Global health research is essential for development. A major issue is the inequitable distribution of research efforts and funds directed towards populations suffering the world's greatest health problems. This imbalance is fostering major attempts at redirecting research to the health problems of low and middle income countries. Following the creation of the Coalition for Global Health Research – Canada (CGHRC) in 2001, the Canadian Society for International Health (CSIH) decided to review the role of non-governmental organizations (NGOs) in global health research. This paper highlights some of the prevalent thinking and is intended to encourage new thinking on how NGOs can further this role.This paper was prepared by members of the Research Committee of the CSIH, with input from other members of the Society. Persons working in various international NGOs participated in individual interviews or group discussions on their involvement in different types of research activities. Case studies illustrate the roles of NGOs in global health research, their perceived strengths and weaknesses, and the constraints and opportunities to build capacity and develop partnerships for research.NGOs are contributing at all stages of the research cycle, fostering the relevance and effectiveness of the research, priority setting, and knowledge translation to action. They have a key role in stewardship , resource mobilization for research, the generation, utilization and management of knowledge, and capacity development. Yet, typically, the involvement of NGOs in research is downstream from knowledge production and it usually takes the form of a partnership with universities or dedicated research agencies.There is a need to more effectively include NGOs in all aspects of health research in order to maximize the potential benefits of research. NGOs, moreover, can and should play an instrumental role in coalitions for global health research, such as the CGHRC. With a renewed sense of purpose and a common goal, NGOs and their partners intend to make strong and lasting inroads into reducing the disease burden of the world's most affected populations through effective research action. Each country needs to be able to generate knowledge relevant to its own situation, to allow it to determine its particular health problems, appraise the measures available for dealing with them, and choose the actions likely to produce the greatest improvement in health. This should not be seen as the exclusive preserve of universities or research councils, but equally of health/public services, non-governmental organizations, etc." ["s, etc." .. NGOs are generally regarded as valued partners in health research for development, research being viewed as a broad process involving not only the production of knowledge, but also up-stream and down-stream activities needed for its relevance and effectiveness, such as priority setting and knowledge translation. NGOs have made and continue to make substantive contributions through supporting relevant and effective research. In her address at the First Steering Committee Meeting of the International Conference on Health Research for Development in 1999, the (then) Director General of the World Health Organization (WHO), Dr. Gro Harlem Brundtland, voiced her appreciation of NGOs as a partner with WHO in health research [Non-governmental organizations (NGOs) have been defined by the World Bank as 'private organizations that pursue activities to relieve suffering, promote the interests of the poor, protect the environment, provide basic social services, or undertake community development'. NGO activities can be local, national or international. NGOs have contributed to the development of communities around the world and are important partners of many governments – while remaining independent from governments. According to the Human Development Report , there wresearch ., that is, the health of individuals and societies in less developed, less resourced, poorer nations and regions of the world.There are several views on what is meant by global health and global health research. In its simplest form, global health is population health on a global scale, and global health research is research which addresses the health of human populations around the globe. Global health also refers to 'inherently global health issues', that is, health-determining phenomena that transcend national borders and political jurisdictions, such as globalization and climate change. In setting global health research priorities, both the burden of disease and inherently global issues should be considered ,5. The vA major global health research issue is the inequitable distribution of research efforts and funds directed towards populations suffering the world's greatest health problems. This situation has been referred to as the 10/90 gap because only a meager 10% of all health research funding is being used to address 90% of the world's burden of disease, suffered primarily in developing countries . Becauseth Global Forum on Health Research, held in Arusha, Tanzania in November 2002, Dr. Gerald Keusch, Director of the Fogarty International Center, listed the scope of health research as including: fundamental discovery research, pathogenesis research, epidemiology research, clinical research, product development research, translational and adaptational research, operational research, health services research, policy research and research on health systems [One of the roles of health research is to ensure that the measures proposed to break out of the vicious cycle of ill health and poverty are based, as far as possible, on evidence, so that the resources available to finance these measures are used in the most efficient and effective way possible . There a systems . NGOs inThe purpose of this paper is to document the role that NGOs have played in global health research and to highlight the need to expand this role. This paper is also intended as a tool to stimulate research activity in NGOs and to advocate for increased NGO involvement in global health research. Following a brief review on the central role of global health research in development, the roles of NGOs at different stages within the research process are discussed and illustrated with a few examples. Key challenges are also identified. The last part of the paper identifies future needs for strengthening the role of NGOs in global health research. World report on knowledge for better health 2004).While research means different things to different people, it may best be defined as 'a knowledge loop' from generation of knowledge to its effective use . Indeed,The call to shift health research priorities from problems of industrialized countries to those affecting populations in developing countries is not new. In 1990, concerns regarding the inequitable distribution of research efforts were first raised in the Report to the Commission on Health Research for Development . Since tSeveral important initiatives have been undertaken to address the global health research agenda. They have been fostered by individuals and groups from local, national and international bodies who shared a common vision in advocating for health research directed towards the low and middle income countries.The Commission on Health Research for Development declared in 1990 that "For the most vulnerable people, the benefits of research offer a potential for change that has gone largely untapped" . The ComForging Links for Health: Perspectives from the Council on Health Research for Development", [Health Research for Development: The Continuing Challenge [Over the years, COHRED has assisted increasing numbers of countries in the exploration and implementation of essential national health research (ENHR) strategies. Networks were created to facilitate national level activities in Africa, Asia, and the Commonwealth Caribbean. For example, AFRO-NETS, the 'African Networks for Health Research and Development', was established in 1997 to facilitate exchange of information among different networks active in this type of research in English-speaking Africa, and to facilitate collaboration in the fields of capacity building, planning and research. Regional and global working groups and projects were established which allowed experiences with ENHR to be shared. Several communication strategies were utilized, including quarterly newsletters, websites and other publications to share experiences and lessons learned. A framework for capacity development, a critical component of ENHR, was established through partnerships and like-minded networks and organizations. The book, 'opment",  and the Several questions remain unanswered:• To what extent have the recommendations been implemented?• Have the recommendations made a real difference in the lives of the countries that carry 90% of the disease burden?• Has 'Essential National Health Research' worked?• What is the current situation with regard to health research for development?• Where and how do we proceed from here?The 2000 International Conference on Health Research for Development provided COHRED and several partner organizations with an opportunity to review and reflect on their experience with health research, its impact on health and equity and to devise a global strategy for the first years of the coming millennium .ad hoc Committee on Health Research Relating to Future Intervention Options [The Global Forum for Health Research, created in 1998 as a response to the Report of the WHO  Options , has pro Options  emphasizIn November 2001, four Canadian federal agencies, Canadian International Development Agency (CIDA), International Development Research Centre (IDRC), Health Canada, and Canadian Institutes of Health Research (CIHR) signed a Memorandum of Understanding to support national consultation regarding Canada's role in global health research. This marked the first time in Canadian history that Canada's two overseas development agencies, Health Canada and Canada's major federal health research funding agency have collaborated to address global health research.The Canadian Coalition for Global Health Research (CCGHR) is developing into a network of health researchers, funding agencies, NGOs, and other stakeholders committed to support the pursuit of effective global health research by ensuring that all these groups work together as effectively as possible with researchers in developing countries. This collaborative approach serves as a framework for future research projects in the area of global health, with each organization bringing its own specific area of expertise to the table. It aims to improve the effectiveness of development assistance and to increase the sustainable health gains per dollar of Canadian funds invested in research.Inequities in health are caused by a number of determinants, including the use of or access to health care facilities. Research which addresses these issues requires an intersectoral approach, involving trans-disciplinary teams and methodologies. Building trans-disciplinary teams requires commitment from the research community to seek out colleagues from other disciplines, from the funding agencies to appreciate innovative initiatives, from the community at large as partners and contributors, and from the policy arena to develop strategies for intersectoral policies and programs which may well have the lead outside ministries of health. Indeed, working outside government altogether may well be a solid and sustainable strategy. Understanding and engaging the broader community on these issues comes naturally to communities unrestricted by bureaucratic boundaries. This is where NGOs excel.NGOs have contributed to all different stages of the research cycle , downstream actions (knowledge management, dissemination and translation), and the advocacy efforts required to connect research with policies, programs and training.Historically, the influence of the biomedical researchers' lobby has been the strongest with regard to agenda-setting and fundraising. Behavioral scientists and social health researchers generally have much weaker potential to influence resource allocation, agenda-setting and policy formulation. Partnerships could be strengthened and supported between NGOs and social science researchers in resource-poor countries to improve influence potential, as the social sector issues that tend to be most relevant to human populations are also of utmost importance to NGOs.Creating a favorable environment for "relevant" research requires a health system that is supportive and provides financing opportunities. It also requires the existence of a culture of "evidence-generating and evidence-based research". There must be a healthy relationship between communities, researchers and policy makers. Networks to share experiences, lessons learned and policy impact can be enhanced by partnerships with NGOs.A disproportionately large number of people living in developing countries suffer large disease burdens. Promoting research and development on neglected diseases or issues of global health significance may contribute to bridging the 90/10 research gap, by stimulating research by public or civil society organizations on issues that do not represent marketable research, and are therefore neglected by the private sector. There is a role for NGOs in advocating for more research on these neglected topics (see under 4.1, example of initiative for neglected disease drugs, Médecins sans Frontières [MSF]).Health research needs to generate knowledge that will facilitate the identification of choices and options to reinforce equity-based policies and programs. In doing so, it also needs to address the difficulties of collecting data that are of primary importance when inequities are discussed. The essential function that data serve will allow tracking and monitoring of resources for research and for improving opportunities for those researchers in more disadvantaged countries. NGOs often have access to information that will highlight inequities and the determinants of inequities.Similarly, NGOs can advocate for formative and evaluative research on programs that address major health problems, but which are generally a low priority for funding agencies. In doing so, they can contribute to making data available for evidence-based decision-making in policy and program planning. Food system-based approaches to reducing micronutrient deficiencies and malnutrition in general are one of these under-researched areas.NGOs are well-placed to foster public participation in decisions about health research, as they are close to communities. They can provide the mechanisms by which such public participation is ensured in decision-making processes. Significant progress has been made over the last decade in health research priority-setting for the implementation of ENHR at the country level. Among the lessons learned, it appears that community involvement is in most cases an unresolved issue . What isDefining the research that needs to be done requires the input of civil society and NGOs as much at the beginning as at the end, in terms of dissemination, communication and action.NGOs assume a range of roles in research, but a thread that runs through all these is their representation and advocacy for the vulnerable. Broad research roles are described in greater detail in other sections of this paper. This section focuses on the role of NGOs in shaping and interpreting ethical frameworks -25, thatAs watchdogs, NGOs actively seek breaches of ethics and hold researchers to account when the principles of respect for persons, beneficence and justice are not upheld, a role they are well positioned to assume given their understanding of and links to marginalized groups. Watchdogs, as they uncover ethical breaches that may be defined by culture or power relations, have assisted in shaping ethical frameworks to better address ethics when research is conducted among vulnerable groups.In the communities where NGOs work, they can act as community partner members of and witnesses to research. In this role they can assist with, for example, interpreting research objectives to participants to ensure that consent is informed and the rights of subjects are respected. They may provide researchers with enumerators or local information to expedite the data collection process. NGOs can also monitor the long-term outcomes arising from research, and make sure that the participants benefit from successful intervention.As knowledge translators, NGOs interpret the knowledge generated by research to their constituents, a key role in working towards the vulnerable having access to the benefits of research that could improve their lives. This may be research conducted in these communities or globally.While current levels of financial resources are not sufficient to adequately respond to the demonstrated need for health research, there are many sources of "funds" for health research. Some are monetary contributions and some are in-kind contributions. NGOs can provide not only direct funding for projects  but, and perhaps equally important, they can provide valuable in-kind funding. Thus, personnel or materials developed by NGOs can be used in health research projects at little or no cost.Some NGOs are directly involved in the administration of research grants. Others may be the fiduciary agent for a grant to a research organization that is exploring an issue related to an NGO program. However, most are organizations that work with communities. A major role is therefore to identify resource gaps using networks to link communities, health providers and managers, and funding agencies in a meaningful way so that financing can appropriately be directed to targeted health issues. NGOs may also contribute by identifying other potential sources of funding, for instance, in the local private sector.Knowledge can be acquired in various ways, by many methods, and by different types of people; there are different cultures of enquiry. Because of their typical 'grass-roots' experience, several NGOs are able to access indigenous knowledge and specific information, which may be less attainable for other types of organizations. This type of knowledge might be very useful when pooled with knowledge acquired by others; in this way, a more comprehensive analysis can occur. NGOs can be particularly adept in conducting formative research (baseline studies, needs assessment), in operational or action research and in process and impact evaluation. This type of research is particularly relevant for setting priorities, for informing intervention, as well as for identifying further research needs.There is a need for NGOs to be more involved in policy research even in Canada" (Interview with B. Tomlinson, CCIC).Although knowledge generation is generally not a primary NGO activity, there may be specific 'knowledge generation' research niches for NGOs. For instance, as suggested by the Canadian Council for International Cooperation (CCIC) and actually carried out by a few NGOs, "Figure While asserting that the production of knowledge is the primary function of research, and that levels of knowledge have increased considerably, a discussion paper for the International Conference on Health Research  also recInadequacies include the inability of developing countries to access pertinent international research literature and knowledge bases (either as contributors or users), the inability to access new information technologies, and the inability to ensure closer links among the research community, health service managers and health policy makers., that is, the conversion of research findings from basic, clinical or epidemiological environmental health science research into information, resources, or tools that can be applied by health care providers and community residents to improve public health outcomes in at-risk neighborhoods.The effective use of research findings and their dissemination is an increasingly important public health policy concern. In 1995, an international research conference was held in Vancouver, Canada, on dissemination research. This type of research is similar to what is now called 'translational research' NGOs are frequently at the interface of applied research and policy-making, at least at the administrative level, and their potential input into research utilization for policy-making needs to be valued. Research can make a substantive contribution in at least three phases of the policy-making process: agenda-setting, policy formulation, and implementation . It is wNGOs often play a critical role in interpreting the evidence and translating its relevance for local communities. Inevitably the level of involvement by the community depends on relevance and opportunity for action and advocacy. Assessing and evaluating opportunities for advocacy and action occur as NGOs work with communities on these issues. Effective involvement of the community and its participation is a "matter of reciprocity and continuing dialogue in which participation takes different forms and influences change in several directions" . Once thet al [et al contend that it may not be relevant to transfer knowledge from individual studies, but rather, from bodies of cumulative research knowledge, and that knowledge transfer brokers are needed for this purpose. This model of specialized roles is probably more relevant at the macro level and in industrialized countries. In resource-poor countries, polyvalent organizations such as NGOs have a key role in sharing, translating and implementing research findings at the community and country level. They provide channels for the use of research results at the community level, as they are closest to the communities themselves. For that very reason, they may also feel more compelled to complete the research cycle, including application of the findings. Third World Network , for instance, an independent non-profit international network of organizations and individuals involved in issues relating to development, conducts and disseminates research to help organizations around the world participate in and influence international economic and social policy. NGOs may also be involved in testing pilot models of intervention and in their subsequent scaling-up.According to Lavis et al , while tThe preliminary examination of the functions performed by the some 125 organizations involved in a significant way in health research reveals that while knowledge generation is a concern shared by most, research capacity strengthening receives relatively little attention [The development and retention of research capacity remains a challenge in many countries . QualityNGOs in the North and in resource-poor countries often have the capacity for facilitating training and for sharing the lessons learned in needed skills. Partnership with NGOs in such capacity-building needs to be valued and reinforced. The Canadian Society for International Health (CSIH) and the Canadian Public Health Association (CPHA) have participated in capacity-building activities in many countries and continue to share their experiences and lessons learned. Support for such sharing and building capacity makes sense and should be facilitated by donor agencies. WHO, through its creation of a Department of Research Policy and Cooperation within the cluster of Evidence and Information for Policy, has defined as one of its objectives: "the development of initiatives aimed at strengthening research capacity in the developing world with the ultimate aim of enshrining research as a foundation for policy".A number of other international initiatives have also attempted to address some of these capacity issues: the International Health Policy Program (IHPP), the Applied Research on Child Health (ARCH) project, the Swiss Commission for Research Partnership with Developing Countries (KAPE) and, in Canada, the IDRC. Since 1970, IDRC has been providing financial and technical assistance to academic institutions, government agencies and NGOs in developing countries, as a means of promoting sustainable and practical development and strengthening indigenous research capacity. IDRC's experience provides important and valuable lessons about implementing applied research in partnership with NGOs , as summThe CPHA, through the CIDA-funded initiative Canada's International Immunization Program – Phase 2 (CIIP2), dedicated 5% of the program's budget to applied research. Part of this funding was used to strengthen primary health care in developing countries through the NGOs that implemented the immunization and primary health care activities through the auspices of CIIP2.NGOs who wish to become more involved in research generally recognize the need for extramural training and support. Partnering with universities and research institutions may provide such training opportunities. Additionally, there are international institutions such as INTRAC  that are specifically geared towards meeting the challenges and needs of NGOs in research. Those NGOs that are part of international networks can draw from the body of research conducted elsewhere.NGOs may also provide substantive input into research training, be it by grounding research methods in reality so that research is more applicable, or by providing research sites and questions for academia and graduate students. NGOs may also be in a good position to identify young scientists and promising investigators in host countries.Stimulating the demand for research by user groups, rather than supply-driven research, is one of the three strategies identified by Harrison & Neufeld  for capaThere is a lack of accessible and centralized information on NGO involvement in health research, although the CPHA CIIP2 applied research publication lists over 20 examples of NGO-related applied research carried out in the 1990s. The examples given below are based on discussions with a limited number of Canadian and international NGOs: CARE, World Vision Canada (WV), CECI , Inter Pares, HKI , and CCIC. In the case of AMREF , ADI , Médecins sans Frontières (MSF) and RITC , most of the information was obtained from their websites and related publications and documents. The interviews and discussions covered the specifics of the implication of the NGO in health research, lessons learned through the experience, and respondents' perceptions on the role of NGOs in global health research, and on the strengths and weaknesses of their organization in this regard. These selected NGOs provide insight into some of the critical issues facing NGO involvement in global health research. It should be kept in mind that this selection is small and not meant to be representative. Nonetheless, all of these NGOs are involved, directly or indirectly, in global health research, and they are all Canadian or present in Canada.The interviews covered a broad range of cases, from NGOs little involved in research to those actually conducting independent research. The types of involvement are briefly described below. A salient observation is that what is considered as research by different NGOs is, for the most part, unclear and highly variable. This suggests the need for NGOs to develop common views on what is research, the various types of research, and the components of the research process. The interviews also revealed that while some NGOs are reluctant to be involved in research, others are eager to strengthen their capacity to do so.CECI has long been involved in health research, although it is reluctant to call this 'research'. A major activity is the undertaking of baseline studies that typically include an assessment of the health and nutritional status of populations. The data are used to orient or reorient programs, and to inform communities. In Cambodia, for instance, it conducted an initial assessment for a project aimed at improving the livelihood of rural poor in two sectors: health/nutrition, and agriculture marketing . One interesting aspect of its recent work is the 'policy feedback' that it conducts in its large projects. The intent of the analysis is to clearly identify the lessons learned, and to discuss these with decision-makers and technical officers. This may be considered as part of 'knowledge translation' and it can be a particularly useful approach in advancing policies and programs. While CECI is also involved in health projects that do not include research even in a broad sense, it conducts research in areas that are indirectly related to health. For instance, in the IDRC-funded project intended to alleviate poverty in Burkina Faso, Viet Nam and Nepal, it collaborates with local universities and research institutions for the research and training components, notably on adapting the assessment of poverty to the specific context.CCIC and its member NGOs are involved in international policy research. For instance, Trade-Related Aspects of Intellectual Property Rights (TRIPS) agreements have implications on access to drugs. In the reorientation of CIDA for improved aid effectiveness, there are obvious health implications, including how to respond to health plans as defined by health ministries, and assist with poverty reduction strategies. CCIC sees research on policies as a critical role of NGOs, and considers that NGOs should be more involved in the policy debate both in Canada and globally.per se; it partners with research institutions, in the field and in Canada. It does not have the capacity to analyze all the data that it collects and therefore it collaborates with academic institutions in Canada. Graduate students can use the data for their theses. WV officers may also sit on graduate students' supervisory or examining committees. The primary use of the research findings is to reorient programs and inform the community. As programs may have to change their operations as a result of such research, the exercise may, at times, be regarded as threatening.World Vision (WV) Canada is active in research, particularly (but not only) in the framework of its MICAH projects  funded by CIDA. It primarily conducts formative and evaluation research  is a technical assistance NGO that is also directly involved in research as part of its mandate. It addresses the causes of preventable blindness. It also provides rehabilitation services to blind people, and helps reduce micronutrient malnutrition which can cause blindness and death in children. It is involved in most stages of the research cycle, focusing on operations and action research. HKI's focus on blindness and micronutrients is a strength in that its research is more focused than that of other NGOs involved in health and nutrition. Its funds for research come from different sources. A research component may be built into programs, some operational research is conducted with funds for surveillance, or funds are provided for R&D specifically (eg. for FRAT studies [Fortification Rapid Assessment Technique]) and for the development of tools to assess the quality of nutrition interventions leading to adoption of relevant strategies . HKI has in-house expertise in research. There are several full-time research positions. In addition, it works with research partners at the local level, as well as with universities in Canada and USA.Forum Afrique Canada , for instance, it is studying Canadian government trade and aid policy after G-8. It has in-house research expertise, particularly in sociology, although there is no research position as such. It usually works with partners, as it is a small NGO. Inter Pares uses research findings mainly for education and advocacy.Inter Pares was created in 1975 to support NGOs from the South and to provide international development education in Canada. Inter Pares uses its own funds to conduct social research on political and economic issues, primarily action research. For instance, it carried out collaborative research with NGOs in the Philippines and of Bangladesh on family planning policy, and in Africa it has carried out research on economic issues. With AMREF has been active since 1957 in the field of applied health research and has an extensive bibliography documenting research results in the form of peer-reviewed publications, theses, manuals, reports, abstracts and conference presentations. The focus of AMREF's research activities has been primarily in the operational and applied domains. Many have addressed the important disease burden caused by communicable diseases such as malaria , an NGO affiliated with WHO, specifically provides support for research among its numerous activities. In particular, it supports the research work of the 10/66 Dementia Research Group . The viMédecins sans Frontières (MSF) was the first NGO to both provide emergency medical assistance and publicly bear witness to the plight of the populations they served. MSF is at the forefront of emergency health care as well as care for populations suffering from endemic diseases and neglect. MSF has undertaken an initiative on drugs for neglected infectious disease which combines advocacy, research and capacity development, and networking. In contrast with private sector research, it is need-driven rather than profit-driven. Five pilot projects are currently underway focusing on capacity building and technology transfer. This initiative started with a review of pharmaceutical research and development outcomes over the last 25 years and of current private and public initiatives. Highlights of the findings and conclusions, published in Lancet , are proResearch for International Tobacco Control (RITC) is an International Secretariat based at IDRC headquarters (Ottawa) that funds multidisciplinary tobacco control research projects in developing countries. Its mission is to create a strong research, funding and knowledge base for the development of effective tobacco control policies and programs, through a combination of research, dissemination, strengthening of capacity and coordination. RITC concentrates on research on psycho-social correlates of tobacco use. It provides support to research projects conducted by NGOs, such as, the Youth and Tobacco Survey conducted in Russia by the Russian Public Health Association, with the technical assistance of the CPHA table .CPHA has also provided technical and financial support through its various international initiatives funded by CIDA to its public health association partners to carry out of the GYTS in Burkina Faso, Niger, Haiti, and Cuba; and in partnership with Institutes of Public Health in Bosnia & Herzegovina, Serbia & Montenegro, and in the UN-administered province of Kosovo. The results from the surveys (carried out in collaboration with the Centers for Disease Control and Prevention [CDC] of USA) are being used to develop tobacco control policy and youth-focused smoking prevention and cessation programs.The experience of CSIH in global health research is described under 5.2.1.The following summary of perceived NGO strengths (and weaknesses) in health research is based primarily on the data from individual and group interviews that were specifically conducted as inputs to this discussion paper.NGOs give a human face to research, and they are in a good position also to build on indigenous capacity" (Interview with S. Baker, HKI Africa). Additionally, they may be more compelled to complete the research cycle and apply the findings. Their involvement in research is perceived as a motivation to use the research to design, develop and respond to circumstances affecting development. Evaluation research, in which they are frequently involved, tells them whether or not they have an impact.It is a common view that NGOs are in a good position to participate in health research because of their knowledge of, and their presence in, local communities. Furthermore, their involvement increases the relevance of research to communities. "Since they are closely connected with communities, they have the ability to see the application of their research results. For this reason, NGO-initiated research is often more likely to be translated into practice in a timely manner as it is almost always directly related to practice. The NGO structure brings concreteness and a style which is guided by values and beliefs with an action orientation.Another major strength of some NGOs is their international networks which give them access to technical information and support. Finally, NGO values of ethics, solidarity and dialogue are important for health research to contribute to reducing inequities and for empowerment.Many reasons that account for the reluctance of NGOs to become (more) involved in research activities pertain to NGO perceptions on research . In the past, research was an academia-driven and based, elitist and theoretical exercise, the results of which are of little use to NGOs and the communities with which they work. Traditional research strategies and approaches were seen as top-down, non-participative and controlled by external actors. Research activities were regarded as requiring special technical expertise, much time and effort, access to professional journals and research literature, and substantial human and financial resources, characteristics not typically found in NGOs. Finally, the scientific rigor demanded by researchers was believed to be difficult to achieve in field-based situations, where unpredictability and subjectivity are the norm.What NGOs perceive as research and as their role in this respect varies widely. For instance, there is some hesitation and even reluctance in including baseline studies or project evaluation under 'research'.Donors do not want to hear that NGOs are doing research as they are implementation organizations" .Another obstacle is the fact that some NGOs that raise funds from the public are afraid to go against the expectations of the donors if the money is reallocated for research, and particularly for policy research in Canada: "Among other barriers, interview respondents mentioned lack of training opportunities, lack of funding owing to their (limited) mandate, priorities of funding agencies, and time constraints.Because of lack of training or of specialized researchers, NGOs may not be in a position to conduct top quality research, and scientific rigor may be lacking in certain instances. Lack of access to scientific literature when in the field can also be a major shortfall.It is often difficult to secure research funding from certain donors. Many Canadian NGOs rely heavily on negotiated contracts with CIDA, which leaves little time and place for research. However, CIDA does fund some research (in a broad sense), particularly formative and summative evaluations. These are encouraging trends, but the aim should be for bilateral agencies to openly fund some research, like in the UK and some Scandinavian countries.NGOs do not have a research mandate, and therefore we do not foresee developing research expertise in-house. Linking for instance with universities is feasible for development-driven research" (Interview with R. Hazel, CECI). NGOs may have to change their structures and priorities in order to support autonomous research.Lack of interest or of a clear view of the whole research process can also be considered as impediments. As several NGOs do not see research as part of their mandate, they may not be willing to get involved in research: "Because NGO research is often conducted on a small scale and is usually of a qualitative nature, it often goes unrecognized by governments, and even by research organizations and funding agencies, which tend to favour large scale quantitative research.NGOs interested in pursuing a research profile require a type of mentorship in terms of standard performance indicators in the research domain. For example, publication has traditionally not been a strength and much NGO research does not reach beyond the gray literature or report level. Because scientific publications are an important means of transfer or dissemination of research results, NGO capacity to publish their findings needs to be strengthened.There is not enough networking and collaboration between NGOs and the international research community, including academia. This has traditionally been due to a dichotomy in the interests of NGOs and the academic community, in that NGOs are more oriented towards a development agenda, while academics tend towards special research interests.In light of the above issues and concerns, and in order to foster greater interest and participation of NGOs in research, the barriers of lack of interest, lack of funds, lack of training and lack of recognition, among others, need to be addressed. We discuss some strategies below.Substantial global health academic capacity has developed within NGOs both in the U.S. and the U.K. For example, Family Health International (FHI) has integrated research, training, and development capacity on an evidence-based foundation. It also embodies many of the competitive aspects of private sector-led initiatives that can allow creativity and innovation.As emphasized by Harrison and Neufeld , howeverA peer-learning process is among the strategies for NGO capacity building in research. NGOs can draw on expertise already developed in research-based NGOs. The learning process should be shared with NGOs from the North and from the South. Additionally, NGOs should consider taking the initiative in organizing scientific activities (seminars, workshops, symposia) on global health research topics, which could serve as a catalyst in bringing together different stakeholders.The creation of networks which have the common goal of supporting global health research is one way to strengthen partnerships and to consolidate valuable resources from each partner. Leadership and governance issues are necessary hurdles which can be overcome by focusing on the ultimate gains in terms of supporting and conducting successful research activities. NGOs can assist in the establishment and functioning of these networks, particularly by providing stable infrastructure support. One of the greatest challenges is in making the network function effectively through different leadership turnovers in the different partner organizations.CSIH has had global health research as part of its mandate since its formation. CSIH is an active member of the Canadian Coalition for Global Health Research (CCGHR), and a key challenge in this capacity is to develop a strong foundation of understanding and mutual respect amongst all players in global health research, including NGOs. CSIH experience in global health research is described in table Partnerships with universities and other research institutions is one means of strengthening the research capacity of NGOs, and also of academia. NGOs and research organizations each have a unique 'value-added' contribution to make to global health research and therefore, partnering among them amplifies their individual strengths. Such partnering may be a real challenge for NGOs, however, as their institutional culture is so different. NGOs may be invited by universities to partner, but plans are often already laid out, so that the NGOs may only be involved in executing the plans. What NGOs want is to be part of the research process from the start. An interesting initiative to document and promote South-Canada health research partnerships is currently underway .NGOs are open to partnerships with academia, but the goal has to be development-oriented. Experience suggests that it is often difficult to reconcile the academic and development framework, for instance when integrating MSc or PhD students in development projects. Nonetheless, integrating graduate students in NGO projects should be a good strategy for balanced and equal partnerships., the prospects of university-NGO research links are constrained by funding. Nevertheless, the recent Global Health Research Initiative of the coalition of Canadian institutions funding global health research is promising as it opens new avenues for research collaboration between the North and the South, and hopefully also between universities and NGOs.As less than 1% of university-based health research in Canada is directed towards the problems of global health according to a Canadian Consultation on Global Health Research held in 2001 During the 1990s, there were several attempts to bridge the NGO/academic gap with respect to health research in developing countries. Save the Children UK, Oxfam and some US-based institutions supported workshops and symposia that aimed at bringing together representatives from both communities as a means of building links and forging partnerships in support of increasing the scale, scope and relevancy of health research in developing countries (Edwards M, Griffiths M: Terms of Reference for the DSA [Development Studies Association] Workshop on the Academic Practitioner Interface. London: DSA, 1994). CPHA, through the CIDA-funded Canada's International Immunization Program – Phase 2 (CIIP2), supported applied research carried out by Laval University, Université de Montreal and University of British Columbia. At the time, these were quite innovative approaches to applied health research, linking the universities with local NGOs. In 1995 CPHA also organized a Symposium on NGO/University Linkages for Health Research .One mechanism to expand the use of research generated by NGOs is to improve the linkage between NGOs and universities. Each complements the other in the area of health research. NGOs offer proximity to people and situations, reality-based and context-specific research environments, opportunities to develop and assess innovative strategies and research methods, a means of disseminating and popularizing the results of research projects, and credibility outside of academia. Universities and other research organizations offer expertise in research design and application, an environment for reflection, access to and knowledge about most recent literature, a tradition of scientific rigor and interest in new, innovative research methods and approaches, and a high degree of credibility. Academics can also provide guidance and advice on how to prepare research proposals and to carry out research studies, guidance in the preparation of reports and publishing of research results, and training for NGO staff in research methods.The participants of the CPHA Symposium on NGO/University Linkages for Health Research in Developing Countries  identifiThe development of innovative North-South research partnerships is the focus of a working paper prepared for the CCGHR by Neufeld et al . As emphFinally, as mentioned earlier, stronger partnerships between NGOs and social science researchers in particular should be sought in resource-poor countries. Lessons learned from these partnerships, in the areas of action and indeed policy and legislation (for example, in the tobacco or environment fields) show how evidence can be transformed into action with the right partnerships between researchers and NGOs.NGOs may benefit in various ways from developing a global health research network. First, many NGOs already operate at national and international levels and understand the challenges of coordination and communication which this entails. There is a need to identify overarching principles of NGO contribution to health research. Second, NGOs must be both proactive and interactive within the framework of the health research agenda. Roles must be clearly understood by each partner. In any case, advocacy for relevant research and use of results would be a critical function of the network (see training modules on advocacy ). Such nIn the framework of an NGO network of this sort, the following discrete activities could be envisaged by the lead NGO:• To invite NGOs to post on a selected website success stories, as well as their experience/opinions/needs/priority research issues, using a template adapted from the one developed for this purpose in the UK• To organize workshops for NGOs who are, or who wish to become, involved in research, with research organizations where deemed appropriate.The purpose would be:• To link these NGOs in order for them to interact on research issues;• To share lessons learned and success stories of research involvement of NGOs and their partners• To enhance understanding of, and collaboration with, potential research partners;• To set-up a core group of NGOs involved in global health research to convey NGO views to global health research fora and organizations.The following are a few key questions that could be addressed by an NGO network:1. How can NGOs contribute to the framing of the research questions if we were to support the necessary equity-based research for improving the overall performance of the health system?2. How do we balance this with the necessity for research which documents and monitors sustained and emerging inequities which may have a greater impact on the health and well being of individuals than the health (care) system will ever have?3. How can we ensure that NGOs influence research priorities so that they are reflective and evaluative of overseas development assistance (ODA) direction and priorities such as national poverty reduction strategies. For example what is the impact of PRSPs on equity and health? How will researchers monitor this? What could be the potential role of NGOs in partnership with researchers to begin to monitor and evaluate this new direction in overall aid policy?4. How can NGOs be best represented within the international research community?The new millennium offers many challenges. In order to maximize the potential benefits of health research, all partners including NGOs must share a common vision and recognize and appreciate the strengths of each. Participation in health research needs to be a coordinated effort. One key challenge will be to establish better communication among all partners in health research. This can only be achieved by a willingness to share in leadership, ownership and in the conduct of health research activities. Another key challenge will be to explore ways in which funding for health research can be strengthened. Leveraging must be seen as a strategic tool of NGOs to maximize dollars allocated to health research.Several NGOs have had impressive track records in global health research. Other NGOs have expressed an interest in becoming more involved in global health research. Their contribution to more equitable, ethical, relevant and effective research is crucial and needs to be strengthened. Research has to be regarded as a broad loop system rather than restricted narrowly to the production of knowledge. This is particularly critical for global health research whose primary goal should be to improve health and its determinants in low and middle income countries. NGOs principal roles in the process pertain to shaping research priorities, advocacy for more relevant research, translating and using research findings, in addition to generating new knowledge in areas where they may have a comparative advantage, notably qualitative, social, action, evaluative, and policy research. NGO partnerships with research organizations should be seen as means of a mutual enhancement of health research capacity and contribution to development. NGOs should be instrumental in building with other stakeholders coalitions for global health research with the aim of closing the 10/90 health research gap.ADI Alzheimer's Disease InternationalAFRO-NETS African Networks for Health Research and DevelopmentAIDS Acquired immunodeficiency syndromeAMREF African Medical and Research FoundationARCH Applied Research on Child Health (ARCH) ProjectCCIC Canadian Council for International CooperationCCISD Centre de coopération internationale en santé et développementCCIH Canadian Conference on International Health (hosted by CSIH)CECI Centre canadien d'étude et de coopération internationaleCCGHR Canadian Coalition for Global Health ResearchCDC Centers for Disease Control and PreventionCHW Community health workerCIDA Canadian International Development AgencyCIHR Canadian Institutes of Health ResearchCIIP2 Canada's International Immunization Program – Phase 2COHRED Council on Health Research for DevelopmentCPHA Canadian Public Health AssociationCSIH Canadian Society for International HealthDFID Department for International Development (UK)DSA Development Studies AssociationENHR Essential National Health ResearchFCTC Framework Convention on Tobacco Control (WHO)FHI Family Health InternationalFRAT Fortification Rapid Assessment TechniqueGYTS Global Youth and Tobacco Survey (WHO and CDC)HKI Helen Keller InternationalIDRC International Development Research CentreIHPP International Health Policy ProgramINTRAC International NGO Training and Research CentreKFPE Commission for Research Partnerships with Developing CountriesLMIC Low and middle-income countriesMICAH Micronutrients and Health in Africa (WV project)MSF Médecins sans frontièresNEPAD New Partnership for Africa's DevelopmentNGO Non-governmental organizationOECD Organization for Economic Co-Operation and DevelopmentODA Overseas Development AssistancePRSP Poverty Reduction Strategy PapersRITC Research for International Tobacco ControlRPHA Russian Public Health AssociationSHARED Scientists for Health and Research for DevelopmentTRIPS Trade-Related Aspects of Intellectual Property RightsUNDP United Nations Development ProgrammeWHO World Health OrganizationWV World Vision (Canada)HD designed the outline of the paper, conducted interviews with NGO representatives, wrote the first complete draft, and coordinated the review process within the CSIH. JHR drafted some sections and provided comments on the successive versions of the papers. MM provided international NGO and a field based perspectives to the paper, in addition to conducting group discussions with NGO personnel. LJ designed the figures, and edited and formatted the text. TG contributed to the conceptualisation and content of the manuscript. She wrote several sections and edited others, and she provided case studies.


Dendritic cells (DCs) are professional antigen-presenting cells that play important roles during human immunodeficiency virus type 1 (HIV-1) infection. HIV-1 derived lentiviral vectors (LVs) transduce DCs at high efficiency but their effects on DC functions have not been carefully studied. Modification of DCs using LVs may lead to important applications in transplantation, treatment of cancer, autoimmune and infectious diseases.Using DCs prepared from multiple blood donors, we report that LV transduction of DCs resulted in altered DC phenotypes and functions. Lentiviral transduction of DCs resulted in down-regulation of cell surface molecules including CD1a, co-stimulatory molecules CD80, CD86, ICAM-1, and DC-SIGN. DCs transduced with LVs displayed a diminished capacity to polarize naive T cells to differentiate into Th1 effectors. This impaired Th1 response could be fully corrected by co-transduction of DCs with LVs encoding interleukin-12 (IL-12), interferon-gamma (IFN-γ), or small interfering RNA (siRNA) targeting IL-10.DCs transduced with LVs in vitro displayed diminished Th1 functions due to altered DC phenotypes. Our study addresses an important issue concerning lentiviral infection and modification of DC functions, and provides a rational approach using LVs for immunotherapy. During HIV-1 infection, an increase in DC-SIGN and CD40 has been reported, as has a decrease in the expression of CD80 and CD86 in dendritic cells (DCs) of lymphoid tissue  others hLVs are useful gene transfer tools that can efficiently target many types of cells including DCs. As important immune modulating cells for immunotherapy and vaccine applications, DCs play critical roles in activating the host immune response. DCs can capture, process, and present foreign antigens, migrate to lymphoid-rich tissues, and stimulate antigen-specific immune responses . DCs pre+ T cells toward Th1 function, which is essential for the clearance of intracellular pathogens. IL10, on the other hand, suppresses IL-12 production from DCs and diminishes the commitment of Th1 differentiation. Besides cytokine signaling, there is accumulating evidence that co-stimulatory molecules and adhesion molecules such as CD80, CD86, and ICAM-1 not only engage in T-cell stimulation, but also direct the differentiation of naive T cells [Engagement between DCs and T cells not only stimulates T-cell proliferation, but also polarizes differentiation of naïve T helper (Th) cells into IFN-γ-producing Th1 or IL-4-producing Th2 effector cells ,7. ProduEfficient gene transfer into DCs without cytotoxicity has always been difficult ,12. LVs 2. On Day 3, 1 ml of fresh AIM-V medium containing 560 u/ml of GM-CSF and 25 ng/ml of IL-4 was added to the culture. On Day 5, the non-adherent cells were harvested by gentle pipetting. After washing, the DCs were frozen for later use or used immediately.Peripheral blood mononuclear cells (PBMCs) from healthy donors (Civitan Blood Center, Gainesville, FL) were isolated from buffy coats by gradient density centrifugation in Ficoll-Hypaque (Sigma-Aldrich, St. Louis, MO) as previously described . DCs werMLV and LVs were constructed as described previously ,20. The The LVs were produced and concentrated as described previously . Lentivi5 per well in a 24-well plate containing 200 μl of medium supplemented with GM-CSF (560 u/ml) and IL-4 (25 ng/ml). DC infection was carried out by adding concentrated LVs to the cells at a multiplicity of infection (MOI) of 50–100 (~105–106 transducing units/ng of p24) as previously described [We plated Day-5 immature DCs at 5 × 10and IL-4  ng/ml. DFor analysis of cell-surface marker expression by flow cytometry, we incubated DCs for 10 min with normal mouse serum and then 30 min with fluorochrome-conjugated anti-human monoclonal antibodies. In different experiments, these antibodies included HLA-ABC ; HLA-DR ; CD1a (HI49, mouse IgG1k, APC-labeled, Becton Dickinson Pharmigen, San Diego, CA); CD80 (L307.4, mouse IgG1k, Cychrome-labeled, Becton Dickinson);CD86 ; ICAM-1 ; DC-SIGN (eB-h209, rat IgG2a, APC-labeled, eBioscience, San Diego, CA); CD11c (Bly-6, mouse IgG1, PE-labeled, BD Pharmigen); CD40 (5C3, mouse IgG1, Cy-chrome-labeled, Becton Dickinson); CD123 (mouse IgG1, PE-labeled, BD Pharmigen); and CD83 (HB15e, mouse IgG1, R-PE-labeled, Becton Dickinson). We included the corresponding isotype control antibody in each staining condition. After two washes, the cells were resuspended and fixed in 1% paraformaldehyde in PBS and analyzed using a FACSCalibur flow cytometer and the CELLQUEST program (Becton Dickinson). The live cells were gated by forward- and side-light scatter characteristics and the percentage of positive cells and the mean fluorescence intensity (MFI) of the population were determined.2O to make an 8:1:1 ddH2O/DMSO/ethanol solution. The cells were incubated in 37°C water bath for 1–1.5 min, and diluted with 10-fold volume of cold medium and kept on ice until FACS sorting.The lentiviral siRNA vector-transduced cells co-expressing nuclear lacZ gene were separated from un-transduced cells by staining with fluorescent LacZ substrate and sorted by FACS. To label the lacZ-positive cells, we resuspended cells in 100 ul medium and added 100 ul of FDG  working solution (2 mM) which was diluted from a 10 × stock FDG solution (20 mM). The stock solution was made by dissolving 5 mg FDG (MW 657, Molecular Probe, Eugene, OR) in a 1:1 mixture of DMSO/ethanol and mixing with ice-cold ddH+ T cells from PBMCs were collected by negative selection, using a CD4+ T cell isolation Rosette cocktail (StemCell Technologies, Vancouver, BC) according to the manufacturer's instructions. Briefly, we centrifuged 45 ml of buffy coat (approximately 5 × 108 PBMCs) in a sterile 200-ml centrifuge tube with 2.25 ml of the CD4+ T cell-enrichment Rosette cocktail at 25°C for 25 min. Thereafter, 45 ml of PBS containing 2% FBS was added to dilute the buffy coat. After gentle mixing, we layered 30 ml of the diluted buffy coat on top of 15 ml of Ficoll Hypaque in a 50-ml centrifuge tube and centrifuged for 25 min at 1,200 g. Non-rosetting cells were harvested at the Ficoll interface and washed twice with PBS (2% FBS), counted, and cryopreserved in aliquots in liquid nitrogen for future use. The purity of the isolated CD4+ T cells was consistently above 95%. The CD4+CD45RA naïve T cells were purified based on negative selection of CD45RO- cells using the MACS (Miltenyi Biotec, Auburn, CA) magnetic affinity column according to the manufacturer's instructions.The CD4in vitro DC:T cell co-culture method was modified based on Caron et al[The ron et al. Briefly5 allogeneic CD4 T cells in a 96-well U-bottomed plate in a total volume of 200 μl for 5 days. The proliferation of T cells was monitored by adding 20 μl of the CellTiter96 solution to each well according to the manufacturer's instruction (Promega). The cells were further cultured for 4 hr before reading the OD490 value using a microplate reader (EL808, BIO-TEK Instrument Inc.,Winooski, VT).We co-cultured serial dilutions of DCs, from 10,000 cells per well to 313 cells per well, with 1 × 10To investigate the effects of lentiviral vectors (LVs) on DCs, we transduced monocyte-derived DCs with LVs encoding different reporter genes. The efficiency of LV transduction of DCs is illustrated by a reporter gene assay Fig. . The DCsTo see if LVs affected DC surface marker expression, we examined the expression profile of surface molecules on DCs by antibody immunostaining. We transduced PBMC-derived imDC with mock (control 293 supernatants) vectors, empty LV particles, LV, and MLV carrying a reporter gene. After induction of maturation with LPS plus TNF-α, we harvested the DCs for antibody staining and FACS. The results are shown in Fig. in vitro immunity assay using co-culture of human DCs and naïve T cells.It has been reported that retroviral infection induces up-regulation of Th2 cytokines including IL-10 and impairs DC maturation ,28. Beca+ T cells from healthy donors' blood and co-cultured the T cells with allogeneic monocyte-derived DCs treated with TNFα and LPS to induce maturation, as illustrated in Fig. We generated DCs from PBMCs and infected the d5 DCs with LV carrying a reporter gene. To characterize the function of DCs, we purified naïve CD4in vivo study [Because T cell co-stimulatory molecules are important mediators of DC functions, the down-regulation of CD80 and CD86 in DCs after LV transduction might contribute to the observed Th1 impairment. To examine this possibility, CD80 and CD86 were up-regulated in DCs using LVs encoding these two genes to see if the impaired Th1 response could be corrected. The LVs encoding human CD80 and CD86 were constructed as shown in Fig. vo study . DCs werTo see if the up-regulation of the T-cell co-stimulatory molecules in DCs could restore the Th1 response, we co-cultured naïve CD4 T cells with DCs transduced with mock, LV-PLAP, LV-PLAP plus LV-CD80, or LV-PLAP plus LV-CD86. After 8 days, the T cells were reactivated and analyzed by ICCS and FACS using anti-IL-4 and anti-IFN-γ antibodies as described earlier. We found that after LV transduction the Th1 population was reduced from 24% to 13%. Moreover, this impairment could not be corrected by the up-regulation of CD80 and CD86 in DCs  has been shown to enhance IL-12 production in DCs . To overIL-10 is a critical immune modulatory gene and modulation of IL-10 gene expression may alter DC function. To test this, we constructed LVs encoding siRNA targeting IL-10. We chose two regions in the IL-10 mRNA as the siRNA target sites Fig. . The siRThe effect of the IL-10 LV siRNAs was then examined in DCs by co-transduction using a reporter LV and the IL-10 LV-siRNAs. The transduced DCs were then treated with LPS and analyzed for IL-10 expression as described above. Again, the empty LV had no effect and LV transduction alone up-regulated IL-10 expression. However, co-transduction with LV-siRNA targeting IL-10 down-regulated IL-10 expression Fig. ; the lowTo examine whether co-transduction of DCs with LVs expressing the IL-10 siRNA could promote a Th1 response, we transduced DCs with LV alone or together with either an LV-siRNA (#2) or a control LV-siRNA (GFPi). For positive control, we incubated DCs with soluble IFN-γ as previously described. After the DCs were co-cultured with naïve T cells for 10 days, the T cells were reactivated and analyzed for Th functions by ICCS to determine intracellular expression of IFN-γ and IL-4. The results clearly demonstrated that the IL-10 LV-siRNA vector, but not the GFPi LV-siRNA vector, enhanced Th1 response at levels comparable to that of the positive control  is a 44-kDa type I membrane protein with an external mannose-binding, C-type lectin domain . It has Polarization of naïve Th cells into Th1 cells is critical for the induction of cellular immunity against intracellular pathogens and cancer cells. The observed impairment of the Th1 response by LV-transduced DCs raises a potential issue with LV-based immunotherapy. We illustrated that co-transduction with LV encoding IL-12 or IFN-γ, but not CD80, CD86, or ICAM-1, in DCs effectively restored Th1 immunity. In addition, co-transduction with LVs expressing small interfering RNA targeting IL-10 could also promote DC-mediated Th1 immunity. In a step toward future generation of vaccines, LVs encoding IL-12 and IL-10-siRNA as potent Th1 adjuvants may be used to enhance the cellular immune response in the prime-and-boost vaccination regimen. In summary, our study has addressed an important immune suppression effect of LVs and presented a solution that is important for future LV-based DC immunotherapy applications.HIV-1 human immunodeficiency virus type 1LV lentiviral vectorDC dendritic cellTh T helperMLV murine leukemia virusRT-PCR reverse transcription-polymerase chain reactionFACS fluorescence activated cell sorterICCS intracellular cytokine stainingIL interleukinPLAP placenta alkaline phosphatasesiRNA small interfering RNAPBMC peripheral blood mononuclear cellsMOI multiplicity of infectionLPS lipopolysaccharideTNFα tumor necrosis factor alphaINF-γ interferon-gammaThe author(s) declare that they have no competing interests.The study was conceived by LJC; XC and LJC participated in designing and coordinating the study; JH carried out some of the lentiviral constructions, siRNA designs and participated in result discussion; XC performed the statistical analysis; LJC and XC carried out detailed analysis of the results and XC drafted and LJC finalized the manuscript. All authors read and approved the final manuscript.


By combining ontologies from different sources the authors developed a novel approach to describing phenotypes of mutant mice in a standard, structured manner. The mouse is an important model of human genetic disease. Describing phenotypes of mutant mice in a standard, structured manner that will facilitate data mining is a major challenge for bioinformatics. Here we describe a novel, compositional approach to this problem which combines core ontologies from a variety of sources. This produces a framework with greater flexibility, power and economy than previous approaches. We discuss some of the issues this approach raises. N-ethyl-N-nitrosourea (ENU) mutagenesis [Mutant mice are the premier genetic models for human diseases. An increasing number of laboratories and companies worldwide are now carrying out detailed analyses of mouse phenotypes that have been generated from large-scale mutagenesis of the mouse genome. Description of mouse phenotypes has not traditionally adhered to predefined rules or been recorded in databases. However, the sheer volume of data from high-throughput screens (such as agenesis ) is now Ontologies have been an important tool for structuring biological information since the time of Linnaeus. With the advent of the Gene Ontology (GO) in 2000 ,4. In thThe term ontology is derived from the Greek and is used in philosophy to mean 'a description of what exists'. There are many definitions of the word, however, and for the purpose of this article, an ontology is 'a specification of entities and their relationships' . The keyAn ontology together with a set of individual instances of the kinds of entities it specifies constitutes a knowledge base . It may An important question here is why do we need to use ontologies; why not simply use a series of unconnected, standard terms such as provided by a controlled vocabulary? The advantages of using ontologies have been argued extensively, but the main reason is that ontologies are attempting to capture the precise meaning of terms. Furthermore, ontologies can be used for reasoning and inference (for example, consistency checking or drawing conclusions from the knowledge). The most important factor from our perspective is the need to combine information from different phenotypes or from different protocols (assays). For example, if a mutant mouse has six digits in each forelimb we will wish to use this information in a variety of ways . For this, we need not just a controlled vocabulary of terms, but also information about how these terms relate to one another .Traditionally, the main source of information for most scientists is the peer-reviewed journal literature. Electronic versions of published information have opened the road to accessing and retrieving information in a much easier and more cost-effective manner. The growth and wider availability of the world-wide web has led to a significant growth in the amount of readily available electronically stored information . With thThe most comprehensive attempt to annotate mammalian phenotypic data so far, the Mammalian Phenotype Ontology (MP) , is currThis approach allows high-level access to the knowledge held in the ontology, but also has certain limitations similar to the GO paradigm. If one attempts to create too much specificity within an ontology of this type it can expand to unmanageable proportions and parentage relationships can be overlooked as their number grows. For example, merely creating new terms by prepending the two qualifiers, 'increased' and 'decreased', everywhere that is applicable, will massively increase the size of the ontology. To allow a systematic approach to the model, combinations would also have to be instantiated that might never be used. Because there is a practical limit to the number of values that can be managed, such an approach is limited. Inevitably, decisions have to be made as to which individual combination describes a particular phenotypic entity best.We note here though that the development of MP is being developed pragmatically, with instances being added as needed to annotate mouse phenotypes, following the paradigm used by GO developers. MP is a cross-product ontology that includes mouse anatomy ontology, GO and other controlled terms as part of the construction of MP terms. Although the cross-reference IDs are not visible, they are part of the design of MP. Some of the work described here reflects insights gained during extensive discussions about the representation of phenotypes at the Phenotype Consortium meeting held in Bar Harbor, ME in September 2003. The developers of the MP ontology are part of this consortium and have intentionally created their ontology in such a way that it can be easily extended to form instances of the compositional approach discussed in the next section.With the objective of capturing information about phenotypes in any organism, Ashburner proposed the Phenotype And Trait Ontology (PATO)  in 2002.The description of mutant phenotypes must provide a practical way to capture the biologically relevant information about the phenotype in machine-readable form . It shouAccording to the schema in Figure According to the schema in Figure This class holds the information (organism attributes) of an organism in which the phenotypic characters are observed . They hold multiple relations to values, qualifiers and free text as well as their own metadata. The slot for free text is included to capture knowledge that cannot be expressed through the ontology as yet.Splitting PATO into two different ontologies, PATO attributes (above) and PATO values, allows the PATO ontology to be incorporated into the schema . Values A central idea in this schema is that of the 'phenotypic character', which we can define as any feature of the organism that is observed or 'assayed'. An example for the mouse is tail length. A phenotypic character is a compound composed of an entity, in this case an anatomical entity 'tail', and an attribute of tail, here 'length'. Similarly the physiological entity 'hearing' (GO:0007605) has the attributes 'sensitivity', 'range', and so on. Thus, 'hearing range' and 'hearing sensitivity' are distinct phenotypic characters. The ideal phenotypic character is one that can be measured independently of others. In practice, however, phenotypic characters are rarely independent. Furthermore, the observations from any particular assay will most probably depend on several different phenotypic characters. For example, the results returned by the click-box test for hearing sensitivity in the mouse actually depend, not only on hearing, but also on the mouse's ability to make a detectable locomotor response (the Preyer reflex ).These multiple dependencies are captured in the schema, enabling the ontology to support the appropriate possible groupings of phenotypes. This will allow us, for example, to group all mutants that have (by direct assay), or may have (for example, those failing the click-box test), an effect on the locomotor system. Conversely, different assays may provide information about a single phenotypic character. For example, an acoustic brain-stem response   can be mThe expressivity of representation languages such as DAML+OIL , OWL 2323 and OBFinally, we note here that it should be possible to link current high-level structures (such as the current MP ontology), which are necessary in many cases for annotation purposes, to the more expressive form we propose here, so that it can also be explored computationally.t(10) = 3.754, p < 0.004]." The authors also describe the assays used to record these observations: "Nesting Patterns: six cages of wild-type and six cages of mutant mice (N = 4 mice per cage) were used to evaluate nesting patterns. A 5 × 5 cm piece of cotton nesting material (Ancare, Bellmore, NY) was placed in each cage. After 45 min, photographs were taken of each nest and the nest depth was measured. Nest height data were analyzed using the Student's t test."In this section we describe an example of the application of the compositional schema. We chose a phenotype example at random from the MP database: 'nest building' [MP:0001447]. Several descriptions of nest-building patterns can be found in the corresponding reference . For exaFor some users/applications the compound term 'abnormal nest building' might be a sufficient description of this particular phenotypic instance, but this would result in information loss. A human would have to retrieve and read the reference to extract further information. Our schema allows the expression of this information in a machine and human readable manner. In Table Nest building {has_attribute} attribute:quality {characterized_by} defined_quality_assay (described in Nesting Patterns [well-formedPatterns ) {returnNest building {has_attribute} attribute:quality {characterized_by} defined_quality_assay (described in Nesting Patterns [poorly-formedPatterns ) {returnNest building {has_attribute} attribute:quality {characterized_by} defined_quality_assay (described in Nesting Patterns [fluffyPatterns ) {returnWe note here that had the value 'fluffy' not been included in the standard values for a quality assay, it could be captured in the free-text field provided by the schema. To express a nest of 50 mm depth or significantly shallower:Nest building {has_attribute} attribute:absolute_depth {characterized_by} undefined_ absolute_depth_assay {returns_value} 50 mmNest building {has_attribute} attribute: relative_depth {characterized_by} undefined_ relative_depth_assay {returns_value} shallow {has_qualifier} significantWith this information one could go back to a higher level and still be able to express a more general characterization of this phenotype as 'abnormal nest building' but obviously the opposite is not possible.An important unresolved issue concerning the use of ontologies to describe phenotypes arises from the fact that all the ontological structures developed so far are designed to describe individual mice. Mutagenesis experiments usually characterize a number of mutant mice to take into account variable penetrance of the mutation and other stochastic effects. A strategy will therefore need to be developed to describe the generalized phenotypic properties of a cohort of mice. This may involve the use of more sophisticated relations such as {usually characterized by} or even quantitative relations such as {80% characterized by}.The assay plays a central role in our schema Figure . Assays eye {has_attribute} attribute:color {characterized_by} visual inspection {returned_value} pinkOn a practical level, assays can add specificity and functionality to the relationship between entities, their attributes and the corresponding values. Most important, an assay vocabulary allows the entire schema to be dynamic by including new assays and capturing explicit differences between assays in different laboratories. The assay will also allow standardization and definition of values for a given phenotypic character, for example, how abnormal is defined in relation to body position.Our schema can be expressed using a variety of modeling tools and knowledge representation (KR) languages . We chosThe schema was designed to be easily populated using extant core ontologies, such as anatomy, and defining attributes related to each entity. The assay vocabulary can be constructed as required. Permitted values are defined in the range of different assay attributes in part devised in the form of a general scheme and in part built from the output of particular assays. Although we include for demonstration purposes three core ontologies, namely behavior, anatomy, and developmental anatomy Figures  and 4, wFigure et al. [Since most of the ontologies we are planning to use were generated using the DAG-edit  format, et al. , with miDecisions will inevitably have to be made to combine a core ontology with its attributes and then define facets of that relationship, for example, cardinality, attribute value type and attribute range. In our schema, the class hierarchy of all ontologies employed represents an 'is-a' relation. So, mouse social behavior 'is-a' mouse behavior, or mouse social behavior is a 'kind-of' mouse behavior and so forth. All other relationships, including PATO and 'part-of' relationships, are modeled as attributes. However, we note here that efforts are currently being made by the GO consortium to define and formalize the 'part-of' relationship, which is considered vital and special in bio-ontologies, especially anatomy .Because our phenotype ontology and PATO need to be the result of a collaborative effort within the communities, we feel that it is important to set out the basic modeling concepts that need to be applied upon allocating attributes to the core ontologies. Deciding whether to introduce a new attribute or represent this functionality through an entity is often quite difficult. Several things need to be considered in order to make the best decision, although it should be noted that there are no clear distinction as to what is a right or wrong decision.The first thing to take into account is that subclasses of a class inherit all properties of the parent and could have additional properties and different restrictions from the latter. PATO should remain as general as possible, and, when possible, care should be taken to avoid making PATO domain specific. For example, in the behavior ontology there is a class named 'reflexes' that contains children such as 'blinking reflex', 'Preyer reflex' and 'righting reflex'. It might be worth considering having one 'attribute of reflex' available in PATO rather than creating a separate attribute 'of' for each individual reflex, such as 'attribute of blinking reflex', 'attribute of Preyer reflex', and so on. Then again, if one wishes to assign different functionalities to these properties, creating separate attributes might be useful. As a rule though, one should consider that PATO needs to be consistent, usable and interoperable if it is to be applied to the general domain of phenotypes. Repetition between core ontologies and PATO should be avoided where possible.What is also often not clear is whether one should add a new class to represent functionality or assign attributes to already existent classes. For example, think of the entity 'body position'. There are several ways to model this entity in the mouse behavior phenotype ontology. One could declare 'body position' as a child of a class called 'posture'. An 'attribute of body position' could then be assigned to this class with a range of values that might be specific to an assay, for example SHIRPA  allows t'Body position' could also be split into an entity of 'body' and an attribute of 'position'. Again, a new class 'body position' should be assigned, if one considers the objects with different attributes as different kind of object and this distinction important in the domain. As a general rule, before assigning new classes and attributes one should consider the functionality and their role in the domain, creating more distinctions as the depth of knowledge that is required to be expressed in the ontology increases.Classes in the hierarchy should not necessarily have to introduce new properties . AlthougWe have presented here an approach to the use of ontologies in describing mouse phenotypes that could provide a platform for the consistent representation of mouse phenotypic data. We have also described in detail a possible methodology to construct applications of this schema across different domains. We have dealt with modeling issues and provide guidelines to deal with semantic and practical problems.We maintain that such modeling efforts in any domain should be done in a collaborative fashion in the community. Repetition between different parts of the mouse phenotype ontologies is unavoidable. However, the use of consistent IDs, synonyms and records for associated annotations could allow seamless integration of ontology products. The nature of the schema proposed, as well as its components, is extremely dynamic; therefore coordination of efforts is vital.The structure allows extensibility and interoperability. Although an ontology should not cover all possible information about a domain, the main idea behind this concept is to allow the phenotype ontology to cope with novel and unpredictable phenotypes and account for new assays, serving scientific autonomy and information validity and integrity. We have built a software system  which in


An increase in the number of attendees due to acute gastroenteritis and fever was noted at one hospital emergency room in Taiwan over a seven-day period from July to August, 2001. Molecular and epidemiological surveys were performed to trace the possible source of infection.An epidemiological investigation was undertaken to determine the cause of the outbreak. Stool and blood samples were collected according to standard protocols per Center for Disease Control, Taiwan. Typing of the Salmonella isolates from stool, blood, and food samples was performed with serotyping, antibiotypes, and pulsed field gel electrophoresis (PFGE) following XbaI restriction enzyme digestion.Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) was isolated from the stool samples of 28 of 32 individuals and from a recalled bread sample. All S. Enteritidis isolates were of the same antibiogram. PFGE typing revealed that all except two of the clinical isolates and the bread isolates were of the same DNA macrorestriction pattern.Comparison of the number of patients with and without acute gastroenteritis (506 and 4467, respectively) during the six weeks before the outbreak week revealed a significant increase in the number of patients during the outbreak week (162 and 942, respectively) (relative risk (RR): 1.44, 95% confidence interval (CI): 1.22–1.70, P value < 0.001). During the week of the outbreak, 34 of 162 patients with gastroenteritis were positive for Salmonella, and 28 of these 34 cases reported eating the same kind of bread. In total, 28 of 34 patients who ate this bread were positive for salmonella compared to only 6 of 128 people who did not eat this bread (RR: 17.6, 95%CI 7.9–39.0, P < 0.001). These breads were produced by the same bakery and were distributed to six different traditional Chinese markets., S. Enteritidis was confirmed as the vehicle of infection. Alertness in the emergency room, surveillance by the microbiology laboratory, prompt and thorough investigation to trace the source of outbreaks, and institution of appropriate control measures provide effective control of community outbreaks.The egg-covered bread contaminated with  Salmonellosis, resulting from the ingestion of contaminated poultry, beef, pork, eggs, and milk , is an iSalmonella species are relatively uncommon in Taiwan [Salmonella food-borne disease outbreaks but serovar Enteritidis has emerged as a new serovar in Taiwan [Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) infections [Food-borne disease outbreaks due to n Taiwan  comparedn Taiwan  and Japan Taiwan . Thirty-n Taiwan . Serovarn Taiwan , consistfections ,24,25.S. Enteritidis that was performed after notification of the rapid increase of the number of patients with febrile gastroenteritis in an emergency room and an unusually high percentage of Group D Salmonella isolated since it comprised only 7.4% of the various Salmonella groups in the hospital one year before the outbreak.We report the findings of the investigation of an outbreak of Salmonella infection. A cohort study of all patients attending the ER with acute gastroenteritis between July 28 and August 3, 2001 was performed in order to test the hypothesis that illness was caused by a specific food.A rapid increase in the number of attendees due to acute gastroenteritis and fever was noted to have begun on July 28, 2001 at the emergency room (ER) of Kaohsiung Municipal Hsiao-Kang Hospital, and this increase continued for the following six days. Clinical and demographic features and the food reported to have been consumed three days before development of gastrointestinal symptoms for all patients with acute gastroenteritis at the ER from July 28 to August 3, 2001 were reviewed by ER personnel and the infection control team. The infection control team conducted patient interviews and reviewed charts using a standardized case record form. Stool and blood cultures were performed once specimens were available. These specimens were further analyzed by the hospital's clinical microbiology laboratory and the laboratory of the Center for Disease Control, Taiwan (CDC, Taiwan) in Kaohsiung, Taiwan. Charts of all patients with and without acute gastroenteritis who visited the ER from six weeks before until two weeks after July 28, 2001 were reviewed by infection control nurses for the baseline data of the ER. An outbreak-associated case was defined as a patient visiting the ER with the diagnosis of acute gastroenteritis and having Methods for sample collection, cultivation and isolation were conducted according to the standard protocol of the CDC, Taiwan for food-borne disease outbreaks as described previously .Salmonella serotypes were determined with the use of antiserum (Difco, Detroit, MI, USA) according to the manufacturer's instructions. Serogrouping and serotyping were performed by the slide agglutination method and tube agglutination method to identify the somatic O antigen and flagellar H antigen, respectively [ectively .Escherichia coli ATCC 25922 was used as the quality control organism.Antimicrobial susceptibility was determined by the disk diffusion method according to the National Committee for Clinical Laboratory Standards . The antXbaI (New England Biolabs, Beverly, MA) was used at the manufacturer's suggested temperature. Restriction fragments were separated by PFGE in 1% agarose gel (Bio-Rad, Hercules, CA.) in 0.5X TBE buffer (45 mM Tris, 45 mM boric acid, 1.0 mM EDTA, pH 8.0) for 25 h at 200 V at a temperature of 14°C, with ramped times of 2 to 40 s using the Bio-Rad CHEF-DRII apparatus (Bio-Rad Laboratories, Richmond, CA). Gels were then stained with ethidium bromide and photographed under ultraviolet light. The resulting genomic-DNA profiles, or "fingerprints," were interpreted according to established guidelines [Total DNA was prepared and PFGE was performed as described previously ,13. The Salmonella infection were suspected as the vector of infection. A sample of the implicated food was collected from a patient's home within six hours of the patient having symptoms of febrile gastroenteritis and it was stored at 4°C. Further tracing the source of the suspected food and the subsequent environmental investigation were undertaken by the Bureau of Health, Kaohsiung County and CDC, Taiwan.Food items having a significant relationship with gastroenteritis cases with t-test. Significance was considered at a P value < 0.05 (Epi Info. Version 3.2.2 Centers for Disease Control and Prevention (CDC), USA).Differences between groups were compared using Chi-Square analysis. Relative risk and 95% confidence intervals were also calculated when Chi-Square analysis was used. Continuous variables were analyzed with Student's An increase in the number of patients admitted due to acute gastroenteritis and fever was noticed beginning from the early morning of July 28, 2001 with a return to normal levels one week later. Reviewing daily case-visits to the ER due to all causes and due to gastroenteritis six weeks before this outbreak period revealed significantly more cases due to all causes visiting the ER on Saturday and Sunday than on weekdays (From Monday to Friday) (mean ± standard deviation (S.D.): 140.3 ± 38.3 compared with 109.6 ± 16.7, P = 0.004). The number of patients visiting due to gastroenteritis on Saturday and Sunday were also higher than those visiting on weekdays (mean ± S.D.: 15.8 ± 7.3 compared with 10.5 ± 3.6, P = 0.032)  of 942 patients visiting the ER during the outbreak period had acute gastroenteritis. Compared with 85 (9.7%) of 872 patients in the week before the outbreak, significantly higher percentages of patients with gastroenteritis were observed in the outbreak week (Relative risk (RR): 1.92, 95% confidence interval (CI): 1.38 – 2.26, P < 0.001). A comparison of the number of patients with and without acute gastroenteritis (506 and 4467, respectively) during the six weeks before the outbreak week and during it also revealed a significant increase in the number of patients of gastroenteritis during the outbreak week .Salmonella compared to only 6 of 128 people who did not eat this bread (RR: 17.6, 95%CI 7.9–39.0, P < 0.001). The association of consuming the kind of bread and having Salmonella infection was significant . No other identified food or restaurant exposure was significantly associated with the outbreak.Investigation of food consumed by gastroenteritis patients during the outbreak period revealed 34 (21%) had consumed the same kind of egg covered bread decorated with mayonnaise and fried seasoned pork fiber from six traditional Chinese markets (three located in Kaohsiung City and three in Kaohsiung County). There were 2, 2, 2, 3, 3, and 22 gastroenteritis patients that consumed the bread purchased from 6 markets, respectively. In total, 28 of 34 patients who ate this bread were positive for Salmonella cases that consumed the implicated bread, 12 were male. Their age ranged from three to 71 years old. Eleven cases (39.3%) were under 18 years old and one case was over 65. The incubation periods ranged from 4 to 17 (median, mean ± S.D. 10, 9.4 ± 3.2) hours after consumption of the bread. Twenty-seven of the 28 patients were hospitalized. The clinical symptoms among the 28 cases included abdominal pain (100%), fever (100%), diarrhea (100%), vomiting (85.7%), chills (35.7%), and headache (7.1%). The laboratory data showed eight (28.6%) cases had white blood cell counts higher than 10000 /mm3. Positive occult blood reaction was found in stool specimens of 24 of 27 (88.9%) patients tested. Colitis was found in all four patients who received colonoscopy examination. The two bacteremia patients' fever subsided one day and two days after admission, respectively, without antimicrobial therapy. No mortality or sequellae occurred among these cases during hospitalization or in the three months' follow-up by infection control nurses.Regarding the 28 Salmonella and surveillance culture of the workplace surfaces were not performed.Further investigation by the city public health administration found the incriminated bread from the six markets was all produced by the same bakery that was prohibited from production of all kinds of bread on August 1, 2001. Because the bakery had stopped their production one day before the official prohibition, no further samples of the components of bread, such as the egg and mayonnaise, were available. Investigation of the bakery staff with stool cultures for Salmonella isolates. Twenty-eight Salmonella isolates were cultured from 32 available stool specimens from 34 patients who consumed the implicated food. These isolates were all of group D and were further identified as S. Enteritidis. This pathogen grew in two of the 34 patients' blood cultures. A Salmonella isolate of the same serovar was cultured from bread provided by a patient. The sample had been stored in the refrigerator because the patient had not finished eating it.In the week of this outbreak, there was an extraordinarily high percentage (94.1%) of group D of Salmonella was isolated from six of the 25 patients' stool specimens. For the six Salmonella isolates from cases not consuming the implicated food during the outbreak period, two were of group B and four were of group D. Further serovar analysis and PFGE analysis of the four group D isolates was not performed. Among the four group D isolates, two were resistant to ampicillin and trimethoprim/sulfamethoxazole, which was different to the antibiogram of the isolates from cases consuming the implicated bread.A total of 30 stool specimens from 25 patients not consuming the implicated bread were collected and All 28 stool isolates, two blood isolates and one food isolate were susceptible to the nine antimicrobial agents tested. Only two stool isolates showed unrelated PFGE patterns with more than six band differences compared to the epidemic PFGE pattern of the other isolates. All the other stool, blood, and food isolates had the same PFGE pattern indicating a clonal relationship  had not consumed the implicated bread, Fig.  suggestiS. Enteritidis [S. Enteritidis [Salmonella isolates.Compared with phage typing  and plaseritidis ,19 thougeritidis . AnalytiSalmonella groups and knowledge that the percentage of group D Salmonella patients was low before. Thus, the unusually high percentage of group D Salmonella seen within the week of the outbreak led to the investigation. The implicated vehicle of infection in the outbreak was an egg-covered bun topped with mayonnaise and fried seasoned pork fiber which had been distributed to different markets after production at the same bakery. Epidemiological study, antibiograms, and serologic and molecular typing patterns revealed that almost all cases of S. Enteritidis infection during the period were the same as that of the bread isolate. Only the implicated baked good from the same bakery caused Salmonella infections while other items from the bakery were not found to be epidemiologically related with the outbreak. This finding suggests contamination of the pathogen did not occur during the common routes of production, transportation, or selling of goods from the bakery. Although the bread sold in the traditional Chinese markets frequently does not conform to sanitary requirements, and although the staff involved in distribution and selling are not subject to the routine hygiene inspections, in food stores, the possibility of contamination of a specific bakery product simultaneously at six markets in the same time period is very low, suggesting that the contamination occurred during the production process. Food contamination in the outbreak was traced to the same bakery but an investigation of infection among the bakery staff and sources of the contents of the bread was not conducted. Although the baking process involves high temperatures sufficient to kill pathogens, the manual addition of toppings or flavors, such as mayonnaise, eggs, and meat products provide possibilities for contamination with food-borne pathogens. In addition, insufficient baking may be a risk factor for human health because it may not destroy microbial contamination.Outbreaks can be missed easily if the increase in case number is not noticed due to the wide distribution of outbreak sources in different areas or if a relatively small outbreak occurs among a large population and cases are exposed to the outbreak source at different times. Recognition of the outbreak reported in this study was aided by the routine surveillance of Salmonella serovars have been related specifically with some foods. S. Enteritidis is particularly related to eggs [S. Enteritidis outbreaks related to baked goods[Salmonella isolated from the bread had the same serovar, antibiogram, and PFGE pattern as isolates from patients and the significant relationship between consumption of this baked good and the isolation of Salmonella implicated this food product as the vehicle of contamination.Different  to eggs -28. In tked goods,5,7,8, aSalmonella outbreaks [This outbreak had a relatively short incubation compared to the 24–72 hour range reported for other utbreaks ,30. WhetSalmonella Enteritidis outbreaks.For many food-borne outbreaks, the pathogens and transmission vehicles are often not identified, usually because of delayed collection of epidemiologic and microbiologic information . InitiatThe author(s) declare that they have no competing interest.Po-Liang Lu and L. K. Siu were in charge of the investigation, data handling and writing of the manuscript. Po-Liang Lu and Shang- Jyh Hwang participated in the design of the study. Po-Liang Lu, In-Jane Hwang, Ya-Lina Tung and Shang-Jyh Hwang were in the investigation team for data collecting and data analysis. Po-Liang Lu performed the statistical analysis. Chun-Lu Lin and L. K. Siu carried out microbiological assays. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here: