The British people voted in 2016 to leave the European Union (EU). The UK has a unique history as a leader in animal protection policy. It has a relatively large economy and significant political power on a global basis. Brexit presents both threats and opportunities to animal protection in the United Kingdom (UK), EU, and internationally. This paper assesses the opportunities Brexit presents for animal protection in terms of five criteria. These are first, the political situation; second, regulatory changes; third, economic and trade factors; fourth, institutional considerations; and fifth, EU and international impacts. Brexit provides the opportunity to reform UK farming to promote high animal welfare outside of the EU Common Agricultural Policy (CAP). Brexit means the UK can ban live animal exports and the import and sale of fur products and foie gras outside of the EU. Leaving the EU permits the UK to have stricter requirements for the Pet Travel Scheme (PETS) to control puppy smuggling. Brexit provides an opportunity for the UK Government to reform policy-making for sentient animals. New sentience legislation could establish a fully independent UK Animal Welfare Advisory body and mandate Government to use animal welfare impact assessments on all policy that affects sentient species. Despite such opportunities, the UK Government appears uncommitted to major reforms. The drafting of the Agriculture Bill does not suggest a progressive animal welfare agenda. For live exports, the Government will consult on how to improve welfare, rather than outright prohibition. Similarly, rather than ban the import and sale of fur, the Government will use its influence to improve the welfare of fur-farmed animals outside the UK. Brexit provides some opportunities for animal protection. Pre-Brexit, the Government has not demonstrated the political will and commitment to realise these opportunities.The British people voted in a 2016 referendum to leave the European Union (EU). Brexit presents threats and opportunities to animal protection in the United Kingdom (UK), the EU, and internationally. This paper discusses opportunities for animal protection in terms of five criteria. These are first, political context; second, regulatory changes; third, economic and trade factors; fourth, institutional- and capacity-related factors; and fifth, EU and international considerations. Brexit permits reform of UK agricultural policy outside of the Common Agricultural Policy (CAP) to reward high welfare as a public good. The Agriculture Bill, however, does not suggest a radical reform agenda for animal welfare. Brexit permits a ban on live exports, but the UK Government is consulting on improving welfare, not prohibition. Brexit provides an opportunity to ban the import and sale of fur, but the UK Government has signalled it will work to improve welfare in fur farming. Brexit permits the UK to prohibit the import and sale of foie gras, but the Government has stated a ban may be challenged at the World Trade Organisation (WTO). Brexit allows more stringent Pet Travel Scheme (PETS) requirements to reduce puppy smuggling. Lucy’s Law and stricter enforcement will also mitigate the problem. New sentience legislation provides the opportunity for a fully independent and properly constituted UK Animal Welfare Advisory body conducting animal welfare impact assessments and ethical appraisal. The Government has proposed sentience legislation but there is a major risk it will not be in place before the UK leaves the EU. The Government has expanded the remit of the Farm Animal Welfare Committee, which is not fully independent and is dominated by veterinary members and agricultural interests. Brexit provides some opportunities for animal protection with radical reform of agricultural policy, prohibition of live exports, and banning the import and sale of fur and foie gras. Pre-Brexit, the Government has not demonstrated the political will and commitment to realise these opportunities. The United Kingdom (UK) has been a member of the European Union (EU) since 1993 and, before that, the European Communities (EC) since 1973. UK and EU animal protection law and policy is highly integrated. The UK, as a powerful EU member state with a history of progressive animal protection regulation, has had substantial influence on the development of EU animal welfare law. Furthermore, around 80% of UK animal welfare laws are based on EU regulations, rules, and directives. Farming policy in the UK has been determined by the EU’s Common Agricultural Policy (CAP) since 1973. There is a substantial volume of trade in agri-products and live animals between the UK and EU. The UK’s departure from the EU, or Brexit, therefore has the potential for major impacts on animal protection in the UK, EU, and internationally.The UK voted narrowly by 51.9% to 48.1% in a June 2016 referendum to leave the EU. David Cameron, who had campaigned to remain in the EU, resigned as Prime Minister of the UK immediately after the result. Theresa May replaced David Cameron as Prime Minister in July 2016. Theresa May’s Government gave notice of Article 50 to leave the EU in March 2017. May’s UK Government then negotiated the Withdrawal Agreement and Political Declaration with the EU. The EU signed a Withdrawal Agreement in October 2018. However, Theresa May was unable to pass her Withdrawal Agreement through Parliament. The EU granted Theresa May’s request for an extension of Article 50 until 31 October 2019. Without a withdrawal deal, the UK would crash out of the EU at the end of March 2019. Theresa May resigned as Prime Minister on 24 May 2019 based on her Government’s failure to ratify the Withdrawal Agreement in Parliament, poor UK local election results for the Conservative Party, and continued opposition within her Parliamentary party.In July 2019, Boris Johnson was elected leader of the Conservative Party and appointed Prime Minister of the UK. Johnson has stated that the UK will leave the EU on 31 October with or without an exit deal with the EU  word, which a few hon. Members have mentioned, we will be considering our future approach to regulation in the context of the negotiations on our future relationship with the EU.In July 2019, the Parliamentary EFRA Committee launched an inquiry into puppy smuggling . The folThe Canine and Feline Sector Group (CFSG)  has asseIn the event of a no deal, the EU plans to treat the UK as an unlisted third country and apply regulations and tariffs. These include checks and controls for sanitary and phytosanitary standards. This would mean increased journey times, which may have a positive impact on reducing puppy smuggling in the short term. In a no-deal situation, the UK could ban the import of puppies, but under WTO rules, it would need to extend this ban to the Republic of Ireland to be compliant .The UK is a relatively large market, with 66 million consumers that are wealthy on a global basis. The EU and non-EU nations therefore have an economic incentive to export goods and services to the UK, just as the UK has the same incentive to export its goods overseas. In this respect, if the UK Government makes a firm commitment to animal welfare and insists on parity of standards with trade partners, it may result in animal welfare reforms as a result of future trade deals. Furthermore, the UK can use soft power to project its progressive animal welfare values on a more global basis.The Government accepted an amendment in the Trade Bill 2017–19 to stipulate that imports meet the welfare standards of the UK. However, this applied only to existing free trade agreements. Furthermore, the Trade Bill 2017–19 fell with the prorogation of the Parliamentary session. The background briefing notes to the Queen’s Speech do not reference animal welfare in the section on the new Trade Bill. Furthermore, despite a further proposal to recognise animal sentience, there is a major risk that the UK will leave the EU without sentience legislation in place. If the UK leaves on no-deal terms, it will revert to WTO trading rules, which means it is far less likely that imports would be required to meet UK animal welfare standards.The development of Brexit policy has shone a light on UK policy making with respect to sentience legislation. As discussed in McCulloch ,19, the If the Government is to pay regard to animal welfare, it should have a robust and systematic process in order to do this. Governments ordinarily use impact assessments to assess the impacts of policy options, for instance, economic impact assessment, environmental impact assessment, and social impact assessment. However, the UK Government has no formal and systematic process to assess the impacts of policy options on sentient species, which is entailed by the Treaty of Lisbon or equivalent sentience legislation. McCulloch and Reiss ,111 haveArticle 13 of the Treaty of Lisbon states that EU member states, and therefore their respective governments, must pay full regard to animal welfare. Governments, however, also have duties to pay regard to other parties, such as human society and the biotic environment. Since governments have a duty to pay regard to various groups or entities that legally deserve consideration, policy makers require a means of deciding on policy that is justifiable in terms of the distribution of positive and negative impacts. For instance, Clause 1.1 of the Government’s Animal Welfare (Sentencing and Recognition of Sentience) Bill states that Ministers of the Crown must have regard to the welfare needs of sentient animals. Clause 1.2 states that in discharging the duty Ministers must also have regard to the public interest. Hence, the Government necessarily requires both a body and a process whereby it can conduct or be advised on how to weigh these interests.McCulloch and Reiss  have argMcCulloch and Reiss have argued that it is the inherent moral dimension of animal health and welfare policy making that impacts sentient species, and a lack of ethical appraisal, that contributes to the ongoing highly controversial nature of public policy that impacts animals , foot and mouth disease, bovine tuberculosis and badger culling). Such an independent Ethics Council for Animal Policy would be similar in concept to the highly respected UK Nuffield Council for Bioethics . It shouA coalition of leading UK-based animal protection organisations, including the RSPCA, CIWF, and WAP, have called for a ‘Better deal for animals’. A UK Government and Parliament petition to recognise animal sentience and require that animal welfare has full regard in law received 103,866 signatures . In the On 1 October 2019, the Government announced that the remit of FAWC was to be expanded to provide research and advice on pets and kept wild animals. FAWC has been renamed the Animal Welfare Committee (AWC) to recognise this change . It is uPost-Brexit, can the UK—as a more independent nation—have a greater opportunity to enhance animal protection in the EU and worldwide? First, the UK will need to maintain its current standards of animal protection. If the Government were to permit the import of lower welfare agri-products, for instance from the US, it is difficult to see how Brexit would lead to a net positive impact on animal welfare in the EU and internationally.Secondly, it will be in negotiating trade deals that the UK can have the most impact on animal welfare. Given that the EU has, on a global basis, very high animal welfare standards, there is arguably more potential for the UK to influence animal protection positively in non-EU nations. Indeed, the Wildlife and Countryside Link and A-Law make the case that post-Brexit, the UK may have more influence if it continues to align itself with EU policy:The UK and the EU will find they have in common their respective citizens’ desire to advance animal welfare; and the UK’s power to influence animal welfare standards worldwide will be so much greater if it seeks to do so in partnership, where appropriate, with the world’s biggest consumer market.(p. 6) Hence, despite Brexit meaning the UK leaving the EU, the UK should still work in concert with the EU to have an impact on animal welfare at the international level. This follows from the market size, and therefore economic and political weight, of the UK and the EU. The UK population, at around 66 million, is considerable. However, the EU population is around 510 million, and the US population around 325 million. Given these disparities, the UK should continue to align itself with the EU to have an impact on animal protection at the international level.Finally, there is potential for more specific UK-based reforms to have an impact at the EU or international level. Successful reform of UK agricultural policy to support high welfare may be emulated in other countries. If the UK prohibits live exports, or the import and sale of fur and foie gras, other nations may follow. Ministers often ask civil servants and experts what other nations do when they consult both informally and formally. For instance, one of the questions in the Animal Welfare (Sentencing and Recognition of Sentience) Bill asked how other states recognised animal sentience. Elected decision-makers generally prefer to follow tried and tested solutions to policy problems, rather than carve out entirely new solutions . The proBrexit is a major political upheaval for the UK, with threats and opportunities to animal protection. Given the unique history of the UK in animal protection and its relative economic size and political power, impacts will not be restricted to the UK, but will reach the EU and international level. This paper has assessed the opportunities that Brexit presents for animal protection. The paper assessed opportunities in terms of five criteria: First, political context; second, regulatory changes; third, economic and trade factors; fourth, institutional- and capacity-related factors; and fifth, EU and international considerations.The Conservative Governments delivering Brexit have made some progressive animal protection policies and some positive policy statements about maintaining and improving animal welfare after the UK’s withdrawal from the EU. The major opportunities Brexit presents for animal protection are as follows: Reform of UK agricultural policy outside of the CAP to reward high welfare; banning live exports; prohibition of the import and sale of fur products; prohibition of the import and sale of foie gras; more stringent regulation of PETS to reduce puppy smuggling; and passing sentience legislation to give powers for a fully independent and properly constituted UK Animal Welfare Council to advise Government through animal welfare impact assessments and ethical appraisal of policy options.The Government’s positions in these policy areas are as follows: The Agriculture Bill 2017–19 would give powers to ministers to support animal welfare as a public good. However, it is difficult to interpret the Bill as an agenda for radical animal welfare reform, and there are threats such as no commitment to funding post-2027 and the objective to increase productivity, which is generally associated with welfare problems. Furthermore, the briefing to the 2019 Queen’s Speech proposing a new Agriculture Bill does not reference animal welfare as a public good.The Government has previously stated that all options are on the table for live exports. However, there are substantial legal and political barriers to a full ban and Government has signalled further control of the trade, rather than an outright ban. There was no Bill to ban live exports in the 2019 Queen’s Speech and the background briefing document stated that Government planned to consult on improving the welfare of exported live animals. The Labour Party has pledged to ban the live export of animals but maintain trade across the Irish border.The Government has indicated it will look to use its influence on the international stage to reform fur farming, and not prohibit the import and sale of fur in the UK. This is disappointing, because the UK has a specific legal ban on fur farming and there is overwhelming and consistent public opposition to the UK fur trade. The scientific consensus is that fur farms cannot meet the welfare needs of species such as mink and foxes, which are not domesticated and remain essentially wild. The Labour Party has pledged to ban all fur in the UK. On foie gras, the Government has no policy position and has stated it will look at the issue after the UK’s departure from the EU. The Labour Party has pledged to ban imports of foie gras to the UK.The Government has introduced Lucy’s Law to ban the third party sale of puppies and kittens in the UK. It has stated that it will review the PETS requirements after Brexit. The Government published the Animal Welfare (Sentencing and Recognition of Sentience) Bill in 2017. The Bill was later withdrawn after criticism from the Parliamentary EFRA Committee. The 2019 Queen’s Speech briefing includes a commitment to recognise animals as sentient and impose a duty on the Government to have all due regard to animal welfare in formulating and implementing policy. However, there remains a major risk that the UK will leave the EU without this crucial legislation in place in the post-Brexit period. This risks importing lower welfare agri-products from nations such as the US.Animal protection groups have lobbied for a fully independent and properly constituted Animal Welfare Advisory body to implement sentience policy post-Brexit. Such a body could conduct mandatory animal welfare impact assessments and ethical appraisal of policy options. However, the Government has expanded the remit of FAWC (now the AWC) to provide advice on companion and kept wild animals. As a committee within Defra, FAWC/AWC is not fully independent and is dominated by veterinary members and agricultural interests. Furthermore, rather than use prospective animal welfare impact assessments the Government has indicated it prefers a retrospective ministerial report mechanism to discharge duties related to sentience. The Labour Party has pledged to strengthen animal welfare in UK law by appointing an Animal Welfare Commissioner to ensure that animal welfare is considered in policy making. It has also stated that it will recognise decapod crustaceans  and cephalopods  as sentient animals.Brexit provides some significant opportunities to reform animal welfare in the UK. The greatest positive impact would be if progressive reforms in the UK were to be replicated, or otherwise have a positive impact on the far larger numbers of sentient animals in the EU and internationally. However, the UK Government will need to commit to a progressive animal welfare agenda in order for positive impacts to materialise. The development of policy in the pre-Brexit period suggests the UK Government is not sufficiently committed for the major animal welfare reforms to be realised.


Good Clinical Laboratory Practice (GCLP) is a standard that ensures quality and reliability of research data by adopting the principles of Good Laboratory Practice and Good Clinical Practice. Even though implementing a quality system in a basic research laboratory is still a contentious issue, it ensures that the research data are accurate, valid, and reliable. GCLP implementation requires proper documented procedures and safety precautions to achieve this objective.This article describes the Kenya Medical Research Institute (KEMRI)–Wellcome Trust Research Laboratories experience in the implementation of GCLP guidelines in a laboratory conducting basic research.The laboratory managed to implement GCLP elements that could be applied to a basic research laboratory, such as standard operating procedures, equipment management, laboratory analytical plans, organization, and personnel. The laboratory achieved GCLP accreditation in October 2015.The methodology, suggestions, and comments that arose from our experience in implementing GCLP guidelines can be used by other laboratories to develop a quality system using GCLP guidelines to support medical research conducted to ensure the research data are reliable and can be easily reconstructed in other research settings. The KEMRI-Wellcome Trust Research Programme was established in 1989 by the collaboration between the Kenya Medical Research Institute (KEMRI) and the Wellcome Trust and the University of Oxford, with the goal of using research to achieve better health for all in Africa working with the local community as well as developing African scientific leadership. The KEMRI-Wellcome Trust Research Laboratories integrated epidemiological, social, laboratory, and clinical research in parallel with results feeding into local and international health policy. It consists of four main state-of-the-art laboratories: clinical trials laboratory (CTL), short-turnaround laboratory (STAT), microbiology laboratory, and immunology basic research laboratory.The immunology basic research laboratory of the KEMRI-Wellcome Trust Research Laboratories conducts research with a focus on host immune responses to malaria and human immunodeficiency virus infections as well as the impact of malnutrition on immune responses. The research mainly performed in this laboratory involves experiments that help advance the scientific knowledge on malaria transmission with the aim of developing a protective vaccine. The work is being done by a group of scientists with various backgrounds in immunology together with a team of several PhD and master’s students as well as technical staff. Because of the numerous scientific work undertaken, several international publications and presentations to international scientific conferences have been produced. As such, the reputation at KEMRI-Wellcome Trust Research Laboratories has been held high, and it continues to attract several collaborators and funding organizations to foster medical research in the field of immunology.Figure 1.In 2014, there was a growing interest from the funding organizations to ensure that the immunology research laboratory get accredited. The funding organizations wanted their research work to be conducted in compliance with GCLP standards. Thus, there was need to develop a quality system in the immunology laboratory using the GCLP guidelines with the aim of it getting accredited. GCLP standards provided the best platform to achieve this because other laboratories within the KEMRI-Wellcome Trust Research Laboratories, such as CTL, STAT, and microbiology laboratories, were already GCLP accredited by Qualogy Accreditation Scheme Ltd . This article describes our experience in implementing the GCLP in an immunology basic research laboratory within the KEMRI-Wellcome Trust Research Laboratories. The laboratory achieved a GCLP accreditation in 2015, as indicated in The main objective of developing a quality system using GCLP standards was to provide research data that are of high quality and reliable while also achieving GCLP accreditation. GCLP was chosen as the best platform to achieve this because samples used for this research were being received from clinical trials. Furthermore, achieving GCLP accreditation would further boost our reputation and provide a better working relationship with our funders and sponsors. Achieving GCLP accreditation also helped us standardize some of the common procedures within a research setting such as equipment management, organization, and standard operating procedures.1 To achieve this, a staffing summary was performed and an organizational chart with reporting lines developed. This was done to meet the requirements of GCLP as well as the sponsors and to enhance communication and coordination, which, if not clearly addressed, can be a major detractor of quality system implementation, as indicated by Sollecito and Johnson.2 A quality assurance (QA) unit was formed to provide guidance and direction on the implementation of the GCLP standards. The organizational chart is described in Figure 2.GCLP guidelines require that laboratory personnel should be qualified by education and experience, as well as have a personnel training file and formal reporting structures be in place to avoid conflict of interests.The laboratory director, who is charged with the overall responsibility of the GCLP compliance, was appointed and a research laboratory manager appointed to assist. The scientists, together with their research staff, were then grouped into their research clusters with the principal investigators (PIs) designated as the head of these groups. The QA unit had the responsibility of ensuring all protocols were approved, approving assay validation reports, developing standard operating procedures (SOPs), and conducting audits to determine compliance. The PIs were responsible for providing leadership and commitment within their research teams/groups. The staff within the research groups were tasked to write SOPs, validate assays, perform equipment management procedures, and develop their personnel training files.3All laboratory staff developed their personnel training files as required by the GCLP standards. Personnel training files contained records of formal qualification, professional qualification, resume, job descriptions, induction and orientation trainings, confidentiality statement, training and competency assessment records, registration with professional bodies, and immunization records. In addition to the above records, all staff were tasked to document trainings of various procedures relevant to their areas of work and competency assessments performed to ensure all staff were competent to perform the assigned procedures.4GCLP guidelines require that all procedures be documented and written in a standard format. This is further elaborated in other standards, such as the Clinical and Laboratory Standards Institute and International Standards for Organization.5 To establish an SOP system, the laboratory quality officer conducted an SOP training to all staff and developed an SOP template to be used to document all the procedures to ensure quality, integrity, and reliability of data generated.3 Each research team was then assigned SOPs to develop using the provided SOP template. The SOP template covered the following sections:Since research is an ever-evolving process aimed at discovering new concepts and enhancing scientific knowledge, most PhD students often keep changing the protocols and perform optimization on most assays to produce consistent results. Thus, it is difficult to develop and implement quality assurance system in a basic research laboratory.Introduction/purposeScope/responsibilitySafety precautions/risk assessmentsDefinitions and abbreviationsSpecimenEquipment/materials/reagentsMethodologyRelated documents References5 All staff from each research group were then required to acknowledge these SOPs by reading and signing the SOPs’ attestation. The SOP review period was designed to be after every 2 years. The laboratory quality officer from the QA unit was responsible for coordinating all SOP revisions, distribution, removing obsolete copies from active use, and archiving. Any deviations from the SOPs were documented on a corrective action preventive action form and signed by the PI.This was only done to routine research procedures that had been optimized, validated, and approved by the laboratory director.Even though the development of analytical plans as a component of a quality system is one of the items considered difficult to implement in a basic research laboratory, its implementation can be enhanced through flexible routines.1 The laboratory analytical plan is a formal document that reports all the study-related procedures within the laboratory. All the staff from the research groups were trained on writing a laboratory analytical plan by the laboratory quality officer, and an analytical plan template was developed to document all the existing and upcoming studies. The laboratory analytical plan template had the title of the study, list of procedures and their SOPs, roles of laboratories involved, a detailed list of the study’s organizational and management structure, description of laboratory procedures, confidentiality, and data analysis. The laboratory analytical plans helped in planning the conduct of the research studies and capturing some of the information about the laboratory that is not always indicated in the study protocol. All these laboratory analytical plans were finalized and approved by the laboratory quality officer and the PIs/study coordinators. Copies of approved laboratory analytical plans and protocols were filed in the laboratory for easy accessibility and reference.GCLP requires that all studies should have a documented laboratory analytical plans.6 To achieve this, numerous experiments were optimized to establish acceptance criteria for the formal validation experiments. Validation plans that include the selected parameters and a statistical analysis plan were developed and approved by the PIs.To produce accurate and reliable research data, these procedures need to be validated. Moreover, one of the GCLP requirements is to have all the research procedures validated. Validated and optimized procedures are easy to transfer from one laboratory to another, as suggested by Ozaki et al.4 Documented evidence of proper installation, operation, maintenance, inspection, and calibration should be maintained to ensure test results are of high quality.4 Equipment assessment was performed to meet these requirements based on risk assessment. Moreover, an equipment inventory was developed to capture all the laboratory equipment available in the laboratory with information about the unique identification numbers of each piece of equipment, model/make, serial number, manufacturer, and location of the equipment.7 An equipment file with the required documentations was developed for each piece of equipment.GCLP guidelines require that the equipment used in the conduct of clinical research must be verified and “fit for purpose.”All materials, kits, and reagents used within immunology were periodically checked to ensure that expired ones were removed from the laboratory. SOPs related to reagents preparation, labeling, and storage were developed and their records maintained by the laboratory quality officer. All reagents were labeled with the name of the reagent, their concentrations, date prepared, storage conditions, expiry date, and initials of the preparer. For those that did not have expiry dates indicated on them, their expiry dates were tracked from the date the reagents were opened. All these procedures ensured that reagents and kits remained stable to produce quality and reliable research data.8Setting up a quality assurance system using GCLP guidelines requires commitment from the management and the technical staff. The commitment demonstrated by the management and the staff was key to the successful implementation of the GCLP guidelines. Moreover, GCLP implementation also requires efficient coordination and communication since some of its aspects can be poorly accepted or misunderstood.9GCLP implementation was successful at our laboratory because of three main reasons. First and most important was the efficient management structure that enhanced communication between the management and the staff. This structure also provided leadership and direction, making the staff develop interest in the implementation process. Second, the foundation of the laboratory best practices and quality culture was established by conducting technical and quality management system-related trainings, which made the implementation of the quality system a reality. The success was majorly attributed to the skilled and competent workforce who, after the trainings, used their knowledge and skills to implement the good laboratory practices, increase quality improvement, and resolve most of the findings raised by the laboratory quality officer. Conducting the trainings like SOP writing, good documentation practices, GCLP, improvement projects, and quality indicator trainings that targeted the implementation of various aspects of the quality system within the laboratory allowed more staff to be trained and helped encourage universal participation in building a competent and skilled workforce. Last, the application of common sense and good science knowledge were equally beneficial to achieve compliance.Despite the successful implementation of the GCLP process, some challenges were experienced. The biggest challenge was to adapt the GCLP guidelines to a basic research laboratory that did not conduct clinical trials. Thus, during implementation, we chose to implement the GCLP elements that could be applied to a basic research laboratory like SOPs, equipment management, laboratory analytical plans, and the organization and personnel. The other challenge was developing a deeper quality system that was simple, flexible, and able to add value to the organization considering good scientific and technical performance. The huge documentation that comes with the implementation of the GCLP guidelines needs to be thought of to ensure all staff can continue complying to GCLP guidelines for sustainability of the GCLP accreditation.The experience at KEMRI-Wellcome Trust Research Immunology basic laboratory during the GCLP implementation process clearly demonstrates what other laboratories that fully dedicate their concerted efforts can achieve in implementing a quality improvement process. Compliance with the GCLP standards, coupled with the periodic inspections, will ensure that the basic research performed meets the international standards of GCLP by producing valid and reliable data that can be reconstructed by other research organizations.


Age-related frailty is a multidimensional dynamic condition associated with adverse patient outcomes and high costs for health systems. Several interventions have been proposed to tackle frailty. This correspondence article describes the journey through the development of evidence- and consensus-based guidelines on interventions aimed at preventing, delaying or reversing frailty in the context of the FOCUS (Frailty Management Optimisation through EIP-AHA Commitments and Utilisation of Stakeholders Input) project (664367-FOCUS-HP-PJ-2014). The rationale, framework, processes and content of the guidelines are described.The guidelines were framed into four questions – one general and three on specific groups of interventions – all including frailty as the primary outcome of interest. Quantitative and qualitative studies and reviews conducted in the context of the FOCUS project represented the evidence base. We followed the GRADE Evidence-to-Decision frameworks based on assessment of whether the problem is a priority, the magnitude of the desirable and undesirable effects, the certainty of the evidence, stakeholders’ values, the balance between desirable and undesirable effects, the resource use, and other factors like acceptability and feasibility. Experts in the FOCUS consortium acted as panellists in the consensus process. Overall, we eventually recommended interventions intended to affect frailty as well as its course and related outcomes. Specifically, we recommended (1) physical activity programmes or nutritional interventions or a combination of both; (2) interventions based on tailored care and/or geriatric evaluation and management; and (3) interventions based on cognitive training . The panel did not support interventions based on hormone treatments or problem-solving therapy. However, all our recommendations were weak  due to the limited available evidence and based on heterogeneous studies of limited quality. Furthermore, they are conditional to the consideration of participant-, organisational- and contextual/cultural-related facilitators or barriers. There is insufficient evidence in favour of or against other types of interventions.We provided guidelines based on quantitative and qualitative evidence, adopting methodological standards, and integrating relevant stakeholders’ inputs and perspectives. We identified the need for further studies of a higher methodological quality to explore interventions with the potential to affect frailty. The term ‘frailty’ has been used to describe an age-related state of decline and vulnerability characterised by weakness and decreased physiological reserve . Frail oAlthough the use of different definitions leads to the identification of different target populations and leads to a variation in the estimate of the actual prevalence of frailty , 6, the To add to the international efforts to reduce the impact of frailty, we conducted the FOCUS (Frailty Management Optimisation through EIP-AHA Commitments and Utilisation of Stakeholders Input) project, funded by the European Union’s Third Health Programme 2014–2020) , 9. The 4–2020 , At the time of designing our project, we recognised the need to offer patients, their formal and informal carers, healthcare practitioners, and decision-makers a scientific support to their actions, by bringing together scientific evidence and stakeholder perspectives into recommendations that responded to the definition of clinical guidelines , 18. In This correspondence article presents the development process of our FOCUS guidelines, providing their justification, with emphasis on their novelty, limitations and implications.We started by formulating the questions to be answered by the guidelines, based on the PICO format  Table 22. While We adopted the GRADE  Working Group system to assess the certainty of evidence and define the strength of related recommendations , 26. In The development of our guidelines was preceded by a series of quantitative and qualitative studies performed within the FOCUS project, reported in separate publications –16, whicThe guideline panel included the FOCUS project investigators with a background in health and social sciences as well as expertise in frailty, aging and health research methods. Most of them are members of the European Innovation Partnership on Active and Healthy Ageing (EIP-AHA) . Table SOf the 13 FOCUS project investigators who were given the role of voting panellists, 11 participated in the voting process. All criteria for all questions were covered by the voting, but with a few missing votes, which were evenly distributed across criteria and questions. We provide an example of the voting outcome, based on the iEtD output and with the addition of the panel’s comments and technical team’s annotations .The recommendation stems from the belief, common among different stakeholders and endorsed by the guideline panel, that frailty as an outcome is important and that interventions explicitly conceived to address it are needed. We found some quantitative evidence for different interventions being associated with delaying the progression of frailty or reverting frailty, although the effects were moderate in their size at maximum. These interventions, based on either a mainly physical concept of frailty or on a more comprehensive view, could be potentially fair, acceptable and feasible from a population perspective. However, the strength of this recommendation is weak/conditional because of the low certainty/quality of the quantitative evidence, due to substantial study limitations, inconsistency and heterogeneity across studies  as well as imprecision of the estimates.The level or extent of frailty was expected to act as an effect modifier of interventions on frailty, i.e. affecting their relative effect, because of several reasons. For instance, some interventions are expected to work only or more in the presence of a certain deficit status. On the other hand, being frailer might affect the opportunity to benefit from interventions that either require an active participation or work only in less severe illness states. In fact, among the studies examined in our systematic review , those tThe panel judged the implementation of strategies to monitor (and then improve) the adherence to the intervention as important, in particular in the case of interventions that require an active participation of the older person. The need to implement an effective evaluation plan of the interventions was also emphasised. In the FOCUS project, we aimed to analyse the initiatives (i.e. commitments or Good Practices) within the EIP-AHA  in orderR1 – We suggest implementing physical interventions, including physical activity/exercise, nutritional interventions, and a combination of exercise and nutritional interventions, to prevent or delay the progression of frailty or to reverse frailty . The recommendation is stronger for group-based supervised exercise programmes, either alone or in association with nutritional supplementation.We found quantitative evidence supporting the success of physical interventions in delaying the progression of or revert frailty, especially when frailty was defined according to a physical paradigm or measures of its physical component. This component, according to older people’s opinions collected through our qualitative studies Table , althougAmong the studies on physical interventions included in our systematic review , those tR2 – We suggest implementing interventions based on tailored care and/or Geriatric Evaluation and Management (GEM), to prevent or delay the progression of frailty or to reverse frailty . The recommendation is stronger for GEM-based interventions involving a multidisciplinary team, especially in inpatient clinical settings, but still conditional to the confirmation from further studies of good quality.We found some quantitative evidence for interventions based on a tailored care concept and/or on GEM being able to delay the progression of or revert frailty, as defined according to either a mainly physical paradigm or a multidomain paradigm. Some studies also reported on the impact of these interventions on frailty-related outcomes like functional ability, quality of life and hospitalisation , and on exercise combined with diet consultation, at least in prefrail populations. At present, the panel does not suggest adopting interventions based on hormone therapy or on problem-solving therapy with the aim of preventing or delaying the progression of frailty or of reverting frailty . Currently, there is no evidence in favour or against other interventions that could potentially be effective on frailty .Interventions included in this last question were assessed separately because of their heterogeneity. Only one study per intervention was available. The panel judgment was mainly based on the quantitative results of these single studies and their quality, along with considerations regarding values, equity, acceptability and feasibility that could apply to each intervention separately. Thus, the evidence was judged as uncertain and the recommendations were made with weak strength, conditional to the confirmation from further studies.All the studies relevant to this question were performed on a population definable as prefrail; therefore, no subgroup considerations could be made.This paper presents evidence- and consensus-based clinical guidelines on interventions to prevent or treat frailty, developed in the context of the FOCUS project. This was a core objective of the project, i.e. an ultimate deliverable in which the quantitative and qualitative research studies conducted in context of the project converged, and also represented the content of the service we intended to deliver in our proposal. To achieve such a pivotal objective, we required a solid methodological structure and the involvement of the entire FOCUS consortium to act as the guideline panel. We developed four guidelines based on one overarching question concerning interventions for frailty in general and three sub-questions on specific groups of interventions. Each guideline includes a recommendation on whether and how interventions should be implemented.the main areas of interest that policy makers at Member State level would need to support in order to tackle frailty” [Our work has distinctive characteristics among other initiatives that have been undertaken to help clinicians and patients make informed decisions in the context of frailty. For instance, in 2015, the EIP-AHA – Action Group A3 produced a Decalogue on Frailty Prevention as a result of the work conducted by the group in the period 2012–2015, which includes ten key messages focusing on “frailty” . Before frailty” . Both offrailty” . Startinfrailty” , 37. At Our work has recognised limitations. The main limitation to the production of definitive guidelines was related to the quantitative evidence base. The evidence available was of low quality due to methodological bias, inconsistencies and imprecision in the existing studies. In addition to the low quality of the collected evidence, there was a high level of methodological heterogeneity, which had already prevented any quantitative synthesis in our systematic review . The divThe ultimate lesson we learned with our 3-year work is that, while stakeholders showed awareness of and place relevance to the challenge represented by age-related frailty, the current scientific quantitative evidence still has important limitations. This was reflected in our guidelines, which could include only weak or conditional recommendations. Even if such types of recommendations might seem less appealing or helpful, they do yield an important message to the clinician and decision-maker, i.e. to be considered when these interventions are adopted, or indeed, to give consideration to when and whether to adopt them. This message reflects the limitations of the scientific literature and important implementation considerations. Our work also conveys an important lesson to the researcher – while the evidence heterogeneity could never be eliminated since it reflects the heterogeneity in the definition of frailty itself, we need more studies which specifically recognise and measure frailty as an outcome as well as greater adherence to high methodological standards. This is true in general, but in particular for studies on comprehensive frailty interventions, which have the potential to be effective, especially when defined holistically, and have so far not been adequately evaluated. We also need more studies to compare different interventions. The fact that most of the studies included in our evidence base compared the experimental intervention with ‘usual practice’, which can differ dramatically from reality to reality, made the findings extremely context bound. This generated the need to include, in our guidelines, not only an answer to the question ‘does it work?’ but also a consideration of the circumstances of success.Herein, we delivered evidence- and consensus-based guidelines on interventions that can affect frailty as an outcome. We recognised that these are not conclusive; rather, they should foster further studies, both on those interventions already investigated in the literature but requiring higher research standards as well as on potentially effective interventions that have not been appropriately studied thus far. On the other hand, our recommendations should start informing practices. In the context of the FOCUS project, we ended our project by testing the feasibility and impact of our guidelines when used to inform clinical decisions and help implement or refine interventions addressing frailty as a patient-important outcome; the results of these proof-of-concept pilots are currently in preparation for publication .Additional file 1. Synthesis of quantitative evidence from the FOCUS systematic review [c review , showingAdditional file 2. Narrative synthesis of the effect of interventions on secondary outcomes, as offered to the guideline panellists.Additional file 3: Table S1. Table that describes the members of the FOCUS Guideline Panel and their role in the development of the guidelines.Additional file 4. Example of the output of the voting process using the Evidence to Decision template, as received and processed by the guidelines technical team.Additional file 5. Detailed justification for the judgement upon each Evidence to Decision criterion, for each guideline.


M = 14.30, SD = 1.71). Latent class analysis revealed three school refusal behavior profiles: non-school refusal behavior, school refusal behavior by negative reinforcements , and school refusal behavior by positive reinforcements (oriented to obtaining the attention of others with significant or tangible reinforcements). The ANOVA found statistically significant differences for all cyberbullying behaviors. Students with school refusal by negative reinforcements had significantly higher mean scores as compared to the other profiles in victimization, aggression, aggression-victimization, and observation behaviors, while the levels of cyberbullying were similar between students without school refusal and students with school refusal behavior by positive reinforcements. These findings underscore the need to consider priority interventions to prevent cyberbullying in children who refuse school for the purpose of avoiding situations of anxiety and negative emotions.Cyberbullying is a common relational problem having negative repercussions on the academic performance of adolescents. Numerous questions remain to be answered with regard to the relationship between cyberbullying and school refusal behavior. This study examines school refusal profiles  and assesses whether these profiles vary with respect to the level of victimization, aggression, aggression-victimization, and observation of cyberbullying (measured with the Screening of Harassment among Peers). The sample consisted of 1,102 Spanish high school students, aged 12–18 ( School refusal behavior (SRB) is defined as the difficulty in attending or remaining in school for the entire day . This phTherefore, it is important to understand the causes that may lead students to reject and not attend school as well as to determine the different profiles in these youth who do not attend school, in order to improve the prevention or intervention strategies employed in the scholastic context. Of the most relevant contemporary theoretical approaches, we find the functional model proposed by Based on the functional classification system, prior studies have attempted to analyze the SRB profiles in children and adolescents . For insIn addition to low psycho-emotional and academic adjustment, SRB has also been strongly associated with variables of social interaction. So, many authors have suggested that poor relationships with schoolmates and bullying are significant factors in determining school rejection and absenteeism . BarbozaHowever, the phenomenon of victimization and bullying between peers is not unique to the school setting. With the widespread and generalized use of the information and communication technologies and the social networks, minors today are immersed in an environment in which they are more likely to suffer from or perpetrate these acts of bullying, better known as “cyberbullying” or “electronic bullying.” Cyberbullying has been defined as an aggressive action carried out repeatedly and deliberately through electronic means, toward an individual who cannot easily defend him/herself . The maivia internet and in person. Male victims of cyberbullying were 3.58 times more likely to be absentee students, with this risk increasing to 6.68 if they were victimized both in the traditional manner and via electronic means , with 509 males (46.2%) and 593 females (53.8%) participating. The sample’s distribution based on academic year was as follows: 184 (7th grade), 193 (8th grade), 190 (9th grade), 182 (10th grade), 208 (11th grade), and 145 (12th grade). The χ2 test was used to analyze the homogeneity of the sample in terms of gender and course, with no statistically significant differences being found between the groups of Gender x Course .Participants were students from secondary-level education of the Valencian Community (Spain) during the 2017–2018 academic years. The Valencian Community approximately served a total of 261,000 secondary education students . Two-stanever; 6: always) and that assess the relative self-perception of the four fundamental factors of SRB: avoidance of school situations that provoke negative affectivity , escape from aversive social o evaluative situations , pursuit of attention from significant others , and pursuit of tangible reinforcement . The scale can be used for students from 8 to 17 years of age. SRAS-R scores have been found to have suitable psychometric properties in adolescents from distinct cultures  is one of the most widely-used questionnaires for the measurement of SRB considering functional conditions . The quecultures  and factcultures  and in Ccultures . In thisvia four subscales: victimization (behavior suffered by the bullying victim), aggression (bullying behavior perpetrated by the aggressor), observation (bullying behavior witnessed by the observer), and aggression-victimization (bullying behaviors that are suffered as a victim and perpetrated as an aggressor) . The reliability of the instrument has been confirmed by the original authors in samples of Spanish adolescents (The Screening of Harassment Among Peers (SPH) is a self-reporting instrument that assesses bullying and cyberbullying behavior in adolescents and youth taking place over the past year, gressor) . The quelescents , 2015. IInitially, the researchers interviewed the management team of the selected schools in order to explain the purpose of the study. Then, an informative letter was sent to the parents of the minors in order to explain the study and to request their informed consent in writing. Questionnaires were responded to collectively and voluntarily in the classrooms during a class session, ensuring the anonymity of the participants and the confidentiality of the data. To do so, identification numbers were assigned on the response sheets of each participant. The researchers were present during the administration of the tests to clarify any potential doubts and to verify the correct completion of the questionnaires, which had a mean completion time of 15 min.post hoc Scheffé tests to determine the groups between which there were statistically significant differences. Finally, the d index (standardized mean difference) proposed by d ≤ 0.50 means a small effect size, while 0.51 ≤ d ≤ 0.79 is moderate, and d ≥ 0.80 is large.The SRB profiles were defined based on the combinational differences of the four functional conditions of the SARS-R, and were established using the latent class analysis (LCA). LCA is considered to be the most appropriate procedure for establishing profiles in large samples and it surpasses the limitations found in other statistical techniques such as the analysis of conglomerates . ConsideThe LCA found that the class made up of three profiles with different levels of SRB, considering the four dimensions of the SRAS-R, ANE, ESE, PA, and PTR see , had thepost hoc tests indicate that the students with a SRB by negative reinforcements profile received significantly higher scores on victimization, aggression, aggression-victimization, and observation of cyberbullying than the non-SRB group and the group of students with SRB by positive reinforcement , whereas the differences between the group of SRB by negative reinforcement and the non-SRB were moderated by the differences in victimization, aggression, and aggression-victimization, and were small for the difference in observation of cyberbullying.As shown in via latent class analysis, of the SRB profiles, taking into account the motives leading students to reject school, according to the four-factor functional model . This class coincides with the findings of Given the results for the three latent classes, the cyberbullying behavior was analyzed, finding inter-class differences in the scores for victimization, aggression, observation, and aggression-victimization. The profile of students with SRB by negative reinforcements had significantly higher scores than the other profiles for all cyberbullying behaviors. These results confirm the second hypothesis which anticipates that the adolescents with a high SRB profile would have higher scores in cyberbullying and reinforces the findings of prior studies that found cybervictimized adolescents to be a population at risk of not attending school because they did not feel safe  and in oHowever, students with SRB by positive reinforcements are not different from the non-SRB group in terms of the four cyberbullying roles. This may be explained by the different impact of cyberbullying according to the school rejection declarations. The results of this work expand upon the results of prior studies, analyzing other important roles in cyberbullying such as that of the aggressor-victimized and the cyberbullying observer. This study found that students with SRB derived from a high negative emotionality and avoidance of evaluation and social situations had higher scores on aggression-victimization and on the observation of cyberbullying behaviors. Like in other studies e.g.,  in whichThis study has certain limitations, including the impossibility of generalizing the results to other education levels and to other countries. Future studies should analyze whether or not the findings differ in other academic levels and in other cultures. Furthermore, the cross-sectional design used in the study makes it impossible to establish causal relationships. Therefore, it is recommended that longitudinal studies be carried out to provide additional information on the evolution of the SRB phenomenon and cyberbullying over the years. In addition, regarding To conclude, this study has found the existence of three profiles of adolescents who reject school, with the most prevalent profile (38.02%) having a negative emotional component whose motives for rejecting school include avoiding negative emotions and social and assessment situations in the school (SRB by negative reinforcements). These students also correspond with the profile of having a higher level of cyberbullying behavior, both as victims and aggressors, aggressor-victim, and observer. Thus, strategies to prevent cyberbullying in academic settings should focus on the identification and intervention of cases, taking SRB into account, especially in adolescents who reject school to avoid situations of anxiety and/or negative emotions.The datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by the University of Alicante. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.BD conceived the study and participated in its design, coordination, statistical analysis, and the manuscript drafting. MM-M participated in the study design and data interpretation, while also assisting in the drafting of the manuscript. CR-E performed a critical review of the manuscript and assisted with interpretation of the findings. ER assisted with the study conception. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Over recent years, there has been an increase in popularity of the acquisition of dermoscopic skin lesion images using mobile devices, more specifically using the smartphone camera. The demand for self-care and telemedicine solutions requires suitable methods to guide and evaluate the acquired images’ quality in order to improve the monitoring of skin lesions. In this work, a system for automated focus assessment of dermoscopic images was developed using a feature-based machine learning approach. The system was designed to guide the user throughout the acquisition process by means of a preview image validation approach that included artifact detection and focus validation, followed by the image quality assessment of the acquired picture. This paper also introduces two different datasets, dermoscopic skin lesions and artifacts, which were collected using different mobile devices to develop and test the system. The best model for automatic preview assessment attained an overall accuracy of 77.9% while focus assessment of the acquired picture reached a global accuracy of 86.2%. These findings were validated by implementing the proposed methodology within an android application, demonstrating promising results as well as the viability of the proposed solution in a real life scenario. Malignant melanoma is the 19th most common cancer among men and women, with nearly 300,000 new cases in 2018 while non-melanoma skin cancer is the 5th most common cancer, with over 1 million diagnoses worldwide in 2018, a number which is considered to be an underestimation . HoweverDermatology is the branch of medicine dealing with the diagnosis, treatment and prevention of skin diseases. Acquisition of dermoscopic images of skin lesions and melanocytic lesions is a standard procedure in dermatological practice, which results in a valuable asset for every clinician . By defiThe recent advances in mobile health , a rising digital health sector that provides healthcare support, delivery and intervention via mobile technologies such as smartphones, led to an increase in the number of available self-care and telemedicine solutions related to the skin. Early detection, surveillance, easier access to health care services, avoiding unnecessary medical appointments or just documenting specific cases, are some of the reasons behind the creation of such systems, particularly in the dermatology field ,9,10,11.This context motivated the development of a new algorithm for automated focus assessment of dermoscopic images, more specifically, by correctly identifying structures of interest, that is, skin moles, while simultaneously assessing the quality of those images. Particularly, an image focus validation approach was developed to perform real-time image quality control. With this work, we aim to contribute to the standardization of image acquisition in dermoscopy via mobile devices, by assisting and guiding the user during the acquisition process of skin lesions, and consequently facilitate both monitoring and diagnosis procedures.This paper is structured as follows: Due to the most recent technological breakthroughs in the area, a new generation of mobile devices has appeared. These devices, such as smartphones, are typically low cost, light weight, portable, and have high computational power, thus, constituting one of the most common forms of image acquisition and processing. Especially in dermatology, where these types of devices are being used to acquire skin lesion images and exchange information among general practitioners, dermatologists or patients ,13. AcquAs can be seen in the recent literature, a wide variety of m-Health solutions are available ,13. ThesRecent studies have studied the influence of several conditions, such as lighting, background color, field of view, image orientation, focus and depth of field, resolution, scale, color calibration and image storage may have on the acquired image as being key aspects in dermatology ,17. ThusIn terms of IQA objective methods, the literature is quite vast and the used methods can be divided in three different groups—full reference methods, where a reference image is present; reduced reference methods, where only partial information about the original image is available together with a set of discriminative features; no reference methods, where no original image is available . For theThe proposed system allows the focus assessment of skin moles in dermoscopic images by using a feature-based machine learning methodology. The system was designed to guide the user throughout the acquisition process by means of an image focus validation approach, followed by the IQA of the acquired picture.The architecture of the developed solution, illustrated in The present work aims to assist the acquisition process of dermoscopic images acquired with smartphones by providing automatic focus assessment. Some public databases of skin lesions contain dermoscopic images that were classified by dermatologists for diagnosis purposes, such as ISIC, PH2 or HAM10000 . These dFor the collection of both datasets, two different dermoscopes were used—Dermlite DL1 (DL1)  and DermRegarding the variance between the acquisition devices, 11 different smartphones with different cameras properties were used during the acquisition. The overall robustness of the proposed solution across different smartphones was addressed in this approach, by including the highest possible number of different devices in the study together with the use of two different dermoscopes. A complete list of the mobile devices used alongside with the major characteristics is available in DermIQA) dataset. This dataset is composed of a total of 1979 images of skin moles from 14 different Caucasian subjects. The images were acquired using the aforementioned dermoscopes together with the 11 different smartphones. The goal of collecting this dataset is to have at least one blurred and one focused image for each skin mole, smartphone and dermoscope. For each acquisition both camera preview image and captured image were saved for the following reasons: (i) in the preview stage the goal is to assess the image in terms of image stabilization and standardization, where as in the acquired image the goal is to check the quality of the image that will be saved in the system and used for monitoring or diagnosis purposes; (ii) also, the preview image has smaller resolution than the acquired images (720 × 1280 vs. 1080 × 1920 px). A summary of the number of collected images and their distribution regarding the focus level is provided in In order to address the problem of assessing image quality and focus of skin moles images in real-time, a dataset of focused and non-focused images was collected, named the Dermoscopic Image Quality Assessement  and non-relevant structures like artifacts, which served as a basis for building the Artifact Detection module referred previously.DermArtifacts) image dataset is composed of a total of 232 camera preview images—131 skin moles images and 131 images with different backgrounds and/or with lenses artifacts, representing the negative class of interest. The skin mole images were arbitrarily chosen from the DermIQA dataset, whereas the 131 images with artifacts were additionally collected. Some of the smartphones from the ones listed in the The Dermoscopic Lens Artifacts . This square region is used by the algorithm to extract the metrics and make decisions accordingly. The square image is then converted to the gray scale colorspace The generation of us study , it beinAdditionally, it should be noted that the magnitude of the absolute value of roscopic  and skinroscopic  images. Following the system architecture diagram, the aim of this work was to find accurate and robust models for three different tasks—(i) artifact detection, (ii) preview focus assessment and iii) acquired picture focus assessment.DermArtifact dataset, this division was performed by keeping 70% and 30% of the data as train and test sets, respectively. As for the DermIQA dataset, this division was performed by keeping all the images from 9 subjects as the train set and images from 5 subjects as the test set, with the purpose of having a wide variety of skin moles both in the training and test set. Since with a random split, the test set could end up with more than two or three similar skin moles, and since the objective is to validate the algorithm by using moles with the highest variety of characteristics as possible, these moles should also be represented in the test set in order to ensure a more general and robust outcome. Also, this division was made for both preview and acquired images separately, enabling the creation of two different classification models, one for assessing only the preview images (ii) and another to assess the final acquired images (iii).In order to train the different models, two datasets were collected (as explained in the previous section), which were then subdivided into train and test datasets according to each correspondent classification task. For the Due to the limited computational capabilities of some smartphone models, and in order to ensure not only real-time computational calculation of focus metrics but also real-time feedback to the user regarding the focus level on camera preview frames, we opted to use a Decision Tree classifier. Furthermore, the subset of values for hyper-parameter optimization used for model optimization during training are further detailed in F1-Score or Informedness/Youden Index metric was used as the classification metric to optimize when evaluating the DermArtifacts or DermIQA datasets, respectively;Run grid search with the previously defined model hyper-parameters using the stratified cross validation technique . Take the best estimator chosen by the search in (1) and evaluate its performance on the correspondent test set;Recall value on test set is greater than 85%;Keep best estimator parameters if the chosen metric to optimize for on train set is greater than the previously saved/stored one and its Repeat the above steps for 5000 iterations.Moreover, and for each aforementioned classification task, the following optimization pipeline was adopted to train and select the best overall model:DermArtifacts dataset. The optimized model found in the training phase, together with its respective best selected features, was assessed on the correspondent test set data, recurring to classification metrics as Accuracy, Recall, Precision, Specificity and F1-Score. The best classification results for this task are presented in The Artifact Detection module in our proposed pipeline was created using the Focus Assessment module on the test set of the DermIQA dataset are presented in the Division between the Sum of the values of the Image Curvature of blur and gray image  and two high-end devices (Samsung S9 and OnePlus 6T) were selected for evaluation. Mean running time for preview and acquired image focus assessment is presented in Acquiring skin lesion images using a smartphone is undoubtedly becoming more frequent in our daily lives, not only by professionals but also by patients seeking clinical guidance. By using handheld and decentralized image acquisition approaches, the time between the identification of a potential lesion and a diagnosis could be drastically shortened. However, if the quality of the acquired images is not assessed during or immediately after an acquisition, the number of unsuitable images for a clinical assessment may dramatically increase. Recent studies point out that professionals do not necessarily need to be trained photographers to ensure an adequate acquisition of quality images . HoweverWith this clear goal in mind, the methodology proposed in this work was revealed to be suitable and robust enough to fulfill this purpose. In particular, accurate results were obtained during this study for the focus assessment of mobile-acquired skin mole dermoscopic images. Additionally, suitable results were also obtained for the detection of artifacts on dermoscopic images, as well as for the focus assessment on camera preview images. Remarkably, only two highly discriminating focus metrics are used to achieve the reported results in each classification task, thus making this approach suitable for real-time usage with mobile devices. Despite these results, when combining this two individual tasks, the overall performance of the proposed solution suffers a significant decrease, as expected. This loss in performance may be due to the inherent complexity of each task. Individual limitations for each type of assessment may be unveiled and more pronounced when looking into these two different tasks as one. Despite this decrease, the overall performance for focus assessment of preview images in terms of artifact detection and focus is quite satisfactory.The proposed pipeline in this study, involving artifact detection, preview and taken image assessment was already deployed in an Android application running on a smartphone. The application allows the manual acquisition of dermoscopic images in an easy and intuitive way, providing real-time feedback about the level of focus of the images being acquired. In Thus, given the importance of such images for diagnosis purposes, we can consider that the ultimate goal of this study was fulfilled, since the process of acquiring dermoscopic images of skin moles can now be simplified and better quality images can be collected for screening and diagnosis purposes. Particularly, by embedding this image quality assessment methodology in handheld image acquisition tools, standardized images can be obtained, which may increase the efficiency in the dermatological clinical flow.Due to the constant increase and demand for telemedicine solutions, more specifically for dermatological purposes, is clear that standardization of the image acquisition process is a crucial step. Image quality evaluation of the acquired image is necessary, as well as providing adequate guidance to the end-user in the image acquisition process. In this paper, we presented a solution that acts on these two premises, being able to guide the user in real-time during the acquisition process, as well as assessing whether a certain mobile-acquired dermoscopic image is properly focused. Particularly, the proposed solution was designed for assisting the process of collecting a skin mole image in real-time, by using any smartphone camera with a dermoscope attached.In terms of the automated analysis of camera preview images, our approach was divided into two different tasks—real-time artifacts detection and real-time preview images focus assessment, with obtained accuracies of 97.3% and 83.7%, respectively. An accuracy rate of 77.9% was achieved for the complete camera preview focus assessment module, which included both previously refferred tasks. Regarding image quality assessment of the mobile-acquired picture, the results obtained also demonstrate the adequacy of the proposed methodology, being achieved an accuracy of 86.2% using an approach that only requires the extraction of two lightweight focus metrics.To finalize, an embedded Android application with the proposed methodology was also developed, in order to test the viability of the proposed approach in a real life scenario. Empirically, the results obtained through the real-time usage of the developed application seem to be in line with the results obtained through the validation datasets. However, further testing in real clinical settings are required, in order to properly evaluate the performance and suitability of the proposed approach for screening and diagnosis purposes.For future work, it would be valuable to have the dataset annotated by specialists and enhanced with more images of different subjects and types of skin lesions in order to increase the robustness of our solution. Regarding the features used, further research should be done in the search and optimization of features capable of being used in real-time. Finally, testing the pipeline used in a real live scenario would be of utmost importance in order to provide more concrete indicators of the quality of the work here described.


Early diagnosis represents the target of contemporary medicine and has an important role in the prognosis and further treatment. Saliva is a biofluid that generated a high interest among researchers due to its multiple advantages over other body fluids. The multitude of components that can act as biomarkers influenced the existing technologies to develop protocols that could allow saliva to become the new noninvasive diagnostic method. Saliva as a diagnostic tool can bring substantial addition to the diagnostic armamentarium, providing important information about oral and general health. The diagnostic applications of saliva extended and had a rapid evolution due to the advancement in salivaomics. The present review summarizes the latest researches in saliva-related studies and explores the information and correlations that saliva can offer regarding the systemic and oral diseases, highlighting its great potential of diagnosis. It is expected that in the future specific guidelines and results regarding the salivary diagnostics are to be available, together with high-sensitivity and specificity tests for multiple systemic and oral diseases. Body fluids provide a wide perspective regarding the biological processes and the health of different organs. The human body is composed of a variety of fluids, such as blood, urine, and saliva, with a high quantity of proteins that can be associated with several systemic and oral diseases. These fluids proved to have found widespread clinical applications in order to diagnose and monitor human health. The high global impact of a large number of diseases including cancer and cardiovascular, metabolic, and neurological diseases challenged the clinicians to provide and improve the diagnosis procedures and clinical evaluation of these patients. One of the most appealing diagnostic tools is thought to be the human saliva, holding the key to an early diagnosis, a better treatment, and an improved prognosis . The earSystemic diseases are very challenging to diagnose without more invasive supplementary investigations. In order to overcome this condition, medical researchers worked into finding molecular disease biomarkers that can be easily identified and where they can successfully implement a noninvasive and fast diagnosis. During this path of research, three main limitations have influenced until recent the late development and research of specific biomarkers for early disease detection: (1) the lack of definitive molecular biomarkers for specific diseases, 2) the lack of an easy and inexpensive sampling method with minimal discomfort, and (3) the lack of an accurate and easy-to-use platform that can facilitate the early detection. Until now, it can be considered that limitations 1 and 3 have found solutions with the help of salivary biomarkers and an ongoing development of salivary diagnosis [ the lackSalivary diagnosis is viewed as a promising modality that can provide an early and accurate diagnosis, an improved prognosis, and a good monitoring post-therapy. The whole saliva is composed of the secretions of the minor and major salivary glands as well as mucosal transudations, gingival crevicular fluid, serum and some blood derivatives, desquamated epithelial cells, bacteria, viruses, fungi, and food debris. Saliva is a complex fluid that also contains a high number of hormones, proteins, enzymes, antibodies, cytokines, and antimicrobial constituents that can facilitate their associations with a variety of systemic diseases . The assThe past research within the last 10 years proves the fact that saliva as a diagnostic tool has gained a lot of attention and has become a translational research method. Saliva has the potential to become a first-line diagnostic tool with the help of the advancement made in early detection and the development of biomolecules that have clinical importance . SalivarSaliva has several advantages over serum and tissue fragments in its use as a diagnostic tool. One of the most appealing characteristics is the noninvasive approach that, combined with the easy collection method and storage, makes it a valuable tool. New technologies have proven their efficacy and unveiled a large number of salivary biomarkers that are connected to several general and oral diseases .The aim of this review is to emphasize the role and importance of saliva as a diagnostic tool for the diagnosis of systemic and oral diseases. The use of this method brings to light an efficient and easy approach that can improve considerably the diagnosis, prognosis, treatment, and post-therapy monitoring. Various components in this fluid can act as biomarkers for multiple diseases providing valuable information regarding the health status. The focus is on providing information about the important salivary constituents, the mechanism of using saliva as a diagnostic tool, and the clinical applications that can influence an early diagnosis.The definition of a biomarker refers to a pharmacological or physiological measurement that can be used to predict a toxic event, in this specific case a molecule that contains particular material that can be used in order to diagnose a disease or measure the progression and treatment outcome. The characteristics of biomarkers make them proper for an alternative diagnostic tool, with or without the help of other methods .The development of mass spectrometric technologies led medicine to a new era in biomarker discovery that will have an important impact on future disease diagnosis and therapy. More studies in salivary proteins showed the fact that saliva contains actually hundreds of minor proteins or peptides that although are present in variable concentrations can have a significant role in the diagnosis of diseases; these proteins can receive the role of biomarkers in relation to specific conditions. Although proteomes play an important role in the diagnosis, the salivary transcriptomic technology succeeded to improve the diagnostic potential of saliva for multiple medical applications .Proteomic technology helped to discover the salivary biomarkers by outlining the importance of the proteome and the analysis of the expressed proteomics. The existence of the proteomes in the body fluids represents a high potential of disease markers. An accurate analysis of the human saliva proteome can be related to the general health status. Many functional alterations of proteins result from posttranslational modifications such as phosphorylation, glycosylation, acetylation, and methylation . These kThe transcriptomic technology allowed researchers to discover the salivary transcriptomes (RNA molecules) that include the molecules the cells use to transport information provided by the DNA for protein production. This opportunity provides medical research with a second diagnostic tool that involves saliva and that can provide more opportunities for salivary diagnostics .The most important and revealing components of the saliva are the proteins. Human saliva has a specific proteomic content that allows researchers to perform assays in order to discover novel saliva biomolecules associated with general health status. Proteomic studies of saliva help with the identification of new proteins and peptides that can help quantify the biological activity in pathological states.http://www.skb.ucla.edu) is the first database that contains all the proteomic data being accessible to the public. The techniques used by researches and biochemists in order to perform the proteome work from saliva are gel electrophoresis, capillary electrophoresis, nuclear magnetic resonance, MS, immunoassay, and LC [The Saliva Proteome Knowledge Base , proteins such as enzymes, immunoglobulins, antimicrobial factors, albumin, polypeptides and oligopeptides, traces of albumin, and mucosal glycoproteins of great importance in maintaining a balance of the oral health. Saliva also contains glucose, urea, and ammonia in various quantities that can interact and be responsible for several general diseases .The oral fluid originates preponderantly from three pairs of major salivary glands  and from numerous minor salivary glands. Parotid glands are serous glands, and their secretion lacks mucin; the submandibular and sublingual glands are mixed ones, with ser-mucous secretion. Minor salivary glands that are situated in the connective tissue below the circumvallate papillae are Von Ebner glands, and the mucous ones are Blandin-Nühm glands .μl/h per tooth and it can be considered as a plasma transudate. The oral fluid also can contain food debris and blood-derivated compounds such as plasmatic proteins, erythrocytes, and leucocytes in case there is inflammation present [The salivary composition varies and depends on the type of the gland, mucous or serous ones . Its com present . The com present , 18  . In thisSaliva is an important fluid, and interest in it has developed due to the wide spectrum of proteins/peptides, electrolytes, hormones, and nucleic acids that are in its composition and can provide important information about the body's health. The delay in the use of saliva as a diagnostic method was mainly because until recent there has been a lack of understanding of the biomolecules that were found in the saliva. As a diagnostic tool, several disadvantages have been reported: the variations due to the diurnal/circadian rhythm, the method of collection that can influence the salivary composition, and the necessity of sensitive detection systems. However, saliva is considered to have an enormous potential of biomarkers that range from changes in biochemical, DNA, RNA, and proteins to the oral environment. As a diagnostic tool, saliva can provide a new and noninvasive perspective in order to obtain a diagnosis, and it can be expected in the future to become a substitute for serum and urine tests . A part Clinical research has developed various protocols in order to assay saliva. At the time, saliva is most frequently used as a diagnostic tool for systemic diseases and the future relies in combinations of different biomarker panels that can be used for screening in order to improve the early diagnosis and the general outcome . The firFor many years now, researchers investigated the importance of the changes that occurred in the saliva, changes that affect the flow rate and composition. The changes in the fluid are valuable regarding the diagnosis of oral and systemic diseases . At firsRegarding the periodontal pathogenic processes, periodontitis can be classified based on the three phases of evolution: inflammation, connective tissue degradation, and bone turnover. There are, associated with each phase of the periodontal disease, different salivary biomarkers that can stage the evolution and the status of the patient. At the beginning of the inflammatory phase, prostaglandin E2, interleukin-1, interleukin-6, and tumor necrosis factor-alpha are found in a high number, released from a variety of cells . As the An important cytokine with a proinflammatory role involved in the inflammation process associated with periodontitis is interleukin-1. IL-1 can be the product of several cells, as epithelial cells, monocytes, polymorphonuclear neutrophils, fibroblasts, endothelial cells, and osteoblasts , 29. IntAnother key biomarker involved in periodontitis is mainly produced by macrophages and is represented by tumor necrosis factor-alpha. It is an important immune mediator, and in relationship with this disease, it influences bone collagen synthesis and induces collagenases, similar to IL-1 , 39. Alsα, matrix metalloproteinase-8, interleukin- (IL-) 1β, IL-6, prostaglandin E2, and tumor necrosis factor- (TNF-) α levels seem to be associated with gingivitis and periodontitis, having a high potential to be used in their diagnosis [A recent study outlined the existence of certain correlations between salivary superoxide dismutase levels and the gingival index, pocket depth, and clinical attachment loss found in patients that were diagnosed with chronic periodontitis. Saliva's potential of diagnosis is seen as a noninvasive and easy way to diagnose patients with premalignant conditions . Also, siagnosis . Based oiagnosis , 47. Moriagnosis .Sjögren's syndrome (SS) is an autoimmune chronic systemic disease that has important symptoms: xerostomia and keratoconjunctivitis. Patients diagnosed with SS have a decreased salivary flow rate and a modified composition of the saliva. It was shown the fact that this syndrome is accompanied with significant changes in the proteome and transcriptome, having also important alterations in the levels of IL-4, IL-5, and cytokine clusters . AnotherEarly diagnosis and treatment is the key to a good prognosis in almost all types of cancer. Saliva has been used in studies as a diagnostic tool for oral squamous cell carcinoma (OSCC) based on the help of salivary analytes  . Oral caThe existence of gene mutations can often be associated and used as biomarkers in order to diagnose oral cancer. In saliva, the tumor-specific DNA was positive in 100% of the patients diagnosed with oral cancer, and 47-70% of the patients with tumors in other places of the body also carry specific tumor DNA markers in the saliva .p53 protein is responsible for tumor suppression, and it is produced in cells as a response to multiple DNA damages. The inactivation of p53 during a mutation is one of the main causes of the development of malignancy. Studies have shown the fact that p53 antibodies were detected in the saliva of patients diagnosed with oral squamous cell carcinoma [The arcinoma . CA 125 arcinoma . Also, aarcinoma .It can be affirmed the fact that all the results prove that saliva has an important charge of biomarkers that can be used successfully in providing a screening and diagnosis of oral cancer.β, IL-6, TNF-α, and prostaglandin E2 are increased in atherosclerosis, suggesting that these cytokines could be potential biomarkers in the diagnosis of atherosclerosis. Other studies concluded the fact that other salivary markers can be C-reactive protein (CRP), myoglobin (MYO), creatine kinase myocardial band (CKMD), cardiac troponins (cTn), and myeloperoxidase. Acute myocardial infarction was predicted by a correlation of an ECG with the CRP levels, proving 80% sensitivity and 100% specificity [α-2-HS-glycoprotein in saliva seem to decrease in patients diagnosed with cardiovascular diseases, suggesting the fact that the peptidome can contribute to the early diagnosis of these patients [Cardiovascular disease (CVD) includes atherosclerosis, coronary heart disease, and myocardial infarction. The studies performed by Kosaka et al.  show thacificity . In salicificity . Also, tpatients .Alzheimer's disease (AD) is one of the most common neurovegetative disorders that occur to the aging population. It is supposed that the process of Alzheimer's is initiated years before it becomes clinically manifest . Until nThe study performed by Carro et al.  investigAhmadi-Motamayel et al.  conducteParkinson's disease is characterized pathologically by progressive degeneration of dopaminergic (DA) neurons in the substantia nigra pars compacta. The formation of a-synuclein- and ubiquitin-containing fibrillar inclusions (Lewy bodies and Lewy neurites) occurs in this cell population as well as variable changes in other neurotransmitter systems . The aimThe existing tests for viral infections are based on salivary biomarkers, basically on viral DNA and RNA, antigens, and antibodies. Currently, several salivary tests are available based on the proteomic analysis of the saliva and the existing antibodies for hepatitis A, B, C viruses, HIV-1, rubella virus, mumps virus, and others . A new sThe HIV (human immunodeficiency virus) is possible through antibody-based screening assays. The diagnostic test is an antibody assay that can be a Western blot test via blood or saliva or a polymerase chain reaction via blood . These sEarly diagnosis is an important aspect regarding this type of cancer knowing the fact it is the most common cause of death in men and women. Until now, conventional diagnosis methods are not suited for screening, with a high false-negative rate , 82. CT The tissues that are found in the orofacial region are heterogeneous, a fact that makes the treatment of pain conditions a challenge for the clinician. The main problem for an adequate treatment option consists in the diversity of conditions for which orofacial pain is a major symptom that makes it hard to differentiate many of these disorders clinically . SeveralOrofacial pain is a sensory experience within a specific anatomical region and can be related to some common chronic orofacial entities: TMJ myalgia and arthralgia, atypical odontalgia, persistent dentoalveolar pain disorder, burning mouth syndrome, persistent idiopathic facial pain, neuralgia of the head and neck, and primary headache syndromes .Collecting saliva in order to identify biomarkers associated with orofacial pain is a painless method and is easy to collect and store. Recently, saliva and synovial fluids have piqued the interest of numerous researchers and clinicians as possible alternatives to serum.Further researches conclude that saliva-based biomarkers are not only preferred but also are accurate in discerning healthy subjects from those afflicted with periodontal disease or burning mouth syndrome –94. SaliThe study performed by Jasim et al. focused on salivary biomarkers related to chronic pain by comparing blood samples and saliva samples of the same subjects and revealed the fact that five specific biomarkers related to pain were targeted. The results showed that they were first to find several isoforms of NGF, CGRP, and BDNF in saliva. The expression showed great variations between different saliva collection methods . GlutamaWith the help of a standardized collection procedure and protocol, the use of salivary biomarkers for different orofacial pain disorders is a promising diagnostic method that will allow for a noninvasive approach.Saliva is an important biological fluid with a wide area of research and applications, having a high potential to become the future in early diagnosis. The effective contribution of genomic and proteomic technologies made possible for saliva to become an attractive solution to other invasive diagnostic methods. Saliva as a diagnostic tool for oral and systemic diseases has multiple advantages over other body fluids and based on specific biomarkers can provide an accurate diagnosis. However, until saliva becomes a certified diagnostic test that can replace the conventional ones, all the research values must be compared with the existing accepted methods. The main problem consists in the fact that a standardized and accurate method of saliva collection needs to be associated with each type of diagnostic test, in order to avoid errors. This review has discussed several oral and systemic diseases that could be diagnosed based on different salivary biomarkers, but research needs to be extended in order for saliva to become an effective and sure diagnostic tool that can be used for screening and uncontestable diagnosis.