Research focusing on management of foot health has become more evident over the past decade, especially related to chronic conditions such as diabetes. The level of methodological rigour across this body of work however is varied and outputs do not appear to have been developed or translated into clinical practice. The aim of this systematic review was to assess the latest guidelines, standards of care and current recommendations relative to people with chronic conditions to ascertain the level of supporting evidence concerning the management of foot health.A systematic search of electronic databases  for literature on recommendations for foot health management for people with chronic conditions was performed between 2000 and 2016 using predefined criteria. Data from the included publications was synthesised via template analysis, employing a thematic organisation and structure. The methodological quality of all included publications was appraised using the Appraisal for Research and Evaluation (AGREE II) instrument. A more in-depth analysis was carried out that specifically considered the levels of evidence that underpinned the strength of their recommendations concerning management of foot health.The data collected revealed 166 publications in which the majority (102) were guidelines, standards of care or recommendations related to the treatment and management of diabetes. We noted a trend towards a systematic year on year increase in guidelines standards of care or recommendations related to the treatment and management of long term conditions other than diabetes over the past decade. The most common recommendation is for preventive care or assessments (e.g. vascular tests), followed by clinical interventions such as foot orthoses, foot ulcer care and foot health education. Methodological quality was spread across the range of AGREE II scores with 62 publications falling into the category of high quality (scores 6–7). The number of publications providing a recommendation in the context of a narrative but without an indication of the strength or quality of the underlying evidence was high (79 out of 166).It is clear that evidence needs to be accelerated and in place to support the future of the Podiatry workforce. Whilst high level evidence for podiatry is currently low in quantity, the methodological quality is growing. Where levels of evidence have been given in in high quality guidelines, standards of care or recommendations, they also tend to be strong-moderate quality such that further strategically prioritised research, if performed, is likely to have an important impact in the field.The online version of this article (10.1186/s13047-017-0232-3) contains supplementary material, which is available to authorized users. Chronic conditions represent a global health burden . Their iDespite this need for the management of foot problems, provision of podiatry services is an area that lacks guidance. Feedback from people who have chronic conditions suggests that they are not accessing care for foot problems , 16, 17 There has been increased attention to evidence informed medicine and the desire for reproducibility in choice of foot-care tailored to individual patient needs , 18–22. Guidelines, standards of care and recommendations for management of foot health have become more evident over the past decade, particularly in the management of foot problems related to the manifestations of diabetes . The impTo provide a narrative synthesis on the specific characteristics of guidelines and recommendations for foot health management using criteria from the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist .To assess the methodological quality of included studies against the Appraisal of Guidelines for Research and Evaluation (AGREE II) document .To determine the level of evidence used in guidelines and recommendations for foot health management.The aim of this investigation was to systematically review the literature related to guidelines, standards of care and current recommendations concerning the management of foot health for people with chronic conditions. The findings of this study will be of interest to podiatrists and health care professionals by identifying the evidence-based behind interventions for foot health management, which can then be used to support and guide practice. Specific objectives were:Search criteria for the systematic review were identified using the “PICO” statement. Search terms were defined by three experts in podiatry  and the final list agreed through a consensus meeting. Electronic databases  were searched for English language publications and translations reporting recommendations for the management of foot health for people with chronic conditions between 2000 and 2016. The year 2000 was considered an appropriate point to commence the literature search, given the establishment of degree education in podiatry from 1993, and the subsequent development of postgraduate and post-doctoral qualifications and research skills within the profession.Searches were performed using the following text word terms and MeSH headings: foot, chronic, chronic disease, long term care, neurodegenerative diseases, multiple sclerosis, arthritis, lung diseases, emphysema, diabetes mellitus, hypertension, cerebrovascular disorders, myocardial ischemia, heart failure, colonic diseases, human immunodeficiency virus, osteoporosis, fibromyalgia, neoplasms, mental disorders, depression, asthma, chronic obstructive pulmonary disease, thyroid diseases, hyperlipidaemia, psoriatic arthritis, gout, ankylosing spondylitis, colitis, scleroderma, practice guidelines, algorithms.Hand searches of bibliographic references identified additional publications. Grey literature was included based on an initial search using the terms foot, feet and podiatry and rerun in conjunction with the terms guideline, recommendation and management to ensure that the search had captured all relevant sources. Publications identified through the grey literature and hand searches were not restricted by date. The systematic literature search was facilitated by a medical librarian at the Bodleian library, University of Oxford. The full search strategy can be seen in Additional file A publication was included if it met all the following criteria:Includes the management of foot healthProvides recommendations for the management of foot health, e.g. guidelines, guidance documents, standards of care, treatment algorithms and clinical pathways..Excluded publications were those that did not fulfil the above criteria on population group, were not related to the management of foot health, such as technical and methodological papers, and were not formal publications presenting guidelines or recommendations . Literature identified from the electronic search strategy was excluded if published outside the date range 2000 to 2015.Two independent reviewers (KE and LM) selected eligible publications based on titles and abstracts. Potentially relevant publications were subject to full-text screening. Disagreement between reviewers was resolved by consultation with a third reviewer from a panel of qualified podiatrists and experts in the field of podiatry and foot and ankle research . Data was extracted from the identified literature based on the criteria given in the PRISMA (2009) checklist . The PRIThree independent expert reviewers  assessed all the included publications July 2016) for quality using the AGREE II checklist . The AGR016 for qData from the included publications was synthesised via template analysis, employing a thematic organisation and structure. Template analysis is a process by which textual data is organised and analysed according to themes, allowing unstructured textual material to be categorised into meaningful datasets. Indexing sections of text relevant to a theme also permits the development of codes which emerge from the data . PredeteWhere stated, the level of evidence supporting the recommendations related to foot health management was extracted and interpreted according to the GRADE  system . The GRAThe search strategy retrieved 5379 citations from electronic and grey literature sources. In total, 1938 duplicate citations were removed between the electronic and grey literature sources. Results from the electronic search were then subject to title screening according to the review selection criteria with 856 citations being excluded; results from the grey literature were not subject to title screening as this was deemed inappropriate due to the use of short titles for the majority of articles, and all were put forward for screening by abstract. Following abstract screening, 2368 citations were found ineligible with 122 citations being included from hand searching the bibliographies of excluded papers. The search process was then repeated for 2015 to 2016 to ensure that the findings of the review would be as up-to-date as possible, and this resulted in the identification of an additional 14 publications. From full-text screening, 187 articles were considered ineligible, and the remaining 166 publications were included in the final review.The abstracts of all publications found were exported from the databases and collated into a Microsoft® Excel (2010) spreadsheet  for scoring for inclusion (1) or exclusion (0).Screening by abstract resulted in the exclusion of 2358 citations. An additional 122 relevant citations were also identified from hand searching bibliographies of any publications excluded at this stage. Of the 339 citations that were put forward for full-text screening, 152 were included in the final review.The full text version of all publications found were exported from the databases and collated into a second Microsoft® Excel 2010) spreadsheet for scoring for inclusion (1) or exclusion (0). Four reviewers  independently screened the full text citations against the inclusion criteria. All publications that met the inclusion criteria were selected, however only the most recent versions were included such that 152 were put forward for review. The literature search process was repeated for any publications that had been released until September 2016 to make the review as up-to-date as possible. An additional 14 publications were identified, bringing the final total up to 166. Fig. 0 spreadsOf the 166 included publications, 74 (45%) were from the grey literature, 65 (39%) were from electronic databases and 27 (16%) were from hand searches.The 166 included papers were published between 1986 and 2016; of these, the majority (11.5%) were published in 2011. Prior to 2000, the studies represented are those identified from the grey literature for which no date criteria were imposed. Importantly, the distribution of guidelines, standards of care and recommendations concerning the management of foot health in people with chronic conditions shows a steadily increasing trend over time  of the 166 included publications were based in, or included, the United Kingdom  of the 166 included publications were reported as guidelines; this included Global or National guidelines, clinical guidelines, best practice guidelines, evidence-based guidelines and guideline summaries Fig. . Among tOf the 166 included publications, the majority 61.5%) were guidelines, standards of care or recommendations related to the treatment and management of diabetes or the diabetic foot , preventative care and assessments along with clinical interventions and foot health education are themes that are covered the most; on the other hand, comparatively little attention is given here to areas such as core podiatry and podiatric surgery. Recommendations for core podiatry tend to feature the most in publications with an AGREE II grade of 3 or below, indicative of low methodological quality, as do those for podiatric surgery, although these are comparatively few in number overall.Of the included papers that have a quality threshold score of 4 and above for reasonable to high methodological quality , the difference between evidence and narrative based publications is more apparent (14+ papers).From those papers that reported levels of evidence alongside recommendations, this data was then extracted and used to populate a data extraction form  were guidelines, standards of care or recommendations that were focussed on the treatment and management of diabetes. Methodological quality of the included publications ranged from high to low according to the AGREE II rating system  suggestiExamining patterns across the 166 publications would suggest that the majority of supporting evidence for foot health management is included within UK guidelines, recommendations or standards of care, followed by the US, Europe and Australia. This is not surprising as our search was limited to English only publications, however of note, within these countries podiatry is a recognised allied health discipline , 33. PodGuidelines, standards of care and recommendations play an important role in synthesising evidence for health care and health policy information however according to the AGREE II consortium  the poteFrom our review, we also established that it was rare that a podiatrist or foot care specialist was included as a stakeholder in the higher scoring publications. This is of concern as the recommendations contained within guidelines are defined by the scope of the stakeholders. Stakeholders advise guideline developers in searching for, selecting, critiquing and combining data . WithoutThe strength of recommendations within guidelines, recommendations or standards of care is informed by the quality of the research evidence on which they are based , 25. FolWhere a level of evidence was given, this tended to be in the higher scoring publications for methodological quality and related to GRADE  system  as stronAccording to GRADE, for those themes with emerging good quality evidence, further research, if performed, is likely to have an important impact on confidence in the estimate of the effect and may change the estimate . As the The findings of this systematic review need to be considered in the context of several limitations. First the scrutiny for evidence for ‘podiatry’ was determined a priori by the authors as being most accessible from guidelines as they usually incorporate research evidence into their clinical recommendations. In this instance it would have been inappropriate to select publications by single interventions due to the wide range of interventions related to management of foot health / podiatry in long term conditions and the multi-professional nature involved in the interventions. As only English-language publications were included in the review this may have resulted in additional relevant, high-quality evidence from non-English publications being omitted.The hand searching process also procured a number of publications not identified from the electronic database search. This may be due to the grey literature and hand searched references not being restricted by date as had the electronic searches. This highlights the differences in approach required in searching the electronic and grey literature, and the challenges inherent in designing an inclusive search strategy. In view of this, the grey literature search in particular may not have captured all relevant articles; however, every attempt was made to minimise the potential of ‘missed’ literature by developing the search strategy with input from experts in the field of podiatry and foot health care, and guided by a specialist in designing literature search strategies.the extensive user testing done on the GRADE system could be one factor that has contributed to its popularity among guideline developers” [The reader needs also be aware that while the GRADE system is widely adopted by medical researchers, it does have its limitations. Often manuscripts retrieved from the grey literature, such as observational studies, narratives and clinical expert opinion, tend to be classified as being low methodological quality; however in the absence of any other more suitable system it was decided to use GRADE as “elopers” .Lastly, quality of evidence was determined by methodological assessment of the publications in accordance with the AGREE II consortium and interpretation of cited evidence  through Gathering evidence for the effectiveness of core podiatry  and the effectiveness of podiatric surgery should be a high priority for the podiatry and foot and ankle research communities.Professional bodies and the foot and ankle research communities should have a coordinated podiatry  representation on key policy and national and international guideline development committees that have a focus on foot and ankle health and management.Professional bodies and the foot and ankle research communities should have coordinated collaboration for collection of outcomes data and evidence that demonstrates the value and impact of podiatry and foot health care on patients.Future guidelines, standards of care and recommendations should follow a recognised framework (e.g. AGREE II) and use one validated standardised system for grading evidence (i.e. GRADE) for podiatry and foot health care or alternatively have a system that will enable comparability between grading systems (as attempted to do in this paper).It is clear that evidence needs to be accelerated and in place to support the future of the podiatry and foot health clinical workforce. In this systematic review, we have found that the field has an emergent evidence base that is becoming more established year on year. We have also discovered small but relevant numbers of high level evidence for the effectiveness of podiatry and foot health management especially concerning assessment for monitoring and prevention of serious foot complications associated with chronic conditions. Where levels of evidence have been given in high quality guidelines, standards of care or recommendations, they also tend to be strong-moderate quality such that further strategically prioritised research, if performed, is likely to have an important impact in the field.The results from this study will help inform the stakeholders and users of podiatry services such as podiatry clinicians, clinical guideline developers or policymakers on evidence for foot health management in their decision making.Additional file 1: Appendix A. Electronic search strategy: an example using Embase. (DOCX 84 kb)Additional file 2: Appendix B. Scoring of included papers against AGREE II criteria for quality assessment. (DOCX 140 kb)Additional file 3: Appendix C. Types of evidence within the included publications. (DOCX 100 kb)Additional file 4: Appendix D. Levels of evidence where reported from included studies. (DOCX 41 kb)Additional file 5: Appendix E. Levels of Evidence for Recommendations concerning Foot Health Management. (DOCX 28 kb)


A range of evidence informs decision-making on innovation in health care, including formal research findings, local data and professional opinion. However, cultural and organisational factors often prevent the translation of evidence for innovations into practice. In addition to the characteristics of evidence, it is known that processes at the individual level influence its impact on decision-making. Less is known about the ways in which processes at the professional, organisational and local system level shape evidence use and its role in decisions to adopt innovations.A systematic scoping review was used to review the health literature on innovations within acute and primary care and map processes at the professional, organisational and local system levels which influence how evidence informs decision-making on innovation. Stakeholder feedback on the themes identified was collected via focus groups to test and develop the findings.Following database and manual searches, 31 studies reporting primary qualitative data met the inclusion criteria: 24 were of sufficient methodological quality to be included in the thematic analysis. Evidence use in decision-making on innovation is influenced by multi-level processes  and interactions across these levels. Preferences for evidence vary by professional group and health service setting. Organisations can shape professional behaviour by requiring particular forms of evidence to inform decision-making. Pan-regional organisations shape innovation decision-making at lower levels. Political processes at all levels shape the selection and use of evidence in decision-making.The synthesis of results from primary qualitative studies found that evidence use in decision-making on innovation is influenced by processes at multiple levels. Interactions between different levels shape evidence use in decision-making . Organisational leaders need to consider whether the environment in which decisions are made values diverse evidence and stakeholder perspectives. Further qualitative research on decision-making practices that highlights how and why different types of evidence come to count during decisions, and tracks the political aspects of decisions about innovation, is needed.The online version of this article (10.1186/s13012-017-0669-6) contains supplementary material, which is available to authorized users. A range of evidence informs decision-making on innovation in health care, including formal research findings , local dThe purpose of this systematic scoping review was to understand how processes at different levels influence the use of evidence in decision-making on health care innovations by (1) mapping processes at the professional, organisational and local system levels which influence how evidence informs decision-making on innovation and (2) collecting stakeholder feedback to validate and develop the findings. The review focussed on primary qualitative studies as these were appropriate for understanding how and why contextual processes at different levels influence evidence use in decision-making. Qualitative studies can capture this context by focusing on processes and experiences of innovation at the professional group, organisational  and local system level (the embedding of organisations in the wider environment or context) .Literature on evidence use in decision-making on innovation was identified, selected and analysed using a scoping review approach –25, whicThis review’s guiding research question was ‘How do decision-making processes at the professional group, organisational, and local system level influence the use of evidence in decisions to adopt innovations in acute and primary health care?’ Selection of these three levels reflects the theorised influence of these aspects of the local context during quality improvement processes , 26, witThe conceptual literature on evidence use highlights that a range of evidence may impact on decisions about innovation or improvement. The evidence-based medicine (EBM) movement, and its extension into other areas, including health care management, has been influential in how evidence is conceptualised. EBM involves providing care by integrating individual clinical expertise, evidence from systematic research and patient choice . Those cInnovation was defined broadly as the development and implementation of new ideas, products, processes or organisational forms , 31. OurThis review included decisions about whether to adopt new innovations or spread existing innovations up to the point of implementation (implementation itself was considered relevant where it influenced adoption decisions). We adopted a ‘processual’ approach to the study of decision-making on innovation, understanding it as an ongoing, often non-linear process that unfolds over time . DiffereThe scoping review aimed to identify examples of evidence use in decisions about innovation (or related improvement activity) from studies conducted in relation to the UK National Health Service (NHS) and health systems internationally. The review’s focus was on the influence of interactions between evidence use and processes at the micro  and meso  level on decisions to introduce or diffuse innovations. Selection criteria were defined a priori and applied by two researchers to the title/abstract, and then full text, of potentially relevant papers. The review focused on decision-making on innovations in health care services  and multi-sectoral studies including health care services. Studies that examined decisions about innovation or other improvement activities, but did not refer to evidence use, were excluded. Only studies conducted in Organisation for Economic Co-operation and Development (OECD) countries were included to aid comparability of health care systems. Only English language references, published since 2006, were included. This date was chosen because it coincided with recognition among policymakers and researchers of the challenges of mobilising evidence in health care, including concerns about traditional models of translating research into practice  and critTo identify relevant literature, social science and biomedical databases were searched in May 2016. A search strategy was created for MEDLINE. Search terms in the title or abstract were ‘innovation or improvement’, ‘decision or decision-making’, ‘evidence’ and ‘health care’. Medical Subject Headings (MeSH) were also used, which included ‘diffusion of innovation’, ‘translational medical research’, ‘Evidence-based practice’, ‘knowledge bases’ and ‘decision making, organizational’. The search was adapted for other databases: Embase, PsycINFO, Scopus, Health Management Information Consortium (HMIC), and EBSCO Business Source Complete , primary care providers (3), service commissioners (3), patient representatives 5) and knowledge intermediaries (3). Reporting of the focus groups  and suggestions by PAG members, including book chapters, identified eight additional studies for inclusion, meaning 31 studies were reviewed.The database search produced 1816 results, after duplicates were excluded. After screening by title and abstract using the inclusion and exclusion criteria, 184 articles were identified for full-text screening, 23 of which were selected for data extraction  Qualitative Checklist , which iA summary of the studies’ characteristics is provided in Additional file There were 24 studies of sufficient quality to be included in the thematic analysis. Thematic analysis examined how processes at different levels  influenced the use of evidence in decision-making on innovation  drew on a range of evidence, including alternative evidence such as patient stories, and prioritised local need for innovations over research evidence , 48. In Some studies highlighted that decisions to develop and adopt innovations reflected professional interests. A study of surgical innovation found that surgeons ‘spoke for’ patients by introducing new techniques that would ‘make sense’ for them, even though supporting data were lacking . A studyPower dynamics between different professional groups influenced evidence use. A study of interventions to improve prescribing practice in primary care found that managers leading the programme privileged scientific evidence, while attempting to marginalise GPs’ clinical and experiential knowledge . SimilarThe stakeholder feedback indicated that professional processes influenced decision-making. They confirmed that professional credibility of those presenting evidence, as well as clinical leadership and ‘soft’ persuasion skills and relationship-building (including ‘endless discussions’), encouraged evidence to be taken seriously and acted upon. There was recognition that preferences for evidence varied by stakeholder and therefore the same evidence often need to be framed differently to influence different stakeholders, particularly the needs of commissioners or funders of potential innovations, ‘as everybody has different buttons’. The ongoing process of building relationships during decision-making was more apparent in the stories of innovation shared in the focus groups than in the literature review  .Organisations contributed to assessing non-clinical aspects of innovation. Along with evidence of clinical need or effectiveness, budgetary and financial issues were important in decision-making , 49. OrgA number of organisational facilitators to evidence use in decisions about innovation were identified. In a study of technology adoption within hospitals, access to and use of research evidence in decision-making was enabled by organisational processes, including infrastructure redevelopment projects and an emphasis on collaboration . In a stUnderlying organisational issues could act as barriers to introducing innovations . A lack Organisational politics influenced the type of evidence accessed and how it was interpreted. The use of economic evaluation by committees making technology coverage decisions was limited by unclear relationships with resource allocators, an explicitly political decision-making process, and poorly specified decision-making criteria . A studyThe stakeholder feedback confirmed that an innovation was more likely to be adopted when it was aligned with organisational needs, e.g. when it is a priority  and it addressed a clear, practical problem. The focus groups elaborated on the influence of the decision-making approach taken in relation to innovations of different scales. There was recognition that large-scale change was difficult because a wide range of stakeholders were often involved and that evidence often showed both pros and cons. The stakeholders discussed different approaches to organisational decision-making; ‘autocratic’ as opposed to ‘democratic’ organisations were better placed to introduce change, but once a decision had been made, there was the challenge of getting a change accepted and having a culture that valued evidence was deemed important for this.External pressures, including system restructuring , meetingPan-regional organisations influenced how evidence was used in decisions about innovation. On the one hand, such organisations had a downward influence on evidence use in local decision-making. A study of a collective primary care organisation showed how it influenced GPs’ prescribing practice by emphasising evidence that appealed to this professional group , while deemphasizing the contribution of the interventions to cost containment which appealed less to GPs . A natioParticipation in external systems or networks enabled a wider range of potential stakeholders to inform decision-making on innovation. However, taking into account a range of external stakeholders’ views could hinder implementing innovations based on formal evidence alone; the politics of decision-making could be more important than evidence, including the assessment of likely public perceptions of decisions taken . DecisioThe stakeholder feedback indicated that organisations at the local system level played an important role in enabling innovation. The backing of research organisations and other knowledge intermediaries, e.g. Academic Health Science Networks and CLAHRCs, provided a facilitating role–one participant referred to them as ‘ambassadors’ for innovation–that could help to bring together relevant stakeholders. The role of intermediaries in mobilising evidence for innovations by brokering social relationships came through more clearly in the focus groups than in the literature review, possibly because studies of knowledge mobilisation tend to consider implementation processes (which were excluded from the review) rather than adoption decisions . The focThis is the first systematic scoping review to examine how processes at multiple levels  influence evidence use in decision-making on innovation. At the professional level, preferences vary by professional group and health service setting. Commissioners favoured evidence derived from contact with colleagues or professional ‘networking’, combined with service user involvement and assessment of local needs rather than research evidence. Doctors in acute settings preferred research evidence, while those working in primary care emphasised clinical and experiential knowledge of patients’ needs. Preferences for non-research evidence were partly due to barriers to using some forms of research, e.g. cost analyses, or a perceived lack of formal evidence for making the decision at hand. Professional interests, and dynamics of power between professional groups, shaped the construction, interpretation and application of evidence during decision-making on innovation. Organisational roles included influencing the culture of evidence use (e.g. encouraging decisions to be data-driven), assessing non-clinical aspects of evidence  and enabling stakeholder involvement. At the local system level, the embedding of pan-regional organisations shaped innovation decision-making at lower levels, while external pressures could encourage particular types of evidence  or inhibited its use. The politics of decision-making, e.g. linked to the financial context in which innovations were being considered, was an important influence on evidence use at all levels. An overview of the themes identified is provided in Table Much of the existing literature on evidence use in decision-making on innovation has focussed on processes at a particular level or not been explicit about the need to study processes at different levels  between conceptual levels shape evidence use in decision-making . We conclude with recommendations for policy and practice in terms of enhancing the use of evidence in decisions about innovation. First, while a range of evidence may inform decision-making, from research evidence through to local data and professional opinion, key decision-makers should reflect on the types of evidence that are routinely used in decision-making and how this influences the outcome . Second, the role of politics and power in decision-making needs to be acknowledged and skilfully managed. Evidence can potentially have an emancipatory role in lending authority to participants beyond other characteristics . To enable this role, organisations need to value challenging evidence and perspectives and build staff and organisational capacity in acquiring and applying evidence. Third, decision-makers need to consider the ways in which the environment in which decisions are made encourages diverse evidence and perspectives. For example, organisational leaders should consider how to mitigate professional interests and power when developing processes for enabling stakeholder involvement in decision-making.Additional file 1:PRISMA checklist. (DOC 59 kb)Additional file 2:Summary of search strategies. (DOCX 97 kb)Additional file 3:Characteristics of primary studies included in full text review and quality assessment , 67–72. Additional file 4:Charting of themes across primary studies included in full text review , 67–72. Additional file 5:Focus group reporting. (DOCX 115 kb)


Eulemur fulvus (brown lemur) in the epidemiology of Rift Valley fever (RVF) in Mayotte, during an interepidemic period, was explored. In February and March 2016, 72 animals were blood sampled and tested for RVF. No evidence of RVF genome or antibodies was found in the samples. The role of other wild mammals on the island should, however, be further investigated.The potential role of  Since then, the virus may have circulated at a low level, indicating a current interepidemic period , rodents (Rattus rattus and Mus musculus), tenrecs (Tenrec ecaudatus), civets (Viverricula indica), shrews (Suncus madagascariensis), and lemurs (Eulemur fulvus) . Previous studies in wildlife indicated that bats and micromammals could be involved in RVF epidemiology  of Mayotte , the Ethics Committee of the CYROI La Reunion , and by the Animal Welfare Ethical and Review Board of the London School of Hygiene and Tropical Medicine (2015/3N).50/mL  of animals were estimated to be <1 year old, 23% (n = 16) were between 1 and 3 years old, 36% (n = 25) were aged 3–6 years, and 27% (n = 19) were estimated to be >6 years old. All samples were found negative by RT-PCR and ELISA, in all age groups, indicating no evidence of current, recent, or past RVF infections in our study animals . The estimated lemur population size of Mayotte is about 10–20,000  and rodents (Family Nesomyidae and Muridae) in neighboring Madagascar showed no evidence of RVF infection during 2008–2010 after the RVF epidemic (Olive et al. There is evidence that lemurs can be exposed to Chikungunya and Flavivirus infections in Mayotte and Madagascar (Fontenille et al.


Patient-reported outcome measures (PROMs) can provide valuable information which may assist with the care of patients with chronic kidney disease (CKD). However, given the large number of measures available, it is unclear which PROMs are suitable for use in research or clinical practice. To address this we comprehensively evaluated studies that assessed the measurement properties of PROMs in adults with CKD.Four databases were searched; reference list and citation searching of included studies was also conducted. The COnsensus-based Standards for the selection of health Measurement INstruments (COSMIN) checklist was used to appraise the methodological quality of the included studies and to inform a best evidence synthesis for each PROM.The search strategy retrieved 3,702 titles/abstracts. After 288 duplicates were removed, 3,414 abstracts were screened and 71 full-text articles were retrieved for further review. Of these, 24 full-text articles were excluded as they did not meet the eligibility criteria. Following reference list and citation searching, 19 articles were retrieved bringing the total number of papers included in the final analysis to 66. There was strong evidence supporting internal consistency and moderate evidence supporting construct validity for the Kidney Disease Quality of Life-36 (KDQOL-36) in pre-dialysis patients. In the dialysis population, the KDQOL-Short Form (KDQOL-SF) had strong evidence for internal consistency and structural validity and moderate evidence for test-retest reliability and construct validity while the KDQOL-36 had moderate evidence of internal consistency, test-retest reliability and construct validity. The End Stage Renal Disease-Symptom Checklist Transplantation Module (ESRD-SCLTM) demonstrated strong evidence for internal consistency and moderate evidence for test-retest reliability, structural and construct validity in renal transplant recipients.We suggest considering the KDQOL-36 for use in pre-dialysis patients; the KDQOL-SF or KDQOL-36 for dialysis patients and the ESRD-SCLTM for use in transplant recipients. However, further research is required to evaluate the measurement error, structural validity, responsiveness and patient acceptability of PROMs used in CKD. Chronic kidney disease (CKD) is a global health issue . It affeEarly symptoms of CKD, such as fatigue, are usually non-specific . HoweverHRQOL can be assessed using self-administered, validated questionnaires known as patient-reported outcome measures (PROMs) . PROMs hIf any benefit is to be derived from the use of PROMs, it is important that they are well validated to ensure that they actually measure what they are supposed to measure, produce consistent results and capture all aspects of the construct(s) under investigation that matter to the target population if any benefit is to be derived from their use .A systematic review by Gibbons and Fitzpatrick  evaluateTherefore, we have evaluated the methodological quality of the selected studies using the COnsensus-based Standards for the selection of health Measurement INstruments (COSMIN) checklist  and usedThis systematic review was conducted and reported according to a registered and published protocol (PROSPERO registration number: CRD42016035554) (See st December 2015 without language restrictions . At. At19]. The search strategy retrieved 3,702 titles/abstracts. After 288 duplicates were removed, 3,414 abstracts were screened and 71 full-text articles were retrieved for further review. Of these, 24 full-text articles were excluded for various reasons . FollowiA total of 25 PROMs were identified from the 66 publications; 20 disease-specific, 3 generic and 2 utility PROMs See . As the Three disease-specific PROMs were used in pre-dialysis populations namely the: Agarwal , KDQOL-3Agarwal. There was limited evidence for test-retest reliability and content validity. Internal consistency and structural validity were rated as 'unknown' [unknown' .Kidney Disease Quality of Life—36 (KDQOL-36). Strong evidence for internal consistency was found [as found  and theras found , 34. Stras found .KDQOL-SF. There was moderate evidence for test-retest reliability [iability  and hypoiability , 36. Intiability , 36.Fourteen PROMS were used in this group of patients. Among the five studies that evaluated KDQOL-36, two had mixed samples , 38. As Klersy et al.  used a sKDQOL-36. Moderate evidence was found for internal consistency [sistency , test-resistency , 42 and sistency , 38. Howsistency .KDQOL-SF. There was strong evidence of internal consistency and structural validity [validity . Moderatvalidity , 45 and validity  while thvalidity . Responsvalidity .Chinese Dialysis Quality of Life Scale (CDQOL). Reliability  for CDQOL was rated as 'unknown' and there was limited evidence for content validity and hypothesis testing [ testing .CHOICE Health Experience Questionnaire (CHEQ). Moderate evidence for hypothesis testing and content validity was found [as found  while inas found , 51.Dialysis Symptom Index (DSI). There was limited evidence for content validity [validity . Reliabivalidity , 53.Modified Edmonton Symptom Assessment System (modified ESAS). There was limited evidence of test-retest reliability, content validity and hypothesis testing. Responsiveness was rated as 'unknown' [unknown' , 55.Kidney Disease Questionnaire (KDQ). There was moderate evidence of hypothesis testing [ testing , 57 whil testing . Structu testing  and inteKDQOL. There was limited evidence for content validity while internal consistency and structural validity were rated as 'unknown' [unknown' .KDQOL (Modified). There was limited evidence for hypothesis testing while internal consistency was rated as 'unknown' [unknown' .Nottingham Health Profile (NHP). There was moderate evidence for internal consistency [sistency , 61 and sistency . Test-reSF-12. Moderate evidence was found for internal consistency and test-retest reliability. There was limited evidence for structural validity and hypothesis testing [ testing .WHOQOL-BREF . There was moderate evidence for internal consistency while test-retest reliability and structural validity were rated as 'unknown' [unknown' .Quality of Life Index (QLI) 3.0. There was moderate evidence for hypothesis testing [ testing , 65. Int testing –67 whileSF-36 version 2. Moderate evidence was found for internal consistency while limited evidence was found for hypothesis testing [ testing .Ten PROMs were evaluated specifically in renal transplant patients and all except the SF-36 and the EQ-5D were disease specific measures.SF-36 version 2. Internal consistency was rated as 'unknown' [unknown' .End-Stage Renal Disease Symptom Checklist–Transplantation Module (ESRD-SCL-TM). There was strong evidence for internal consistency [sistency , 71, modsistency , hypothesistency  and strusistency  while reEQ-5D. Moderate evidence for hypothesis testing was found [as found .Modified Transplant Symptom Occurrence and Symptom Distress Scale (MTSOSD). Moderate evidence for hypothesis testing was found for MTSOSD [r MTSOSD .Gastrointestinal Symptom Rating Scale (GSRS). Moderate evidence for hypothesis testing was found while internal consistency was rated as 'unknown' [unknown' .Gastrointestinal Quality of Life Index (GIQLI). Moderate evidence for hypothesis testing was found while internal consistency was rated as 'unknown' [unknown' .KDQOL-SF. There was limited evidence for hypothesis testing and limited evidence against test-retest reliability. Internal consistency was rated as 'unknown' [unknown' .Kidney Transplant Questionnaire (KTQ). There was moderate evidence for internal consistency [sistency  and hyposistency , 79. Limsistency  and contsistency  Structursistency  and respReTransQoL (RTQ) version 1. There was moderate evidence of content validity [validity , conflicvalidity , 81 and validity . Internavalidity .RTQ version 2. This revised version had moderate evidence for internal consistency and structural validity. There was still limited evidence for hypothesis testing [ testing .The study by Almutary et al.  evaluateCKD-Symptom Burden Index (CKD-SBI)Limited evidence was found for hypothesis testing while internal consistency was rated as 'unknown' .Modified Time Trade-Off (TTO)There was limited evidence for test-retest reliability and hypothesis testing .Internal consistency: Of the 58 studies that assessed internal consistency, 42 were scored 'poor' for methodological quality. This was due to one of 3 reasons:Some studies did not conduct a factor analysis and did not reference a relevant study that conducted one , 75, 82.Some studies conducted a factor analysis but had inadequate sample sizes by COSMIN standards , 78, 80.Some studies referenced a relevant study that conducted a factor analysis but the sample size used for the study was inadequate by COSMIN standards , 84–95.Test-retest reliability: Thirty-four studies conducted test-retest reliability and most of them reported internal correlation coefficients (ICC). The study by Duarte et al.  was the Measurement error: The included studies did not provide adequate information on parameters such as the minimal important change (MIC) , the staContent validity: This was assessed by development studies for RTQ , modifieConstruct validity (Hypothesis testing): Of the 66 studies evaluated, only 13 reported clearly formulated a priori hypotheses or expectations regarding the magnitude and direction of correlations. For this reason, most of the studies were rated as 'fair' for the methodological quality of their hypothesis testing.The absence of clear a priori hypotheses make it difficult to determine whether any results reported for construct validity and responsiveness was due to chance or not , 101.Structural validity: A number of measures were rated poorly for structural validity due to issues with factor analysis . Out of the 19 studies that conducted a factor analysis, 8 were scored 'poor' , 78, 80 Criterion validity: This was not assessed for any study as the COSMIN Delphi panel does not regard any PROM as true 'gold standard'  and the Cross-cultural validity/Translations: Twenty-five studies translated PROM instruments and adapted them to varying degrees for their study population. As none of these studies performed a multi-group confirmatory factor analysis or assessed differential item functioning (DIF) between language groups, the decision was made not to assess cross-cultural validity. Therefore, only the quality of their translations was assessed according to the provisions specified in the COSMIN manual .The translations conducted by 20 studies , 96, 102Responsiveness was only assessed by 8 of the included studies , 78–80 aA number of the PROMs included in this review had significant floor and ceiling effects which might indicate a reduction in their ability to discriminate between patients with the lowest or highest possible scores and detect changes over time .Ten instruments  had floor and ceiling effects > 15% while the RTQ v2 and SF-12 , 81 had The ‘work status’ dimension of the KDQOL-SF had a 70% floor effect , 86 whilEight studies namely; RTQ v1 , RTQv2 , GSRS & This was difficult to assess for the included studies as less than a third reported the average time needed to complete the questionnaires, few reported the recall period used and none reported administrative requirements for collection and analysis of data. However, most of the studies reported good response rates which suggest that patients might find the use of PROMs acceptable.The fact that majority of the studies failed to report the level and/or the method used for handling missing data meant there might be a risk of bias . QuestioPatient involvement in the process of item generation and/or item selection was reported in the development of the RTQ , KDQ 5656, KTQ , ESRD-SCAll the studies that translated and adapted measures reported pre-testing their translations in patients to assess a combination of comprehension, cultural relevance and acceptability except Rebollo et al.This is the first review to use the COSMIN checklist  to evaluIn the pre-dialysis population, the KDQOL-36 exhibited strong evidence for internal consistency and moderate evidence for construct validity (hypothesis testing). It should be noted that the evidence for this measure was obtained from studies conducted in Taiwanese patients , and a cIn dialysis patients, we found evidence to support the use of both the KDQOL-SF and the KDQOL-36. The KDQOL-SF demonstrated strong evidence for internal consistency and structural validity and moderate evidence for test-retest reliability and construct validity (hypothesis testing) while the KDQOL-36 had moderate evidence for internal consistency, test-retest reliability and construct validity (hypothesis testing). Again it should be noted that this evidence was obtained from a significant number of non-English studies, further validation work would be needed before these measures could be confidently utilised in an English speaking population.In renal transplant patients, the ESRD-SCLTM demonstrated strong evidence for internal consistency and moderate evidence for test-retest reliability, structural validity and construct validity (hypothesis testing).Consistent with the review by Gibbons and Fitzpatrick , much ofIn line with our findings, Gibbons and Fitzpatrick  found evThere were methodological issues with the majority of the PROMs included in this review. These included: sample sizes smaller than current recommendations, a lack of clearly described a priori hypotheses and inadequate reporting of missing data, and little or no information on measurement error and responsiveness. Similar issues were reported by Gibbons and Fitzpatrick  and highGiven that the COSMIN standards only became available within the last decade, it is unsurprising that most of the earlier studies fared poorly when judged against these exacting methodological and reporting standards even though they might meet the minimum standards recommended by the International Society for Quality of Life Research (ISOQOL) . HoweverThis systematic review provides a basis for identifying PROMs with potential utility in clinical practice. There is evidence that the use of PROMs in clinical practice could enhance communication –112 betwWhilst effective management of risk factors can slow CKD progression  many patWhilst a systematic review can provide valuable evidence on psychometric properties of PROMs, clinicians and researchers need to consider a number of issues such as the domains covered by different measures, the available supporting evidence, and the suitability for the target population and use . As no single measure covers all the domains that might be of interest, there might be a need to administer more than one measure. Patient acceptability is also a key issue. Therefore, it is important that patients are involved not just in the development of measures but also in the selection for research and/or practice.During the course of this review, we became aware of the existence of the IPOS-Renal and contacted its developers . This meThe key strengths of this review are the use of the COSMIN standards and criteria for evidence synthesis which ensured that our assessments of the included PROMs were robust, the stratification of the review by stage of CKD and the absence of language restrictions which strengthened our findings. The main limitation is the fact that the included studies did not adequately report their assessments for a number of measurement properties thus making it difficult and sometimes impossible to evaluate these properties.all measurement properties and some lack validation in English-speaking populations. Future work should be undertaken to address these gaps. For all measures, it is vital that content validity is established according to FDA guidelines to ensure that they actually measure the concept(s) of interest. This should be conducted before other measurement properties are fully evaluated and adequately reported in order to facilitate their subsequent evaluation. Investigators may use this review to identify the gaps in evidence and design studies to address these issues in future.At present, we suggest the use of the KDQOL-36 in pre-dialysis patients though initial validation might be required. We recommend the KDQOL-SF and the KDQOL-36 for use in dialysis patients as we found evidence supporting both of these measures. The shorter 36-item KDQOL-36 may be more practical for use in routine clinic settings, while the longer 80-item KDQOL-SF might be preferred for research purposes where more detailed information may be required. We suggest using the ESRD-SCLTM in renal transplant recipients to assess issues pertaining to renal transplantation and immunosuppression therapy. These measures are recommended based on the fact that they currently possess the best evidence available according to COSMIN standards in these populations and meet the minimum standards recommended by ISOQOL . HoweverS1 Text(PDF)Click here for additional data file.S2 Text(DOC)Click here for additional data file.S3 Text(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


During 2010–2012, the percentage of adults aged ≥18 years who had no natural teeth was higher in nonmetropolitan areas than in metropolitan areas for all age groups. The percentage of adults with no natural teeth also increased steadily with age in metropolitan and nonmetropolitan locations. Among persons aged ≥85 years in nonmetropolitan locations, 40% had lost all their natural teeth, compared with 31% of those in metropolitan areas. Among adults aged 18–44 years, the percentages were 3.8% in nonmetropolitan areas and 2.1% in metropolitan areas.Sources: National Health Interview Survey, 2010–2012. Available at http://www.cdc.gov/nchs/nhis.htm.http://www.cdc.gov/nchs/hdi.htm.CDC. Health Data Interactive. Available at Reported by: Ellen A. Kramarow, PhD, ekramarow@cdc.gov, 301-458-4325.


Influenza subtypes such as H7 have pandemic potential since they are able to infect humans with severe consequences, as evidenced by the ongoing H7N9 infections in China that began in 2013. The diversity of H7 viruses calls for a broadly cross-protective vaccine for protection. We describe the construction of recombinant modified vaccinia virus Ankara (MVA) vectors expressing the hemagglutinin (HA) or neuraminidase (NA) from three H7 viruses representing both Eurasian and North American H7 lineages – A/mallard/Netherlands/12/2000 (H7N3), A/Canada/rv444/2004 (H7N3), and A/Shanghai/02/2013 (H7N9). These vectors were evaluated for immunogenicity and protective efficacy against H7N3 virus in a murine model of intranasal challenge. High levels of H7-, N3-, and N9-specific antibodies, including neutralizing antibodies, were induced by the MVA-HA and MVA-NA vectors. Mice vaccinated with MVA vectors expressing any of the H7 antigens were protected, suggesting cross-protection among H7 viruses. In addition, MVA vectors expressing N3 but not N9 elicited protection against H7N3 virus challenge. Similar outcomes were obtained when immune sera from MVA vector-immunized mice were passively transferred to naïve mice prior to challenge with the H7N3 virus. The results support the further development of an MVA vector platform as a candidate vaccine for influenza strains with pandemic potential. Nevertheless, construction, manufacture, and evaluation of a new strain-specific MVA vector in response to an emerging pandemic influenza virus will require a considerable amount of time and may not be faster than a conventional vaccine response. On the other hand, if vectors are capable of generating significant cross-protective immune responses, candidate vaccine vector development and evaluation can be done prior to the emergence of a novel influenza virus, shortening the time needed for a successful vaccination response to the outbreak.Modified vaccinia virus Ankara (MVA) vectors expressing influenza virus genes have shown promise as potential candidate vaccines, particularly for potential pandemic influenza viruses. The advantages of an MVA vector platform as a vaccine against influenza have been described previously6, and one MVA vector expressing the HA from the H5N1 A/Vietnam/1194/2004 virus has been assessed for safety and immunogenicity in a clinical trial in humans7. Evaluation of potential cross-protection has been encouraging. Not only have MVA vectors designed to enhance cross-reactivity, such as those expressing a mosaic H5 HA8 or multiple H5 HAs6, been shown to induce cross-protective responses, but relatively broad cross-protective responses have been demonstrated from MVA vectors expressing a single H5 HA10.Pandemic preparedness for avian influenza has resulted in the construction and evaluation in various animal models of several MVA candidate vaccine vectors for H5 influenza viruses. Most of the characterized MVA vectors for avian H5 express the virus hemagglutinin (HA)12 but with yearly epidemics continuing13, is a vivid reminder that other avian subtypes of influenza also pose a serious public health threat and should be included in vaccine preparedness planning. Recently, an MVA vector expressing the H7 hemagglutinin was shown to protect ferrets against an H7N9 challenge14. But, as for H5 MVA vectors, more work is needed to define cross-reactivity and evaluate potential candidate H7 MVA vectors.The emergence of the novel H7N9 virus in China, beginning in 2013Here, we describe the construction of MVA vectors expressing the HA or neuraminidase (NA) from H7N3 and H7N9 viruses. MVA vectors were evaluated for their ability to elicit protective immunity in a mouse challenge model of H7N3. The results provide evidence for cross-protective responses to MVA-expressed HA from Eurasian and North American H7 lineages. In addition, the protective effect of MVA-expressed NA was demonstrated.http://www.who.int/influenza/vaccines/virus/en/). H7N3 A/mallard/Netherlands/12/2000 NIBRG-60  was developed by the National Institute for Biological Standards and Control (NIBSC) (United Kingdom); H7N3 A/Canada/rv444/2004 (A/Can) was developed at the St. Jude Children’s Research Hospital ; H7N9 A/Shanghai/02/2013 (IDCDC-RG32A) (A/Shang) was developed at the Centers for Disease Control and Prevention . Influenza viruses were propagated in 9-day-old specific pathogen-free embryonated chicken eggs. For the isolation of genomic RNA for the cloning of viral genes, the three H7 viruses were propagated in Madin-Darby canine kidney (MDCK) cells. The modified vaccinia virus Ankara (MVA) was obtained from Dr. Bernard Moss, NIAID/NIH, and was propagated in DF-1 cells (ATCC # CRL-12203). The purification of MVA and recombinant MVA vectors from infected DF-1 cells by ultracentrifugation through a 36% sucrose cushion was performed as previously described16. All experiments, including animal experiments, were conducted under biosafety level 2 plus (BSL-2+) laboratory conditions.The H7 viruses used in these studies were reassortant candidate vaccine viruses  at a multiplicity of infection (MOI) of 1.0. After 24 hours, cells were scrapped and harvested by low speed centrifugation at 1000 rpm for 10 min at 4 °C. Total RNA was isolated from each cell pellet using the RNeasy mini kit and protocol described by the manufacturer . Viral cDNAs were synthesized from total RNAs using the Omniscript reverse transcription kit (Qiagen) and the 12-mer Uni-12 influenza universal primer18 by LR recombination (ThermoFisher Scientific) to generate pLW44-HA or pLW44-NA shuttle vectors. In making recombinant MVA, monolayers of DF-1 cells were infected with MVA at an MOI of 0.1 for 1 hour, followed by transfection with pLW44-HA or pLW44-NA. Recombinant MVA expressing HA (MVA-HA) or NA (MVA-NA) were obtained from the infected/transfected cells by serial plaque purification (3 to 6 times) of recombinant plaques on DF-1 cells. Genetic stability of recombinant vectors was confirmed by plaque assay and the absence of parental MVA in the purified recombinant vectors was confirmed by PCR assay. Expression of authentic HAs and NAs by the recombinant MVA vectors was confirmed by Western blotting. For downstream use in animal inoculation, recombinant MVA-HA and MVA-NA were further pelleted on 36% sucrose cushion as previously described16.Each PCR product was ligated with pENTR-D-TOPO vector  to generate entry clones. The authenticity of the HA and NA nucleotide sequences in the pENTR-D-TOPO vector was confirmed by nucleotide sequencing. Each entry clone was recombined with plasmid pLW44attR2 PFU–108 PFU per dose in 50–100 µL) in sterile, endotoxin-free phosphate-buffered saline (PBS). Mice were inoculated subcutaneously at the base of the tail, and evaluated daily for up to 7 days post-vaccination. Blood samples were obtained from mice three weeks post-vaccination.All immunizations and blood draws were performed in accordance with an animal protocol approved by the FDA White Oak Consolidated Animal Program. Six to eight week-old male BALB/cByJ mice were purchased from the Jackson Laboratory . For immunization, MVA-HA or MVA-NA vectors, as well as MVA control, were diluted to the appropriate dose in plaque-forming units (PFU)  for the detection of specific immunoglobulin G (IgG) responses to H7 was developed and optimized using recombinant H7 protein of A/Anhui/1/2013 (H7N9) produced in baculovirus . Immulon 2HB plates were coated with the recombinant H7 protein at 1 µg/mL in PBS, and stored at 4 °C overnight. Excess coating solution was removed from plates, and blocking buffer  was added to wells, and plates were incubated for two hours at 37 °C. Plates were washed in wash buffer (PBS containing 0.05% Tween-20) and incubated with serial two-fold dilutions of each test serum sample diluted in wash buffer containing 10% FBS as the diluent. After incubation for 1.5 to 2 hours, plates were washed and incubated with a 1:5,000 dilution of goat anti-mouse IgG, horseradish peroxidase (HRP) conjugate  for 1 hour. Plates were washed and incubated with the HRP substrate 2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid] hydrogen peroxide  for 30 minutes. After stopping the HRP/substrate reaction with 1% sodium dodecyl sulfate (SDS), the ODSimilar ELISAs were set-up for the detection of the IgG response to influenza NA (N3 and N9) in test serum samples, except that plates were coated with recombinant N3 protein of A/Canada/rv444/2004 (H7N3)  or recombinant N9 protein of A/Shanghai/2/2013 (H7N9)  for the quantitation of N3-specific and N9-specific antibodies, respectively. In calculating mean antibody titers, a serum sample with no detectable IgG was assigned a titer of 10 .8 PFU of recombinant MVA-HA vectors or with parental MVA, via the subcutaneous route. A control group was left as untreated (naïve). On day 7 post-inoculation, all mice were sacrificed, dissected, and spleen isolated from each mouse as previously described19. Lymphocytes were prepared from each mouse, cultured in Roswell Park Memorial Institute 1640 (RPMI-1640) medium at 106 cells/well in 24-well tissue culture plates, and stimulated in vitro by live virus infection with candidate vaccine viruses A/Can or A/mal/NL or A/Shang. Culture supernatants were collected after 72 hours incubation, clarified by centrifugation, and tested for secreted IFN-γ, as previously described19.Groups of naïve mice (3 mice per group) were inoculated with 1050) of A/mallard/Netherlands/12/2000 (H7N3) which proved to be more lethal than A/Can and A/Shang vaccine viruses, as determined in preliminary LD50 experiments. Challenge virus at the appropriate dose was re-suspended in sterile PBS, and each anesthetized mouse received 10 µL of the suspension per nostril. Following challenge, mice were evaluated and weighed daily for two weeks. The study endpoint was a 25% decrease in body weight from the weight on the day of challenge. A mouse that reached this study endpoint was sacrificed and considered to have succumbed to infection.The intranasal challenge route was used for the assessment of protective immune responses in mice. Mice were weighed and anesthetized by intraperitoneal injection of ketamine/xylazine (0.5 mg/0.1 mg per gram body weight), and challenged with 5 to 10 50% lethal dose . For passive transfer, each antiserum was diluted 1:1.5 in sterile PBS, and inoculated into groups of naïve mice (5 mice per group) with 0.5 mL per mouse , via the intraperitoneal (i.p.) route. After 6 hours, mice were weighed, anesthetized, and challenged with 5 LD50 of A/mallard/Netherlands/12/2000 (H7N3) as described above, and evaluated over two weeks.Hyperimmune antisera were prepared by immunizing groups of mice (5 to 10 mice per group) with 1020. Since the recombinant MVA expressed NA alone, the homologous H7N3 influenza virus preparations could be used as the source of NA in the ELLA without any concern of interference by HA-specific antibodies. An H6N9 reassortant virus generated by reverse genetics as previously described21 was used to measure N9-specific antibody titers.The enzyme-linked lectin assay (ELLA) was used for the detection of NA-inhibiting antibodies in immune antisera as previously described23. HA-pseudovirus neutralization was tested as described24. The antibody dilution causing a 95% reduction of pseudovirus-expressed luciferase compared to control was used as the neutralization endpoint titer , and was calculated with nonlinear regression using Prism software . Data reported were from at least two independent experiments, with each sample run in duplicate.H7 HA from A/mallard/Netherlands/12/2000, A/Shanghai/2/2013 and A/Canada/rv444/2004 were used to construct retroviral pseudoviruses. HA-pseudoviruses were produced in 293 T cells essentially as described previouslyDifferences in antibody responses to HA or NA between treatment groups inoculated with different doses of MVA-HA or MVA-NA were compared using a statistical t-test of the log10-transformed IgG titers or the Mann-Whitney rank sum test where an initial test of normality failed. In all cases a p-value < 0.05 was considered statistically significant.All data generated or analyzed during this study are included in the text, tables, and figures.25; the second H7N3, A/Canada/RV444/2004 (A/Can), is a North American lineage H7 influenza isolate. The H7N9 virus, A/Shanghai/2/2013 (A/Shang), is an H7 virus of Eurasian origin (group I lineage) from the recent 2013 H7N9 outbreak in China. The construction and purification of the recombinant viruses are described in Materials and Methods.Recombinant MVA vectors were constructed expressing the HA or NA from two H7N3 viruses and one H7N9 virus. One H7N3 virus, A/mallard/Netherlands/12/2000  is from the group II H7 Eurasian lineageRecombinant proteins expressed by the MVA vectors were characterized by Western blotting. Lysates of cells infected with the recombinant viruses were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and the blotted proteins on the membrane matrix were detected with antibodies specific to H7 or N3 or N9 Fig. . The H7 50 of 1.6 × 104 PFU when inoculated into mice by the intranasal route. In order to determine whether MVA vectors expressing the A/mal/NL HA or NA would elicit protective responses against the A/mallard/Netherlands/12/2000 challenge virus, dose-ranging studies of the recombinant vectors were performed. Mice were vaccinated with a range of doses of the MVA vector expressing the A/mal/NL HA (102–105 PFU) or NA (103–105 PFU). A control group was vaccinated with 105 PFU of MVA. Three weeks post-vaccination, serum samples obtained from the animals were tested for H7-specific IgG (for the MVA-HA treatment cohort) or N3-specific IgG (for the MVA-NA treatment cohort) by ELISA. Mice in the MVA control group had no quantifiable H7-specific or N3-specific IgGs  of 3.5 ± 0.3 and 3.4 ± 0.4 for the 105 and 104 PFU MVA-HA groups, respectively  and 3/5 (mean titer = 1.8 ± 0.8) in the MVA-HA and MVA-NA, respectively, had measurable levels of specific IgG; these IgG titers were significantly lower than those in the 104 PFU groups.To evaluate the immunogenicity and protective effect of recombinant vectors expressing HA and NA, a lethal challenge model was established using A/mal/NL. This virus, which can be handled under BSL-2+ conditions, resulted in morbidity, as measured by weight loss and mortality, with an LDgGs Fig. . Similarely Fig. ; these I50 of A/mal/NL; disease progression post-challenge is shown in Fig. 2 PFU MVA-HA group lost weight dramatically and succumbed to infection, with a mean time-to-death (MTD) of 7.2 and 8 days, respectively. All mice vaccinated with ≥104 PFU of MVA-HA or MVA-NA survived challenge, with the MVA-HA treatment cohort losing less weight than the MVA-NA treatment cohort. This set of data indicates that recombinant H7 or N3 expressed from MVA vectors elicit protective responses against the homologous H7N3 virus in mice, and shows that doses ≥104 PFU are protective in this model.All mice were challenged at 3-weeks post-immunization with 5 LD3 PFU and 104 PFU of the MVA vectors expressing the HA or NA of A/mal/NL were selected, since our data above indicated that a dose of 103 PFU of either MVA-HA or MVA-NA provided only partial protection against A/mal/NL challenge and weight loss was also observed at the 104 PFU dose. Four groups of mice (5 per group) were vaccinated with either MVA-HA only (103 PFU and 104 PFU) or MVA-NA only (103 PFU and 104 PFU). Two other groups were vaccinated with a mixture of 103 PFU MVA-HA and 103 MVA-NA or a mixture of 104 PFU MVA-HA and 104 MVA-NA. For the groups that received 103 or 104 PFU of MVA-HA or MVA-NA only, the equivalent dose of MVA was mixed with each dosage in order to normalize for the MVA background among all six treatment groups. One group of mice was vaccinated with 106 PFU of MVA-HA as a positive control, and one group was vaccinated with 106 PFU of MVA as a negative control. Three weeks post-vaccination, sera were obtained from all mice and tested for H7-specific IgG and N3-specific IgG by ELISA  and those vaccinated with a combination of MVA-HA/MVA-NA. In the 104 group, 3/5 mice had detectable H7 IgG titers and 5/5 mice had anti-N3 IgG. Again, there were no statistical difference between the single antigen groups and the antigen combination group. H7-specific and N3-specific IgGs were not detected in the sera of mice in the MVA control group, but all mice in the high-dose 106 MVA-HA group had high titers of anti-H7 IgG. Data from these experiments confirm a dose response to vaccination with the MVA vectors, with antibody titers increasing with dose escalation. Further, co-administration of vectors expressing H7 and N3 did not appear to interfere with the IgG response to each antigen.In the 1050 of A/mal/NL, and evaluated daily. Figure 6 PFU MVA-HA positive control group lost weight, and all survived virus challenge. In the MVA (negative control) group, 3/5 mice succumbed to infection, and although two mice survived, their respective peak weight loss  was close to the study endpoint of 25%. At low-dose vector immunization, 3/5 mice in the 103 PFU MVA-HA and 2/5 of those in the 103 PFU MVA-NA survived challenge and maximum weight loss of the survivors in these groups was near the study endpoint. Adding 103 MVA-HA and 103MVA-NA did not confer any obvious protective advantage against challenge. One mouse in the combined 103 PFU MVA-HA/103 MVA-NA treatment group was found dead a day after challenge; the death was considered unrelated to the virus challenge. Of the remaining four evaluable mice in the group, three succumbed to infection with a mean time-to-death of 8 days.In order to assess the effect of co-expression of H7 and N3 on the protection of mice from influenza, mice in this study were challenged with 5 LD4 PFU of MVA-HA only or 104 MVA-NA only survived challenge, albeit with significant peak mean weight losses of 9.9% (on day 6) and 12.7% (on day 6), respectively. All mice vaccinated with a mixture of 104 PFU MVA-HA/104 MVA-NA also survived, but the peak mean weight loss (10.8% on day 6) was similar to those of the 104 PFU MVA-HA only or MVA-NA only treatment groups, again suggesting a lack of additive effect against challenge. The results of this virus challenge study corroborated our previous observation that animals vaccinated with ≥104 PFU of MVA vectors expressing either influenza virus H7 or N3 were protected from lethal challenge with the H7N3 homologous virus. Although, immunization with a combination of MVA-HA and MVA-NA vectors did not seem to enhance protection in this mouse model, there appeared to be no inhibitory effects of combining the two vectors.In a pattern consistent with our previous observations, all mice vaccinated with 107 PFU of MVA-HA or MVA-NA . Three weeks post-vaccination, sera were obtained from mice and tested for H7- or N3 or N9-specific IgGs by ELISA. No mice inoculated with the empty MVA vector had detectable IgG against HA or NA  protected against H7N3 virus challenge, but the immune response against the heterologous N9 subtype A/Shang was not protective Fig. . Among t8 PFU of each HA- and NA-expressing MVA vector along with a control group vaccinated with 108 PFU of MVA. Mice were bled three weeks after each immunization, the immune serum obtained from mice in each treatment group was pooled, and each serum pool tested for HA neutralizing and NA inhibiting antibodies.As described above, serum samples obtained from mice immunized with MVA vectors expressing the HA or NA of influenza A/H7 viruses induced H7-, or N3- or N9-subtype-specific IgG responses. In further characterizing the antibody response to HA and NA following vaccination with these MVA vectors, sera from vector-immunized mice were tested for strain-specific functional antibody responses. Groups of mice were vaccinated twice at an interval of three weeks with 10The serum samples from MVA-HA vector immunized mice were evaluated in a cross-neutralization assay using lentiviral pseudoviruses expressing the HA of A/mal/NL, A/Shang, and A/Can Table . AntiserA parallel evaluation of the inhibiting antibody titer in sera from MVA-NA immunized mice was performed in a neuraminidase-inhibition (NI) assay, using homologous and heterologous viruses Table . Antiserin vitro and are functional in vivo in providing protection against H7N3 virus challenge.In assessing the functional contribution of the antibody response elicited by MVA-HA and MVA-NA vectors to protection against H7N3 virus challenge, high-titered antisera were passively transferred into naïve mice, which were then challenged with A/mal/NL. Mice in the different treatment groups were inoculated i.p. with 0.33 mL of antiserum in a total volume of 0.5 mL, and six hours after the passive serum inoculation were challenged with A/mal/NL. Influenza morbidity in the various treatment groups in the days after challenge is shown in Fig. 8 PFU of MVA vectors expressing the HA of A/Can, A/mal/NL or A/Shang. A control group was vaccinated with MVA and a group was left untreated (naïve). Spleen cells were harvested 7 days post-vaccination, lymphocytes were cultured in vitro and re-stimulated by live virus infection with A/Can, A/mal/NL or A/Shang at an MOI of 1.0. Culture supernatants harvested after 72 hours of incubation were tested for the presence and quantity of secreted IFN-γ. There was no detectable IFN-γ in cells from the naïve animals and very low levels of IFN-γ in cells from the MVA and MVA-HA (Shang) animals regardless of the virus used for re-stimulation  was also protective, but the MVA vector expressing the N9 from the H7N9 virus (45.0% identity) was not. Since antibody to HA and NA have been shown to be independent correlates of protection37, it was somewhat surprising that co-administration of MVA-HA and MVA-NA did not result in obvious improved protection in our challenge experiments. This may be due to limitations in the challenge model used. For example, at doses of MVA vector of 104 or greater, all mice responded with detectable HA-specific antibody and all were protected from challenge. At the lower immunization doses used for the co-administration experiments, antibody responses were not evident in all animals, suggesting that there might be a “take” threshold that must be reached for successful immunization. Additional measurements of pathogenesis such as measuring the viral load in the lungs might add additional information. But, it is also possible that a challenge model that employs higher immunization and challenge doses might be able to demonstrate an additive effect of HA and NA MVA vector immunization.Very few studies using MVA vectors to express influenza NA have been reported. In one study, an MVA vector expressing the H1N1pdm2009 NA was only partially protective against a homologous influenza virus challenge38. In our limited analysis, immunization with recombinant MVA vectors expressing HA of A/H7 viruses also appeared capable of stimulating cellular immune responses, but the role of cell-mediated immunity to HA and NA in the context of a robust protective antibody response to those proteins is difficult to determine. Additional immunization approaches employing MVA vectors expressing H7N9 HA and NA, along with vectors expressing conserved proteins, could be explored as a means to maximize a broad protective response to H7 viruses.Since the antibody response to influenza virus infection is known to be critical for effective control of influenza disease, and since most influenza vaccines contain HA as the vaccine antigen, most of our analyses of the immune response to MVA vector immunization focused on measuring and understanding the antibody response to HA and NA. Nevertheless, cell-mediated immunity is known to be an important component of the immune response to influenza virus infection, and indeed, other MVA vectors designed to elicit cell-mediated immunity to conserved proteins such as NP have been described, including some that have been evaluated for protection against H7 virus challenge1, and the MVA vector-mediated cross-protection demonstrated in our study, as well as other studies, seems to indicate that an MVA HA or NA-expressing vector might not need to be perfectly matched to an emerging virus strain in order to provide protection. The promising results with MVA NA vectors provide another option for an MVA vector repository since the drift of HA and NA does not occur at the same rate. Future steps in the development of MVA vectors as a potential pandemic influenza response will include a more complete characterization of the protective response to vaccination, including a finer analysis of both antibody and cell-mediated immune responses, and a more thorough evaluation of the broadness of the protective response. In addition, studies that compare the immunity and protection afforded by MVA vectors to that elicited by other types of influenza vaccines, particularly traditional inactivated vaccines, will be important. Further, additional studies using MVA vectors expressing HA and NA glycoproteins and conserved viral proteins, as well as prime-boost studies that evaluate a mismatched vector or vaccine followed by a matched vaccine, will be important for developing optimal immunizations strategies. Finally, to be truly feasible as a pandemic response, licensure of an MVA vector vaccine should be pursued in advance of the emergence of a novel influenza virus.The results from the current study add support to the concept of an MVA vector platform as a reasonable and feasible vaccine strategy for pandemic influenza. It has been suggested that repositories of MVA vectors be established for potential pandemic influenza strainsSupplementary Info File for Fig 1


The aim of this study was to assess the mean value of spleen stiffness measured by Shear wave elastography in healthy patients and its dependence on age, sex, and spleen dimensions, and to evaluate the repeatability of this method.The final study group included 59 healthy volunteers without any clinical evidence of liver disease, portal hypertension, hematological disorders, and without any pathological ultrasonographic spleen findings. Each patient underwent abdominal ultrasound examination and elastography of the liver and the spleen.N = 25), it was 17.3 ± 2.7 kPa, and in the group of women (N = 34), it was 16.1 ± 2.2 kPa. The study confirmed no correlation between spleen stiffness and sex, age of patients, and spleen size. Coefficient of repeatability and correlation coefficient between the results of the first and the second measurement showed good but not ideal repeatability of the measurement results.The mean value of spleen stiffness was 16.6 ± 2.5 kPa. In the group of men (Our outcomes may be a reference point for evaluating spleen stiffness in research on patients with various illnesses. Elastography is a relatively new diagnostic method used to assess stiffness (elasticity) of soft tissues . Its useShear wave elastography (SWE) is, next to transient elastography (TE) or acoustic radiation force impulse (ARFI), one of the most advanced elastography techniques. All of them rely on measuring shear waves propagation and allow to assess tissue stiffness in a quantitative way. Shear waves in TE are induced mechanically by the “thump” at the skin surface, whereas in ARFI technique it is a result of ultrasound-induced radiation force at selected depth , 3.The concept of SWE relies on generating transverse waves by using an acoustic radiation force created by a focused ultrasound beam at different depths. The waves create a new, transverse cone-shaped shear wave. To estimate propagation of the shear wave, an unusually quick data acquisition by the ultrasound (US) probe is required, whereas slower techniques are sufficient in TE and ARFI methods. The SWE device must be able to register at least 5000 frames per second . Due to Single studies of spleen elastography have been published so far. They have shown a potential usefulness of spleen elastography in estimating the risk of esophageal varices in patients with liver cirrhosis , 6 or poHowever, it is necessary to provide information about spleen elasticity in healthy people, without chronic liver diseases, hematological disorders, and viral infections.The results of two studies on the value of SWE spleen stiffness in healthy volunteers have been published. In 2011, Arda et al. reported mean values of SWE spleen stiffness as 3.1 ± 1.9 kPa among men and ±2.9 kPa among women without a statistically significant difference in elasticity values between sexes . The meaThe aims of this research were to estimate the average value of SWE spleen stiffness, its correlation with age, sex, spleen size, and to evaluate repeatability of this method, and therefore estimate the groundwork for further investigations concerning spleen sonoelastography.The study was conducted in accordance with the guidelines of the local University Ethics Committee for conducting research involving humans. Each patient signed his/her informed consent before participation in the examination.A group of 70 adult volunteers without any history of chronic liver diseases, portal hypertension, or hematological disorders underwent abdominal US examination and SWE liver elastography. The studied group consisted mainly of the hospital staff, students, or their relatives. Patients with focal splenic lesions, portal vein or bile tract dilatation, significant liver steatosis , and SWE liver elasticity value higher than 6.5 kPa were excluded from the next examination stage. As a result, 63 volunteers were selected for further research. The examination was performed using an Aixplorer device . The ultrasound examination, as well as elastography, was performed by an experienced radiologist. Spleen SWE examinations were performed through intercostal spaces with the patient lying supine with the arms behind his head. In order to minimize breathing motion, the patient was instructed to inhale deeply and hold his breath. This position eliminates artifacts and allows one to visualize the spleen most precisely. All elastographic measurements were performed with a convex transducer (Supersonic Imaging System) with a frequency range 1–6 MHz. Transverse and longitudinal dimensions of the spleen were also measured. It was impossible to measure SWE spleen stiffness in four patients due to significant artifacts as shown in Fig. The final study group included 59 volunteers. For more information, see Table N = 40; and patients aged 45 and above, N = 19. In the second way, volunteers were divided into three subgroups: patients aged up to 34, N = 24; patients aged from 35 up to 49, N = 17; and above 49, N = 18.To analyze age correlation, subjects were divided in two ways. In the first one, two subgroups were created—patients aged up to 45, The dimension correlation was analyzed after dividing the acquired values of transverse and longitudinal spleen dimensions into two groups based on a median. The median for a longitudinal dimension was 9.6 cm, and for a transverse dimension—4.5 cm.To verify the normal distribution, the Shapiro–Wilk test was used.U Mann–Whitney test was used to identify correlations between spleen stiffness and sex as well as spleen size and age in two age subgroups: up to 45 years and above.The To assess correlation between spleen stiffness and age in three subgroups—up to 34 years, from 35 to 49 years, and over 49—the Dunn’s test was applied.To estimate the repeatability of spleen SWE, three statistical methods were used: the Bland–Altman test, the R&R method, and calculation of correlation coefficient. The confidence intervals on the difference between the result of the first and the second measurement were stated at the 95% confidence level.All statistical calculations were carried out using statistical computing software Statistica v. 10 .p = 0.102). For more information, see Table The mean value of SWE spleen stiffness was 16.6 ± 2.5 kPa. In the group of men, it was 17.3 ± 2.7 kPa, and in the group of women, it was 16.1 ± 2.2 kPa. There was no statistically significant difference between these two groups (p = 0.820). For more information, see Table In the subgroup including patients up to the age of 45, the mean value of a spleen stiffness was 16.5 ± 2.4 kPa, while in patients over 45 it was 16.8 ± 2.6 kPa. No statistically significant difference between these two groups was observed (p = 0.943). For more information, see Table The mean values of spleen stiffness in three subgroups, up to the age of 34, from the age of 34 to 49, and over 50 are as follows: in the first subgroup, it was 16.4 ± 2.3 kPa; in the second subgroup, 16.6 ± 2.6 kPa; and in the third subgroup, 16.9 ± 2.6 kPa. No statistically significant difference between these groups was observed (N = 31), the mean spleen stiffness was 16.5 ± 2.2 kPa; in the group with bipolar spleen dimension above 9.6 cm (N = 28), it was 16 ± 2.7 kPa. There was no statistically significant difference between these groups (p = 0.836). For more information, see Table In the group with bipolar spleen dimension up to 9.6 cm (N = 30), the average spleen stiffness was 16.7 ± 2.7 kPa; in the group with transverse spleen dimension above 4.5 cm (N = 29), it was 16.5 ± 2.2 kPa. There was no statistically significant difference between these groups (p = 0.102). For more information, see Table In the group with transverse spleen dimension up to 4.5 cm (r = 0.608 (0.417–0.748), p < 0.0001.Coefficient of Repeatability was calculated as a percentage of the two measurements whose difference exceeded the limit of agreement, rAB = 3/59 = 5.1%  can be conditionally accepted); Repeatability: EV (equipment variation) = 4.37; Reproducibility: AV (appraiser variation) = 1.01; Repeatability and Reproducibility: R&R = 4.48.The results of this analysis show a high repeatability of the spleen stiffness measurements and indicate that two good quality measurements acquired from the same area of the splenic parenchyma could be sufficient Figs. , 4.Although manual palpation has been an important element of physical examination for centuries, it was only at the end of the 20th century that brought us the possibility of a graphic and quantitative assessment of tissue stiffness due to elastography. The constant development of this branch of science has led to the creation of many new elastographic techniques. To evaluate the stiffness of the parenchymal organs, usually quantitative techniques, such as TE, ARFI, pSWE, SWE, and elasto-MR, are used. Each of these methods has its advantages and disadvantages , 3. The Elastography, as a very innovative method, has brought a lot of enthusiasm in the scientific world in the last century. A lot of studies about this method have been published. It might be said that elastography has completely changed the diagnostic process of liver fibrosis. Elastography is also used as a complementary method in diagnosing focal changes in, e.g., the breast(s) and thyroid . The splThe mean value of spleen stiffness obtained in our research was 16.6 ± 2.5 kPa and it was similar to the result of Leung et al. (17.3 ± 2.6 kPa) . Our stuWe strongly believe that our results may be a reference point for evaluating spleen stiffness in research on patients with different illnesses. Our study demonstrated good but not ideal repeatability of measurement results (5.1%). It suggests a necessity to perform at least two measurements of spleen stiffness in an SWE elastography examination.