Salmonella disease (iNTS) is a major cause of morbidity and mortality globally, particularly as a cause of bloodstream infection in children and immunocompromised adults in sub-Saharan Africa. Vaccines to prevent non-typhoidal Salmonella (NTS) would represent a valuable public health tool in this setting to avert cases and prevent expansion of antimicrobial resistance. Several NTS and combination typhoidal-NTS vaccine candidates are in early-stage development, although the pathway to licensure is unclear due to challenges in conducting large phase III field trials.Invasive non-typhoidalSalmonella CHIMs have been conducted safely and have played pivotal roles in progressing vaccine candidates to pre-qualification and licensure. The Challenge Non-TyphoidalSalmonella (CHANTS) consortium has been formed with funding from the Wellcome Trust, to deliver the first NTS CHIM, which can act as a platform for future vaccine evaluation. Controlled human infection models (CHIM) present an opportunity to accelerate vaccine development for a range of enteric pathogens. Several recent typhoidal This paper reports the conclusions of a consultation group workshop convened with key stakeholders. The aims of this meeting were to: (1) define the rationale for an NTS CHIM (2) map the NTS vaccine pipeline (3) refine study design and (4) establish potential future use cases. The views expressed in this article are those of the author(s). Publication in Wellcome Open Research does not imply endorsement by Wellcome.. Studies involving the controlled exposure of healthy volunteers to pathogenic or attenuated pathogens have been used for several decades to better understand the pathophysiology of disease – complementing insights from pre-clinical and field studies. Historically, a major use of such models has been in the field of vaccine development, in which a CHIM can be used to generate an early indication of vaccine efficacy and to suggest immune correlates of protection. This is particularly true for enteric pathogens for which, to date, CHIM studies have had a pivotal role in accelerating vaccine development for pathogens such asVibrio cholerae andSalmonella Typhi, and are predicted to have an important role in vaccine development forShigella spp.Controlled human infection models (CHIM) provide a valuable method to study infectious diseasesSalmonella (iNTS). Over 2600 non-typhoidal Salmonella (NTS) serovars exist, which affect a broad host-range and are associated with a spectrum of clinical phenotypes in human infection, exhibiting significant geographic variation. In high-income settings, NTS infections primarily cause a self-limiting enterocolitis, termed diarrhoeal NTS (dNTS). In contrast, invasive non-typhoidalSalmonella disease (iNTS) typically presents as a non-specific febrile illness – often without gastrointestinal symptoms – following invasion into sterile sites causing bloodstream infection, meningitis, or sepsis. iNTS disease is a major cause of morbidity and mortality in sub-Saharan Africa (SSA), primarily affecting immunocompromised hosts., The most recent GBD 2019 data estimate iNTS were responsible for 535,000 cases, 77,500 deaths, and 4·26 million lost disability-adjusted life years (DALYs) in 2017. The precise burden of disease is, however, relatively poorly understood and estimates vary depending on case-definitions and methods of surveillance. The global burden of diarrhoeal disease attributable to non-typhoidalSalmonella serovars (dNTS) is less well characterised, but likely is responsible for a larger case load – preliminary estimates from the Institute of Health Metrics and Evaluation (IHME) give estimates of 73.9 million cases and 61,600 deaths attributable to dNTS in 2019. The World Health Organisation (WHO) has recently listedSalmonella enterica as a priority pathogen, identified as one of 12 families of bacteria that pose the greatest risk to human health through rising antimicrobial resistance.There is growing interest and awareness by regulatory agencies of the utility of CHIM studies in vaccine development for diseases which occur sporadically, or for which field efficacy trials are difficult to conduct. One such disease is invasive non-typhoidal. Vaccine development for iNTS is hampered, in part, by an incomplete understanding of mechanisms and determinants of immunity during natural infection, as well as a lack of commercial incentive. In addition, the epidemiology of iNTS disease dictates that the large-scale phase III trials of vaccine candidates needed to establish efficacy will require a large financial and time commitment. The most efficient pathway to vaccine licensure and deployment is unclear.Vaccines for iNTS would represent a valuable public-health tool in sub-Saharan Africa, in part because other effective methods for disease control are – arguably – slow and cost prohibitive in many endemic countries in the short-term. Several candidate vaccines are in development, including conjugate vaccines, live-attenuated oral vaccines, and those using the generalised modules for membrane antigens (GMMA) platform, typically in multivalent formulationsSalmonella serovars. The use-case for an NTS CHIM in vaccine assessment is controversial. Recently, the Wellcome Trust has funded the establishment of the Challenge Non-TyphoidalSalmonella (CHANTS) consortium, to develop a first-in-human model ofSalmonella Typhimurium infection using contemporary strains. This consortium brings together stakeholders with expertise inSalmonella biology and controlled human infection studies to design a safe clinical study that de-risks the development of an NTS CHIM. The vision of this program is to develop an iNTS CHIM that dovetails with future iNTS vaccine efficacy studies, whilst simultaneously addressing fundamental questions on the immunological basis of susceptibility to iNTS disease.Unlike other enteric pathogens, there is relatively limited experience of controlled human infection studies of non-typhoidalA new CHIM for NTS will only significantly advance vaccine development if careful consideration is given to study design at an early stage, particularly with regards to selection of clinically meaningful endpoints and setting appropriate efficacy thresholds. Additionally, understanding the needs of key stakeholders is necessary to map out a potential use case in a vaccine product development pathway.th July 2022, with the aim of gathering input into the design and use-case of the proposed NTS CHIM. The consultation group meeting aimed to link the CHANTS consortium with key stakeholders in the NTS vaccine field, including regulators, academic researchers, funders, and vaccine developers. This meeting report summarises the key discussion points and outcomes from the workshop. We begin by reviewing the rationale for the development of an NTS CHIM and identify key learning points from other human challenge models for enteric diseases. This report focuses on priority topics discussed in the meeting, including challenge strain selection; choice of endpoints; clinical considerations and mapping out a future use-case.To address these issues, a consultation group workshop meeting was convened on 5Salmonella Typhi, NTS serovars typically have a broad host range. Several animal models have been developed to assess NTS candidate vaccines. Many of these models have provided valuable pre-clinical insights into host-pathogen interactions but do not always replicate the manifestations of disease in a context relevant for vaccine development. Historically, human challenge with wild-type or attenuated NTS serovars has been performed in a small number of volunteers, but a large-scale programme of activities for vaccine assessment has not previously been established.Unlike human host-restrictedIn the absence of an established NTS CHIM, there are several unknowns that hamper vaccine development. An NTS CHIM provides opportunities to understand NTS disease pathogenesis  and the mechanisms that drive these distinct clinical phenotypes. Investigation of immune responses to the challenge agent at time points before and after exposure will provide insights into mechanisms of protective immunity and identification of immune correlates of protection. Once established, an NTS CHIM would provide a platform to evaluate vaccine efficacy and support licensure of vaccine candidates currently in early phase evaluation. Furthermore, controlled challenge with NTS can elucidate novel target antigens for future vaccine development, identify diagnostic biomarkers, and facilitate assessment of the impact of infection on the host gastrointestinal microbiome. Whilst the anticipated benefits of conducting an NTS CHIM are many, there are several unknowns in relation to clinical responses to challenge. In particular, an NTS CHIM in healthy, immunocompetent adults from high-income settings is unlikely to accurately replicate an invasive disease phenotype most frequently observed in immunocompromised children with risk factors, including prematurity, malnutrition, and malaria infection, in lower- and middle-income countries (LMICs). . Acknowledging the gaps which remain in this field, it was concluded that to address these questions, a CHIM could be established to characterise the response to wild type NTS challenge in order to understand if – and how – a human challenge model of NTS disease could be used for future vaccine assessment.These uncertainties have been discussed at several expert meetings in the preceding years and have been discussed elsewhereThe CHANTS consortium has since been funded by Wellcome Trust, including key partners from Imperial College London, PATH, University of Oxford, Malawi-Liverpool-Wellcome Trust Clinical Research Programme, and the University of Liverpool. This consortium aims to establish an NTS CHIM that offers maximum insight from controlled human infection, whilst being underpinned by the central principle of volunteer safety.In the first session, the workshop discussed the overarching considerations pertaining to the safety and ethical acceptability of an NTS challenge model. The principal tension in the development of an NTS CHIM is developing a framework that prioritises safety and ethical considerations, whilst simultaneously providing a clinically meaningful way to test vaccines that protect against invasive disease. Specifically, the development of a dNTS challenge model would pose fewer safety and ethical challenges but would not necessarily help to accelerate vaccines designed to address iNTS disease.Salmonella Typhi and Paratyphi CHIMs are good examples of this, as BSI occurs following challenge with these serovars in healthy, immunocompetent adults and responds rapidly to effective antibiotic treatment. In contrast, BSI withSalmonella enterica subspeciesenterica serovars Typhimurium and Enteritidis is almost exclusively observed in clinically vulnerable populations with impaired host immunity. This includes a high mortality rate (5–15%) and the potential to seed deep-seated infections. It is plausible that silent or pauci-symptomatic BSI  could be seen after challenge of healthy volunteers with NTS, but this is unlikely to represent the same pathophysiological processes as those associated with iNTS disease in the target population for vaccination.A CHIM that reliably achieves bloodstream infection (BSI) would have the greatest utility when viewed solely from the perspective of vaccine efficacy assessments against invasive disease. PreviousSalmonella. Most CHIMs typically start by challenging exceptionally healthy volunteers in a narrow age range . It was acknowledged outright this population are not representative of the population at greatest risk of iNTS and are unlikely to develop invasive disease.Well recognised host-risk factors associated with invasive disease includes extremes of age, recent malaria infection, sickle cell disease, malnutrition, advanced HIV infection in adults and other defects in the immune system. Volunteers with these characteristics could arguably never be safely or ethically challenged with virulentSalmonella Typhimurium ST313 isolates have been found in both invasive and diarrhoeal infections. The relative contribution of pathogen specific factors in achieving invasive disease within the context of the model was discussed by the consultation group and is summarised below (Study Design). Importantly, it was noted thatThe consortium had a high degree of confidence in the establishment of a colonisation and gastrointestinal NTS disease model in immunocompetent adults. It was acknowledged that the prospect of achieving an invasive disease model representative of iNTS is less secure. The session finished by highlighting the key safety considerations which would be adopted to de-risk the model for participants, namely: establishment of an independent data and safety monitoring committee; thresholds for initiation of antimicrobial therapy; quality assurance of strain manufacture and administration, and close liaison with public health authorities including the local health protection team and the UK Health Security Agency (UKHSA) in view of potential secondary household or community transmission.. Development of effective vaccines for NTS may contribute to reduced case numbers, thus reducing antibiotic use and the expansion of antimicrobial resistance. The next session aimed to put an NTS CHIM within the context of vaccines currently under assessment. Key questions to address included where a CHIM would sit within the vaccine product development pathway, and what additional data could be generated that would meaningfully accelerate vaccine development.As listed WHO priority pathogens, invasive NTS isolates are increasingly associated with multi-drug resistance, significantly limiting treatment optionsIn this workshop, Professor Cal MacLennan outlined the NTS vaccine landscape as of July 2022. He defined several key considerations in NTS vaccine development. These included:•    Whether an NTS vaccine could be developed as a stand-alone product or formulated in combination with other serovars•    Whether such a vaccine could provide protection against both invasive and diarrhoeal disease•    What would be the most appropriate age of administration and alignment within the Expanded Programme on Immunisation (EPI) schedule?•    What is the expected duration and putative mechanisms of protection?•    What would be the efficacy of a vaccine in individuals with comorbidities at higher risk of developing NTS?S. Typhimurium andS. Enteritidis. The most advanced include a bivalent conjugate vaccine comprising core and O-polysaccharides conjugated to serovar specific phase I flagellin protein (FliC) (University of Maryland/Bharat Biotech), where Phase I studies are complete. Additionally, a bivalent vaccine developed by GSK Vaccines for Global Health (GVGH), based on the generalised modules for membrane antigens (GMMA) platform, is approaching Phase I. Several other vaccines are in pre-clinical testing including a bivalent NTS vaccine developed by Boston Children’s Hospital based on the multiple antigen presenting system (MAPS) technology.Several NTS vaccines are currently under development which aim to targetSalmonella vaccines candidates are also in pre-clinical testing. These include a trivalent NTS ; a trivalent NTS + Vi-CRM197 (GVGH); and a trivalent NTS + Vi-DT (IVI/SK). In addition, hypothetical quadrivalent vaccines for enteric fever (S. Typhi and Paratyphi A) withSalmonella Typhimurium and Enteritidis have been proposed.Several expanded-valency formulations of NTS and typhoidalSalmonella serovars, as the priority for protection is a time-limited period where the burden of disease is highest – specifically the first three years of life. Cost effectiveness would rely on having the capacity to mass produce and roll-out a vaccine at low-cost.Analyses indicate that iNTS vaccines would be cost-effective in resource-limited countries, particularly when combined with otherAs phase I data from vaccine trials become available, it was acknowledged there is a need to engage with vaccine developers early, particularly with respect to where a CHIM would fit in an already well-defined development pipeline. Until readouts from a pilot NTS CHIM are known, the future utility of the model for vaccine assessment remain uncertain.Having considered the rationale for an NTS CHIM and its potential utility for supporting vaccine development, the consultation group reviewed different proposed challenge study designs, based on a draft template protocol. Salmonella Typhimurium administered in sodium bicarbonate in a dose escalation model targeting an attack rate of 60–75%.The originally proposed study was to perform oral challenge with two wild-type strains ofThis study proposes to use two challenge strains belonging to ST19 and ST313 sequence types. This approach would directly address the contribution of African ST313 lineages to systemic disease compared with non-invasive isolates.in vitro andin vivo; be capable of causing the disease of interest; and possess a representative array of antigens and virulence factors.Dr Blanca Perez Sepulveda (University of Liverpool) outlined the rationale for the choice of strains used in this model. All human challenge models are limited in the number of strains that can be used and are frequently criticised for failing to replicate the global diversity of strains responsible for infection in endemic settings. Where possible, bacterial strains used for challenge should: be representative of circulating strains; be sensitive to multiple antibiotics used for treatment; have a traceable history from isolation; have been well characterised phenotypically and genotypicallySalmonella serovars Typhimurium and Enteritidis.S. Typhimurium accounts for approximately two-thirds of iNTS in Africa. Sequencing of isolates from across Africa has identified multilocus sequence type (MLST) 313 as the major cause of invasive disease, which is distinct from the sequence types 19 and 34, which are more frequently associated with gastrointestinal disease in high-income settings. ST313 appears to have undergone sequential evolutionary changes that converge towards a genotype associated with invasive disease, in the same way as reported forSalmonella Typhi and Paratyphi serovars. The key changes are characterized by stepwise genomic degradation typified by multiple gene deletions and pseudogene formation, often in genes associated with gastrointestinal survival. The data suggests that ST313 has adapted to a human niche and can evade mucosal immunity. Similar findings have been observed in invasiveS. Enteritidis ST11 isolates from Africa. Transmission of invasive isolates is complex and likely involves a combination of zoonotic reservoirs (particularly forS. Enteritidis) as well as human-to-human transmission. Invasive NTS isolates are increasingly associated with multi-drug resistance, significantly limiting treatment options.The NTS serovars commonly isolated in BSI varies by location, but usually include. Consensus was reached that challenge with highly characterised pathogens is advantageous from a participant safety perspective. This study proposes to manufacture challenge stocks from the ST313 lineage 2 reference strain D23580, which can be considered as an archetypal isolate representative of strains causing invasive disease in sub-Saharan Africa. D23580 was originally isolated in 2004 from a blood culture taken from a 24-month-old HIV-negative child at Queen Elizabeth Central Hospital, Blantyre, Malawi. D23580 harbours four plasmids, including pSLT-BT, which encodes drug resistance to chloramphenicol, ampicillin, streptomycin, sulphonamides, and trimethoprim. The presence of drug resistant mobile genetic elements within D23580 was felt not to represent a significant risk when balanced against the benefits of using a well-characterised strain that is reliably sensitive to fluoroquinolones and more representative of isolates detected from endemic settings. In parallel, it was proposed to manufacture a challenge stock of the ST19 4/74 strain, originally isolated from a calf in England in 1974. Genomic analysis indicates that it is an archetypal ST19 strain, representative of current clinical infections. Both strains have undergone detailed genotypic, transcriptomic, proteomic, and phenotypic characterisation. Whilst challenge withS. Enteritidis would be valuable, resource limitations currently preclude stock development, and there was agreement that the most valuable data, within resource constraints, would be generated from aS. Typhimurium CHIM.These proposals are predicated on the hypothesis that challenge of healthy volunteers with ST313 will be associated with a distinct clinical phenotype, as compared to challenge with ST19. There is a substantial body of phenotypic, genomic, and epidemiological evidence to indicate that infection with specific African lineages of ST313 will be associated with less gastrointestinal disease as compared with ST19 pathovariants3 CFU was rational, based upon prior experience withS. Typhi and Paratyphi A challenge administered with sodium bicarbonate.The consultation group were invited to consider challenge dosing. It was raised that the dose needed to achieve 60–75% attack rates might vary between strains and depend on what clinical endpoints are selected. For example, a relatively low dose of ST19 may cause a higher proportion of volunteers developing diarrhoeal disease compared with ST313. In view of the novelty of this model, it was suggested that a starting dose of 10Salmonella studies. It was recommended that stability testing should be performed at least annually, and more frequently at the start of the study to ensure the challenge agent remains stable. Characterisation tests should include antibiotic susceptibility testing, whole genome sequencing and growth characteristics in different media and survival tests as a minimum. All strains will be manufactured to GMP standard at the pilot biomanufacturing facility of the Walter Reed Army Institute of Research , following protocols previously established for typhoidalThe selection of appropriate endpoints is critical to ensure utility of the model in future vaccine efficacy studies. An NTS CHIM could be designed to result in colonisation, diarrhoeal disease, systemic illness and/or bacteraemia. It was emphasised that these endpoints aren’t mutually exclusive and that a participant may progress from one state to another. Exposure to the gastrointestinal tract is a prerequisite for the development of invasive disease, but kinetics of invasion after exposure are poorly understood. The consultation group discussed the relative merits of developing a colonisation/early infection model, or a disease model. It was acknowledged that the primary goal of vaccine development is to protect against invasive disease, but that protection against diarrhoeagenic disease would also be beneficial.The consultation group was invited to consider the following possible endpoint definitions of Salmonellosis, outlined inThe consultation group discussed the merits and demerits of defining a composite endpoint or co-primary endpoints for each definition, generating attack rates for each of the definitions. Secondary and exploratory endpoints would also be generated, including duration/magnitude of diarrhoea, shedding, seroconversion, symptom profile and blood culture clearance.Colonisation was thought to be insufficient as a surrogate marker for invasive disease. The primary concern was that colonisation as a primary endpoint will likely be achieved at a low infecting dose, preventing further dose escalation. Bloodstream infection and/or fever would be favourable in terms of model utility; however, care would be needed to minimise risks, as outlined above. It was acknowledged that the dose required to achieve invasive infection in healthy participants may be many orders of magnitude higher than in natural exposure, setting an unachievable threshold to demonstrate vaccine efficacy. The consultation group agreed that endpoints should have some flexibility, as the outcome following challenge is unknown.There was consensus that a composite endpoint comprising colonisation, diarrhoeal disease and bacteraemia would not be informative. Co-primary endpoints would be preferable, to provide attack rates for colonisation, gastroenteritis and systemic disease and highlight strain differences. The challenge doses used could be escalated until safety signals require the escalation to be halted. Diarrhoea without fever could be designated as a secondary/exploratory endpoint so this would not preclude further dose escalation. The group highlighted the risk of prematurely ending the study if the target attack rate is achieved using a primary endpoint that is not representative of iNTS disease. There was broad consensus in having the scope to dose escalate with appropriate oversight by a data safety monitoring committee (DSMC).Dose ranges and ceilings were also discussed. A stage might be reached where the investigators and DSMC consider the challenge dose to be grossly unrepresentative of natural exposure – at which point, consideration should be given to terminating the study. The consortium agreed that double-blind randomisation of participants is of value to maintain integrity in assessment of key subjective symptomatic outcome measures.In the final session related to study design, the consortium was invited to consider practical clinical aspects of study delivery. Key considerations included location, inpatient vs. outpatient model; inclusion and exclusion criteria (with safety as priority); pre-enrolment tests; infection control considerations; strategies to mitigate complicated disease and treatment protocols.Key clinical risks and risk mitigation strategies that should inform study design are highlighted inSalmonella gastroenteritis is typically self-limiting in healthy immunocompetent adults and that antibiotic treatment is not always indicated in this patient group – in part due to proposed risk of prolonged faecal shedding coupled with antibiotic related adverse events. Prolonged faecal shedding is less likely when fluoroquinolones are used for treatment of sensitive strains, when compared with other classes of antimicrobials. To protect participant safety and to minimise risk of prematurely terminating infection, treatment criteria should be distinct from diagnostic criteria. Signs of bloodstream infection or persistent fever will result in immediate antibiotic treatment, as will a combination of gastrointestinal symptoms that exceed a pre-defined threshold. Asymptomatic individuals who establish colonisation (positive stool culture detected on >2 occasions) could also receive antibiotic therapy at day 14. Whilst the use of antimicrobial therapy for uncomplicated NTS infection is contentious, the consortium agreed that antibiotic treatment could be justified within the context of a volunteer CHIM study with close post-challenge monitoring and assessment of clearance cultures. The consultation group also agreed that the study information booklet and screening procedures should counsel participants of the risks and benefits of antibiotic treatment.The consultation group discussed antibiotic treatment at length. Opinions differed on the value of offering antibiotic treatment to all participants regardless of symptoms. All members agreed that antibiotic treatment would be indicated in cases of invasive disease. It was acknowledged thatSalmonella CHIMs conducted as out-patient studies, the balance of risks, in conjunction with the novelty of challenge with NTS, led the group to favour an in-patient model. This followed from in-depth discussion regarding quarantine, balancing participant safety/public health with over-burdensome restrictions and the risk to mental well-being of participants. Discharge criteria and infection control considerations were developed with current public health guidance in mind. The UKHSA considers people non-infectious 48 hours after diarrhoea has ceased and clearance stool samples are not routinely required for NTS. In this study, participants will be quarantined for at least seven days, until resolution of diarrhoea for at least 48 hours, negative blood clearance cultures if bacteraemia develops, and be medically fit as assessed by a study clinician.Whilst extensive safety data are available from priorScreening and pre-enrolment testing will consider inclusion and exclusion criteria to mitigate risk of adverse outcomes. Only healthy participants will be considered for participation, with targeted investigations undertaken to exclude occult conditions which may predispose to complications . We will also exclude individuals with existing mild/sub-clinical irritable bowel syndrome (IBS) to avoid long-term complications post-infection. Salmonella exposure was not viewed as being feasible, owing to lack of standardised assays at the time of writing. Instead, a post-challenge analysis could be stratified by baseline sero-status to inform future studies. Practically, a clinical history and/or microbiological evidence of documentedSalmonella infection and/or prior Typhoid oral Ty21a vaccination, would exclude participation due to potential cross-reactivity.Serological pre-screening for priorThe final workshop session focussed on defining the future use case for an NTS CHIM. The consultation group agreed that any model would preferably have a clear use in NTS vaccine assessment whilst simultaneously allowing investigators a platform to better understand NTS infection biology. It was acknowledged that these are not mutually exclusive aims but that trade-offs may be required given feasibility and budgetary constraints. Shigella spp. andVibrio cholerae, respectively. Whilst this study offers the opportunity to investigate a wide range of immune responses to infection, the consortium agreed certain assays should be prioritised, and standardised, to align with those targeted by NTS vaccine candidates currently in development. The study proposes to characterise antigen-specific and functional antibody responses at baseline, during acute disease, and during convalescence. O-antigen ELISA and serum bactericidal assays will be utilised to characterise the humoral response to NTS challenge. Saliva and stool samples will be collected to characterise mucosal IgA responses at baseline and following challenge, whilst peripheral blood mononuclear cells will be used to measure antibody-secreting-cell and memory B-cell activity (via ELISpot assays). High throughput functional antibody assays, biophysical antibody profiling and Fc-glycosylation studies could also be undertaken as part of so-called systems serology platforms.A primary application of CHIMs is to define immune correlates of infection- or vaccine-derived immunity. Prior CHIMs for enteric pathogens have played a key role in elucidating serum anti-LPS IgG and vibriocidal antibodies as correlates of protection againstIt was acknowledged that correlates of protection may differ for colonisation, gastroenteritis, and invasive disease. Careful consideration is needed to balance de-risking phase 3 studies by providing early efficacy signals, and undermining confidence in vaccine candidates based on surrogate endpoints that might not be relevant to invasive disease. It was voiced that identification of correlates should be hypothesis driven and follow a well-constructed sample collection schedule. Exploratory measurements will include whole blood and/or single cell transcriptomics, which could also help to identify the establishment of invasive disease in study participants.The target population for any future NTS vaccine is anticipated to be children <5 years of age, although other groups including adults living with HIV may also benefit. Invasive disease is strongly linked to immune susceptibility; either in very young infants who lack protective antibody responses, or children with recent malaria infection, who may have macrophage and/or complement deficiencies. It was agreed that the main role of a CHIM for vaccine development would be increasing confidence that a candidate vaccine would work in these susceptible populations. Whilst a challenge study involving these groups could be considered ethically unjustifiable, it may be informative to have adult participants with recent malaria infection in an NTS challenge study and compare the data to immunogenicity studies from children administered a vaccine candidate as part of a field trial. From discussions held about the model described, there were no immediate concerns or additional issues perceived about future transfer of an NTS CHIM to endemic settings.The CHANTS workshop has successfully generated key recommendations to inform the establishment of an NTS challenge model. The aim of this workshop was to provide an expert consultation platform at an early stage of trial design. It is hoped that these contributions increase the likelihood that an NTS CHIM can be appropriately used to accelerate, and not hinder, vaccine development. Future consultation groups will likely be convened to report on preliminary data and refine the future use case of the model. Inputs from the expert consultation group will be considered alongside the outcome of public engagement and involvement meetings to inform the final trial protocol, which will be published once all approvals are in place. Continuing engagement with stakeholders will be key to defining and prioritising appropriate immunological endpoints to measure, specifically to ensure these are appropriately aligned and calibrated with assays currently being applied in iNTS vaccine trials.Vibrio cholerae andSalmonella Typhi. We contend that a safe, reproducible, and well-designed CHIM – with clinically meaningful endpoints – will allow the field to build upon insights from animal-models and could have an important role in accelerating vaccine development for iNTS.Our vision is to develop an iNTS CHIM programme that dovetails with future iNTS vaccine efficacy studies, whilst simultaneously addressing fundamental questions on the immunological basis of susceptibility to iNTS disease. Several iNTS candidate vaccines are in development, but the epidemiology of iNTS disease is such that phase-III trials require a large financial and time commitment before efficacy readout is obtained. There are several instances where CHIM studies have accelerated vaccine candidates through to licensure, in particular for enteric pathogens such as No further comments. Authors have included requested revisions.Does the article adequately reference differing views and opinions?YesAre all factual statements correct, and are statements and arguments made adequately supported by citations?YesIs the Open Letter written in accessible language?YesWhere applicable, are recommendations and next steps explained clearly for others to follow?YesIs the rationale for the Open Letter provided in sufficient detail?YesReviewer Expertise:Vaccine development, Salmonella, extracellular vesiclesI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. This article is an open letter detailing the conclusions of a group workshop regarding the development of a NTS vaccine. Several key challenges are made clear, including the host, ethical, and pathogenesis factors. The need for a NTS vaccine is made clear; however, what is not clear is the focus on NTS vs iNTS. In the safety and ethics section, it is suggested that the vaccine be tested against the ability to protect against invasive disease; however, the reasons why this is preferred to any NTS clinical symptoms were not clear. Since the host and bacterial factors that cause an NTS infection to become invasive are not well known, a serious barrier exists to the development of this CHIM. Additionally, over 2600 serovars of NTS exists, but the article does not make that clear. Of note, the selected challenge strain of ST313 is an excellent choice for creating a iNTS vaccine and the rationale written in the article is excellent. I recommend the acceptance of the article given that the authors revise the letter to clearly state 1) why protection against iNTS is preferred vs all NTS infections 2) the serovars the developed vaccine would protect against 3) the distinction between NTS, iNTS, and dNTSDoes the article adequately reference differing views and opinions?YesAre all factual statements correct, and are statements and arguments made adequately supported by citations?YesIs the Open Letter written in accessible language?YesWhere applicable, are recommendations and next steps explained clearly for others to follow?YesIs the rationale for the Open Letter provided in sufficient detail?YesReviewer Expertise:Vaccine development, Salmonella, extracellular vesiclesI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Reviewer 2 Thank you for your comprehensive review and feedback. "This article is an open letter detailing the conclusions of a group workshop regarding the development of a NTS vaccine. Several key challenges are made clear, including the host, ethical, and pathogenesis factors. The need for a NTS vaccine is made clear; however, what is not clear is the focus on NTS vs iNTS. In the safety and ethics section, it is suggested that the vaccine be tested against the ability to protect against invasive disease; however, the reasons why this is preferred to any NTS clinical symptoms were not clear. Since the host and bacterial factors that cause an NTS infection to become invasive are not well known, a serious barrier exists to the development of this CHIM."S. Typhimurium and/orS. Enteritidis – serovars which are known to account for >90% of iNTS disease causing isolates. The ideal vaccine candidate would offer effective protection against both iNTS and dNTS. However, in these early stages, the priority is to develop vaccines which offer effective protection against invasiveSalmonella disease. The rationale for this is highlighted in the stark differences in morbidity and mortality observed between iNTS and dNTS . Furthermore, increasing multi-drug resistance amongst iNTS disease causing serovars  highlights the urgent need for effective vaccination strategies in endemic settings to avert further expansion of antimicrobial resistance. It is anticipated that successful candidates could be in bivalent formulations, or trivalent, providing coverage for both NTS and typhoidal serovars in relevant settings.  We wholly agree our understanding of the bacterial and host factors leading to differential clinical phenotypes (iNTS vs. dNTS) is incomplete. We aim to gain a deeper understanding of these factors through this first-in-human NTS CHIM, and this is a primary rationale for the study. Thank you. The primary focus of this meeting was to discuss the potential merits of developing a NTS CHIM, and the possible applications such a model could have in supporting candidate vaccine evaluation and licensure. Development of these vaccine candidates is undertaken by other stakeholders and is not within the remit of the CHANTS consortium. Vaccines currently in early-stage development are reviewed in the ‘NTS vaccine landscape’ section. Candidates utilise differing vaccine platforms and technologies; however, all are targeted at providing protection against"Additionally, over 2600 serovars of NTS exists, but the article does not make that clear."   Thank you, this has been clarified in the introductory section."Of note, the selected challenge strain of ST313 is an excellent choice for creating a iNTS vaccine and the rationale written in the article is excellent.I recommend the acceptance of the article given that the authors revise the letter to clearly state 1) why protection against iNTS is preferred vs all NTS infections 2) the serovars the developed vaccine would protect against 3) the distinction between NTS, iNTS, and dNTS." Thank you. These clarifications have been highlighted in response to comments above and in the main body of the manuscript. Salmonella (NTS) controlled human infection model (CHIM).Thank you for the opportunity to review this Open Letter reporting on a 2022 CHANTS consortium workshop discussing a range of topics relevant to development of a non-typhoidal The authors and workshop participants are well-qualified to have pursued this initiative and their broad expertise adds weight to the discussion described in the report and its recommendations. The report itself is a mature and thoughtful work, addressing contentious questions and areas of uncertainty with the even-handed seriousness that has become characteristic of modern investigator-initiated human challenge research (a standard set by a number of the authors and attendees at the workshop). At the outset, there is a familiar tension here, as for many CHIM initiatives, in balancing the experimental aims to address unanswered questions related to NTS infection and disease in humans with the vision of the CHIM as a safe and robust clinical trial platform. Again, I think the authors have done a good job here, making the case that an NTS CHIM meets a “safe enough and useful enough” threshold to pursue further, looking forward to the use case for a successful CHIM without pre-judging the outcomes of the initial efforts to establish the CHIM. The focus on study endpoints was strong, reminiscent of the 2018 publication by Chad Porter and colleagues regarding clinical endpoints in the Shigella CHIM (PMID: 29590182). The point that CHIM endpoints must be selected with specific consideration of vaccine indications and endpoints in later phase clinical trials was well made. Similar points are made in the Discussion section of the typhoid CHIM trial that evaluated the Vi-TT vaccine  and the linked editorial commentary . As with most well-written meeting reports, there are few targets here for a specific critique. As the authors describe, there will be important decisions in developing the final protocol for the first NTS CHIM study. The devilish details of the final protocol may inspire more specific criticisms. The meeting report outlines a cautious general approach, for an inpatient trial in extensively screened volunteers  with a relatively low threshold for antibiotic therapy and a relatively stringent requirement for microbiological eradication afterwards. The risk assessment table is excellent and a model for other CHIM investigators. I’m not certain that clinical examination can truly ‘exclude valvular heart disease’ given the significant subclinical fraction revealed by screening echocardiography studies (e.g. for rheumatic heart disease). I’m unconvinced that there’s a specific need for further screening on this front though, if all other ‘healthy adult’ requirements are met. Somewhat similar, I wonder what is the prevalence of subclinical aortic and biliary tract pathology in otherwise healthy young adults? That said, so long as a standardised approach is taken to performing and reporting the abdominal ultrasounds, and members of the clinical trial team are trained to complete these targeted ultrasounds, they may not be too onerous, too expensive, and won’t exclude too many otherwise eligible volunteers. The term ‘double-blind randomisation’ will eventually need to be defined in more detail. Who exactly will be blinded? Presumably only the volunteers, the clinical trials facility staff, and those charged with assessing endpoints? Too much blinding too early in the development of a CHIM may be overly restrictive and prevent the almost inevitable flexible adjustments the protocol will require as early lessons emerge. I understand the choice to exclude volunteers previously vaccinated against typhoid from the initial trial. Although resource constraints may preclude challenging some of these volunteers at a dose level that achieves a high attack rate in unvaccinated volunteers, I do wonder if samples from these participants could make a meaningful contribution to the immunological work to follow the clinical trial. Given the investments required to launch a CHIM study, this idea may not happen at all if it doesn’t happen early and in parallel to another study. The final reference regarding systems serology introduces these methods in relation to HIV research. The same others  have since published systems serology investigations specifically related to CHIM studies  that would seem to be even more appropriate references here? I know that these authors have a strong record of work with clinicians and investigators in low- and middle-income countries. As they point out, the global burden of NTS disease is concentrated in LMICs and especially in Africa. However, there is limited representation from LMICs including African countries (besides Prof. Gordon) in the author group for this open letter and the workshop attendees. I do trust the consortium has plans to boost its engagement in this regard. Nonetheless, it does bear mentioning. I do note the authors have made a general comment in the conclusion regarding plans for stakeholder engagement. The rationale for this Open Letter is clear: to introduce the CHANTS consortium and its mission to a broader audience, describing the rationale for developing an NTS CHIM and the seriousness with which the consortium is considering the safety of prospective participants and how to design the model in a way that will boost its contribution to accelerating NTS vaccine development. It is never possible to predict 100% of all impacts that will flow from the successful establishment of a new CHIM. The contributors to this meeting have made a strong case that a CHIM can be pursued with sensible safety guardrails (safe enough) and that it is a worthwhile objective that is more likely than not to meaningfully contribute to NTS science and vaccine development beyond what has been achieved to date with in vitro studies, animal models, and studies of naturally infected humans (useful enough). I look forward to news of the study protocol and the early findings that emerge from a new NTS CHIM.Does the article adequately reference differing views and opinions?YesAre all factual statements correct, and are statements and arguments made adequately supported by citations?YesIs the Open Letter written in accessible language?YesWhere applicable, are recommendations and next steps explained clearly for others to follow?YesIs the rationale for the Open Letter provided in sufficient detail?YesReviewer Expertise:I am a paediatric infectious diseases physician and human infectious challenge researcher. In my PhD I led the CHIVAS-M75 study to establish a Streptococcus pyogenes pharyngitis CHIM in healthy adult volunteers. I am now undertaking S. pyogenes challenge trials to evaluate vaccines and accelerate development of improved long-acting penicillin formulations (primarily for secondary prevention of rheumatic heart disease). I am also involved in efforts towards developing new human models of bacterial and mycobacterial skin infections and sexually-transmitted infections.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Reviewer 1 Thank you for your comprehensive review and feedback. "I’m not certain that clinical examination can truly ‘exclude valvular heart disease’ given the significant subclinical fraction revealed by screening echocardiography studies (e.g. for rheumatic heart disease). I’m unconvinced that there’s a specific need for further screening on this front though, if all other ‘healthy adult’ requirements are met. Somewhat similar, I wonder what is the prevalence of subclinical aortic and biliary tract pathology in otherwise healthy young adults? That said, so long as a standardised approach is taken to performing and reporting the abdominal ultrasounds, and members of the clinical trial team are trained to complete these targeted ultrasounds, they may not be too onerous, too expensive, and won’t exclude too many otherwise eligible volunteers."Salmonella endocarditis is thought to represent a less likely complication even amongst the small proportion who may develop bacteraemia. Timely antibiotic therapy will be commenced immediately upon development of sustained fever and/or bacteraemia, significantly reducing the probability of such complications developing. Thank you. We wholly agree on points raised regarding potential subclinical aortic, valvular, and biliary tract disease. Our intended participant cohort will be comprised of healthy adults (aged 18-50) without chronic disease or risk factors for severe Salmonellosis. Screening protocols are designed to select such individuals through comprehensive medical history, physical examination, and review of primary care records. We recognise the pre-test probability of subclinical biliary or aortic disease in this cohort is very low. However, we have agreed on abdominal ultrasonography with standardised assessment and reporting protocols as an appropriate measure to exclude these specific risk factors for complicated infection. Abdominal USS is accessible, non-invasive, and can be performed and reported in a timely manner. Transthoracic echocardiography was carefully considered but felt unnecessary due to burden of imaging and reporting times – again, in the context of a low pre-test probability for subclinical valvular disease. Furthermore,"The term ‘double-blind randomisation’ will eventually need to be defined in more detail. Who exactly will be blinded? Presumably only the volunteers, the clinical trials facility staff, and those charged with assessing endpoints? Too much blinding too early in the development of a CHIM may be overly restrictive and prevent the almost inevitable flexible adjustments the protocol will require as early lessons emerge." Thank you. As suggested, blinding will be applied to participants, clinical study staff delivering direct patient care, and the same clinical team members responsible for assessment of clinical endpoints. Unblinding will occur at study conclusion, or as deemed necessary by the Data Safety and Monitoring Committee in response to safety thresholds.  "I understand the choice to exclude volunteers previously vaccinated against typhoid from the initial trial. Although resource constraints may preclude challenging some of these volunteers at a dose level that achieves a high attack rate in unvaccinated volunteers, I do wonder if samples from these participants could make a meaningful contribution to the immunological work to follow the clinical trial. Given the investments required to launch a CHIM study, this idea may not happen at all if it doesn’t happen early and in parallel to another study."Salmonella infection). As Ty21a is a live attenuated vaccine, this would be considered prior ‘oral challenge’ with a clinically relevantSalmonella serovar. No other prior formulation of typhoid vaccination would preclude participation. Immunological assays will be applied following the clinical study on samples obtained from all participants, irrespective of attack rate. Thank you. Participants will only be excluded if they have received prior vaccination with oral typhoid Ty21a vaccine  have since published systems serology investigations specifically related to CHIM studies  that would seem to be even more appropriate references here?" Thank you. The final references have been updated to reflect your appropriate suggestions."I know that these authors have a strong record of work with clinicians and investigators in low- and middle-income countries. As they point out, the global burden of NTS disease is concentrated in LMICs and especially in Africa. However, there is limited representation from LMICs including African countries (besides Prof. Gordon) in the author group for this open letter and the workshop attendees. I do trust the consortium has plans to boost its engagement in this regard. Nonetheless, it does bear mentioning. I do note the authors have made a general comment in the conclusion regarding plans for stakeholder engagement."   Thank you for this important comment. As suggested, we aim to maximise engagement with key stakeholders from LMICs, where the greatest burden of iNTS disease is concentrated. Members from academic and research institutions in sub-Saharan Africa have been consulted throughout the protocol development stages of this study, and also form central roles within our Data Safety and Monitoring Committee. Future applications and use case of the model will involve ongoing collaboration with representatives from SSA, including potential transfer of the model to an endemic setting, and in planning future vaccine efficacy studies.


We appreciated the comments of De Luca and colleagues  regardinWe agree with the authors regarding the surgical approach of pulsating intraoral metastases from renal cell carcinoma (RCC); preoperative vascular embolization should be performed in order to prevent massive hemorrhage during biopsy and surgery. However, in our case, the mass was not pulsatile and only a modest contrast enhancement was reported on imaging exams; indeed, a desmoid-type benign lesion was suspected.Cutaneous and subcutaneous RCC metastases, as reported by Al Abdrabalnabi et al. , can mimMetastasectomy in patients with RCC has never been analyzed in clinical randomized studies.  However,The relation between targeting agents and metastasectomy is not defined at present. However, in selected patients with a limited metastatic burden after systemic therapy, metastasectomy seems feasible with an acceptable morbidity . In a muMost of all, we thank the authors for sharing their therapeutic approach to solitary, accessible RCC metastatic lesion in head and neck and look forward to learning more about their relevant results; moreover, we agree that patients with a history of RCC should undergo proper monitoring even after a long disease-free survival.


Reading is a high-order cognitive process that is unique in human beings. There is a prolonged developmental course and a wide range of proficiency levels associated with reading. In this review, I focus on brain changes underlying Chinese reading development in both typical readers and readers with reading disability. Reading development in typical readers is characterized by a shift from dorsal phonological reading to ventral orthographic reading in the brain and increased interactive specialization in the reading network. Even though some individuals with reading disability may be able to catch up with typical readers on phonological reading by adulthood, they cannot reach fluent orthographic reading. In the brain, the reduction of brain activation in the left inferior frontal gyrus associated with reading disability disappears by adulthood, suggesting that this is a developmental delay, while there is a greater reduction of brain activation in the left inferior temporal gyrus in adults than children with reading disability. It suggests a greater deficit in the dorsal phonological reading pathway in children and a greater deficit in the ventral orthographic reading pathway in adults with reading disability. This review provides insights about the developmental trajectories in typical and atypical reading. Learning to read is one of the most important missions in child development. Significant structural and functional brain changes are accompanied with learning to read, and studying reading development in the developing brain offers a great opportunity to understand how learning experience interacts with brain maturation. Moreover, in comparison to typical readers, individuals with reading disability experience an atypical developmental trajectory of reading both behaviorally and neurologically. It is of great interest to understand how learning and development take place in the atypical brain. However, very few studies and reviews have concerned brain changes during reading development in readers with reading disability after formal schooling, and there is even less such research in Chinese reading disability. Chinese, a very contrastive language comparing to alphabetic languages, offers a chance to understand the generality and specificity in reading development and disorders across different languages. Chinese is a morpho-syllabic language, in which each character represents a morpheme and a syllable rather than a word. Chinese characters have more complex visual-spatial layout than alphabetic languages, in which strokes are packed in a square with top-down, left-right and inside-outside spatial arrangement of the strokes. Moreover, strokes do not map to phonemes in the syllable and the whole character has to be mapped to the whole syllable. Taken together, morphological skills and holistic visual orthographic configuration skills might be more essential than phonological skills in Chinese reading development and disability due to the specific features of Chinese.Reading is essentially mapping visual symbols to the spoken language system to acquire meaning. According to No developmental models have been proposed to explain Chinese reading development; however, studies have shown developmental changes in Chinese reading. For example, two proof-reading studies have found that children show a greater reliance on phonology while adults show a greater reliance on orthography during Chinese reading , suggestFurthermore, there is an even greater reliance on the direct mapping from orthography to semantics in proficient reading in Chinese than in English, due to the morpho-syllabic nature of the Chinese writing system. Each Chinese character represents a syllable and a morpheme with the existence of substantial homophones. Therefore, phonology is not reliable in mapping to meaning and the direct mapping from orthography to semantics is more efficient. Consistent with these features of Chinese, studies have also found that morphological awareness plays a more important role in reading development than phonological awareness, especially in later stage of Chinese reading development . AnotherConsistent with the reading developmental shift from phonological reading to orthographic reading, there is a dorsal to ventral shift in the reading brain . One impThe interactive specialization hypothesis  has beenIn addition, studies have also suggested increased interactivity between brain regions with reading development. For example, a study found greater connectivity in the prefrontal-superior temporal network in adults than children in audiovisual integration during Chinese reading . AnotherUnfortunately, about 7% of the population experience reading disability across different languages in the world according to a recent meta-analysis study , which iNeuroimaging research has revealed altered brain functions associated with RD. For example, reduced brain activation in the left occipito-temporal and left temporo-parietal areas has been reported , suggestAs for the left inferior frontal gyrus (IFG), however, there is less consensus about its abnormality in the literature of RD . Some stIn addition to the task difficulty and age issues, cross-linguistic differences may also explain the inconsistent findings about the left IFG across studies. A recent meta-analysis study on hundreds of previous studies has focused on language differences in brain structural and functional alterations associated with RD . It reveThe differences between children and adults with RD have been overlooked. Would they show a shift from phonological reading to orthographic reading? Would they show an increased interactive specialization with age? According to the limited studies, phonological awareness deficits seem to be a persistent problem in individuals with RD . Even thEven though, in Chinese, no published studies have compared children and adults with RD either behaviorally or neurologically, one recent study has compared Chinese dyselxic children and control children on behavioral tests from grade 2 to grade 6 . It was There have been very few studies that have actually concerned brain developmental changes in individuals with RD. A meta-analysis study has collected studies on children and adults with RD and made a comparison . It was No published studies have compared brain alterations in Chinese children and adults with RD. However, a recent study  has examper se. For typical children, even those younger ones with a lower reading skill, brain activation at this region was higher than RD children. Taken together, RD readers show a significant increase in phonological reading at the left IFG over development; however, they do not show adequate increase in orthographic reading in the left ITG.According to this study , it appeMoreover, the catch-up at the left IFG seems to be driven by over-connectivity with other brain regions especially in the right hemisphere in adults with RD . PreviouFindings of developmental changes in individuals with RD have important implications in the diagnosis and treatment of RD. For example, diagnosis and treatment may be focused on word reading accuracy in children but reading fluency in adults since we found children and adults with RD showed the most significant deficits in word reading accuracy and reading fluency, respectively. During reading treatment, brain stimulation at age-specific regions, such as the left IFG for children and left ITG for adults may be helpful, because we found reduced brain activation in the left IFG in children with RD and in the left ITG in adults with RD. Our findings provide important insights in developing age-specific and more individualized diagnosis and treatment programs for individuals with RD.Typical reading development is characterized by increased involvement of the ventral orthographic reading pathway and decreased involvement of the dorsal phonological reading pathway. It is also characterized by increased interactive specialization in the reading network, so that each region is only involved in certain computations and there is an increased connectivity between regions. Reading development in individuals with RD is characterized by developmental delay in word decoding accuracy, but persistent deficits in phonological awareness and word decoding fluency, and increased deficits in sentence reading fluency over development. In the brain, reading development in RD readers is characterized by developmental delay in phonological reading at the left IFG and a failure in shifting to the ventral orthographic reading in the left ITG. Atypical reading development is also characterized by a lack of developmental increase in task specialization, suggesting possible alternative learning mechanisms in the atypical brain.FC: Conceptualization, Funding acquisition, Writing – original draft, Writing – review and editing.


This study aimed at comparing several health outcomes in young adulthood among child refugees who settled in the different immigration and integration policy contexts of Denmark, Norway and Sweden.The study population included refugees born between 1972 and 1997 who immigrated before the age of 18 and settled in the three Nordic countries during 1986–2005. This population was followed up in national registers during 2006–2015 at ages 18–43 years and was compared with native-born majority populations in the same birth cohorts using sex-stratified and age-adjusted regression analyses.Refugee men in Denmark stood out with a consistent pattern of higher risks for mortality, disability/illness pension, psychiatric care and substance misuse relative to native-born majority Danish men, with risk estimates being higher than comparable estimates observed among refugee men in Norway and Sweden. Refugee men in Sweden and Norway also demonstrated increased risks relative to native-born majority population men for inpatient psychiatric care, and in Sweden also for disability/illness pension. With the exception of increased risk for psychotic disorders, outcomes among refugee women were largely similar to or better than those of native-born majority women in all countries.The observed cross-country differences in health indicators among refugees, and the poorer health outcomes of refugee men in Denmark in particular, may be understood in terms of marked differences in Nordic integration policies. However, female refugees in all three countries had better relative health outcomes than refugee men did, suggesting possible sex differentials that warrant further investigation. Throughout the past several decades, the Nordic countries have experienced demographic changes to their populations due to international migration. Since the 1970s, refugee and family reunification migration have been among the most common reasons for migration to the Nordic region. Children comprise a substantial proportion of this refugee population, with recent figures showing that up to one third of all refugees seeking asylum in the Nordic countries are younger than 18 [The health and well-being of immigrants is influenced by a confluence of risk and protective factors related to the pre-migration context, the migration experience and post-migration living conditions. Yet, given the nature of forced migration, refugees may experience difficulties that differ from those of other immigrants, including exposure to traumatic events in the country of origin, such as war, violence and perilous migration journeys ,3, whichRefugee children have similarly been shown to have increased risks for poor mental-health outcomes associated with exposure to violence  and postThe extent to which refugees who migrated as children can overcome traumatic pre- and peri-migration experiences as well as post-migration challenges is strongly impacted by the social context in the country of settlement and the resources available to them . The welUnlike pre- and peri-migration factors, those related to the post-migration context are modifiable determinants of immigrants’ health. Cross-country comparative studies of child refugees are particularly relevant for disentangling the role of the post-migration context for several reasons. First, young immigrants arriving to the country of settlement early in life have been exposed to the post-migration policy context for a prolonged period of time during their formative years. As such, comprehensive policy environments that facilitate integration can serve as a buffer against poor health outcomes, while more restrictive environments may create institutional barriers and exclusion that can contribute to the formation of poor health outcomes . Second,The aim of this study was to compare health outcomes of child refugees in young adulthood across three Nordic countries with diverse immigration and integration polices using MIPEX ratings as a platform for discussion of how different immigration and integration policies may directly and indirectly influence health outcomes.Throughout the remainder of the text, the term ‘native-born’ will be used to describe the native-born majority populations of the Nordic countries. The terms ‘refugees’, ‘refugee men’ and ‘refugee women’ will be used to describe individuals who migrated to the Nordic region during childhood as refugees. This includes both accompanied and unaccompanied minors who themselves were granted asylum on refugee grounds, as well as children who arrived as family relations to parents who had been granted asylum on such grounds.The refugee study population consisted of 27,413 refugees in Denmark, 17,160 in Norway and 109,408 in Sweden who were born between 1972 and 1997 and who were granted residency as children (0–17 years) between 1986 and 2005 (1990–2005 for refugees in Norway). Refugees’ health outcomes were studied from age 18 onwards during the period 2006–2015 and compared to the native-born population birth cohorts in the country of residence.National population registers in the three countries were used to compile information on demographics, income, education, medical care and cause of death . These dSix health outcomes were defined using ICD-10 diagnostic codes and included (a) all-cause mortality (between the ages of 18 and 43); (b) disability/illness pension due to disability or chronic illness at the age of 30 (limited to birth cohorts born between 1972 and 1985); and (c) four indicators of psychiatric care (between the ages of 18 and 43), including any inpatient psychiatric care, any outpatient psychiatric care, any inpatient care with a diagnosis of a psychotic disorder and any inpatient care with a diagnosis related to substance misuse.The health indicators used in this study depend to a considerable degree on the national context. As such, relative measures that make comparisons with the native-born populations in each country were used for cross-country comparisons. Cox regression was used to analyse risk of mortality and psychiatric care in the refugee populations relative to the native-born comparison populations. These analyses were based on person-time in the study, which was calculated from 1 January 2006 or the 18th birthday (whichever came last) to 31 December 2015, date of death or first outcome (whichever came first). These results are presented as hazard ratios (HR) with 95% confidence intervals (CI). Logistic regression was used to standardise disability/illness pension receipt at the age of 30, with results presented as percentages and odds ratios (OR). All analyses were sex stratified and adjusted for year of birth.For psychiatric outpatient care, refugee men in Denmark had the highest risk compared to the native-born . Relative to their native-born counterparts, refugee men in Norway also showed elevated risk , while refugee men from Sweden and refugee women from all countries had lower or similar risks, respectively.Refugee men and women had higher risks for inpatient care for psychotic disorders relative to the native-born in all countries. Elevated risks relative to the native-born were higher among refugee men than they were for refugee women in Denmark and Sweden, but the sex discrepancy was particularly pronounced when comparing refugee men  and women  in Denmark.Refugee men in Denmark had the highest risk of hospital admissions with a main or complimentary substance misuse diagnosis relative to the native-born , but refugee men in Sweden also showed elevated risk . Refugee men and women from Norway and refugee women from Denmark had risks similar to the native-born populations, while refugee women in Sweden showed decreased risk .This study of national cohorts of refugees who immigrated to Denmark, Norway and Sweden as children between 1986 and 2005 has described mortality, disability/illness pension and psychiatric care outcomes among refugees in young adulthood relative to their native-born counterparts. In all three countries, refugee men had higher risks of inpatient psychiatric care compared to native-born men, and refugees of both sexes had higher risks for inpatient care for psychotic disorders relative to native-born comparison populations. Refugee women generally demonstrated health outcomes that were more similar to the native-born populations than did refugee men in all countries. Refugee men from Denmark showed the highest risks for mortality, disability/illness pension, outpatient psychiatric care and inpatient care for substance misuse relative to the native-born.There are three main findings of this study: (a) with some notable exceptions, the risk for adverse health outcomes was generally higher among refugees compared to the native-born, (b) this pattern was largely driven by higher risks among refugee men than refugee women and (c) refugee men in Denmark had considerably higher risks compared to refugee men in Norway and Sweden.The poorer health outcomes observed among refugee men in Denmark could potentially be interpreted as better access to health-care services in Denmark. However, this seems unlikely, given the overall pattern of poorer health outcomes observed, including increased risk of mortality and disability/illness pension, indicators that are not confounded by barriers to care. In addition, the pattern of poorer health outcomes coincides with poorer educational attainment and labour market outcomes in Denmark, suggesting that social determinants such as these may have a negative influence on refugees’ health and well-being. It is also unlikely that these findings can be attributed to negative health selection. Despite some socio-demographic heterogeneity among the refugee study populations, previous population-based studies have shown comparable proportions of mental-health symptoms among refugee children who migrated to the Nordic region during the same time as those included in our study ,18, provPolicy differences were not a subject of empirical investigation in this study. Yet, the results may be considered in terms of the lower Danish MIPEX scores, indicative of an unfavourable policy environment for refugee integration. The policy environment may affect refugees’ health through multiple pathways . First, Second, Denmark’s lower MIPEX scores with regard to family reunification, anti-discrimination, access to nationality and a secure future are also important to consider. Experiences of discrimination , social Clear sex differentials in the health indicators were also observed. While refugee men in all countries had equal or poorer health outcomes relative to their male native-born counterparts, the differences between refugee women and native-born women were generally less pronounced, and refugee women also tended to show lower risks for poor health than refugee men. The extent to which differences between refugee men and women could be attributed to the policy environment is uncertain. However, the mechanisms through which the post-migration context influences health may be different for refugee men and women. Previous research has pointed to sex differences in achievement–expectation mismatch, social marginalisation and substance use as possible explanations for elevated risk of psychotic disorders among refugee men relative to refugee women . Other sDespite our use of total population cohorts, the low frequency of the outcomes assessed limited our ability to adjust for multiple covariates or further stratify the analyses. In addition, with the exception of mortality, we assessed indicators of health care and social benefits usage rather than health outcomes themselves. There was also some degree of heterogeneity by origin among refugees settled in each of the compared Nordic countries, which may limit cross-country comparability. The inpatient mentalhealth indicators used in this study reflected care for severe psychiatric disorders, which tend to be more common in men; it is possible that other types of health indicators (e.g. self-report) would have shown different patterns by sex and country of residence. The observational study design utilised also entails that the causal mechanisms between immigration and integration policy contexts and health can only be speculated upon. Further studies are needed to investigate whether restrictive policy changes have had a detrimental impact on the health of refugees and to address the limitations described above.This cross-country comparative study of three Nordic welfare states demonstrates that young adult refugee men in Denmark – the country with the most restrictive immigration policy – have poorer health than native-born Danish men, as well as young refugee men in Sweden and Norway. Recent trends towards more restrictive immigration policies  in all t


Introduction and importance: This case report highlights the multidisciplinary approach required to achieve successful anatomical and functional outcomes, in an eye with total limbal stem cell deficiency (LSCD) associated with underlying corneal scarring and thinning.Presentation of case: A 59-year-old gentleman had poor visual recovery in the right eye (RE) following accidental carbide blast, 1-year before presenting to us. The visual acuity was counting fingers and clinical examination revealed cicatricial entropion involving the upper eyelid, total LSCD, corneal scarring with a central descemetocele and cataract in the RE. Prior to ocular surface reconstruction, entropion correction was performed. Three months later, penetrating keratoplasty combined with cataract surgery and intraocular lens implantation (penetrating keratoplasty (PK) triple), with autologous simple limbal epithelial transplantation (SLET) was performed. The visual acuity was 20/100, 18 months after the surgery, with a clear well-epithelized corneal graft and stable ocular surface.Discussion: LSCD is caused by a decrease in the population and /or function of the limbal epithelial stem cells. Limbal stem cell transplantation (LSCT) is warranted in eyes with total LSCD. In eyes with coexisting corneal scarring, LSCT alone may be inadequate to restore the vision. These eyes require simultaneous or sequential lamellar or full-thickness corneal transplantation for visual rehabilitation. Though, the existing literature favors a sequential approach, where LSCT is performed first followed by corneal transplantation, under certain circumstances such as a thin underlying cornea like in our case, corneal transplantation may have to be combined with LSCT to achieve optimal outcomes.Conclusion: Combining autologous SLET with PK can be performed for visual rehabilitation in eyes with unilateral total LSCD and underlying corneal thinning. Corneal and limbal graft survival is prolonged if existing adnexal comorbidities are addressed before any surgical intervention is planned and adequate time interval is allowed for the surface inflammation to subside. Visual rehabilitation in these eyes is extremely challenging. To treat LSCD, limbal stem cell transplantation (LSCT) is required. This procedure can be autologous in cases of unilateral LSCD where the limbal epithelial stem cells (LESCs) are harvested from the contralateral eye or this can be allogeneic where the LESCs are harvested from a living-related donor or a cadaveric cornea.– In eyes with total LSCD and underlying clear corneal stroma, only performing LSCT may stabilize the ocular surface and provide adequate visual outcomes. However, in eyes with underlying full-thickness corneal scarring, performing only LSCT may not be sufficient to achieve optimal functional outcomes. In such eyes, a deep anterior lamellar keratoplasty (DALK) or a penetrating keratoplasty (PK) is warranted to restore the corneal transparency. However, in the presence of LSCD, performing a PK or DALK alone is known to have very poor outcomes with a higher risk of graft rejection and failure associated with ocular surface inflammation causing persistent epithelial defects. In such eyes, optimal visual recovery can be achieved by performing a corneal transplantation either simultaneously with LSCT or sequentially after LSCT.Ocular chemical injuries are often associated with poor long-term visual outcomes due to chronic sequelae such as limbal stem cell deficiency (LSCD), corneal scarring, and vascularization., Poor outcomes have been documented with simultaneous PK and LSCT, with the corneal graft survival ranging from 35% to 64%., At present, there are no existing guidelines supporting the relative timing of the two transplant procedures and whether they can be combined as a single stage procedure. In certain cases, performing LSCT and PK becomes inevitable as LSCT alone cannot be performed due to residual corneal thickness being inadequate. We report a case where PK was performed simultaneously with autologous LSCT in an eye with total unilateral LSCD with descemetocele with surrounding corneal scarring and cataract.The published literature recommends that a corneal transplant should be performed once the surface inflammation has decreased, the corneal epithelium is stable and regular, and there is regression of stromal vascularization.A 59-year-old Asian Indian male, a farmer by occupation, presented with gradual loss of vision in the right eye (RE) over a period of 1 year, associated with pain, watering and redness following accidental carbide blast. In the acute phase, amniotic membrane transplantation (AMT) was performed twice by the primary ophthalmologist. At presentation, the best corrected visual acuity (BCVA) was counting fingers close to face (CFCF) and 20/20 in the right and left eye (LE), respectively. On clinical examination, the RE showed cicatricial entropion involving the upper eyelid . Underly A bandage contact lens was placed over the corneal surface. Lateral paramedian permanent tarsorrhaphy was performed to promote epithelization and to protect the ocular surface. Postoperatively, prednisolone acetate (1%) eye drops 1 hourly and moxifloxacin eye drops (0.5%) four times daily were prescribed. After 1 week, antibiotic eye drop was discontinued and topical steroid drop was tapered gradually over the subsequent weeks to four times daily after 6 weeks, which was continued for 6 months of follow-up after which this was further stepped down to thrice daily.Prior to performing any surface reconstruction, entropion correction of the upper eyelid with mucous membrane grafting was performed in the RE  of 0.8:1. A diagnosis of secondary glaucoma was established, and the patient has been managed on topical anti-glaucoma medications  with good intra-ocular pressure control until the last follow-up visit. Patient was also started on topical cyclosporine 0.1% two times a day in view of having a high-risk graft. The patient has completed 18 months of postoperative follow-up and maintains a BCVA of 20/100 in the RE. Postoperatively, the graft is clear and well epithelized, with a stable ocular surface . The AS- To reduce the risk of graft rejection and failure, it is important to address these existing comorbidities prior to performing any transplant procedure. In our case, prior to performing ocular surface reconstruction for visual rehabilitation, entropion correction with mucous membrane grafting was performed. This procedure of entropion correction with anterior lamellar recession and labial mucous membrane graft (MMG) is a well-established technique with modifications in the suturing technique. The risk of surgical failure increases in eyes with cicatricial entropion secondary to non-infectious etiology such as Stevens–Johnson syndrome, burns, and trauma. The modified technique used in this patient, has been described by Singhet al., where labial MMG is used for spacing the anterior lamella and reconstruction of the lid margin and the posterior lamella. This technique has shown promising long-term outcomes, particularly in eyes with cicatricial entropion secondary to ocular surface cicatricial disorders.The outcome of a corneal or limbal transplant is determined by presence of comorbidities such as tear film abnormalities, lid and lid margin related disease, uncontrolled intraocular pressure and presence of active surface inflammation.,, However, limitations exist with different procedures such as the risk of iatrogenic LSCD in the contralateral healthy donor eye with CLAU, difficulty in the surgical procedure with the need for life long systemic immunosuppression and need for a donor cornea with KLAL, and the need for an expensive laboratory setting to allowex vivo expansion of LESCs in CLET. Autologous simple limbal epithelial transplantation (SLET) introduced in 2012, has quickly gained popularity as it uses minimal limbal tissue from the contralateral healthy eye, allowsin vivo expansion of stem cells without the risk of iatrogenic LSCD in the donor eye, and also alleviates the need for systemic immunosuppression. However, in eyes with LSCD and corneal scarring, the results of LSCT procedures combined with PK have not been encouraging. Shimazakiet al. and Solomonet al. published outcomes of simultaneous KLAL and PK in eyes with total LSCD., Inferior outcomes were seen in eyes where simultaneous procedure was performed as compared to those eyes where two-staged surgery was performed (KLAL followed by PK) with higher rate of corneal graft rejection. Corneal graft rejection has been attributed to increased exposure of the host immune system to the donor corneal antigens through the limbal allograft antigens, which are derived from the same donor as the central graft. Also, simultaneous PK increases the surface inflammation and the wound healing response. Both studies recommended a time interval of 3–6 months after KLAL to allow stabilization of the ocular surface before performing keratoplasty.,Over several years, conjunctival limbal autograft (CLAU), cultivated limbal epithelial transplantation (CLET), keratolimbal allograft (KLAL) have been successfully performed in eyes with LSCD.et al. compared anatomical and functional outcomes of simultaneous PK and CLET with two-staged procedure in eyes with unilateral total LSCD secondary to ocular burns. They concluded that a two-staged surgical approach of first transplanting the cultivated limbal stem cells, followed by PK, resulted in superior outcomes in restoring the ocular surface stability and vision, as compared to single-staged procedure. Also, once the surface was well epithelized, which usually takes around 6 weeks, the time interval between CLET and PK did not affect the corneal graft survival. This was in accordance with the outcomes published by Baradaran-Rafiiet al. and Sangwanet al., where a two-staged sequential surgical approach showed favorable outcomes in terms of corneal graft survival and final visual acuity, as compared to simultaneous surgeries.,In 2011, Basu, The cause for poor visual recovery in eyes is complications like secondary glaucoma, amblyopia and infectious keratitis, rather than the surgical procedure itself. Encouraging outcomes have been reported in eyes where sequential ocular surface reconstruction by SLET followed by keratoplasty was performed in eyes with unilateral total LSCD., Simultaneous allogeneic SLET with PK has been described in a case of sterile keratolysis following severe ocular chemical burn, as an emergency procedure to preserve the globe tectonicity.The existing literature reporting outcomes of simultaneous autologous SLET and PK have also been discouraging. Simultaneous PK is an established risk factor for recurrence of LSCD and failure of SLET in eyes with total unilateral LSCD in adults and children. Also, when BKpro1 is performed in a patient with a contralateral healthy eye, these patients may not be aware of the decreased vision in the operated eye, secondary to any complication, as the contralateral eye maintains a good vision.Surgical decision making in such cases depends on the status of the contralateral eye. Of the various techniques described earlier for ocular surface reconstruction, in our case, autologous SLET was preferred over CLAU, CLET or allogeneic SLET. The need for a CAG or CLAU was eliminated in our case as there was no co-existing symblepharon. As our patient had a healthy contralateral eye, autologous LSCT was preferred to avoid the need for systemic immunosuppression. Similarly, for visual rehabilitation, PK was preferred over Boston type 1 keratoprosthesis (BKpro1), which is often reserved in patients who are bilaterally blind. Though BKpro1 has shown to provide rapid visual recovery in eyes with LSCD secondary to chemical burns, glaucomatous optic neuropathy is common in these eyes and may reduce the long-term visual potential post BKpro1. The central area of the corneal graft may have healthy donor corneal epithelium which need not be debrided. Hence, AMG may not be required centrally, and in our case, a central opening was created in the AMG, thus allowing for better visual acuity. Also, it has been reported that AMG may persist as a subepithelial membrane in some cases leading to haze and poor visual acuity post-operatively. This central opening is not created in SLET cases as the underlying cornea is devoid of epithelium and if the AMG does not cover this area, then this area of the corneal stroma may not get epithelized as there is no continuous scaffold to grow over.Hence, in our case, we chose to perform autologous SLET combined with PK. Although sequential surgery would have been ideal, this was not possible in our case due to the underlying cornea being thin. Corneal thinning increases the risk of frank perforation of the cornea during pannus dissection for SLET, thus necessitating PK. Thus, performing SLET alone was not a possibility. Although performing PK alone could have been a possibility, this would have led to recurrence of LSCD over the graft, thus, leading to failure of the PK procedure. Also, in our case, there were certain modifications to the surgical procedure. Pannus dissection was not performed 360 degrees, as is performed in SLET as a complete pannus was not seen over the cornea. Hence pannus dissection was performed only in the areas of the pannus nasally and temporally and this conjunctiva was allowed to recess. Sutures were used to anchor the AMG on the perilimbal sclera as the underlying cornea was epithelized and it is possible that the AMG would not have been stable otherwise. Also, as the pannus dissection was not performed 360 degrees, it was not possible to tuck the AMG under the surrounding conjunctiva, as is performed in SLET. Hence, sutures were deemed to be necessary. Additionally, while performing SLET, the explants were placed peripherally over the host cornea. This surgical modification can prevent the loss of explants, in eyes where a repeat penetrating keratoplasty is required. Also, in these cases, it may be more important to support the paracentral area and the limbal area in the form of a doughnut AMG. Another option would be choosing to take a larger graft and include the limbus from the donor graft, however, this would also require systemic immunosuppression. Simultaneous intervention in our case also allowed for faster visual recovery, reduced multiple follow-up visits for the patient, reduced the cost of surgery, and allowed for an earlier diagnosis and management of secondary glaucoma. We also believe that addressing the adnexal abnormalities prior to ocular surface reconstruction helped us achieve the desired outcome of a clear graft with a stable ocular surface which was maintained for a follow-up period of 18 months.An allogeneic SLET was not performed as the recipient will then be exposed to antigens from two donors versus a single donor.Written informed consent for publication of their clinical details and clinical images was obtained from the patient. The authors have addressed adequately all the comments. Congratulations for successful management of this challenging case!Are enough details provided of any physical examination and diagnostic tests, treatment given and outcomes?YesIs the case presented with sufficient detail to be useful for other practitioners?PartlyIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment?YesIs the background of the case’s history and progression described in sufficient detail?YesReviewer Expertise:CorneaI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The authors present a very interesting case highlighting the advantages of autologous simple limbal epithelial transplantation combined with penetrating keratoplasty as a single-stage procedure. Despite the evidence supporting a two-stage procedure, the authors clearly explained and justified the use of combined surgery by a multidisciplinary team and an organized surgical approach in a well-selected patient such as the one described in this case report. Underscoring the importance of addressing the adnexal abnormalities before any ocular surface reconstruction is key. The authors used the “Doughnut” amniotic membrane transplantation, my suggestion is that they could explain the benefits of using this technique for retaining a clear central cornea after AMT transplantation.Are enough details provided of any physical examination and diagnostic tests, treatment given and outcomes?YesIs the case presented with sufficient detail to be useful for other practitioners?PartlyIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment?YesIs the background of the case’s history and progression described in sufficient detail?YesReviewer Expertise:Cornea, Ocular Surface, Dry Eye.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. This case report nicely documents the sequence of rehabilitative procedures that need to be done in order to restore vision in an eye with cicatricial entropion, corneal thinning, cataract and total LSCD following thermal injury. They should be congratulated for a great outcome on an eye that would otherwise be lost. Although the cited literature suggests that a two stage procedure has better long-term outcomes in terms of graft survival, in certain cases, such as the one described by the authors, this cannot be done due to the significant corneal thinning and the risk of perforation during pannus dissection for the LSCT. The authors should discuss in more detail why corneal thinning precludes a sequential procedure. Moreover, they should discuss how they modified their technique. It seems that they did not perform a 360 peritomy as is commonly done in SLET and pannus dissection, but rather performed the PK and then secured the AMT over the peripheral conjunctiva. Finally, the authors should discuss why they performed a central opening in the ΑΜΤ and whether, in their experience, this leads to better visual quality in the long term. Why did they choose to do that in this case? Or is it something they always perform in their SLET procedures?Are enough details provided of any physical examination and diagnostic tests, treatment given and outcomes?YesIs the case presented with sufficient detail to be useful for other practitioners?PartlyIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment?YesIs the background of the case’s history and progression described in sufficient detail?YesReviewer Expertise:CorneaI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.