2. As suThe ScandiDos Delta4 Discover  is a transmission detector consisting of 4040 p-type diodes that affixes directly to the treatment head of the TrueBeam  linac via a hook system, similar to the accessory mount. Diodes are spaced every 2.5 mm along the Multi-leaf Collimator (MLC) trajectory and coincide with MLC spacing perpendicularly for a standard collimator TrueBeam machine for a maximum detection area of 20 cm x 25 cm. Although the detector resides on the head of the linac and remains directly in the beam path, it has been shown to have minimal impact on perturbing the treatment beam . CurrentIn order to quantify the output using the Discover, a conversion factor must first be obtained. With the monthly output phantom, consisting of a 20 cm x 20 cm x 30 cm slab of solid water, and an accredited dosimetry calibration laboratory (ADCL) calibrated ion chamber (IC) at 5 cm depth, multiple measurements were taken with the IC, and no Discover in the beam path, and the Discover over a wide range of simulated outputs, from 0.95 cGy/monitor unit (MU) to 105 cGy/MU, for both the IC and Discover, using a dose-rate of 600 MU/min for all energies, with the exception of 10FFF, where 800 MU/min was used. By means of the ScandiDos Delta4 software (version 8), the raw signal from the Discover was exported to a spreadsheet, where the central 5 mm x 5 mm portion of a 10 cm x 10 cm field was averaged for each delivered output mentioned. The IC-measured output was then divided by the average raw signal to obtain an output-per-raw signal factor for each measurement. The output-per-raw signal factors for each delivered output mentioned were averaged to obtain a global calibration factor to convert Discover raw signal to output. This was done for each photon energy, including FFF beams  as a function of gantry angle by delivering 100 MU to each set up at the four cardinal gantry angles. MU linearity was measured and compared with ion chamber measurements for 6x photon energy by delivering 2 MU to 800 MU at a dose rate of 600 MU/min. Additionally, the Discover was used to take output measurements approximately every hour throughout the course of a treatment day, and compared with IC output measurements at the beginning and end of the treatment day for 6x, in order to demonstrate linac output variations throughout the course of a typical treatment day.Output measurements for each photon energy were comparable between IC-measured values and Discover-measured values. The maximum difference observed for flattened beams was -0.57% for 6x, and the maximum difference observed for FFF beams was 0.21% for 10FFF. Figure Figure Figure The Delta4 Discover is able to accurately detect linear accelerator outputs, when compared to IC-measured outputs, for all relevant photon energies. It offers the potential for improved efficiency for performing linear accelerator QA by greatly simplifying detection setup geometry and by its versatility, as it connects directly to the head of the linear accelerator and remains in the beam path throughout treatment. This allows it to efficiently and accurately detect linear accelerator output continuously throughout the day. With an accurate understanding of the temporal changes in linear accelerator output performance, there is potential to influence the medical physicist's determination of when output baselines should be set and adjustments made. Additionally, it is a vendor-independent technique that helps provide an added layer of confidence in the system.In this work, we explored only a part of Discover’s potential to perform linear accelerator QA; focusing specifically on output. However, there are conceivably many other applications that Discover may be well suited to perform. One such application is for mechanical QA of the collimator and gantry angles due to the built-in gyroscope and inclinometer. Another potential application is that of MLC QA. Sarkar et al. have shown that Discover is able to detect submillimeter deviations in leaf positions in dynamic leaf plans . TherefoLimitations to implementing Discover as a viable QA tool exist and must be considered by the user. One such limitation is the upfront time required to obtain an appropriate calibration factor to convert the Discover raw signal to output. This process can be time-consuming on the front end of the implementation. However, this is also true for setting up any output check device. The simplicity of the Discover and the fact that there would be no set-up time, given that it is already on the head of the linac, may mitigate this limitation and be a potential advantage over other output check modalities. The setup for output vs. gantry angle would also be very fast for the same reason and much less cumbersome than using an IC and phantom while attempting to account for the couch at 180 degrees. Another such limitation is that of the analysis process. Currently, only a non-commercial version of the ScandiDos Delta4 software allows the user to obtain the raw signal from Discover’s detectors. Once the raw signal is obtained, the analysis can be cumbersome; requiring the user to manually average the diode signal and convert it to output. We would recommend that the vendor add QA analysis to the software to improve the functionality and efficiency of performing QA with Discover. Additionally, with diode degradation over time being a potential issue, the calibration factor should be checked against IC measurements periodically, and recalibration be performed as needed to ensure a current calibration factor. However, according to the manufacturer's specifications, the long-term sensitivity decrease of the diodes is <0.1%/kGy for 6x.In this work, we measure and report the linear accelerator output using the ScandiDos Delta4 Discover and compare it to IC-measured output. The Discover is able to accurately detect linear accelerator output for all relevant photon energies while greatly simplifying setup geometry and uncertainties. Potential for other QA applications for Discover also exists to further improve efficiency.


Osteoarthritis (OA) is a chronic degenerative joint disorder for which there is no known cure. Non-surgical management for people with mild-to-moderate hip OA focuses mainly on alleviating pain and maximising function via the National Institute for Health and Care Excellence (NICE) recommended combination of education and advice, exercise, and, where appropriate, weight loss. The CHAIN (Cycling against Hip pAIN) intervention is a group cycling and education intervention conceived as a way of implementing the NICE guidance.CycLing and EducATion (CLEAT) is a pragmatic, two parallel arm, randomised controlled trial comparing CHAIN with standard physiotherapy care for the treatment of mild-to-moderate hip OA. We will recruit 256 participants referred to the local NHS physiotherapy department over a 24-month recruitment period. Participants diagnosed with hip OA according to NICE guidance and meeting the criteria for GP exercise referral will be eligible to participate. Primary outcome is the difference in Hip Disability and Osteoarthritis Outcome Score (HOOS) function, daily living subscale between those receiving CHAIN and standard physiotherapy care. Secondary outcomes include performance-based functional measures , ability for patient to self-care (patient activation measure) and self-reported health-related resource use including primary and secondary care contacts. The primary economic endpoint is the number of quality adjusted life years (QALYs) at 24 weeks follow-up. The study is funded by the National Institute for Health Research, Research for Patient Benefit PB-PG-0816-20033.The literature identifies a lack of high-quality trials which inform on the content and design of education and exercise in the treatment of patients with hip OA and explore cost-effectiveness. CLEAT is a pragmatic trial which seeks to build further evidence of the clinical benefits of the CHAIN intervention compared to standard physiotherapy care within a randomised, controlled trial setting, and examine its cost-effectiveness.ISRCTN19778222. Protocol v4.1, 24th October 2022.The online version contains supplementary material available at 10.1186/s12891-023-06456-0. Osteoarthritis (OA) is a long-term health condition for which there is no cure. Mechanical and systemic factors may result in joint pain leading to a reduction in quality of life and activities of daily living . Data coGiven its prevalence, OA represents a significant economic burden, due to the impact on people’s ability to work and their need for social care and welfare benefits as well as on-going healthcare . AccordiA systematic and network meta-analysis review concluded that low impact single exercise interventions, such as aerobic exercise , were the most effective therapies for improving pain and function in hip OA . Indeed,A review of potential mechanisms underpinning the benefits of exercise therapy on pain and function in OA found that an increase in upper leg strength, a decrease of extension impairments, and an improvement in proprioception, were all possible mediators of the link between exercise and a decrease in OA symptoms in the lower limb; another review highlighted the need to address muscle weakness in people with hip OA , 13. CycAs a way of implementing the NICE guidelines for hip OA, a cycling and education intervention, the CHAIN (Cycling against Hip pAIN) programme, was developed in 2013 through discussions with local orthopaedic teams and physiotherapists  and was The primary clinical objective is to determine whether there is a difference in the change in self-reported function from baseline to post-treatment assessment between those receiving the 8 weeks cycling and education intervention compared to those who received routine physiotherapy care. Self-reported function is assessed using the Hip Disability and Osteoarthritis Outcome Score (HOOS) function, daily living subscale . The priThe secondary objectives are to estimate cost-effectiveness of the CHAIN intervention by comparing health-resource use of the participants on the two arms of the study through completion of a questionnaire; and to compare changes between the groups in:Self-reported level of hip pain assessed with the Pain subscale of the Hip Disability and Osteoarthritis Outcome Scale (HOOS) .Performance assessments: 40 m walking test, 30s chair stand test, Stair Climb test .Function level in performing five important activities they have difficulty with, assessed by the Patient-Specific Functional Scale  ParticipParticipant’s knowledge, skill, and confidence to manage their own health assessed using the Patient Activation Measure questionnaire .Quality of life assessed with the EQ-5D-5 L and EQ-5D VAS questionnaire .Data will be collected within two weeks of those on the CHAIN intervention arm completing their treatment and collected by post/email or telephone at 3 months post-treatment (with the exception of the objective function assessments).This pragmatic trial follows a successful feasibility study of the intervention , 19 and A flow diagram showing the design of this trial is shown in Fig. CLEAT is a pragmatic, two parallel arm, randomised controlled trial comparing a cycling and education intervention (CHAIN) with standard physiotherapy care reflecting standard practice in the National Health Service (NHS) in the UK . The proThis trial is open to patients who are referred to the NHS Physiotherapy Department with hip pain. For participants in the intervention arm, the trial will be conducted at a leisure centre, which was involved in the feasibility study. Participants in the (standard physiotherapy care) control arm, will be treated by the NHS Physiotherapy team they have been referred to.We intend to recruit participants with hip pain or hip OA. OA will be diagnosed by a clinician using the NICE criteria  to reviePotential participants will be identified from Physiotherapy waiting lists by senior physiotherapists and screened by the research team as per exclusion criteria Table . EligiblPrior to the baseline visit, the Trial Manager will contact participants by telephone to discuss the trial, offer participation and receive verbal informed consent , blood pressure, resting heart rate and range of motion of both hips. Performance assessments as recommended by the Osteoarthritis Research Society International (OARSI) for core performance tests will be collected [Baseline assessments (Visit 2) will take place on the same day as randomisation. The participant will be asked to sign the informed consent form by the trial manager, or a delegated member of the research team, and the remaining baseline data will be collected by the trial assessors (see the Outcome measures section) and will include a review of participants’ medical history; physical measurements such as height, weight, body composition  will randomise the participant, using the randomisation system incorporated within the trial database, to receive the next treatment allocation from the randomised list. If a participant does not complete the screening/baseline procedures or meet the required eligibility criteria, they will not be randomised and will be removed from further involvement with the study.Assessors undertaking assessments at baseline and follow-up will be blinded to the randomisation, and participants will be educated to ensure that they do not inform the assessors of which treatment arm they participated in. The nature of the intervention means that participants and treatment providers will not be blinded.Treatment will commence within 2 weeks of randomisation at Baseline Assessment.For 8 weeks following randomisation, participants in groups of up to 15, will attend a weekly, one hour education and cycling session at a local leisure centre. Each session will typically involve a 30 min education session which aims to motivate participants to exercise, reassure them that it is safe to do so, and facilitate positive lifestyle/activity change, followed by a 30 min indoor static cycling class (35 and 40 min for the last two sessions). An overview of CHAIN week-by-week is shown in Fig. The Trust Physiotherapy Department will allocate treatment slots (within two weeks of randomisation) for participants in the control arm. Once randomised to this arm, at their baseline visit participants are given the next available date and time for their first treatment session. Thereafter the physiotherapy appointments are made between the participant and the Physiotherapy Department directly as per standard care. Over 8 weeks, participants in the control group will attend up to four one-to-one sessions of physiotherapy (as per NHS standard care ) for up There will be two follow up assessments, the first within 2 weeks of the end of the CHAIN intervention (or end of 8 weeks treatment period for the control arm) (Visit 4) and the second (Visit 5) occurring approximately 3 months after the post-treatment visit. Visit 4 will be undertaken by trained, qualified physiotherapists and will consist of the same physical and functional assessments performed at Visit 2, completion of patient questionnaires as well as a review of participants’ activity diaries and a feedback questionnaire for those participants that were randomised to the CHAIN intervention; Visit 5 consists of 5 patient questionnaires (Hip Disability and Osteoarthritis Outcome Score (HOOS); Patient Specific Functional Scale (PSFS); Patient Activation Measure (PAM); EQ-5D-5 L; and resource use) sent to participants by email or post. Participants will be contacted by text/telephone/email to help ensure completion and return of questionnaires. Where this is difficult to achieve, we will prioritise collection of the primary outcome at Visit 4. The data to be collected at each of these follow-up appointments are summarised in Table The primary outcome is the function, daily living component from the Hip Disability and Osteoarthritis Outcome Scale (HOOS) , measureWe are measuring a number of secondary outcomes including participant reported outcome measures, resource use and QALYs at both Visit 4 and visit 5; and changes in physical and functional measurements at Visit 4. These are summarised in the SPIRIT diagram Table , but in HOOS function, daily living score measured out of 100, after completion of the HOOS Questionnaire at Visit 2 and Visit 5 (score at Visit 4 is primary outcome);HOOS pain score measured at Visits 2, 4 and 5; HOOS symptoms score measured at Visits 2, 4 and 5;HOOS stiffness score measured at Visits 2, 4 and 5;HOOS sports and recreational activities score measured at Visits 2, 4 and 5;HOOS Quality of Life score measured at Visits 2, 4 and 5;Function assessed by 30-second chair stand test measured at Visit 2 and 4;Function assessed by stair climb test measured at Visit 2 and 4;Function assessed by 40-metre walk test measured at Visit 2 and 4;Patient activation measured by responses to PAM Questionnaire at Visits 2, 4 and 5;BMI, body composition, blood pressure and resting heart rate measured at Visits 2 and 4;Analgesia use collected from the Resource Use Questionnaire during Visits 2, 4 and 5;Patient-perceived general health measured using EQ-5D Visual Analogue Score (VAS) at Visits 2, 4 and 5;Patient-perceived quality of life measured by EQ-5D-5 L Questionnaire at Visits 2, 4 and 5, used to derive Quality Adjusted Life Years (QALYs).Self-reported resource use measured by responses to the Resource Use Questionnaire during Visits 2, 4 and 5.Hip OA is a non-life-threatening condition typified by on-going progression of symptoms and deterioration of function. From the time the participant signs the consent form until the day the participant has completed their post-treatment assessment (Visit 4), the only adverse events recorded will be those for which participants seek advice from a health care professional, such as an unplanned visit to a General Practitioner for an acute condition. Participants will be asked at the start of each CHAIN session or physiotherapy session whether they have had any adverse events, and they will be asked again at the assessment post-treatment.Based on our feasibility study  as well et al’s study [The sample size calculation is based on the primary outcome measure, the physical function score from the HOOS function scale at Visit 4. This has identical items to the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) function scale , with to’s study , for whi’s study  that ass’s study . Using a’s study , 37) of ’s study –39 and t’s study ) of 13 o’s study ) the recPrior to completing data collection, a detailed Statistical Analysis Plan will be written and approved by the Trial Steering Committee. Participants’ data will be analysed in the group they were randomised to, and we will attempt to collect outcome data on all participants unless they requested to be withdrawn from the study . Analysis and initial write-up will be blinded to treatment groups and reported according to the Consolidated Standards of Reporting Trials. Baseline characteristics of the sample in each arm of the trial  will be summarised using descriptive methods. The primary outcome of the function component of HOOS measured at Visit 4 will be compared between trial arms using a multi-level/mixed model approach, considering baseline function and CHAIN group (cluster).We will conduct a per protocol analysis where participants will be excluded from the analysis if (a) there was a protocol violation (including swapping treatment arms) or (b) adherence to exercise was poor  or adherence to physiotherapy was poor in the control arm .The economic evaluation will use data collected within the trial to establish the resources required to provide the intervention, estimate its cost, and provide a full cost-effectiveness analysis (CEA). In order to comply with NICE guidance  the econCost and outcome data will be synthesised to present incremental cost-effectiveness ratios (ICERs) - the incremental cost per unit of additional outcome - for the primary outcome (cost per unit change on the HOOS measure) and the primary economic endpoint of policy relevance (cost per QALY).Intervention resource requirements and costs  will be collected at an individual participant level, using case report forms. Resource use data  will be collected at Visits 1/2, 4 and 5, using a self-report Resource Use Questionnaire. This questionnaire will draw on measures used with similar populations in the Database of Instruments for Resource Use Management (DIRUM) repository , 43, aloWe will follow the internationally recognised Consolidated Health Economic Evaluation Reporting Standards (CHEERS) guidelines for reporting cost-effectiveness studies . DescripA cost-consequences analysis will also present key costs and outcomes in a disaggregated, tabular format , 52. ThiA public and participant involvement forum, comprising six people who had undertaken the pilot CHAIN programme has informed this protocol, providing feedback on the study design and documentation, the design of the intervention, the main outcome of interest and how the data would be disseminated . The forThe sponsor for this study is the University Hospitals Dorset NHS Foundation Trust, and they have overall responsibility for the initiation and management of the trial. The Chief Investigator will be responsible for trial design, data analysis and interpretation, manuscript writing and dissemination. The Trial Management Group will meet approximately 4 times per year to ensure all practical details of the trial are progressing and working well. The Trial Steering Committee will conduct oversight of the study and will meet at least yearly. It will comprise an independent Chair, independent statistician, and member of the Patient Advisory Group, along with the Chief Investigator (CI) and a member of the Sponsor team. As this is a low-risk intervention and there are no plans to undertake interim analysis, the Trial Steering Committee will also act as the Data Monitoring Committee in this study.https://www.uhd.nhs.uk/directory/name/28-services/bournemouth/1480-cycling-and-education-cleat .All data collected in this trial will be entered into a secure cloud based independently run trial database. All data will be anonymised, and participants given a unique trial number. The research team will be responsible for capturing relevant data onto paper source documents and entering the data into the electronic case report form. Results will be disseminated through open-access peer-reviewed publications, presented at relevant conferences and on our CLEAT website This trial will be conducted in accordance with Good Clinical Practice principles and guidelines, the Declaration of Helsinki, University Hospitals Dorset NHS Foundation Trust standard operating procedures, relevant UK legislation and the trial protocol. Ethical approval was granted on the 5th Nov 2019 (19/SC/0502), by the South Central Oxford C Research Ethics Committee . The trial will be reported in line with the CONSORT statement , 54 and Information about study patients will be kept confidential and managed in accordance with data protection legislation, the UK Policy Framework for Health and Social Care Research and Research Ethics Committee Approval.Information enabling direct/immediate identification  will only be accessible to a limited number of personnel within the research team and will only be used where necessary for the purposes of the study or safeguarding participants. No personal identifiable information will be recorded on any data collection documentation. Only authorized members of the research team will have access to the research data, and any data shared from the participants’ medical records, and patients will be anonymised with regards to any future publications relating to this study. All research data will be stored securely in adherence with the data protection legislation in force, the Sponsor’s data management policies, and the patient information sheet. Consent will be obtained to allow authorised staff employed by the Sponsor to review identifiable data to ensure the study is monitored / audited to assess compliance with the protocol.The Cochrane Collaboration’s 2014 review of studies of exercise for hip OA found evidence supporting the use of exercise to reduce pain and improve physical function . FurtherThis trial aims to generate findings that are directly applicable to routine hospital care, and as such the standard care arm is pragmatic representing a mixture of approaches in the treatment of OA . To compThe trial takes place at a single high-volume site; this is mainly due to reducing the burden of travel for the participants in the CHAIN arm because the intervention will be delivered at a leisure centre with previous experience of hosting CHAIN, and to simplify the operational running of the study. Dorset Council has one of the oldest populations in the United Kingdom , and thParticipant recruitment commenced on 29th January 2020; however recruitment was paused until June 2021 due to the global pandemic, and participants recruited before the pandemic had to be withdrawn as they had not completed treatment. Recruitment should be completed by May 2023 and completion of the study by Spring 2024.To date, there have been three substantial amendments:Amendment 1 9th June 2020. Changes to study patient documentation due to pause during Covid pandemic.Amendment 2 25th April 2021. Changes made to study timings and location due to Covid restrictions, extra demographics collected, exclusion criteria on steroid use adapted to omit use for asthma.Amendment 4 7th October 2022. Exclusion criteria on age and steroid use updated to increase recruitment, study patient documentation updated to include report sent to CHAIN participants on change in outcomes, and leaflets giving participants details on where to attend treatment.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3Supplementary Material 4Supplementary Material 5


Mothers with a history of symptoms of depression had significantly more worry-related sleep problems during the pandemic (p = 0.020) than those without a history of depressive symptoms. Our study underlines the negative impact of the COVID-19 pandemic on diverse aspects of everyday life of mothers in Nepal.The COVID-19 pandemic has affected many aspects of daily life worldwide, but the impact may be higher for impoverished populations. The main aim of this study is to describe the impact of the COVID-19 pandemic on different aspects of daily life in mothers in Nepal. We included 493 mothers of children aged 54–71 months participating in a randomized controlled trial on vitamin B12 supplementation. Mothers answered questions regarding the exposure and impact of the pandemic on their daily lives, and pandemic-related worries and sleep problems. We examined the extent to which worry, and sleep problems differed between mothers according to their exposure to COVID-19, socioeconomic status, and previous symptoms of depression. The mean age (SD) of the mothers was 32.3 (4.6) years and 54% had education below the secondary level. Of the mothers, 5.4% had either been exposed to someone who had tested positive or who had a family member with COVID-19. One-third of the participants responded that the pandemic had affected their economic situation, employment, and family life to a great deal. Both mothers and fathers with educational levels above 10 years or households with higher socioeconomic status had significantly higher average worry scores (maternal  The first infection with the SARS-CoV-2 virus was identified at the end of 2019 in Wuhan city, China . While tIn Nepal, the first case of COVID infection was identified in January and the second case in March 2020. The government of Nepal declared a strict lockdown from the end of March 2020. The level of restrictions to control the spread of the virus varied until August . The resA pandemic also has considerable psychological impact such as increase in worry, fear, distress, and anxiety in the population , 10. DurWorry and worry-related sleep problems may be more severe for people in high-risk groups. Underlying chronic conditions such as diabetes and cancer affect the severity of the COVID condition , and maySocioeconomic factors may have an impact on worries related to the COVID-19 . The socIn August 2020, there were 39,460 confirmed cases in Nepal, which increased to 77,817 in September 2020, 170,743 in October and 274,143 when reaching February 2021. The number of deaths from COVID-19 was 404 in September 2020 (the start of our study) and slightly increased to 646 in October and 839 in November the same year. There was, however, a drastic decreasing trend from December with only 52 death cases in February 2021  when we The present study was conducted in the Bhaktapur municipality of Nepal. The municipality has a population of 83000  and boarThe participants included in the current study were mothers of children participating in a double-blinded clinical trial  entitled “The effect of Vitamin B12 supplementation in Nepali Infants on Growth and Neurodevelopment” . A totalFig 1.A total of 67 children from the original study of 600 children were lost to follow up  when we initiated the COVID-19 exposure interviews with the mothers. Of the remaining 533 participants, we were not able to complete the COVID-19 exposure questionnaire with 40 mothers and thus the final sample consisted of 493 mothers. The flow of participants is depicted in th years  and above. For occupation, we categorized the occupation groups of both mothers and fathers as no formal work, agriculture, carpet worker, daily wage earner, self-employed, working in the service, and working abroad. Trained staffs measured height and weight in order to calculate body mass index (BMI) for the nutritional status of the mothers using a stadiometer for height  and an electronic scale for weight .At enrollment, mothers answered questions on demographics and household characteristics. Information on the family members included mother’s age, age category of family members, caste/ethnicity of family, level of education, occupation group, and nutritional status of the mother. Family members were categorized in age groups for which we considered children <16 and adults >60 more vulnerable to consequences of a COVID-19 infection. Caste/ethnicity of the families was categorized as Newar, Brahmin, Chhetri, Tamang and others. The educational level was assessed as illiterate, primary school, secondary school, School Leaving Certificate/intermediate school, bachelor’s degree, and above. For our analysis, we dichotomized education up to 10rd percentile of our study sample.Socioeconomic status was measured by a multidimensional measure, the WAMI-index with the components; water and sanitation, household assets, maternal education, and income . In contIn follow-ups of the original study when the children were 30–35 months old, the mothers answered the Self-Reporting Questionnaire-20 (SRQ-20). The scores from SRQ-20 were from 2 years prior to the current study and covered previous maternal symptoms of depression.The COVID-19 follow-up was completed during a visit to the study clinic when the children were in between 54 and 71 months old. Trained study staff administered the questionnaires with the mothers in a private room at the study clinic. Women who did not attend this follow-up visit for the present study (approximately 50%) were interviewed through phone calls due to restrictions of movement (lockdown).We assessed COVID-19 exposure through a questionnaire particularly developed for this study See . The preWe also asked if the mothers believed that the life of someone close to her or her own life was in danger . These items of the questionnaire are similar to previous traumatic exposure questionnaires , 28. TheWorry of contracting the corona virus was measured by two items, of which one was worry about contracting the corona virus yourself, and one was the worry that someone in the family would contract the corona virus. The worry items are based on a previous questionnaire on worry for infections . Three iThe Self-Reporting Questionnaire-20 (SRQ-20)  is a briThe instruments were translated into Nepali by a psychologist and medical doctors and then back-translated by a qualified person independent from the study who was fluent in both Nepali and English language, following the standard guidelines for translation processes . DiscrepEthical approvals were obtained from Nepal Health and Research Council  and from the Regional Committee for Medical and Health Research Ethics  in Norway. Mothers were asked to visit the central clinic and we obtained informed written consent for the extended follow ups.th years vs. above 10th years), symptoms similar to COVID-19 (absence vs. presence of at least one symptom), chronic disease of mother (absence vs. presence of at least one chronic disease), chronic disease in family member (absence vs. presence of at least one chronic disease), previous symptoms of depression (scores above 10 vs. 10 and under), and the WAMI-index score (≤33rd percentile vs. >33rd percentile of the WAMI score). The missing values were handled by listwise deletion. Data was analyzed using the STATA 16.0 software.All the continuous variables are presented in means and standard deviations, and categorical variables in numbers and percentages. Exposure variables were categorized into a three-point scale: “Not at all” , “Some”  and “A great deal” . We compared the mean worry and sleep scores between groups using the student’s t-test. The high risk groups were dichotomized as follows: education of mother and father (up to 10Table 1).The mean age of the mothers was 32.3 (SD: 4.6) years, more than 75% belonged to the Newar ethnic group, and approximately 50% resided in a joint family. Half of the families owned land, and 57% lived in their own house. Around 55% of both the mothers and fathers had an educational level up to 10 years (Table 2).Among all mothers, 26 (5.4%) had either a positive COVID-19 case in the family or had been exposed to someone who had tested positive in the previous two weeks. Among these 26, the mean (SD) number of days of the quarantine was 15.7 (10.1) (Table 3).Among all mothers, 188 participants, either herself or her family members, had chronic diseases and among these, 1.4% were concerned about the shortage of essential medicines due to the pandemic. Less than 10% believed to a great deal that their life or the life of someone close to them was in danger. Approximately 22% reported that the pandemic had to a great deal a negative effect on family life. More than 29% reported that they had economic problems, and 33% reported that their employment was greatly affected by the pandemic. Approximately 9% were to a great deal concerned that they would experience health related issues. About 16% of the mothers felt that food security and daily life were greatly affected by the pandemic (The mean (SD) worry and sleep score were 5.4 (2.5) and 4.9 (2.8), respectively. The distributions of all the response categories for each item of worry and sleep are shown in The present study revealed a considerable impact on the everyday life of the Nepalese mothers in relation to the COVID pandemic between 9 September 2020 and 10 February 2021. Approximately one third of the mothers were heavily affected by the COVID-19 pandemic in terms of their economy and employment, and for 16% their daily life and food security were perceived as severely affected. Only a small group reported major impact on their health. Worry about contracting the virus showed a social gradient, with more worry in the higher socioeconomic status groups. Sleep problems related worry was not common, but higher for those with a previous history of symptoms of depression.The mothers reported that the economic and employment status were greatly affected by the pandemic. At baseline for the original study, many reported to have no formal work, being in agricultural work, carpet work and being a daily wage earner and about half of their husbands had the same occupations signifying that the type of work in Bhaktapur is mainly based on daily wage earnings. These were occupations that were almost impossible to keep functioning during lock down or other restrictions. Other aspects of the daily life, like food insecurity, health related issues, shortage of essential medicines were also marginally prevalent. These results are in line with a previous review of published articles related to the psychosocial impact of COVID-19 in Nepal showing substantial impact on economy, agriculture, employment and the health sector . We founAlthough many mothers reported that they or someone in their family had a chronic disease, the mothers were not so much concerned about supplies of essential medicines. This could be related to the fact that the study setting is close to the capital city of Nepal where the government made more efforts to prevent the pandemic compared to other regions of the country. In another study from Eastern less urban areas of Nepal, people perceived that medical supply was the most affected aspect of the health care system in Nepal during the pandemic .Around 10% of the participants believed that their own life or the lives of someone close to them were in danger. Approximately 40% of the participants reported being worried that they themselves or their family members would contract the corona virus. A high rate of worry is expected during a pandemic and in line with previous studies that demonstrated an increase in public anxiety during epidemics or pandemics such as the 2003 SARS pandemic, the 2009/2010 H1N1 pandemic and the 2014/2016 Ebola pandemic –37. WorrThe rate of worry related sleep problems were low in the present study. Despite the major impact of the pandemic on their daily life, the mothers reported that the social, economic and health-related consequences due to the COVID-19 pandemic did not negatively affect their sleep and less than ten percent of the mothers reported these problems. We do not have any possible explanation to the low prevalence of sleep problems since we lack specific information about the sleep patterns of Nepalese women. However, the low prevalence might be due to taking COVID-19 very lightly since most of the people who were infected during the study period were asymptomatic or had mild symptoms.The mothers who had a previous history of depression reported more sleep problems due to concerns with the COVID-19 pandemic. This finding is in accordance with previous studies showing that depressive symptoms is associated with poorer or disrupted quality of sleep during the pandemic , 39, andThere was a social gradient in worry, but in the opposite direction than expected from previous studies in other parts of the world. In the present study, there were higher levels of worry for contracting the virus among those who had an educational level higher than secondary. This group with the lowest educational qualifications being least concerned about contracting COVID-19, might be the challenging factor in controlling the pandemic due to less safety measures to protect them from virus infection. Hence, public health practitioners should consider the level of education while addressing the psychological impacts of pandemics since our results suggests that education can be the deciding factor for psychological impact of the individuals. Further, and also in contrast to our expectations and the established social gradient of health that has been evident during the pandemic , we founst variant of SARS-Cov-2 in Nepal at the time of the data collection . All PLOS journals now require all data underlying the findings described in their manuscript to be freely available to other researchers, either 1. In a public repository, 2. Within the manuscript itself, or 3. Uploaded as supplementary information.This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If your data cannot be made publicly available for ethical or legal reasons , please explain your reasons by return email and your exemption request will be escalated to the editor for approval. Your exemption request will be handled independently and will not hold up the peer review process, but will need to be resolved should your manuscript be accepted for publication. One of the Editorial team will then be in touch if there are any issues.7. Please amend your] detailed Financial Disclosure statement. This is published with the article, therefore should be completed in full sentences and contain the exact wording you wish to be published.i) Please include all sources of funding  for your study. List the grants (with grant number) or organizations (with url) that supported your study, including funding received from your institution. ii). State the initials, alongside each funding source, of each author to receive each grant.iii). State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”Additional Editor Comments (if provided):This is an important contribution to our understanding of the COVID-19 pandemic on daily life and well-being. In addition to the suggestions of the reviewers, I would add:1) Since your framing of the paper and discussion focus on worry, please title the paper accordingly. This is a very interesting framing, and the title should reflect that. Please further incorporate in the introduction that you are considering "worry" not just about COVID-19 itself but many of its sequelae in Nepal .2) Please indicate the age range of children in the abstract. This is very clear in the methods, but after reading the abstract, I thought certainly outcomes would vary by age of children (which turned out to not be relevant as I saw the study design).3) In the discussion, please include more thinking about the gendered experience of women.Thank you for this submission![Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to Questions  Comments to the Authorpublication criteria? Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe methodologically and ethically rigorous research with conclusions that are appropriately drawn based on the data presented.  1. Does this manuscript meet PLOS Global Public Health’s Reviewer #1: YesReviewer #2: Yes**********  2. Has the statistical analysis been performed appropriately and rigorously?  Reviewer #1: YesReviewer #2: Yes**********  3. Have the authors made all data underlying the findings in their manuscript fully available (please refer to the Data Availability Statement at the start of the manuscript PDF file)?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: YesReviewer #2: Yes**********  4. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS Global Public Health does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: No**********  5. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: This study uses cross-sectional survey data to assess the impact of the COVID-19 pandemic on various aspects of daily life for mothers in Bhaktapur, Nepal. While previous studies in Nepal have assessed psychological impacts during the early pandemic period, this study centers on a later phase of the pandemic . Findings of higher pandemic-associated worry in mothers with higher educational attainment and socioeconomic status are concordant with a prior, national-level study assessing pandemic distress in April-May 2020 . Readers of PLOS Global Public Health may be interested in implications of these findings for health education efforts in Nepal, as well as suggestions for addressing these psychological impacts.Major and minor comments for the authors are provided below.INTRODUCTIONThe authors provide a comprehensive overview of previous literature on the impact of COVID-19 on psychological health in general and in Nepal specifically.Additional contextual information about Bhaktapur municipality  would be appreciated in the Introduction, as well as in the Discussion of the study findings.METHODSThe authors have clearly described the participant recruitment strategy and reasons for loss to follow up.Definition of variables is easily understood, but what is meant by “age category of family members” is not immediately clear.The methods for statistical analysis are technically sound, although authors should discuss explicitly their strategy for handling any missing data.The authors received ethical approvals from the Nepal Health and Research Council (NHRC) and Regional Committee for Medical and Health Research Ethics (REC) and described obtaining written consent from study participants. Authors have made data available upon request in accordance with requirements of NHRC and REC.RESULTSResults are communicated clearly.Did the authors note any difference in key findings (negative impacts of COVID-19) by mode of data collection (phone vs. in-person interview)?TABLESTables 1-5 and Supplemental Table 1 are well-organized and easily comprehensible.DISCUSSION/CONCLUSIONSThe authors appropriately situate their findings within a broader discussion of previous literature on pandemic distress in Nepal and in other LMICs.Authors have thoughtfully considered the Strengths and Limitations of the study, including generalizability, variability in the mode of data collection (phone and in-person interviews), and inability to collect data on depressive symptoms through clinical interview. Authors should additionally address how the study setting in Bhaktapur affects generalizability.Authors should expound on implications of their findings for readers of PLOS Global Public Health. What are the implications for health education efforts in Nepal if those least concerned about COVID-19 are those who with the lowest educational attainment? What should public health practitioners consider when aiming to address the psychological impacts the findings highlight?FIGURESFigure 1 is easily comprehensible. The title should be reworded to reflect the participants of the current study, e.g. “Recruitment of mothers in Bhaktapur, Nepal”.MINOR COMMENTS:Use of the term “Corona” should be replaced with “Coronavirus” or “COVID-19,” including in the title of Table 2.This article satisfies the below criteria for acceptance to PLOS Global Public Health:1. The study presents the results of primary scientific research.2. Results reported have not been published elsewhere.3. Experiments, statistics, and other analyses are performed to a high technical standard and are described in sufficient detail.4. Conclusions are presented in an appropriate fashion and are supported by the data.5. The article is presented in an intelligible fashion and is written in standard English.6. The research meets all applicable standards for the ethics of experimentation and research integrity.7. The article adheres to appropriate reporting guidelines and community standards for data availability.Reviewer #2: The authors present findings of a study that assessed effect of COVID-19 in an urban district of Nepal. The article is largely coherent however it would benefit with some specific detail in few places. I would strongly recommend the article to be professionally edited for language as there are typos and grammatical errors in the article .Line number Comments103 Suggest to include some information on characteristics of respondents who were lost to follow up. Was it different from the final sample?106 Need to specific and consistent in using the term. Caregivers or mothers. Grandparents, father or other members of an extended family can also be a caregiver113 What was the definition for” no work” specially for mothers (57.8%)? Did it also include housewife or household work? Strongly suggest to use alternative word135 Recommend including information tool development of Corona Exposure questionnaire. Was this questionnaire pre-tested?213-215 Convoluted sentence. Please make this sentence easier to read269 What could be the possible explanation about the low prevalence of sleep problem – What do authors think about that? - more resilience because of repeated stressors? or taking COVID-19 lightly?287-289 Argument here is really not clear. Please add more reasoning about why they could be less sensitive**********  6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.Do you want your identity to be public for this peer review? If you choose “no”, your identity will remain anonymous but your review may still be made public.Privacy Policy.  For information about this choice, including consent withdrawal, please see our Reviewer #1: NoReviewer #2: No**********https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  20 Feb 2022AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 2 Mar 2022Impact of the COVID-19 pandemic on daily life and worry among mothers in Bhaktapur, NepalPGPH-D-21-00572R1Dear Mr. Ranjitkar,We are pleased to inform you that your manuscript 'Impact of the COVID-19 pandemic on daily life and worry among mothers in Bhaktapur, Nepal' has been provisionally accepted for publication in PLOS Global Public Health.Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.globalpubhealth@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they'll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Global Public Health.Best regards,Rachel Hall-CliffordAcademic EditorPLOS Global Public Health***********************************************************Reviewer Comments :


Most investigations of geographical within-species differences are limited to focusing on a single species. Here, we investigate global differences for multiple bacterial species using a dataset of 757 metagenomics sewage samples from 101 countries worldwide. The within-species variations were determined by performing genome reconstructions, and the analyses were expanded by gene focused approaches. Applying these methods, we recovered 3353 near complete (NC) metagenome assembled genomes (MAGs) encompassing 1439 different MAG species and found that within-species genomic variation was in 36% of the investigated species (12/33) coherent with regional separation. Additionally, we found that variation of organelle genes correlated less with geography compared to metabolic and membrane genes, suggesting that the global differences of these species are caused by regional environmental selection rather than dissemination limitations. From the combination of the large and globally distributed dataset and in-depth analysis, we present a wide investigation of global within-species phylogeny of sewage bacteria. The global differences found here emphasize the need for worldwide data sets when making global conclusions. A metagenomic analysis of 757 sewage samples from 101 countries suggests that global differences in some species might be caused by regional environmental selection. Very recently, sewage samples have been used in the surveillance of the Covid-19 pandemic7. In supplement to such surveillance activities, understanding of the microbial community residing in sewage is important, because sewage has been suggested to comprise a reservoir of AMR and at the same time, provide an environment for potential genetic transfer between the bacteria in the community8. Several studies have examined the bacterial composition of sewage samples by 16 S rRNA investigations11 or mapping to reference databases12. However, such investigations are limited to species previously identified and furthermore, it can be difficult to distinguish closely related species from 16S rRNA analysis, thus, species living in sewage that are closely related to species from the human gut could be confused. Within the last decade, investigations of microbiomes in human hosts, soil, plants, and more have found that differences in bacterial communities correlates with geography16. Among bacterial species isolated from clinical infections such as Staphylococcus aureus, Streptococcus pneumoniae, and Escherichia coli, within-species diversity correlating with geography has been observed in multiple studies19. Some of these geographical differences could be a result of local environmental selection but may also be due to the effect of dispersal limitations on local prevalence. These findings challenge the long-standing, Baas Becking ecological hypothesis that “Everything is everywhere, but the environment selects”20.Sewage samples have proven useful for surveillance of antimicrobial resistance (AMR)Eubacterium rectale and Candidatus Pelagibacter, from marine metagenomes21 and human gut microbiomes22. Another study did not identify significant geographical differences within subspecies of for instance Bacteroides vulgatus and Alistipes putredinis from the human gut23. However, the subjects included in that study were limited to North Americans and Europeans. It has often been difficult to obtain comparable samples in a standardized way across large geographies. The Global Sewage dataset2, containing samples from 101 different countries, serves as an ideal candidate for a broader investigation of regional within-species diversity. A phylogenetic analysis of 79 samples focusing only on reference mapping to known bacterial species has previously been performed12 and found geographical clustering for environmental and human commensal bacteria.In the attempt to disentangle the effect of environmental selection and/or dispersal limitations, researchers have studied the within-species diversity not only from isolates, but also in metagenomics data. Correlations between diversity within species and geography have been identified in bacterial species, such as In this study, we aim to investigate the microbial community of sewage by constructing metagenome assembled genomes (MAGs) and determine the within-species phylogeny on a global scale, using 757 sewage samples from 241 sites and a total of 101 different countries. With these phylogenies, we increase the depth of the analysis by comparing geographical clustering between different genes when stratified by the cellular localization of the encoded proteins. Both in terms of sample size and global reach this study is the most comprehensive investigation of within species diversity among sewage species to date. From our analysis, we identified 3353 near complete (NC) MAGs from 1439 different MAG species and found that variation within some species (12 species out of 33 investigated) correlated with geographical separation. Furthermore, we found that, for a selection of species, genes associated with organelles displayed on average 10% less geographical variation compared to other groups of genes, suggesting that the geographical clustering is primarily due to environmental selection. Thus, we confirm the fundamental microbial ecology doctrine that microbes are globally dispersed but selected by the environment.24 and MetaBAT225). From 757 samples across 101 different countries . Lower assembly quality and higher strain diversity has been suggested to reduce binning performance26, which could be why the binning of the sewage metagenomes is more complicated than binning of the human gut samples. Additionally, we found that only 1.2% of all the NC MAGs could be identified as human gut microbiome species  catalogue27), similar to a previous study based on mapping of reads from a subset of the Global Sewage samples, where 3.7% of the reads were found to be associated with the human microbiome2. In contrast to this, other studies using 16 S rRNA marker genes have found a higher proportion (15% and 4.3–28.7%)11. The difference in these results could be due to the limitations of each of the different methods for bacterial identification. There are advantages and disadvantages for different methods for bacterial identification and genome binning can be used to detect prevalent, including novel, bacterial genomes.As the sewage was collected from urban areas, we were interested in knowing how large a fraction of the MAGs that could be associated with the human gut microbiome. The 3353 NC MAGs, we identified, were less than the number of NC MAGS (5036) found from binning of 1000 human faecal samples11 and shotgun sequencing in Portugal28. However, the phylum compositions differed significantly from 1000 diverse human gut metagenomes  regions and a pool of human gut samples Fig. . The phyWhen mapping all our reads back to the collection of classified MAGs from this study and human gut, we confirmed similar regional taxonomic profiles Fig. . AdditioTogether our results suggest a major shift in taxonomy, enrichment for aerophilic organisms and a lower genome retrieval efficiency, due to higher alpha diversity and multi-host strain heterogeneity, in the sewage compared to human guts.30, however, ANI boundaries could vary between species and these results are debated32.To infer the phylogeny and identify geographical clustering within single species, we identified MAG species that were present across multiple samples. From the 1439 different MAG species, only 41 contained MAGs retrieved from ten or more different samples. Altering the 95% species Average Nucleotide Identity (ANI) threshold to 90% or 97.5% only resulted in a few variations to the originally identified MAG species cluster sizes and only a single new MAG species, found in ten or more samples, was identified  were in risk of being mixed into one MAG and that such mixed MAGs were placed exactly as or very similar to the most identical reference strain in the species trees, thus not affecting the results from our analyses , which we identified as belonging to the ees Fig. . Potenti2 value from the PERMANOVA test, which describes how much of the variation in the data that can be explained from the regional origin of the samples. Of the 33 tested trees, 12 clustered significantly  according to genome sample region . The portion of variation explained by geography is much higher than the average of 19% variation in the significant trees found by a similar study of the human gut microbiome24, suggesting that more of the genomic variation found in environmental bacteria correlates with geography than the variation found in gut microbes. The geographical clustering suggests that either dispersal limitations or local selection can cause localized divergence.To assess the degree of geographical clustering and potential dispersal limitation, we performed a PERMANOVA test on the distance matrices from the ASTRAL trees with more than one genome from at least two regions, leading to a total of 33 tested species  (Supplementary Table 2), with the exception of one species (C26), which we therefore excluded from further analysis.To investigate whether the observed geographical clustering was the result of dispersal limitations or local selection, we examined whether the clustering varied between different groups of genes. We hypothesised that proteins on the surface of the bacteria would be subject to selective pressure from the environment, because of their direct interaction with the surroundings, whereas the intracellular proteins, not directly interacting with the environment, would vary as a consequence of genetic drift. We therefore tested whether the variation explained by geography differed as a function of overall gene product cellular location . As we found that organelle genes varied less than other genes in 94% of species  from the three gene groups , we found nine MAG species with significantly  different geographical clustering (R2) between the groups after Benjamini Hochberg (BH) correction of p values. The significant MAG species included E. coli, E. flexneri, Sebaldella termitidis and unknown species in the Veillonella, Brachymonas, and Neisseria genera and the Desulfobulbaceae, Saccharofermentanaceae, and Thiotrichaceae families. One of the MAG species with significant differences was C5 (Brachymonas), in which the trees of genes associated with organelles had significantly lower geographical clustering (R2), than trees of genes from the other two groups  in organelle gene trees were also found for the eight other significant MAG species  in organelle genes for the majority (16 of 23) of the remaining MAG species without significant difference.With a Kruskal-Wallis Rank-Sum test on geographical clustering . For all the nine MAG species, the dN/dS ratios were larger for membrane genes than organelle genes, suggesting positive selection of the membrane genes  the geographical clustering is primarily due to regional selection, rather than dispersal limitations.8. To prevent global transmission of these genes, it is important to better understand how sewage bacteria are globally disseminated.Here, we present an investigation of how sewage bacteria are dispersed globally. We found that in general the bacteria identified by metagenomic binning in untreated sewage, from the inlet to the wastewater treatment plants, showed a degree of geographical clustering for the within-species diversity. Interestingly, the genomic variation in organelle genes showed less regional clustering than genes involved in metabolic and membrane functions, suggesting that the clustering observed is primarily due to selection rather than dispersal limitations. Thus, the bacteria residing in sewage can spread globally, but are under evolutionary pressure to adjust to the different environments across the world. This selection pressure combined with the co-existence of multiple bacterial species and the presence of antimicrobial resistance genes (ARGs) and antimicrobial drugs in sewage create a high probability for transferral of ARGs between bacteria33. We have previously suggested that sewage sampling is a desirable strategy for such surveillance activities34. Metagenomics binning can be used to identify novel, bacterial genomes that are not present in reference databases. Here, we found that binning of shotgun sequences could identify a fraction of the bacteria residing in the sewage samples and that the bacteria originating from the human gut were a small subgroup of the microbiome found in these samples. This is a reminder that when using sewage samples for surveillance activities, the detection method should be selected carefully. If monitoring human-derived pathogens in sewage, one needs to consider that DNA from these organisms is a very small fraction of the total pool of microbial DNA. Mapping to reference genomes can be useful for the monitoring of the global spread or the local levels of a particular species, like we have seen for the covid-19 pandemic7. However, metagenomic assembly and binning could be used to identify potential candidates for surveillance and possibly to clarify the genomic context in which ARGs emerged and disseminate. Thus, there is more potential for new and important discoveries using metagenomics binning of sewage samples.The WHO has a goal of delaying the dissemination and emergence of AMR through monitoringIn conclusion, this study shows a clear geographical phylogenetic clustering of 12 bacterial species from sewage and suggests that this could be caused by global differences in the selection pressure in wastewater and the corresponding adaptation of sewage bacteria. Thus, demonstrating the importance of more understanding of the dynamics in the microbial life residing in sewage. Furthermore, the worldwide differences found with-in different bacterial species underpin the importance of including samples from the entire world if global conclusions must be made.35 . In brief, untreated sewage samples were collected before the inlet to the wastewater treatment plant at sample sites. DNA was extracted and fragmented from the untreated sewage and libraries were sequenced using Illumina paired-end sequencing to an average sequencing depth of approximately 42 mio reads. BBduk was used for adaptor removal and quality trimming of reads, using a quality threshold of 20 and a minimum read length of 50 bp. In total, 757 samples from 241 sites spanning 101 different countries across the world were included in this project. Of the 757 different samples, 56 were re-sequenced to reach a sufficient sequencing depth, the handling of these duplicates is described under the relevant methods. A complete list of samples included can be found in Supplementary Data Samples were collected and handled as part of the Global Sewage project25, we filtered contigs to a minimum size of 1500 bp and performed single-sample binning with MetaBAT2 using default settings. For binning with VAMB (v3.0.1)24, we combined contigs >2000 bp from all samples into one catalogue. From a pilot run with VAMB24 binning, we found no increase in the number of NC MAGs when using contigs >1500 bp, therefore we chose a minimum contig size of 2000 bp to reduce mapping time. We mapped reads from each sample to the contig catalogue using Minimap2 (v2.6)36. Afterwards, jgi_summarize_bam_contig_depths from MetaBAT (v2.10.2)25 was used to calculate abundances of contigs in each sample. The output abundances were combined into a matrix, normalized using vambtools24, and used as input to VAMB. We calculated and normalized Tetra Nucleotide Frequencies (TNFs) using vambtools as well. From the contig catalogue, we obtained contig names and lengths and used them as input to VAMB along with the normalized TNFs. We ran VAMB using the memory mapping mode available at the github repository. VAMB was run using a GPU with a mini-batch size of 256 and a network of 48 latent and 1024 hidden neurons.We assembled forward, reverse, and singleton reads with metaSpades (v3.13) using kmer sizes between 27 and 127 bp with an interval of 20 bp. Scaffolds above 1000 bp were saved for further analyses. For binning with MetaBAT2 (v2.10.2)37. We defined the quality of MAGs as in Almeida et al. 201938 where NC MAGs were defined as >0.9 completeness and <0.05 contamination. We used NC MAGs in our further analysis. To group similar MAGs  into clusters, we used dRep compare (v2.2.3)39 with a mash threshold of 90% and an Average Nucleotide Identity (ANI) threshold of 95% on all identified NC MAGs. dRep dereplication was run with the same thresholds on each of the dRep clusters identified from the comparing and the resulting score was used to select between MAGs from the same sample within one cluster, to avoid redundancy if a bin was identified both by MetaBAT and VAMB. Additionally, the dereplication results were used to select the best MAG species representative for each cluster of MAGs. We assessed the taxonomy of all NC MAGs with GTDB-Tk classify_wf (v0.3.2)40.We assessed the quality of all MAGs with a size above 1 Mbp using CheckM (v1.1.3) lineage_wf24 were used. Difference in phylum distributions between human and sewage samples were tested for significance with a two-sample Kolmogorov-Smirnov test in R. Additionally, these human gut MAGs were dereplicated like the MAGs from the sewage data and used together with reads from 15 randomly selected human samples (no infant or diseased hosts) from the dataset from Almeida et al. 201938 for abundance comparisons. The read count abundances of MAG species were obtained using CoverM (v 0.6.1)41, by mapping to the best representative MAG species genomes. In the mapping process reads from duplicated samples were pooled into a single fastq file, and prior to abundance investigations the counts from all samples were rarefied. The rrarefy function from the Vegan R package was used to rarefy the counts to the minimum number of counts (431.564) found in a sample, afterwards the abundance matrix was filtered based on the expected/covered ratio of a genome (>=0.5) and Transcripts Per Kilobase Million (TPM) were calculated from the read counts. The expected coverage (c) of a genome was calculated as:For comparison of the taxonomic distribution of sewage MAGs to human gut species taxonomy, the MAGs created in Nissen et al. 202142. The fraction of sewage MAGs likely to originate from the human gut microbiome was found by using MASH (v2.0)43 to map to the Unified Human Gastrointestinal Genome (UHGG) catalogue27 and identified as within a mash distance of 0.05 to any genomes in this catalogue.Where l is the average read length, g is the genome length, and r is the number of counts mapped. This calculation is simplified from the calculation described in Rasmussen et al. 201544 and rooted on a Thermotogae, because this is the bacterial phylum most closely related to Archaea45. We visualized the trees using iTol (v1.0)46. For the 41 dRep clusters spanning ten or more samples, a separate tree of the MAGs belonging to each cluster was inferred. For this, we used Prodigal (v2.6.3)47 protein predictions from GTDB-Tk as input to Sonicparanoid (v1.3.4)48, to identify orthologous genes, using the fast mode. To align DNA sequences of all identified orthologous, we used MAFFT (v7.453)49. Samples were excluded from the alignments if they had more than one copy of an orthologous gene, to avoid uncertainty of which gene was used to infer phylogeny. We used TrimAl (v1.4)50 to convert alignments to phylip format, prior to building a separate phylogenetic tree for each gene using IQ-TREE (v1.6.8)51 with automatic model selection. Trees were created if a gene was observed in at least three samples, which is the lowest possible number of samples that a tree can be inferred from with IQ-TREE. To infer the overall species tree phylogeny, we used all the gene trees from a specific MAG species as input to ASTRAL (v5.7.4)52. In this tree, IQ-TREE was used to correct branch lengths with the ASTRAL tree as constrained tree input. We used the ggtree (v2.0.4) package in R53 for visualization of species trees. To validate this method for investigation of within-species differences, we applied CheckM and dRep compare with same settings as described to a dataset of 50 human gut samples spiked with one to three different Salmonella strains and furthermore, created an ASTRAL species tree as described above for the MAG species found from this analysis.We reconstructed phylogenetic trees containing all the identified MAGs using the marker gene set from GTDB-Tk. One tree was created including the GTDB-Tk reference species and another without these. Both trees were inferred with FastTree (v2.1.11)54. From the InterProScan output, we then extracted the GO-term annotation and used the GO.db-package (v2.1)55 in R to get the annotations within the Cellular Component category. We grouped the genes into the top-level annotations within this category, to get overall groupings for comparisons between gene groups. We selected the groups membrane and organelle for further analysis, and the remaining genes were combined into one collapsed group. The organelle-associated genes were mostly coding for proteins that were a part of the ribosome (85%) and to a lesser extend proteins bound to the chromosome (9%), acting as part of the flagellum (5%), or polyhedral organelles (1%). For many of the orthologous genes (up to 91%), it was not possible to annotate them to any cellular component GO term. To include these unannotated genes in the analysis, we grouped them with gene groups other than membrane and organelles. When investigating the biological process GO term annotation of this gene group, the largest fraction (on average 45%) of genes were annotated to be part of a metabolic pathway and we therefore considered this group to represent metabolic genes. The genes involved in oxygen tolerance were likewise identified from the InterProScan output, by identifying the genes involved in the KEGG pathway map00190, oxidative phosphorylation.To assign functional annotation to the genes, we used the Prodigal protein predictions from GTDB-Tk as input to InterProScan (v5.36-75.0)56) was used for genewise dN/dS calculation. Genes with genetic variation between samples were identified with the snppos_analyzer from CSI phylogeny (v1.4)57 and only these genes were input to codeml. Furthermore, to make sure that gene alignments were in frame, only alignments starting with a start codon  were included. The phylip format gene alignments from the phylogeny reconstruction were converted to fasta files using TrimAl (v1.4)50 and stop codons were removed prior to dN/dS calculation. Along with the alignments, the gene tree files from IQ-TREE (v1.6.8)51 were used as input to codeml paml (v.4.9j)56. One dN/dS ratio per gene was calculated with codeml by setting the model option to 0 and the seqtype option to codons. Additionally, optimization was performed one branch at the time (method: 1) and ambiguous sites were removed from the calculation (cleandata: 1), otherwise default settings were used.Codeml (paml (v.4.9j)58 to perform a Permutational multivariate analysis of variance (PERMANOVA) according to geography. Geographical clustering was identified as the R2 value from the PERMANOVA test, which describes how much of the variation in the data that can be explained from the regional origin of the samples. Prior to the testing, multiple MAGs from the same city within one tree were limited to one representative MAG based on the dRep score, this filtering also excluded any duplicate MAGs from duplicated samples. In addition to this, a MAG was removed from the tree if it was the only representative of a region in this tree. To adjust for multiple testing, we corrected the p values from these tests using Benjamini & Hochberg (BH) algorithm59, adjusting for the number of ASTRAL species trees tested with PERMANOVA. For some short genes with low variance, the geographical R2-values outputted from the PERMANOVA test were negative 2- or dN/dS-values between gene groups, we grouped the values of the gene trees according to the Cellular Component annotations and used a Kruskal-Wallis test on the values from the different groups. Afterwards, we applied a Wilcoxon Rank Sum Test on the groups from MAG species displaying significance  from the Kruskal-Wallis test, to identify which of the three groups that were differing from each other. P values from the Wilcoxon- and Kruskal-Wallis tests were adjusted using the BH algorithm59, adjusting for the number of gene group tests.To identify species with any significant differences in geographical R2) between gene groups, we investigated if different gene qualities could bias the geographical R2 value. This was done by calculating the Pearson Correlation Coefficient (PCC) for geographical R2 values according to the number of samples in the tree, gene variation, gene length, and regional entropy. Number of MAGs was counted in a tree after removing duplicate city and single region samples, as it was done prior to the PERMANOVA. Gene variation was obtained as the mean fraction of varying sites across all pairwise sequences (mean pi). Gene lengths were identified as the number of positions in the fasta output from Sonicparanoid. Regional entropy was calculated as:n was the total number of regions in the tree, and pi was the proportion of samples belonging to a specific region. We performed these calculations on all gene trees tested with PERMANOVA. P values, test statistics, and 95% confidence intervals of Kruskal-Wallis-, Wilcoxon-, and species tree PERMANOVA tests can be found in Supplementary Data To support the comparisons of geographical clustering R between Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Reporting Summary


Laser interstitial thermal therapy (LITT) is increasingly being used in the treatment of brain tumors, whereas high-quality evidence of its effectiveness is lacking. This pilot examined the feasibility of conducting a randomized controlled trial (RCT) in patients with irresectable newly diagnosed glioblastoma (nGBM), and generated data on technical feasibility and safety.We included patients with irresectable nGBM with KPS ≥ 70 and feasible trajectories to ablate ≥ 70% of the tumor volume. Patients were initially randomized to receive either biopsy combined with LITT or biopsy alone, followed by chemoradiation (CRT). Randomization was stopped after 9 patients as the feasibility endpoint with respect to willingness to be randomized was met. Main endpoints were feasibility of performing an RCT, technical feasibility of LITT and safety. Follow-up was 3 months.A total of 15 patients were included, of which 10 patients received a biopsy followed by LITT and 5 patients a biopsy. Most patients were able to complete the follow-up procedures . Patients were planned within 3 weeks after consultation  and no delay was observed in referring patients for CRT . Two CD ≥ 3 complications occurred in the LITT arm and none in the biopsy arm.An RCT to study the effectiveness of LITT in patients with an irresectable nGBM seems feasible with acceptable initial safety data. The findings from this pilot study helped to further refine the design of a larger full-scale multicenter RCT in the Netherlands.Protocol and study identifier: The current study is registered at clinicaltrials.gov .The online version contains supplementary material available at 10.1007/s11060-023-04371-x. Glioblastoma (GBM) is the most common and most aggressive primary malignant brain tumor, with an incidence of 3–5 cases per 100.000 people in Europe . The oveAlthough many efforts have been made to improve current therapies, patients with GBM face a poor prognosis, with a median survival of just 9–18 months , 6. CurrIn recent years, laser interstitial thermal therapy (LITT) has been developed as a minimally invasive treatment for brain tumors, especially in cases where surgery is not deemed safe . SeveralThe aim of this pilot study was to examine the feasibility of conducting a randomized controlled trial (RCT) investigating LITT in patients with an irresectable nGBM and to identify any safety or technical feasibility issues to further refine the design of a larger full-scale multicenter RCT.The study was initially designed as a prospective randomized pilot study, with randomization between biopsy and LITT in the same session (n = 10) and biopsy alone (n = 10). The main goal of randomization in the pilot study was to determine whether patients with glioblastoma were willing to be randomized for biopsy and LITT versus biopsy only. After including the first 11 patients and treating the first 9 patients, randomization was stopped as the feasibility endpoint with respect to willingness to be randomized was met. From then onwards patients were only included in the LITT arm until the target number of 10 patients in the LITT arm was reached.This study is reported in accordance with the extension of the Consolidated Standards of Reporting Trials (CONSORT) for randomized pilot and feasibility trials .Inclusion criteria were defined as follows:Patients aged ≥ 18 with radiologically suspected diagnosis of supratentorial glioblastoma for whom the multidisciplinary tumor board advised biopsy only and/or patient wished no resection.Maximal volume ≤ 70 cc on post-contrast T1 magnetic resonance imaging (MRI).Safe trajectory/trajectories possible for ablation of at least 70% of the tumor, avoiding eloquent structures or transgression of a ventricle or vessel.Karnofsky Performance Score (KPS) ≥ 70.Exclusion criteria included:Contra-indication for general anesthesia or MRI.Lesion > 70 cc on post-contrast MRI on the day before intervention.Non-glioblastoma diagnosis as per frozen section analysis or final histology.Pregnancy.Multifocality and cystic aspect of the tumor were initially defined as exclusion criteria, but the 70% ablation criterium already ensured that highly multifocal and cystic tumors were excluded from the study. Hence these were removed as separate exclusion criteria.An extensive description of the study procedures can be found in the supplements. In short, patients were eligible for the study when suspected of a nGBM and the local multidisciplinary tumor board advised biopsy only  or patients did not wish a surgical resection. When eligible, patients were informed about the study and, after written informed consent was obtained, formally included and randomized to receive either biopsy alone or biopsy and LITT, both followed by standard adjuvant treatment. After treating the first 9 patients randomization stopped and the remaining patients were planned for LITT. All LITT procedures were performed with the Visualase™ MRI-guided laser ablation system (Medtronic) using the 10-mm diffusing tip LITT probes. Follow-up consisted of scheduled meetings with the treating neurosurgeons 6 weeks and 3 months after surgery. Follow-up MRI was performed at 3 months after surgery. Patients filled in two quality of life questionnaires (QoL) (EQ-5D and EORTC QLQ - BN20) before and 3 months after surgery.Primary outcomes of the study were practical feasibility of a randomized trial in patients with nGBM; safety of the LITT procedure in combination with biopsy; technical feasibility of LITT at our center.Secondary endpoints of the study were 3-months survival, tumor volume change and change in QoL before and 3 months after treatment.A detailed description of the chosen outcomes measures is reported in the study protocol  and in the supplement of this paper.Descriptive statistics (mean ± standard deviation (SD) or median with an interquartile range for continuous variables, and frequencies or percentages for categorical variables) were computed using SPSS  to characterize the sample and examine feasibility outcomes. No comparative analysis was performed due to low sample size.The study was approved by the Institutional Review Board (METC Oost Nederland). Written informed consent was obtained from all participants before enrolment.The trial CONSORT flow-chart of the inclusion process is depicted in Fig. From November 2020 to February 2022, 27 patients were prospectively screened for eligibility. Of these, 21 met the inclusion criteria (78%), 18 (66%) were included and 15 (55%) were treated.Until September 2021, 19 patients were screened and 6 did not meet the inclusion criteria (KPS < 70 (n = 3), ablation not feasible (n = 3). Of the 13 patients invited to the study, 11 signed informed consent and were randomized (85%). Two patients decided not to be enrolled; one refused any treatment and one wanted to be treated elsewhere. After randomization 2 patients were excluded, one (randomized in LITT arm) withdrew consent for any treatment and one (randomized in the biopsy arm) developed a psychosis and was considered not mentally competent to continue in the study. With these findings, we considered the randomization feasible in this population and randomization was stopped to maximize experience with LITT. After September 2021, 8 additional patients were assessed for eligibility, and all met the inclusion criteria. One patient refused any treatment and one patient wanted to be treated elsewhere. In total, 15 patients were treated in the study, of which 5 in the biopsy group and 10 in the LITT group. As one patient in the biopsy group was excluded after treatment because of a non-GBM definitive histological diagnosis, 14 patients were included in the analyses. Baseline characteristics are shown in Table The clinical 3-months follow-up was completed by 13 out of 14 patients (93%), 12 patients (86%) filled in the QoL questionnaires, and 11 patients (78%) received a 3-months postoperative MRI.All patients in the study were treated within three weeks from inclusion and median time from procedure to CRT was 37 days, which in accordance with current national guidelines. Two patients did not start adjuvant treatment, one patient was deceased and the other had a KPS < 70. The median procedure time for biopsy followed by LITT was 206 min. In one case, intra-operative revision was necessary due to malposition. In our series, no disagreement between frozen section and definitive histology was found.A total of 13 adverse events (AEs) occurred in 8 patients in the LITT arm and in 1 patient in the biopsy arm . Nine AEs were considered not related to the procedure. Four AEs were considered related to the procedure: one patient in the biopsy arm developed a bleeding after biopsy, leading to a mild dysphasia. One patient in the LITT arm developed post-operative edema leading to hydrocephalus (necessitating shunting) and increased neurological deficits (leading to a KPS < 70). One patient in the LITT arm died as a result of progressive bleeding after biopsy. Overall major complication rate (CD ≥ 3) was 20% and one case of post-operative mortality occurred in in the LITT arm. No major complications occurred in the biopsy arm.An overview of the primary outcomes is depicted in Table At 3-months follow-up 13 out of 14 patients were alive and 1 patient in the LITT arm died after a complication. Median KPS worsened in both control and LITT group at 6 weeks and 3-months follow-up.All patients completed the online QoL questionnaire at screening and 12 out of 14  at 3-months follow-up. Patients in both groups showed a worse QLQ-BN20 Mean Total Score and lower EQ-5D Mean Index Value and Mean Visual Analogue Score.Eleven patients  underwent a 3-month follow-up MRI. Mean tumor volume increased on 3-months post-operative MRI in both groups, both in T2 and T1 with contrast.An overview of the secondary outcomes is depicted in Table The main findings of this pilot study are: (1) randomization seems feasible in this patient population and adherence to the study protocol and follow-up procedures was good; (2) combining stereotactic biopsy with LITT in the same session is feasible and does not cause any delay in planning surgery or starting adjuvant therapy; (3) the safety profile of the procedure appears in line with results reported in literature for LITT  and cranThe inclusion rate of screened patients was 66%, and most of them (85%) were willing to be randomized, which shows that patients are motivated to participate to the study. None of the patients withdrew after the treatment, which suggests that the follow-up procedures were well tolerated. As inclusions were considerably hindered at the beginning of the trial due to the COVID-19 pandemic, our recruitment rate was lower than anticipated with 18 patients in 15 months instead of 20 patients in 12 months.Two serious complications (CD ≥ 3) occurred in two LITT patients. One case of post-ablation edema leading to hydrocephalus necessitating shunting and new neurological deficits, and one case of progressive post-operative hemorrhage resulting in death. Post-ablation edema is a known complication in LITT (8.82–35.5%) , 16, 17,Other reported AEs such as DVT and epilepsy were not considered directly related to the procedure as all of them presented more than 30 days after ablation and are often described in patients with glioblastoma , 27.Median KPS and mean QoL scores seem to worsen and mean tumor volumes seem to increase over 3 months in both groups. This probably reflects the aggressive and progressive nature of the disease. No conclusion on differences between the two groups can be drawn given the number of included patients. Even though a trend toward less growth with stable median KPS and quality of life was noticed, an appropriate powered study is necessary to determine the effect of LITT on disease progression.This feasibility study helped the design of a full-scale multicenter RCT. Five key lessons emerged from our pilot study.First, multifocality and cystic degeneration were initially considered as exclusion criteria, in line with previously published series , 28. In Second, after the post-operative hemorrhage complication, an amendment to the study protocol was made to interrupt the procedure in case of significant bleeding after biopsy. We are aware that in some centers biopsy and ablation are performed in two different sessions, but in our opinion this approach causes several disadvantages, exposing patients twice to a surgical procedure and general anesthesia, delaying the start of adjuvant therapy and incrementing costs.Third, we noted that prevention of peroperative CSF leakage is essential to avoid laser misplacement. In one case an intraoperative revision was necessary because of an inadequate laser probe position. The inaccuracy was probably due to a combination of a navigation registration error and perioperative cerebrospinal fluid (CFS) leakage, and we were able to correct the position by navigating on the intraoperative MRI. We therefore advise to use the intraoperative MRI scan in case of replacing the probes.Fourth, evaluation of real ablation volume on post-operative MRI is challenging in patients with glioblastoma, as ablation zone appears as a peripherally enhancing rim on T1-weighted contrast-enhanced images , and is Fifth, 2 out of 14 patients (14%) were unable to complete the QoL questionnaires since their clinical condition was too poor or because the patient had died. In future studies, it should be considered that data on QoL from the clinically most compromised patients might be incomplete.The major strength of our study is that, as far as we are aware, this is the first prospectively controlled (feasibility) study for LITT in patients with newly diagnosed glioblastoma. As opposed to previous non controlled series, we provide more reliable insights in general applicability of this technique and practicability of designing a larger clinical trial in this population. The most important limitation of this pilot study is the small sample size collected from a single center limiting any precise comparison between groups or extrapolation to other centers. Furthermore, the recruitment rate was affected by the COVID-19 pandemic, so some uncertainty remains about the inclusion rate in the future.In conclusion, our pilot study showed that patients with irresectable nGBM showed high willingness to participate in a randomized study investigating LITT in comparison to standard of care, with good adherence to the study procedures. The study protocol was feasible and initial safety data appear acceptable when compared to craniotomy for tumor resection. To study the effectiveness of LITT in patients with nGBM a larger trial is needed. The outcomes of this pilot study helped to further refine the design of multicenter RCT, which is currently recruiting patients in the Netherlands.Supplementary material 1 (DOCX 214.7 kb)Below is the link to the electronic supplementary material.